COMPOSITIONS AND METHODS FOR THE TREATMENT OF BACTERIAL INFECTIONS
20230190950 · 2023-06-22
Inventors
- Suzanne AKERS-RODRIGUEZ (La Mesa, CA, US)
- James Michael BALKOVEC (Martinsville, NJ, US)
- Daniel C. BENSEN (Carlsbad, CA, US)
- Timothy Blizzard (Princeton, NJ)
- Allen Borchardt (San Diego, CA, US)
- Thomas Patrick BRADY (San Diego, CA, US)
- Zhi-Yong Chen (La Jolla, CA, US)
- Quyen-Quyen Thuy DO (San Diego, CA, US)
- Joanne M. FORTIER (San Diego, CA, US)
- Wanlong Jiang (San Diego, CA)
- Thanh Lam (San Diego, CA, US)
- Alain NONCOVICH (La Jolla, CA, US)
- Leslie W. Tari (Solana Beach, CA, US)
Cpc classification
A61K47/65
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
A61K47/6811
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
A61K47/68
HUMAN NECESSITIES
A61K47/65
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
Abstract
Compositions and methods for the treatment of bacterial infections include conjugates containing an Fc domain covalently linked to one or more monomers of cyclic heptapeptides or one or more dimers of cyclic heptapeptides. In particular, conjugates can be used in the treatment of bacterial infections caused by Gram-negative bacteria.
Claims
1.-34. (canceled)
35. A conjugate described by formula (D-Ia): ##STR01141## wherein each M1 comprises a first cyclic heptapeptide comprising a linking nitrogen and each M2 comprises a second cyclic heptapeptide comprising a linking nitrogen; L′ in each M2-L′-M1 is a linker covalently attached to the Fc domain monomer and to the linking nitrogen in each of M1 and M2; each E is an Fc domain monomer; n is 1 or 2; T is an integer from 1 to 20, or a pharmaceutically acceptable salt thereof.
36. (canceled)
37. The conjugate of claim 35, wherein L′ in each M2-L′-M1 is described by formula (D-L): ##STR01142## wherein L is a remainder of L′; A1 is a 1-5 amino acid peptide covalently attached to the linking nitrogen in each M1 or is absent; and A2 is a 1-5 amino acid peptide covalently attached to the linking nitrogen in each M2 or is absent.
38. The conjugate of claim 35, wherein the conjugate is described by formula (D-II): ##STR01143## wherein L is a remainder of L′; each of R.sup.1, R.sup.12, R′.sup.1, and R′.sup.12 is, independently, a lipophilic moiety, a polar moiety, or H; each of R.sup.11, R.sup.13, R.sup.14, R′.sup.11, R′.sup.13, and R′.sup.14 is, independently, optionally substituted C1-C5 alkamino, a polar moiety, a positively charged moiety, or H; each of R.sup.15 and R′.sup.15 is, independently, a lipophilic moiety or a polar moiety; each of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R′.sup.2, R′.sup.3, R′.sup.4, R′.sup.5, R′.sup.6, R′.sup.7, R′.sup.8, R′.sup.9, and R′.sup.10 is, independently, a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted C1-C5 alkamino, optionally substituted C1-C20 alkyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C5-C15 aryl, optionally substituted C1-C20 heteroalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C2-C15 heteroaryl, optionally substituted C6-C35 alkaryl, or optionally substituted C6-C35 heteroalkaryl; or two R groups on the same or adjacent atoms join to form an optionally substituted 5-8 membered ring; each of a′, b′, c′, a, b, and c is, independently, 0 or 1; each of N.sup.1, N.sup.2, N.sup.3, N.sup.4, N′.sup.1, N′.sup.2, N′.sup.3, and N′.sup.4 is a nitrogen atom; each of C.sup.1, C.sup.2, C.sup.3, C′.sup.1, C′.sup.2, and C′.sup.3 is a carbon atom, or a pharmaceutically acceptable salt thereof.
39.-52. (canceled)
53. The conjugate of claim 38, wherein the conjugate is described by formula (D-IV): ##STR01144## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2, or a pharmaceutically acceptable salt thereof.
54.-57. (canceled)
58. The conjugate of claim 53, wherein the conjugate is described by formula (D-V): ##STR01145## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; each of R.sup.2, R.sup.6, R′.sup.2, and R′.sup.6 is, independently, a positively charged moiety, a polar moiety, optionally substituted C1-C5 alkamino, optionally substituted C1-C20 alkyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C5-C15 aryl, optionally substituted C1-C20 heteroalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C2-C15 heteroaryl, optionally substituted C6-C35 alkaryl, or optionally substituted C6-C35 heteroalkaryl, or a pharmaceutically acceptable salt thereof.
59. The conjugate of claim 58, wherein the conjugate is described by formula (D-V-1): ##STR01146## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; or a pharmaceutically acceptable salt thereof.
60. (canceled)
61. The conjugate of claim 58, wherein the conjugate is described by formula (D-V-3): ##STR01147## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; or a pharmaceutically acceptable salt thereof.
62. (canceled)
63. (canceled)
64. The conjugate of claim 58, wherein the conjugate is described by formula (D-V-6): ##STR01148## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; each of R′.sup.16 and R.sup.16 is, independently, a lipophilic moiety, optionally substituted C1-C20 alkyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C5-C15 aryl, optionally substituted C1-C20 heteroalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C2-C15 heteroaryl, optionally substituted C6-C35 alkaryl, or optionally substituted C6-C35 heteroalkaryl; or a pharmaceutically acceptable salt thereof.
65. (canceled)
66. The conjugate of claim 58, wherein the conjugate is described by formula (D-V-8): ##STR01149## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; each of R′.sup.16 and R.sup.16 is, independently, a lipophilic moiety, optionally substituted C1-C20 alkyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C5-C15 aryl, optionally substituted C1-C20 heteroalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C2-C15 heteroaryl, optionally substituted C6-C35 alkaryl, or optionally substituted C6-C35 heteroalkaryl; or a pharmaceutically acceptable salt thereof.
67.-69. (canceled)
70. The conjugate of claim 38, wherein the conjugate is described by formula (D-VI): ##STR01150## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; each of R.sup.2, R.sup.6, R.sup.8, R′.sup.2, R′.sup.6, and R′.sup.8 is, independently, a positively charged moiety, a polar moiety, optionally substituted C1-C5 alkamino, optionally substituted C1-C20 alkyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C5-C15 aryl, optionally substituted C1-C20 heteroalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C2-C15 heteroaryl, optionally substituted C6-C35 alkaryl, or optionally substituted C6-C35 heteroalkaryl, or a pharmaceutically acceptable salt thereof.
71. The conjugate of claim 70, wherein the conjugate is described by formula (D-VI-1): ##STR01151## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; or a pharmaceutically acceptable salt thereof.
72.-81. (canceled)
82. The conjugate of claim 70, wherein the conjugate is described by formula (D-VI-3): ##STR01152## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; or a pharmaceutically acceptable salt thereof.
83. (canceled)
84. (canceled)
85. The conjugate of claim 70, wherein the conjugate is described by formula (D-VI-6): ##STR01153## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; each of R′.sup.16 and R.sup.16 is, independently, a lipophilic moiety, optionally substituted C1-C20 alkyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C5-C15 aryl, optionally substituted C1-C20 heteroalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C2-C15 heteroaryl, optionally substituted C6-C35 alkaryl, or optionally substituted C6-C35 heteroalkaryl; or a pharmaceutically acceptable salt thereof.
86.-92. (canceled)
93. The conjugate of claim 70, wherein the conjugate is described by formula (D-VI-8): ##STR01154## wherein each of R′.sup.1 and R.sup.1 is, independently, benzyl or CH.sub.2CH(CH.sub.3).sub.2; each of R′.sup.16 and R.sup.16 is, independently, a lipophilic moiety, optionally substituted C1-C20 alkyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C5-C15 aryl, optionally substituted C1-C20 heteroalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C2-C15 heteroaryl, optionally substituted C6-C35 alkaryl, or optionally substituted C6-C35 heteroalkaryl; or a pharmaceutically acceptable salt thereof.
94.-135. (canceled)
136. The conjugate of claim 35, wherein L′, L, or L.sup.1 comprises one or more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino, wherein R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl.
137.-141. (canceled)
142. The conjugate of claim 35, wherein each L is described by formula (D-L-I): ##STR01155## wherein L.sup.A is described by formula G.sup.A1-(Z.sup.A1).sub.g1—(Y.sup.A1).sub.h1—(Z.sup.A2).sub.i1—(Y.sup.A2).sub.j1—(Z.sup.A3).sub.k1—(Y.sup.A3).sub.l1—(Z.sup.A4).sub.m1—(Y.sup.A4).sub.n1—(Z.sup.A5).sub.O1-G.sup.A2; L.sup.B is described by formula G.sup.B1-(Z.sup.B1).sub.g2—(Y.sup.B1).sub.h2—(Z.sup.B2).sub.i2—(Y.sup.B2).sub.j2—(Z.sup.B3).sub.k2—(Y.sup.B3).sub.l2—(Z.sup.B4).sub.m2—(Y.sup.B4).sub.n2—(Z.sup.B5).sub.O2-G.sup.B2; L.sup.C is described by formula G.sup.C1-(Z.sup.C1).sub.g3—(Y.sup.C1).sub.h3—(Z.sup.C2).sub.i3—(Y.sup.C2).sub.j3—(Z.sup.C3).sub.k3—(Y.sup.C3).sub.l3—(Z.sup.C4).sub.m3—(Y.sup.C4).sub.n3—(Z.sup.C5).sub.O3-GC.sup.2; G.sup.A1 is a bond attached to Q in formula (L-I); G.sup.A2 is a bond attached to A1 or M1 if A1 is absent; G.sup.B1 is a bond attached to Q in formula (L-I); G.sup.B2 is a bond attached to A2 or M2 if A2 is absent; G.sup.C1 is a bond attached to Q in formula (L-I); G.sup.C2 is a bond attached to E; each of Z.sup.A1, Z.sup.A2, Z.sup.A3, Z.sup.A4, Z.sup.A5, Z.sup.B1, Z.sup.B2, Z.sup.B3, Z.sup.B4, Z.sup.B5, Z.sup.C1 Z.sup.C2, Z.sup.C3, Z.sup.C4, and Z.sup.C5 is, independently, optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene, or optionally substituted C2-C15 heteroarylene; each of Y.sup.A1, Y.sup.A2, Y.sup.A3, Y.sup.A4, Y.sup.B1, Y.sup.B2, Y.sup.B3, Y.sup.B4, Y.sup.C1, Y.sup.C2, Y.sup.C3, and Y.sup.C4 is, independently, O, S, NR.sup.i, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino; R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl; each of g1, h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, l2, m2, n2, o2, g3, h3, i3, j3, k3, l3, m3, n3, and o3 is, independently, 0 or 1; Q is a nitrogen atom, optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene, or optionally substituted C2-C15 heteroarylene.
143. (canceled)
144. The conjugate of claim 35, wherein E has the sequence of any one of SEQ ID NOs: 1-31.
145. The conjugate of claim 35, wherein when n is 2, E dimerizes to form an Fc domain.
146.-532. (canceled)
533. A pharmaceutical composition comprising a conjugate of claim 35, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
534.-538. (canceled)
539. A method of protecting against or treating a bacterial infection in a subject, said method comprising administering to said subject a conjugate of claim 35.
540.-569. (canceled)
Description
DESCRIPTION OF THE DRAWINGS
[0456]
[0457]
[0458]
[0459]
[0460]
[0461]
[0462]
[0463]
[0464]
[0465]
[0466]
[0467]
[0468]
[0469]
[0470]
[0471]
[0472]
[0473]
[0474]
[0475]
[0476]
[0477]
[0478]
[0479]
[0480]
[0481]
[0482]
[0483]
[0484]
[0485]
[0486]
[0487]
[0488]
[0489]
[0490]
[0491]
[0492]
[0493]
[0494]
[0495]
[0496]
[0497]
[0498]
[0499]
[0500]
[0501]
[0502]
[0503]
[0504]
[0505]
[0506]
[0507]
[0508]
[0509]
[0510]
[0511]
[0512]
[0513]
[0514]
[0515]
[0516]
[0517]
[0518]
[0519]
[0520]
[0521]
[0522]
[0523]
[0524]
[0525]
[0526]
[0527]
[0528]
[0529]
[0530]
[0531]
[0532]
[0533]
[0534]
[0535]
[0536]
[0537]
[0538]
[0539]
[0540]
[0541]
[0542]
[0543]
[0544]
[0545]
[0546]
[0547]
[0548]
[0549]
[0550]
[0551]
[0552]
[0553]
[0554]
[0555]
[0556]
[0557]
[0558]
[0559]
[0560]
DETAILED DESCRIPTION
[0561] The disclosure features conjugates, compositions, and methods for the treatment of bacterial infections (e.g., Gram-negative bacterial infections). The conjugates disclosed herein include monomers or dimers of cyclic heptapeptides (e.g., two polymyxin cores) conjugated to Fc domains. The dimers of cyclic heptapeptides are linked to each other through a linker and/or one or two peptides (e.g., a peptide including a 1-5 amino acid residue(s)). The monomers or dimers of cyclic heptapeptides in the conjugates described herein bind to lipopolysaccharides (LPS) in the cell membrane of Gram-negative bacteria to disrupt and permeabilize the cell membrane, leading to cell death and/or sensitization of the Gram-negative bacteria to other antibiotics. The Fc domains in the conjugates bind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells, e.g., neutrophils, to activate phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of bacterial cells by immune cells and further enhancing the antibacterial activity of the conjugates.
I. Bacterial Infections
[0562] Pathogenic bacteria cause bacterial infections and diseases such as tuberculosis, pneumonia, and foodborne illnesses. Bacteria may be categorized into two major types: Gram-positive bacteria and Gram-negative bacteria. Gram-positive bacteria possess a thick cell wall containing multiple layers of peptidoglycan and teichoic acids, while Gram-negative bacteria have a relatively thin cell wall containing fewer layers of peptidoglycan that are surrounded by a second lipid membrane containing lipopolysaccharides (LPS) and lipoproteins. LPS, also called endotoxins, are composed of polysaccharides and lipid A. These differences in bacterial cell wall structure can produce differences in antibiotic susceptibility. Examples of Gram-positive bacteria include, but are not limited to, bacteria in the genus Streptococcus (e.g., Streptococcus pyogenes), bacteria in the genus Staphylococcus (e.g., Staphylococcus cohnii), bacteria in the genus Corynebacterium (e.g., Corynebacterium auris), bacteria in the genus Listeria (e.g., Listeria grayi), bacteria in the genus Bacillus (e.g., Bacillus aerius), and bacteria in the genus Clostridium (e.g., Clostridium acetium). Examples of Gram-negative bacteria include, but are not limited to, bacteria in the genus Escherichia (e.g., Escherichia coli), bacteria in the genus Klebsiella (e.g., Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella terrigena, and Klebsiella variicola), bacteria in the genus Acinetobacter (e.g., Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter kookii, and Acinetobacter junii), bacteria in the genus Pseudomonas (e.g., Pseudomonas aeruginosa), bacteria in the genus Neisseria (e.g., Neisseria gonorrhoeae), bacteria in the genus Yersinia (e.g., Yersinia pestis), bacteria in the genus Vibrio (e.g., Vibrio cholerae), bacteria in the genus Campylobacter (e.g., Campylobacter jejuni), and bacteria in the genus Salmonella (e.g., Salmonella enterica).
[0563] Bacteria may evolve to become more or fully resistant to antibiotics. Resistance may arise through natural resistance in certain types of bacteria, spontaneous random genetic mutations, and/or by inter- or intra-species horizontal transfer of resistance genes. Resistant bacteria are increasingly difficult to treat, requiring alternative medications or higher doses, which may be more costly or more toxic. Bacteria resistant to multiple antibiotics are referred to as multidrug resistant (MDR) bacteria. For example, the mcr-1 gene encodes a phosphoethanolamine transferase (MCR-1) which confers resistance to colistin, a natural polymyxin, through modification of LPS. This is the first known horizontally-transferable resistance determinant for the polymyxin class of antibiotics. The mcr-1 gene has also been found in bacterial strains which already possess resistance to other classes of antibiotics, such as in carbapenem-resistant Enterobacteriaceae (CRE). An mcr-1 resistance plasmid refers to a bacterial plasmid that carries mcr-1 alone or in combination with other antibiotic resistance genes. A mcr-1 resistance plasmid refers to a bacterial plasmid that carries one or more antibiotic resistance genes. Examples of mcr-1 resistance plasmids include, but are not limited to, pHNSHP45, pMR0516mcr, pESTMCR, pAF48, pAF23, pmcr1-IncX4, pmcr1-Incl2, pA31-12, pVT553, plCBEC72Hmcr, pE15004, pE15015, and pE15017.
[0564] In another example, the mcr-2 gene also confers resistance to colistin. The mcr-2 gene was identified in porcine and bovine colistin-resistance E. coli that did not contain mcr-1 (Xavier et al., Euro Surveill 21(27), 2016). The mcr-2 gene is a 1,617 bp phspoethanolamine transferase harbored on an IncX4 plasmid. The mcr-2 gene has 76.7% nucleotide identity to mcr-1. Analysis of mcr-2 harboring plasmids from E. coli isolates shows that the mobile element harboring mcr-2 is an IS element of the IS.sub.1595 superfamily, which are distinguished by the presence of an ISXO.sub.2-like transposase domain (Xavier et al., supra). The MCR-2 protein was predicted to have two domains, with domain 1 (1-229 residues) as a transporter and domain 2 (230-538 residues) as a transferase domain. An mcr-2 resistance plasmid refers to a bacterial plasmid that carries mcr-2 alone or in combination with other antibiotic resistance genes. A mcr-2 resistance plasmid refers to a bacterial plasmid that carries one or more antibiotic resistance genes. Mcr-2 resistance plasmids include, but are not limited to, pKP37-BE and pmcr2-IncX4.
[0565] Furthermore, resistant strain E. coli BAA-2469 possesses the New Delhi metallo-β-lactamase (NDM-1) enzyme, which makes bacteria resistant to a broad range of β-lactam antibiotics. Additionally, E. coli BAA-2469 is also known to be resistant to penicillins (e.g., ticarcillin, ticarcillin/clavulanic acid, piperacillin, ampicillin, and ampicillin/sulbactam), cephalosporins (e.g., cefalotin, cefuroxime, cefuroxime, cefotetan, cefpodoxime, cefotaxime, ceftizoxime, cefazolin, cefoxitin, ceftazidime, ceftriaxone, and cefepime), carbapenems (e.g., doripenem, meropenem, ertapenem, imipenem), quinolones (e.g., nalidixic acid, moxifloxacin, norfloxacin, ciprofloxacin, and levofloxacin), aminoglycosides (e.g., amikacin, gentamicin, and tobramycin), and other antibiotics (e.g., tetracycline, tigecycline, nitrofurantoin, aztreonam, trimethoprim/sulfamethoxazole).
[0566] In some embodiments, a resistant strain of bacteria possesses the mcr-1 gene, the mcr-2 gene, and/or a chromosomal mutation conferring polymyxin resistance. In some embodiments, a resistant strain of bacteria is a resistant strain of E. coli (e.g., E. coli BAA-2469).
II. Conjugates of the Disclosure
[0567] Provided herein are synthetic conjugates useful in the treatment of bacterial infections (e.g., Gram-negative bacterial infections). The conjugates disclosed herein include an Fc domain conjugated to one or more monomers of cyclic heptapeptides or one or more dimers of two cyclic heptapeptides. The dimers of two cyclic heptapeptides include a first cyclic heptapeptide (e.g., a first polymyxin core) and a second cyclic heptapeptide (e.g., a second polymyxin core). The first and second cyclic heptapeptides are linked to each other by way of a linker and/or one or two peptides (e.g., each peptide including a 1-5 amino acid residue(s)).
[0568] Without being bound by theory, in some aspects, conjugates described herein bind to the cell membrane of Gram-negative bacteria (e.g., bind to LPS in the cell membrane of Gram-negative bacteria) through the interactions between the cyclic heptapeptides in the conjugates and the cell membrane of Gram-negative bacteria. The binding of the conjugates to the cell membrane of Gram-negative bacteria disrupt and permeabilize the cell membrane, leading to cell death and/or sensitization of the Gram-negative bacteria to other antibiotics. In some embodiments, the initial association of the conjugates with the bacterial cell membrane occurs through electrostatic interactions between the cyclic heptapeptides in the conjugates and the anionic LPS in the outer membrane of Gram-negative bacteria, disrupting the arrangement of the cell membrane. Specifically, conjugates described herein may bind to lipid A in the LPS. More specifically, the cyclic heptapeptides in the conjugates described herein may bind to one or both phosphate groups in lipid A. In some embodiments, antibiotic-resistant bacteria (e.g., antibiotic-resistant, Gram-negative bacteria) has one phosphate group in lipid A. In some embodiments, conjugates described herein may bind to multiple Gram-negative bacterial cells at the same time. The binding of the conjugates described herein to the LPS may also displace Mg.sup.2+ and Ca.sup.2+ cations that bridge adjacent LPS molecules, causing, e.g., membrane permeabilization, leakage of cellular molecules, inhibition of cellular respiration, and/or cell death. Furthermore, in addition to disrupting the cell membrane of Gram-negative bacteria, the Fc domain in the conjugates described herein binds to the FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells and serves as a gradient against which immune cells chemotax to the site of bacterial infection and/or growth. The binding of the Fc domain in the conjugates described herein to the FcγRs on immune cells activates phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of Gram-negative bacterial cells by immune cells and further enhancing the antibacterial activity of the conjugates.
[0569] Conjugates provided herein are described by any one of formulas (I)-(XXXIII). In some embodiments, the conjugates described herein include one or more monomers of cyclic heptapeptides conjugated to an Fc domain. In some embodiments, the conjugates described herein include one or more dimers of cyclic heptapeptides conjugated to an Fc domain. In some embodiments, when n is 2, E (an Fc domain monomer) dimerizes to form an Fc domain. Conjugates described herein may be synthesized using available chemical synthesis techniques in the art. In some embodiments, available functional groups in the cyclic heptapeptides and the linker, e.g., amines, carboxylic acids, maleimides, bis-sulfones, azides, alkynes, and/or hydroxyl groups, may be used in making the conjugates described herein. For example, the linking nitrogen (described further herein) in a cyclic heptapeptide may form an amide bond with the carbon in a carboxylic acid group in the linker. A peptide including one or more (e.g., 1-3; 1, 2, or 3) amino acid residues (e.g., natural and/or non-natural amino acid residues) may be also be covalently attached to the linking nitrogen of the cyclic heptapeptide through forming an amide bond between the carbon in a carboxylic acid group in the peptide and the linking nitrogen. In cases where a functional group is not available for conjugation, a molecule may be derivatized using conventional chemical synthesis techniques that are well known in the art. In some embodiments, the conjugates described herein contain one or more chiral centers. The conjugates include each of the isolated stereoisomeric forms as well as mixtures of stereoisomers in varying degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers, enantiomers, and tautomers that can be formed.
[0570] Cyclic Heptapeptide or Polymyxin Core
[0571] In some aspects, a cyclic heptapeptide or polymyxin core, as used herein, refers to certain compounds that bind to lipopolysaccharides (LPS) in the cell membrane of Gram-negative bacteria to disrupt and permeabilize the cell membrane, leading to cell death and/or sensitization to other antibiotics. In some aspects, cyclic heptapeptide, as used herein, refers to certain compounds that kill or inhibit the growth of Gram-negative bacteria as determined by measuring the minimum inhibitory concentration (MIC) against at least one Gram-negative bacteria as known in the art, wherein the MIC is 128 μg/mL or less.
[0572] Cyclic heptapeptides are composed of, at least, amino acid residues, each of which may, independently, may have a D- or L-configuration, assembled as a cyclic heptapeptide ring. A cyclic heptapeptide includes seven natural or non-natural amino acid residues attached to each other in a closed ring. The ring contains six bonds formed by linking the carbon in the α-carboxyl group of one amino acid residue to the nitrogen in the α-amino group of the adjacent amino acid residue and one bond formed by linking the carbon in the α-carboxyl group of one amino acid residue to the nitrogen in the γ-amino group in the side chain of the adjacent amino acid residue. For the amino acid residue whose nitrogen in the γ-amino group in the side chain participates in forming the ring, the nitrogen in the α-amino group of this amino acid residue does not participate directly in forming the ring and serves as the linking nitrogen (thus, referred to as the “linking nitrogen” herein) that links one cyclic heptapeptide or polymyxin core to an Fc domain by way of a linker and/or one or two peptides (e.g., a peptide including a 1-5 amino acid residue(s)), or in the case of a conjugate including an Fc domain covalently linked to one or more dimers of cyclic heptapeptides or polymyxin cores, the linking nitrogen serves to link one cyclic heptapeptide or polymyxin core to another cyclic heptapeptide or polymyxin core. Conjugates described herein are separated into two types: (1) one or more dimers of cyclic heptapeptides conjugated to an Fc domain and (2) one or more monomers of cyclic heptapeptides conjugated to an Fc domain. The dimers of cyclic heptapeptides (e.g., two polymyxin cores) are linked to each other at their linking nitrogens through a linker and/or one or two peptides (e.g., a peptide including a 1-5 amino acid residue(s)).
[0573] In some embodiments, a peptide including one or more (e.g., 1-5; 1, 2, 3, 4, or 5) amino acid residues (e.g., natural and/or non-natural amino acid residues) may be covalently attached to the linking nitrogen of the cyclic heptapeptide or the polymyxin core.
[0574] Cyclic heptapeptides or polymyxin cores may be derived from polymyxins (e.g., naturally existing polymyxins and non-natural polymyxins) and/or octapeptins (e.g., naturally existing octapeptins and non-natural octapeptins). In some embodiments, cyclic heptapeptides may be compounds described in Gallardo-Godoy et al., J. Med. Chem. 59:1068, 2016 (e.g., compounds 11-41 in Table 1 of Gallardo-Godoy et al.), which is incorporated herein by reference in its entirety. Examples of naturally existing polymyxins and their structures are shown in Table 1A. Examples of non-natural polymyxins and their structures are shown in Table 1B.
TABLE-US-00017 TABLE 1A Natural polymyxins and their structures
TABLE-US-00018 TABLE 1B Non-natural polymyxins and their structures (Dab: diaminobutyric acid; Dap: diaminopropionic acid; Orn: ornithine; Abu: 2-aminobutyric acid; Nle: norleucine)
[0575] Conjugates of Dimers of Cyclic Heptapeptides Linked to an Fc Domain
[0576] The conjugates described herein an Fc domain covalently linked to one or more dimers of cyclic heptapeptides. The dimers of two cyclic heptapeptides include a first cyclic heptapeptide (e.g., a first polymyxin core) and a second cyclic heptapeptide (e.g., a second polymyxin core). The first and second cyclic heptapeptides are linked to each other by way of a linker and/or one or two peptides (e.g., each peptide including a 1-5 amino acid residue(s)). In some embodiments of the dimers of cyclic heptapeptides, the first and second cyclic heptapeptides are the same. In some embodiments, the first and second cyclic heptapeptides are different. The disclosure provides a conjugate, or a pharmaceutically acceptable salt thereof, described by formula (I):
[0577] In some embodiments, the disclosure provides a conjugate, or a pharmaceutically acceptable salt thereof, described by the formulae below:
##STR00736## ##STR00737## ##STR00738## ##STR00739## ##STR00740## ##STR00741## ##STR00742## ##STR00743## ##STR00744## ##STR00745## ##STR00746## ##STR00747## ##STR00748## ##STR00749## ##STR00750## ##STR00751## ##STR00752## ##STR00753## ##STR00754## ##STR00755## ##STR00756## ##STR00757## ##STR00758## ##STR00759## ##STR00760## ##STR00761##
[0578] In some embodiments, when a is 1 in the conjugate of formula (II), (i) each of R.sup.2, R.sup.3, and R.sup.4 is, independently, a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (ii) R.sup.2, R.sup.3, and C.sup.1 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted cycloalkyl, optionally substituted heterocycloalkyl comprising 1 or 2 heteroatoms independently selected from N, O, and S, and R.sup.4 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (iii) R.sup.3, R.sup.4, N.sup.1, and C.sup.1 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted heterocycloalkyl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, or optionally substituted heteroaryl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, and R.sup.2 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl.
[0579] In some embodiments, when b is 1 in the conjugate of formula (II), (i) each of R.sup.5, R.sup.6, and R.sup.7 is, independently, a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (ii) R.sup.5, R.sup.6, and C.sup.2 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted cycloalkyl, optionally substituted heterocycloalkyl comprising 1 or 2 heteroatoms independently selected from N, O, and S, and R.sup.7 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (iii) R.sup.6, R.sup.7, N.sup.2, and C.sup.2 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted heterocycloalkyl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, or optionally substituted heteroaryl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, and R.sup.5 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl.
[0580] In some embodiments, when c is 1 in the conjugate of formula (II), (i) each of R.sup.8, R.sup.9, and R.sup.10 is, independently, a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (ii) R.sup.8, R.sup.9, and C.sup.3 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted cycloalkyl, optionally substituted heterocycloalkyl comprising 1 or 2 heteroatoms independently selected from N, O, and S, and R.sup.10 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (iii) R.sup.9, R.sup.10, N.sup.3, and C.sup.3 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted heterocycloalkyl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, or optionally substituted heteroaryl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, and R.sup.8 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl.
[0581] In some embodiments, when a′ is 1 in the conjugate of formula (II), (i) each of R′.sup.2, R′.sup.3, and R′.sup.4 is, independently, a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (ii) R′.sup.2, R′.sup.3, and C′.sup.1 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted cycloalkyl, optionally substituted heterocycloalkyl comprising 1 or 2 heteroatoms independently selected from N, O, and S, and R′.sup.4 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (iii) R′.sup.3, R′.sup.4, N′.sup.1, and C′.sup.1 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted heterocycloalkyl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, or optionally substituted heteroaryl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, and R′.sup.2 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl.
[0582] In some embodiments, when b′ is 1 in the conjugate of formula (II), (i) each of R′.sup.5, R′.sup.6, and R′.sup.7 is, independently, a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (ii) R′.sup.5, R′.sup.6, and C′.sup.2 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted cycloalkyl, optionally substituted heterocycloalkyl comprising 1 or 2 heteroatoms independently selected from N, O, and S, and R′.sup.7 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (iii) R′.sup.6, R′.sup.7, N′.sup.2, and C′.sup.2 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted heterocycloalkyl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, or optionally substituted heteroaryl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, and R′.sup.5 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl.
[0583] In some embodiments, when c′ is 1 in the conjugate of formula (II), (i) each of R′.sup.8, R′.sup.9, and R′.sup.10 is, independently, a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (ii) R′.sup.8, R′.sup.9, and C′.sup.3 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted cycloalkyl, optionally substituted heterocycloalkyl comprising 1 or 2 heteroatoms independently selected from N, O, and S, and R′.sup.10 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl; or (iii) R′.sup.9, R′.sup.10, N′.sup.3, and C′.sup.3 together form a ring (e.g., an optionally substituted 5-8 membered ring) comprising optionally substituted heterocycloalkyl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, or optionally substituted heteroaryl comprising an N heteroatom and additional 0-2 heteroatoms independently selected from N, O, and S, and R′.sup.8 is a lipophilic moiety, a positively charged moiety, a polar moiety, H, optionally substituted alkamino, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenylene, optionally substituted cycloalkynylene, optionally substituted aryl, optionally substituted heteroalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkenylene, optionally substituted heterocycloalkynylene, optionally substituted heteroaryl, optionally substituted alkaryl, or optionally substituted heteroalkaryl.
[0584] In the conjugates described herein, the squiggly line connected to E indicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) dimers of cyclic heptapeptides may be attached to an Fc domain monomer or an Fc domain. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) dimers of cyclic heptapeptides may be attached to an Fc domain monomer. In some embodiments, when n is 2, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) dimers of cyclic heptapeptides may be attached to an Fc domain. The squiggly line in the conjugates described herein is not to be construed as a single bond between one or more dimers of cyclic heptapeptides and an atom in the Fc domain monomer. In some embodiments, when T is 1, one dimer of cyclic heptapeptides may be attached to an atom in the Fc domain monomer or Fc domain. In some embodiments, when T is 2, two dimers of cyclic heptapeptides may be attached to an atom in the Fc domain monomer or Fc domain.
[0585] As described further herein, a linker in a conjugate described herein (e.g., L′, L, or L.sup.1) may be a branched structure. As described further herein, a linker in a conjugate described herein (e.g., L′, L, or L.sup.1) may be a multivalent structure, e.g., a divalent or trivalent structure having two or three arms, respectively. In some embodiments when the linker has three arms, two of the arms may be attached to the first and second cyclic heptapeptides and the third arm may be attached to the Fc domain monomer or Fc domain.
[0586] In conjugates having an Fc domain covalently linked to one or more dimers of cyclic heptapeptides, as represented by the formulae above, when n is 2, two Fc domain monomers (each Fc domain monomer is represented by E) dimerize to form an Fc domain.
[0587] Conjugates of Monomers of Cyclic Heptapeptides Linked to an Fc Domain
[0588] In some embodiments, the conjugates described herein include an Fc domain covalently linked to one or more monomers of cyclic heptapeptides. Conjugates of an Fc domain and one or more monomers of cyclic heptapeptides may be formed by linking the Fc domain to each of the monomers of cyclic heptapeptides through a linker and/or one or more amino acids (e.g., a peptide including a 1-5 amino acid residue(s)).
[0589] In the conjugates having an Fc domain covalently linked to one or more monomers of cyclic heptapeptides described herein, the squiggly line connected to E indicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers of cyclic heptapeptides may be attached to an Fc domain monomer or an Fc domain. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) monomers of cyclic heptapeptides may be attached to an Fc domain monomer. In some embodiments, when n is 2, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers of cyclic heptapeptides may be attached to an Fc domain. The squiggly line in the conjugates described herein is not to be construed as a single bond between one or more monomers of cyclic heptapeptides and an atom in the Fc domain monomer or Fc domain. In some embodiments, when T is 1, one monomer of cyclic heptapeptide may be attached to an atom in the Fc domain monomer or Fc domain. In some embodiments, when T is 2, two monomers of cyclic heptapeptides may be attached to an atom in the Fc domain monomer or Fc domain.
[0590] As described further herein, a linker in a conjugate having an Fc domain covalently linked to one or more monomers of cyclic heptapeptides described herein (e.g., L′ or L) may be a divalent structure having two arms. One arm in a divalent linker may be attached to the monomer of cyclic heptapeptide and the other arm may be attached to the Fc domain monomer or Fc domain.
[0591] In some embodiments, a conjugate containing an Fc domain covalently linked to one or more monomers of cyclic heptapeptides provided herein is described by any one of formulae below:
##STR00762## ##STR00763## ##STR00764## ##STR00765## ##STR00766## ##STR00767## ##STR00768## ##STR00769## ##STR00770## ##STR00771## ##STR00772## ##STR00773## ##STR00774## ##STR00775## ##STR00776## ##STR00777##
[0592] In conjugates having an Fc domain covalently linked to one or more monomers of cyclic heptapeptides, as represented by the formulae above, when n is 2, two Fc domain monomers (each Fc domain monomer is represented by E) dimerize to form an Fc domain.
III. Fc Domain Monomers and Fc Domains
[0593] An Fc domain monomer includes a hinge domain, a C.sub.H2 antibody constant domain, and a C.sub.H3 antibody constant domain. The Fc domain monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. The Fc domain monomer can also be of any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain monomer can also be of any species, e.g., human, murine, or mouse. A dimer of Fc domain monomers is an Fc domain that can bind to an Fc receptor, which is a receptor located on the surface of leukocytes.
[0594] In some embodiments, an Fc domain monomer in the conjugates described herein may contain one or more amino acid substitutions, additions, and/or deletion relative to an Fc domain monomer having a sequence of any one of SEQ ID NOs: 1-14, 1-29, or 1-31. In some embodiments, an Asn in an Fc domain monomer in the conjugates as described herein may be replaced by Ala in order to prevent N-linked glycosylation (see, e.g., SEQ ID NOs: 11-14, where Asn to Ala substitution is labeled with *). In some embodiments, an Fc domain monomer in the conjugates described herein may also containing additional Cys additions (see, e.g., SEQ ID NOs: 9 and 10, where Cys additions are labeled with *).
[0595] In some embodiments, an Fc domain monomer in the conjugates as described herein includes an additional moiety, e.g., an albumin-binding peptide, a purification peptide (e.g., a hexa-histidine peptide (HHHHHH (SEQ ID NO: 32)), or a signal sequence (e.g., IL2 signal sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 33)) attached to the N- or C-terminus of the Fc domain monomer. In some embodiments, an Fc domain monomer in the conjugate does not contain any type of antibody variable region, e.g., V.sub.H, V.sub.L, a complementarity determining region (CDR), or a hypervariable region (HVR).
[0596] In some embodiments, an Fc domain monomer in the conjugates as described herein may have a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 1-14, 1-29, or 1-31 shown below. In some embodiments, an Fc domain monomer in the conjugates as described herein may have a sequence of any one of SEQ ID NOs: 1-14, 1-29, or 1-31 shown below.
TABLE-US-00019 SEQ ID NO: 1: murine Fc-IgG2a with IL2 signal sequence at the N-terminus (bold) MYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM YSKLRVEKKNWWERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 2: mature murine Fc-IgG2a PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWWERNSYSC SVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 3: human Fc-IgG1 with IL2 signal sequence at the N-terminus (bold) MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3) SEQ ID NO: 4: mature human Fc-IgG1 MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 5: murine Fc-IgG2a with IL2 signal sequence (bold) at the N-terminus and hexa- histidine peptide (italicized) at the C-terminus MYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHH SEQ ID NO: 6: mature murine Fc-IgG2a with hexa-histidine peptide (italicized) at the C-terminus PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSC SVVHEGLHNHHTTKSFSRTPGKHHHHHH SEQ ID NO: 7: human Fc-IgG1 with IL2 signal sequence (bold) at the N-terminus and hexa- histidine peptide (italicized) at the C-terminus MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 8: mature human Fc-IgG1 with hexa-histidine peptide (italicized) at the C-terminus MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 9: human Fc-IgG1 with IL2 signal sequence (bold) at the N-terminus, two additional cysteines in the hinge region (*), and hexa-histidine peptide (italicized) at the C-terminus MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 10: mature human Fc-IgG1 with two additional cysteines in the hinge region (*) and hexa-histidine peptide (italicized) at the C-terminus MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 11: murine Fc-IgG2a with IL2 signal sequence (bold) at the N-terminus, Asn to Ala substitution (*), and hexa-histidine peptide (italicized) at the C-terminus MYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYA*STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTI SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF MYSKLRVEKKNWVERNSYSCSWHEGLHNHHTTKSFSRTPGKHHHHHH SEQ ID NO: 12: mature murine Fc-IgG2a with Asn to Ala substitution (*) and hexa-histidine peptide (italicized) at the C-terminus PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYA*STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEE EMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHHTTKSFSRTPGKHHHHHH SEQ ID NO: 13: human Fc-IgG1 with IL2 signal sequence (bold) at the N-terminus, Asn to Ala substitution (*), and hexa-histidine peptide (italicized) at the C-terminus MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYA*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 14: mature human Fc-IgG1 with Asn to Ala substitution (*) and hexa-histidine peptide (italicized) at the C-terminus MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYA*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 15: human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N- terminus MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 16: human IgG1 Fewith Human Serum Albumin Signal Sequence (bold) at the N- terminus then terminal K removed, G4S linker (italicized) and then c-Myc tag MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 17: mature human IgG1 Fewith terminal K removed, G4S linker (italicized) and then c-Myc tag ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNH YTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 18: human IgG1 Fc with Human Serum Albumin Signal Sequence (bold), with lysine to serine modification (*) to prevent lysine conjugation at this site MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS*DTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 19: mature human IgG1 Fc with lysine to serine modification (*) to prevent lysine conjugation at this site ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS*DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 20: human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N- terminus with lysine to serine modification (*) to prevent lysine conjugation at this site, G4S linker (italicized) and C-Myc tag MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS(*)DTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 21: mature human IgG1 Fc with lysine to serine modification (*) to prevent lysine conjugation at this site, G4S linker (italicized) and C-Myc tag ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS(*)DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 22: human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N- terminus, Asn to Ala substitution (*), G4S linker (italicized) and C-myc tag MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 23: mature human IgG1 Fcwith Asn to Ala substitution (*), G4S linker (italicized) and C-myc tag ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 24: human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N-terminus, with H310A (*) and H435A (*) mutations to impede FcRn binding G4S (italicized) and C-myc tag MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLA(*)QDWLN GKEYKCKVSNKALPAPIEKTISKA(*)KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNA YTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 25: mature human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N- terminus, with H310A (*) and H435A (*) mutations to impede FcRn binding G4S (italicized) and C- myc tag ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLA(*)QDWLNGKEYKCKVSNKALPAPIEKTISKA(*)KGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNAYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 26: human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N- terminus, G4S linker (italicized) and mutated (lysine to phenylalanine, bold) C-myctag MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGGSEQFLISEEDL SEQ ID NO: 27: mature human IgG1 Fcwith G4S linker (italicized) and mutated (lysine to phenylalanine, bold) C-myc tag ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQFLISEEDL SEQ ID NO: 28: human IgG1 Fcwith Human Serum Albumin Signal Sequence (bold) at the N- terminus, Asn to Ala substitution (*), G4S linker (italicized) and mutated (lysine to phenylalanine, bold) C-myc tag MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGGSEQFLISEEDL SEQ ID NO: 29: mature human IgG1 Fc with Asn to Ala substitution (*), G4S linker (italicized) and mutated (lysine to phenylalanine, bold) C-myc tag ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQFLISEEDL SEQ ID NO: 30: human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N- terminus, removal of the residues ISAMVRS from the N-terminus of the mature human IgG1 Fc, Glu to Asp substitution (*), Met to Leu substitution (**), G4S linker (italicized), and mutated (lysine to phenylalanine, bold) C-myc tag MKWVTFISLLFLFSSAYSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRD(*)EL(**)TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQFLISEEDL SEQ ID NO: 31: human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N- terminus, removal of the residues ISAMVRS from the N-terminus of the mature human IgG1 Fc, Glu to Asp substitution (*), and Met to Leu substitution (**) MKWVTFISLLFLFSSAYSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRD(*)EL(**)TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0597] As defined herein, an Fc domain includes two Fc domain monomers that are dimerized by the interaction between the C.sub.H3 antibody constant domains, as well as one or more disulfide bonds that form between the hinge domains of the two dimerizing Fc domain monomers. An Fc domain forms the minimum structure that binds to an Fc receptor, e.g., Fc-gamma receptors (i.e., Fcγ receptors (FcγR)), Fc-alpha receptors (i.e., Fcα receptors (FcαR)), Fc-epsilon receptors (i.e., Fcε receptors (FcεR)), and/or the neonatal Fc receptor (FcRn). In some embodiments, an Fc domain of the present invention binds to an Fcγ receptor (e.g., FcRn, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and/or FcγRIV and/or the neonatal Fc receptor (FcRn).
[0598] As used herein, a sulfur atom “corresponding to” a particular cysteine residue of a particular SEQ ID NO. should be understood to include the sulfur atom of any cysteine residue that one of skill in the art would understand to align to the particular cysteine of the particular sequence. The protein sequence alignment of human IgG1 (UniProtKB: P01857), human IgG2 (UniProtKB: P01859), human IgG3 (UniProtKB: P01860), and human IgG4 (UniProtKB: P01861) is provided below (aligned with Clustal Omega Multiple Pairwise Alignment). The alignment indicates cysteine residues (e.g., sulfur atoms of cysteine residues) that “correspond to” one another (in boxes and indicated by the * symbol). One of skill in the art would readily be able to perform such an alignment with any IgG variant of the invention to determine the sulfur atom of a cysteine that corresponds to any sulfur atom of a particular cysteine of a particular SEQ ID NO. described herein. For example, one of skill in the art would readily be able to determine that Cys10 of SEQ ID NO: 10 (the first cysteine of the conserved CPPC motif of the hinge region of the Fc domain) corresponds to, for example, Cys109 of IgG1, Cys106 of IgG2, Cys156 of IgG3, Cys29 of SEQ ID NO: 1, Cys9 of SEQ ID NO: 2, Cys30 of SEQ ID NO: 3, or Cys10 of SEQ ID NO: 10.
[0599] As used herein, a nitrogen atom “corresponding to” a particular lysine residue of a particular SEQ ID NO. should be understood to include the nitrogen atom of any lysine residue that one of skill in the art would understand to align to the particular lysine of the particular sequence. The protein sequence alignment of human IgG1 (UniProtKB: P01857), human IgG2 (UniProtKB: P01859), human IgG3 (UniProtKB: P01860), and human IgG4 (UniProtKB: P01861) is provided below (aligned with Clustal Omega Multiple Pairwise Alignment). The alignment indicates lysine residues (e.g., nitrogen atoms of lysine residues) that “correspond to” one another (in boxes and indicated by the * symbol). One of skill in the art would readily be able to perform such an alignment with any IgG variant of the invention to determine the nitrogen atom of a lysine that corresponds to any nitrogen atom of a particular lysine of a particular SEQ ID NO. described herein. For example, one of skill in the art would readily be able to determine that Lys35 of SEQ ID NO: 10 corresponds to, for example, Lys129 of IgG1, Lys126 of IgG2, Lys176 of IgG3, Lys51 of SEQ ID NO: 1, Lys31 of SEQ ID NO: 2, Lys50 of SEQ ID NO: 3, or Lys30 of SEQ ID NO: 10.
Protein Sequence Alignment of IgG1, IgG2, IgG3, and IgG4
[0600]
TABLE-US-00020
[0601] Activation of Immune Cells
[0602] Fc-gamma receptors (FcγRs) bind the Fc portion of immunoglobulin G (IgG) and play important roles in immune activation and regulation. For example, the IgG Fc domains in immune complexes (ICs) engage FcγRs with high avidity, thus triggering signaling cascades that regulate immune cell activation. The human FcγR family contains several activating receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) and one inhibitory receptor (FcγRIIb). FcγR signaling is mediated by intracellular domains that contain immune tyrosine activating motifs (ITAMs) for activating FcγRs and immune tyrosine inhibitory motifs (ITIM) for inhibitory receptor FcγRIIb. In some embodiments, FcγR binding by Fc domains results in ITAM phosphorylation by Src family kinases; this activates Syk family kinases and induces downstream signaling networks, which include PI3K and Ras pathways.
[0603] In the conjugates described herein, the portion of the conjugates including monomers or dimers of cyclic heptapeptides bind to lipopolysaccharides (LPS) in the cell membrane of Gram-negative bacteria to disrupt and permeabilize the cell membrane, leading to cell death and/or sensitization to other antibiotics, while the Fc domain portion of the conjugates bind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells and activate phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of bacterial cells by immune cells and further enhancing the antibacterial activity of the conjugates. Examples of immune cells that may be activated by the conjugates described herein include, but are not limited to, macrophages, neutrophils, eosinophils, basophils, lymphocytes, follicular dendritic cells, natural killer cells, and mast cells.
IV. Linkers
[0604] A linker refers to a linkage or connection between two or more components in a conjugate described herein (e.g., between two cyclic heptapeptides in a conjugate described herein, between a cyclic heptapeptide and an Fc domain in a conjugate described herein, and between a dimer of two cyclic pentapeptide and an Fc domain in a conjugate described herein).
[0605] Linkers in Conjugates Having an Fc Domain Covalently Linked to Dimers of Cyclic Heptapeptides
[0606] In a conjugate containing an Fc domain covalently linked to one or more dimers of cyclic heptapeptides as described herein, a linker in the conjugate (e.g., L′, L, or L.sup.1) may be a branched structure. As described further herein, a linker in a conjugate described herein (e.g., L′, L, or L.sup.1) may be a multivalent structure, e.g., a divalent or trivalent structure having two or three arms, respectively. In some embodiments when the linker has three arms, two of the arms may be attached to the first and second cyclic heptapeptides and the third arm may be attached to the Fc domain monomer or Fc domain. In some embodiments when the linker has two arms, one arm may be attached to an Fc domain and the other arm may be attached to one of the two cyclic heptapeptides. In other embodiments, a linker with two arms may be used to attach the two cyclic heptapeptides on a conjugate containing an Fc domain covalently linked to one or more dimers of cyclic heptapeptides.
[0607] In some embodiments, the linker L′ in the conjugate described by formula (I) is described by formula (L):
##STR00785##
in which L is a remainder of L′; A1 is a 1-5 amino acid peptide (e.g., a 1-4, 1-3, or 1-2 amino acid peptide) covalently attached to the linking nitrogen in each M1 or is absent; and A2 is a 1-5 amino acid peptide (e.g., a 1-4, 1-3, or 1-2 amino acid peptide) covalently attached to the linking nitrogen in each M2 or is absent. In some embodiments, a conjugate described herein may contain a linker that has a trivalent structure (e.g., a trivalent linker; a linker of formula (L-I)). A trivalent linker has three arms, in which each arm is conjugated to a component of the conjugate (e.g., a first arm conjugated to a first cyclic heptapeptide, a second arm conjugated to a second heptapeptide, and a third arm conjugated to an Fc domain in the conjugate described herein). In some embodiments, the one or more dimers of cyclic heptapeptides in the conjugates described herein may each be, independently, connected to an atom in the Fc domain.
[0608] In some embodiments, a linker in a conjugate having an Fc domain covalently linked to one or more dimers of cyclic heptapeptides is described by formula (L-I):
##STR00786##
wherein L.sup.A is described by formula G.sup.A1-(Z.sup.A1).sub.g1—(Y.sup.A1).sub.h1—(Z.sup.A2).sub.i1—(Y.sup.A2).sub.j1—(Z.sup.A3).sub.k1—(Y.sup.A3).sub.l1—(Z.sup.A4).sub.m1—(Y.sup.A4).sub.n1—(Z.sup.A5).sub.O1-G.sup.A2; L.sup.B is described by formula G.sup.B1-(Z.sup.B1).sub.g2—(Y.sup.B1).sub.h2—(Z.sup.B2).sub.i2—(Y.sup.B2).sub.j2—(Z.sup.B3).sub.k2—(Y.sup.B3).sub.l2—(Z.sup.B4).sub.m2—(Y.sup.B4).sub.n2—(Z.sup.B5).sub.O2-G.sup.B2; L.sup.C is described by formula G.sup.C1-(Z.sup.C1).sub.g3—(Y.sup.C1).sub.h3—(Z.sup.C2).sub.i3—(Y.sup.C2).sub.j3—(Z.sup.C3).sub.k3—(Y.sup.C3).sub.l3—(Z.sup.C4).sub.m3—(Y.sup.C4).sub.n3—(Z.sup.C5).sub.O3-GC.sup.2; G.sup.A1 is a bond attached to Q in formula (L-I); G.sup.A2 is a bond attached to the first cyclic heptapeptide or a peptide (e.g., a peptide including 1-5 amino acid residue(s)) attached to the first cyclic heptapeptide, if the peptide is present; G.sup.B1 is a bond attached to Q in formula (L-I); G.sup.B2 is a bond attached to the second cyclic heptapeptide or a peptide (e.g., a peptide including 1-5 amino acid residue(s)) attached to the second cyclic heptapeptide, if the peptide is present; G.sup.C1 is a bond attached to Q in formula (L-I); G.sup.C2 is a bond attached to an Fc domain; each of Z.sup.A1, Z.sup.A2, Z.sup.A3, Z.sup.A4, Z.sup.A5, Z.sup.B1, Z.sup.B2, Z.sup.B3, Z.sup.B4, Z.sup.B5, Z.sup.C1, Z.sup.C2, Z.sup.C3, Z.sup.C4, and Z.sup.C5 is, independently, optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene, or optionally substituted C2-C15 heteroarylene; each of Y.sup.A1, Y.sup.A2, Y.sup.A3, Y.sup.A4, Y.sup.B1, Y.sup.B2, Y.sup.B3, Y.sup.B4, Y.sup.C1, Y.sup.C2, Y.sup.C3, and Y.sup.C4 is, independently, O, S, NR.sup.i, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino; R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl; each of g1, h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, l2, m2, n2, o2, g3, h3, i3, j3, k3, l3, m3, n3, and o3 is, independently, 0 or 1; Q is a nitrogen atom, optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene, or optionally substituted C2-C15 heteroarylene.
[0609] In some embodiments, depending on the structure and chemical formula of L.sup.C, L.sup.C may have two points of attachment to the Fc domain (e.g., two G.sup.C2)
[0610] Linkers of formula (L-I) that may be used in conjugates described herein include, but are not limited to,
##STR00787## ##STR00788## ##STR00789## ##STR00790## ##STR00791##
[0611] Linkers in Conjugates Having an Fc Domain Covalently Linked to Monomers of Cyclic Heptapeptides
[0612] In a conjugate containing an Fc domain covalently linked to one or more monomers of cyclic heptapeptides as described herein, a linker in the conjugate (e.g., L′, or L) may be a divalent structure having two arms. One arm in a divalent linker may be attached to the monomer of cyclic heptapeptide and the other arm may be attached to the Fc domain. In some embodiments, the one or more monomers of cyclic heptapeptides in the conjugates described herein may each be, independently, connected to an atom in the Fc domain.
[0613] In some embodiments, a linker is described by formula (L-I):
J.sup.1-(Q.sup.1).sub.g-(T.sup.1).sub.h-(Q.sup.2).sub.i-(T.sup.2).sub.j-(Q.sup.3).sub.k-(T.sup.3).sub.l-(Q.sup.4).sub.m-(T.sup.4).sub.n-(Q.sup.5).sub.o-J.sup.2
wherein J.sup.1 is a bond attached to a cyclic heptapeptide or a peptide (e.g., a peptide including 1-5 amino acid residue(s)) attached to the cyclic heptapeptide, if the peptide is present; J.sup.2 is a bond attached to an Fc domain; each of Q.sup.1, Q.sup.2, Q.sup.3, Q.sup.4 and Q.sup.5 is, independently, optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene, or optionally substituted C2-C15 heteroarylene; each of T.sup.1, T.sup.2, T.sup.3, T.sup.4 is, independently, O, S, NR.sup.i, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino; R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl; and each of g, h, i, j, k, l, m, n, and o is, independently, 0 or 1.
[0614] In some embodiments, depending on the structure and chemical formula of L-I, J.sup.2 may have two points of attachment to the Fc domain (e.g., two J2).
[0615] Linkers of formula (L-I) that may be used in conjugates described herein include, but are not limited to,
##STR00792## ##STR00793##
wherein each of d and e is, independently, an integer from 1 to 26.
[0616] In some embodiments, a linker provides space, rigidity, and/or flexibility between the cyclic heptapeptides and the Fc domain in the conjugates described here or between two cyclic heptapeptides in the conjugates described herein. In some embodiments, a linker may be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a C—O bond, a C—N bond, a N—N bond, a C—S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation. In some embodiments, a linker (L′, L, or L.sup.1 as shown in any one of formulas (I)-(XVII)) includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). In some embodiments, a linker (L′, L, or L.sup.1) includes no more than 250 non-hydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 non-hydrogen atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-hydrogen atom(s)). In some embodiments, the backbone of a linker (L′, L, or L.sup.1) includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). The “backbone” of a linker refers to the atoms in the linker that together form the shortest path from one part of the conjugate to another part of the conjugate. The atoms in the backbone of the linker are directly involved in linking one part of the conjugate to another part of the conjugate. For examples, hydrogen atoms attached to carbons in the backbone of the linker are not considered as directly involved in linking one part of the conjugate to another part of the conjugate.
[0617] Molecules that may be used to make linkers (L′, L, or L.sup.1) include at least two functional groups, e.g., two carboxylic acid groups. In some embodiments of a trivalent linker, two arms of a linker may contain two dicarboxylic acids, in which the first carboxylic acid may form a covalent linkage with the first cyclic heptapeptide in the conjugate and the second carboxylic acid may form a covalent linkage with the second cyclic heptapeptide in the conjugate, and the third arm of the linker may for a covalent linkage (e.g., a C—O bond) with an Fc domain in the conjugate. In some embodiments of a divalent linker, the divalent linker may contain two carboxylic acids, in which the first carboxylic acid may form a covalent linkage with one component (e.g., a cyclic heptapeptide) in the conjugate and the second carboxylic acid may form a covalent linkage (e.g., a C—S bond or a C—N bond) with another component (e.g., an Fc domain) in the conjugate. In some embodiments, when the cyclic heptapeptide is attached to a peptide (e.g., a peptide including a 1-5 amino acid residue(s)) at the linking nitrogen, the linker may form a covalent linkage with the peptide.
[0618] In some embodiments, dicarboxylic acid molecules may be used as linkers (e.g., a dicarboxylic acid linker). For example, in a conjugate containing an Fc domain covalently linked to one or more dimers of cyclic heptapeptides, the first carboxylic acid in a dicarboxylic acid molecule may form a covalent linkage with the linking nitrogen of the first cyclic heptapeptide and the second carboxylic acid may form a covalent linkage with the linking nitrogen of the second cyclic heptapeptide.
[0619] In some embodiments, when the first and/or second cyclic heptapeptide is attached to a peptide (e.g., a peptide including a 1-5 amino acid residue(s)) at the linking nitrogen, the first carboxylic acid in a dicarboxylic acid linker may form a covalent linkage with the terminal amine group at the end of the peptide that is attached to the first cyclic heptapeptide and the second carboxylic acid in the dicarboxylic acid linker may form a covalent linkage with the terminal amine group at the end of the peptide that is attached to the second cyclic heptapeptide.
[0620] Examples of dicarboxylic acids molecules that may be used to form linkers include, but are not limited to,
##STR00794## ##STR00795## ##STR00796## ##STR00797##
[0621] Other examples of dicarboxylic acids molecules that may be used to form linkers include, but are not limited to,
##STR00798## ##STR00799## ##STR00800## ##STR00801##
[0622] In some embodiments, dicarboxylic acid molecules, such as the ones described herein, may be further functionalized to contain one or more additional functional groups.
[0623] In some embodiments, when the cyclic heptapeptide is attached to a peptide (e.g., a peptide including a 1-5 amino acid residue(s)) at the linking nitrogen, the linking group may comprise a moiety comprising a carboxylic acid moiety and an amino moiety that are spaced by from 1 to 25 atoms. Examples of such linking groups include, but are not limited to,
##STR00802## ##STR00803##
[0624] In some embodiments, a molecule containing an azide group may be used to form a linker, in which the azide group may undergo cycloaddition with an alkyne to form a 1,2,3-triazole linkage. In some embodiments, a molecule containing an alkyne group may be used to form a linker, in which the alkyne group may undergo cycloaddition with an azide to form a 1,2,3-triazole linkage. In some embodiments, a molecule containing a maleimide group may be used to form a linker, in which the maleimide group may react with a cysteine to form a C—S linkage. In some embodiments, a molecule containing one or more sulfonic acid groups may be used to form a linker, in which the sulfonic acid group may form a sulfonamide linkage with the linking nitrogen in a cyclic heptapeptide. In some embodiments, a molecule containing one or more isocyanate groups may be used to form a linker, in which the isocyanate group may form a urea linkage with the linking nitrogen in a cyclic heptapeptide. In some embodiments, a molecule containing one or more haloalkyl groups may be used to form a linker, in which the haloalkyl group may form a covalent linkage, e.g., C—N and C—O linkages, with a cyclic heptapeptide.
[0625] In some embodiments, a linker (L′, L, or L.sup.1) may comprise a synthetic group derived from, e.g., a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, a linker may comprise one or more amino acid residues. In some embodiments, a linker may be an amino acid sequence (e.g., a 1-25 amino acid, 1-10 amino acid, 1-9 amino acid, 1-8 amino acid, 1-7 amino acid, 1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid, 1-2 amino acid, or 1 amino acid sequence). In some embodiments, a linker (L′, L, or L.sup.1) may include one or more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene (e.g., a PEG unit), optionally substituted C2-C20 alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene (e.g., C6 arylene), optionally substituted C2-C15 heteroarylene (e.g., imidazole, pyridine), O, S, NR.sup.i (R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.
[0626] Covalent conjugation of two or more components in a conjugate using a linker may be accomplished using well-known organic chemical synthesis techniques and methods. Complementary functional groups on two components may react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine. Site-specific conjugation to a polypeptide (e.g., an Fc domain monomer or an Fc domain) may accomplished using techniques known in the art. Exemplary techniques for site-specific conjugation of a small molecule to an Fc domain are provided in Agarwall. P., et al. Bioconjugate Chem. 26:176-192 (2015).
[0627] Other examples of functional groups capable of reacting with amino groups include, e.g., alkylating and acylating agents. Representative alkylating agents include: (i) an α-haloacetyl group, e.g., XCH.sub.2CO— (where X═Br, Cl, or I); (ii) a N-maleimide group, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group; (iii) an aryl halide, e.g., a nitrohaloaromatic group; (iv) an alkyl halide; (v) an aldehyde or ketone capable of Schiff's base formation with amino groups; (vi) an epoxide, e.g., an epichlorohydrin and a bisoxirane, which may react with amino, sulfhydryl, or phenolic hydroxyl groups; (vii) a chlorine-containing of s-triazine, which is reactive towards nucleophiles such as amino, sulfhydryl, and hydroxyl groups; (viii) an aziridine, which is reactive towards nucleophiles such as amino groups by ring opening; (ix) a squaric acid diethyl ester; and (x) an α-haloalkyl ether.
[0628] Examples of amino-reactive acylating groups include, e.g., (i) an isocyanate and an isothiocyanate; (ii) a sulfonyl chloride; (iii) an acid halide; (iv) an active ester, e.g., a nitrophenylester or N-hydroxysuccinimidyl ester; (v) an acid anhydride, e.g., a mixed, symmetrical, or N-carboxyanhydride; (vi) an acylazide; and (vii) an imidoester. Aldehydes and ketones may be reacted with amines to form Schiff's bases, which may be stabilized through reductive amination.
[0629] It will be appreciated that certain functional groups may be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; conversion of thiols to carboxyls using reagents such as α-haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.
V. Antibacterial Agents
[0630] In some embodiments, one or more antibacterial agents may be administered in combination (e.g., administered substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions) or administered separately at different times) with a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)).
[0631] Antibacterial agents may be grouped into several classes, e.g., quinolones, carbapenems, macrolides, DHFR inhibitors, aminoglycosides, ansamycins (e.g., geldanamycin, herimycin, and rifaximin), carbacephem (e.g., loracarbef), cephalosporins (e.g., cefadroxil, cefaolin, cefalotin, cefalothin, cephalexin, e.g., cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, and ceftobiprole), glycopeptides (e.g., teicoplanin, vancomycin, telavancin, dalbavancin, and oritavancin), lincosamides (e.g., clindamycin and lincomycin), lipopeptides (e.g., daptomycin), monobactams (e.g., aztreonam), nitrofurans (e.g., furazolidone and nitrofurantoin), oxazolidinones, pleuromutilins, penicillins, sulfonamides, and tetracyclines (e.g., eravacycline, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline). Quinolones include, but are not limited to, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin. Carbapenems include, but are not limited to, ertapenem, doripenem, imipenem/cilastatin, and meropenem. Macrolides include, but are not limited to, solithromycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spiramycin. In some embodiments, a macrolide is solithromycin. DHFR inhibitors include, but are not limited to, diaminoquinazoline, diaminopyrroloquinazoline, diaminopyrimidine, diaminopteridine, and diaminotriazines. Aminoglycosides include, but are not limited to, plazomicin, amikacin, gentamicin, gamithromycin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, and spectinomycin. Oxazolidinones include, but are not limited to, linezolid, tedizolid, posizolid, radezolid, and furazolidone. Pleuromutilins include, but are not limited to, retapamulin, valnemulin, tiamulin, azamulin, and lefamulin. Penicillins include, but are not limited to, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, and ticarcillin. Sulfonamides include, but are not limited to, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (Co-trimoxazole) (TMP-SMX), and sulfonamidochrysoidine.
[0632] In some embodiments, the antibacterial agent used in combination with a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) is selected from the group consisting of linezolid, tedizolid, posizolid, radezolid, retapamulin, valnemulin, tiamulin, azamulin, lefamulin, plazomicin, amikacin, gentamicin, gamithromycin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin, clindamycin, lincomycin, daptomycin, solithromycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, penicillin g, temocillin, ticarcillin, amoxicillin clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate, bacitracin, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (tmp-smx), sulfonamidochrysoidine, eravacycline, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol(bs), ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, thiamphenicol, tigecycline, tinidazole, and trimethoprim, prodrugs thereof, and pharmaceutically acceptable salts thereof.
[0633] In some embodiments, the antibacterial agent used in combination with a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) is tedizolid, azithromycin, meropenem, amikacin, levofloxacin, rifampicin, linezolid, erythromycin, or solithromycin. In some embodiments, the antibacterial agent used in combination with a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) is tedizolid, azithromycin, meropenem, amikacin, or levofloxacin. In some embodiments, the antibacterial agent used in combination with a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) is tiamulin. In some embodiments, the antibacterial agent used in combination with a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) is solithromycin.
VI. Methods
[0634] Methods described herein include, e.g., methods of protecting against or treating a bacterial infection (e.g., a Gram-negative bacterial infection) in a subject and methods of preventing, stabilizing, or inhibiting the growth of bacteria, or killing bacteria (e.g., Gram-negative bacteria). A method of treating a bacterial infection (e.g., a Gram-negative bacterial infection) in a subject includes administering to the subject a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) or a pharmaceutical composition thereof. In some embodiments, the bacterial infection is caused by Gram-negative bacteria. In some embodiments, the bacterial infection is caused by a resistant strain of bacteria. In some embodiments, the resistant strain of bacteria is a resistant strain of E. coli. A method of preventing, stabilizing, or inhibiting the growth of bacteria, or killing bacteria (e.g., Gram-negative bacteria) includes contacting the bacteria (e.g., Gram-negative bacteria) or a site susceptible to bacterial growth (e.g., Gram-negative bacterial growth) with a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) or a pharmaceutical composition thereof. In some embodiments, the bacterial infection is caused by Gram-negative bacteria. In some embodiments, the bacterial infection is caused by a resistant strain of bacteria. In some embodiments, the resistant strain of bacteria is a resistant strain of E. coli. In some embodiments, a conjugate used in any methods described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) may bind to LPS in the cell membrane of Gram-negative bacteria to disrupt and permeabilize the cell membrane, leading to cell death and/or sensitization to other antibiotics. Furthermore, in addition to disrupting the cell membrane of Gram-negative bacteria, the Fc domain in the conjugates described herein binds to the FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells and serves as a gradient against which immune cells chemotax to the site of bacterial infection and/or growth. The binding of the Fc domain in the conjugates described herein to the FcγRs on immune cells activates phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of Gram-negative bacterial cells by immune cells and further enhancing the antibacterial activity of the conjugates.
[0635] Moreover, methods described herein also include methods of protecting against or treating sepsis in a subject by administering to the subject a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)). In some embodiments, the method further includes administering to the subject an antibacterial agent. Methods described herein also include methods of preventing lipopolysaccharides (LPS) in Gram-negative bacteria (e.g., a resistant strain of Gram-negative bacteria or a resistant strain of E. coli (e.g., E. coli BAA-2469)) from activating a immune system in a subject by administering to the subject a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)). In some embodiments of the method, the method prevents LPS from activating a macrophage. In some embodiments, the method further includes administering to the subject an antibacterial agent. In some embodiments, a conjugate used in any methods described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) may bind to LPS in the cell membrane of Gram-negative bacteria to disrupt and permeabilize the cell membrane, leading to cell death and/or sensitization to other antibiotics.
[0636] In some embodiments, the methods described herein may further include administering to the subject an antibacterial agent in addition to a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)). Methods described herein also include methods of protecting against or treating a bacterial infection in a subject by administering to said subject (1) a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) and (2) an antibacterial agent. Methods described herein also include methods of preventing, stabilizing, or inhibiting the growth of bacteria, or killing bacteria, by contacting the bacteria or a site susceptible to bacterial growth with (1) a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) and (2) an antibacterial agent.
[0637] In some embodiments, the conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) is administered first, followed by administering of the antibacterial agent alone. In some embodiments, the antibacterial agent is administered first, followed by administering of the conjugate described herein alone. In some embodiments, the conjugate described herein and the antibacterial agent are administered substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the conjugate described herein or the antibacterial agent is administered first, followed by administering of the conjugate described herein and the antibacterial agent substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions). In some embodiments, the conjugate described herein and the antibacterial agent are administered first substantially simultaneously (e.g., in the same pharmaceutical composition or in separate pharmaceutical compositions), followed by administering of the conjugate described herein or the antibacterial agent alone. In some embodiments, when a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) and an antibacterial agent are administered together (e.g., substantially simultaneously in the same or separate pharmaceutical compositions, or separately in the same treatment regimen), the MIC of each of the conjugate and the antibacterial agent may be lower than the MIC of each of the conjugate and the antibacterial agent when each is used alone in a treatment regimen.
VII. Pharmaceutical Compositions and Preparations
[0638] A conjugate described herein may be formulated in a pharmaceutical composition for use in the methods described herein. In some embodiments, a conjugate described herein may be formulated in a pharmaceutical composition alone. In some embodiments, a conjugate described herein may be formulated in combination with an antibacterial agent in a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes a conjugate described herein (e.g., a conjugate described by any one of formulas (I)-(XXXIII)) and pharmaceutically acceptable carriers and excipients. Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acid residues such as glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and sorbitol.
[0639] Examples of other excipients include, but are not limited to, antiadherents, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, sorbents, suspensing or dispersing agents, or sweeteners. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
[0640] The conjugates herein may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the conjugates herein be prepared from inorganic or organic bases. Frequently, the conjugates are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.
[0641] Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.
[0642] Depending on the route of administration and the dosage, a conjugate herein or a pharmaceutical composition thereof used in the methods described herein will be formulated into suitable pharmaceutical compositions to permit facile delivery. A conjugate (e.g., a conjugate of any one of formulas (I)-(XXXIII)) or a pharmaceutical composition thereof may be formulated to be administered intramuscularly, intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally (e.g., a tablet, capsule, caplet, gelcap, or syrup), topically (e.g., as a cream, gel, lotion, or ointment), locally, by inhalation, by injection, or by infusion (e.g., continuous infusion, localized perfusion bathing target cells directly, catheter, lavage, in cremes, or lipid compositions). Depending on the route of administration, a conjugate herein or a pharmaceutical composition thereof may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.
[0643] A conjugate described herein may be formulated in a variety of ways that are known in the art. For use as treatment of human and animal subjects, a conjugate described herein can be formulated as pharmaceutical or veterinary compositions. Depending on the subject (e.g., a human) to be treated, the mode of administration, and the type of treatment desired, e.g., prophylaxis or therapy, a conjugate described herein is formulated in ways consonant with these parameters. A summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott Williams & Wilkins (2012); and Encyclopedia of Pharmaceutical Technology, 4th Edition, J. Swarbrick and J. C. Boylan, Marcel Dekker, New York (2013), each of which is incorporated herein by reference. Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. The formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, and preservatives. The conjugates can be administered also in liposomal compositions or as microemulsions. Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for conjugates herein. Suitable forms include syrups, capsules, and tablets, as is understood in the art.
[0644] The pharmaceutical compositions can be administered parenterally in the form of an injectable formulation. Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Formulations may be prepared as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagles Medium (α-MEM), F-12 medium). Such injectable compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, such as sodium acetate and sorbitan monolaurate. Formulation methods are known in the art, see e.g., Pharmaceutical Preformulation and Formulation, 2nd Edition, M. Gibson, Taylor & Francis Group, CRC Press (2009).
[0645] The pharmaceutical compositions can be prepared in the form of an oral formulation. Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
[0646] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavoring agents, plasticizers, humectants, and buffering agents. Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
[0647] Dissolution or diffusion controlled release of a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) or a pharmaceutical composition thereof can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of the conjugate, or by incorporating the conjugate into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.
[0648] The pharmaceutical composition may be formed in a unit dose form as needed. The amount of active component, e.g., a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)), included in the pharmaceutical compositions are such that a suitable dose within the designated range is provided (e.g., a dose within the range of 0.01-100 mg/kg of body weight).
VIII. Routes of Administration and Dosages
[0649] In any of the methods described herein, conjugates herein may be administered by any appropriate route for treating or protecting against a bacterial infection (e.g., a Gram-negative bacterial infection), or for preventing, stabilizing, or inhibiting the growth of bacteria, or killing bacteria (e.g., Gram-negative bacteria). Conjugates described herein may be administered to humans, domestic pets, livestock, or other animals with a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, administering comprises administration of any of the conjugates described herein (e.g., conjugates of any one of formulas (I)-(XXXIII)) or compositions intramuscularly, intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally (e.g., a tablet, capsule, caplet, gelcap, or syrup), topically (e.g., as a cream, gel, lotion, or ointment), locally, by inhalation, by injection, or by infusion (e.g., continuous infusion, localized perfusion bathing target cells directly, catheter, lavage, in cremes, or lipid compositions). In some embodiments, if an antibacterial agent is also administered in addition to a conjugate described herein, the antibacterial agent or a pharmaceutical composition thereof may also be administered in any of the routes of administration described herein.
[0650] The dosage of a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) or pharmaceutical compositions thereof depends on factors including the route of administration, the disease to be treated (e.g., the extent and/or condition of the bacterial infection), and physical characteristics, e.g., age, weight, general health, of the subject. Typically, the amount of the conjugate or the pharmaceutical composition thereof contained within a single dose may be an amount that effectively prevents, delays, or treats the bacterial infection without inducing significant toxicity. A pharmaceutical composition may include a dosage of a conjugate described herein ranging from 0.01 to 500 mg/kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg) and, in a more specific embodiment, about 0.1 to about 30 mg/kg and, in a more specific embodiment, about 1 to about 30 mg/kg. In some embodiments, when a conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) and an antibacterial agent are administered together (e.g., substantially simultaneously in the same or separate pharmaceutical compositions, or separately in the same treatment regimen), the dosage needed of the conjugate described herein may be lower than the dosage needed of the conjugate if the conjugate was used alone in a treatment regimen.
[0651] A conjugate described herein (e.g., a conjugate of any one of formulas (I)-(XXXIII)) or a pharmaceutical composition thereof may be administered to a subject in need thereof, for example, one or more times (e.g., 1-10 times or more; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) daily, weekly, monthly, biannually, annually, or as medically necessary. Dosages may be provided in either a single or multiple dosage regimens. The timing between administrations may decrease as the medical condition improves or increase as the health of the patient declines. The dosage and frequency of administration may be adapted by the physician in accordance with conventional factors such as the extent of the infection and different parameters of the subject.
EXAMPLES
Example 1—Preparation of Fc Constructs
[0652] Reverse translations of the amino acids comprising the protein constructs (SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13) were synthesized by solid-phase synthesis. The oligonucleotide templates were cloned into pcDNA3.1 (Life Technologies, Carlsbad, Calif., USA) at the cloning sites BamHI and XhoI (New England Biolabs, Ipswich, Mass., USA) and included signal sequences derived from the human Interleukin-2 or human albumin. The pcDNA3.1 plasmids were transformed into Top 10 E. coli cells (LifeTech). DNA was amplified, extracted, and purified using the PureLink® HiPure Plasmid Filter Maxiprep Kit (LifeTech). The plasmid DNA is delivered, using the ExpiFectamine™ 293 Transfection Kit (LifeTech), into HEK-293 cells per the manufacturer's protocol. Cells were centrifuged, filtered, and the supernatants were purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 2—Synthesis of Intermediate 1 (Int-1)
[0653] Degradation of Polymyxin B to Tri-Boc Polymyxin B Cycloheptapeptide
##STR00804##
[0654] Polymyxin B (100 g, 72.2 mmol) was dissolved in acetonitrile (1000 mL) and water (500 mL) and stirred at room temperature for 10 mins. TEA (58.5 g, 8.0 eq) was added and the mixture stirred for a further 10 mins. Boc.sub.2O (94.6 g, 6.0 eq) was subsequently added in one portion and the mixture stirred for 6 hrs at 20° C. Savinase® (300 mL) was then added. The pH of the resulting mixture was adjusted to 9.0 with aq 4M sodium hydroxide solution (10 mL) and the reaction mixture stirred at 25° C. Additional Savinase® (100 mL) was added after 17 hrs, and another quantity of Savinase® (100 mL×2) was added after 26 hrs. After an overall reaction time of 80 hrs, the mixture was diluted with ethyl acetate (2000 mL). After separation of the layers, the organic phase was washed with 0.1 M NaOH solution (1000 mL×2, 10V×2), then water (1000 mL, 10V). The organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and the solvent evaporated at reduced pressure. The residue was purified by silica gel chromatography eluting with 80% (EtOAc:MeOH:H.sub.2O.Math.NH.sub.3=40:10:1) in ethyl acetate to give the title compound (49.8 g, 65.0%). LCMS: m/z (M+H).sup.+ calcd for C.sub.50H.sub.83N.sub.11O.sub.14:1061.61; found: 1062.5.
Example 3—Synthesis of Int-2 (Tripeptide (Dab-Thr-Dab))
[0655] Step a.
##STR00805##
[0656] NH.sub.2-Dab(Boc)-OMe (HCl salt) (5.000 g, 1 eq.), Z—NH—Thr-OH (5.049 g, 1.05 eq.), EDCl (5.350 g, 1.5 eq.), HOBt (3.733 g, 1.5 eq.) and NaHCO.sub.3 (3.095 g, 2 eq.) were weighed into a 100-mL round bottom flask. 24 mL of DCM/DMF (4:1) was added into the flask. The mixture was stirred at room temperature for less than 3 hrs. (TLC or LC/MS monitoring). After completion, EtOAc (200 mL) was added to dilute the reaction mixture and washed with 1N aq. HCl, saturated NaHCO.sub.3 and brine. Dried with Na.sub.2SO.sub.4 and condensed. The residue was purified with normal phase silica (2˜7% MeOH/DCM) to give 8.24 g pure desired product (>95%).
[0657] Step b.
##STR00806##
[0658] Starting material (SM) (8.24 g) was dissolved in 100 mL of MeOH/EtOAc. And 5% Pd/C (3.75 g, 0.1 eq.) was added. Under H.sub.2 balloon, the reaction mixture was stirred for 2 h. Checked LC/MS. Celite filtration and MeOH wash. Dried to give 5.87 g of free amine (>99%). The material was used in the next step without purification.
[0659] Step c.
##STR00807##
[0660] NH.sub.2-Thr-Dab(Boc)-OMe (1.46 g, 1 eq.), Z—NH-Dab(Boc)-OH(DCHA salt) (2.463, 1.05 eq.), EDCl (1.260 g, 1.5 eq.), HOBt (0.888 g, 1.5 eq.) and NaHCO.sub.3 (0.738, 2 eq.) were weighed into a 100-mL round bottom flask. 20 mL of DCM/DMF (4:1) was added into the flask. The mixture was stirred at room temperature for less than 3 hrs with TLC or LC/MS monitoring. After the completion, EtOAc (100 mL) was added to dilute the reaction mixture and washed with 1N aq. HCl, saturated NaHCO.sub.3 and brine. Dried with Na2SO4 and condensed. The residue was purified with normal phase silica (2-7% MeOH/DCM) to give 2.78 g pure desired product (>95% isolated yield). (The reaction was repeated at 4.4 g scale and gave the similar result.)
[0661] Step d.
##STR00808##
[0662] A solution of the Cbz-tripeptide (1.55 g, 2.32 mmol) in methanol (10 mL) was charged with 5% Pd/C (0.300 g) and flushed with hydrogen from a balloon. After stirring overnight under hydrogen atmosphere, LCMS showed complete conversion. Pd/C was removed by filtration through Celite. The filtrate was concentrated and used in the next Step without further purification.
Example 4—Synthesis of Int-3 (Tripeptide Acid)
[0663] ##STR00809##
[0664] A solution of the Cbz-tripeptide-OMe (7.85 g, 11.76 mmol, described in Step 3 of Int-2 synthesis), dissolved in THF (30 mL), and water (30 mL), was treated with powdered LiOH (0.338 g, 14.1 mmol) and stirred at room temperature. After 15 minutes LCMS showed consumption of starting material. The reaction was made slightly acidic by adding concentrated HCl (aq), then extracted into ethyl acetate, dried over sodium sulfate, concentrated, and purified by reversed phase liquid chromatography (RPLC) using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using no modifier. Yield 7.2 g, 94%. Ion(s) found by LCMS: (M+H)+=654.2.
Example 5—Synthesis of Int-4
[0665] ##STR00810## ##STR00811##
[0666] Step a.
##STR00812##
[0667] A stirring solution of methyl (2S)-2-[(N-{(2S)-2-amino-4-[(tert-butoxycarbonyl)amino]butanoyl}-L-threonyl)amino]-4-[(tert-butoxycarbonyl)amino]butanoate (0.399 g, 0.748 mmol), 2,2′-{[(benzyloxy)carbonyl]azanediyl}diacetic acid (0.100, 0.374 mmol), and DIEA (0.261 mL, 1.50 mmol), in DMF (1 mL), were treated with a solution of HATU (0.285 g, 0.748 mmol), dropwise over 30 minutes, at room temperature. The desired product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using no modifier. Yield 0.200 g, 41%. Ion(s) found by LCMS: (M+H)+=1298.7.
[0668] Step b.
##STR00813##
[0669] A solution of dimethyl (2S,5S,8S,16S,19S,22S)-12-[(benzyloxy)carbonyl]-2,8,16,22-tetrakis{2-[(tert-butoxycarbonyl)amino]ethyl}-5,19-bis[(1R)-1-hydroxyethyl]-4,7,10,14,17,20-hexaoxo-3,6,9,12,15,18,21-heptaazatricosane-1,23-dioate (0.380 g, 0.242 mmol), in methanol (1 mL) was treated with a solution of lithium hydroxide (0.028 g, 1.171 mmol), in water (1 mL), then stirred at room temperature for 30 minutes. The reaction was made slightly acidic (pH=5) with concentrated HCl (several drops). The desired product was isolated directly by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using no modifier. Yield 0.164 g, 44%. Ion(s) found by LCMS: [(M−2Boc)/2]+H+=535.8, [(M−3Boc)/2]+H+=485.8
[0670] Step c.
##STR00814##
[0671] A solution of (2S,5S,8S,16S,19S,22S)-12-[(benzyloxy)carbonyl]-2,8,16,22-tetrakis{2-[(tert-butoxycarbonyl)amino]ethyl}-5,19-bis[(1R)-1-hydroxyethyl]-4,7,10,14,17,20-hexaoxo-3,6,9,12,15,18,21-heptaazatricosane-1,23-dioic acid (0.164 g, 0.129 mmol), tri-Boc polymyxin heptapeptide (0.329 g, 0.310 mmol, Int-1), DIEA (0.146 mL, 0.839 mmol), and DMF (1 mL), was treated with a solution of HATU (0.175 g, 0.460 mmol) in DMF (1 mL), dropwise over 30 minutes. The crude reaction mixture was taken on to the next Step without purification. Ion(s) found by LCMS: [(M−2Boc)/3]+H+=1053.3, [(M−3Boc)/3]+H+=1019.9, [(M−4Boc)/3]+H+=986.6.
[0672] Step d.
##STR00815##
[0673] Crude Cbz deca Boc intermediate (DMF solution) was diluted with methanol (10 mL), charged with 5% Pd/C (0.150 g), and hydrogen from a balloon. The reaction was monitored by LCMS. After 2 hr the mixture was filtered through celite, concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using no modifier. Yield 0.252 g, 61% (two steps). Ion(s) found by LCMS: [M/3]+H+=1075.3.
[0674] Step e. Preparation of Deca Boc Int-4
##STR00816##
[0675] A solution of Step d product (0.135 g, 0.042 mmol), 3-(2-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}ethoxy)propanoic acid (0.015 g, 0.050 mmol), and DIEA (0.026 mL, 0.150 mmol), in DMF (1 mL), were treated with HATU (0.019 g, 0.050 mmol), while stirring at room temperature. After 30 minutes, the product was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using no modifier. Yield 0.112 g, 76% yield. Ion(s) found by LCMS: [(M−3Boc)/3]+H+=1069.6, [(M−4Boc)/3]+H+=1036.3, [(M−5Boc)/3]+H+=1003.0.
[0676] Step f.
##STR00817##
[0677] A solution of Deca Boc Int-4 (0.112 g, 0.032 mmol), dissolved in DCM (1 mL), was treated with TFA (1 mL), while stirring at room temperature. After 5 minutes, the product was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 0% to 100% acetonitrile and water, using trifluoroacetic acid as the modifier. Yield of Int-4 was 0.071 g, 88% yield. Ion(s) found by LCMS: [M/3]+H+=836.1, [M/4]+H+=627.3, [M/5]+H+=502.1.
Example 6—Synthesis of Int-5
[0678] ##STR00818## ##STR00819##
[0679] Step a. Synthesis of Int-5a
##STR00820##
[0680] HATU (1.56 g, 4.11 mmol) in DMF (1.5 mL) was added, dropwise, to a solution of 2,2′-{[(benzyloxy)carbonyl]azanediyl}diacetic acid (0.5 g, 1.87 mmol), norleu-OMe hydrochloride salt (0.71 g, 3.93 mmol), and triethylamine (1.13 g, 11.23 mmol) in DMF (5 mL) over a period of 30 minutes. The mixture was stirred for an additional 20 minutes then applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the CBZ-protected di-ester as a clear oil, ion found by LC/MS [M+H]+=522.6. The di-ester intermediate was stirred in a 1/1/2 mixture (10 mL) of THF/methanol/water containing LiOH (0.18 g, 7.48 mmol) for 20 minutes. The mixture was applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford (2S,2'S)-2,2′-({[(benzyloxy)carbonyl]azanediyl}bis[(1-oxoethane-2,1-diyl)azanediyl])dihexanoic acid as a white solid. Yield: 43%, 2 Steps. Ion found by LC/MS [m-H]—=492.3.
[0681] Step b. Synthesis of Int-5b.
##STR00821##
[0682] EDC (0.38 g, 2.0 mmol) was added to a solution of Int-1 (2.0 g, 1.88 mmol), Cbz-D-Ser-OH (0.48 g, 2.0 mmol), and HOBt (0.31 g, 2.0 mmol) in a 5/1 mixture of DCM/DMF (15 mL). The mixture was stirred for an additional 2 hours and applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the CBZ-protected intermediate a white solid. Ion found by LC/MS [m/2+H]+=542.4 (loss of 1 Boc group). The CBZ-protected intermediate was stirred in methanol (50 mL) containing 1 g of 5% Pd/C under 1 atmosphere of hydrogen for 6 hours. The mixture was filtered and concentrated to afford the Int-5b (free amine) as a white solid. Yield: 73%, 2 Steps. Ion found by LC/MS [m/2+H]+=525.6 (loss of 1 Boc group).
[0683] Step c. Synthesis of Int-5c.
##STR00822##
[0684] HATU (370 mg, 0.97 mmol) in DMF (2 mL) was added, dropwise over 30 minutes, to a stirring solution of Int-5b (1.10 g, 0.95 mmol), CBZ-Thr-OH (253 mg, 1.0 mmol), and triethylamine (300 mg, 2.97 mmol) in DMF (6 mL). The reaction was stirred for 30 minutes and then applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford 1.1 g of the CBZ-protected intermediate a white solid. LC/MS [m/2+H].sup.+=593.0 (loss of 2 Boc groups). The CBZ-protected intermediate was stirred in methanol (50 mL) containing 0.30 g of 5% Pd/C under 1 atmosphere of hydrogen for 6 hours. The mixture was filtered and concentrated to afford the Int-5c (free amine) as a white solid. Yield: 66%, 2 Steps. Ion found by LC/MS [m/2+H].sup.+=525.8 (loss of 2 Boc groups).
[0685] Step d. Synthesis of Int-5d.
##STR00823##
[0686] The title compound was prepared from Int-5c and Z-(γBoc)-Dab-OH in an analogous manner as described in Step c. Yield: 78%. Ion found by LC/MS [M/2+H].sup.+ 676.2 (loss of 1 Boc group).
[0687] Step e. Synthesis of Int-5e.
##STR00824##
[0688] HATU (0.11 g, 0.30 mmol) in DMF (0.5 mL) was added, dropwise, to a stirring mixture of Int-5d (0.44 g, 0.30 mmol), and INT-A1 (0.70 g, 0.14 mmol) in DMF (1.5 mL) over a period of 20 minutes. The mixture was applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 30% to 95% methanol and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the CBZ-protected intermediate, LC/MS [m/3+H].sup.+ 1020.0 (loss of 3 Boc groups). The CBZ-intermediate was stirred in methanol (10 mL) in the presence of 5% Pd/C (100 mg) under 1 atmosphere of hydrogen for 2 hours. The mixture was filtered and concentrated then purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 30% to 95% acetonitrile and water using no modifier. The pure fractions were pooled and lyophilized to afford the title compound as a white solid. Yield: 51%, 2 Steps. Ion found by LC/MS [m/2+H].sup.+=1562.6 (loss of 2 Boc groups).
[0689] Step f. Synthesis of Int-5.
##STR00825##
[0690] HATU (57 mg, 0.15 mmol) in DMF (1 mL) was added, dropwise, to a stirring mixture of propargyl-peg-4-carboxylic acid (42 mg, 0.16 mmol), and Int-5e (345 mg, 0.11 mmol) in DMF (1.5 mL) over a period of 20 minutes. The mixture was applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 30% to 95% methanol and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the octa Boc-protected intermediate. The octa Boc-protected intermediate was stirred in a 1/1 mixture of TFA/DCM (5 mL) containing thioanisole (106 mg, 0.85 mmol) for 20 minutes at ambient temperature. The mixture was concentrated then purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 0% to 70% acetonitrile and water using no modifier. The pure fractions were pooled and lyophilized to afford the title compound Int-5 as a white solid. Yield: 36%, 2 Steps. LC/MS [m/4+H].sup.+=667.2.
Example 7—Synthesis of Int-6 (azido-peg4-hexyl pyrolidine-3,4-dicarboxamide)
[0691] Step a. Synthesis of Int-6a (More Polar Diastereomer)
##STR00826##
[0692] HATU (3.1 g, 8.1 mmol) in DMF (5 mL) was added, dropwise, to a solution of racemic-trans1-(tert-butoxycarbonyl)pyrrolidine-3,4-dicarboxylic acid (1 g, 3.9 mmol), H-Norleu-OMe-hydrochloride (1.5 g, 8.1 mmol), and triethylamine (4.0 g, 39.5 mmol) in DMF (10 mL) over a period of 20 minutes. The mixture was stirred for an additional 20 minutes then applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The two diasteromers were separated, pooled and lyophilized separately into the more polar diasteromers (a) and the less polar diastereomer (b): Ion found by LC/MS [M+H]+=414.2 (loss of 1 Boc group) for both Boc-protected intermediates. Each diastereomer was stirred in a 1/1 mixture of DCM/TFA (8 mL) for 20 minutes then concentrated and purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 0% to 75% acetonitrile and water using no modifier. Yield: 88%, 2 Steps. Ion found by LC/MS [M+H]+=414.2.
(The Stereochemistry of the Polar Diastereomer was Assigned Using Single Crystal x-Ray Crystallography)
[0693] Step b. Synthesis of Int-6
##STR00827##
[0694] Azido-PEG4-NHS ester (254 mg, 0.65 mmol) in DMF (1 mL) was added, dropwise to a mixture of Int-6a (more polar diastereomer from Step A), TFA salt (265 mg, 0.50 mmol) and triethylamine (66 mg, 0.65 mmol) in DMF (1 mL) and the mixture was stirred at ambient temperature for 2 hours. The mixture was applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the di-ester intermediate. LC/MS [M+H].sup.+=687.4. The di-ester intermediate was stirred in a 1/1/2 mixture of MeOH/THF/DI water containing LiOH (36 mg, 1.5 mmol) at ambient temperature for 5 minutes. The mixture was acidified with glacial acetic acid, concentrated and applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the title compound Int-6 as a clear oil. Yield: 69%, 2 Steps. Ion found by LC/MS [M+H].sup.+=659.4.
Example 8—Synthesis of Int-7
[0695] ##STR00828##
[0696] Step a. penta-Boc-PMB-decapeptide (Int-7a)
[0697] Boc polymyxin B heptapeptide (0.464 g, 0.437 mmol, Int-1), and tripeptide acid (0.316 g, 0.398 mmol, Int-3), were dissolved in DMF (1 mL), DIEA (0.229 mL, 1.31 mmol), then treated with HATU (0.166 g, 0.437 mmol). After stirring for 30 min LCMS showed complete conversion. The crude Cbz-product was diluted with methanol (4 mL), and charged with 5% Pd/C (0.250 g) and placed under a hydrogen atmosphere. After 2 hr the mixture was filtered through Celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using no modifier. Yield 0.640 g, 94% (two steps). Ion(s) found by LCMS: [(M−1Boc)/2]+H+=1054.6, [(M−2Boc)/2]+H+=1004.6.
[0698] Step b. Coupling of Int-6 and Int-7a to give Int-7
[0699] HATU (150 mg, 0.40 mmol) in DMF (1 mL) was added, dropwise, to a stirring mixture of Int-6 (120 mg, 0.30 mmol), and penta-Boc-PMB-decapeptide (626 mg, 0.40 mmol, Int-7a) in DMF (2 mL) over a period of 20 minutes. The mixture was applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 30% to 95% methanol and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the Boc-protected intermediate, ion found by LC/MS [m/3+H].sup.+ 1150.4 (loss of 3 Boc groups). The Boc-protected intermediate was stirred in a 1/1 mixture of TFA/DCM (6 mL) containing thioanisole (225 mg, 1.81 mmol) for 20 minutes at ambient temperature. The mixture was concentrated then purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 0% to 70% acetonitrile and water using no modifier. The pure fractions were pooled and lyophilized to afford the title compound (Int-7) as a white solid. Yield: 68%, 2 Steps. Ion found by LC/MS [m/3+H].sup.+=917.2.
Example 9—Synthesis of Int-8
[0700] ##STR00829##
Step a. Synthesis of benzyloxycarbonyl-3,4-bis{[(2S)-1-methoxy-1-oxooctan-2-yl]carbamoyl}pyrrolidine
[0701] A solution of racemic 1-[(benzyloxy)carbonyl]pyrrolidine-3,4-dicarboxylic acid (3.69 g, 12.6 mmol), methyl (2S)-2-aminooctanoate (4.58 g, 26.4 mmol), and DIEA (13.8 mL, 79.3 mmol), in DMF (20 mL) was treated with HATU (10.0 g, 26.4 mmol) at room temperature for 30 minutes. The crude product was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using no modifier. Two diastereomers where observed and were separable. The desired more polar isomer (Int-8a) had the shorter retention time and was taken on to Step b. Yield 1.44 g, 19%.
Step b. Synthesis of Int-8
[0702] A solution of benzyloxycarbonyl-3,4-bis{[(2S)-1-methoxy-1-oxooctan-2-yl]carbamoyl}pyrrolidine (1.44 g, 2.38 mmol, more polar isomer from Step A, Int-8a) in THF (10 mL) and water (10 mL) was treated with powdered lithium hydroxide (0.143 g, 5.96 mmol) at room temperature. LCMS after 10 minutes showed complete conversion. The reaction was made slightly acidic by adding concentrated HCl, and concentrated to an oil. The oil was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using TFA as the modifier. Yield 1.14 g, 83% yield.
Example 10—Synthesis of aminooctanoyl penta Boc polymyxin B decapeptide (Int-18)
[0703] ##STR00830##
Step a. Synthesis of penta Boc polymyxin B decapeptide (Int-7a)
[0704] Tri-Boc polymyxin B heptapeptide (0.464, 0.437 mmol, Int-1), and tripeptide acid (0.316, 0.398 mmol, Int-3), were dissolved in DMF (1 mL), DIEA (0.229 mL, 1.31 mmol), then treated with HATU (0.166 g, 0.437 mmol). After stirring for 30 min LCMS showed complete conversion. The crude Cbz-product was diluted with methanol (4 mL), and charged with 5% Pd/C (0.250 g) and placed under a hydrogen atmosphere. After 2 hr the mixture was filtered through Celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using no modifier. Yield of Int-7a was 0.640 g, 94% (two steps). Ion(s) found by LCMS: [(M−1 Boc)/2]+H+=1054.6, [(M−2Boc)/2]+H+=1004.6.
Step b. Synthesis of aminooctanoyl penta Boc polymyxin B decapeptide (Int-18)
[0705] A solution of Int-7a (3.00 g, 1.918 mmol), Cbz-aminooctanoic acid (0.591 g, 2.01 mmol), and DIEA (1.05 mL, 6.04 mmol), in DMF (10 mL), was treated with a solution of HATU (0.766 g, 2.01 mmol) dissolved in DMF (3 mL), dropwise over 60 minutes. After 2 h, LCMS showed complete consumption of starting material. The crude mixture was treated with 5% Pd/C (1.7 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere for 2 hr or until complete by LCMS. The crude reaction was filtered through Celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 20% to 100% methanol and water, using no modifier. Yield of Int-18 was 2.59 g, 79% (two steps). Ion(s) found by LCMS: [M/2]+H+=853.4.
Example 11—Synthesis of Int-9
[0706] ##STR00831##
[0707] Step a. Synthesis of Penta-Boc Dimer
##STR00832##
[0708] A solution of Int-8 (0.401 g, 0.70 mmol), Int-7a (2.18 g, 1.39 mmol), and DIEA (0.76 mL, 4.39 mmol) in DMF, was treated with a solution of HATU (0.557 g, 1.46 mmol) in DMF (5 mL), dropwise over 1 h. After stirring for an additional 30 minutes the reaction was charged with 5% Pd/C (1.5 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere for 2 h. The resulting mixture was filtered through celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 1.39 g, 56% yield.
[0709] Step b.
##STR00833##
[0710] A solution of product from Step a (0.300 g, 0.085 mmol), Cbz-PEG8-acid (0.049 g, 0.085 mmol), and DIEA (0.049 mL, 0.280 mmol), in DMF (2 mL), was treated with a solution of HATU (0.036 g, 0.0934 mmol) in DMF (1 mL), dropwise over 1 h. After stirring an additional 30 minutes the reaction was charged with 5% Pd/C (0.35 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere for 2 h. The resulting mixture was filtered through celite, concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.315 g, 94% yield. Ion(s) found by LCMS: [M−2(Boc)/3]+H+=1252.4.
[0711] Step c.
##STR00834##
[0712] A solution of product from Step b (0.315 g, 0.080 mmol), bis-sulfone acid (0.044 g, 0.088 mmol), and N-methylmorpholine (0.020 mL, 0.175 mmol), in DMF (3 mL), was treated with a solution of HATU (0.033 g, 0.088 mmol), in DMF (1 mL), dropwise over 1 h. After stirring for an additional 30 minutes, the reaction was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.243 g, 69% yield. Ion(s) found by LCMS: did not ionize.
[0713] Step d.
##STR00835##
[0714] A solution of product from Step c (0.216 g, 0.049 mmol), suspended in DCM (2 mL) was treated with TFA (2 mL) at room temperature. After stirring for 5 minutes, the reaction was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using TFA as the modifier. Yield of Int-9 was 0.226 g, quantitative yield. Ion(s) found by LCMS: [M/3]+H+=1146.3, [M/4]+H+=860.0, [M/5]+H+=688.2.
Example 12—Synthesis of Int-10
[0715] ##STR00836## ##STR00837## ##STR00838##
Step a. Synthesis of Amino Peg 8 penta Boc polymyxin B undecapeptide
[0716] A solution of Int-18 (0.600 g, 0.352 mmol), Cbz-amino Peg 8 acid (0.223 g, 0.387 mmol), and DIEA (0.202 mL, 1.161 mmol), in DMF (3 mL) was treated with a solution of HATU (0.147 g, 0.387 mmol), in DMF (1 mL), dropwise over 1 hr. After stirring for an additional 30 minutes, the reaction was charged with 5% Pd/C (0.400 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere until complete by LCMS (2 h). The resulting mixture was filtered through celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.450 g, 64%. Ion(s) found by LCMS: [M/2]+H+=1064.6, [M−1(Boc)/2]+H+=1014.6.
[0717] Step b. Synthesis of deca Boc PEG8 polymyxin B dimer
[0718] A solution of amino PEG8 penta Boc polymyxin B undecapeptide (0.746 g, 0.350 mmol), Cbz iminodiacetic acid (0.046 g, 0.171 mmol), and DIEA (0.197 mL, 1.13 mmol), in DMF (5 mL), was treated with a solution of HATU (0.133 g, 0.350 mmol) in DMF (2 mL), dropwise over 1 h. After stirring an additional 30 minutes, the reaction was charged with 5% Pd/C (0.350 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere until complete by LCMS. The crude reaction was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.465 g, 62% (two steps). Ion(s) found by LCMS: [M−3(Boc)/4]+H+=897.3, [M−4(Boc)/4]+H+=872.3.
[0719] Step c. Synthesis of deca Boc PEG 8 polymyxin B dimer bis sulfone
[0720] A solution of deca Boc PEG 8 polymyxin B dimer (0.465 g, 0.107 mmol), bis sulfone acid (0.059 g, 0.117 mmol), and N-methylmorpholine (0.026 mL, 0.235 mmol), in DMF (5 mL) was treated with a solution of HATU (0.045 g, 0.117 mmol) and DMF (1 mL), dropwise over 1 h. The resulting solution was stirred for an additional 30 minutes, then concentrated to a viscous oil. The crude oil was used in the next Step without further purification. Yield 0.465 g, 62% (two steps). Ion(s) found by LCMS: did not ionize.
[0721] Step d. Synthesis of Int-10
[0722] A solution of crude deca Boc PEG 8 polymyxin B dimer bis sulfone, suspended in DCM (4 mL), was treated with TFA (4 mL), and stirred at room temperature for 5 min. The resulting solution was concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using TFA as the modifier. Int-10 was obtained in a yield of 0.099 g, 27% (three steps). Ion(s) found by LCMS: [M/2]+H+=1917.5, [M/3]+H+=1278.7.
Example 13—Synthesis of Int-11
[0723] ##STR00839##
[0724] Step a. Synthesis of Deca Boc Amino PEG 8 Dimer
##STR00840##
[0725] A solution of Int-18 (0.500 g, 0.291 mmol), Cbz-amino PEG 8 diacid (0.089 g, 0.138 mmol), and DIEA (0.167 mL, 0.960 mmol), in DMF (4 mL), was treated with a solution of HATU (0.122 g, 0.320 mmol), in DMF (1 mL), dropwise over 1 h. After stirring an additional 30 minutes, the reaction was charged with 5% Pd/C (0.5 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere until complete by LCMS. The crude mixture was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.212 g, 37% (two steps). Ion(s) found by LCMS: [M−3(Boc)/4]+H+=897.3, [M−4(Boc)/4]+H+=872.3.
[0726] Step b. Synthesis of Bis Sulfone Deca Boc Amino PEG 8 Dimer
##STR00841##
[0727] A solution of deca Boc amino PEG 8 dimer (0.212 g, 0.0545 mmol), bis-sulfone acid (0.033 g, 0.0654 mmol), and N-methylmorpholine (0.022 mL, 0.196 mmol), in DMF (3 mL), were treated with a solution of HATU (0.025 g, 0.065 mmol), in DMF (1.0 mL), dropwise over 1 h. The resulting crude reaction mixture was concentrated to an oil and used in the next Step without purification. The product did not ionize by LCMS.
[0728] Step c. Synthesis of Int-11
##STR00842##
[0729] The crude oil was dissolved in DCM (5 mL), and treated with TFA (5 mL), for 5 minutes at room temperature. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using 0.1% TFA as the modifier. Yield 0.099 g, 40% (two steps). Ion(s) found by LCMS: [M/3]+H+=1123.3, [M/4]+H+=842.7.
Example 14—Conjugate 1 (Conjugate of h-IgG1 Fc-2(Int-4))
[0730] A solution of h-IgG1 Fc (SEQ ID NO: 4) (4.66 mg in 175 μL of pH 7.4 PBS, MW=54,731 Da), Tris pH=8.0 (155 μL), and TCEP (68 μL of 10 mM stock in pH 8.0 Tris) were mixed at room temperature. LCMS after 1 hr showed complete reduction. Int-4 was added as solution (153 μL of 8.04 mg dissolved in 250 μL of pH 8.0 Tris, 8 eq/disulfide). LCMS after 12 h showed that all the reduced Fc was consumed, and a product peak with mass=32,380 Da (DAR=2). The solution was diluted with 15 mL pH 7.4 PBS and concentrated using a centrifugal concentrator (10,000 MWCO) to a volume of 1 mL, two times. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Yield 0.001 g, 38% after purification. Product peak found by LCMS 32,380 Da (DAR=2/Fc monomer, 4/Fc dimer). Final product: Conjugate 1.
Example 15—Conjugate 2 (Conjugate of m-IgG2a Fc-3(Int-4))
[0731] A solution of m-IgG2a Fc (SEQ ID NO: 2) (4.5 mg in 315 μL pH 7.4 PBS, MW˜54,000 Da), and TCEP (66 μL of 10 mM stock in pH 8.0 Tris) were mixed at room temperature. LCMS after 1 hr showed complete reduction. Int-4 was added as powder (7.9 mg, 8 eq/hinge disulfide). LCMS after 12 h showed that all the reduced Fc was consumed, and a product peak with mass=36,512 Da (DAR=3). The solution was diluted with 15 mL pH 7.4 PBS and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Yield 0.004 g, 69% after purification. Product peak found by LCMS 36,512 Da (DAR=3/Fc monomer, 6/Fc dimer). Final product: Conjugate 2.
Example 16—Conjugate 3 (Conjugate of m-IgG2a Fc-8(Int-5))
[0732] Peg4-azidoNHS ester (2.2 mg in 2.2 μL of pH 7.4 PBS 1× buffer solution) was added to a solution of m-IgG2a Fc (SEQ ID NO: 2) (28 mg in 300 μL pH 7.4 PBS, MW˜54,000 Da) and the mixture was shaken gently for 12 hours at ambient temperature.
[0733] The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA). Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Int-5 (28 mg, 7.8 μmol) in a solution of 10 nM sodium ascorbate, 10 mM Copper (II) sulfate and 10 mM tris(3-hydroxylpropyltriazolylmethyl)amine in pH 7.4 PBS 1×buffer (3 mL) was added to the purified azido-m-IgG2a conjugate in pH 7.4 PBS 1× buffer and the mixture was shaken gently at ambient temperature for 24 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 17—Conjugate 4 (Conjugate of m-IgG2a(His).SUB.6 .Fc-3(Int-9))
[0734] A solution of m-IgG2a(His).sub.6 Fc (SEQ ID NO: 6) (6.15 mg in 0.5 mL pH 7.4 PBS, MW˜59,880 Da), and TCEP (1.85 μL of 500 mM stock in pH 7.4 PBS, 3 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS with 2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-9 was added as powder (2.11 mg, 1.5 eq/hinge disulfide). LCMS after 1 h at room temperature showed all of the reduced Fc was consumed, and a broad peak with masses corresponding to DAR=2, 3, and 4. The solution was treated with DTT (0.142 mg, 9 eq) and stored at 4 C for 48 h. LCMS showed DAR 3 prior to purification. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 18—Conjugate 5 (Conjugate of h-IgG1 Fc-2(Int-9))
[0735] A solution of h-IgG1 Fc (SEQ ID NO: 2) (10.5 mg in 0.6 mL pH 7.4 PBS, MW˜54,731 Da), and TCEP (4.6 μL of 500 mM stock in pH 7.4 PBS, 6 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS with 2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-9 was added as powder (5.2 mg, 3.0 eq/hinge disulfide). LCMS after 1 h at room temperature showed all of the reduced Fc was consumed, and a broad product peak with a mass=61,306 Da(DAR=2). The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 19—Conjugate 6 (Conjugate of m-IgG2a(His).SUB.6 .Fc-2(Int-5))
[0736] Peg4-azido-NHS ester (4.4 mg in 2.2 mL of pH 7.4 PBS 1× buffer solution) was added to a solution of m-IgG2a(His).sub.6 Fc (SEQ ID NO: 6) (30 mg in 2.4 mL pH 7.4 PBS, MW˜55,600 Da) and the mixture was shaken gently for 12 hours at ambient temperature. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA). Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Int-5 (28 mg, 7.8 μmol) in a solution of 10 nM sodium ascorbate, 10 mM Copper (II) sulfate and 10 mM tris(3-hydroxylpropyltriazolylmethyl)amine in pH 7.4 PBS 1× buffer (3 mL) was added to the purified azido-m-IgG2a conjugate in pH 7.4 PBS 1× buffer and the mixture was shaken gently at ambient temperature for 24 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 20—Conjugate 7 (Conjugate of h-IgG1(His).SUB.6 .Fc-2(Int-10))
[0737] A solution of h-IgG1(His).sub.6 Fc (SEQ ID NO: 8) (10.0 mg in 1.0 mL pH 7.4 PBS, MW˜56,633 Da), and TCEP (6.3 μL of 500 mM stock in pH 7.4 PBS, 9 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS with 2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-10 was added as powder (5.3 mg, 3.0 eq/hinge disulfide). LCMS after 2 h at room temperature showed all of the reduced Fc was consumed, and a broad product peak, mass=63,682 Da(DAR=2). The reaction was treated with DTT (0.272 mg, 10 eq) and stored at 4 C. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 21—Conjugate 8 (Conjugate of h-IgG1(His).SUB.6 .Fc-2(Int-7))
[0738] DBCO-peg4-NHS ester (14.4 mg in 2.5 mL of pH 7.4 PBS 1× buffer solution) was added to a solution of h-IgG1(His).sub.6 Fc (SEQ ID NO: 8) (30 mg in 3 mL pH 7.4 PBS, MW˜56,633 Da) and the mixture was shaken gently for 12 hours at ambient temperature. Some precipitate was observed during at the end of this time period. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA). Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Int-7 (28 mg, 7.1 μmol) in a solution of in pH 7.4 PBS 1× buffer (2 mL) was added to the purified azido-h-IgG1 conjugate (4.97 mg) in pH 7.4 PBS 1× buffer (3 mL). Acetonitrile (2 mL) was added when solution began to turn cloudy. The mixture was shaken gently at ambient temperature for 24 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 22—Conjugate 9 (Conjugate of h-IgG1(His).SUB.6 .Fc-4(Int-10))
[0739] A solution of engineered h-IgG1(His).sub.6 Fc (SEQ ID NO: 10) (10.0 mg in 0.84 mL pH 7.4 PBS, MW˜57,676 Da), and TCEP (12.5 μL of 500 mM stock in pH 7.4 PBS, 9 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS with 2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-10 was added as powder (6.87 mg, 2.0 eq/hinge disulfide). LCMS after 24 h at room temperature showed all the reduced Fc was consumed, and a broad product peak with masses=64,726 Da(DAR=2), 71,915 Da(DAR=4), and 78,818 Da (DAR=6). The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 23—Conjugate 10 (Conjugate of h-IgG1(His).SUB.6 .Fc-4(Int-9))
[0740] A solution of h-IgG1(His).sub.6 Fc (SEQ ID NO: 10) (10.0 mg in 0.84 mL pH 7.4 PBS, MW˜57,676 Da), and TCEP (12.5 μL of 500 mM stock in pH 7.4 PBS, 9 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS with 2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-9 was added as powder (6.87 mg, 2.0 eq/hinge disulfide). LCMS after 1 h at room temperature showed all of the reduced Fc was consumed, and a broad product peak with a mass=70,223 Da(DAR=4). The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 24—Conjugate 11 (Conjugate of h-IgG1(His).SUB.6 .Fc-4(Int-11))
[0741] A solution of h-IgG1(His).sub.6 Fc (SEQ ID NO: 10) (10.0 mg in 0.84 mL pH 7.4 PBS, MW˜57,676 Da), and TCEP (12.5 μL of 500 mM stock in pH 7.4 PBS, 9 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS with 2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-11 was added as powder (6.31 mg, 2.0 eq/hinge disulfide). LCMS after 1 h at room temperature showed all of the reduced Fc was consumed, and a broad product peak with a mass=69,913 Da(DAR=4). The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 25—Synthesis of Int-12 (maleimide-PEG 8-amino-polymyxin B)
[0742] ##STR00843##
[0743] Step a. Synthesis of Penta Boc Polymyxin B Decapeptide (Int-7a)
##STR00844##
[0744] Tri-Boc polymyxin B heptapeptide (0.464 g, 0.437 mmol, Int-1), and tripeptide acid (0.316 g, 0.398 mmol, Int-3), were dissolved in DMF (1 mL), DIEA (0.229 mL, 1.31 mmol), then treated with HATU (0.166 g, 0.437 mmol). After stirring for 30 min LCMS showed complete conversion. The crude Cbz-product was diluted with methanol (4 mL), and charged with 5% Pd/C (0.250 g) and placed under a hydrogen atmosphere. After 2 hr the mixture was filtered through Celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using no modifier. Yield of Int-7a was 0.640 g, 94% (two steps). Ion(s) found by LCMS: [(M−1 Boc)/2]+H+=1054.6, [(M−2Boc)/2]+H+=1004.6.
[0745] Step b. Synthesis of Aminooctanoyl Penta Boc Polymyxin B Decapeptide (Int-18)
##STR00845##
[0746] A solution of Int-7a (3.00 g, 1.918 mmol), Cbz-aminooctanoic acid (0.591 g, 2.01 mmol), and DIEA (1.05 mL, 6.04 mmol), in DMF (10 mL), was treated with a solution of HATU (0.766 g, 2.01 mmol) dissolved in DMF (3 mL), dropwise over 60 minutes. After 2 h, LCMS showed complete consumption of starting material. The crude mixture was treated with 5% Pd/C (1.7 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere for 2 hr or until complete by LCMS. The crude reaction was filtered through Celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 20% to 100% methanol and water, using no modifier. Yield of Int-18 was 2.59 g, 79% (two steps). Ion(s) found by LCMS: [M/2]+H+=853.4.
[0747] Step c. Synthesis of Maleimide Penta Boc Polymyxin B Undecapeptide
##STR00846##
[0748] A solution of aminooctanoyl penta Boc polymyxin B undecapeptide (0.5 g, 0.293 mmol), maleimide-acid (0.168 g, 0.323 mmol), DIEA (0.153 mL, 0.880 mmol), in DMF (3 mL), was treated with a solution of HATU (0.123 mg, 0.323 mmol) in DMF (1 mL) dropwise over 60 minutes. The resulting mixture was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 20% to 100% methanol and water, using no modifier. Yield 0.595 g, 92%. Ion(s) found by LCMS: [M−1(Boc)/2]+H+=1054.6, [M−2(Boc)/2]+H+=1004.6
[0749] Step d. Synthesis of Int-12
##STR00847##
[0750] A solution of maleimide penta Boc polymyxin B undecapeptide (0.595 g, 0.269 mmol), dissolved in DCM (2 mL), was treated with TFA (2 mL) while stirring for 5 minutes at room temperature. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using TFA (0.1%) as the modifier. Yield 0.560 g, 73%. Ion(s) found by LCMS: [M/3]+H+=570.0, [M/4]+H+=427.7.
Example 26—Synthesis of Int-13 (Bis Sulfone Peg 8 Polymyxin B)
[0751] ##STR00848##
Step a. Synthesis of Amino PEG 8 Penta Boc Polymyxin B Undecapeptide
[0752] ##STR00849##
[0753] A solution of aminooctanoyl penta Boc polymyxin B decapeptide (0.600 g, 0.352 mmol, Int-18), Cbz-amino Peg 8 acid (0.223 g, 0.387 mmol), and DIEA (0.202 mL, 1.161 mmol), in DMF (3 mL) was treated with a solution of HATU (0.147 g, 0.387 mmol), in DMF (1 mL), dropwise over 1 hr. After stirring for an additional 30 minutes, the reaction was charged with 5% Pd/C (0.400 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere until complete by LCMS (2 h). The resulting mixture was filtered through celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.450 g, 64%. Ion(s) found by LCMS: [M/2]+H+=1064.6, [M−1(Boc)/2]+H+=1014.6.
Step b. Synthesis of PEG 8 Penta Boc Polymyxin B Undecapeptide
[0754] ##STR00850##
[0755] A solution of amino Peg 8 penta Boc polymyxin B undecapeptide (0.450 g, 0.211 mmol), bisulfone acid (0.116 g, 0.233 mmol), and N-methylmorpholine (0.051 mL, 0.465 mmol) in DMF (4 mL), was treated with a solution of HATU (0.0884 g, 0.232 mmol), in DMF (1.0 mL), dropwise over 1 hr. After stirring for an additional 30 minutes the reaction was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.475 g, 86%. Ion(s) found by LCMS: No ions observed.
Step c. Synthesis of Bis Sulfone PEG 8 Polymyxin B (Int-13)
[0756] ##STR00851##
[0757] A solution of Peg 8 penta Boc polymyxin B undecapeptide (0.475 mg, 0.182 mmol), suspended in DCM (3 mL) was treated with TFA (3 mL) for 5 minutes at room temperature, then concentrated to an oil. The crude oil was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using TFA (0.1%) as the modifier. Yield 0.475 g, 97%. Ion(s) found by LCMS: [M/3]+H+=704.0, [M/4]+H+=528.3.
Example 27—Synthesis of Int-14 (Bis Sulfone Peg 4 Polymyxin B)
[0758] ##STR00852##
[0759] The title compound was prepared analogously to Int-13, where Cbz-amino PEG 4 acid was used in place of Cbz-amino Peg 8 acid in the first Step of that sequence. Ion(s) found by LCMS: [M/2]+H+=967.5, [M/3]+H+=645.3.
Example 28—Conjugate 12 (Conjugate of m-IgG2a Fc-3(Int-12))
[0760] A solution of m-IgG2a Fc (SEQ ID NO: 2) (10 mg in 169 μL pH 7.4 PBS, MW˜54,000 Da), and TCEP (2.8 μL of 50 mM stock in pH 8.0 Tris) were mixed at room temperature. LCMS after 1 hr showed complete reduction. Int-12 was added as powder (19.0 mg, 6 eq/hinge disulfide). LCMS after 48 h at 4 C showed that all the reduced Fc was consumed. The solution was diluted with 15 mL pH 7.4 PBS and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 29—Conjugate 13 (Conjugate of h-IgG1 Fc-2(Int-13))
[0761] A solution of h-IgG1 Fc (SEQ ID NO: 4) (25 mg in 1 mL pH 7.4 PBS, MW=54,731 Da), and TCEP (9.1 μL of 50 mM stock in pH 7.4 PBS, 5 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS w/2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-13 was added as powder (14.7 mg, 6 eq/hinge disulfide). LCMS after 12 h at room temperature showed all the reduced Fc was consumed, and a product peak with mass=58,329 Da(DAR=2). The solution was diluted with 15 mL pH 7.4 PBS and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 30—Conjugate 14 (Conjugate of h-IgG1 Fc-2(Int-14))
[0762] A solution of h-IgG1 Fc (SEQ ID NO: 4) (25 mg in 1 mL pH 7.4 PBS, MW=54,731 Da), and TCEP (3.6 μL of 500 mM stock in pH 7.4 PBS, 2 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS w/2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-14 was added as powder (13.7 mg, 6 eq/hinge disulfide). LCMS after 12 h at room temperature showed all the reduced Fc was consumed, and a broad product peak with mass=57,977 Da(DAR=2). The solution was diluted with 15 mL pH 7.4 PBS and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 31—Conjugate 15 (Conjugate of m-IgG2a(His).SUB.6 .Fc-3(Int-13))
[0763] A solution of m-IgG2a(His).sub.6 Fc (SEQ ID NO: 6) (12.8 mg in 1 mL pH 7.4 PBS, MW-59,880 Da), and TCEP (3.8 μL of 500 mM stock in pH 7.4 PBS, 3 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS w/2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-13 was added as powder (3.4 mg, 2 eq/hinge disulfide). LCMS after 1 h at room temperature showed all of the reduced Fc was consumed, and a product peak with mass=65,438 Da(DAR=3). The solution was diluted with 15 mL pH 7.4 PBS and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 32—Conjugate 16 (Conjugate of h-IgG1(His).SUB.6 .Fc-2(Int-5))
[0764] Peg4-azido-NHS ester (38.4 mg in 150 uL of DMF) was added to a solution of h-IgG1(His).sub.6 Fc (SEQ ID NO: 8 (140 mg in 15 mL pH 7.4 PBS 1× buffer, MW-55,111 Da) and the mixture was shaken gently for 12 hours at ambient temperature.
[0765] The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA). Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Int-5 (150 mg, 41.9 μmol) was added to the purified azido-h-IgG1 conjugate (56.9 mg in 7 mL of pH 7.4 PBS 1× buffer, MW˜58703 Da by MALDI) followed by 2 mL of a solution of 10 mM sodium ascorbate, 10 mM Copper (II) sulfate and 10 mM tris(3-hydroxylpropyltriazolylmethyl)amine in pH 7.4 PBS 1× buffer and the mixture was shaken gently at ambient temperature for 4 hours at which point an additional 0.6 mL of the solution of 10 mM sodium ascorbate, 10 mM Copper (II) sulfate and 10 mM tris(3-hydroxylpropyltriazolylmethyl)amine in pH 7.4 PBS 1× buffer was added and the mixture was shaken gently for 12 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 33—Conjugate 17 (Conjugate of Aglycosylated h-IgG1(his).SUB.6 .Fc-2(Int-5))
[0766] Peg4-azido-NHS ester (20.4 mg in 82 uL of DMF) was added to a solution of aglycosylated h-IgG1(His).sub.6 Fc (SEQ ID NO: 14) (74 mg in 8 mL pH 7.4 PBS 1× buffer, MW˜53,637 Da) and the mixture was shaken gently for 12 hours at ambient temperature. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA). Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Int-5 (150 mg, 41.9 μmol) was added to the purified azido-h-IgG2a conjugate(azido-CTP-125) (56.9 mg in 7.5 mL of pH 7.4 PBS 1× buffer, MW˜57,118 Da by MALDI) followed by 2 mL of a solution of 10 mM sodium ascorbate, 10 mM Copper (II) sulfate and 10 mM tris(3-hydroxylpropyltriazolylmethyl)amine in pH 7.4 PBS 1× buffer and the mixture was shaken gently at ambient temperature for 4 hours at which point an additional 0.6 mL of the solution of 10 mM sodium ascorbate, 10 mM Copper (II) sulfate and 10 mM tris(3-hydroxylpropyltriazolylmethyl)amine in pH 7.4 PBS 1× buffer was added and the mixture was shaken gently for an additional 12 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 34—Synthesis of Int-15
[0767] ##STR00853## ##STR00854## ##STR00855## ##STR00856## ##STR00857## ##STR00858## ##STR00859##
[0768] Step a.
##STR00860##
[0769] A solution of Cbz-iminodiacetic acid (0.149 g, 0.557 mmol), Int-18 (2.00 g, 1.173 mmol, described in Example 10 Step b.), and DIEA (0.644 mL, 3.69 mmol) in DMF, was treated with a solution of HATU (0.468 g, 1.231 mmol) in DMF (5 mL), dropwise over 1 h. After stirring for an additional 30 minutes the reaction was charged with 5% Pd/C (1.0 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere for 2 h. The resulting mixture was filtered through celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 1.0 g, 48% yield (two steps).
[0770] Step b.
##STR00861##
[0771] Product from Step a (1.03 g, 0.294 mmol), Cbz-N-amido-PEG4-acid (0.117 g, 0.294 mmol) and DIEA (0.110 mL, 0.617 mmol) in DMF (15 mL), was treated HATU (0.117 g, 0.308 mmol). After stirring for an additional 30 minutes the reaction was charged with 5% Pd/C (1.0 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere for 2 h. The resulting mixture was filtered through celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.450 g, 41% yield (two steps).
[0772] Step c.
##STR00862##
[0773] Product from Step b (0.450 g, 0.114 mmol), 4-(3-tosyl-2-(tosylmethyl)propanoyl)benzoic acid (0.023 g, 0.229 mmol) in DMF (10 mL), was treated HATU (0.043 g, 0.114 mmol). After stirring for 30 minutes the reaction was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.158 g, 31% yield.
[0774] Step d.
##STR00863##
[0775] Product from Step c (0.158 g, 0.037 mmol) was treated with TFA (5 mL) for 5 minutes then concentrated to an oil. The oil was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using 0.1% TFA as the modifier. Yield 0.47 g, 27% yield.
Example 35—Conjugate 18 (Conjugate of h-IgG1(His).SUB.6 .Fc-2(Int-15))
[0776] A solution of h-IgG1(His).sub.6 Fc (SEQ ID NO: 8) (857 μL, 10.20 mg, 0.1757 μmol, 11.90 mg/mL, MW=58,046 Da) in pH 7.4 PBS was treated with a water solution of TCEP (12.6 μL, 6.324 μmol, 500 mM) for 1 h, when Q-TOF analysis returned MW=28,773 Da. Excess TCEP was removed by buffer exchange with a 10,000 MWCO centrifugal filter (2×15 mL pH 7.4 PBS). The resulting solution was treated with Int-15 (6.15 mg, 1.405 μmol, 8 eq), then agitated gently for 1.5 h, when Q-TOF analysis showed full disappearance of the starting protein. Freshly prepared DTT water solution (54 μL, 3.513 μmol, 1 mg/100 μL) was added. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 36—Conjugate 19 (Conjugate of m-IgG2a(His).SUB.6 .Fc-3(Int-15))
[0777] A solution of mlgG2a(His).sub.6 Fc (SEQ ID NO: 6) (900 μL, 8.69 mg, 0.1408 μmol, 9.658 mg/mL, MW=61,739 Da) in pH 7.4 PBS was treated with a water solution of TCEP (7.6 μL, 3.802 μmol, 500 mM) for 1 h, when Q-TOF analysis returned MW=29,998 Da. Excess of TCEP was removed by buffer exchange with a 10,000 MWCO centrifugal filter (2×15 mL pH 7.4 PBS). The resulting solution was treated with Int-15 (3.70 mg, 0.8447 μmol, 6 eq), then agitated gently for 1.5 h, when Q-TOF analysis showed full disappearance of the starting protein. Freshly prepared DTT water solution (43 μL, 2.816 μmol, 1 mg/100 μL) was added. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 37—Synthesis of Int-16
[0778] ##STR00864## ##STR00865## ##STR00866## ##STR00867## ##STR00868##
[0779] Step a.
##STR00869##
[0780] A solution of Cbz-iminodiacetic acid (0.149 g, 0.557 mmol), Int-18 (2.00 g, 1.173 mmol, described in Example 10 Step b.), and DIEA (0.644 mL, 3.69 mmol) in DMF, was treated with a solution of HATU (0.468 g, 1.231 mmol) in DMF (5 mL), dropwise over 1 h. After stirring for an additional 30 minutes the reaction was charged with 5% Pd/C (1.0 g), vacuum flushed with hydrogen, and stirred under a hydrogen atmosphere for 2 h. The resulting mixture was filtered through celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 1.0 g, 48% yield (two steps).
[0781] Step b.
##STR00870##
[0782] Product from Step a (0.800 g, 0.228 mmol), alkyne-peg4-acid (0.0833 g, 0.274 mmol) and DIEA (0.131 mL, 0.753 mmol) in DMF (5 mL), was treated HATU (0.0954 g, 0.251 mmol). After stirring for 30 minutes the reaction was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.859 g, 99% yield.
[0783] Step c.
##STR00871##
[0784] Product from Step b (0.859 g, 0.229 mmol) was treated with TFA (5 mL) for 5 minutes then concentrated to an oil. The oil was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using 0.1% TFA as the modifier. Yield 0.863 g, 97% yield.
Example 38—Synthesis of Int-17
[0785] ##STR00872##
[0786] Step a.
##STR00873##
[0787] A solution of Int-18 (0.800 g, 0.469 mmol, described in Example 10 Step b.), alkyne-peg4 acid (0.134 g, 0.516 mmol), and DIEA (0.294 mL, 1.69 mmol) in DMF (3 mL), was treated with HATU (0.214 g, 0.563 mmol). After stirring for 30 minutes the reaction was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield 0.83 g, 91% yield.
[0788] Step b.
##STR00874##
[0789] A solution of penta Boc product from the previous Step (0.830 g, 0.426 mmol), was treated with TFA (5 mL). After stirring for 5 minutes the reaction was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using TFA as the modifier. Yield 0.689 g, 80% yield.
Example 39—Conjugate 20 (Conjugate of m-IgG2a(His).SUB.6 .Fc-3(Int-16))
[0790] A solution of mlgG2a(His).sub.6 Fc (SEQ ID NO: 6) (42.6 mg, 0.0707 μmol, 10.65 mg/mL, MW=60,252da) in pH7.4 PBS was treated with azido-peg4-NHS ester (32.9 mg, 84.8 μmol, 120 eq), then agitated gently overnight at RT. Unreacted azido-peg4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS). The final volume of the azide functionalized Fc was 3.0 mL. Maldi TOF analysis gave a molecular weight of 64,396 (azide DAR=15.1). To this was added freshly prepared solutions of CuSO.sub.4 (300 μL of 20 mM), THPTA (300 μL of 50 mM, Tris(3-hydroxypropyltriazolylmethyl)amine), ascorbic acid (300 μL of 100 mM), and alkyne derivatized small molecule (109.9 mg, 28.3 μmol, Int-16)). The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 40—Conjugate 21 (Conjugate of h-IgG1(His).SUB.6 .Fc-2(Int-17))
[0791] A solution of hlgG1(His).sub.6 Fc (SEQ ID NO: 8) (67.2 mg, 1.19 μmol, 16.8 mg/mL, MW=56,633 Da) in pH7.4 PBS was treated with azido-peg4-NHS ester (55.0 mg, 142.5 μmol, 120 eq), then agitated gently overnight at RT. Unreacted azido-peg4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS). The final volume of the azide functionalized Fc was 5.0 mL. To this was added freshly prepared solutions of CuSO.sub.4 (500 μL of 20 mM), THPTA (500 μL of 50 mM, Tris(3-hydroxypropyltriazolylmethyl)amine), ascorbic acid (500 μL of 100 mM), and alkyne derivatized small molecule (95.7 mg, 47.5 μmol, Int-17). The resulting solution was agitated gently overnight. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 41—Synthesis of Int-19, Int-20, Int-21 and Int-22
[0792] ##STR00875## ##STR00876## ##STR00877##
[0793] Synthesis of Int-19 and Int-20
[0794] HATU (198 mg, 0.52 mmol, in 1 mL of DMF) was added to a stirring mixture of propargyl-PEG4-acid (148 mg, 0.57 mmol), Int-6a, TFA salt (250 mg, 0.47 mmol) and triethylamine (158 mg, 1.57 mmol) in DMF (2 mL) and the mixture was stirred at ambient temperature for 30 minutes. The mixture was applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the di-ester intermediate. LC/MS [M+H]+=656.4. The di-ester intermediate was stirred in a 1/1/2 mixture of MeOH/THF/DI water containing LiOH (36 mg, 1.5 mmol) at ambient temperature for 5 minutes. The mixture was acidified with glacial acetic acid, concentrated and applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the title compound Int-19 as a clear oil. Yield: 74%, 2 Steps. Ion found by LC/MS [M−H]−=626.2.
[0795] Int-20 was synthesized in a similar way as described for Int-19. Isolated yield was 73%. Positive mass ions were found by LC/MS at tr=4.10 with 8-minute 5-95% CAN/water method: (M+2H.sup.+)/2=402.5.
Synthesis of Int-21
[0796] A solid mixture of PMBD (example 10 Step b., 7.444 g, 4.760 mmol), Int-19 1.494 g, 2.380 mmol), EDCl (1.141 g, 5.950 mmol) and HOAt (0.8099 g, 5.950 mmol) were dissolved in 20 mL of dry DMF immediately followed by adding triethylamine (1.66 mL, 9.52 mmol). The mixture was stirred for overnight (˜15 hours) at room temperature. HPLC analysis showed the completion of the reaction. Most of the solvent (DMF) of the reaction mixture was removed by reduced pressure rotovap to give a viscous oily residue. To this residue methanol was added with gentle stirring to triturate the reaction product. After some crystalline solid started to precipitate the flask was put standstill for more than 3 hours. Simple filtration collected the crystalline white solid with methanol wash (3×5 mL). The solid was dried to give 6.500 grams of pure product (73% isolated yield). The solid was dissolved with 100% TFA and stirred for 30 minutes. TFA was removed by rotovap. The residue was dissolved in minimum amount of water and purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 35% acetonitrile and water using 0.1% TFA modifier. The pure fractions were lyophilized to afford the title product Int-21 as a white powder (TFA salt, 6.08 g, 90% isolated yield, Positive mass ions were found (M+6H+)/6=453.8 and (M+5H.sup.+)/5=544.2.
[0797] Synthesis of Int-22
[0798] A solid mixture of PMBD (example 10 Step b, 2.505 g, 1.602 mmol), Int-20 (0.644 g, 0.801 mmol), EDCl (0.3839 g, 2.003 mmol) and HOAt (0.0.2726 g, 2.003 mmol) were dissolved in 20 mL of dry DMF immediately followed by adding triethylamine (0.56 mL, 3.2 mmol). The mixture was stirred for overnight (˜15 hours) at room temperature. HPLC analysis showed the completion of the reaction. Most of the solvent (DMF) of the reaction mixture was removed by reduced pressure rotovap to give a viscous oily residue. To this residue methanol was added with gentle stirring to triturate the reaction product. After some crystalline solid started to precipitate the flask was put standstill for more than 3 hours. Simple filtration collected the crystalline white solid with methanol wash (3×5 mL). The solid was dried to give 2.183 grams of pure product (70% isolated yield). The solid (2.020 g) was dissolved with 100% TFA and stirred for 30 minutes. TFA was removed by rotovap. The residue was dissolved in minimum amount of water and purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 35% acetonitrile and water using 0.1% TFA modifier. The pure fractions were lyophilized to afford the product Int-22 as a white powder (TFA salt, 2.297 g, 90% isolated yield,
[0799] Positive mass ions were found by LC/MS at tr=1.87 with 8-minute 5˜95% CAN/water method: (M+7H.sup.+)/7=414.2, (M+6H.sup.+)/6=483.0, (M+5H.sup.+)/5=579.5, (M+4H.sup.+)/4=724.1.
Example 42—Conjugate 22 (Conjugate of h-IgG1(His).SUB.6.Fc-2(Int-21))
[0800] A solution of hlgG1(His).sub.6Fc (SEQ ID NO: 8) (15.0 mL, 2.54 μmol, 9.33 mg/mL, MW=55111 Da) in pH 7.4 PBS×1 was treated with azido-peg4-NHS ester (0.0384 g in 150 μL of DMF, 96.9 μmol, 38 eq), then agitated gently overnight at RT. Unreacted azido-peg4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS×1). The final volume of the azide functionalized Fc was 9.00 mL with concentration at 5.16 mg/mL, MW=60302. To this Fc-azido PBS×1 buffer solution (0.0781 g, 1.50 μmol, 15.15 mL) were added alkyne derivatized small molecule (TFA salt, 209.7 mg, 54.4 μmol, Int-21) and freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (4.49 mL of 10.0 mM, 30 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 4.49 mL of 10.0 mM, 30 eq), and ascorbic acid (4.49 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 4 hours at room temperature. Another portion of freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (2.25 mL, of 10.0 mM, 15 eq) was added and the reaction mixture was gently agitated for overnight (˜15 hours) at room temperature, then the final portion of freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (2.25 mL, of 10.0 mM, 15 eq) was added and the reaction mixture was gently agitated for 2 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
[0801] Example 43—Conjugate 23 (Conjugate of h-IgG1(c-myc) Fc-2(Int-21))
[0802] A solution of hlgG1(c-myc) Fc (SEQ ID NO: 17) (9.50 mL, 1.68 μmol, 10.3 mg/mL, MW=58,065 Da) in pH 7.4 PBS×1 was treated with azido-peg4-NHS ester (98%, 6.66 mg, 16.8 μmol, 10 eq), then agitated gently overnight at RT. Unreacted azido-peg4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS×1). The final volume of the azide functionalized Fc was 9.00 mL with concentration at 8.91 mg/mL, MW=59006. To this Fc-azido PBS×1 buffer solution were added freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (2.04 mL of 10.0 mM, 15 eq), Tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 2.04 mL of 10.0 mM, 15 eq), ascorbic acid (2.04 mL of 10.0 mM, 15 eq), and alkyne derivatized small molecule (TFA salt, 209.7 mg, 54.4 μmol, Int-21). The resulting solution was agitated gently for 4 hours at room temperature. Another portion of freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (1.02 mL, of 10.0 mM, 7.5 eq) was added and the reaction mixture was gently agitated for overnight (˜15 hours) at room temperature, then the final portion of freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (1.02 mL, of 10.0 mM, 7.5 eq) was added and the reaction mixture was gently agitated for 2 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 44—Synthesis of Int-23
[0803] ##STR00878##
[0804] Step a. Synthesis of Int-1-Gly Conjugate
##STR00879##
[0805] To a mixture of Int-1 (4.24 g, 4 mmol) and Z-Gly-OH (1 g, 4.8 mmol) in anhydrous DMF (8 mL) was added HATU (1.87 g, 4.9 mmol) in portions over 20 minutes, followed by DIPEA (936 mg, 7.2 mmol). After the reaction mixture was stirred for 15 minutes, it was poured into water (100 mL). The white solid product was collected by filtration and washed with water. The material was re-dissolved in MeOH (50 mL) and treated with Pd/C (1 g). The mixture was stirred under hydrogen overnight. Pd/C was then filtered off, and the filtrate was concentrated and purified through RPLC (150 g, 15 to 75% MeOH and water). Yield 4.02 g, 89.8%. Ion found by LCMS: [(M−Boc+2H)/2].sup.+=510.4, [(M−3Boc+2H)/2].sup.+=410.2.
[0806] Step b.
##STR00880##
[0807] To a mixture of the step-a product (4.02 g, 3.592 mmol) and Z-Thr-OH (980.3 mg, 3.87 mmol) in anhydrous DMF (5 mL) was added HATU (1.47 g, 3.87 mmol) in portions over 10 minutes, followed by DIPEA (755 mg, 5.8 mmol). After the addition, the reaction was stirred for 20 minutes and then poured into water (100 mL). The white solid product was collected by filtration and washed with water. The material was re-dissolved in MeOH (50 mL) and added with Pd/C (1 g). The mixture was stirred under hydrogen overnight. Pd/C was then filtered, and the filtrate was concentrated and purified through RPLC (150 g, 15 to 80% MeOH and water). Yield 3.68 g, 83.9%. Ions found by LCMS: [(M−2Boc+2H)/2].sup.+=511.
[0808] Step c.
##STR00881##
[0809] A mixture of the step-b product (1.2 g, 0.984 mmol) and Z-Dab(Boc)-OH.Math.DCHA (605 mg, 1.13 mmol) was dissolved in anhydrous NMP (3 mL). It was added with HATU (430 mg, 1.13 mmol) in portions over 5 minutes, followed by DIPEA (150 mg, 1.13 mmol). The reaction was stirred for 30 minutes and then directly purified through RPLC (100 g, 40 to 100% MeOH and water). The collected fractions were concentrated by rotary evaporation to a white solid (Ion found by LCMS: [M−2Boc+2H)/2].sup.+=678). The material was re-dissolved in MeOH (30 mL) and treated with Pd/C. The mixture was stirred under hydrogen overnight. Pd/C was filtered, and the filtrate was concentrated by rotary evaporation and further dried under high vacuum. Yield 1.07 g, 76.6%. Ion found by LCMS: [(M−2Boc+2H)/2].sup.+=611.
[0810] Step d. Synthesis of Octa-Boc Int-23
##STR00882##
[0811] A mixture of the step-c product (313.6 mg, 0.2207 mmol) and INT-8 alkyne-PEG4-Pyrrolidine-(NLeu-CO2H)2 (65.9 mg, 0.105 mmol) was dissolved in anhydrous DMF (1 mL). It was added with DIPEA (65 mg, 0.5 mmol) followed by HATU (83.9 mg, 0.2207 mmol) in portions over 5 minutes. The reaction was stirred for 30 minutes and then directly purified through RPLC (50 g, 40 to 100% MeOH and water). Yield 165 mg, 49.6%. Ions found by LCMS: [M−3Boc+3H)/3].sup.+=1044.6, [M−4Boc+3H)/3].sup.+=1011.3, [M−6Boc+3H)/3].sup.+=944.4.
[0812] Step e. Synthesis of Int-23
##STR00883##
[0813] The step-d product (165 mg, 0.0521 mmol) was dissolved in TFA/DCM (1:1, 1 mL), and the solution was stirred for 30 minutes. It was directly purified by RPLC (50 g, 5 to 30% acetonitrile and water, using 0.1% TFA as modifier). Yield 160 mg, 86.6%. Ions found by LCMS: [M+4H)/4].sup.+=658.4, [M+5H)/5].sup.+=527.1, [M+6H)/6].sup.+=439.4, [M+7H)/7].sup.+=376.8.
Example 45—Conjugate 24 (Conjugate of h-IgG1(c-myc) Fc-2(Int-23))
[0814] Int-21 (60 mg, 0.0169 mmol) was added to h-IgG1(c-myc) Fc (SEQ ID NO: 17)-(azido).sub.5 conjugate (0.0002079 M, 2 mL) solution, followed by 0.832 mL of 0.01 M CuSO.sub.4 solution, 0.832 mL of 0.01 M sodium ascorbate solution, and 0.832 mL of 0.01 M THPTA solution. The reaction was gently shaken at room temperature of 4 hours, then added with an additional amount of freshly prepared CuSO.sub.4 solution (0.01M, 0.416 mL). The reaction was shaken overnight. Additional CuSO.sub.4 solution (0.01 M, 0.416 mL, freshly prepared) was added, and the reaction mixture was shaken for another 2 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 46—Conjugate 25 (Conjugate of h-IgG1(c-myc) Fc-2(Int-22))
[0815] A solution of hlgG1(His).sub.6Fc (SEQ ID NO: 17) (20.0 mL, 3.62 μmol, 10.5 mg/mL, MW=57810 Da) in pH 7.4 PBS×1 was treated with azido-peg4-NHS ester (98%, 6.66 mg, 16.8 μmol, 10 eq), then agitated gently overnight at RT. Unreacted azido-peg4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS×1). The final volume of the azide functionalized Fc was 9.00 mL with concentration at 12.4 mg/mL, MW=59641. To this Fc-azido PBS×1 buffer solution (0.0248 g, 0.416 μmol, 2.00 mL were added alkyne derivatized small molecule (TFA salt, 67.1 mg, 16.6 μmol, Int-22) and freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (0.832 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.832 mL of 10.0 mM, 20 eq), ascorbic acid (0.832 mL of 10.0 mM, 20 eq). The resulting solution was agitated gently for 4 hours at room temperature. Another portion of freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (0.420 mL, of 10.0 mM, 10 eq) was added and the reaction mixture was gently agitated for overnight (˜15 hours) at room temperature, then the final portion of freshly prepared pH 7.4 PBS×1 solutions of CuSO.sub.4 (0.420 mL, of 10.0 mM, 20 eq) was added and the reaction mixture was gently agitated for 2 hours. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 47—Conjugate 26 (Conjugate of m-IgG2a(His).SUB.6 .Fc-3(Int-13))
[0816] A solution of mlgG2a(His).sub.6 Fc (SEQ ID NO: 6) (3.1 mg in 0.6 mL pH 7.4 PBS, MW-53,782 Da), and TCEP (2.1 μL of 500 mM stock in pH 7.4 PBS, 9 eq/disulfide) were mixed at room temperature. LCMS after 1 hr showed complete reduction. The solution was diluted with 15 mL pH 7.8 PBS w/2 mM EDTA and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times. Int-10 (described in Example 12) was added as powder (1.16 mg, 2 eq/hinge disulfide). LCMS after 1 h at room temperature showed all of the reduced Fc was consumed. The crude mixture was diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of m-IgG2a(His).sub.6), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards (
Example 48—Minimum Inhibitory Concentration (MIC) Assays
[0817] Generation of E. coli BW25113+pUC18-mcr-1.
[0818] An mcr-1 expression plasmid was constructed (GenScript; Piscataway, N.J.) using the pUC18 high copy number plasmid (Yanisch-Perron, et al., 1985). A 1649-bp fragment was synthesized containing, in order from 5′ to 3′: EcoR1 restriction site (5′-GAATTC-3′), a ribosomal binding site (5′-AGGAGG-3′), a 5-bp native start codon upstream sequence (5′-TTCTC-3′) from the mcr-1-bearing plasmid pHNSHP45 (Liu, Y Y, et al., 2015; GenBank Accession #KP347127), the 1626-bp mcr-1 open reading frame with stop codon (“orf00073” of GenBank Accession #KP347127), and finally an Xba1 restriction site sequence (5′-TCTAGA-3′). Restriction digestion and subsequent ligation into the multiple cloning site of the pUC18 backbone resulted in directional integration of the mcr-1 gene. The pUC18-mcr-1 plasmid was transformed into chemically-competent E. coli BW25113 (Coli Genetic Stock Center #7636) as described previously (Chung, et al., PNAS. 1989), recovered for 1 h shaking at 37° C. in Super Optimal broth with Catabolite repression (SOC) media and then aliquots were plated on Luria-Bertani (LB) media containing 100 μg/mL of carbenicillin. Putative transformant colonies were then verified in MIC assays.
Strains and Culture Conditions.
[0819] Bacterial strains were stored as glycerol stocks at −80° C. prior to culturing on cation-adjusted Mueller-Hinton agar (MHA; BD cat. no. 211438) at 37° C. Antibacterial activity was assessed against a panel consisting of Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae strains (see Table 2).
TABLE-US-00021 TABLE 2 Strains used in MIC assays Abbreviated Alternative name Species Strain ID ID Source Notes Ec BW Escherichia coli BW25113 7636 CGSC Ec BW + FBS Escherichia coli BW25113 7636 CGSC MIC run in the presence of 50% heat-inactivated FBS Ec BW Escherichia coli BW25113 + 7636 In- COL-R; contaions (mcr-1) pUC18- house mcr-1 expression mcr-1 plasmid, pUC18-mcr-1 Ec BW4X-12 Escherichia coli BW25113 7636 In- COL-R; spontaneous (COL-R) 4X-12 house mutant (PmrB- Leu10Pro) Ec 25922 Escherichia coli ATCC ATCC 25922 Ec 25922 + Escherichia coli ATCC ATCC MIC run in the FBS 25922 presence of 50% heat-inactivated FBS Ec 25922 Escherichia coli ATCC In- COL-R; spontaneous 64X-1 25922 house mutant (PmrB- (COL-R) 64X-1 Ala159Val) Ec AR0349 Escherichia coli AR0349 CDC COL-R; mcr-1; MDR (mcr-1) Ec 2469 Escherichia coli ATCC 1001728 ATCC MDR BAA-2469 Ab 19606 Acinetobacter ATCC ATCC baumannii 19606 Ab ABS075 Acinetobacter ABS075- NR-49900 BEI MDR baumannii UW Ab 17978 Acinetobacter ATCC ATCC baumannii 179778 Ab 8990 Acinetobacter MMX Micro COL-R (COL-R) baumannii 8990 myx, LLC Pa 27853 Pseudomonas ATCC ATCC aeruginosa 27853 Pa PAO1 Pseudomonas PAO1 ATCC ATCC aeruginosa BAA-47 Pa LES431 Pseudomonas LES431 BCCM BCCM MDR aeruginosa 276224 Kp10031 Klebsiella ATCC PCI 602 ATCC pneumoniae 10031 Kp 43816 Klebsiella ATCC ATCC pneumoniae 43816 Kp 6951 Klebsiella MMX Micro COL-R; MDR (COL-R) pneumoniae 6951 myx, LLC Abbreviations: CGSC-Coli Genetic Stock Center; FBS-fetal bovine serum; COL-R-colistin-resistant; ATCC-American Type Culture Collection; MDR-multidrug-resistant; CDC-Centers for Disease Control and Prevention; BEI-Biodefense and Emerging Infections Research Resources Repository; BCCM-Belgian Co-Ordinated Collections of Micro-Organisms
Minimum Inhibitory Concentration (MIC) assays.
[0820] MIC assays were performed according to CLSI broth microdilution guidelines (M07-A9, M100-S23) with the exception of using a 100 μL assay volume, using RPMI media instead of Mueller-Hinton media and preparing stock compounds at 50× final concentration. Briefly, stock solutions of all antibacterial agents were prepared fresh in appropriate solvents (i.e. PBS, pH 7.4, DMSO, DI, etc.). Stock concentrations were made at 50× the highest final assay concentration and serially diluted 2-fold, 8 or 12 times in a 96-well PCR plate (VWR cat. no. 83007-374). Bacterial cell suspensions generated from MHA plate cultures were prepared in 0.85% saline and adjusted to ˜0.1 OD.sub.600 nm. Next, cell suspensions were diluted 1:200 in RPMI containing 5% Luria Broth (LB) to improve bacterial growth (BD Difco, cat. no. 244620) to a concentration of ˜5×10.sup.5 CFU (colony-forming units)/mL. Due to poor solubility in Mueller-Hinton broth media, conjugates 1-15, 18-19 and 26 were evaluated initially in RPMI 1640 medium (+) L-glutamine, (−) sodium bicarbonate (“RPMI A”; MP Biomedicals, cat. no. 1060124), adjusted to pH 7.0 with NaOH and buffered with MOPS (Sigma, cat. no. M3183-500G) as is specified per CLSI guidelines for antifungal susceptibility testing (M27-A3, M27-S4). Later, conjugates 16 and 17, and 20-25 were evaluated in RPMI 1640 medium (+) L-glutamine, (+) sodium bicarbonate, pH 7.4 (“RPMI B”; Gibco, cat. no. 11875-085) as has been described as a more physiologically-relevant media for susceptibility testing of antibacterial compounds. Dorschner R A et al., The mammalian ionic environment dictates microbial susceptibility to antimicrobial defense peptides. FASEB J. 20(1):35-42 (2017). Ninety-eight microliters of each cell suspension in MHB were added to test wells in a 96-well assay plate (Costar cat. no. 3370). A Beckman Multimek 96 liquid handling robot was used to dispense 2 μL of each 50× stock compound into the plate containing 98 μL of each strain in MHB (2% final solvent concentration). E. coli strains BW25113 and ATCC 25922 were run in the presence and absence of 50% heat-inactivated fetal bovine serum (FBS; Sigma, cat. no. F4135-100 mL). Plates were mixed by shaking then incubated at 37° C. overnight (16-20 h). MIC values were read visually at 100% growth inhibition for all conjugates and control drugs (Tables 3A and 3B).
TABLE-US-00022 TABLE 3A MIC values for conjugates MIC (μg/ml) Ec Ec Ec BW Ec 25922 Ec Assay Ec BW + Ec BW 4X-12 Ec 25922 + COL-R AR0349 Ec media Compound BW FBS (mcr-1) (COL-R) 25922 FBS 64X-1 (mcr-1) 2469 RPMI A COL (colistin) 1 0.125 8 32 4 0.25 ND ND 4 RPMI B COL (colistin) ND ND ND ND 0.25 0.125 0.25 0.5 0.25 RPMI A Conjugate 1 102.4 12.8 ND ND ND ND ND ND ND RPMI A Conjugate 2 >128 >128 ND ND ND ND ND ND ND RPMI A Conjugate 3 16 32 ND ND 64 64 ND ND ND RPMI A Conjugate 4 32 32 >128 >128 >128 64 ND ND >128 RPMI A Conjugate 5 16 64 256 >256 >256 128 ND ND 256 RPMI A Conjugate 6 4 8 8 16 32 16 ND ND 8 RPMI A Conjugate 7 16 32 64 128 64 64 ND ND 32 RPMI A Conjugate 8 16 32 >256 >256 16 32 ND ND 16 RPMI A Conjugate 9 16 16 >256 32 16 128 ND ND 16 RPMI A Conjugate 10 16 64 128 256 16 128 ND ND 16 RPMI A Conjugate 11 16 64 128 >256 32 256 ND ND 32 RPMI A Conjugate 12 16 16 ND ND 64 256 ND ND ND RPMI A Conjugate 13 64 128 >128 >128 >128 >128 ND ND 128 RPMI A Conjugate 14 32 >128 >128 >128 >128 >128 ND ND >128 RPMI A Conjugate 15 >128 >128 >128 >128 >128 >128 ND ND >128 RPMI B Conjugate 16 8 8 16 16 16 32 ND ND 16 RPMI B Conjugate 17 8 4 16 16 16 32 ND ND 16 RPMI A Conjugate 18 16 32 32 64 32 64 ND ND 16 RPMI A Conjugate 19 16 8 32 64 32 32 ND ND 16 RPMI B Conjugate 20 8 8 16 16 16 16 ND ND 16 ND-not determined; RPMI A-RPMI 1640 (+) L-glutamine, (−) sodium bicarbonate, containing 5% LB, adjusted to pH 7.0 with NaOH, buffered with MOPS: RPMI B-RPMI 1640 (+) L-glutamine, (+) sodium bicarbonate, pH 7.4, containing 5% LB.
TABLE-US-00023 TABLE 3B MIC values for conjugates MIC (μg/ml) Ec Ec Ec BW Ec 25922 Ec Assay Ec BW + Ec BW 4X-12 Ec 25922 + COL-R AR0349 Ec media Compound BW FBS (mcr-1) (COL-R) 25922 FBS 64X-1 (mcr-1) 2469 RPMI B Conjugate 21 8 8 16 16 64 128 ND ND 32 RPMI B Conjugate 22 8 2 8 8 8 4 ND ND 8 RPMI B Conjugate 23 8 ND 8 8 16 8 8 16 8 RPMI B Conjugate 24 ND ND ND ND 32 64 16 64 16 RPMI B Conjugate 25 ND ND ND ND 16 16 16 16 168 RPMI A Conjugate 26 16 32 >128 >128 >128 128 ND ND 32 RPMI B Conjugate 27 ND ND ND ND 256 >256 256 256 256 RPMI B Conjugate 28 ND ND ND ND 32 64 32 32 32 RPMI B Conjugate 29 ND ND ND ND 64 256 64 256 64 RPMI B Conjugate 30 ND ND ND ND 16 32 16 16 16 RPMI B Conjugate 31 ND ND ND ND 16 32 16 32 16 RPMI B Conjugate 32 ND ND ND ND 256 256 128 256 256 RPMI B Conjugate 33 ND ND ND ND 32 64 32 32 32 RPMI B Conjugate 34 ND ND ND ND 32 64 32 32 32 RPMI B Conjugate 35 ND ND ND ND 16 32 16 16 16 RPMI B Conjugate 36 ND ND ND ND 64 128 32 64 64 RPMI B Conjugate 37 ND ND ND ND 64 128 64 128 64 RPMI B Conjugate 38 ND ND ND ND 16 16 8 16 8 RPMI B Conjugate 39 ND ND ND ND 32 256 128 256 32 RPMI B Conjugate 40 ND ND ND ND 32 64 16 32 32 RPMI B Conjugate 41 ND ND ND ND 16 16 16 16 16 RPMI B Conjugate 42 ND ND ND ND 64 256 64 128 128 ND-not determined; RPMI A-RPMI 1640 (+) L-glutamine, (−) sodium bicarbonate, containing 5% LB, adjusted to pH 7.0 with NaOH, buffered with MOPS: RPMI B-RPMI 1640 (+) L-glutamine, (+) sodium bicarbonate, pH 7.4, containing 5% LB.
TABLE-US-00024 TABLE 3C MIC values for conjugates MIC (μg/ml) Ec Ec Ec BW Ec 25922 Ec Assay Ec BW + Ec BW 4X-12 Ec 25922 + COL-R AR0349 Ec media Compound BW FBS (mcr-1) (COL-R) 25922 FBS 64X-1 (mcr-1) 2469 RPMI B Conjugate 43 ND ND ND ND 16 256 16 32 32 RPMI B Conjugate 44 ND ND ND ND 16 32 16 16 16 RPMI B Conjugate 45 ND ND ND ND 64 >128 64 128 64 RPMI B Conjugate 46 ND ND ND ND 64 >128 64 128 128 RPMI B Conjugate 47 ND ND ND ND 32 64 16 32 32 RPMI B Conjugate 48 ND ND ND ND 16 32 16 32 16 RPMI B Conjugate 49 ND ND ND ND 16 16 32 32 16 RPMI B Conjugate 50 ND ND ND ND 64 64 64 64 8 RPMI B Conjugate 51 ND ND ND ND 16 32 16 16 16 RPMI B Conjugate 52 ND ND ND ND 16 16 16 16 16 RPMI B Conjugate 53 ND ND ND ND 16 16 16 32 16 RPMI B Conjugate 54 ND ND ND ND 64 64 64 128 64 RPMI B Conjugate 55 ND ND ND ND >128 128 128 >128 >128 RPMI B Conjugate 56 ND ND ND ND 32 32 32 32 32 RPMI B Conjugate 57 ND ND ND ND 16 32 16 16 16 RPMI B Conjugate 58 ND ND ND ND 16 64 16 32 32 RPMI B Conjugate 59 ND ND ND ND 16 32 16 16 16 RPMI B Conjugate 60 ND ND ND ND 16 32 16 16 16 RPMI B Conjugate 61 ND ND ND ND 64 256 64 64 64 RPMI B Conjugate 62 ND ND ND ND 32 64 16 32 32 RPMI B Conjugate 63 ND ND ND ND 256 >256 128 256 256 RPMI B Conjugate 64 ND ND ND ND 32 128 32 32 32 ND-not determined; RPMI A-RPMI 1640 (+) L-glutamine, (−) sodium bicarbonate, containing 5% LB, adjusted to pH 7.0 with NaOH, buffered with MOPS: RPMI B-RPMI 1640 (+) L-glutamine, (+) sodium bicarbonate, pH 7.4, containing 5% LB.
TABLE-US-00025 TABLE 3D MIC values for conjugates MIC (μg/ml) Ec Ec Ec BW Ec 25922 Ec Assay Ec BW + Ec BW 4X-12 Ec 25922 + COL-R AR0349 Ec media Compound BW FBS (mcr-1) (COL-R) 25922 FBS 64X-1 (mcr-1) 2469 RPMI B Conjugate 65 ND ND ND ND 32 64 16 32 32 RPMI B Conjugate 66 ND ND ND ND 64 >128 64 64 64 RPMI B Conjugate 67 ND ND ND ND 64 128 64 64 64 RPMI B Conjugate 68 ND ND ND ND 32 128 32 32 32 RPMI B Conjugate 69 ND ND ND ND 32 128 32 64 32 RPMI B Conjugate 70 ND ND ND ND 64 256 64 128 64 RPMI B Conjugate 71 ND ND ND ND 128 >128 64 128 128 RPMI B Conjugate 72 ND ND ND ND 32 128 32 32 32 RPMI B Conjugate 73 ND ND ND ND 32 128 32 32 32 RPMI B Conjugate 74 ND ND ND ND 32 64 16 32 32 RPMI B Conjugate 75 ND ND ND ND 32 64 16 32 32 RPMI B Conjugate 76 ND ND ND ND 64 128 64 128 64 RPMI B Conjugate 77 ND ND ND ND 32 32 32 32 32 RPMI B Conjugate 78 ND ND ND ND 64 128 32 64 64 RPMI B Conjugate 79 ND ND ND ND 32 64 32 64 64 ND-not determined; RPMI A-RPMI 1640 (+) L-glutamine, (−) sodium bicarbonate, containing 5% LB, adjusted to pH 7.0 with NaOH, buffered with MOPS: RPMI B-RPMI 1640 (+) L-glutamine, (+) sodium bicarbonate, pH 7.4, containing 5% LB.
TABLE-US-00026 TABLE 3E MIC values for conjugates MIC (μg/ml) Assay Ab Ab Ab Ab 8990 Pa Pa Pa Kp Kp Kp 6951 media Compound 19606 AB5075 17978 (COL-R) 27853 PAO1 LES431 10031 43816 (COL-R) RPMI A COL (colistin) 2 4 ND ND 1 4 ND 1 2 ND RPMI B COL (colistin) 1 1 ND 64 2 2 1 0.5 0.5 8 RPMI A Conjugate 1 ND ND ND ND ND 6.4 ND 102.4 ND ND RPMI A Conjugate 2 ND ND ND ND ND 32 ND >128 ND ND RPMI A Conjugate 3 ND ND ND ND ND 32 ND 64 ND ND RPMI A Conjugate 4 32 32 ND ND ND 16 ND 64 >128 ND RPMI A Conjugate 5 16 32 ND ND ND 16 ND 32 256 ND RPMI A Conjugate 6 8 8 ND ND ND 8 ND 16 128 ND RPMI A Conjugate 7 64 64 ND ND 32 32 ND 128 64 ND RPMI A Conjugate 8 16 16 ND ND 16 16 ND 32 32 ND RPMI A Conjugate 9 16 16 ND ND 16 16 ND 32 32 ND RPMI A Conjugate 10 32 16 ND ND 16 16 ND 32 32 ND RPMI A Conjugate 11 32 32 ND ND 16 16 ND 64 64 ND RPMI A Conjugate 12 ND ND ND ND ND 16 ND 64 ND ND RPMI A Conjugate 13 >128 128 ND ND ND 16 ND 64 >128 ND RPMI A Conjugate 14 >128 128 ND ND ND 128 ND 32 >128 ND RPMI A Conjugate 15 >128 >128 ND ND ND 16 ND >128 >128 ND RPMI B Conjugate 16 8 16 16 ND >128 >128 ND 128 >128 ND RPMI B Conjugate 17 8 16 16 ND >128 >128 ND 128 >128 ND RPMI A Conjugate 18 32 16 16 ND 16 16 ND 32 64 ND RPMI A Conjugate 19 16 16 16 ND ND 32 16 32 32 ND RPMI B Conjugate 20 8 16 8 ND 128 >128 ND 32 32 ND ND-not determined; RPMI A-RPMI 1640 (+) L-glutamine, (−) sodium bicarbonate, containing 5% LB, adjusted to pH 7.0 with NaOH, buffered with MOPS: RPMI B-RPMI 1640 (+) L-glutamine, (+) sodium bicarbonate, pH 7.4, containing 5% LB.
TABLE-US-00027 TABLE 3F MIC values for conjugates MIC (μg/ml) Assay Ab Ab Ab Ab 8990 Pa Pa Pa Kp Kp Kp 6951 media Compound 19606 AB5075 17978 (COL-R) 27853 PAO1 LES431 10031 43816 (COL-R) RPMI B Conjugate 21 16 16 16 ND >128 >128 ND 128 128 ND RPMI B Conjugate 22 8 8 8 ND >128 >128 ND 8 8 ND RPMI B Conjugate 23 16 16 ND 64 64 >64 32 16 16 16 RPMI B Conjugate 24 32 16 ND >256 >256 >256 64 128 >256 128 RPMI B Conjugate 25 16 16 ND 128 128 256 32 32 32 32 RPMI A Conjugate 26 128 32 64 ND 16 64 ND 32 >128 ND RPMI B Conjugate 27 256 256 ND >256 >256 >256 256 256 256 >256 RPMI B Conjugate 28 32 32 ND 64 128 >128 64 64 64 64 RPMI B Conjugate 29 >256 >256 ND >256 256 256 128 128 64 256 RPMI B Conjugate 30 16 16 ND 128 128 256 32 32 32 32 RPMI B Conjugate 31 16 16 ND 128 128 >128 32 64 64 32 RPMI B Conjugate 32 256 128 ND >256 >256 >256 128 256 >256 >256 RPMI B Conjugate 33 32 32 ND 64 128 128 64 64 64 64 RPMI B Conjugate 34 32 32 ND 256 256 256 64 128 64 32 RPMI B Conjugate 35 16 16 ND 64 64 128 32 32 16 32 RPMI B Conjugate 36 32 32 ND >256 >256 >256 256 256 256 128 RPMI B Conjugate 37 32 32 ND >256 >256 >256 256 256 256 256 RPMI B Conjugate 38 8 16 ND 64 64 128 16 32 32 16 RPMI B Conjugate 39 >256 64 ND >256 256 >256 32 64 64 >256 RPMI B Conjugate 40 16 16 ND >128 >128 >128 32 64 128 32 RPMI B Conjugate 41 16 16 ND 64 128 >128 32 32 16 16 RPMI B Conjugate 42 32 32 ND >256 >256 >256 64 >256 >256 256 ND-not determined; RPMI A-RPMI 1640 (+) L-glutamine, (−) sodium bicarbonate, containing 5% LB, adjusted to pH 7.0 with NaOH, buffered with MOPS: RPMI B-RPMI 1640 (+) L-glutamine, (+) sodium bicarbonate, pH 7.4, containing 5% LB.
TABLE-US-00028 TABLE 3G MIC values for conjugates MIC (μg/ml) Assay Ab Ab Ab Ab 8990 Pa Pa Pa Kp Kp Kp 6951 media Compound 19606 AB5075 17978 (COL-R) 27853 PAO1 LES431 10031 43816 (COL-R) RPMI B Conjugate 43 16 32 ND 32 64 64 16 64 64 32 RPMI B Conjugate 44 16 16 ND 128 256 256 16 32 64 32 RPMI B Conjugate 45 32 32 ND >128 >128 >128 32 >128 >128 >128 RPMI B Conjugate 46 128 64 ND >128 >128 >128 128 >128 >128 >128 RPMI B Conjugate 47 16 16 ND 256 >256 >256 64 256 256 128 RPMI B Conjugate 48 16 16 ND 128 128 >128 32 32 64 32 RPMI B Conjugate 49 16 32 ND >128 >128 >128 64 64 64 32 RPMI B Conjugate 50 32 32 ND >128 >128 >128 >128 >128 >128 128 RPMI B Conjugate 51 16 16 ND 128 128 >128 64 32 32 32 RPMI B Conjugate 52 32 16 ND 128 256 >256 32 32 32 32 RPMI B Conjugate 53 16 16 ND 64 128 >128 64 32 32 32 RPMI B Conjugate 54 32 32 ND >128 >128 >128 >128 >128 >128 >128 RPMI B Conjugate 55 128 >128 ND >128 >128 >128 >128 >128 >128 >128 RPMI B Conjugate 56 64 32 ND 128 >128 >128 64 128 128 64 RPMI B Conjugate 57 16 16 ND 128 128 >128 32 32 32 32 RPMI B Conjugate 58 64 16 ND 256 256 >256 64 64 64 64 RPMI B Conjugate 59 16 16 ND 256 256 >256 64 32 32 32 RPMI B Conjugate 60 16 16 ND >256 256 >256 64 64 128 32 RPMI B Conjugate 61 64 64 ND >512 >512 >512 64 256 256 128 RPMI B Conjugate 62 32 16 ND >512 512 >512 64 64 64 64 RPMI B Conjugate 63 32 64 ND >256 >256 >256 >256 >256 >256 >256 RPMI B Conjugate 64 64 32 ND >512 >512 >512 64 128 64 128 ND-not determined; RPMI A-RPMI 1640 (+) L-glutamine, (−) sodium bicarbonate, containing 5% LB, adjusted to pH 7.0 with NaOH, buffered with MOPS: RPMI B-RPMI 1640 (+) L-glutamine, (+) sodium bicarbonate, pH 7.4, containing 5% LB.
TABLE-US-00029 TABLE 3H MIC values for conjugates MIC (μg/ml) Assay Ab Ab Ab Ab 8990 Pa Pa Pa Kp Kp Kp 6951 media Compound 19606 AB5075 17978 (COL-R) 27853 PAO1 LES431 10031 43816 (COL-R) RPMI B Conjugate 65 16 16 ND >128 >128 >128 32 128 128 64 RPMI B Conjugate 66 32 32 ND >128 >128 >128 64 >128 >128 128 RPMI B Conjugate 67 >128 128 ND >128 >128 >128 64 >128 128 >128 RPMI B Conjugate 68 32 32 ND >128 >128 >128 64 64 64 64 RPMI B Conjugate 69 32 32 ND >128 >128 >128 64 128 128 128 RPMI B Conjugate 70 256 256 ND >256 >256 >256 >256 256 >256 256 RPMI B Conjugate 71 >128 128 ND >128 >128 >128 >128 >128 >128 >128 RPMI B Conjugate 72 32 32 ND 128 >128 >128 32 128 128 64 RPMI B Conjugate 73 32 32 ND 64 128 >128 32 128 128 64 RPMI B Conjugate 74 32 32 ND >256 >256 >256 256 128 64 64 RPMI B Conjugate 75 32 32 ND >128 >128 >128 128 64 64 64 RPMI B Conjugate 76 64 64 ND >128 256 256 64 128 128 128 RPMI B Conjugate 77 32 16 ND 256 256 256 32 64 64 32 RPMI B Conjugate 78 32 32 ND 64 >128 >128 32 128 128 64 RPMI B Conjugate 79 32 32 ND >256 256 256 64 128 128 64 ND-not determined; RPMI A-RPMI 1640 (+) L-glutamine, (−) sodium bicarbonate, containing 5% LB, adjusted to pH 7.0 with NaOH, buffered with MOPS: RPMI B-RPMI 1640 (+) L-glutamine, (+) sodium bicarbonate, pH 7.4, containing 5% LB.
Example 49—Antibacterial Activity of Conjugates in Human Blood
[0821] Overnight stationary-phase cultures were centrifuged at 3,500×g for 10 min at room temperature and resuspended in 1×PBS to an optical density at 600 nm (OD.sub.600) of 0.4 (1×10.sup.8 colony forming units [CFU]/mL) in RPMI with L-glutamine and phenol red (Life Technologies). Bacteria were diluted 1:25 in RPMI (4×10.sup.6 CFU/mL) and 10 μl added to wells of a non-binding 96-well plate (Corning 3641) containing 80 ul of 50% hirudin anti-coagulated blood (complement active) or 50% heparin anti-coagulated blood (complement inactive) in RPMI. After adding 5 ul of 20× solutions of each conjugate diluted in RPMI (128-2 ug/mL final concentration), and 5 ul RPMI for a final blood concentration 40%, the plate was incubated under static conditions at 37° C. for 90 min. Minimum bactericidal concentration (MBC) was determined by 10-fold serial dilution in 1×PBS, then plating 4 ul spots onto rectangular LB agar plates and overnight incubation at 37° C. MBC was defined as the minimum conjugate concentration required for complete bacterial clearance. An untreated growth control (40% blood, 0 μg/mL conjugate) was included in all assays. Conjugate 23 MBCs are lower in complement active hirudin-treated blood compared to complement inactive heparin-treated blood, indicating that conjugate 23-mediated killing is complement dependent (Table 4).
TABLE-US-00030 TABLE 4 Conjugate MBCs (μg/mL) in complement active hirudin-treated human blood vs. (complement inactive heparin-treated human blood). Bacterial strain KP AB PA EC EC AB 6269 6338 7874 AR0349 25922 5075 PA 01 Conjugate COL.sup.R COL.sup.R COL.sup.R COL.sup.R COL.sup.S COL.sup.S COL.sup.S 22 8 (>32) 4 (32) >32 (>32) 4 (32) 2 (16) 4 (32) >32 (>32) 23 4 (32) 1 (16) 32 (>32) 1 (8) <0.5 (4) 1 (8) 32 (>32) Abbreviations: KP, Klebsiella pneumoniae; AB, Acinetobacter baumannii; PA, Pseudomonas aeruginosa; EC, Escherichia coli; COL, colistin; .sup.R, resistant; .sup.S, sensitive.
Example 50—Binding of Conjugates to Bacteria Measured by Flow Cytometry
[0822] Binding of conjugates to gram negative bacteria was measured via flow cytometry. Gram negative bacteria (E. coli ATCC 25922) were grown overnight in Luria-Bertani (LB) (BD Difco™ Cat. 244620) at 37° C. while shaking. The following day, bacteria were grown to log-phase in LB until reaching an OD.sub.600 of 0.4 (˜10.sup.8 CFU/mL). 1 mL of bacteria per condition were washed in DPBS pH 7.4 (Gibco 10010-023) and treated with 5 μM of Conjugate 3 (
[0823] Furthermore,
Example 51—Binding of Conjugates to FcRn
[0824] Binding of Conjugates Conjugate 15 or Conjugate 4 compared to unconjugated control Fc molecule (m-IgG2a type; SEQ ID NO: 2), to neonatal Fc receptor (FcRn) was analyzed by ELISA to determine the impact of drug conjugation on interaction with FcRn.
[0825] 96-well ELISA plates (Fisher, Cat. No. 12-565-136) were coated with 0.25 μg/well of recombinant mouse FcRn (R&D, Cat. No. 6775-FC-050) or recombinant human FcRn (R&D, Cat. No. 8639-FC-050) in carbonate-bicarbonate buffer (Sigma, Cat. No. C34041) at pH 6.25 overnight at 37° C. A pH of 6.25 was maintained during the complete experiment. Blocking was carried out with 5% skim milk in PBS with 0.05% Tween 20. Purified conjugates were added at 20 μg/mL starting concentration and serially 2-fold diluted in blocking buffer. For detection, HRP-labeled secondary anti-mouse IgG-HRP (GE Healthcare, Cat. No. NA931V) or anti-human IgG-HRP (GE Healthcare, Cat. No. NA933V) was used to detect the bound conjugates. Absorbance was measured at 450 nm with a 96-well microplate reader and data were analyzed using GraphPad Prism software,
Example 52—Pharmacokinetics of Conjugate 23
[0826] The pharmacokinetics of conjugate 23 was evaluated in male CD-1 mice (N=3 animals/group) after 10 mg/kg intravenous (IV) or intraperitoneal (IP) administration. Whole blood samples (by tail snip) were collected and the plasma harvested at 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 48 and 72 hours post injection. Plasma concentrations of conjugate 23 were measured by sandwich ELISA using an Anti-Myc tag capture antibody (Abcam ab9132) at and an HRP-conjugated anti-hlgG-Fcg F(ab′).sub.2 (Jackson ImmunoResearch 709-036-098) for detection. The amount of conjugate 23 in plasma samples was quantified using a conjugate 23 standard curve. Data were analyzed using GraphPad Prism 6 and non-linear regression, Sigmoidal 4PL. Plasma pharmacokinetic parameters were then calculated by non-compartmental analysis using Phoenix WinNonlin software (
Example 53—Complement-Dependent Cytotoxicity (CDC)
[0827] To demonstrate that the conjugates can facilitate immune-mediated killing of compound bound bacteria CDC assays were performed. Gram-negative bacteria were cultured overnight in Luria-Bertani (LB) (BD Difco™ Cat. 244620) at 37° C. under constant agitation. The next day, bacteria were grown to log phase until the OD.sub.600 reached 0.4 (˜10.sup.8 CFU/mL). Bacteria were washed in DPBS pH 7.4 (Gibco 10010-023) resuspended in RPMI 1640 (Gibco 11-835-055) at 10.sup.6 CFU/mL and 100 mL/well was added to the wells of a 96-well, TC-treated, polystyrene plate (Fisher 08-772-2C). Then 10% normal human serum ((NHS) Innovative research, IPLA-CSER), 10% heat-inactivated human serum (HIS), which is complement inactive (Innovative research, IPLA-CSER, 56 C for 30 minutes) or RPMI was added to wells. Finally, Conjugate 7 (cyclic heptapeptide hlgG1-Fc) or control (cyclic heptapeptide negative hlgG1-Fc aglycosylated hlgG1-Fc negative control molecule) were diluted to 0.2 uM final in RPMI and added to wells.
[0828] The plates were incubated at 37° C. for 90 minutes. At the end of the assay, wells were serially diluted in sterile water and remaining bacteria were cultured overnight at 37° C. on agar plates. Colonies were counted the next day and CFU/mL and % survival normalized to RPMI control was determined using the following equations: CFU/mL=(colonies*dilution factor)/volume and % inhibition normalized to RPMI=([CFU/mL of test condition]/[CFU/mL of molecule in RPMI])*100.
[0829]
Example 54—In Vivo Efficacy Testing of Conjugates in a Mouse Model of Septicemia
[0830] In order to better determine the potency of the conjugates, several were tested in a mouse model of peritonitis/septicemia. These studies were conducted using immunocompetent C57BL/6 mice (an inbred strain). Table 5 outlines the experimental design for a study with Conjugate 12. In this experiment the challenge pathogen was a mouse virulent isolate of E. coli (ATCC 25922) which was administered IP with 5% mucin to inhibit local macrophages (0.5 mL total volume with 0.9% saline as the vehicle; 1.4E3 CFUs of bacteria delivered). Test articles were dissolved in 0.9% saline which also served as the vehicle only control and dosed IP once as indicated in the table. The end point for the study was survival over 4-days.
TABLE-US-00031 TABLE 5 In Vivo Efficacy Testing of Conjugate 12 in a Mouse Model of Septicemia E. coli Compound Compound C57BL/ (ATCC 25922) (mg/kg) (IP) (mg/kg) (IP) End Point Group 6 (n) (IP) Test Article (T − 1 hr.) (T + H + hr.) (Survival) 1 10 1E3 Vehicle — — 4 day 2 10 1E3 Colistin — 1 mortality 3 10 1E3 Conjugate 12 5 — 4 10 1E3 Conjugate 12 10 — 5 10 1E3 Conjugate 12 20 —
[0831] The study results were plotted as a Kaplan-Meier graph (
Expanded Dose Titration of In Vivo Conjugate Activity in a Mouse Model of Septicemia.
[0832] In a follow up to the first efficacy study a dose titration study was run using Conjugate 3. This study was essentially performed as the previous study (Table 4) except the final challenge inoculum was 3.4E3 and all test articles/controls were dosed IV, 1 hour prior to bacterial challenge. Study results are illustrated in
Screening and Dose Titration of Conjugate 23 in a Mouse Model of Septicemia.
[0833] The activity of conjugate 23 was evaluated in vivo using an established mouse septicemia model. In this model CD-1 mice (Charles River Labs; females, 20 g) were challenged intraperitoneally (0.2 mL) with approximately 1×10.sup.7 CFU of E. coli 25922 grown in LB media. Prior to challenge bacteria were diluted (1:1) in fresh LB+PBS with 5% gastric hog mucin to locally inhibit macrophages.
[0834] In the first study (Table 6, study 1) conjugate 23 was administered as a single dose therapeutically 1 hr. following bacterial challenge. All animals treated with the ADC survived over the course of the study (5 days) while mice treated with vehicle only (1×PBS) reached 100% mortality. Based on the strength of these results conjugate 23 was further evaluated in this model (Table 6, study 2). In this shorter experiment an IgG1 Fc only negative control was included to exclude the possibility that the efficacy achieved in study 1 was the result of non-specific priming of the immune system by the Fc component of the ADC. As expected 4 of 5 mice treated with the negative control reached mortality by day 1 of the study. In comparison, a dose titration of conjugate 23 ranging from 6.25 to 50 mg/kg demonstrated a Stepwise (dose dependent) increase in survival with increasing conjugate 23 concentration.
[0835] Collectively, Conjugates 3, 12 and 23 showed significant efficacy in lethal models of E. coli sepsis.
TABLE-US-00032 TABLE 6 Screening of Conjugate 23 in a lethal mouse peritonitis/septicemia model Route Group Test Article Schedule (n) mg/kg % Survival Study 1 IP @ T + 1 1 Vehicle (PBS) 3 — 0 2 Colistin 3 0.3 100 3 Conjugate 23 5 50 100 Study 2 4 Vehucle (PBS) 3 — 0 5 Colistin 3 0.3 100 6 Human IgG1 5 50 20 7 Conjugate 23 5 50 80 8 Conjugate 23 5 25 60 9 Conjugate 23 5 12.5 40 10 Conjugate 23 5 6.25 0
Example 55—Synergy of ADC Conjugates with Azithromycin (AZM) in an A. baumannii Lung Infection Model
[0836] The murine pneumonia model was adapted from Lin et al., 2015 (PMID: 26288841). A. baumannii AB5075 was grown to log-phase in LB at 37° C., 200 rpm shaking. Bacteria were washed once with PBS via centrifugation at 4,000 rpm. Bacteria were adjusted to desired concentration in PBS.
[0837] Prior to infection, 8-week-old female C57BI/6J mice (Jackson Labs) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Using an otoscope, mice were infected directly into the trachea. For CFU burden studies mice were infected with 2×10.sup.7 CFU in 30 μL PBS. At 24 h post-infection mice were sacrificed by CO.sub.2 and both lung lobes were harvested. Lungs were homogenized with 1 mm silica beads in 1 mL PBS using a MagNA Lyser (Roche). Homogenization was carried out at 6,000 rpm for 60 s and chilled on ice for 60 s in-between runs. Lung homogenate was diluted in PBS ranging from 10.sup.−1 to 10.sup.−8, plated on LB plates and incubated at 37° C. overnight. The next day, CFU were enumerated and surviving CFU calculated relative to weight of the lung. Mice were treated intraperitoneally with colistin (10 mpk) or PBS at t=+1 h and 5 h post-infection. Conjugates or hlgG1 (50 mpk) were administered intraperitoneally at t=−12 h pre-infection and t=+1 h post-infection. Azithromycin (Fresenius-Kabi) was administered subcutaneously once at t=+1 h post-infection. Efficacy of ADC conjugates (50 mpk) was determined upon intratracheal infection of A. baumannii AB5075. For CFU burden studies, mice (n=5 per group) were infected with 3×10.sup.7 bacteria, sacrificed and lung CFU burden determined at 24 h time point. All ADC conjugates tested reduced CFU burden of A. baumannii AB5075—conjugate 6 by 1.12 logs (
Example 56—Efficacy of ADC Conjugates in a Lethal A. baumannii Lung Infection Model
[0838] The murine pneumonia model was adapted from Lin et al., 2015 (PMID: 26288841). A. baumannii AB5075 was grown to log-phase in LB at 37° C., 200 rpm shaking. Bacteria were washed once with PBS via centrifugation at 4,000 rpm. Bacteria were adjusted to desired concentration in PBS. Prior to infection, 8-week-old female C57BI/6J mice (Jackson Labs) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Using an otoscope, mice were infected directly into the trachea. Mice were treated intraperitoneally with colistin (10 mpk) or PBS at t=+1 h and 5 h post-infection. Conjugates or hlgG1 (50 mpk) were administered intraperitoneally at t=−12 h pre-infection and t=+1 h post-infection. For survival studies mice were infected with 5×10.sup.7 CFU in 30 μL of PBS. Mice were treated intraperitoneally with colistin (10 mpk) or PBS at t=+1 h, 5 h, 24 h, 36 h, 48 h and 60 h post-infection. Conjugates or hlgG1 (50 mpk) were administered intraperitoneally at t=−12 h pre-infection and t=+1 h post-infection. For the survival model, survival of mice (n=5 per group) was monitored for 5 days. ADC conjugate 22 showed 100% protection which is en par with colistin as positive control that was dosed at t=+1 h, t=+7 h, t=+24 h and t=+48 h. In comparison, control IgG1 resulted in only 20% protection (
Example 57—Measurement of Binding of ADC Conjugates to LPS Using an LPS Displacement Assay
[0839] To assess the ability of ADC conjugates to bind to LPS, we incubated purified LPS with dansyl-PMB, which becomes fluorescent upon binding to LPS. Next, we measured the change in fluorescence intensity upon addition of ADC conjugates. A decrease in fluorescence intensity by ADC conjugates reflects displaced dansyl-PMB from LPS and correlates with binding of ADC conjugates to LPS. LPS displacement was determined with fluorescent dansyl-polymyxin B (PMB). For that, 2 μL purified LPS from E. coli O111::B4 (Sigma) at 1 mg/mL was added to 45 μL of PBS and incubated with 3 μL of 200 μM dansyl-PMB (in-house) in a black 96 well plate (Costar) for 5 min at room temperature (RT) in the dark. Test articles were added to corresponding wells in 25 μL at concentrations ranging from 20 μM to 0.2 μM. After 5 min incubation at RT in the dark, the relative fluorescence unit (RFU) is determined at excitation at 340 nm and emission at 485 nm on EnSpire plate reader (Perkin Elmer).
[0840] The % LPS displacement is calculated relative to vehicle control. % LPS displacement=(1−(RFU test article−dansyl-PMB in PBS)/(RFU no test article−dansyl-PMB in PBS))×100.
ADC conjugate 23 binds to purified LPS from E. coli O111::B4 in dose-dependency tested from 0.05-5 μM (
Example 58—Measurement of Binding of ADC Conjugates to LPS Using a Macrophage Activation Assay
[0841] To address the question if binding to LPS by ADC as described in
[0842] The % inhibition is calculated relative to PBS as vehicle control. % Inhibition=(1−(NO produced in μM test article)/(NO produced in μM vehicle control))×100. We tested the impact of ADC conjugate 23 to macrophages that were stimulated with 10 μg/mL LPS from E. coli O111::B4 to maximally induce NO production. We found that ADC conjugate 23 prevented LPS-induced macrophage activation in dose-dependency from 0.05-5 μM (
Example 59—Synthesis of Int-24
[0843] ##STR00884## ##STR00885## ##STR00886## ##STR00887## ##STR00888##
[0844] Step a.
##STR00889##
[0845] A solution of Boc-piperazine (0.316 g, 1.70 mmol), propargyl-PEG-4-bromide (0.500 g, 1.70 mmol), and potassium carbonate (0.352 g, 2.55 mmol) dissolved in acetonitrile (8 mL) was heated in a 65° C. oil bath for 15 h. The reaction was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of mono-TFA salt 0.837 g, 96% yield. Ion(s) found by LCMS: (M+H).sup.+=401.4
[0846] Step b.
##STR00890##
[0847] A solution of Boc-piperazine-PEG-4-alkyne (0.837 g, 1.63 mmol), and TFA (5 mL) was stirred for 5 minutes, concentrated under vacuum, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of bis-TFA salt 0.783 g, 91% yield. Ion(s) found by LCMS: (M+H).sup.+=301.4
[0848] Step c.
##STR00891##
[0849] A solution of racemic trans-cyclopentanone-3,4-dicarboxylic acid (0.500 g, 2.90 mmol), L-norleucine methylester HCl (1.16 g, 6.39 mmol), DIEA (3.03 mL, 17.4 mmol), and HATU (2.43 g, 6.39 mmol) in DMF (5 mL) was stirred at room temperature for 1 hr. The diastereomeric products were separated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using 0.1% TFA as the modifier. The diastereomer with the shorter retention time was collected and taken on in the sequence. Yield of desired diastereomer 0.419 g, 34% yield. Ion(s) found by LCMS: did not ionize.
[0850] Step d.
##STR00892##
[0851] A solution of product from the previous step (0.280 g, 0.657 mmol), piperazine-PEG4-alkyne (0.416 g, 0.788 mmol), acetic acid (75 μL, 1.313 mmol), and dichloromethane (2 mL) was stirred for 30 min at room temperature, then treated with sodium triacetoxyborohydride (0.278 g, 1.31 mmol) in two equal portions 60 minutes apart. The reaction was stirred overnight, then purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of product was 0.275 g, 45% yield. Ion(s) found by LCMS: (M+H).sup.+=711.4
[0852] Step e.
##STR00893##
[0853] A solution of product from the previous step (0.275 g, 0.293 mmol) dissolved in THF (4 mL), was treated with a solution of lithium hydroxide (0.042 g, 1.76 mmol) dissolved in water (2 mL). LCMS after 30 min showed complete hydrolysis. The reaction was made slightly acidic with concentrated HCl, then purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using no modifier. Yield of product was 0.170 g, 85% yield. Ion(s) found by LCMS: (M+H).sup.+=683.0
[0854] Step f.
##STR00894##
[0855] A solution of the di-acid product from the previous step (0.073 g, 0.107 mmol), INT-7a (0.351 g, 0.225 mmol, Example 8, Step a), and DIEA (0.123 mL, 0.708 mmol) dissolved in DMF (1 mL) was treated with a solution of HATU (0.090 g, 0.236 mmol), in DMF (1 mL), dropwise over 60 minutes. Product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using 0.1% TFA as the modifier. Yield of 0.350 g, 82% yield. Ion(s) found by LCMS: [M−2(Boc)/3]+H+=1191.7, [M−3(Boc)/3]+H+=1158.4, [M−3(Boc)/4]+H+=869.0, [M−4(Boc)/4]+H+=843.7.
[0856] Step g.
##STR00895##
[0857] A solution of the deca-boc-protected product from the previous step (0.350 g, 0.093 mmol), was stirred in TFA (3 mL) for five minutes, and then stripped of TFA using a rotary evaporator. Product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using 0.1% TFA as the modifier. Yield of the product (Int-24) as a TFA salt was 0.340 g, 88% yield. Ion(s) found by LCMS: [M/4]+H+=924.6, [M/5]+H+=694.1, [M/6]+H+=555.3, [M/7]+H+=463.0.
Example 60—Synthesis of Int-25
[0858] ##STR00896##
[0859] Step a.
##STR00897##
[0860] A flask charged with L-dimethyltartrate (1.25 g, 7.02 mmol), 5-pentynal (1.23 mL, 10.5 mmol), p-toluenesulfonic acid (0.120 g, 0.702 mmol), and toluene (35 mL), was equipped with a Dean-Stark trap and heated in a 155° C. oil bath. After 30 minutes, approximately 0.4 mL of water was collected. The reaction was cooled down, then stripped of toluene and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% methanol and water, using no modifier. Yield of product was 0.854 g, 47% yield. Ion(s) found by LCMS: (M+H).sup.+=257.2
[0861] Step b.
##STR00898##
[0862] A solution of the bis-ester from the previous step (0.850 g, 3.32 mmol), dissolved in THF (6 mL), was treated with a solution of lithium hydroxide (0.238 g, 9.95 mmol) dissolved in water (3 mL). LCMS after 30 minutes indicated complete reaction. The reaction was made slightly acid with conc. HCl, stripped of THF, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using no modifier. Yield of product was 0.817 g, 108% yield. The product was contaminated with salt, and was used in the next step without further purification. Ion(s) found by LCMS: (M+H).sup.+=229.2
[0863] Step c.
##STR00899##
[0864] A solution of di-acid product from the previous step (0.035 g, 0.153 mmol), Int-7a (0.505 g, 0.323 mmol, Example 8, Step b), and DIEA (0.177 mL, 1.02 mmol) dissolved in DMF (2 mL) was treated with a solution of HATU (0.129 g, 0.339 mmol), in DMF (1 mL), dropwise over 60 minutes. The product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield of product was 0.361 g, 67% yield. Ion(s) found by LCMS: [M−3(Boc)/3]+H+=1007.4, [M−4(Boc)/3]+H+=973.7, [M−5(Boc)/3]+H+=940.3.
[0865] Step d.
##STR00900##
[0866] A solution of the deca-boc-protected product from the previous step (0.361 g, 0.109 mmol), was stirred in TFA (3 ml) for five minutes and then stripped of TFA using a rotary evaporator. Product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using 0.1% TFA as the modifier. Yield of Int-25 as a TFA salt was 0.350 g, 93% yield. Ion(s) found by LCMS: [M/3]+H+=773.3, [M/4]+H+=580.4, [M/5]+H+=464.7.
Example 61—Synthesis of Int-26
[0867] ##STR00901##
[0868] Step a.
##STR00902##
[0869] A solution of diacid (0.200 g, 0.876 mmol, Example 60, step b), L-norleucine methyl ester hydrochloride (0.255 g, 1.753 mmol), and DIEA (0.962 mL, 5.52 mmol), in DMF (4 mL) was treated with HATU (0.700 g, 1.841 mmol) in one portion, at room temperature. After stirring for 30 minutes, the reaction was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using no modifier. The product containing fractions were combined and concentrated to yield 0.210 g, 50%. Ion(s) found by LCMS: (M+H).sup.+=483.3
[0870] Step b.
##STR00903##
[0871] A solution of the diester from Step a (0.210 g, 0.435 mmol), in THF (2 mL) was treated with a solution of lithium hydroxide (0.026 g, 1.088 mmol) dissolved in water (1 mL). LCMS after 30 minutes showed complete hydrolysis. Product was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using no modifier. Yield of the desired diacid was 0.195 g, 98%. Ion(s) found by LCMS: (M+H).sup.+=455.2
[0872] Step c.
##STR00904##
[0873] A solution of diacid product from the previous step (0.050 g, 0.110 mmol), Int-7a (0.326 g, 0.232 mmol, Example 8, Step b), and DIEA (0.121 mL, 0.695 mmol) dissolved in DMF (2 mL) was treated with a solution of HATU (0.093 g, 0.243 mmol), in DMF (1 mL), dropwise over 60 minutes. Product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield of the desired product was 0.303 g, 74% yield. Ion(s) found by LCMS: [M−2(Boc)/3]+H+=1115.5, [M−3(Boc)/3]+H+=1082.7, [M−4(Boc)/3]+H+=1049.2.
[0874] Step d.
##STR00905##
[0875] A solution of the deca-boc-protected product from the previous step (0.303 g, 0.085 mmol), was stirred in TFA (2 mL) for five minutes, and then stripped of TFA using a rotary evaporator. The product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using 0.1% TFA as the modifier. Yield of Int-26 as a TFA salt was 0.274 g, 87% yield. Ion(s) found by LCMS: [M/3]+H+=849.1, [M/4]+H+=637.2, [M/5]+H+=510.0.
Example 62—Synthesis of Int-27
[0876] ##STR00906## ##STR00907## ##STR00908## ##STR00909##
[0877] Step a.
##STR00910##
[0878] Neat 4-chloro butanal dimethyl acetal (1.85 g, 12.1 mmol), L-dibenzyl tartrate (1.0 g, 3.03 mmol), and methanesulfonic acid (0.029 mL, 0.454 mmol) were heated in an 80° C. oil bath for 1 hr. The crude reaction was purified directly by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% acetonitrile and water, using no modifier. Yield 0.122 g, 9.6% yield. Ion(s) found by LCMS: (M+H).sup.+=419.2
[0879] Step b.
##STR00911##
[0880] A solution of the diester from Step a (0.244 g, 0.583 mmol), Boc-piperazine (0.434 g, 2.33 mmol), potassium iodide (0.10 g, 0.583 mmol), and sodium bicarbonate (0.098 g, 1.17 mmol) dissolved in DMF (0.5 mL) was heated in an 80° C. oil bath for 3 hours. The crude reaction was purified directly by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using no modifier. Yield 0.134 g, 40% yield. Ion(s) found by LCMS: (M+H).sup.+=569.4
[0881] Step c.
##STR00912##
[0882] Boc protected diester from Step b (0.215 g, 0.378 mmol) was treated with TFA (2 mL) for 5 minutes, then stripped of TFA under vacuum, and purified by directly by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using no modifier. Yield 0.116 g, 65% yield. Ion(s) found by LCMS: (M+H).sup.+=469.2.
[0883] Step d.
##STR00913##
[0884] A solution of product from the previous step (0.116 g, 0.248 mmol), propargyl-PEG4-bromide (0.110 g, 0.371 mmol), and sodium bicarbonate (0.062 g, 0.743 mmol) were dissolved in DMF (2 mL) and heated in an 80° C. oil bath for 4 hr. Product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using no modifier. Yield 0.072 g, 43% yield. Ion(s) found by LCMS: (M+H).sup.+=683.3
[0885] Step e.
##STR00914##
[0886] A solution of the dibenzyl ester from Step d (0.072 g, 0.105 mmol), in THF (1 mL) was treated with a solution of lithium hydroxide (0.0076 g, 0.316 mmol) dissolved in water (0.5 mL). LCMS after 30 minutes showed complete hydrolysis. The reaction was made slightly acidic with conc. HCl then purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using no modifier. Yield 0.052 g, 98%. Ion(s) found by LCMS: (M+H).sup.+=503.5.
[0887] Step f.
##STR00915##
[0888] A solution of di-acid product from the previous step (0.065 g, 0.089 mmol), Int-75 (0.326 g, 0.232 mmol, Example 104, step e), and DIEA (0.155 mL, 0.890 mmol) dissolved in DMF (2 mL) was treated with a solution of HATU (0.117 g, 0.306 mmol), in DMF (1 mL), dropwise over 60 minutes. Product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30% to 100% methanol and water, using no modifier. Yield of 0.228 g, 57% yield. Ion(s) found by LCMS: [M−3(Boc)/4]+H+=880.6, [M−4(Boc)/4]+H+=855.5, [M−5(Boc)/4]+H+=830.5.
[0889] Step g.
##STR00916##
[0890] A solution of deca-boc-protected product from the previous step (0.228 g, 0.060 mmol), was stirred in TFA (2 mL) for five minutes, and then stripped of TFA using a rotary evaporator. Product was isolated by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% methanol and water, using 0.1% TFA as the modifier. The yield of Int-27 as a TFA salt was 0.157 g, 66% yield. Ion(s) found by LCMS: [M/3]+H+=940.4, [M/4]+H+=705.5, [M/5]+H+=564.8.
Example 63—Synthesis of Int-28
[0891] ##STR00917##
[0892] Step a.
##STR00918##
[0893] To a 60° C. stirring solution of L-dimethyltartrate (1.64 g, 9.22 mmol), 4-ethynylbenzaldehyde (600 mg, 4.61 mmol) and trimethylorthoformate (1.01 mL, 9.22 mmol), was added methane sulfonic acid (0.021 mL, 0.323 mmol) and the temperature was raised to 100° C. After 2 h, the reaction was cooled to room temperature and diluted with 20 mL of ethyl acetate. The organic layer was carefully added to a saturated aqueous solution of sodium bicarbonate (50 mL). Upon gas evolution ceased (bubble monitoring) the organic layer was separated and washed with water (50 mL), then dried with brine (50 mL) and solid magnesium sulfate. The resulting mixture was filtered through celite, concentrated, and purified by normal phase liquid chromatography (silica gel) using an Isco CombiFlash liquid chromatograph eluted with 1% to 25% hexanes and ethyl acetate. Yield 0.416 g of (4R,5R)-dimethyl 2-(4-ethynylphenyl)-1,3-dioxolane-4,5-dicarboxylate, 31% yield. .sup.1H NMR (Methanol-d.sub.4) δ: 7.57 (d, J=8.3 Hz, 2H), 7.50 (d, J=8.3 Hz, 2H), 6.08 (s, 1H), 5.05 (d, J=3.8 Hz, 1H), 4.91 (d, J=3.8 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.56 (s, 1H).
[0894] Step b.
##STR00919##
[0895] To a stirring solution of (4R,5R)-dimethyl 2-(4-ethynylphenyl)-1,3-dioxolane-4,5-dicarboxylate (0.416 g, 1.433 mmol) in tetrahydrofuran (5.0 mL) and water (5.0 mL) it was added lithium hydroxide (0.103 g, 4.299 mmol) and stirring was continued overnight. The reaction was concentrated to about half of its volume per rotatory evaporation. Ethyl acetate (15 mL) was added and subsequently a 1.0 M solution of sulfuric acid (5.0 mL), while the mixture was stirred vigorously for 10 minutes. The organic phase was separated and then washed with water (10 mL) and brine (10 mL). The organic layer was then dried with sodium sulfate, filtered, and concentrated to dryness per vacuum techniques. HPLC analysis for the residues revealed one component in high purity, so it was used in the next step without further purification. Yield 0.306 g of (4R,5R)-2-(4-ethynylphenyl)-1,3-dioxolane-4,5-dicarboxylic acid, 82% yield.
[0896] Step c.
##STR00920##
[0897] To a 0° C. stirring solution of (4R,5R)-2-(4-ethynylphenyl)-1,3-dioxolane-4,5-dicarboxylic acid (0.045 g, 0.172 mmol), Int-7a (0.537 g, 0.343 mmol, described in Example 8 step a), and DIEA (0.126 mL, 0.721 mmol) in DMF (5.0 mL), was treated with a solution of HATU (0.134 g, 0.352 mmol) in DMF (3.0 mL), dropwise. Temperature was slowly allowed to reach ambient temperature after 15 min. Upon completion, all the volatiles were rotary evaporated. The residue was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 50% to 100% water and methanol, using no modifier. Yield 0.400 g, 69% yield. .sup.1H NMR diagnostic peaks (Methanol-d.sub.4) δ: 7.61 (d, J=8.3 Hz, 2H), 7.51 (d, J=8.3 Hz, 2H), 7.27 (m, 10H), 6.14 (s, 1H), 1.44 (br s, 90H), 1.21 (t, J=5.1 Hz, 12H), 0.69 (d, J=18.8 Hz, 12H).
[0898] Step d.
##STR00921##
[0899] Product from step c (0.400 g, 0.119 mmol) was treated with dichloromethane (4.0 mL), 2-methyl-2-butene (0.25 mL), and TFA (2.0 mL) until gas evolution ceased (bubbler monitoring), then concentrated to an oil. The oil was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% water and methanol, using 0.1% TFA as the modifier. Yield 0.085 g, 20% yield desired product Int-28. Ion(s) found by LCMS: (M+3H)/3=784.5, (M+4H)/4=588.8, (M+5H)/5=471.2.
Example 64—Synthesis of Int-29
[0900] ##STR00922##
[0901] Step a.
##STR00923##
[0902] To a 0° C. stirring solution of (4R,5R)-2-(4-ethynylphenyl)-1,3-dioxolane-4,5-dicarboxylic acid (0.254 g, 0.969 mmol), L-norleucine methyl ester hydrochloride (0.370 g, 2.034 mmol), and DIEA (1.05 mL, 6.006 mmol) in DMF (5.0 mL), was treated with a solution of HATU (0.134 g, 0.352 mmol) in DMF (5.0 mL), dropwise. Temperature was slowly allowed to reach ambient after 15 min. Upon completion, all the volatiles were evaporated. The residue was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% water and methanol, using no modifier. Yield 0.217 g, 43% yield of bis-(dimethyl ester). .sup.1H NMR (Methanol-d.sub.4) δ: 7.62 (d, J=8.3 Hz, 2H), 7.53 (d, J=8.3 Hz, 2H), 6.13 (s, 1H), 4.89 (d, J=4.4 Hz, 1H), 4.84 (d, J=4.4 Hz, 1H), 4.49 (td, J=9.1, 8.5, 5.0 Hz, 2H), 3.75 (s, 1H), 3.74 (s, 3H), 3.58 (s, 1H), 1.79 (m, 4H), 1.34 (m, 8H), 0.91 (m, 6H).
[0903] Step b.
##STR00924##
[0904] To a stirring solution of bis-(dimethyl ester) product from step a (0.217 g, 0.420 mmol) in tetrahydrofuran (4.0 mL) and water (1.0 mL) it was added lithium hydroxide (0.030 g, 1.260 mmol) and stirring was continued overnight. The reaction was concentrated to about 1 mL per rotatory evaporation. Dichloromethane (15 mL) was added and subsequently a 1.0 M solution of sulfuric acid (5.0 mL), while the mixture was stirred vigorously for 10 minutes. The organic phase was separated and then washed with water (10 mL) and brine (10 mL). The organic layer was then dried with sodium sulfate, filtered, and concentrated to dryness per vacuum techniques. HPLC analysis for the residues revealed one component in high purity that was used in the next step. Yield 0.207 g of dicarboxylic acid, quantitative yield.
[0905] Step c.
##STR00925##
[0906] To a 0° C. stirring solution of dicarboxylic acid product from step b (0.047 g, 0.096 mmol), Int-7a (0.300 g, 0.192 mmol, described in Example 8 step a), and DIEA (0.104 mL, 0.595 mmol) in DMF (5.0 mL), was treated with a solution of HATU (0.076 g, 0.201 mmol) in DMF (3.0 mL), dropwise. Temperature was slowly allowed to reach ambient temperature after 15 min. Upon completion, all the volatiles were evaporated. The residue was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 50% to 100% water and methanol, using no modifier. Yield 0.287 g, 84% yield. .sup.1H NMR for the diagnostic peaks (Methanol-d.sub.4) δ: 7.63 (d, J=8.2 Hz, 2H), 7.53 (d, J=8.2 Hz, 2H), 7.35-7.15 (m, 10H), 6.15 (s, 1H), 4.85 (br s, 2H), 1.44 (s, 90H), 1.20 (br d, J=6.0 Hz, 12H), 0.99-0.83 (m, 6H), 0.70 (br d, J=17.6 Hz, 12H).
[0907] Step d.
##STR00926##
[0908] Product from step c (0.287 g, 0.080 mmol) was treated with dichloromethane (4.0 mL), 2-methyl-2-butene (0.25 mL), and TFA (2.0 mL) until gas evolution ceased (bubbler monitoring), then concentrated to an oil. The oil was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% water and methanol, using 0.1% TFA as the modifier. Yield 0.185 g, 62% yield of desired product Int-29. Ion(s) found by LCMS: (M+4H)/4=645.4, (M+5H)/5=516.4.
Example 65—Synthesis of Int-30
[0909] ##STR00927##
[0910] Step a.
##STR00928##
[0911] To a 0° C. stirring solution of 4-formylbenzoic acid (0.260 g, 1.729 mmol), propargyl-PEG4-amine (0.400 g, 0.729 mmol), and DIEA (0.632 mL, 3.632 mmol) in DMF (2.0 mL), was treated with HATU (0.671 g, 0.1.764 mmol). Temperature was slowly allowed to reach ambient after 15 min. Upon completion, all the volatiles were evaporated. The residue was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% water and methanol, using no modifier. Yield 0.570 g of N-(2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)ethyl)-4-formylbenzamide, 91% yield. .sup.1H NMR (Methanol-d.sub.4) δ: 10.07 (s, 1H), 8.01 (s, 4H), 4.16 (d, J=2.4 Hz, 2H), 3.76-3.51 (m, 16H), 2.84 (t, J=2.4 Hz, 1H).
[0912] Step b.
##STR00929##
[0913] To a 60° C. stirring solution of L-dimethyltartrate (0.838 g, 4.706 mmol), N-(2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)ethyl)-4-formylbenzamide (0.570 mg, 1.568 mmol) and trimethyl orthoformate (1.03 mL, 9.412 mmol), it was added methane sulfonic acid (0.005 mL, 0.078 mmol) and the temperature was raised to 100° C. After 18 h, the reaction was cooled to room temperature and diluted with 20 mL of ethyl acetate. The organic layer was carefully added to a saturated aqueous solution of sodium bicarbonate (50 mL). Upon gas evolution ceased (bubble monitoring) the organic layer was separated and washed with water (50 mL), then dried with brine (50 mL) and solid magnesium sulfate. The resulting mixture was filtered through celite, concentrated, and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 25% to 100% water and methanol, using no modifier. Yield 0.360 g of (4R,5R)-dimethyl 2-(4-(2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)ethylcarbamoyl)phenyl)-1,3-dioxolane-4,5-dicarboxylate, 44% yield. .sup.1H NMR (Methanol-d.sub.4) δ: 8.56 (br s, 1H), 7.87 (d, J=8.4 Hz, 2H), 7.69 (d, J=8.4 Hz, 2H), 6.14 (s, 1H), 5.07 (d, J=3.7 Hz, 1H), 4.94 (d, J=3.7 Hz, 1H), 4.15 (d, J=2.4 Hz, 2H), 3.86 (s, 3H), 3.81 (s, 3H), 3.72-3.50 (m, 16H), 2.84 (t, J=2.4 Hz, 1H).
[0914] Step c.
##STR00930##
[0915] To a stirring solution of (4R,5R)-dimethyl 2-(4-(2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)ethylcarbamoyl)phenyl)-1,3-dioxolane-4,5-dicarboxylate (0.360 g, 0.688 mmol) in tetrahydrofuran (5.0 mL) and water (2.5 mL) it was added lithium hydroxide (0.066 g, 2.751 mmol) and stirring was continued overnight. The reaction was concentrated to about 2.5 mL per rotatory evaporation. Ethyl acetate (15 mL) was added and subsequently a 1.0 M solution of sulfuric acid (4.0 mL), while the mixture was stirred vigorously for 10 minutes. The organic phase was separated and then washed with water (10 mL) and brine (10 mL). The organic layer was then dried with sodium sulfate, filtered, and concentrated to dryness. HPLC analysis revealed one component in high purity, that was used in next step without further purification. Yield 0.342 g of (4R,5R)-2-(4-(2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)ethylcarbamoyl)phenyl)-1,3-dioxolane-4,5-dicarboxylic acid, quantitative yield. .sup.1H NMR (Methanol-d.sub.4) δ: 7.87 (d, J=8.2 Hz, 2H), 7.71 (d, J=8.2 Hz, 2H), 6.14 (s, 1H), 4.98 (d, J=3.9 Hz, 1H), 4.87 (d, J=3.9 Hz, 1H), 4.16 (d, J=2.4 Hz, 2H), 3.73-3.52 (m, 16H), 2.83 (t, J=2.4 Hz, 1H).
[0916] Step d.
##STR00931##
[0917] To a 0° C. stirring solution of (4R,5R)-2-(4-(2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)ethylcarbamoyl)phenyl)-1,3-dioxolane-4,5-dicarboxylic acid (0.400 g, 0.807 mmol), L-norleucine methyl ester hydrochloride (0.246 g, 1.695 mmol), and DIEA (0.703 mL, 4.036 mmol) in DMF (5.0 mL), was treated with a solution of HATU (0.645 g, 1.695 mmol) in DMF (5.0 mL), dropwise. Temperature was slowly allowed to reach ambient temperature after 15 min. Upon completion, all the volatiles were evaporated. The residue was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% water and methanol, using no modifier. Yield 0.328 g, 54% yield of bis-(dimethyl ester). .sup.1H NMR (Methanol-d.sub.4) δ: 7.91 (d, J=8.3 Hz, 2H), 7.75 (d, J=8.3 Hz, 2H), 6.18 (s, 1H), 4.93 (d, J=4.5 Hz, 1H), 4.86 (d, J=4.5 Hz, 1H), 4.50 (td, J=8.1, 7.6, 5.2 Hz, 2H), 4.16 (d, J=2.4 Hz, 2H), 3.76 (s, 3H), 3.74 (s, 3H), 3.70-3.51 (m, 16H), 2.84 (t, J=2.4 Hz, 1H), 1.97-1.63 (m, 4H), 1.46-1.22 (m, 8H), 0.91 (dt, J=9.5, 7.1 Hz, 6H)
[0918] Step e.
##STR00932##
[0919] To a stirring solution of bis-(dimethyl ester) product from step d (0.328 g, 0.437 mmol) in tetrahydrofuran (5.0 mL) and water (2.5 mL) it was added lithium hydroxide (0.031 g, 1.312 mmol) and stirring was continued overnight. The reaction was concentrated to about 2.5 mL per rotatory evaporation. Ethyl acetate (15 mL) was added and subsequently a 1.0 M solution of sulfuric acid (4.0 mL), while the mixture was stirred vigorously for 10 minutes. The organic phase was separated and then washed with water (10 mL) and brine (10 mL). The organic layer was then dried with sodium sulfate, filtered, and concentrated to dryness. HPLC analysis revealed one component in high purity that was used in the next step without further purification. Yield 0.325 g of dicarboxylic acid, quantitative yield.
[0920] Step f.
##STR00933##
[0921] To a 0° C. stirring solution of dicarboxylic acid product from step e (0.058 g, 0.080 mmol), Int-7a (0.250 g, 0.160 mmol, described in Example 8 step a), and DIEA (0.070 mL, 0.400 mmol) in DMF (3.0 mL), was treated with a solution of HATU (0.063 g, 0.168 mmol) in DMF (1.5 mL), dropwise. Temperature was allowed to reach ambient temperature over a 15 min period. Upon completion, all the volatiles were evaporated. The residue was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 50% to 100% water and methanol, using no modifier. Yield 0.232 g, 76% yield. .sup.1H NMR (Methanol-d.sub.4) δ: 7.90 (d, J=7.9 Hz, 2H), 7.73 (d, J=7.9 Hz, 2H), 7.25 (m, 10H), 6.19 (s, 1H), 3.64 (br d, J=7.8 Hz, 16H), 2.74 (br s, 1H), 1.43 (br d, J=2.7 Hz, 90H), 1.19 (br d, J=6.2 Hz, 12H), 0.89 (br d, J=7.9 Hz, 6H), 0.70 (br d, J=18.6 Hz, 12H).
[0922] Step g. Deprotection to give Int-30
##STR00934##
[0923] Product from the previous step (0.232 g, 0.061 mmol) was treated with dichloromethane (4.0 mL), 2-methyl-2-butene (0.25 mL), and TFA (2.0 mL) until gas evolution ceased (bubbler monitoring), then concentrated to an oil. The oil was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% water and methanol, using 0.1% TFA as the modifier. Yield of Int-30 was 0.208 g, 86%. Ion(s) found by LCMS: (M+4H)/4=703.5, (M+5H)/5=563.2.
Example 66—Synthesis of Int-31
[0924] ##STR00935## ##STR00936## ##STR00937## ##STR00938## ##STR00939##
[0925] Step a.
##STR00940##
[0926] In a sealed tube, a stirring mixture of methyl 3-bromo-5-(bromomethyl)benzoate (3.08 g, 10.00 mmol) and sodium azide (0.715 g, 11.00 mmol) in DMF (10 mL) was heated at 80° C. for 1 h, when HPLC analysis showed full conversion of the bromide. All the volatiles were evaporated. The residue was taken up in DCM (40 mL) and water (40 mL). The water layer was washed with additional DCM (2×30 mL). The combined organic layers were dried with brine and solid sodium sulfate. Upon filtration, all the volatiles were evaporated, affording 2.73 g of the crude azide derivative in high purity, as confirmed per .sup.1H-NMR and LC-MS analysis. .sup.1H NMR (DMSO-d.sub.6) δ: 8.03 (t, J=1.6 Hz, 1H), 7.98-7.93 (m, 1H), 7.90 (t, J=1.5 Hz, 1H), 4.60 (s, 2H), 3.88 (s, 3H). This azide (2.73 g) was dissolved under stirring in THF (30 mL) and water (2 mL), and triphenylphosphine (3.148 g, 12.00 mmol) was added. The reaction was stirred at room temperature overnight, allowing gas evolution through a bubbler. All the volatiles were evaporated per vacuum techniques. The desired product was isolated by normal phase liquid chromatography using an Isco CombiFlash liquid chromatograph eluted with 1% to 20% hexanes and ethylacetate. From the desired fractions, the desired compound was obtained still contaminated with triphenylphenylphosphine oxide. This material was therefore dissolved in DCM (50 mL) and extracted an acidic solution (12 mL of 1.0 M H.sub.2SO.sub.4 and 30 mL of water). The water layer was added dropwise to a vigorously stirring solution of saturated sodium bicarbonate (60 mL) and stirring was continued overnight. The obtained mixture was extracted with DCM (3×30 mL). The combined organic layers were dried with brine and solid sodium sulfate. Upon filtration, all the volatiles were evaporated per vacuum techniques, affording 1.31 g, 54% of 3-(aminomethyl)-5-bromobenzoate. Ions found by LCMS: (M+H)+=244.0; 246.0.
[0927] Step b.
##STR00941##
[0928] A solution of methyl 3-(aminomethyl)-5-bromobenzoate (1.306 g, 5.350 mmol) and methyl 3-bromo-5-formylbenzoate (1.238 g, 5.096 mmol) in DCM (10 mL) was evaporated and left over high vacuum overnight. The residue was taken up in THF (30 mL) and treated under vigorous stirring with sodium triacetoxyborohydride (3.240 g, 15.29 mmol). This mixture was quenched after 18 h by the addition of a saturated aqueous solution of ammonium chloride (50 mL). The obtained mixture was extracted with DCM (3×30 mL). The combined organic layers were dried with brine and solid sodium sulfate. Upon filtration, all the volatiles were evaporated. The desired product was isolated by normal phase liquid chromatography using an Isco CombiFlash liquid chromatograph eluted with 1% to 50% hexanes and ethylacetate. Yield 1.611 g, 67% of 3-((N-(1′-(3′-carboxymethyl-5-bromo)phenylene)methylene)methyl)-5-bromobenzoate. Ions found by LCMS: (M+H)+=470.0, 472.1, 474.0.
[0929] Step c
##STR00942##
[0930] To a 0° C. stirring solution of methyl 3-((N-(1′-(3′-carboxymethyl-5-bromo)phenylene)methylene)methyl)-5-bromobenzoate (1.611 g, 3.419 mmol) and DIPEA (0.953 mL, 5.471 mmol) in THF (20 mL) it was added Cbz-Cl (0.732 mL, 5.129 mmol) dropwise. The temperature was raised to ambient temperature after 10 minutes and stirring was continued until complete disappearance of the starting amine by LC-MS analysis. All the volatiles were removed by rotatory evaporation. The desired product was isolated by normal phase liquid chromatography using an Isco CombiFlash liquid chromatograph eluted with 1% to 20% hexanes and ethylacetate. Yield 2.070 g, quantitative yield of N-Cbz protected methyl 3-((N-(1′-(3′-carboxymethyl-5-bromo)phenylene)methylene)methyl)-5-bromobenzoate. Ions found by LCMS: (M+H)+=603.0, 605.0, 607.0.
[0931] Step d
##STR00943##
[0932] Under nitrogen, in a capped microwave reaction vessel stirring mixture of N-Cbz protected methyl 3-((N-(1′-(3′-carboxymethyl-5-bromo)phenylene)methylene)methyl)-5-bromobenzoate (0.299 g, 0.494 mmol), potassium phenyltrifluoroborate (0.200 g, 1.087 mmol), sodium carbonate (0.209 g, 1.976 mmol) and bis(triphenylphosphine)palladium (II) dichloride (0.035 g, 0.049 mmol) in MeCN (4 mL) and water (4 mL) were irradiated with microwaves for 10 min at 130° C. Upon cooling, the reaction was treated under stirring with SiliaMetS® (0.200 g, 0.240 mmol) for 1 h, and TFA was added (0.380 mL, 5.0 mmol). Upon filtration, all the volatiles were evaporated per vacuum techniques. The desired product was isolated as a dodeca-TFA salt by RPLC using an Isco CombiFlash liquid chromatograph eluted with 0% to 100% methanol and water, using TFA as the modifier. Yield 0.072 g, 25% yield of N-Cbz protected 3-((N-(1′-(3′-carboxy-5-phenyl)phenylene)methylene)methyl)-5-phenylbenzoic acid. .sup.1H NMR (0.500 mL Chloroform-d+0.050 mL TFA) δ: 8.18 (d, J=5.5 Hz, 2H), 7.82 (d, J=16.6 Hz, 2H), 7.60 (d, J=33.5 Hz, 2H), 7.52-7.27 (m, 15H), 5.32 (s, 2H), 4.70 (d, J=19.9 Hz, 4H).
[0933] Step e
##STR00944##
[0934] To a 0° C. stirring solution of N-Cbz protected 3-((N-(1′-(3′-carboxy-5-phenyl)phenylene)methylene)methyl)-5-phenylbenzoic acid (0.075 g, 0.131 mmol), Int-7a (0.466 g, 0.262 mmol, described in Example 8 step a), and DIEA (0.137 mL, 0.787 mmol) in DMF (5.0 mL), was treated with a solution of HATU (0.102 g, 0.269 mmol) in DMF (1.5 mL), dropwise. Temperature was slowly allowed to reach ambient temperature after 15 min. Upon completion, all the volatiles were evaporated. The residue was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 50% to 100% water and methanol, using no modifier. Yield 0.451 g, 94% yield. .sup.1H NMR for the diagnostic peaks .sup.1H NMR (Methanol-d.sub.4) δ: 8.12-7.09 (m, 31H), 5.25 (s, 2H), 1.41 (br d, J=10.2 Hz, 90H), 1.24-1.04 (m, 12H), 0.69 (br d, J=18.7 Hz, 12H).
[0935] Step f
##STR00945##
[0936] Product from step e was dissolved in methanol (25 mL) and suspended with SiliaCatPd(0)® (0.123 g, 0.025 mmol) under hydrogen atmosphere (˜1 atm). When all the starting material was consumed by HPLC analysis, the mixture was filtered over a short Celite pad, and all the volatiles were evaporated per vacuum techniques. HPLC analysis for the residues revealed one component in high purity, so it was used in the next step without further purification. Yield 0.431 g, 99% yield. .sup.1H NMR for the diagnostic peaks .sup.1H NMR (Methanol-d.sub.4) δ: 8.07 (d, J=9.0 Hz, 2H), 7.87 (d, J=8.4 Hz, 4H), 7.74-7.70 (m, 4H), 7.55-7.11 (m, 18H), 1.42 (d, J=11.1 Hz, 90H), 1.20 (br t, J=6.7 Hz, 12H), 0.69 (br d, J=17.9 Hz, 12H)
[0937] Step g
##STR00946##
[0938] To a 0° C. stirring solution of propargyl-PEG4-acid (0.032 g, 0.122 mmol), product from step f (0.431 g, 0.122 mmol), and DIEA (0.064 mL, 0.366 mmol) in DMF (5.0 mL), was treated with a solution of HATU (0.049 g, 0.128 mmol) in DMF (1.5 mL), dropwise. Temperature was slowly allowed to reach ambient after 15 min. Upon completion, all the volatiles were evaporated per vacuum techniques. The residue was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 50% to 100% water and methanol, using no modifier. Yield 0.326 g, 71% yield.
[0939] Step h
##STR00947##
[0940] Product from step g (0.341 g, 0.086 mmol) was treated with dichloromethane (4.0 mL), 2-methyl-2-butene (0.25 mL), and TFA (2.0 mL) until gas evolution ceased (bubbler monitoring), then concentrated to an oil. The oil was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% water and methanol, using 0.1% TFA as the modifier. Yield of Int-31 was 0.251 g, 74% yield.
[0941] Ion(s) found by LCMS: (M+4H)/4=693.3, (M+5H)/5=554.8.
Example 67—Synthesis of Int-32
[0942] The general method for the preparation of the extended Threonines is described in the following reference: David Aiker, Giles Hamblett, Laurence M. Harwood, Sarah M. Robertson, David J. Watkin and Eleri Williams, Tetrahedron 54 (1998) 6089-6098.
##STR00948##
[0943] Step a.
##STR00949##
[0944] Propionaldehyde (7.4 g, 127 mmol, 3 eq.) was added to a solution of the 5-phenyl-morpholin-2-one (7.5 g, 42 mmol, 1 eq.) in 100 mL toluene along with 15 grams of activated 4 Å sieves and a condenser. The reaction mixture was heated to reflux for 24 hours under nitrogen. The reaction was filtered and concentrated to give the crude product as an oil, which was used in the next step without any purification. Ion(s) found by LCMS: (M+H).sup.+=276.2.
[0945] Step b.
##STR00950##
[0946] To the crude material from step a in methanol (50 mL) was added 2 M HCl (10 mL). The reaction mixture was heated to reflux under nitrogen until LCMS indicated the absence of starting material and the presence of a new compound (1-2 h). The solvent was removed in vacuo to yield a yellow oil, which was then purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of the product as yellow oil: 5.6 g, 42% yield in two steps. Ion(s) found by LCMS: (M+H)+=267.2.
[0947] Step c.
##STR00951##
[0948] The compound from step b (1.3 g, 5 mmol) was dissolved into 30 mL methanol, Pearlman's catalyst (1.3 g) and ammonium formate (3.1 g, 10 eq.) were added to the reaction vessel and the solution degassed and heated to reflux for 4 hours. The solution was cooled and filtered to remove the catalyst. Solvent was removed in vacuo and the crude mixture was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of product as yellow oil as TFA salt, 1.30 g, 100% yield. Ion(s) found by LCMS: (M+H).sup.+=147.1.
[0949] Step d.
##STR00952##
[0950] Lithium hydroxide (120 mg, 5 mmol) in 2 mL water was added into a solution of the amino acid ester from the previous step (0.3 g, 2 mmol) in 2 mL water, 2 mL methanol and 2 mL THF. The reaction was monitored by LCMS. After completion, the solution was treated with Amberlite® IRN-77, ion exchange resin to adjust to pH 1, then filtered, concentrated, and used in next step without further purification.
[0951] Step e.
##STR00953##
[0952] The amino acid from the previous step (0.3 g, 2 mmol) was dissolved in 5 mL methanol and 5 mL Sat. NaHCO.sub.3 solution, followed added CbzCl (510 mg, 3 mmol, 1.5 eq.), the solution was stirred for 1 hour then neutralized by 1N HCl. The solution was extracted with ethyl acetate and dried over Na.sub.2SO.sub.4. The solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of the product as a yellow oil, 450 mg, 92% yield. Ion(s) found by LCMS: (M+H)+=268.1.
General Method for the Preparation of a Decapeptide Containing an Extended L-Threonine
[0953] ##STR00954## ##STR00955##
[0954] Step f.
##STR00956##
[0955] To a solution of Int-1 (2.1 g, 2 mmol, Example 2) and (2S)-2-{[(benzyloxy) carbonyl]-amino}-4-[(tert-butoxycarbonyl)amino]butanoic acid (740 mg, 2 mmol) in DMF (30 mL) was added EDC (0.6 g, 3 mmol), HOBT (0.45 g, 3 mmol), and DIEA (0.7 mL, 5 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and re-dissolved into 5 mL methanol, then added drop-wise to 200 mL vigorously stirred water, this heterogeneous solution was stirred for 1 hour, then filtered with Buchner funnel with Filter paper. The collected crude product was re-dissolved into 20 mL methanol, then 1 g of 5% Pd/C was added to the above solution, and the mixture was stirred at room temperature under a hydrogen atmosphere overnight. Palladium was removed by filtration after the reaction was complete as determined by LCMS. The filtrate was concentrated and used next step without further purification. Ion(s) found by LCMS: [M/2]+H+=631.9, [M/3]+H+=421.6.
[0956] Step g.
##STR00957##
[0957] To a solution of product from the previous step f and (3R)—N-[(benzyloxy)carbonyl]-3-hydroxy-L-norvaline (540 mg, 2 mmol) in DMF (20 mL) was added EDC (0.6 g, 3 mmol), HOBT (0.45 g, 3 mmol), DIEA (0.56 mL, 4 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and re-dissolved into 5 mL methanol and added dropwise into 100 mL vigorously stirred water, this heterogeneous solution was stirred for 1 hour, then filtered with Buchner funnel with filter paper. The collected crude product was re-dissolved in 20 mL methanol, then treated with 1 g of 5% Pd/C. The mixture was stirred at room temperature under a hydrogen atmosphere overnight. The palladium charcoal was removed by filtration. The filtrate was concentrated and used next step without further purification. Ion(s) found by LCMS: [M/2]+H+=689.4, [M/3]+H+=459.9.
[0958] Step h.
##STR00958##
[0959] To a solution of PMB nonapeptide-NH2 from the previous step g and (2S)-2-{[(benzyloxy) carbonyl]-amino}-4-[(tert-butoxycarbonyl)amino]butanoic acid (720 mg, 2 mmol) in DMF (20 mL) was added EDC (0.6 g, 3 mmol), HOBT (0.45 g, 3 mmol), DIEA (0.56 mL, 4 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and re-dissolved into 5 mL methanol, then added drop-wise into 100 mL vigorously stirred water. This heterogeneous solution was stirred for 1 hour, then filtered with Buchner funnel with filter paper. The collected crude product was dissolved in 20 mL methanol, then treated with 1 g of 5% Pd/C. The mixture was stirred at room temperature under a hydrogen atmosphere overnight. The palladium charcoal was removed by filtration. The filtrate was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 10% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of product as a TFA salt: 2.3 g, 73% yield. Ion(s) found by LCMS: [M/2]+H+=789.5, [M/3]+H+=526.7, [M/4]+H+=395.3.
General Method for the Preparation of the Corresponding PMB Dimer
[0960] ##STR00959## ##STR00960##
[0961] Step i.
##STR00961##
[0962] To a solution of cbz-diacetic acid (2 g, 7 mmol), and methyl L-norleucine HCl salt (2.1 g, 14.2 mmol) in DMF (40 mL) was added EDC (4.0 g, 20 mmol), HOBT (3.1 g, 20 mmol), and DIEA (4.2 mL, 30 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of the product as an oil 2.7 g, 72% yield. Ion(s) found by LCMS: (M+H)+=536.3.
[0963] Step j.
##STR00962##
[0964] The compound from step i (536 mg, 1 mmol) was dissolved in 10 mL methanol, then 200 mg of 5% Pd/C was added to the solution. The mixture was stirred at room temperature under a hydrogen atmosphere overnight. The palladium on charcoal was filtered, concentrated, and used in next step without further purification.
[0965] Step k.
##STR00963##
[0966] A solution of the compound from step j (450 mg, 1 mmol), propargyl-PEG4-NH.sub.2 (230 mg, 1 mmol) in DMF (5 mL) was treated with EDC (300 mg, 1.5 mmol), HOBT (230 mg, 1.5 mmol), and DIEA (0.3 mL, 1.5 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of the product as an oil, 520 mg, 78% yield. Ion(s) found by LCMS: (M+H)+=659.4.
[0967] Step l.
##STR00964##
[0968] Lithium hydroxide (120 mg, 5 mmol) dissolved in 2 mL water was added to a solution of the amino acid ester from step k (0.65 g, 1 mmol) in 2 mL methanol and 2 mL THF. The progress of the reaction was monitored by LCMS. After completion, the solution was treated with Amberlite® IRN-77 ion exchange resin to adjust the pH to 1. The resulting solution was filtered and concentrated then used in next step without further purification. Ion(s) found by LCMS: (M+H)+=631.4.
[0969] Step m.
##STR00965##
[0970] The compound from the previous step l (56 mg, 0.089 mmol), and penta-Boc-polymyxin B decapeptide (295 mg, 0.18 mmol, Example 8, step a) in DMF (5 mL) were added EDC (60 mg, 0.3 mmol), HOBT (45 mg, 0.3 mmol), and DIEA (0.14 mL, 1.0 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and re-dissolved into 2 mL methanol, The resulting solution was added dropwise into 50 mL vigorously stirred water, this heterogeneous solution was stirred for 1 hour, then filtered with Buchner funnel with filter paper. The crude product was used in next step without further purification.
[0971] Step n. Deprotection to Give Int-32
##STR00966##
[0972] Product from step m was treated in 2 mL DCM and 2 mL TFA at room temperature. The solution was stirred 10 min, then concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 0% to 100% acetonitrile and water, using formic acid as the modifier. Yield of Int-32 was 0.120 g, 67% yield. Ion(s) found by LCMS: [M/4]+H+=691.4, [M/5]+H+=553.3, [M/6]+H+=461.3, [M/7]+H+=395.5.
Example 68—Synthesis of Int-33
[0973] ##STR00967##
[0974] The title compound Int-33 was prepared analogously to Example 67, where propionaldehyde was substituted with butyraldehyde in step a of the sequence. Ion(s) found by LCMS: [M/4]+H+=698.4, [M/5]+H+=558.9, [M/6]+H+=465.9, [M/7]+H+=399.5.
Example 69—Synthesis of Int-34
[0975] ##STR00968##
[0976] The title compound Int-34 was prepared analogously to Example 67, where propionaldehyde was substituted with heptanal in step a of the sequence. Ion(s) found by LCMS: [M/4]+H+=719.4.4, [M/5]+H+=575.7, [M/6]+H+=479.9, [M/7]+H+=411.5.
Example 70—Synthesis of Int-35
[0977] ##STR00969##
[0978] The title compound Int-35 was prepared analogously to Example 67, where propionaldehyde was substituted with 3-phenylpropionaldehyde in step a of the sequence. Ion(s) found by LCMS: [M/4]+H+=729.4, [M/5]+H+=582.7, [M/6]+H+=486.6, [M/7]+H+=417.2.
Example 71—Synthesis of Int-36
[0979] ##STR00970##
[0980] The title compound Int-36 was prepared analogously to Example 68, where the di-acid intermediate was substituted with the di-acid described in Example 41, step m. Ion(s) found by LCMS: [M/4]+H+=693.2, [M/5]+H+=554.5, [M/6]+H+=463.1, [M/7]+H+=397.1.
Example 72—Synthesis of Int-37
[0981] ##STR00971##
[0982] The title compound Int-37 was prepared analogously to Example 69, where the di-acid intermediate was substituted with di-acid described in the example 41, step m. Ion(s) found by LCMS: [M/4]+H+=714.2, [M/5]+H+=572.3, [M/6]+H+=477.1, [M/7]+H+=409.1.
Example 73—Synthesis of Int-38
[0983] ##STR00972##
[0984] The title compound Int-38 was prepared analogously to Example 67, where penta-Boc-polymyxin B decapeptide containing an extended threonine was substituted with the Int-7a (described in Example 8). Ion(s) found by LCMS: [M/4]+H+=684.4, [M/5]+H+=547.7, [M/6]+H+=456.6.
Example 74—Synthesis of Int-39
[0985] ##STR00973##
[0986] The title compound Int-39 was prepared analogously to Example 69, where the di-acid intermediate was replaced with the di-acid intermediate described in step a of Example 90. Ion(s) found by LCMS: [M/4]+H+=662.9, [M/5]+H+=530.5, [M/6]+H+=442.3, [M/7]+H+=379.2.
Example 75—Synthesis of Int-40
[0987] ##STR00974##
[0988] The title compound Int-40 was prepared analogously to Example 68, where the di-acid intermediate was replaced with the di-acid intermediate described in step a of Example 90. Ion(s) found by LCMS: [M/4]+H+=641.9, [M/5]+H+=513.7, [M/6]+H+=428.3, [M/7]+H+=367.2.
Example 76—Synthesis of Int-41
[0989] ##STR00975## ##STR00976##
[0990] Step a.
##STR00977##
[0991] Methyl N-(oxomethylidene)-L-norleucinate (510 mg, 3 mmol) was added into a solution of benzyl tert-butyl 2,2′-azanediyldiacetate (830 mg, 3 mmol) and triethyl amine (0.52 mL, 3 mmol) in 5 mL DMF at room temperature, then stirred overnight. The reaction was concentrated then purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of the products as an oil, 920 mg, 69% yield. Ion(s) found by LCMS: M+H=452.3.
[0992] Step b.
##STR00978##
[0993] Product from the previous step (450 mg, 1 mmol) was dissolved into 10 mL methanol containing 5% water, then treated with 200 mg of 5% Pd/C and stirred at room temperature under a hydrogen atmosphere for 2 hours. The palladium charcoal was removed by filtration, and the filtrate was concentrated and used in next step without further purification.
[0994] Step c.
##STR00979##
[0995] The compound from step b (450 mg, 1 mmol), methyl L-norleucinate (230 mg, 1 mmol) in DMF (5 mL) was added EDC (300 mg, 1.5 mmol), HOBT (230 mg, 1.5 mmol), DIEA (0.3 mL, 1.5 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of the product as an oil, 420 mg, 86% yield. Ion(s) found by LCMS: (M+H)+=488.3
[0996] Step d.
##STR00980##
[0997] The compound from step c (500 mg, 1 mmol) was treated with 5 mL TFA at room temperature, after the completion the reaction, the solution was concentrated and used in next step without further purification.
[0998] Step e.
##STR00981##
[0999] The compound from step d (430 mg, 1 mmol), propargyl-PEG-4-NH2 (230 mg, 1 mmol) in DMF (5 mL) was added EDC (300 mg, 1.5 mmol), HOBT (230 mg, 1.5 mmol), DIEA (0.3 mL, 1.5 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of the product as an oil, 470 mg, 73% yield. Ion(s) found by LCMS: (M+H)+=645.4.
[1000] Step f.
##STR00982##
[1001] Lithium hydroxide (24 mg, 1 mmol) in 1 mL water was added into a solution of the compound from step e (70 mg, 1 mmol) in 1 mL methanol and 1 mL THF. The reaction was monitored by LCMS. After completion, the solution was adjusted to pH 1 with Amberlite® IRN-77, then the resulting solution was filtered and concentrated and used in next step without further purification. Ion(s) found by LCMS: (M+H)+=616.4.
[1002] Step g.
##STR00983##
[1003] The compound from step f (62 mg, 0.1 mmol), and penta-Boc-polymyxin B decapeptide (320 mg, 0.2 mmol, 2 eq) in DMF (5 mL) were added EDC (60 mg, 0.3 mmol), HOBT (45 mg, 0.3 mmol), DIEA (0.14 mL, 1.0 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and re-dissolved into 2 mL methanol, the resulting solution was added drop wise into 50 mL vigorously stirred water, this heterogeneous solution was stirred for 1 hour, then filtered with Buchner funnel with Whatman filter paper. The crude product was used for next step without purification.
[1004] Step h.
##STR00984##
[1005] The compound from step g was treated in 2 mL DCM and 2 mL TFA at room temperature, the solution was stirred 10 min, then concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 0% to 100% acetonitrile and water, using formic acid as the modifier. Yield of Int-41 was 0.070 g, 26% yield. Ion(s) found by LCMS: [M/4]+H+=694.9, [M/5]+H+=555.1, [M/6]+H+=463.6, [M/7]+H+=397.5.
Example 77—Synthesis of Int-42
[1006] ##STR00985## ##STR00986##
[1007] Step a.
##STR00987##
[1008] t-butyl Bromo-acetate (1.2 mg, 6 mmol) was added into a solution of propargyl-PEG-4-NH.sub.2 (460 mg, 2 mmol) and DIPEA (1.4 mL, 10 mmol) in 20 mL DMF, The resulting solution was stirred at room temperature for overnight. The reaction solution was concentrated and purified by flash chromatography to provide products. Yield of product AS AN OIL, 700 mg, 76%. Ion(s) found by LC/MS [M+H]+=460.3.
[1009] Step b.
##STR00988##
[1010] tert-butyl 3-(2-tert-butoxy-2-oxoethyl)-6,9,12,15-tetraoxa-3-azaoctadec-17-yn-1-oate (230 mg, 0.5 mmol) was treated with 5 mL TFA at room temperature, after the completion the reaction, the solution was concentrated and used in next step without further purification.
[1011] Step c.
##STR00989##
[1012] The compound from step b (700 mg, 2 mmol), methyl L-norleucinate (630 mg, 4.2 mmol) in DMF (40 mL) was added EDC (1.0 g, 5 mmol), HOBT (0.9 g, 6 mmol), DIEA (1.0 mL, 7 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of product as an oil, 910 mg, 75%. Ion(s) found by LCMS: (M+H)+=602.4.
[1013] Step d.
##STR00990##
[1014] Lithium hydroxide 24 mg, 1 mmol) in 2 mL water was added into a solution of the amino acid ester from step c (0.06 g, 0.1 mmol) in 2 mL methanol and 2 mL THF. The reaction was monitored by LCMS. After completion, the solution was adjusted to pH 1 with Amberlite® IRN-77, then the resulting solution was filtered and concentrated and used in next step without further purification. Ion(s) found by LCMS: (M+H)+=574.4.
[1015] Step e.
##STR00991##
[1016] The compound from step d (57 mg, 0.1 mmol), and penta-Boc-polymyxin B decapeptide (350 mg, 0.22 mmol, 2.1 eq) in DMF (5 mL) were added EDC (60 mg, 0.3 mmol), HOBT (45 mg, 0.3 mmol), DIEA (0.14 mL, 1.0 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and re-dissolved into 2 mL methanol, the resulting solution was added drop wise into 50 mL vigorously stirred water, this heterogeneous solution was stirred for 1 hour, then filtered with Buchner funnel with Whatman filter paper. The crude product was used for next step without purification.
[1017] Step f.
##STR00992##
[1018] Product from step e was treated in 2 mL DCM and 2 mL TFA at room temperature, the solution was stirred 10 min, then concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 0% to 100% acetonitrile and water, using formic acid as the modifier. Yield of Int-42 was 0.120 g, 67% yield. Ion(s) found by LCMS: [M/4]+H+=666.6, [M/5]+H+=533.5, [M/6]+H+=444.7, [M/7]+H+=381.4.
Example 78—Synthesis of Int-43
[1019] ##STR00993## ##STR00994## ##STR00995##
[1020] Step a.
##STR00996##
[1021] Propargyl-PEG4-acid (850 mg, 3.2 mmol), tert-butyl [2-(piperazin-1-yl)ethyl]carbamate (750 mg, 3.2 mmol) in DMF (20 mL) was added EDC (1.0 g, 5 mmol), HOBT (0.9 g, 6 mmol), DIEA (1.0 mL, 7 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of product as an oil, 850 mg, 56%. Ion(s) found by LCMS: (M+H).sup.+=472.3.
[1022] Step b.
##STR00997##
[1023] Intermediate from the previous step (470 mg, 1 mmol) was treated with 5 mL TFA at room temperature. After completion of the reaction, the solution was concentrated and used in next step without further purification.
[1024] Step c.
##STR00998##
[1025] Benzyl Bromo-acetate (0.8 mg, 3.5 mmol) was added into a solution of compounds from step b (470 mg, 1 mmol, TFA salt) and DIPEA (1.0 mL, 7 mmol) in 10 mL DMF, The resulting solution was stirred at room temperature for overnight. The reaction solution was concentrated and purified by flash chromatography to provide products. Yield of product as an oil, 620 mg, 92%. Ion(s) found by LC/MS [M+H].sup.+=668.3.
[1026] Step d.
##STR00999##
[1027] Lithium hydroxide (120 mg, 5 mmol) in 2 mL water was added into a solution of the amino acid ester from step d (0.500 g, 0.74 mmol) in 2 mL methanol and 2 mL THF. The reaction was monitored by LCMS. After completion, the solution was adjusted to pH 1 with Amberlite® IRN-77, then the resulting solution was filtered and concentrated and used in next step without further purification. Yield of product as an oil 480 mg, 100% yield. Ion(s) found by LCMS: (M+H).sup.+=488.3.
[1028] Step e.
##STR01000##
[1029] To a solution of intermediate from the previous step (360 mg, 0.73 mmol) in DMF (20 mL) was added EDC (300 mg, 1.5 mmol), HOBT (230 mg, 1.5 mmol), DIEA (0.28 mL, 2 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of product as an oil, 420 mg, 77%. Ion(s) found by LCMS: (M+H).sup.+=742.5.
[1030] Step f.
##STR01001##
[1031] Lithium hydroxide (24 mg, 1 mmol) in 2 mL water was added into a solution of the amino acid ester from step d (0.150 g, 0.2 mmol) in 2 mL methanol and 2 mL THF. The reaction was monitored by LCMS. After completion, the solution was adjusted to pH 1 with Amberlite® IRN-77, then the resulting solution was filtered and concentrated and used in next step without further purification. Yield of product as an oil, 140 mg, 100% yield. Ion(s) found by LCMS: (M+H).sup.+=714.4.
[1032] Step g.
##STR01002##
[1033] The compound from step f (50 mg, 0.067 mmol), and penta-Boc-polymyxin B decapeptide (240 mg, 0.15 mmol, 2.1 eq) in DMF (5 mL) were added EDC (60 mg, 0.3 mmol), HOBT (45 mg, 0.3 mmol), DIEA (0.14 mL, 1.0 mmol) at room temperature. The solution was stirred overnight then concentrated and re-dissolved into 2 mL methanol. This solution was added dropwise to 50 mL vigorously stirred water. This heterogeneous solution was stirred for 1 hour, then filtered with a Buchner funnel with Whatman filter paper. The crude product was used for next step without purification.
[1034] Step h.
##STR01003##
[1035] Intermediate from step g was treated with 2 mL DCM and 2 mL TFA at room temperature. The solution was stirred 10 min, then concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 0% to 100% acetonitrile and water, using formic acid as the modifier. Yield of Int-43 was 0.080 g as TFA salt, 67% yield. Ion(s) found by LCMS: [M/4]+H+=715.7, [M/5]+H+=572.7, [M/6]+H+=477.5, [M/7]+H+=409.4.
Example 79—Synthesis of Int-44
[1036] ##STR01004## ##STR01005##
[1037] Step a.
##STR01006##
[1038] To a solution propargyl-PEG-4-acid (0.52 g 2 mmol), and methyl L-norleucinate (0.35 g, 2 mmol) in DMF (20 mL) was added EDC (0.60 g, 3 mmol), HOBT (0.45 g, 3 mmol), DIEA (0.56 mL, 4 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 100% acetonitrile and water, using 0.1% TFA as the modifier. Ion(s) found by LCMS: (M+H)+=416.2.
[1039] Step b.
##STR01007##
[1040] Lithium hydroxide (60 mg, 2.5 mmol) in 2 mL water was added into a solution of the amino acid ester from the previous step (0.4 g, 1 mmol) in 2 mL methanol and 2 mL THF. The reaction was monitored by LCMS. After completion, the solution was adjusted to pH 1 with Amberlite® IRN-77, then the resulting solution was filtered and concentrated and used in next step without further purification. Ion(s) found by LCMS: (M+H)+=402.2.
[1041] Step c.
##STR01008##
[1042] The compound from step d (40 mg, 0.1 mmol), and penta-Boc-polymyxin B decapeptide (160 mg, 0.1 mmol, 1 eq) in DMF (5 mL) were added EDC (40 mg, 0.2 mmol), HOBT (30 mg, 0.2 mmol), DIEA (0.14 mL, 1.0 mmol) at room temperature. The solution was stirred overnight. The resulting solution was concentrated and re-dissolved into 2 mL methanol. The resulting solution was added dropwise into 50 mL vigorously stirred water, this heterogeneous solution was stirred for 1 hour, then filtered with Buchner funnel with Filter paper. The crude product was used in next step without further purification.
[1043] Step d.
##STR01009##
[1044] Intermediate form the previous step was treated with 2 mL DCM and 2 mL TFA at room temperature, stirred for 10 min, then concentrated and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 0% to 100% acetonitrile and water, using formic acid as the modifier. Yield of Int-44 was 0.060 g, 42% yield. Ion(s) found by LCMS: [M/2]+H+=737.9, [M/3]+H+=492.3, [M/4]+H+=369.5.
Example 80—Synthesis of Int-45
[1045] ##STR01010##
[1046] The title compound Int-45 was prepared analogously to Example 79, where the where penta-Boc polymyxin B decapeptide was substituted with decapeptide described in Example 69, step c of the sequence. Ion(s) found by LCMS: [M/2]+H+=758.9, [M/3]+H+=506.3, [M/4]+H+=380.0.
Example 81—Synthesis of Int-46
[1047] ##STR01011##
[1048] The title compound Int-46 was prepared analogously to Examples 80, where the acid intermediate was substituted by propargyl-PEG-4-acid in step c of the sequence. Ion(s) found by LCMS: [M/2]+H+=681.4, [M/3]+H+=454.6.
Example 82—Synthesis of Int-47
[1049] ##STR01012##
[1050] Step a.
##STR01013##
[1051] A flask was charged with and (S)-Methyl 2-amino-5-methylhexanoate (0.010 g, 0.627 mmol), diacid (0.115 g, 0.285 mmol, described in Example 90, step a), DIEA (0.40 mL, 2.28 mmol) in anhydrous DMF (1 mL). To this reaction mixture was added a solution of HATU (0.238 g, 0.627 mmol) in DMF (1 mL) at a rate of 2.5 mL/h. After the addition, the reaction mixture was stirred for an addition hour then was purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 95% acetonitrile and water, using 0.1% TFA as modifier. Yield of 0.126 g, 64%. Ion(s) found by LCMS: (M+H)+=687.4
[1052] Step b.
##STR01014##
[1053] A solution of bis-ester product from the previous step (0.126 g, 0.183 mmol), dissolved in MeOH:THF:H.sub.2O (1:1:2, 8 mL), was treated with lithium hydroxide solid (0.026 g, 1.10 mmol). The reaction mixture was continued stirring overnight. After the reaction was completed by monitoring with LCMS, the reaction was quenched with glacial acetic acid to pH 4, stripped of methanol and tetrahydrofuran then purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 55% acetonitrile and water, using 0.1% TFA as modifier. Yield of 0.088 g, 72%. Ion(s) found by LCMS: (M+H).sup.+=659.3
[1054] Step c.
##STR01015##
[1055] A flask was charged with di-acid product from the previous step (0.048 g, 0.073 mmol), HOAt (0.030 g, 0.220 mmol), EDC (0.042 g, 0.220 mmol) and Int-7a (0.252 g, 0.161 mmol, Example 25, step a) in DMF (2 mL). The reaction mixture was stirred at room temperature for 10 minutes followed by addition of DIEA (0.08 mL, 0.439 mmol) then continued to stir overnight. The deca-Boc-protected intermediate was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 25% then 100% methanol and water, using 0.1% TFA as modifier. Pure fractions were combined and concentrated to yield deca-boc-protected intermediate.
[1056] A solution of deca-Boc-protected intermediate from the previous step was stirred in TFA/CH.sub.2Cl.sub.2 (1:1, 6 mL) at ambient temperature for 1 hour then stripped of TFA and CH.sub.2Cl.sub.2 using the rotary evaporator. The product was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 30% acetonitrile and water, using 0.1% TFA as modifier. Yield of 0.087 g as TFA salt, 31%, 2 steps. Ion(s) found by LCMS: [(M+5H)/5]+=551.20, [(M+6H)/6]+=458.9
Example 83—Synthesis of Int-48
[1057] ##STR01016##
[1058] The title compound Int-48 was prepared from the di-acid product from the Example 82, step a (0.04 g, 0.061 mmol) and the extended threonine decapeptide (0.213 g, 0.134 mmol, Example 67, step h) as described in Example 67, step m. Yield 0.0767 g as TFA salt, 32%, 2 steps. Ion(s) found by LCMS: [(M+5H)/5]+=561.8, [(M+6H)/6]+=468.6.
Example 84—Synthesis of Int-49
[1059] ##STR01017## ##STR01018##
[1060] Step a.
##STR01019##
[1061] A flask was charged with L-valine methyl ester HCl (0.970 g, 5.786 mmol), racemic-trans-(tert-butoxycarbonyl)pyrrolidine-3,4-dicarboxylic acid (0.500 g, 1.929 mmol), DIEA (2.0 mL, 11.57 mmol) in anhydrous DMF (8 mL). To this reaction mixture was added a solution of HATU (2.2 g, 5.86 mmol) in DMF (8 mL) at a rate of 4 mL/h. After stirring overnight solvent was stripped to half of the volume then purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 45% acetonitrile and water, using 0.1% TFA as modifier. Yield of 0.0845 g polar diastereomer 9% (used in the next step). Yield of less polar isomer 0.18 g, 14% yield (discarded). Ion(s) found by LCMS: (M−Boc)+=386.2
[1062] Step b.
##STR01020##
[1063] A solution of the Boc-Pyrrolidine product from the previous step (0.085 g, 0.174 mmol) in dioxane (3 mL) was charged with HCl (4M in dioxane, 2 mL). The reaction mixture was stirred at ambient temperature for 1 hour then concentrated under reduced pressure. The residue was rinsed with ethyl acetate (20 mL, 2×) then concentrated and dried under reduced pressure. Yield 0.160 g as HCl salt, 189%. Ion(s) found by LCMS: [M+H]+=386.0.
[1064] Step c.
##STR01021##
[1065] A flask was charged with propargyl-PEG4-acid (0.054 g, 0.19 mmol), the product from the previous step (0.073 g, 0.173 mmol) and DIEA (0.09 mL, 0.519 mmol) was stirred at ambient temperature in DMF (1 mL). To this solution was added a solution of HATU (0.072 g, 0.19 mmol) in DMF (0.6 mL) via a syringe pump at a rate of 2.5 mL/hr. After stirring for 1 hr product was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 50% acetonitrile and water, using 0.1% TFA as modifier. Yield 0.114 g, 105%. Ion(s) found by LCMS: (M+H)+=628.4
[1066] Step d.
##STR01022##
[1067] A solution of bis-ester from the previous step (0.10 g, 0.159 mmol), dissolved in MeOH:THF:H.sub.2O (1:1:2, 8 mL), was treated with lithium hydroxide solid (0.011 g, 0.478 mmol). The reaction mixture was continued stirring overnight. After the reaction was completed by monitoring with LCMS, the reaction was quenched with glacial acetic acid to pH 4, stripped of methanol and tetrahydrofuran then purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 45% acetonitrile and water, using 0.1% TFA as modifier. Yield of 0.073 g, 77%. Ion(s) found by LCMS: (M+H)+=598.2
[1068] Step e.
##STR01023##
[1069] A 4 mL scintillation vial was charged with di-acid product from the previous step (0.073 g, 0.122 mmol), HOAt (0.051 g, 0.367 mmol), EDC (0.070 g, 0.367 mmol) and PMB-decapeptide-D-Ser (0.390 g, 0.269 mmol, Example 6, Int-5d) in DMF (2 mL). The reaction mixture was stirred at room temperature for 10 minutes followed by addition of DIEA (0.13 mL, 0.733 mmol), then continued to stir overnight. Crude product was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 25% then 100% methanol and water, using 0.1% TFA as modifier. Pure fractions were combined and concentrated to yield deca-boc-protected intermediate.
[1070] A solution of deca-Boc-protected intermediate from the previous step was stirred in TFA/CH.sub.2Cl.sub.2 (1:1, 6 mL) at ambient temperature for 1 hour then stripped of TFA and CH.sub.2Cl.sub.2 using a rotary evaporator. The product was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 30% acetonitrile and water, using 0.1% TFA as modifier. Yield of Int-49 was 0.131 g as a TFA salt, 30%, 2 steps. Ion(s) found by LCMS: [(M+4)/4]+=667.0, [(M+5H)/5]+=534.0, [(M+6H)/6]+=445.0
Example 85—Synthesis of Int-50
[1071] ##STR01024## ##STR01025##
[1072] Step a.
##STR01026##
[1073] A flask was charged with L-Valine methyl ester HCl (0.776 g, 4.63 mmol), racemic-trans1-(tert-butoxycarbonyl)pyrrolidine-3,4-dicarboxylic acid (0.40 g, 1.543 mmol), HOAt (0.861 g, 6.326 mmol), and EDC (1.21 g, 6.326 mmol) in anhydrous DMF (6 mL). The reaction mixture was stirred at ambient temperature for 10 minutes following by addition of DIEA (2.42 mL, 13.89 mmol) then continued to stir overnight. The reaction mixture was reduced by half using a rotatory evaporator, then purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 50% acetonitrile and water, using 0.1% TFA as modifier. Yield 0.144 g, 19% of the polar diastereomer (less polar isomer discarded) as a clear oil. Ion(s) found by LCMS: (M−Boc)+=386.2 and (M−H)+=484.2.
[1074] Steps b, c & d.
[1075] The diacid was prepared as described in Example 84, steps b, c, d. Yield 0.17 g, 135% yield; ion(s) found by LCMS: [M+H]+=385.2 (Step b). Yield 0.201 g, 79% yield; ion(s) found by LCMS: [M+H]+=628.4 (Step c). Yield 0.138 g, 72% yield; ion(s) found by LCMS: [M+H]+=601.3 (Step d).
[1076] Step e.
##STR01027##
[1077] The title compound Int-50 was prepared from the di-acid product of step d (0.069 g, 0.115 mmol) and Int-7a (0.395 g, 0.253 mmol, Example 10, step a) following the procedure described in Example 84, step e. Yield 0.042 g as TFA salt, 10%, 2 steps. Ion(s) found by LCMS: [(M+5H)/5]+=539.6, [(M+6H)/6]+=449.4.
Example 86—Synthesis of Int-51
[1078] ##STR01028## ##STR01029## ##STR01030## ##STR01031##
[1079] Step a.
##STR01032##
[1080] A flask was charged with tri-Boc-PMBH-(NH.sub.2) (2.00 g, 1.883 mmol, Example 2, Int-1), 1-Cbz-aminocyclopropane carboxylic acid (0.542 g, 2.259 mmol) and DIEA (0.98 mL, 5.65 mmol) in DMF (19 mL). To this solution was added a solution of HATU (0.877 g, 2.259 mmol) in DMF (3.5 mL) via a syringe pump at rate of 2.5 mL/hr. The reaction was stirred overnight at ambient temperature. The reaction mixture concentrated to half of its volume using the rotary evaporator then purified by RPLC using an Isco CombiFlash eluting with 5% to 25% to 90% methanol and water, using 0.1% TFA as modifier. Yield 2.159 g, 90%. Ion(s) found by LCMS [(M−2Boc+2H)/2]+=540.5.
[1081] Step b.
##STR01033##
[1082] A solution of the product from the previous step (2.08 g, 1.62 mmol) in methanol (15 mL) was charged with Pd/SiO.sub.2 (0.405 g, 0.081 mmol) and hydrogen gas balloon. The reaction mixture was stirred at ambient temperature overnight. It was filtered through a pad of Celite® and washed with methanol, then concentrated to the title compound as white solid. Yield 1.94 g, 104%. Ion(s) was found by LCMS: [(M−2Boc+2H)/2]+=473.4, [(M−3Boc+2H)/2]+=423.3
[1083] Step c.
##STR01034##
[1084] A flask was charged with product from the previous step (0.979 g, 0.855 mmol), N-Cbz-L-threonine (0.227 g, 0.898 mmol), HOAt (0.175 g, 1.282 mmol) and EDC (0.246 g, 1.28 mmol) in DMF (2.6 mL) then stirred for 10 minutes following by addition of DIEA (0.45 mL, 2.56 mmol). The reaction mixture was stirred at ambient temperature overnight. It was purified by RPLC using an Isco CombiFlash eluting with 5% to 25% then 100% acetonitrile and water, using 0.1% TFA as modifier. Yield 0.84 g, 71%. Ion(s) found by LCMS: [(M−2Boc+2H)/2]+=590.8
[1085] Step d.
##STR01035##
[1086] The title compound was prepared from the product of the previous step using the procedure described in Step b of this example. Yield 0.725 g, 96%. Ion(s) found by LCMS: [M−2Boc+2H)/2]+=523.6
[1087] Step e.
##STR01036##
[1088] The title compound was prepared from the product of the previous (0.725 g, 0.581 mmol) and Z-dab(□Boc)-OH DCHA salt (0.341 g, 0.639 mmol) as described in procedure from Step c. Yield 0.626 g, 68%. Ion(s) found by LCMS: [(M−2Boc+2H)/2]+=690.4
[1089] Step f.
##STR01037##
[1090] The title compound was prepared from the product of the previous step (0.626 g, 0.396 mmol) and Pd/SiO.sub.2 (0.099 g, 0.02 mmol) as described in Step b of this example. Yield 0.575 g. 100%. Ion(s) found by LCMS: (M+Na)+=646.0
[1091] Step g.
##STR01038##
[1092] The title compound Int-51 was prepared from intermediate from Example 84, step d (0.069 g, 0.115 mmol) and intermediate from the previous step (0.366 g, 0.253 mmol), following the procedure from Example 84, step e. Yield 0.0478 g as TFA salt, 12%, 2 steps. Ion(s) found LCMS: [(M+4H)/4]+=664.8, [(M+5H)/5]+=532.0, [(M+6H)/6]+=443.6.
Example 87—Synthesis of Int-52
[1093] ##STR01039## ##STR01040##
[1094] Step a.
##STR01041##
[1095] The diester was prepared from methyl (2S)-2-amino-5-methylhexanoate HCl salt (0.557 g, 2.85 mmol) and racemic-trans-1-(tert-butoxycarbonyl)pyrrolidine-3,4-dicarboxylic acid (0.352 g, 1.36 mmol) as described in Example 84, step a. Yield 0.303 g of the polar diastereomer 41%. Ion(s) found by LCMS: [M−Boc+H]+=442.2.
[1096] Step b.
##STR01042##
[1097] The title compound was prepared from the product of the previous step (0.303 g, 0.56 mmol) and HCl (4N in dioxane, 6.40 mL, 46.0 mmol) as described in Example 84, step b. Yield 0.29 g, 108%. Ion(s) found by LCMS: [M+H]+=442.4
[1098] Step c.
##STR01043##
[1099] The title compound was prepared from the product of the previous (0.29 g, 0.657 mmol) and propargyl-PEG4-acid (0.188 g, 0.722 mmol) as described in Example 84, step c. Yield 0.133 g, 30%. Ion(s) found by LCMS: (pos) (M+H)+=684.4, and (neg) (M−H)−=682.3.
[1100] Step d.
##STR01044##
[1101] The title compound was prepared from the bis-methyl esters product of the previous step (0.133 g, 0.194 mmol) and lithium hydroxide (0.014 g, 0.582 mmol) as described by the Example 84, step d. Yield 0.12 g, 94%. Ion(s) found by LCMS: (pos) (M+H)+=656.3 and (neg) (M−H)+=655.3
[1102] Step e.
##STR01045##
[1103] The title compound Int-52 was prepared from the di-acid product of the previous step (0.06 g, 0.092 mmol), and the product described in Example 6, Int-5d (0.292 g, 0.201 mmol) using the procedure described in Example 84, step e. Yield 0.135 g as TFA salt, 41%, 2 steps. Ion(s) found by LCMS: [(M+5H)/5]+=544.9, [(M+6H)/6]+=454.3
Example 88—Synthesis of Int-53
[1104] ##STR01046##
[1105] The title compound Int-53 was prepared from the di-acid product described in Example 87, step d (0.06 g, 0.092 mmol) and Int-7a (0.315 g, 0.212 mmol, Example 8, step a) using the procedure described in Example 84, step e. Yield 0.116 g as TFA salt, 35%, 2 steps. Ion(s) found by LCMS: [(M+4H)/4]+=687.2, [(M+5H)/5]+=550.1, [(M+6H)/6]+=458.6.
Example 89—Synthesis of Int-54
[1106] ##STR01047## ##STR01048##
[1107] Step a.
##STR01049##
[1108] The title compound was prepared from methyl (2S,3R)-2-amino-3-methylpentanoate (0.242 g, 1.66 mmol) and racemic-trans1-(tert-butoxycarbonyl)pyrrolidine-3,4-dicarboxylic acid (0.210 g, 0.811 mmol) prepared analogously to Example 84, step a. Yield 0.121 g of polar isomer, 29%. Ion(s) found by LCMS: (M+H)+=514.2. Note: Only the polar isomer was taken forward to the next steps.
[1109] Step b.
##STR01050##
[1110] The title compound was prepared from the product of the previous step (0.121 g, 0.235 mmol) and HCl (4M in Dioxane, 0.59 mL, 2.35 mmol) as described in the procedure for Example 84, step b. Yield 0.122 g, 116%. Ion(s) found by LCMS: (M+H)+=414.2
[1111] Step c.
##STR01051##
[1112] The title compound was prepared from the product of the previous step (0.118 g, 0.261 mmol) and propargyl-PEG4-acid (0.188 g, 0.287 mmol) as described in Example 84, step c. Yield 0.075 g, 44%. Ion(s) found by LCMS: (pos) (M+H)+=656.4, (M+Na)+=679.3 and (neg) (M−H)+=654.2
[1113] Step d.
##STR01052##
[1114] The title compound was prepared from bis-methyl ester product in the previous step (0.075 g, 0.114 mmol) and lithium hydroxide (0.008 g, 0.34 mmol) as described by a procedure of Example 84 Step d. Yield 0.056 g, 78%. Ion(s) found by LCMS: (M+H)+=628.3
[1115] Step e.
##STR01053##
[1116] The title compound Int-54 was prepared from the di-acid product of the previous step (0.056 g, 0.088 mmol) and Int-7a (0.305 g, 0.195 mmol, Example 8, step a) using the procedure from Example 84, step e. Yield of 0.0326 g as TFA salt, 10%. Ion(s) found LCMS: [(M+5H)/5]+=544.7, [(M+6H)/6]+=454.3
Example 90—Synthesis of Int-55
[1117] ##STR01054## ##STR01055## ##STR01056##
[1118] EDC (0.77 g, 4.0 mmol) was added to a stirring mixture of diester (1.0 g, 4.0 mmol), HOBt (0.62 g, 4.0 mmol), and propargyl-peg4-amine (0.94 g, 4.0 mmol) in DMF (8 mL) and stirred for 2 hours. The mixture was reduced by half on a rotary evaporator and then purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the di-ester as a clear oil. Positive ions found: by LCMS (M+H.sup.+)=461.2.
[1119] The di-ester was stirred in a 1:1:2 mixture (5 mL) of THF/MeOH/DI water containing LiOH (0.39 g, 16.2 mmol) for 1 hour. The mixture was acidified with glacial acetic acid (˜0.7 mL) and concentrated to half the volume on the rotary evaporator. The di-acid was purified RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the di-acid as a viscous clear oil. Yield 953 mg, 59.8%, 2 steps. Positive ions found by LCMS (M+H.sup.+)=405.2.
[1120] Step b.
##STR01057##
[1121] EDC (0.12 g, 0.65 mmol) was added to a stirring mixture of Isoleucine-methyl ester hydrochloride salt (0.17 g, 0.93 mmol), HOBt (0.099 g, 0.65 mmol), intermediate from the previous step (0.13 g, 0.31 mmol), and triethylamine (0.097 g, 0.96 mmol) in DMF (1.5 mL) and the reaction was stirred for 12 hours. The mixture was applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 20% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the 200 mg of the di-ester as a white solid. Positive ions found: by LCMS (M+H.sup.+)=659.4. The di-acid was hydrolyzed by stirring in a 1:1:2 mixture THF/MeOH/DI water (8 mL) containing LiOH (0.029 g, 1.2 mmol) for 30 minutes at ambient temperature. Glacial acetic acid (˜1 mL) was added and the volume was reduced by 70% on the rotary evaporator. The residue was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 85% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford di-acid as a clear oil. Yield 73%, 2 steps. Positive ions found: by LCMS (M+H.sup.+)=631.4.
[1122] Step c.
##STR01058##
[1123] EDC (89 mg, 0.47 mol) was added to a stirring mixture of intermediate from previous step (140 mg, 0.22 mmol), Int-7a (760 mg, 0.49 mmol), and HOBt (71 mg, 0.47 mmol) in 1.5 mL of DMF. The reaction was stirred for 12 hours at ambient temperature. The mixture was then applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 30% to 100% methanol and water using 0.1% TFA modifier. The fractions containing the positive ion mass observed by LCMS [(M−(3Boc)+3H.sup.+)/3]=1141.2 were pooled and concentrated. The Boc-protected dimer was stirred in a 1/1 mixture of TFA/DCM (10 mL) at ambient temperature for 30 minutes. The solvent was removed by rotary evaporator and dried under high vacuum. The crude deprotected dimer was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 0% to 90% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford desired product as a white solid deca-TFA salt. Yield of Int-55 was 405 mg, 67%, 2 steps. Positive mass ions were found by LCMS (M+4H.sup.+)/4=680.8, LCMS (M+5H.sup.+)/5=544.6, and LCMS (M+6H.sup.+)/6=454.2.
Example 91—Synthesis of Int-56
[1124] ##STR01059##
[1125] The title compound Int-56 was prepared analogously to Example 90, where L-leucine was substituted for L-isoleucine in step A of that example. Yield 430 mg, 66%, 2 steps. Positive ions found by LCMS (M+4H.sup.+)/4=680.8, (M+5H.sup.+)/5=544.6, and (M+6H.sup.+)/6=454.2.
Example 92—Synthesis of Int-57
[1126] ##STR01060##
[1127] The title compound Int-57 was prepared analogously to Example 90 where Int-7a was substituted with Int-7a described in Example 67, step h and the di-acid intermediate described in step b of Example 90. Yield of 210 mg, 69%, 2 steps. Ions found by LCMS (M+3H.sup.+)/3=926.2, LCMS (M+4H.sup.+)/4=695.2, and LCMS (M+5H.sup.+)/5=556.4.
Example 93—Synthesis of Int-58
[1128] ##STR01061## ##STR01062##
[1129] Step a.
##STR01063##
[1130] EDC (3.2 g, 17 mmol) was added to a stirring mixture of racemic-trans1-(tert-butoxycarbonyl)pyrrolidine-3,4-dicarboxylic acid (2 g, 7.7 mmol), Isoluecine methyl ester HCl salt (3.5 g, 19.3 mmol), HOBt (2.6 g, 17 mmol), and triethylamine (2 g, 20 mmol) in 15 mL of DMF. The reaction was stirred for 12 hours, diluted with aqueous 1N HCl (100 mL), extracted into ethyl acetate, dried over sodium sulfate and concentrated. The diastereomers were separated by RPLC using an Isco Combiflash liquid chromatograph eluted with 15% to 95% acetonitrile and water using 0.1% TFA modifier. The polar isomer was pooled and lyophilized to afford 1.2 g of the boc-protected ester. Yield, combined 59%. LCMS [M−(1 boc)+H.sup.+)=414.2. The polar isomer boc-protected ester (1.2 g, 2.3 mmol)) was stirred in 30 mL of 4M HCl (g) in dioxane for 30 minutes. The solvent was removed by rotary evaporator and dried under high vacuum to afford the di-ester-Ile-pyrolidine HCl salt as a white solid. Yield of 0.60 g, 75%. LCMS [M+H.sup.+)=414.2.
[1131] Step b.
##STR01064##
[1132] HATU (389 mg, 0.87 mmol) was added to a stirring mixture of the trans-di-methyl ester-Ile-pyrolidine HCl salt (325 mg, 0.72 mmol), propargyl-peg4 acid (226 mg, 0.86 mmol), and triethylamine (291 mg. 2.9 mmol) and stirred at ambient temperature for 30 minutes. The mixture was applied directly to RPLC using an Isco Combiflash liquid chromatograph eluted with 20% to 95% acetonitrile and water using 0.1% TFA modifier. Fractions showing desired ions by LCMS (M+H.sup.+)=656.4 were pooled and concentrated. The residue was stirred in a 1:1:2 mixture of methanol/THF/DI water containing LiOH (52 mg, 2.2 mmol) at ambient temperature for 30 minutes. The reaction mixture was acidified with a few drops of acetic acid and the volume was reduced by half on the rotary evaporator. The mixture was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. The pure fractions were pooled and lyophilized to afford the propargyl peg4 di-acid intermediate as a clear oil. Yield of 375 mg, 83%, 2 steps. Positive ions were found by LCMS [M+H.sup.+)=626.2.
[1133] Step c.
##STR01065##
[1134] The title compound Int-58 was prepared analogously to Example 90 where Int-7a was substituted with tetra Boc PMBH-Gly-Thr-Dab (described in Example 100, step c), and the intermediate described in step b of this example. Yield; 54%, 2 steps. Positive mass ions were found by LCMS (M+3H.sup.+)/3=877.8, LCMS (M+4H.sup.+)/4=658.8, LCMS (M+5H.sup.+)/5=527.4, and LCMS (M+6H.sup.+)/6=439.8.
Example 94—Synthesis of Int-59
[1135] ##STR01066##
[1136] The title compound Int-59 was prepared analogously to Example 90 step c from the intermediate from Example 93, step b and Int-5d Example 6. Yield; 44%, 2 steps. Positive mass ions were found by LCMS (M+3H.sup.+)/3=898.0, LCMS (M+4H.sup.+)/4=673.8, LCMS (M+5H.sup.+)/5=539.4, and LCMS (M+6H.sup.+)/6=449.8.
Example 95—Synthesis of Int-60
[1137] ##STR01067## ##STR01068##
[1138] The title compound Int-60 was prepared analogously to Example 90, step c from Int-7a (Example 2, step a) and the intermediate from Example 93, step b. Yield of 195 mg, 49%, 2 steps.
[1139] Positive mass ions were found by LCMS (M+3H.sup.+)/3=906.4, LCMS (M+4H.sup.+)/4=680.3, LCMS (M+5H.sup.+)/5=544.5.
Example 96—Synthesis of Int-61
[1140] ##STR01069##
[1141] Step a.
##STR01070##
[1142] The dimethyl ester was prepared analogously to intermediate Example 93, step a substituting L-leucine for L-isoleucine. Yield of 0.85 g, 51%, 2 steps. LCMS (M+H)+=414.2.
[1143] Step b.
##STR01071##
[1144] The diacid was prepared analogously to Example 93, step b. Yield of 0.82 g, 77%, 2 steps. Negative Ion found by LCMS [M−H]−=626.2.
##STR01072##
[1145] The title compound Int-61 was prepared analogously to Example 90, step c from the previous intermediate and Int-7a. Yield of 179 mg, 39%, 2 steps. Positive mass ions were found by LCMS (M+3H.sup.+)/3=906.6, LCMS (M+4H.sup.+)/4=680.3, LCMS (M+5H.sup.+)/5=544.5.
Example 97—Synthesis of Int-62
[1146] ##STR01073##
[1147] The title compound Int-62 was prepared analogously to Example 90, step c from the intermediates described in Example 96 step b, and Example 6, Int-5d. Yield of 225 mg 36%, 2 steps. Positive mass ions were found by LCMS (M+3H.sup.+)/3=897.8, LCMS (M+4H.sup.+)/4=674.0, LCMS (M+5H.sup.+)/5=539.4.
Example 98—Synthesis of Int-63
[1148] ##STR01074##
[1149] Step a.
##STR01075##
[1150] EDC (175 mg, 0.92 mmol) was added to a stirring mixture of di acid (synthesis described in Example 60, step b, 100 mg, 0.43 mmol), isoleucine methyl ester hydrochloride (239 mg, 1.3 mmol), HOBt (141 mg, 0.92 mmol), and triethylamine (132 mg, 1.3 mmol) in 1.5 mL of DMF. The reaction was stirred at ambient temperature for 12 hours then purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 10% to 95% acetonitrile and water using 0.1% TFA modifier. Pure fractions were pooled and concentrated to afford an oil; ion(s) found by LCMS (M+H.sup.+)=483.3. The oil was taken up in a 1:1:2 mixture of methanol/THF/DI water (5 mL) containing LiOH (31 mg, 1.3 mmol) and stirred at ambient temperature for 30 minutes. The mixture was acidified (pH-5) with a few drops of glacial acetic acid and concentrated to half volume. The mixture was purified by RPLC using an Isco Combiflash liquid chromatograph eluted with 5% to 80% acetonitrile and water using 0.1% TFA modifier. Pure fractions were pooled and lyophilized to afford the di-acid intermediate as a clear oil. Yield of 147 mg, 69%, 2 steps. Positive Ion found by LCMS (M+H.sup.+)=455.3.
[1151] Step b.
##STR01076##
[1152] The title compound Int-63 was prepared analogously from Int-7a and the previous intermediate from this example. Yield, 260 mg, 77%, 2 steps. Positive mass ions were found by LCMS (M+3H.sup.+)/3=848.8, LCMS (M+4H.sup.+)/4=637.0, LCMS (M+5H.sup.+)/5=509.9.
Example 99: Synthesis of Int-64
[1153] ##STR01077## ##STR01078##
[1154] Step a.
##STR01079##
[1155] EDC (2.04 g, 10.7 mmol) was added to a stirring mixture of racemic-4-oxocyclopentane-1,2-dicarboxylic acid (0.8 g, 4.6 mmol), HOBt (1.6 g, 10.7 mmol), and triethylamine (1.5 g, 14.4 mmol) in DMF (12 mL) and the reaction was stirred for 12 hours. The mixture was diluted with ethyl acetate (75 mL) washed with 1N HCl (100 mL). The aqueous phase was back extracted with ethyl acetate (2×, 50 mL). The combined organics were washed with brine (100 mL), dried over sodium sulfate and concentrated. The residue was purified by ISCO Combi Flash reversed phase HPLC (acetonitrile in DI water with 0.1% TFA as modifier, 10-95% acetonitrile, 40 minute gradient. The 2 diastereomers were separated and the more polar diastereomer (by HPLC) was taken forward in subsequent reactions. Yield of 1.7 g, 85%. LCMS (M+H.sup.+)=427.8. The shown stereochemistry was assigned by X-ray crystallography.
[1156] Step b.
##STR01080##
[1157] Tert-butyl [ethoxy(ethyl)phosphoryl]acetate (0.43 g, 1.7 mmol) was dissolved in dry THF (15 mL) and cooled to 0° C. under an atmosphere of nitrogen. NaHMDS (1.7 mL, 1.7 mmol) was added and the mixture was stirred for 30 minutes. The intermediate from step a of this example (0.36 g, 0.85 mmol, in 10 mL of THF) was added dropwise and the reaction was stirred for 30 minutes. The reaction was quenched with 1N aqueous HCL (100 mL) and extracted into ethyl acetate (3×, 50 mL). The combined organics were dried over sodium sulfate and concentrated. The residue was purified by ISCO Combi Flash reversed phase HPLC (25-95% acetonitrile in DI water with 0.1% TFA as modifier, 40 minute gradient. The pure fractions were pooled and lyophilized to afford 425 mg of di-ester cyclopentyl acetic acid as a clear oil. Positive ions found by LCMS [M+H]+=525.4. The alkene was reduced by stirring in methanol (25 mL) in the presence of 5% Pd/C (150 mg) under 1 atmosphere of hydrogen for 4 hours. The mixture was filtered through celite and concentrated. The residue was stirred in TFA (15 mL) at ambient temperature for 12 hours. The TFA was removed by rotary evaporator and the residue was purified by ISCO Combi Flash reversed phase HPLC (5-95% acetonitrile in DI water with 0.1% TFA as modifier, 40 minute gradient. The pure fractions were pooled and lyophilized to afford di methyl ester cyclopentyl acid as a clear oil. Yield of 334 mg, 83%, 3 steps. Positive ions found by LCMS [M+H]+=471.2
[1158] Step c.
##STR01081##
[1159] A flask was charged with the intermediate from the previous step (0.303 g, 0.644 mmol), propagyl-PEG4-amine (0.179 g, 0.773 mmol) and DIEA (0.34 mL, 1.93 mmol) in DMF (2 mL) at room temperature. To this solution was added a prepared solution of HATU (0.294 g, 0.773 mmol) in DMF (1.22 mL) via a syringe pump at a rate of 2.5 mL/hr. The reaction mixture was continued stirring overnight then purified by RPLC without aqueous work up using Isco CombiFlash reversed phase HPLC (5-60% acetonitrile in DI water with 0.1% TFA as modifier). The pure fractions were combined and lyophilized to give propargyl-peg4-di-ester intermediate (0.41 g, 93%). Positive ions found by LCMS: [M+H]+=684.4
[1160] To a solution of dimethyl ester (0.41 g, 0.60 mmol) from a previous step in methanol/tetrahydrofuran/DI water (1:1:2, 15 mL) was added lithium hydroxide (0.043 g, 1.80 mmol). The reaction mixture was stirred at ambient temperature for 1 hour then acidified with glacial acetic acid to pH4. Methanol and tetrahydrofuran were stripped under reduced pressure. The mixture was purified by RPLC using Isco CombiFlash reversed phase HPLC (5-40% acetonitrile and DI water, using 0.1% TFA as modifier) to yield the title di-acid compound after lyophilizing, 0.411 g, 94%. Positive ions found by LCMS: [M+H]+=656.4.
[1161] Step d.
##STR01082##
[1162] The title compound Int-64 was prepared from the previous di-acid intermediate and Int-7a with Thr2 extended (described in Example 67, step h), analogously to Example 90 step C. Yield 125 mg, 49%, 2 steps. Positive ions found by LCMS (M+3H.sup.+)/3=934.6, LCMS (M+4H.sup.+)/4=701.3, LCMS (M+5H.sup.+)/5=561.5.
Example 100—Synthesis of Int-65
[1163] ##STR01083## ##STR01084##
[1164] Step a.
##STR01085##
[1165] To a mixture of PMB heptapeptide (4.24 g, 4 mmol, Example 2) and Z-Gly-OH (1 g, 4.8 mmol) in anhydrous DMF (8 mL) was added HATU (1.87 g, 4.9 mmol) in portions over 20 minutes, followed by DIPEA (936 mg, 7.2 mmol). After the reaction mixture was stirred for 15 minutes, it was poured into water (100 mL). The white solid product was collected by filtration and washed with water. The material was re-dissolved in MeOH (50 mL) and treated with Pd/C (5%) (1 g), then stirred under hydrogen overnight. Pd/C (5%) was then filtered, concentrated and purified by RPLC (150 g, 15 to 75% MeOH and water). Yield 4.02 g, 90%. Ions found by LCMS: [(M−Boc+2H)/2].sup.+=510.4, [(M−3Boc+2H)/2].sup.+=410.2
[1166] Step b.
##STR01086##
[1167] To a mixture of the step-a product (4.02 g, 3.592 mmol) and Z-Thr-OH (980.3 mg, 3.87 mmol) in anhydrous DMF (5 mL) was added HATU (1.47 g, 3.87 mmol) in portions over 10 minutes, followed by DIPEA (755 mg, 5.8 mmol). After the addition, the reaction was stirred for 20 minutes and then poured into water (100 mL). The white solid product was collected by filtration and washed with water. The material was re-dissolved in MeOH (50 mL) and treated with Pd/C (5%) (1 g), and stirred under hydrogen overnight. Pd/C was then filtered, and the filtrate was concentrated and purified by RPLC (150 g column, 15 to 80% MeOH and water). Yield 3.68 g, 84%. Ion found by LCMS: [(M−2Boc+2H)/2].sup.+=511.0
##STR01087##
[1168] A mixture of the step-b product (1.2 g, 0.984 mmol) and Z-Dab(Boc)-OH DCHA salt (605 mg, 1.13 mmol) was dissolved in anhydrous NMP (3 mL). It was treated with HATU (430 mg, 1.13 mmol) in portions over 5 minutes, followed by DIPEA (150 mg, 1.13 mmol). The reaction was stirred for 30 minutes and then directly purified by RPLC (100 g column, 40 to 100% MeOH and water). The collected fractions were concentrated by rotary evaporation to a white solid (Ion found by LCMS: [M−2Boc+2H)/2].sup.+=678). The material was re-dissolved in MeOH (30 mL) and treated with Pd/C (5%), then stirred under hydrogen overnight. Pd/C was filtered, and the filtrate was concentrated by rotary evaporation and further dried under high vacuum. Yield 1.07 g, 76.6%. Ion found by LCMS: [(M−2Boc+2H)/2].sup.+=611.
[1169] Step d.
##STR01088##
[1170] A mixture of the step-c product (313.6 mg, 0.2207 mmol) and diacid central linker (65.9 mg, 0.105 mmol, Example 41, INT-19) was dissolved in anhydrous DMF (1 mL). DIPEA (65 mg, 0.5 mmol) and HATU (83.9 mg, 0.2207 mmol) were added in portions over 5 minutes. The reaction was stirred for 30 minutes and then directly purified by RPLC (50 g, 40 to 100% MeOH and water). Yield 165 mg, 50%. Ions found by LCMS: [M−3Boc+3H)/3].sup.+=1044.6, [M−4Boc+3H)/3].sup.+=1011.3, [M−6Boc+3H)/3].sup.+=944.4.
[1171] Step e.
##STR01089##
[1172] The step-d product (165 mg, 0.0521 mmol) was dissolved in TFA/DCM (1:1, 1 mL), and the solution was stirred for 30 minutes. It was concentrated and directly purified by RPLC (50 g column, 5 to 30% acetonitrile and water, using 0.1% TFA as modifier). Yield of Int-65 was 160 mg, 87%. Ions found by LCMS: [M+4H)/4].sup.+=658.4, [M+5H)/5].sup.+=527.1, [M+6H)/6].sup.+=439.4, [M+7H)/7].sup.+=376.8.
Example 101—Synthesis of Int-66
[1173] ##STR01090##
[1174] Step a.
##STR01091##
[1175] To a solution of intermediate from Example 7, INT-6a (228.1 mg, 0.507 mmol) in anhydrous ethanol (2 mL) was added DIPEA (198 mg, 1.52 mmol) and propargyl-PEG4-bromide (180 mg, 0.608 mmol). After the mixture was heated at 80° C. for 30 hours, it was cooled to room temperature and then placed in an ice-water bath. THF (1 mL) and a solution of LiOH (105 mg, 2.5 mmol) in water (2 mL) were added. The reaction was stirred for 3 hours. It was acidified by 4N HCl solution in dioxane (0.7 mL) and directly purified by RPLC (50 g, 5 to 70% acetonitrile and water). Yield 92.9 mg, 30.6%. Ion found by LCMS: [M+H].sup.+=600.
[1176] Step b.
##STR01092##
[1177] To a solution of the step-a product (32 mg, 0.05336 mmol) and Int-7a (Example 8, step a) (183.6 mg, 0.1174 mmol) in anhydrous DMF (1 mL), was added DIPEA (42 mg, 0.4 mmol) and HATU (40.6 mg, 0.107 mmol). The resulting solution was stirred for 30 minutes and was purified by RPLC (50 g column, 20 to 100% MeOH and water). Yield 189 mg, 96%. Ion found by LCMS: [(M−2Boc+3H)/3].sup.+=1164.4, [(M−3Boc+3H)/3].sup.+=1131, [(M−4Boc+3H)/3].sup.+=1097.
[1178] Step c.
##STR01093##
[1179] The step-b product (189 mg, 0.0512 mmol) was dissolved in TFA (0.5 mL) and stirred for 15 minutes. It was directly purified by RPLC (50 g column, 0 to 50% acetonitrile and water, using 0.1% TFA as modifier). Yield of Int-66 was 103 mg, 51%. Ions found by LCMS: [(M+5H)/5].sup.+=538.6, [(M+6H)/6].sup.+=449.2, [(M+7H)/7].sup.+=385.0, [(M+8H)/8].sup.+=337.0.
Example 102—Synthesis of Int-67
[1180] ##STR01094## ##STR01095##
[1181] Step a.
##STR01096##
[1182] To a solution of dimethyl itaconate (1.58 g, 10 mmol) in MeOH (8 mL) was treated with t-butyl piperazine-1-carboxylate (1.86 g, 10 mmol). The mixture was heated at 60° C. for 1 day. It was then purified by HPLC (0 to 20% acetonitrile and water, using 0.1% TEA as modifier). Yield 1.175 g, 34.1%. Ion found by LCMS: [M+H.sup.+=345.0.
[1183] Step b.
##STR01097##
[1184] The step-b product (1.175 g, 3.41 mmol) was dissolved in MeOH (5 mL) and cooled in an ice-water bath. It was treated with a solution of LiOH monohydrate (378 mg, 9 mmol) in water (9 mL). After the reaction was stirred at 0° C. to room temperature overnight, it was re-cooled in an ice-water bath and acidified by 4N HCl solution in dioxane (2 mL). The mixture was partially concentrated by rotary evaporation at room temperature. The residue was purified by RPLC (150 g column, 0 to 80% MeOH and water). Yield 336.5 mg, 31.2%. Ion found by LCMS: [M+H].sup.+=317.2.
[1185] Step c.
##STR01098##
[1186] A mixture of the step b product (336.5 mg, 1.064 mmol) and H-Nle-OMe HCl (464 mg, 2.552 mmol) was dissolved in anhydrous NMP (3 mL) and DIPEA (520 mg, 4 mmol). To this was added HATU (810 mg, 2.13 mmol) in portions over 10 minutes. The reaction was stirred for another 30 minutes, then purified by RPLC (100 g column, 10 to 75% acetonitrile and water, using 0.1% TFA as modifier). Yield 261.7 mg, 43.2%. Ion found by LCMS: [M+H].sup.+=571.4.
[1187] Step d.
##STR01099##
[1188] The step c product (261.7 g, 0.459 mmol) was dissolved in THF (5 mL), and 4N HCl in dioxane (2.5 mL). The mixture was stirred at room temperature for 4 hours and then directly purified by RPLC (50 g, 0 to 80% acetonitrile and water). Yield 144.3 mg, 66.9%. Ion found by LCMS: [M+H].sup.+=471.4.
[1189] Step e.
##STR01100##
[1190] A mixture of the step d product (144.3 mg, 0.307 mmol) and propargyl-PEG4-acid (104 mg, 0.4 mmol) was dissolved in anhydrous DMF (1 mL) and DIPEA (130 mg, 1 mmol). HATU (152 mg, 0.4 mmol) was added, and the reaction was stirred for 30 minutes. It was then purified by RPLC (100 g, 5 to 100% acetonitrile and water). Yield 165 mg, 75.6%. Ion found by LCMS: [M+H].sup.+=713.4.
[1191] Step f.
##STR01101##
[1192] The step e product (165 mg, 0.232 mmol) was dissolved in MeOH (2 mL) and cooled in an ice-water bath. It was treated with a solution of LiOH monohydrate (21 mg, 0.5 mmol) in water (1 mL). After the reaction was stirred for 4 hours, it was acidified by 4N HCl solution in dioxane (0.2 mL) and directly purified by HPLC (5 to 35% acetonitrile and water). Yield 46.4 mg, 29.2%. Ion found by LCMS: [M+H].sup.+=685.
[1193] Step g.
##STR01102##
[1194] The title compound was prepared analogously to Example 101, step-b, using the step-f diacid and Int-7a (Example 8, step a). Yield 246 mg, 96.1%. Ions found by LCMS: [(M−2Boc+3H)/3].sup.+=1192.4, [(M−3Boc+3H)/3].sup.+=1159.1, [(M−4Boc+3H)/3].sup.+=1125.8.
[1195] Step h.
##STR01103##
[1196] The title compound Int-67 was prepared analogously to Example 101, step-c, using the product from step-g of this example. Yield 115 mg, 43.6%. Ions found by LCMS: [(M+4H)/4].sup.+=694.4, [(M+4H)/5].sup.+=555.8, [(M+6H)/6].sup.+=463.2, [(M+7H)/7].sup.+=397.2.
Example 103—Synthesis of Int-68
[1197] ##STR01104##
[1198] Step a.
##STR01105##
[1199] A mixture of 3-(carboxymethyl)pentanedioic acid (190.2 mg, 1.0 mmol) and H-Nle-OMe HCl (400 mg, 2.2 mmol) was dissolved in anhydrous NMP (2 mL) and DIPEA (546 mg, 4.2 mmol). HATU (760.4 mg, 2.0 mmol) was added in portions over 10 minutes, and the reaction was stirred for 1 hour. It was then purified by RPLC (100 g column, 5 to 80% acetonitrile and water). Yield 162.5 mg, 36.6%. Ion found by LCMS: [M+H].sup.+=445.2.
[1200] Step b.
##STR01106##
[1201] A mixture of the step a product (162.5 mg, 0.366 mmol) and propargyl-PEG4-amine (101.8 mg, 0.392 mmol) was dissolved in anhydrous DMF (1 mL) and DIPEA (78 mg, 0.6 mmol). HATU (140 mg, 0.366 mmol) was added, and the reaction was stirred for 30 minutes. It was then purified by RPLC (50 g column, 10 to 80% acetonitrile and water). Yield 159.1 mg, 66.1%. Ion found by LCMS: [M+H].sup.+=658.4.
[1202] Step c.
##STR01107##
[1203] The step-b product (159.1 mg, 0.242 mmol) was dissolved in MeOH (2 mL) and cooled in an ice-water bath. It was treated with a solution of LiOH monohydrate (25.4 mg, 0.6 mmol) in water (1 mL). After the reaction stirred for 4 hours, it was acidified with 4N HCl solution in dioxane (0.3 mL) and directly purified by RPLC (50 g, 5 to 60% acetonitrile and water). Yield 67.1 mg, 44%. Ion found by LCMS: [M+H].sup.+=630.0.
[1204] Step d.
##STR01108##
[1205] The title compound was prepared analogously to Example 101, step-b, using the diacid from step c and Int-7a (described in Example 8, step a). Yield 247.4 mg, 62.4%. Ions found by LCMS: [(M−2Boc+3H)/3].sup.+=1174, [(M−3Boc+3H)/3].sup.+=1140.7, [(M−4Boc+3H)/3].sup.+=1107.4, [(M−5Boc+3H)/3].sup.+=1074, [(M−6Boc+3H)/3].sup.+=1040.7.
[1206] Step e.
##STR01109##
[1207] The title compound Int-68 was prepared analogously to Example 101, step-c, using the step-d product from this example. Yield 197.5 mg, 76.9%. Ions found by LCMS: [(M+4H)/4].sup.+=680.6, [(M+4H)/5].sup.+=544.6, [(M+6H)/6].sup.+=454.2, [(M+7H)/7].sup.+=389.4, [(M+8H)/8].sup.+=340.8.
Example 104—Synthesis of Int-69
[1208] ##STR01110## ##STR01111##
[1209] Step a.
##STR01112##
[1210] To a suspension of (4S)-Boc-amino-L-Proline methyl ester HCl salt (1.222 g, 5 mmol) in DCM (6 mL) and DMF (2 mL) was added benzyl 2-bromoacetate (1.375 g, 6 mmol) and DIPEA (975 mg, 7.5 mmol). The mixture was stirred at room temperature for 3 days. It was then concentrated and purified by RPLC (100 g column, 15 to 75% acetonitrile and water, using 0.1% TFA as the modifier). Yield 1.71 g, 87.2%. Ion found by LCMS: [M+H].sup.+=393.2.
[1211] Step b.
##STR01113##
[1212] The step a product was dissolved in DCM (5 mL) and treated with 4N HCl solution in dioxane (5 mL). The mixture was heated at ˜ 40° C. for 30 minutes. It was then concentrated and re-dissolved in acetonitrile and water for lyophilization. Yield 1.6 g as di-HCl salt. Ion found by LCMS: [M+H].sup.+=293.2.
[1213] Step c.
##STR01114##
[1214] To a solution of propargyl-PEG4 acid (520.6 mg, 2 mmol) in anhydrous DMF (3 mL) was added HATU (760.4 mg, 2 mmol). After the solid dissolved, the step b product (804 mg, 2.2 mmol) was added, followed by DIPEA (910 mg, 7 mmol). The resulting mixture was stirred for 1 hour and then directly purified by RPLC (100 g, 15 to 67% acetonitrile and water). Yield 629 mg, 58.9%. Ion found by LCMS: [M+H].sup.+=535.2.
[1215] Step d.
##STR01115##
[1216] The step c product (629.1 mg, 1.178 mmol) was dissolved in MeOH (3 mL). This solution was cooled in an ice-water bath then treated with a solution of LiOH monohydrate (126 mg, 3 mmol) in water (3 mL). After the mixture was stirred for 3 hours, it was then acidified by 4N HCl solution in dioxane (1 mL). The organic solvents were removed by rotary evaporation, and the residue was purified by RPLC (100 g column, 0 to 50% acetonitrile and water). Yield 478 mg, 94.3%. Ion found by LCMS: [M+H].sup.+=431.2
[1217] Step e. Preparation of Int-75
##STR01116##
[1218] To a mixture of Int-7a (2 g, 1.28 mmol, Example 8, step a) and Z-L-norleucine (407.1 mg, 1.535 mmol) in anhydrous DMF (3 mL) was added HATU (583.6 g, 1.535 mmol) in portions over 10 minutes, followed by DIPEA (390 mg, 3 mmol). After the reaction mixture was stirred for 1 hour, it was poured into water (60 mL). The white solid product was collected by filtration and washed with water. The material was re-dissolved in MeOH (30 mL) and treated with Pd/C (5%) (1 g). The mixture was stirred under hydrogen overnight. Pd/C was then filtered, and the filtrate was concentrated and purified by RPLC (150 g, 40 to 95% MeOH and water). Yield of Int-75 was 2.02 g, 94.3%. Ion found by LCMS: [(M−Boc+2H)/2]+=789.2.
[1219] Step f.
##STR01117##
[1220] The title compound was prepared analogously to Example 101, step-b, using the step-e and step-d intermediates described in this example. Yield 244.4 mg, 65.3%. Ion found by LCMS: [(M−2Boc+3H)/3].sup.+=1183.0, [(M−3Boc+3H)/3].sup.+=1149.7, [(M−4Boc+3H)/3].sup.+=1116.4, [(M−5Boc+3H)/3].sup.+=1083.3, [(M−6Boc+3H)/3].sup.+=1049.2, [(M−7Boc+3H)/3].sup.+=1016.2.
[1221] Step g.
##STR01118##
[1222] The title compound Int-69 was prepared analogously to Example 101, step-c, using the step-f product from this example. Yield 177.8 mg, 67.9%. Ions found by LCMS: [(M+4H)/4].sup.+=687.5, [(M+5H)/5].sup.+=550.1, [(M+6H)/6].sup.+=458.6, [(M+7H)/7].sup.+=393.2.
Example 105—Synthesis of Int-70
[1223] ##STR01119## ##STR01120## ##STR01121##
[1224] Step a.
##STR01122##
[1225] To a solution of Z-L-norleucine (1.062 g, 4 mmol) in anhydrous NMP (5 mL) was added (S)-methyl 2-amino-4-((tert-butoxycarbonyl)amino)butanoate HCl salt (1.182 g, 4.4 mmol) and DIPEA (1.14 g, 8.8 mmol). The resulting mixture was treated with HATU (760.4 mg, 2.0 mmol) in portions over 10 minutes, and then stirred for 1 hour. It was then purified by RPLC (100 g column, 20 to 75% acetonitrile and water). Yield 1.9 g, 99.4%. Ion found by LCMS: [M−Boc+H].sup.+=380.2.
[1226] Step b.
##STR01123##
[1227] The step-a product (1.9 g, 3.975 mmol) was dissolved in MeOH (5 mL) and THF (10 mL). After the solution was cooled in an ice-water bath, it was treated with a solution of LiOH monohydrate (250 mg, 6 mmol) in water (10 mL). After the reaction was stirred for 30 minutes, DCM (100 mL) was added, and the reaction was continued stirring for 30 minutes. It was then acidified with 4N HCl solution in dioxane (1.5 mL), and diluted with water (30 mL). Two layers were separated, and the aqueous layer was back-extracted with EtOAc (80 mL×2). The combined organic layers were dried over Na.sub.2SO.sub.4, concentrated by rotary evaporation, and further dried under high vacuum. Yield 1.72 g, 93%. Ion found by LCMS: [M−Boc+H].sup.+=366.
[1228] Step c.
##STR01124##
[1229] To a solution of the step-b product (535.9 mg, 1.2 mmol) and PMB heptapeptide (1.06 g, 1 mmol, Example 2) in anhydrous DMF (2 mL) was added HATU (456.2 mg, 1.2 mmol) in portions over 10 minutes, followed by DIPEA (312 mg, 2.4 mmol). After the reaction was stirred for 1 hour, it was purified by RPLC (100 g, 40 to 95% MeOH and water). The collected fractions were concentrated by rotary evaporation to a white solid (Ion found by LCMS: [(M −2Boc+2H)/2].sup.+=655.6). The material was re-dissolved in MeOH (30 mL) and treated with Pd/C (5%). The mixture was stirred at room temperature overnight. Pd/C was filtered, and the filtrate was concentrated by rotary evaporation to dryness. Yield 1.177 g, 85% over two steps. Ion found by LCMS: [(M−Boc+2H)/2].sup.+=638.4.
[1230] Step d.
##STR01125##
[1231] To a solution of the step-b product (475.1 mg, 1.02 mmol) and the step-c product (1.177 g, 0.851 mmol) in anhydrous DMF (2 mL) was added HATU (388 mg, 1.02 mmol) in portions over 10 minutes, followed by DIPEA (260 mg, 2 mmol), After the reaction was stirred for 1 hour, it was purified by RPLC (100 g, 40 to 95% MeOH and water). The collected fractions were concentrated by rotary evaporation to a white solid (Ion found by LCMS: [(M−2Boc+2H)/2].sup.+=821.1). The material was re-dissolved in MeOH (30 mL) and treated with Pd/C (5%). The mixture was stirred at room temperature overnight. Pd/C was filtered, and the filtrate was concentrated by rotary evaporation to dryness. Yield 1.196 g, 83.3% over two steps. Ion found by LCMS: [(M−Boc+2H)/2].sup.+=795.
[1232] Step e.
##STR01126##
[1233] To a mixture of diacid (40.4 mg, 0.1 mmol, described in Example 67, step i) and the step-d product from this example (371.6 mg, 0.22 mmol) in anhydrous DMF (2 mL) was added HATU (76 mg, 0.2 mmol) and DIPEA (52 mg, 0.4 mmol). The resulting mixture was stirred for 30 minutes and was purified by RPLC (50 g, column 40 to 95% MeOH and water, using 0.1% TFA as modifier). The collected fractions were concentrated to a white solid (Ions found by LCMS: [(M−2Boc+3H)/3].sup.+=1182.0, [(M−4Boc+3H)/3].sup.+=1116.0, [(M−5Boc+3H)/3].sup.+=1083. The material was re-dissolved in TFA (0.5 mL) and DCM (˜1 mL). The solution was stirred for 30 minutes and directly purified through RPLC (50 g column, 3 to 50% acetonitrile and water, using 0.1% TFA as modifier). Yield of Int-70 was 102.4 mg, 25.6% over two steps. Ions found by LCMS: [(M+5H)/5].sup.+=549.6, [(M+6H)/6].sup.+=458.2, [(M+7H)/7].sup.+=393.0.
Example 106—Synthesis of Int-71
[1234] ##STR01127## ##STR01128##
[1235] Step a.
##STR01129##
[1236] The compound was prepared analogously to Example 101, step-b, using intermediates from Example 105, step-d, and Example 104, step-d. Yield 171 mg, 45.3%. Ions found by LCMS: [(M−2Boc+3H)/3].sup.+=1191.0, [(M−3Boc+3H)/3].sup.+=1157.6, [(M−5Boc+3H)/3].sup.+=1091.2.
[1237] Step b.
##STR01130##
[1238] The title compound Int-71 was prepared analogously to Example 101, step-d, using the step-a intermediate described in this example. Yield 147 mg, 81.6%. Ions found by LCMS: [(M+5H)/5].sup.+=554.9, [(M+6H)/6].sup.+=462.6, [(M+7H)/7].sup.+=396
Example 107—Syntheses of Int-72
[1239] ##STR01131## ##STR01132##
[1240] Step a.
##STR01133##
[1241] A solid mixture of norleucine methyl ester HCl salt (2.110 g, 11.62 mmol), racemic trans-4-oxocyclopentane-1,2-dicarboxylic acid (1.000 g, 5.81 mmol), EDCl (2.783 g, 14.52 mmol), HOAt (1.976 g, 14.52 mmol) and NaHCO.sub.3 (1.952 g, 23.23 mmol) were mixed in 20 mL of DCM/DMF (5:1). The mixture was stirred for overnight (˜15 hours) at room temperature. LC/MS analysis showed the completion of the reaction. Two diastereomers were separated by reversed phase HPLC. The polar diastereomer and less polar diastereomer have the retention time at 4.83 min. and 5.04 min. respectively with 8-minute 5-95% ACN/water method: (M+H).sup.+=427.0 and M−H.sup.+=425.0. The reaction mixture was diluted with EtOAc (200 mL) and washed with 1N aq. HCl, saturated NaHCO.sub.3 and brine respectively. The solution was dried with anhy. Sodium sulfate and the solvents were removed. The residue was dissolved in minimum amount of NMP and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30-60% acetonitrile and water. The pure polar diastereomer dimethyl ester, 1.071 g, was obtained in 46% yield (50% in theory). The absolute stereochemistry of the polar and less polar isomers was determined by X-ray. The polar isomer was used in the next step.
[1242] Step b.
##STR01134##
[1243] The polar dimethyl ester (1.071 g, 2.511 mmol) was treated with LiOH (0.1302 g, 5.273 mmol) in 20 mL of THF/Water (1:1). Stirred for less than 1 hrs. 1N HCl to acidify. Removed THF and extracted with EtOAc to give diacid (1.000 g, 100%). Positive mass ions was found as (M+H.sup.+): 399.0, tr=1.3 minutes with 5-minute 40-95% ACN/water method.
[1244] A solid mixture of diacid (1.000 g, 2.510 mmol), Dab([ ]-Boc) methyl ester HCl salt (1.349 g, 5.020 mmol), EDCl (1.203 g, 6.274 mmol), HOAt (0.8540 g, 6.274 mmol) and NaHCO.sub.3 (0.8424 g, 10.04 mmol) were mixed in 20 mL of DCM/DMF (5:1). The mixture was stirred for overnight (˜15 hours) at room temperature. LC/MS analysis showed the completion of the reaction. The reaction mixture was diluted with EtOAc (200 mL) and washed with 1N aq. HCl, saturated NaHCO.sub.3 and brine respectively. The solution was dried with anhy. Sodium sulfate and the solvents were removed. The residue was dissolved in minimum amount of NMP and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30-60% acetonitrile and water. The cyclopentanonyl central linker, 1.760 g, was obtained in 85% yield. Ions found by LCMS: (M−2Boc+2H.sup.+)/2=314.0.
[1245] Step c.
##STR01135##
[1246] A mixture of cyclopentanonyl central linker (0.8360 g, 1.011 mmol), aminooxy-PEG4-propargyl (0.500 g, 2.022 mmol), and potassium acetate (0.2977 g, 3.033 mmol) were dissolved in 20 mL of methanol/water (5:1). The mixture was stirred for overnight (˜15 hours) at 60° C. HPLC analysis showed the completion of the reaction. Most of the solvents of the reaction mixture was removed by reduced pressure rotovap to give a viscous oily residue. The residue was dissolved in minimum amount of NMP and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30-60% acetonitrile and water. The pure PEG4-oxime central linker dimethyl ester, 1.0457 g, was obtained in 98% yield. The dimethyl ester has the retention time at 5.47 min. with 8-minute 5-95% ACN/water method: (M−2Boc+2H.sup.+)/2=428.6.
[1247] The dimethyl ester (1.0457 g, 0.9900 mmol) was treated with LiOH (0.0.0513 g, 2.079 mmol) in 10 mL of THF/Water (1:1). Stirred for less than 1 hrs. 1N HCl to acidify. Removed THF and extracted with EtOAc to give PEG4-oxime central linker (1.011 g, 99%). Positive mass ions was found as (M −2Boc+2H.sup.+)/2=414.6, tr=4.50 minutes with 8-minute 5-95% ACN/water method.
[1248] Step d.
##STR01136##
[1249] A solid mixture of Int-7a (0.6631 g, 0.4863 mmol, Example 8, step a), PEG4-oxime central linker (0.250 g, 0.2432 mmol), EDCl (0.1165 g, 0.6079 mmol) and HOAt (0.0827 g, 0.608 mmol) were dissolved in 5 mL of dry DMF immediately followed by adding triethylamine (0.17 mL, 0.97 mmol). The mixture was stirred for overnight (˜15 hours) at room temperature. HPLC analysis showed the completion of the reaction. Most of the solvent (DMF) of the reaction mixture was removed by reduced pressure rotovap to give a viscous oily residue. The residue was triturated with methanol. After some crystalline solid started to precipitate the flask was put standstill for more than 3 hours. Simple filtration collected the crystalline white solid with methanol wash (3×5 mL). The solid was dried to give 0.675 grams of pure product, 75% isolated yield. The solid (0.440 g) was dissolved with 100% TFA and stirred for 30 minutes. TFA was removed by rotovap. The residue was dissolved in minimum amount of water and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 35% acetonitrile and water using 0.1% TFA modifier. The pure fractions were lyophilized to afford the product Int-72 as a white powder (TFA salt, 0.360 g, 65% isolated yield, Positive mass ions were found by LC/MS: (M+6H.sup.+)/6=454.0, (M+5H.sup.+)/5=544.6, (M+4H.sup.+)/4=680.4.
Example 108—Syntheses of Int-73
[1250] ##STR01137##
[1251] Step a.
##STR01138##
[1252] A mixture of the polar dimethyl ester (from Synthesis of, Step 1, 0.9959 g, 2.335 mmol), aminooxy-PEG4-propargyl (0.6745 g, 2.916 mmol) and acetic acid (0.26 mL, 4.5 mmol) were dissolved in 20 mL of DCM. The mixture was stirred for 1 hour at room temperature followed by addition of sodium triacetatoxyborohydride (0.9509 g, 4.486 mmol). The reaction mixture was stirred for overnight (˜15 hours) at room temperature. HPLC analysis showed the completion of the reaction. Positive mass ion was found by LC/MS: (M+H).sup.+=642.4. Most of the solvents of the reaction mixture was removed by reduced pressure rotovap to give a viscous oily residue. The residue was dissolved in 20 mL of THF/10% NaHCO.sub.3 (1:1) and treated with Boc anhydride (0.71 mL, 3.1 mmol) and diisopropylethylamine (0.54 mL, 3.1 mmol). The mixture was stirred for overnight (˜15 hours) at room temperature. Most of THF was removed and the residue was extracted with EtOAc. After removal of EtOAc, the residue was dissolved in minimum amount of NMP and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 30-60% acetonitrile and water. The pure PEG4-amino cyclopentanyl dimethyl ester, 0.7405 g, was obtained in 65% yield. The dimethyl ester has the retention time at 2.88 min. with 5-minute 40-95% ACN/water method: (M+H).sup.+=742.4 and M−Boc+H.sup.+=643.0.
[1253] The dimethyl ester (0.7405 g, 0.9980 mmol) was treated with LiOH (0.0.0616 g, 2.495 mmol) in 10 mL of THF/Water (1:1). Stirred for less than 1 hrs then acidified with 1N HCl. The solution was concentrated and extracted with EtOAc to give Boc-PEG4-central linker (0.705 g, 99%). Positive mass ions was found as (M+H).sup.+=714.4, tr=3.30 minutes with 5-minute 5-95% ACN/water method.
[1254] Step b.
##STR01139##
[1255] A solid mixture of Int-5d (0.6544 g, 0.4511 mmol), Boc-PEG4 central linker (0.1610 g, 0.2255 mmol), EDCl (0.1081 g, 0.5636 mmol) and HOAt (0.07675 g, 0.5638 mmol) were dissolved in 5 mL of dry DMF immediately followed by adding triethylamine (0.16 mL, 0.90 mmol). The mixture was stirred for overnight (˜15 hours) at room temperature. HPLC analysis showed the completion of the reaction. Most of the solvent (DMF) of the reaction mixture was removed by reduced pressure rotovap to give a viscous oily residue. To this residue methanol was added with gentle stirring to triturate the reaction product. After some crystalline solid started to precipitate the flask was put standstill for more than 3 hours. Simple filtration collected the crystalline white solid with methanol wash (3×5 mL). The solid was dried to give 0.607 grams of pure product, 75% isolated yield. Positive ions found: (M−3Boc+3H.sup.+)/3=1093.8. The solid (0.440 g) was dissolved with 100% TFA and stirred for 30 minutes. TFA was removed by rotovap. The residue was dissolved in minimum amount of water and purified by RPLC using an Isco CombiFlash liquid chromatograph eluted with 5% to 35% acetonitrile and water using 0.1% TFA modifier. The pure fractions were lyophilized to afford the desired product Int-73 as a white powder (TFA salt, 0.670 g, 87% isolated yield). Positive ions found by LC/MS: (M+7H.sup.+)/7=383.4, (M+6H.sup.+)/6=447.2, (M+5H.sup.+)/5=536.2, (M+4H.sup.+)/4=670.1.
Example 109—Synthesis of Conjugate 27
[1256] A solution of azido-hlgG1(Myc)Fc (50 mg, 0.85 μmol, 15.9 mg/mL, MW=58,743 Da, DAR=3.3) in pH 7.4 PBS was treated with freshly prepared PBS 7.4 solutions of alkyne derivatized small molecule (37.0 mg, 25.5 μmol, Example 38), THPTA (5.5 mg, 12.8 μmol, tris(3-hydroxypropyltriazolylmethyl)amine), sodium ascorbate (5.0 mg, 25.5 μmol), and CuSO.sub.4 (2.0 mg, 12.8 μmol). The resulting solution was agitated on a rocker table overnight at room temperature, then purified by affinity chromatography over a protein-A column (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 63,234 Da (DAR=3.1). Yield 33 mg, 61% yield.
Example 110—Synthesis of Conjugate 38
[1257] A solution of azido-hlgG1(Myc)Fc (30 mg, 0.50 μmol, 15.9 mg/mL, MW=58,743 Da, DAR=4.0) in pH 7.4 PBS was treated with freshly prepared PBS 7.4 solutions of alkyne derivatized small molecule (52.0 mg, 12.6 μmol, See Example 59, Synthesis of int-24), THPTA (3.3 mg, 7.5 μmol, tris(3-hydroxypropyltriazolylmethyl)amine), sodium ascorbate (3.0 mg, 15.1 μmol), and CuSO.sub.4 (1.2 mg, 7.5 μmol). The resulting solution was agitated on a rocker table overnight at room temperature, then purified by affinity chromatography over a protein-A column (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 67,320 Da (DAR=3.8 calculated from the tallest peak). Yield 17.8 mg, 59% yield.
Example 111—Synthesis of Conjugate 39
[1258] A solution of azido-hlgG1(Myc)Fc (30 mg, 0.50 μmol, 15.9 mg/mL, MW=59,641 Da, DAR=4.0) in pH 7.4 PBS was treated with freshly prepared PBS 7.4 solutions of alkyne derivatized small molecule (43.4 mg, 12.6 μmol, See Example 60), THPTA (3.3 mg, 7.5 μmol, tris(3-hydroxypropyltriazolylmethyl)amine), sodium ascorbate (3.0 mg, 15.1 μmol), and CuSO.sub.4 (1.2 mg, 7.5 μmol). The resulting solution was agitated on a rocker table overnight at room temperature, then purified by protein-A affinity chromatography, followed by size exclusion chromatography (See Example 166 for purification procedure). Maldi TOF analysis of the purified final product gave an average mass of 68,535 Da (DAR=3.8 calculated from a weighted average of the Maldi peaks). Yield 16.7 mg, 48% yield.
Example 112—Synthesis of Conjugate 40
[1259] A solution of azido-hlgG1(Myc)Fc (30 mg, 0.50 μmol, 15.9 mg/mL, MW=59,641 Da, DAR=4.0) in pH 7.4 PBS was treated with freshly prepared PBS 7.4 solutions of alkyne derivatized small molecule (46.3 mg, 12.6 μmol, See Example 61), THPTA (3.3 mg, 7.5 μmol, tris(3-hydroxypropyltriazolylmethyl)amine), sodium ascorbate (3.0 mg, 15.1 μmol), and CuSO.sub.4 (1.2 mg, 7.5 μmol). CuSO.sub.4 was added last and caused formation of a cloudy white mixture. The resulting solution was agitated on a rocker table overnight at room temperature, then purified by protein-A affinity chromatography, followed by size exclusion chromatography (See Example 166 for purification procedure). Maldi TOF analysis of the purified final product gave an average mass of 69,074 Da (DAR=3.7 calculated from a weighted average of the Maldi peaks). Yield 12.3 mg, 41% yield.
Example 113—Synthesis of Conjugate 74
[1260] A solution of hlgG1(Myc)Fc (30 mg, 0.51 μmol, 15.9 mg/mL, MW=58,270 Da) in pH 7.4 PBS was treated with TCEP (18.5 μL, 0.5 M, 9.3 μmol) at room temperature. LCMS after 1 h showed complete reduction of the disulfides. This solution was buffer exchanged with PBS, pH=7.8, EDTA 2 mM (3×15 mL) using a 10,000 mw cutoff filter (final volume 4 mL). To this was added a freshly prepared solution of azido-PEG-4-bissulfone linker (1.44 mg, 20.6 μmol, in 0.2 mL DMF) resulting in a cloudy solution.
Azido-PEG-4-bissulfone Linker:
##STR01140##
[1261] Addition of 1 mL methanol removed the cloudiness. The resulting reaction was agitated overnight on a rocker table at room temperature. The azido Fc was purified by protein-A affinity chromatography (See Example 166). LCMS showed clean incorporation of two bissulfone linker units (mass=59,050, azide DAR=2.0).
[1262] A solution of cysteine-azido-hlgG1(Myc)Fc (28.6 mg, 0.48 μmol, 11.7 mg/mL, DAR=2.0) in pH 7.4 PBS was treated with freshly prepared PBS 7.4 solutions of alkyne derivatized small molecule (22.4 mg, 5.8 μmol, See Example 41, INT-21), THPTA (2.1 mg, 4.8 μmol, tris(3-hydroxypropyltriazolylmethyl)amine), sodium ascorbate (2.9 mg, 14.5 μmol), and CuSO.sub.4 (0.77 mg, 4.8 μmol). CuSO.sub.4 was added last and caused formation of a slightly cloudy solution. The resulting solution was agitated on a rocker table overnight at room temperature, then purified by protein-A affinity chromatography, followed by size exclusion chromatography (See Example 166 for purification procedure). Maldi TOF analysis of the purified final product gave an average mass of 64,485 Da (DAR=3.7 calculated from a weighted average of the Maldi peaks). Yield 16 mg, 52% yield.
Example 114—Synthesis of Conjugate 75
[1263] A solution of azido-hlgG1(Myc)Fc (30 mg, 0.50 μmol, 15.9 mg/mL, MW=59,641 Da, DAR=2.8) in pH 7.4 PBS was treated with freshly prepared PBS 7.4 solutions of alkyne derivatized small molecule (15.8 mg, 5.6 μmol, See Example 62), THPTA (3.3 mg, 7.5 μmol, tris(3-hydroxypropyltriazolylmethyl)amine), sodium ascorbate (3.0 mg, 15.1 μmol), and CuSO.sub.4 (1.2 mg, 7.5 μmol). CuSO.sub.4 was added last and caused formation of a cloudy white mixture. The resulting solution was agitated on a rocker table overnight at room temperature, then purified by protein-A affinity chromatography, followed by size exclusion chromatography (See Example 166 for purification procedure). Maldi TOF analysis of the purified final product gave an average mass of 69,074 Da DAR=3.7 calculated from a weighted average of the Maldi peaks). Yield 12.3 mg, 41% yield.
Example 115—Synthesis of Conjugate 28
[1264] To a room temperature solution of alkyne functionalized small molecule (Example 64, 0.050 mg, 13.5 μmol) in pH 7.4 PBS (500 μL), was added in order: a 10 mM solution of THPTA in pH 7.4 PBS (700 μL, 7.0 μmol), a 20 mM solution of sodium ascorbate in pH 7.4 PBS (700 μL, 14.0 μmol), and last a 10 mM solution of copper sulfate in water (700 μL, 7.0 μmol). Upon mixing, a solution of azido-hlgG1(Myc)Fc (29.4 mg, 0.500 μmol, 10 mg/mL, MW=58,837.00 Da, DAR=2.7) in pH 7.4 PBS was gently dispersed. The resulting solution was agitated on a rocker table overnight at room temperature, then purified by affinity chromatography over a protein-A column (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 63,812 Da (DAR=2.4). Yield 10.35 mg, 52% yield.
Example 116—Synthesis of Conjugate 29
[1265] To a room temperature solution of material described in Example 63 (0.047 mg, 13.5 μmol) in pH 7.4 PBS (500 μL), was added in order: a 10 mM solution of THPTA in pH 7.4 PBS (700 μL, 7.0 μmol), a 20 mM solution of sodium ascorbate in pH 7.4 PBS (700 μL, 14.0 μmol), and last a 10 mM solution of copper sulfate in water (700 μL, 7.0 μmol). Upon mixing, a solution of azido-hlgG1(Myc)Fc (29.4 mg, 0.500 μmol, 10 mg/mL, MW=58,837.00 Da, DAR=2.7) in pH 7.4 PBS was gently dispersed. The resulting solution was agitated on a rocker table overnight at room temperature, then purified by affinity chromatography over a protein-A column (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 60,951 Da (DAR=1.6). Yield 15.75 mg, 78% yield.
Example 117—Synthesis of Conjugate 55
[1266] To a room temperature solution of alkyne functionalized small molecule (Example 65, 0.054 mg, 13.6 μmol) in pH 7.4 PBS (500 μL), was added in order: a 20 mM solution of THPTA in pH 7.4 PBS (340 μL, 6.8 μmol), a 20 mM solution of sodium ascorbate in pH 7.4 PBS (2,720 μL, 54.4 μmol), and last a 20 mM solution of copper sulfate in water (340 μL, 6.8 μmol). Upon mixing, the solution of azido-hlgG1(Myc)Fc (29.4 mg, 0.340 μmol, 10 mg/mL, MW=58,821.00 Da, DAR=2) in pH 7.4 PBS was gently dispersed. The resulting solution was agitated on a rocker table overnight at room temperature, then purified by affinity chromatography over a protein-A column (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 61,939 Da (DAR=1.1). Yield 5.92 mg, 30% yield.
Example 118—Synthesis of Conjugate 76
[1267] To a room temperature solution of alkyne functionalized small molecule (Example 66, 0.056 mg, 10.2 μmol) in pH 7.4 PBS (500 μL), it was added in the order: a 20 mM solution of THPTA in pH 7.4 PBS (340 μL, 6.8 μmol), a 20 mM solution of sodium ascorbate in pH 7.4 PBS (680 μL, 13.6 μmol), and last a 20 mM solution of copper sulfate in water (340 μL, 6.8 μmol). Upon mixing, a solution of azido-hlgG1(Myc)Fc (0.020 mg, 0.340 μmol, 10 mg/mL, MW=58,833.00 Da, DAR=3) in pH 7.4 PBS was gently dispensed. The resulting solution was agitated on a rocker table overnight at room temperature, then purified by affinity chromatography over a protein-A column (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 63,911 Da (DAR=1.7). Yield 10.81 mg, 54% yield.
Example 119—Synthesis of Conjugate 69
[1268] A solution of azido-hlgG1(Myc)Fc in PBS buffer (20 mg, 0.34 μmol, 1.40 mL, MW=58,721 Da, DAR=2.7) was treated with alkyne derivatized small molecule (TFA salt, 36 mg, 9.2 μmol, Example 67) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.7 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.02 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 64,845 Da (DAR=2.3). Yield 9.36 mg, 67% yield.
Example 120—Synthesis of Conjugate 34
[1269] A solution of azido-hlgG1(Myc)Fc in PBS buffer (30 mg, 0.50 μmol, 2.0 mL, MW=58,548 Da, DAR=2.7) was treated with alkyne derivatized small molecule (TFA salt, 54 mg, 13.5 μmol, Example 68) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (1.0 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 1.0 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.4 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 64,717 Da (DAR=2.26). Yield 12.7 mg, 42% yield.
Example 121—Synthesis of Conjugate 43
[1270] A solution of azido-hlgG1(Myc)Fc in PBS buffer (30 mg, 0.50 umol, 2.0 mL, MW=58,935 Da, DAR=5) was treated with alkyne derivatized small molecule (TFA salt, 60 mg, 15 μmol, Example 69) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (1.0 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 1.0 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.5 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 70,947 Da (DAR=4.4). Yield 27 mg, 90% yield
Example 122—Synthesis of Conjugate 78
[1271] A solution of azido-hlgG1(Myc)Fc in PBS buffer (20 mg, 0.34 umol, 1.78 mL, MW=58,833 Da, DAR=3) was treated with alkyne derivatized small molecule (TFA salt, 41 mg, 15 μmol, Example 70) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.68 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.68 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.0 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 69,330 Da (DAR=3.3). Yield 10 mg, 50% yield.
Example 123—Synthesis of Conjugate 31
[1272] A solution of azido-hlgG1(Myc)Fc in PBS buffer (30 mg, 0.50 μmol, 2.0 mL, MW=58,743 Da, DAR=3.3) was treated with alkyne derivatized small molecule (TFA salt, 54 mg, 13.5 μmol, Example 71) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (1.0 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 1.0 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.4 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 64462 Da (DAR=2.17). Yield 26.1 mg, 65% yield.
Example 124—Synthesis of Conjugate 33
[1273] A solution of azido-hlgG1(Myc)Fc in PBS buffer (30 mg, 0.50 μmol, 2.0 mL, MW=58,548 Da, DAR=2.7) was treated with alkyne derivatized small molecule (TFA salt, 54 mg, 13.5 μmol, Example 72) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (1.0 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 1.0 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.4 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 64,223 Da (DAR=2.05). Yield 14.5 mg, 48% yield.
Example 125—Synthesis of Conjugate 44
[1274] A solution of azido-hlgG1(Myc)Fc in PBS buffer (30 mg, 0.50 μmol, 2.0 mL, MW=58,935 Da, DAR=3.88) was treated with alkyne derivatized small molecule (TFA salt, 58 mg, 15 μmol, Example 73) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (1.0 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 1.0 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.5 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 69,725 Da (DAR=4). Yield 27 mg, 91% yield.
Example 126—Synthesis of Conjugate 66
[1275] A solution of azido-hlgG1(Myc)Fc in PBS buffer (20 mg, 0.34 μmol, 2.0 mL, MW=58,721 Da, DAR=2.75) was treated with alkyne derivatized small molecule (TFA salt, 35 mg, 9.2 μmol, Example 74) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.7 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.0 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 64,614 Da (DAR=2.3). Yield 15.3 mg, 77% yield.
Example 127—Synthesis of Conjugate 67
[1276] A solution of azido-hlgG1(Myc)Fc in PBS buffer (20 mg, 0.34 μmol, 2.0 mL, MW=58,721 Da, DAR=2.75) was treated with alkyne derivatized small molecule (TFA salt, 34 mg, 9.2 μmol, Example 75) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.7 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.0 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 64,792 Da (DAR=2.4). Yield 14.4 mg, 72% yield.
Example 128—Synthesis of Conjugate 68
[1277] A solution of azido-hlgG1(Myc)Fc in PBS buffer (20 mg, 0.34 μmol, 1.40 mL, MW=58,721 Da, DAR=2.7) was treated with alkyne derivatized small molecule (TFA salt, 36 mg, 9.2 μmol, Example 76) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.7 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.02 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 65,317 Da (DAR=2.4). Yield 13.4 mg, 67% yield.
Example 129—Synthesis of Conjugate 56
[1278] A solution of azido-hlgG1(Myc)Fc in PBS buffer (20 mg, 0.34 μmol, 1.40 mL, MW=58526 Da, DAR=2.59) was treated with alkyne derivatized small molecule (TFA salt, 36 mg, 9.2 μmol, Example 77) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.7 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.02 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 63,622 Da (DAR=2.21). Yield 19.2 mg, 96% yield.
Example 130—Synthesis of Conjugate 79
[1279] A solution of azido-hlgG1(Myc)Fc in PBS buffer (20 mg, 0.34 μmol, 1.40 mL, MW=58833 Da, DAR=3.16) was treated with alkyne derivatized small molecule (TFA salt, 43 mg, 10.2 μmol, Example 78) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.7 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.02 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 65,326 Da (DAR=2.1). Yield 11.8 mg, 59% yield.
Example 131—Synthesis of Conjugate 32
[1280] A solution of azido-hlgG1(Myc)Fc in PBS buffer (40 mg, 0.67 μmol, 2.50 mL, MW=58743 Da, DAR=3.3) was treated with alkyne derivatized small molecule (TFA salt, 46 mg, 9.2 μmol, Example 79) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (1.4 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 1.4 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.4 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 62,465 Da (DAR=2.65). Yield 13.8 mg, 35% yield.
Example 132—Synthesis of Conjugate 45
[1281] A solution of azido-hlgG1(Myc)Fc in PBS buffer (22.5 mg, 0.38 μmol, 1.50 mL, MW=58,835 Da, DAR=5) was treated with alkyne derivatized small molecule (TFA salt, 23 mg, 11 μmol, Example 80) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.75 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.75 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.13 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 65,241 Da (DAR=4.18). Yield 15 mg, 75% yield.
Example 133—Synthesis of Conjugate 46
[1282] A solution of azido-hlgG1(Myc)Fc in PBS buffer (20 mg, 0.32 μmol, 2.50 mL, MW=59641 Da, DAR=3.9) was treated with alkyne derivatized small molecule (TFA salt, 19 mg, 10 μmol, Example 81) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.65 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.65 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.0 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for 12 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 64,789 Da (DAR=4.26). Yield 17.88 mg, 89% yield.
Example 134—Synthesis of Conjugate 42
[1283] A solution was freshly prepared with CuSO.sub.4 (10 mM, 16.07 mg), sodium ascorbate (20 mM, 39.7 mg) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM, 43.86 mg) in PBS 7.4 buffer solution of (10 mL).
[1284] A solution of azido-hlgG1(Myc)Fc (40 mg, 0.679 μmol, 15.0 mg/mL, MW=58,935 Da, DAR=5.0) in pH 7.4 PBS was treated with freshly prepared PBS 7.4 solutions (1.68 mL) of alkyne derivatized small molecule TFA salt (97.0 mg, 27.1 μmol Example 84). To this solution was added a freshly prepared mixed solution of CuSO.sub.4 [(10 mM), sodium ascorbate (20 mM) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM), 1.4 mL]. The resulting solution was gently agitated on a rocker table at room temperature for 3 hours then another 0.5 mL of the mixed solution [CuSO.sub.4 (10 mM), sodium ascorbate (20 mM) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM)]. The resulting solution was continued to agitate overnight then purified by affinity chromatography over a protein-A column. See Example 166 for purification procedure. Maldi TOF analysis of the purified final product gave an average mass of 69,638 Da (DAR=4.0). Yield 37.53 mg, 79% yield.
Example 135—Synthesis of Conjugate 53
[1285] A solution was freshly prepared with CuSO.sub.4 (10 mM, 15.96 mg), sodium ascorbate (20 mM, 39.6 mg) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM, 43.86 mg) in PBS 7.4 buffer solution of (10 mL).
[1286] A solution of azido-hlgG1(Myc)Fc (20 mg, 1.43 mL, 0.342 μmol, 14.0 mg/mL, MW=58,526 Da, DAR=2.6) in pH 7.4 PBS was diluted with PBS 7.4 solutions (0.57 mL) to make a 10.0 mg/mL solution of (2 mL).
[1287] A solution of alkyne derivatized small molecule TFA salt (27.5 mg, 7.08 μmol, Example 88) in PBS 7.4 solutions (1.5 mL) was prepared in a 15 mL centrifuge tube. To this solution was added A solution of (0.68 mL, 10 mM of CuSO.sub.4, 20 mM of sodium ascorbate and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine)) followed by addition of a solution of azido-hlgG1(Myc)Fc (20 mg, 2.0 mL, 0.342 μmol, 10.0 mg/mL, MW=58,526 Da, DAR=2.59) in pH 7.4 PBS. The resulting solution was gently agitated on a rocker table at room temperature overnight then purified by affinity chromatography over a protein-A column. See Example 166 for purification procedure. Maldi TOF analysis of the purified final product gave an average mass of 65,320 Da (DAR=2.5). Yield 19.15 mg, 87% yield.
Example 136—Synthesis of Conjugate 54
[1288] A solution was freshly prepared with CuSO.sub.4 (10 mM, 15.96 mg), sodium ascorbate (20 mM, 39.6 mg) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM, 43.86 mg) in PBS 7.4 buffer solution of (10 mL).
[1289] A solution of azido-hlgG1(Myc)Fc (20 mg, 1.43 mL, 0.342 μmol, 14.0 mg/mL, MW=58,526 Da, DAR=2.59) in pH 7.4 PBS was diluted with PBS 7.4 solutions (0.57 mL) to make a 10.0 mg/mL solution of (2 mL).
[1290] A solution of alkyne derivatized small molecule TFA salt (25.7 mg, 7.08 μmol, Example 87) in PBS 7.4 solutions (1.5 mL) was prepared in a 15 mL centrifuge tube. To this solution was added A solution of (0.68 mL, 10 mM of CuSO.sub.4, 20 mM of sodium ascorbate and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine)) followed by addition of a solution of azido-hlgG1(Myc)Fc (20 mg, 2.0 mL, 0.342 μmol, 10.0 mg/mL, MW=58,526 Da, DAR=2.59) in pH 7.4 PBS. The resulting solution was gently agitated on a rocker table at room temperature overnight then purified by affinity chromatography over a protein-A column. See Example 166 for purification procedure. Maldi TOF analysis of the purified final product gave an average mass of 64,708 Da (DAR=2.3). Yield 19.32 mg, 88% yield.
Example 137—Synthesis of Conjugate 62
[1291] A solution was freshly prepared with CuSO.sub.4 (10 mM, 15.96 mg), sodium ascorbate (20 mM, 39.6 mg) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM, 43.86 mg) in PBS 7.4 buffer solution of (10 mL).
[1292] A solution of azido-hlgG1(Myc)Fc (20 mg, 1.43 mL, 0.342 μmol, 14.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS was diluted with PBS 7.4 solutions (0.57 mL) to make a 10.0 mg/mL solution of (2 mL).
[1293] A solution of alkyne derivatized small molecule TFA salt (36.1 mg, 9.36 μmol, Example 89) in PBS 7.4 solutions (1.5 mL) was prepared in a 15 mL centrifuge tube. To this solution was added A solution of (0.68 mL, 10 mM of CuSO.sub.4, 20 mM of sodium ascorbate and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine)) followed by addition of a solution of azido-hlgG1(Myc)Fc (20 mg, 2.0 mL, 0.342 μmol, 10.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS. The resulting solution was gently agitated on a rocker table at room temperature overnight then purified by affinity chromatography over a Superdex200 column. See Example 166 for purification procedure. Maldi TOF analysis of the purified final product gave an average mass of 66,835 Da (DAR=3.0). Yield 9.86 mg, 41% yield.
Example 138—Synthesis of Conjugate 63
[1294] A solution was freshly prepared with CuSO.sub.4 (10 mM, 15.96 mg), sodium ascorbate (20 mM, 39.6 mg) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM, 43.86 mg) in PBS 7.4 buffer solution of (10 mL).
[1295] A solution of azido-hlgG1(Myc)Fc (20 mg, 1.43 mL, 0.342 μmol, 14.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS was diluted with PBS 7.4 solutions (0.57 mL) to make a 10.0 mg/mL solution of (2 mL).
[1296] A solution of alkyne derivatized small molecule TFA salt (32.8 mg, 9.2 μmol, Example 86) in PBS 7.4 solutions (1.5 mL) was prepared in a 15 mL centrifuge tube. To this solution was added A solution of (0.68 mL, 10 mM of CuSO.sub.4, 20 mM of sodium ascorbate and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine)) followed by addition of a solution of azido-hlgG1(Myc)Fc (20 mg, 2.0 mL, 0.342 μmol, 10.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS. The resulting solution was gently agitated on a rocker table at room temperature overnight then purified by protein-A affinity chromatography. See Example 166 for purification procedure. Maldi TOF analysis of the purified final product gave an average mass of 66,609 Da (DAR=2.9). Yield 9.55 mg, 41% yield.
Example 139—Synthesis of Conjugate 64
[1297] A solution was freshly prepared with CuSO.sub.4 (10 mM, 15.96 mg), sodium ascorbate (20 mM, 39.6 mg) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM, 43.86 mg) in PBS 7.4 buffer solution of (10 mL).
[1298] A solution of azido-hlgG1(Myc)Fc (20 mg, 1.43 mL, 0.342 μmol, 14.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS was diluted with PBS 7.4 solutions (0.57 mL) to make a 10.0 mg/mL solution of (2 mL).
[1299] A solution of alkyne derivatized small molecule TFA salt (35.9 mg, 9.37 μmol, Example 85) in PBS 7.4 solutions (1.5 mL) was prepared in a 15 mL centrifuge tube. To this solution was added A solution of (0.68 mL, 10 mM of CuSO.sub.4, 20 mM of sodium ascorbate and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine)) followed by addition of a solution of azido-hlgG1(Myc)Fc (20 mg, 2.0 mL, 0.342 μmol, 10.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS. The resulting solution was gently agitated on a rocker table at room temperature overnight then purified by affinity chromatography over a Superdex200 column. See Example 166 for purification procedure. Maldi TOF analysis of the purified final product gave an average mass of 66,863 Da (DAR=3.0). Yield 8.62 mg, 36% yield.
Example 140—Synthesis of Conjugate 65
[1300] A solution was freshly prepared with CuSO.sub.4 (10 mM, 15.96 mg), sodium ascorbate (20 mM, 39.6 mg) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM, 43.86 mg) in PBS 7.4 buffer solution of (10 mL).
[1301] A solution of azido-hlgG1(Myc)Fc (20 mg, 1.43 mL, 0.342 μmol, 14.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS was diluted with PBS 7.4 solutions (0.57 mL) to make a 10.0 mg/mL solution of (2 mL).
[1302] A solution of alkyne derivatized small molecule TFA salt (36.4 mg, 9.37 μmol, Example 82) in PBS 7.4 solutions (1.5 mL) was prepared in a 15 mL centrifuge tube. To this solution was added A solution of (0.68 mL, 10 mM of CuSO.sub.4, 20 mM of sodium ascorbate and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine)) followed by addition of a solution of azido-hlgG1(Myc)Fc (20 mg, 2.0 mL, 0.342 μmol, 10.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS. The resulting solution was gently agitated on a rocker table at room temperature overnight then purified by protein-A affinity chromatography. See Example 166 for purification procedure. Maldi TOF analysis of the purified final product gave an average mass of 65,998 Da (DAR=2.7). Yield 10.48 mg, 46% yield.
Example 141—Synthesis of Conjugate 73
[1303] A solution was freshly prepared with CuSO.sub.4 (10 mM, 8.2 mg), sodium ascorbate (20 mM, 22.27 mg) and tris(3-hydroxypropyltriazolylmethyl)amine) (10 mM, 22.0 mg) in PBS 7.4 buffer solution of (5 mL).
[1304] A solution of azido-hlgG1(Myc)Fc (20 mg, 1.43 mL, 0.342 μmol, 14.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS was diluted with PBS 7.4 solutions (0.57 mL) to make a 10.0 mg/mL solution of (2 mL).
[1305] A solution of alkyne derivatized small molecule TFA salt (36.96 mg, 9.37 μmol, Example 83) in PBS 7.4 solutions (1.5 mL) was prepared in a 15 mL centrifuge tube. To this solution was added A solution of (0.68 mL, 10 mM of CuSO.sub.4, 20 mM of sodium ascorbate and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine)) followed by addition of a solution of azido-hlgG1(Myc)Fc (20 mg, 2.0 mL, 0.342 μmol, 10.0 mg/mL, MW=58,721 Da, DAR=2.75) in pH 7.4 PBS. The resulting solution was gently agitated on a rocker table at room temperature overnight then purified by affinity chromatography over a protein-A column. See Example 166 for purification procedure. Maldi TOF analysis of the purified final product gave an average mass of 68,785 Da (DAR=3.5). Yield 16.64 mg, 58% yield.
Example 142—Synthesis of Conjugate 35
[1306] Alkyne derivatized small molecule TFA salt (53 mg, 0.13 mmol, See Example 95) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (30 mg, 51 μmol, MW=58548, DAR 2.7) in 4 mL of PBS 7.4 buffer followed by (1.02 mL) of a premixed solution which was 20 mM sodium ascorbate (0.02 mmol), 10 mM Copper (II) sulfate (0.01 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.01 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 19.9 mg, 60%. MALDI MS analysis showed a range of masses (58264-72608) with an average of mass of 64013. Average DAR=2.4.
Example 143—Synthesis of Conjugate 36
[1307] Alkyne derivatized small molecule TFA salt (49 mg, 0.13 mmol, See Example 93) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (30 mg, 51 μmol, MW=58548, DAR 2.7) in 4 mL of PBS 7.4 buffer followed by (1.02 mL) of a premixed solution which was 20 mM sodium ascorbate (0.02 mmol), 10 mM Copper (II) sulfate (0.01 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.01 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 18.9 mg, 61%. MALDI MS analysis showed a range of masses (58270-72456) with an average of mass of 63816. Average DAR=2.3.
Example 144—Synthesis of Conjugate 37
[1308] Alkyne derivatized small molecule TFA salt (49 mg, 0.13 mmol, See Example 94) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (30 mg, 51 μmol, MW=58548, DAR 2.7) in 4 mL PBS 7.4 buffer followed by (1.02 mL) of a premixed solution which was 20 mM sodium ascorbate (0.02 mmol), 10 mM Copper (II) sulfate (0.01 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.01 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 23.4 mg, 71%. MALDI MS analysis showed a range of masses (58273-72500) with an average of mass of 63921. Average DAR=2.1.
Example 145—Synthesis of Conjugate 49
[1309] Alkyne derivatized small molecule TFA salt (53 mg, 0.0085 mmol, Example 90) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (20 mg, 34 μmol, MW=58526, DAR 2.6) in 3 mL PBS 7.4 buffer followed by (0.68 mL) of a premixed solution which was 20 mM sodium ascorbate (0.014 mmol), 10 mM Copper (II) sulfate (0.0068 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.0068 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 17.9 mg, 81%. MALDI MS analysis showed a range of masses (58344-72906) with an average of mass of 64071. Average DAR=2.4.
Example 146—Synthesis of Conjugate 50
[1310] Alkyne derivatized small molecule TFA salt (31 mg, 0.0085 mmol, See Example 97) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (20 mg, 34 μmol, MW=58526, DAR 2.6) in 3 mL PBS 7.4 buffer followed by (0.68 mL) of a premixed solution which was 20 mM sodium ascorbate (0.014 mmol), 10 mM Copper (II) sulfate (0.0068 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.0068 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 20.6 mg, 94%. MALDI MS analysis showed a range of masses (58346-72992) with an average of mass of 64001. Average DAR=2.5.
Example 147—Synthesis of Conjugate 51
[1311] Alkyne derivatized small molecule TFA salt (33 mg, 0.0085 mmol, See Example 96) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (20 mg, 34 μmol, MW=58526, DAR 2.6) in 3 mL PBS 7.4 buffer followed by (0.68 mL) of a premixed solution which was 20 mM sodium ascorbate (0.014 mmol), 10 mM Copper (II) sulfate (0.0068 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.0068 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 19.3 mg, 88%. MALDI MS analysis showed a range of masses (58289-72667) with an average of mass of 64060. Average DAR=2.6.
Example 148—Synthesis of Conjugate 57
[1312] Alkyne derivatized small molecule TFA salt (33 mg, 0.0085 mmol, See Example 91) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (20 mg, 34 μmol, MW=58526, DAR 2.6) in 3 mL PBS 7.4 buffer followed by (0.68 mL) of a premixed solution which was 20 mM sodium ascorbate (0.014 mmol), 10 mM Copper (II) sulfate (0.0068 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.0068 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 9.96 mg, 44%. MALDI MS analysis showed a range of masses (58980-76291) with an average of mass of 64526. Average DAR=3.0.
Example 149—Synthesis of Conjugate 61
[1313] Alkyne derivatized small molecule TFA salt (32 mg, 0.0092 mmol, See Example 98) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (20 mg, 34 μmol, MW=58721, DAR 2.8) in 3 mL PBS 7.4 buffer followed by (0.68 mL) of a premixed solution which was 20 mM sodium ascorbate (0.014 mmol), 10 mM Copper (II) sulfate (0.0068 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.0068 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 9.86 mg, 42%. MALDI MS analysis showed a range of masses (58258-75079) with an average of mass of 66516. Average DAR=2.9.
Example 150—Synthesis of Conjugate 72
[1314] Alkyne derivatized small molecule TFA salt (34 mg, 0.0092 mmol, See Example 99) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (20 mg, 34 μmol, MW=58721, DAR 2.8) in 3 mL PBS 7.4 buffer followed by (0.68 mL) of a premixed solution which was 20 mM sodium ascorbate (0.014 mmol), 10 mM Copper (II) sulfate (0.0068 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.0068 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 14.7 mg mg, 63%. MALDI MS analysis showed a range of masses (58687-76383) with an average of mass of 67633. Average DAR=3.4.
Example 151—Synthesis of Conjugate 77
[1315] Alkyne derivatized small molecule TFA salt (34 mg, 0.0092 mmol, See Example 92) was dissolved in 1 mL of pH 7.4 PBS buffer and added to azido-hlgG1(Myc)Fc (20 mg, 34 μmol, MW=58721, DAR 2.8) in 3 mL PBS 7.4 buffer followed by (0.68 mL) of a premixed solution which was 20 mM sodium ascorbate (0.015 mmol), 10 mM Copper (II) sulfate (0.0076 mmol) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (0.076 mmol) and the reaction was shaken gently at ambient temperature for 16 hours. See Example 166 for purification procedure. Yield of 16.42 mg, 63%. MALDI MS analysis showed a range of masses (58521-76178) with an average of mass of 68384. Average DAR=3.1.
Example 152—Synthesis of Conjugate 24
[1316] Note: all the solutions were prepared in PBS 7.4 buffer solution. Product described in Example 100 (60 mg, 0.0169 mmol) was added to azido-hlgG1(Myc)Fc (25 mg, 0.42 μmol, 12.4 mg/mL, MW=59,641 Da, DAR=5) solution, followed by 0.832 mL of 0.01 M CuSO.sub.4 solution, 0.832 mL of 0.01 M sodium ascorbate solution, and 0.832 mL of 0.01 M THPTA solution. The reaction was gently shaken at room temperature for 4 hours, then treated with an additional amount of freshly prepared CuSO.sub.4 solution (0.01 M, 0.416 mL). The reaction was shaken overnight. Additional CuSO.sub.4 solution (0.01 M, 0.416 mL, freshly prepared) was added, and the reaction mixture was shaken for another 2 hours. The resulting solution was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 71,189. Da (DAR=4.2). Yield 24.5 mg, 99% yield.
Example 153—Synthesis of Conjugate 48
[1317] Note: All the solutions were prepared in PBS 7.4 buffer solution. The material described in Example 101 (16 mg, 0.004043 mmol) was added to azido-hlgG1(Myc)Fc (30 mg, 0.51 μmol, 13.6 mg/mL, MW=59367 Da, DAR=4), followed by 1.263 mL of 0.01 M CuSO.sub.4, 2.021 mL of 0.01 M sodium ascorbate, and 1.263 mL of 0.01 M THPTA solutions. The reaction was gently shaken at room temperature overnight. It was then treated with an additional amount of alkyne (48 mg, 0.01213 mmol), followed by 1.263 mL of 0.01 M CuSO.sub.4, 2.021 mL of 0.01 M sodium ascorbate, and 1.263 mL of 0.01 M THPTA solutions. The reaction was shaken for 1 more day. The resulting solution was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 68611. Da (DAR=3.6). Yield 28.56 mg, 95% yield.
Example 154—Synthesis of Conjugate 58
[1318] Note: All the solutions were prepared in PBS 7.4 buffer solution. To a solution of azido-hlgG1(Myc)Fc (26 mg, 0.44 μmol, 14 mg/mL, MW=58526 Da, DAR=2.59) was added product from Example 102 (37.1 mg, 0.009205 mmol, 0.5 mL). After the resulting mixture was gently mixed, it was treated with 1.1106 mL of 0.01 M CuSO.sub.4, 1.777 mL of 0.01 M sodium ascorbate, and 1.1106 mL of 0.01 M THPTA solutions. The reaction was gently shaken at room temperature overnight. It was then purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 66062. Da (DAR=2.7). Yield 23.46 mg, 90% yield.
Example 155—Synthesis of Conjugate 59
[1319] The title compound was prepared analogously to Example 155 substituting product from Example 103. Maldi TOF analysis of the purified final product gave an average mass of 66111. Da (DAR=2.7). Yield 22.72 mg, 84% yield.
Example 156—Synthesis of Conjugate 60
[1320] The title compound was prepared analogously to Example 155 using product from Example 104. Maldi TOF analysis of the purified final product gave an average mass of 65,962. Da (DAR=2.6). Yield 16.37 mg, 82% yield
Example 157—Synthesis of Conjugate 70
[1321] Note: all the solutions were prepared in PBS 7.4 buffer solution. To a solution of product from Example 105 (30 mg, 0.007518 mmol, 0.5 mL) was added azido-hlgG1(Myc)Fc (20 mg, 0.34 μmol, 14.3 mg/mL, MW=58721 Da, DAR=2.75). The resulting mixture was gently mixed and then treated with 0.3 mL of 0.05 M CuSO.sub.4 and 0.6 mL of 0.05 M sodium ascorbate solutions. After gently mixed, the reaction mixture was treated with 0.3 mL of 0.05 M THPTA solution and gently shaken at room temperature for 4 hours. It was then treated with an additional amount CuSO.sub.4 (2 mg, 0.0125 mmol), and the reaction was continued shaken overnight. The resulting solution was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). Maldi TOF analysis of the purified final product gave an average mass of 65391. Da (DAR=2.5). Yield 5.27 mg, 39% yield.
Example 158—Synthesis of Conjugate 71
[1322] The title compound was prepared analogously to Example 158 using the product from Example 106. Maldi TOF analysis of the purified final product gave an average mass of 64,733. Da (DAR=2.2). Yield 4.07 mg, 42% yield.
Example 159—Synthesis of Azido-hlgG1(Myc)Fc with Specific DAR Values
[1323] Preparation of solutions of azido-PEG4 NHS ester in DMF/PBS solution: 100.0 mg of Azido-PEG4 NHS ester was dissolved in 1.00 mL of DMF and diluted with 2.57 mL of PBS buffer solution in ice bath to give 0.1 M azido-PEG4 NHS ester solution. 1.00 mL of 0.1 M azido-PEG4 NHS ester was diluted to 0.050M PBS buffer solution. A solution of hlgG1(Myc)Fc (described in Example 1) in pH 7.4 PBS solution (2.31 mL, 5.17 μmol, 13.0 mg/mL, MW=58009 Da) was treated with azido-PEG4-NHS ester as shown in the table below, then agitated gently overnight at RT to give azido-hlgG1(Myc)Fc with specific DAR values. Unreacted azido-PEG4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS). The final average molecular weight of the azido functionalized Fc was measured by MALDI-TOF (Table 7 and
TABLE-US-00033 TABLE 7 Equivalents Azido PEG4 of Azido hlgG1(Myc) Fc NHS ester Azido-Fc PEG4-NHS Batch MW (Da) Solution (μL) MALDI ESI ester Name MALDI ESI 0.1M 0.05M MW (Da) DAR MW (Da) DAR 5 Azido 58009 58275 52 58350 1.3 58825 2 10 Fc 103 58947 3.4 59371 4 20 103 60020 7.4 60465 8 40 207 60944 10.7 61831 13 100 517 61868 14.1
Example 160—Synthesis of Conjugate 30
[1324] A solution of hlgG1(Myc)Fc (22.00 mL, 4.68 μmol, 12.3 mg/mL, MW=57812 Da) in pH 7.4 PBS was treated with azido-PEG4-NHS ester (0.0107 g, 562 μL of 0.050 M solution in DMF/PBS (1:3), 281 μmol, 6 eq), then agitated gently overnight at RT. Unreacted azido-PEG4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS). The final volume of the azide functionalized Fc (143-4A-azido-Fc) was 19 mL with concentration at 14.7 mg/mL, MW (MALDI)=58548, DAR (MALDI)=2.7. To this azido-Fc PBS buffer solution (0.0294 g, 0.502 μmol, 2.00 mL) were added alkyne derivatized small molecule (TFA salt, 52.3 mg, 13.56 μmol, Example 107) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (1.004 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (1.004 mL of 10.0 mM, 20 eq), and sodium ascorbate (1.004 mL of 10.0 mM, 20 eq). The resulting solution was agitated gently for 4 hours at room temperature. Another portion of freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.500 mL, of 10.0 mM, 10 eq) was added and the reaction mixture was gently agitated for overnight (˜15 hours) at room temperature, then the final portion of freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.500 mL, of 10.0 mM, 10 eq) was added and the reaction mixture was gently agitated for 2 hours. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 63146 Da (DAR=2.0). Yield 25.0 mg, 83% yield.
Example 161—Synthesis of Conjugate 41
[1325] A solution of aglycosylated hlgG1(Myc)Fc (CTP-146-2A), 3.80 mL, 0.603 μmol, 8.8 mg/mL, MW=54868 Da) in pH 7.4 PBS was treated with azido-PEG4-NHS ester (0.0019 g, 512 μL of 0.010 M solution in DMF/PBS (1:9), 5.121 μmol, 8.5 eq), then agitated gently overnight at RT. Unreacted azido-PEG4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS). The final volume of the azide functionalized Fc was 7.5 mL with concentration at 4.30 mg/mL, average MW (MALDI)=55634, DAR (MALDI)=2.8. To this Fc-azido PBS buffer solution (0.0275 g, 0.495 μmol, 6.40 mL) were added alkyne derivatized small molecule (TFA salt, 53.4 mg, 13.91 μmol, Example 41, INT-21) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (1.484 mL of 10.0 mM, 30 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 1.484 mL of 10.0 mM, 30 eq), sodium ascorbate (2.226 mL of 10.0 mM, 45 eq). The resulting solution was agitated gently for −15 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166 for purification procedure). MALDI TOF analysis of the purified final product gave an average mass of 61,747 Da (DAR=2.3). Yield 17.8 mg, 65% yield.
Example 162—Synthesis of Conjugate 47
[1326] A solution of aglycosylated hlgG1(Myc)Fc (CTP-143-5A), 17.8 mL, 5.20 μmol, 16.9 mg/mL, MW=57,874 Da) in pH 7.4 PBS was treated with azido-PEG4-NHS ester (0.0019 g, 512 μL of 0.010 M solution in DMF/PBS (1:9), 5.121 μmol, 8.5 eq), then agitated gently overnight at RT. Unreacted azido-PEG4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (6×15 mL pH 7.4 PBS). The final volume of the azide functionalized Fc was 20 mL with concentration at 14.9 mg/mL, MW (MALDI)=59303, DAR (MALDI)=5.2. To this Fc-azido PBS buffer solution (0.0300 g, 0.503 μmol, 2.00 mL) were added alkyne derivatized small molecule (TFA salt, 55.9 mg, 15.1 μmol, Example 108) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (2.012 mL of 10.0 mM, 40 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 2.012 mL of 10.0 mM, 40 eq), sodium ascorbate (3.018 mL of 10.0 mM, 60 eq). The resulting solution was agitated gently for ˜15 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 69833 Da (DAR=4.1). Yield 26.5 mg, 88% yield.
Example 163—Synthesis of Conjugate 52
[1327] A solution of aglycosylated BE-0096hlgG1(Myc)Fc (BE-0096, 8.50 mL, 0.603 μmol, 9.73 mg/mL, MW=54420 Da) in pH 7.4 PBS was treated with azido-PEG4-NHS ester (0.0412 g, 1056 μL of 0.010 M solution in DMF/PBS (1:9), 10.61 μmol, 7 eq), then agitated gently overnight at RT. Unreacted azido-PEG4-NHS ester was removed by buffer exchange with a 10,000 MWCO centrifugal filter (4×15 mL pH 7.4 PBS). The final volume of the azide functionalized Fc was 5.5 mL with concentration at 15.2 mg/mL, MW (MALDI)=55409, DAR (MALDI)=3.6. To this Fc-azido PBS buffer solution (0.0200 g, 0.261 μmol, 1.315 mL) were added alkyne derivatized small molecule (TFA salt, 40.4 mg, 10.511 μmol, (C-801, WJ-052-048)) and freshly prepared pH 7.4 PBS solutions of CuSO.sub.4 (0.721 mL of 10.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.721 mL of 10.0 mM, 20 eq), sodium ascorbate (1.082 mL of 10.0 mM, 30 eq). The resulting solution was agitated gently for ˜15 hours at room temperature. The reaction mixture was purified by affinity chromatography over a protein A column, followed size exclusion chromatography (See Example 166). MALDI TOF analysis of the purified final product gave an average mass of 63851 Da (DAR=3.1). Yield 18.8 mg, 94% yield.
Example 164—ADC Purification Protocols
[1328] The crude mixtures were diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified material was quantified using a Nanodrop™ UV visible spectrophotomer (using a calculated extinction coefficient based on the amino acid sequence of h-IgG1), and concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight ladder with the indicated molecular weight standards.
Example 165—Efficacy of ADC Conjugate 23 in Mouse Pneumonia Model Vs. AB5075
[1329] The murine pneumonia model was adapted from Lin et al., 2015 (PMID: 26288841). A. baumannii AB5075 was grown to log-phase in LB at 37° C., 200 rpm shaking. Bacteria were washed once with PBS via centrifugation at 4,000 rpm. Bacteria were adjusted to the desired concentration in PBS.
[1330] Prior to infection, 8-week-old female C57BI/6J mice (Jackson Labs) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Using an otoscope, mice were infected non-invasively directly into the trachea.
[1331] Mice were infected with 5×10.sup.7 CFU in 30 μL of PBS. Mice were treated intraperitoneally with different doses of conjugate 23 or hlgG1 (50 mpk) at t=−12 h pre-infection and t=+1 h post-infection or with colistin (10 mpk) or PBS at t=+1 h, 5 h, 24 h, 36 h, 48 h and 60 h post-infection.
[1332] Survival of mice (n=5 per group) was monitored for 5 days. ADC conjugate 23 showed a dose-dependent protection: 0% protection at 1 mpk, 20% protection at 10 mpk and 80% protection at 25 and 50 mpk. Colistin was used as positive control resulting in 80% protection. In comparison, control IgG1 and PBS were not protective and mice succumbed to infection between day 1.5-2 (
Example 166—Efficacy of ADC Conjugate 34 in Mouse Pneumonia Model Vs. PAO1
[1333] The murine pneumonia model was adapted from Lin et al., 2015 (PMID: 26288841). P. aeruginosa PAO1 was grown to log-phase in LB at 37° C., 200 rpm shaking. Bacteria were washed once with PBS via centrifugation at 4,000 rpm. Bacteria were adjusted to the desired concentration in PBS. Prior to infection, 8-week-old female C57BI/6J mice (Jackson Labs) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Using an otoscope, mice were infected non-invasively directly into the trachea.
[1334] Mice were infected with 5×10.sup.6 CFU in 30 μL of PBS. Mice were treated intraperitoneally with conjugate 34 (25 mpk) or hlgG1 (50 mpk) at t=−12 h prior to infection or with colistin (10 mpk) or PBS at t=+1 h, 5 h, 24 h, 36 h, 48 h and 60 h post-infection.
[1335] Survival of mice (n=5 per group) was monitored for 5 days. A single dose of ADC conjugate 34 was protective (80% survival) against a lethal challenge with P. aeruginosa PAO1. Colistin was used as positive control resulting in complete protection. In comparison, control IgG1 and PBS were not protective and mice succumbed to infection between day 2-2.5 (
Example 167—Efficacy of ADC Conjugate 34 in Mouse Pneumonia Model Vs. AB5075
[1336] The murine pneumonia model was adapted from Lin et al., 2015 (PMID: 26288841). A. baumannii AB5075 was grown to log-phase in LB at 37° C., 200 rpm shaking. Bacteria were washed once with PBS via centrifugation at 4,000 rpm. Bacteria were adjusted to the desired concentration in PBS. Prior to infection, 8-week-old female C57BI/6J mice (Jackson Labs) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Using an otoscope, mice were infected non-invasively directly into the trachea.
[1337] Mice were infected with 5×10.sup.7 CFU in 30 μL of PBS. Mice were treated intraperitoneally with conjugate 34 or hlgG1 (50 mpk) at t=−12 h pre-infection.
[1338] Survival of mice (n=4 per group) was monitored for 5 days. A single dose of ADC conjugate 34 was protective (75% survival) against a lethal challenge with A. baumannii AB5075. Control IgG1 was not protective and mice succumbed to infection on day 2 (
Example 168—Efficacy of ADC Conjugate 23 in Mouse Pneumonia Model Vs. PAO1
[1339] The murine pneumonia model was adapted from Lin et al., 2015 (PMID: 26288841). P. aeruginosa PAO1 was grown to log-phase in LB at 37° C., 200 rpm shaking. Bacteria were washed once with PBS via centrifugation at 4,000 rpm. Bacteria were adjusted to desired concentration in PBS. Prior to infection, 8-week-old female C57BI/6J mice (Jackson Labs) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Using an otoscope, mice were infected non-invasively directly into the trachea.
[1340] Mice were infected with 5×10.sup.6 CFU in 30 μL of PBS. Mice were treated intraperitoneally with conjugate 23 or hlgG1 (50 mpk) at t=−12 h prior to infection or with colistin (10 mpk) or PBS at t=+1 h and 5 h post-infection.
[1341] Survival of mice (n=5 per group) was monitored for 5 days. A single dose of ADC conjugate 23 was completely protective against challenge with P. aeruginosa PAO1. Positive control, colistin, was also protective (100%). Controls IgG1 (60% survival) and PBS (20% survival) were only partially protective (
Example 169—Efficacy of ADC Conjugate 23 in Mouse Pneumonia Model Vs. PAO1
[1342] The murine pneumonia model was adapted from Lin et al., 2015 (PMID: 26288841). P. aeruginosa PAO1 was grown to log-phase in LB at 37° C., 200 rpm shaking. Bacteria were washed once with PBS via centrifugation at 4,000 rpm. Bacteria were adjusted to the desired concentration in PBS. Prior to infection, 8-week-old female C57BI/6J mice (Jackson Labs) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Using an otoscope, mice were infected non-invasively directly into the trachea.
[1343] Mice were infected with 5×10.sup.6 CFU in 30 μL of PBS. Mice were treated intraperitoneally with conjugate 23, 34, 35, 38, 44 or hlgG1 (25 mpk) at t=−12 h prior to infection or with colistin (10 mpk) or PBS at t=+1 h and 5 h post-infection.
[1344] Survival of mice (n=5 per group) was monitored for 5 days. Single doses of ADC conjugates 23, 34, 35, 38 or 44 were completely protective against challenge with P. aeruginosa PAO1. Positive control, colistin, was also protective (100%). The IgG1 control (60% survival) was only partially protective and mice in the PBS group all succumbed to infection on day 2 (
Example 170—Protective Effect of Conjugate 23 Against Endotoxemia
[1345] Septic shock, resulting in systemic organ failure, is an important component of many bacterial infections. In infections caused by Gm-negative pathogens, or in some mixed infections, LPS (aka endotoxin) is the dominate mediator of this process which results from hyper-stimulation of the immune system. In a mouse model of endotoxemia we were able to demonstrate the protective effect of Conjugate 23 to rescue mice given a lethal dose of LPS.
[1346] Mouse model of endotoxemia. These studies used female immune competent C57BL/6 mice between 8 and 10 weeks old. After acclimation for 7 days mice were challenged IP with 12.5 mg/kg of purified LPS (Escherichia coli O111::B4) in PBS from Sigma (#L4130) in a volume of 0.2 ml. Previously this dose was shown to result in 100% mortality of challenged mice within 5 days. Study mice were housed and cared for following standard IACUC approved protocols, and clearly moribund mice were humanely euthanized and scored as a death.
[1347] Efficacy of Conjugate 23 in endotoxemia. C57BL/6 mice were challenged with a lethal dose of LPS as described above. Immediately following LPS challenge 2 groups of animals received vehicle (PBS) or colistin (10 mg/kg) as a separate IP injection. Due to the short half-life of colistin both groups were dosed twice daily (bid, separated by 4-6 hours) for 2 days. In contrast, conjugate 23 was administered as a single IP dose at 25 mg/kg 4 hours prior to LPS challenge. The Fc-only control for conjugate 23 which lacks the LPS recognition moiety was dosed following the same procedure (hlgG1).
[1348] As illustrated in
Example 171—Conjugate Activity in Human Blood
[1349] Overnight stationary-phase cultures were centrifuged at 3,500×g for 10 min at room temperature and resuspended in 1×PBS to an optical density at 600 nm (OD.sub.600) of 0.4 (1×10.sup.8 colony forming units [CFU]/ml) in RPMI with L-glutamine and phenol red (Life Technologies). Bacteria were diluted 1:25 in RPMI (4×10.sup.6 CFU/ml) and 10 ul added to wells of a non-binding 96-well plate (Corning 3641) containing 80 ul of 50% hirudin anti-coagulated blood (complement active) in RPMI. After adding 5 ul of 20× conjugate diluted in RPMI (128-2 ug/ml final concentration), and 5 ul RPMI for a final blood concentration of 40% in 100 ul, the plate was incubated under static conditions at 37° C. for 90 min. The minimum bactericidal concentration (MBC) was determined by 10-fold serial dilution in 1×PBS, then plating 4 ul spots onto rectangular LB agar plates for overnight incubation at 37° C. MBC was defined as the minimum conjugate concentration required for bacterial clearance (Table 8A and Table 8B). An untreated growth control (40% blood, 0 ug/ml conjugate) was included in all assays as a growth comparator.
TABLE-US-00034 TABLE 8A MBC values of conjugates in blood MBC (μg/ml) in human hirudin-anticoagulated blood E. coli ATCC A. baumannii P. aeruginosa P. aeruginosa Compound 25922 AB5075 PAO1 PA7874 hIgG1 Fc >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 >128 Conjugate 21 6 6 >52 >52 Conjugate 17 5 1.8 >49 49 Conjugate 22 4 ≤2 >128 64 4 4 >32 >32 Conjugate 23 ≤0.5 1 32 32 4 2 64 Conjugate 24 16 <2 >128 128 8 <2 >128 >128 8 2 >32 >32 Conjugate 25 2 ≤2 32 32 ≤2 ≤2 64 32 1 ≤0.5 >32 16 Conjugate 27 32 32 >32 >32 Conjugate 28 16 2 >32 32 Conjugate 29 32 >32 >32 >32 Conjugate 30 2 1 >32 32 Conjugate 31 2 2 >32 32 Conjugate 32 >32 16 >32 >32 Conjugate 33 8 16 >32 >32 Conjugate 34 ≤0.5 0.5 4 4 0.5 ≤0.5 16 8 0.5 ≤0.5 8 8 1 1 8 8 Conjugate 35 1 1 >32 >32 Conjugate 36 16 4 >32 >32 Conjugate 37 16 4 >32 >32 Conjugate 38 2 1 >32 32 1 1 32 16 Conjugate 39 1 1 32 16 1 2 16 8 Conjugate 40 ≤0.5 ≤0.5 16 8 ≤0.5 ≤0.5 8 8 Conjugate 41 2 2 >32 32 2 2 >32 16 Conjugate 42 32 8 >32 >32 32 8 >32 >32 Conjugate 43 4 8 >32 32 4 16 >32 >32 Conjugate 44 4 1 >32 32 1 2 32 16
TABLE-US-00035 TABLE 8B MBC values of conjugates in blood MBC (μg/ml) in human hirudin-anticoagulated blood E. coli ATCC A. baumannii P. aeruginosa P. aeruginosa Compound 25922 AB5075 PAO1 PA7874 Conjugate 45 >32 16 >32 >32 16 32 >32 >32 Conjugate 46 16 16 >32 >32 32 32 >32 >32 Conjugate 47 8 4 >32 >32 4 4 >32 >32 Conjugate 48 2 2 >32 >32 2 2 >32 32 Conjugate 49 2 2 >32 32 Conjugate 50 16 8 >32 >32 Conjugate 51 4 2 >32 32 Conjugate 52 4 4 >32 32 Conjugate 53 2 2 >32 32 Conjugate 54 32 16 >32 >32 Conjugate 55 32 16 >32 >32 Conjugate 56 4 8 >32 32 Conjugate 57 2 2 >32 32 Conjugate 58 8 4 >32 >32 Conjugate 59 2 2 >32 >32 Conjugate 60 4 4 >32 >32 Conjugate 61 8 4 >32 NT Conjugate 62 16 4 >32 NT Conjugate 63 >32 16 >32 NT Conjugate 64 16 8 >32 NT Conjugate 65 16 8 >32 NT Conjugate 66 32 8 >32 NT Conjugate 67 32 16 >32 NT Conjugate 68 16 4 >32 NT Conjugate 69 16 8 >32 NT Conjugate 70 32 >32 >32 NT Conjugate 71 32 32 >32 NT Conjugate 72 4 ≤2 128 64 Conjugate 73 4 2 128 64 Conjugate 74 8 8 >32 NT Conjugate 75 16 4 >32 NT
Example 172—Conjugate Activity in Human Serum
[1350] Overnight stationary-phase cultures were centrifuged at 3,500×g for 10 min at room temperature and resuspended in 1×PBS to an optical density at 600 nm (OD.sub.600) of 0.4 (1×10.sup.8 colony forming units [CFU]/ml) in RPMI with L-glutamine and phenol red (Life Technologies). Bacteria were diluted 1:25 in RPMI (4×10.sup.6 CFU/ml) and 10 uL added to wells of a non-binding 96-well plate (Corning 3641) containing 80 ul of 50% normal human serum (complement-active; Innovative Research IPLA-CSER) in RPMI, or 50% heat-inactivated human serum (56° C. for 30 min; complement inactive) in RPMI. After adding 5 ul of 20× conjugate diluted in RPMI (128-2 ug/ml final concentration), and 5 ul RPMI for a final serum concentration of 40% in 100 ul, the plate was incubated under static conditions at 37° C. for 90 min. The minimum bactericidal concentration (MBC) was determined by 10-fold serial dilution in 1×PBS, then plating 4 ul spots onto rectangular LB agar plates for overnight incubation at 37° C. MBC was defined as the minimum conjugate concentration required for bacterial clearance (Table 9). An untreated growth control (40% serum, 0 ug/ml conjugate) was included in all assays as a growth comparator. MBCs are typically lower in complement active normal human serum compared to complement inactive heat-inactivated human serum, indicating that conjugate-mediated killing is complement dependent (Table 9).
TABLE-US-00036 TABLE 9 MBC values of conjugates in serum MBC (μg/ml) in normal human serum (heat-inactivated human serum) A. baumannii P. aeruginosa AB5075 PAO1 Conjugate 46 32 (>32) >32 (>32) Conjugate 47 8 (16) >32 (>32) Conjugate 48 8 (16) >32 (>32) Conjugate 49 4 (8) >32 (>32) Conjugate 50 8 (32) >32 (>32) Conjugate 51 4 (8) >32 (>32) Conjugate 52 2 (8) >32 (>32) Conjugate 53 4 (8) >32 (>32) Conjugate 54 32 (>32) >32 (>32) Conjugate 55 32 (>32) >32 (>32) Conjugate 56 8 (32) >32 (>32) Conjugate 57 4 (32) >32 (>32) Conjugate 58 8 (32) >32 (>32) Conjugate 59 16 (16) >32 (>32) Conjugate 60 16 (32) >32 (>32) Conjugate 72 4 (16) >128 (>128) Conjugate 73 4 (32) >128 (>128)
Example 173—In Vitro Neutrophil Killing Assay
[1351] 5×10.sup.4 log phase E. coli ATCC 25922 were added to 96-well, round-bottom, non-treated tissue culture plates in the presence or absence of 25 nM ADC. 5×10.sup.4 polymorphprep (AXIS-SHIELD) purified human neutrophils were added to a portion of the wells and plates were incubated at 37° C. shaking at 800 rpm for the indicated time points. Aliquots were removed at each time point, serially diluted in water to lyse neutrophils and incubated overnight on agar plates at 37° C. Surviving CFU/ml were calculated by manually counting colonies and data were graphed using the following formula: % of inoculum=([CFU/mL of test condition]/[CFU/mL of inoculum])*100 (
Example 174—Neutrophil Phagocytosis of FITC-Labeled E. coli (K-12) BioParticles
[1352] 10.sup.5 FITC-labeled E. coli BioParticles (Invitrogen) were opsonized with 250 nM ADC or media only by co-incubation for 25 min at 37° C. shaking (650 rpm). 2×10.sup.5 polymorphprep purified human neutrophils were then added and plates were incubated at 37° C. shaking at 650 rpm for 10 min. Neutrophils were placed on ice for 5 minutes to stop phagocytosis and then washed in cold PBS followed by fixation in 2% PFA. Neutrophils were incubated in 0.2% trypan blue for 2 minutes to quench extracellular fluorescence, washed once and analyzed on a FACSCalibur (BD Biosciences) in the presence of residual trypan blue. Fluorescence was measured in the Blu FL1 channel (
Other Embodiments
[1353] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth.
[1354] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.