Antibody drug conjugate for anti-inflammatory applications

Abstract

Antibody drug conjugates (ADCs) comprising an antibody conjugated to an anti-inflammatory therapeutic agent via a phosphate-based linker with tunable extracellular and intracellular stability are described.

Claims

1. A composition comprising a compound having formula (I) ##STR00051## wherein V is selected from O and S; W is selected from O, N, and CH.sub.2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR.sub.2, O, or S; D is an anti-inflammatory agent; Antibody is an antibody that binds a CD74 protein and comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:7; Z is a linkage formed between (i) a reactive functional group selected from the group consisting of maleimide and strained cycloalkyne, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the anti-inflammatory agent comprises a glucocorticoid receptor agonist.

3. The composition of claim 1, wherein the anti-inflammatory agent comprises Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, or mometasone.

4. The composition of claim 1, wherein the antibody comprises the heavy chain comprising SEQ ID NO: 69 or SEQ ID NO: 70 and the reactive functional group comprises the strained cycloalkyne.

5. A method for treating an inflammatory disease or disorder by providing to a subject having the disease or disorder a composition comprising a compound having formula (I) ##STR00052## wherein V is selected from O and S; W is selected from O, N, and CH.sub.2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR.sub.2, O, or S; D is an anti-inflammatory agent; Antibody is an antibody that binds a CD74 protein and comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73; Z is a linkage formed between (i) a reactive functional group selected from the group consisting of maleimide and strained cycloalkyne and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and a pharmaceutically acceptable carrier to treat the inflammatory disease or disorder.

6. The method of claim 5, wherein the anti-inflammatory agent comprises a glucocorticoid receptor agonist.

7. The method of claim 5, wherein the anti-inflammatory agent comprises Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, or mometasone.

8. The method of claim 5, wherein the antibody comprises the heavy chain comprising SEQ ID NO: 69 or SEQ ID NO:70 and the reactive functional group comprises the strained cycloalkyne.

9. The method of claim 5, wherein the inflammatory disease or disorder comprises Alzheimer's disease, ankylosing spondylitis arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis), asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis, Parkinson's disease, or ulcerative colitis.

10. An antibody drug conjugate comprising (a) an antibody that binds a CD74 protein, the antibody comprising a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69 or 70 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73, wherein the antibody comprises a para-azidophenylalanine (pAzF) conjugated to a molecule selected from the group of molecules consisting of ##STR00053## ##STR00054## ##STR00055## ##STR00056##

11. The composition of claim 4, wherein the strained cycloalkyne is cyclooctyne.

12. The method of claim 8, wherein the strained cycloalkyne is cyclooctyne.

13. The antibody drug conjugate of claim 10, wherein the molecule ##STR00057##

14. The antibody drug conjugate of claim 10, wherein the molecule is ##STR00058##

15. The antibody drug conjugate of claim 10, wherein the molecule is ##STR00059##

16. The antibody drug conjugate of claim 10, wherein the molecule is ##STR00060##

17. The antibody drug conjugate of claim 10, wherein the molecule is ##STR00061##

18. The antibody drug conjugate of claim 10, wherein the molecule is ##STR00062##

19. The antibody drug conjugate of claim 10, wherein the molecule is ##STR00063##

20. The antibody drug conjugate of claim 10, wherein the molecule is ##STR00064##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a scheme for synthesis of ADC 12-4.

(2) FIG. 2 is a graph showing stability of ADC 12-1 in mouse serum. (aDEX is ADC 12-1).

(3) FIG. 3 is a graph showing deconvoluted intact mass spectrum for ADC 12-1 stock solution.

(4) FIG. 4A is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37° C. for 1 hour.

(5) FIG. 4B is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37° C. for 8 hours.

(6) FIG. 4C is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37° C. for 14 days.

(7) FIG. 4D is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37° C. for 21 days.

(8) FIG. 5 is a graph showing In vivo stability of ADC 12-1 versus “naked” antibody (non-conjugated) following IV dosing to DBA1 mice.

(9) FIG. 6A is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: sample B1 at 1 hour.

(10) FIG. 6B is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: sample H3 at 5 days.

(11) FIG. 6C is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: sample B7 at 1 hour.

(12) FIG. 6D is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: sample H9 at 5 days.

(13) FIG. 7 is a graph showing In vitro activity of ADC 12-2 versus “naked” antibody (non-conjugated) in 786-O cells.

(14) FIG. 8 shows the activity of anti-hC74 (LL1) ADCs in Hut78 cells.

(15) FIG. 9 shows the activity of anti-hC74 (LL1) ADCs in SUDHL-6 cells.

(16) FIG. 10 shows the activity of anti-hC74 (LL1) ADCs in 786-O cells.

DETAILED DESCRIPTION OF THE INVENTION

(17) The present invention provides antibody-drug conjugates (ADCs) for use in treatments where it is desirable that the treatment include an anti-inflammatory therapeutic component. The antibody-drug conjugates comprise an antibody that targets or binds the human CD25, human CD70, human CD74 protein, or human CD163 protein (anti-CD25 antibody, anti-CD70 antibody, anti-CD74, or anti-CD163 antibody, respectively) conjugated to an anti-inflammatory therapeutic agent via a phosphate-based linker with tunable stability for intracellular delivery of the therapeutic agent. The phosphate-based linker comprises a monophosphate, diphosphate, triphosphate, or tetraphosphate group (phosphate group) covalently linked to the distal end of a linker arm comprising from the distal to the proximal direction a tuning element, optionally a spacer element, and a reactive functional group. The phosphate group of the phosphate-based linker is capable of being conjugated to the therapeutic agent and the reactive functional group is capable of being conjugated the antibody. The phosphate-based linker has a differentiated and tunable stability in blood vs. an intracellular environment (e.g., lysosomal compartment). The inventors have discovered that the rate at which the phosphate group is cleaved in the intracellular environment to release the payload in its native or active form may be affected by the structure of the tuning element with further effects mediated by substitutions of the phosphate group as well as whether the phosphate group is a monophosphate, diphosphate, triphosphate, or tetraphosphate.

(18) The phosphate-based linkers comprise a monophosphate, diphosphate, triphosphate, or tetraphosphate group and a linker arm comprising a tuning element, an optional spacer element, and a reactive functional group. The phosphate-based linkers have a distal end and a proximal end. The distal end of the phosphate-based linker comprises a monophosphate, diphosphate, or triphosphate group (phosphate group) linked to the distal end of the tuning element comprising the linker arm. The phosphate group is covalently linked to an anti-inflammatory therapeutic agent. The proximal end of the linker arm comprises a reactive functional group capable of reacting with a group (side chain of an amino acid) on an antibody to covalently link the phosphate-based linker to the antibody. Interspersed between the tuning element and the reactive functional group of the linker arm may be an optional spacer element. Such a compound comprises formula (I)

(19) ##STR00010##

(20) Wherein V is selected from O and S; W is selected from O, N, and CH.sub.2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin K sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR.sub.2, O, or S; D is an anti-inflammatory therapeutic agent; wherein the antibody binds the human CD25, human CD70, human CD74 protein, or human CD163 protein (anti-CD25 antibody, anti-CD70 antibody, anti-CD74, or anti-CD163 antibody, respectively); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) a side chain of an amino acid of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

(21) The phosphate-therapeutic agent is stabile extracellularly and labile intracellularly, for example, when present in the lysosomal compartment of the target cell. The tuning element provides a tunable stability to the phosphate-therapeutic agent linkage when the conjugate is within the lysosomal compartment of the target cell. The intracellular stability of the phosphate-therapeutic agent linkage or rate of intracellular release of the therapeutic agent from the conjugate may be adjusted or tuned by the particular tuning element adjacent to the phosphate group and/or by adjusting the number of the phosphate groups.

(22) The link between the antibody and the anti-inflammatory therapeutic agent plays an important role in an antibody drug conjugate (ADC), as the type and structure of the linker may significantly affect the potency, selectivity, and the pharmacokinetics of the resulting conjugate (Widdeson et al, J. Med. Chem. 49: 4392-4408 (2006); Doronina et al., Bioconj. Chem. 17: 114-124 (2006); Hamann et al., Bioconj. Chem. 16: 346-353 (2005); King et al., J. Med. Chem. 45: 4336-4343 (2002); Alley et al., Bioconj. 19: 759-765 (2008); Blattler et al., Biochem. 24: 1517-1524 (1985). ADC delivery of a drug moiety to its intracellular target occurs via a multistep sequence of events: binding to the cell surface, endocytosis, trafficking (within an endosome) to a lysosome, proteolytic degradation of the conjugate, and diffusion of the released drug moiety across the lysosomal or endosomal membrane toward its intracellular target and its interaction with the target. Therefore, the linker should be sufficiently stable while in circulation to allow delivery of the intact ADC to the target cell but, on the other hand, sufficiently labile to allow release of the drug moiety from the ADC once inside the targeted cell. In general, four types of linkers have been used for preparation of ADCs that have currently entered the clinic: (a) acid-labile linkers, exploiting the acidic endosomal and lysosomal intracellular microenvironment (Hamann et al., op. cit.; Blattler et al., op. cit.); (b) linkers cleavable by lysosomal proteases (Dronina et al. op. cit.; King et al. op. cit.); (c) chemically stable thioether linkers that release a lysyl adduct after proteolytic degradation of the antibody inside the cell; (Lewis et al Cancer Res. 68: 9280-9290 (2008); Erickson et al., Cancer Res. 66: 4426-4433 (2006) and (d) disulfide containing linkers (Chari, Adv. Drug Delivery Rev. 31: 89-104 (1998); Widdeson et al., op. cit.), which are cleaved upon exposure to an intracellular thiol. While U.S. Pat. No. 5,094,848 discloses conjugates comprising a diphosphate or amidated diposphate group and a linker arm wherein the linker arm may preferably be an oligopeptide having preferably 2-10 amino acids, in particular embodiments the tuning element of the phosphate-based linkers disclosed herein may include a di-peptide.

(23) The linker-therapeutic agent conjugates of the present invention wherein the therapeutic agent is covalently linked to a tuning element of the linker via a monophosphate, diphosphate, triphosphate, or tetraphosphate linkage have a differentiated and tunable stability of the phosphate linkage in blood vs. an intracellular environment (e.g. lysosomal compartment). Due to location of enzymes that recognize the phosphate linkage, conjugates that have a phosphate group linking a therapeutic agent to a tuning element of the linker are stable in circulation (plasma or blood) but reactive in intracellular compartments (e.g., lysosomes) making them suitable for intracellular delivery of therapeutic agent conjugates. The exemplary therapeutic agent-phosphate-based linker conjugates in the Examples show that the therapeutic agent-phosphate-based linker conjugates of the present invention are stable in blood, which is advantageous for extending the half-life and to prevent premature release of therapeutic agent from the conjugates.

(24) Importantly, the inventors have discovered that by modifying the tuning element and/or V and/or W, and/or the number of phosphate groups, the ability to tune reactivity or cleavage of the phosphate linkage in a lysosomal environment so as to release the therapeutic agent from the conjugate. In general, the rate of release of the therapeutic agent is dependent on the proximal substitution of the tuning element. The ability to cleave the phosphate linkage between the payload and the tuning element efficiently in a lysosome is advantageous for the release of the therapeutic agent from the conjugate once it has been delivered to a cell and internalized through an endosomal pathway. Of note is that unlike other linkers known in the art, there is no need to for the phosphate-based linkers of the present invention to be self-immolative. In addition, the excellent solubility of the therapeutic agent-phosphate-based linker facilitates conjugation to a ligand or cell-targeting moiety and minimizes aggregation of the conjugates. In addition, the phosphate contributes to retention of the therapeutic agent to the conjugate within cell until phosphate linkage is fully cleaved and limits permeability of conjugates containing the payload from entering non-target cells.

(25) The phosphate-based linkers provide greater solubility relative to disulfide linkers, cathepsin B-cleavable linkers, esters and acid-sensitive linkers such as hydrazones. They enable the release of the payload in its parent or unadultered form unlike some of the alternative linkers, and may offer an improved blood/lysosome stability profile. Specifically, these phosphate-based linkers will provide superior blood stability relative to esters and disuflides. Phosphate-based linkers, following lysosomal cleavage will release an alcohol or amine-containing payload whereas the other linker formats may require self-immolative tethers to accomplish this or leave residual linker on the therapeutic agent after lysosomal cleavage. The phosphate-based linker may have greater blood stability relative to the self-immolative cathepsin B linkers in the art, particularly when attached via the oxygen atom of a hydroxyl group of an alcohol-containing therapeutic agent. The enzymatic hydrolysis of the phosphate linkage may be more rapid than the acid-hydrolysis of hyrdazones. The phosphate-based linkers disclosed herein minimize the propensity for conjugates comprising particular therapeutic agent to aggregate. Thus, the phosphate-based linkers disclosed herein are particularly useful for conjugating therapeutic agents that are prone to forming aggregates to a cell-specific targeting ligand to provide a conjugate with a reduced or no detectable propensity for aggregation.

(26) Phosphate Group

(27) The phosphate group comprising the phosphate-based linkers disclosed herein may comprise 1, 2, 3, or 4 phosphate atoms. In particular embodiments, the phosphate group may be a phosphate ester

(28) ##STR00011##
pyrophosphate ester

(29) ##STR00012##
triphosphate ester

(30) ##STR00013##
or tetraphosphate ester

(31) ##STR00014##

(32) In further embodiments the phosphate group may be a phosphoramidate

(33) ##STR00015##
pyrophosphoramidate

(34) ##STR00016##
triphosphophoramidate

(35) ##STR00017##
or tetraphosphoramidate

(36) ##STR00018##
In further still embodiments, the phosphate group may be a phosphonate

(37) ##STR00019##
a diposphonate

(38) ##STR00020##
a phosphorthioate

(39) ##STR00021##
or a diphosphorthioate

(40) ##STR00022##

(41) The wavy lines shown indicate the covalent attachment sites to the payload at the distal end (left) and the tuning element on the proximal end (right).

(42) Anti-Inflammatory Therapeutic Agent

(43) Anti-inflammatory therapeutic agents comprise glucocorticoid receptor agonists, which include but are not limited to glucocorticoids such as Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, and mometasone.

(44) Linker Arm

(45) The linker arm of the phosphate-based linkers disclosed herein comprises a tuning element at the distal end covalently linked to a phosphate group and a functional reactive group at the proximal end capable of covalent linkage to a cell-targeting ligand. Optionally, the linker arm may further include a spacer element interposed between the tuning element and the reactive functional group. Examples of tuning elements include but are not limited to

(46) ##STR00023##

(47) R.sub.1 and R.sub.2 each independently any amino acid

(48) ##STR00024##
The wavy lines indicate the covalent attachment sites to the phosphate group at the distal end (left) and the functional reactive group on the proximal end (right), or optionally, a spacer element.

(49) Further examples of tuning elements include but are not limited to

(50) ##STR00025##
The wavy lines indicate the covalent attachment sites to the phosphate group at the distal end (left) and the functional reactive group on the proximal end (right), or optionally, a spacer element.

(51) In general, the spacer element is to allow for distance control away from the cell-targeting ligand. In some embodiments, this distance may have an impact on the stability/cleavability of the linker. Examples of spacer elements include straight polyethylglycol (PEG) chains (of a defined length) and straight carbon chains with or without solubilizing groups attached thereto.

(52) Targeting Antibody

(53) The linker arm and anti-inflammatory therapeutic agent may be linked to an antibody that selectively delivers the therapeutic agent to a cell, organ, or region of the body that expresses the human CD25 protein, human CD74 protein, human CD74 protein, or human CD163 protein. Antibodies may be either polyclonal or monoclonal, but most preferably are monoclonal and may be human, humanized, or human chimeric antibodies. The antibody may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 and IgA.sub.2). The term “CD” refers to “cluster of differentiation”.

(54) The antibody may be attached to the linker arm by any available reactive group that can react with the reactive functional group on the proximal end of the linker arm. For example, the antibody may be attached through an amine, carboxyl, sulfhydryl, or hydroxyl group. Such a group may reside at N-terminus or at a site internal to the protein chain, for example, the side chain of an amino acid. The antibody may be further derivatized at one or more sites to allow for the attachment of appropriate reactive groups onto the peptide or protein. See, Chrisey et al. Nucleic Acids Res. 24:3031-3039 (1996). In addition, the antibody may be synthesized to contain one or more non-natural amino acids, the side chain thereof which may then serve as a site for attachment of the linker arm comprising the payload-phosphate-based linker. Antibodies comprising non-natural amino acids for conjugation and methods for making such antibodies have been disclosed in U.S. Pat. No. 7,632,924, which is incorporated herein by reference. As exemplified herein the antibody may comprise a substitution of an amino acid residue in the heavy chain or light chain with the non-natural amino acid para-azidophenylalanine (pAzF). The azido group on the side chain of the pAzF residue may be conjugated to a reactive functional group of the therapeutic agent-linker such as a strained cycloalkyne, for example, cyclooctyne.

(55) In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSSYLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO: 2), and CDR3 (QQYSSSPLT; SEQ ID NO: 3) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYIIN; SEQ ID NO: 4), CDR2 (RIIPILGVENYAQKFQG; SEQ ID NO: 5), and CDR3 (KDWFDY; SEQ ID NO: 6). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSSFLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO: 2), and CDR3 (QQYSSSPLT; SEQ ID NO: 3) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYPIN; SEQ ID NO: 7), CDR2 (RIIPILGIADYAQRFQG; SEQ ID NO: 8), and CDR3 (RDWGDY; SEQ ID NO: 9). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSGSSSYLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO: 2), and CDR3 (QQYGSSPIT; SEQ ID NO: 10) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYAIN; SEQ ID NO: 11), CDR2 (RIIPILDIADYAQKFQD; SEQ ID NO: 12), and CDR3 (KDWFDP; SEQ ID NO: 13). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSSFLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO:2), and CDR3 (QQYSSSPLT; SEQ ID NO:3) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYPIN; SEQ ID NO: 14), CDR2 (RIIPILGIADYAQRFQG; SEQ ID NO:8), and CDR3 (RDWGDY; SEQ ID NO:9). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO: 14. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD25 antibody that competes with any one of the aforementioned antibodies for binding to the CD25. The aforementioned anti-CD70 antibodies comprising said CDR sequences have been disclosed in U.S. Pat. No. 7,438,907, which is incorporated herein by reference.

(56) In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSYLA; SEQ ID NO: 15), CDR2 (YDASNRAT; SEQ ID NO: 16), and CDR3 (QQRTNWPLT; SEQ ID NO: 17) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (SYIMH; SEQ ID NO: 18), CDR2 (VISYDGRNKYYADSVK; SEQ ID NO: 19), and CDR3 (DTDGYDFDY; SEQ ID NO:20). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSALA; SEQ ID NO:21), CDR2 (DASSLES; SEQ ID NO:22), and CDR3 (QQFNSYPFT; SEQ ID NO:23) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (YYAMH; SEQ ID NO:24), CDR2 (VISYDGSIKYYADSVK; SEQ ID NO:25), and CDR3 (EGPYSNYLDY; SEQ ID NO:26). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSWLA; SEQ ID NO:27), CDR2 (AASSLQS; SEQ ID NO:28), and CDR3 (QQYNSYPLT; SEQ ID NO:29) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (DYGMH; SEQ ID NO:30), CDR2 (VIWYDGSNKYYADSVK; SEQ ID NO:31), and CDR3 (DSIVMVRGDY; SEQ ID NO:32). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSWLA; SEQ ID NO:33), CDR2 (AASSLQS; SEQ ID NO:34), and CDR3 (QQYNSYPLT; SEQ ID NO:35) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (DHGMH; SEQ ID NO:36), CDR2 (VIWYDGSNKYYADSVK; SEQ ID NO:37), and CDR3 (DSIMVRGDY; SEQ ID NO:38). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSYLA; SEQ ID NO: 15), CDR2 (DASNRAT; SEQ ID NO:39), and CDR3 (QQRSNWPLT; SEQ ID NO:40) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (SDYYYWS; SEQ ID NO:41), CDR2 (YIYYSGSTNYDPSLKS; SEQ ID NO:42), and CDR3 (GDGDYGGNCFDY; SEQ ID NO:43). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD70 antibody that competes with any one of the aforementioned antibodies for binding to the CD70. The aforementioned anti-CD70 antibodies comprising said CDR sequences have been disclosed in U.S. Pat. No. 8,124,738, which is incorporated herein by reference.

(57) In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising the light chain complementarity-determining region (CDR) sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:44), CDR2 (TVSNRFS; SEQ ID NO:45), and CDR3 (SQSSHVPPT; SEQ ID NO:46) and the heavy chain CDR sequences CDR1 (NYGVN; SEQ ID NO:47), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:48), and CDR3 (SRGKNEAWFAY; SEQ ID NO:49). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) selected from SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD74 antibody that competes with any one of the aforementioned antibodies for binding to the CD74. Antibodies comprising said CDR sequences have been disclosed in U.S. Pat. No. 7,772,373, which is incorporated herein by reference. In a particular aspect, the anti-CD74 antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73.

(58) In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising the light chain complementarity-determining region (CDR) sequences CDR1 (QGISSW; SEQ ID NO:50), CDR2 (AAS), and CDR3 (QQYNSYPLT; SEQ ID NO:51) and the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSNK; SEQ ID NO:53), and CDR3 (ASGRYYGSGSYSSYFD; SEQ ID NO:54); or the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSIK; SEQ ID NO:55), and CDR3 (ARGREYTSQNIVILLD; SEQ ID NO:56); or the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSNK; SEQ ID NO:53), and CDR3 (ARGREITSQNIVILLD; SEQ ID NO:57); or the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (IWYDGSNK; SEQ ID NO:58), and CDR3 (ARGGTLVRGAMYGTDV; SEQ ID NO:59). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from AAS, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD74 antibody that competes with any one of the aforementioned antibodies for binding to the CD74. Antibodies comprising said CDR sequences have been disclosed in U.S. Patent Application Publication No. 20140030273, which is incorporated herein by reference. In a particular aspect, the anti-CD74 antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:74, 75, 76, and 77 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:78.

(59) In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD163 antibody comprising the light chain CDR sequences CDR1 (ASQSVSSDV; SEQ ID NO:60), CDR2 (YAS), and CDR3 (QDYTSPRT; SEQ ID NO:61) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63); or the light chain CDR sequences CDR1 (ASQSVSHDV; SEQ ID NO:54), CDR2 (YTS), and CDR3 (QDYSSPRT; SEQ ID NO:65) and the heavy chain CDR sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD163 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from YYAS, YSG, YTS, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD163 antibody that competes with any one of the aforementioned antibodies for binding to the CD163. Antibodies comprising said CDR sequences have been disclosed in U.S. Patent Application Publication No. 20120258107 and 20120276193, which are incorporated herein by reference.

(60) In particular embodiments, the antibody has reduced effector function or lacks effector function compared to a wild-type or native IgG.sub.1 antibody. Reducing or eliminating effector function may be achieved by providing an antibody with an IgG.sub.4 framework or constant domain. In one embodiment, the IgG.sub.4 constant domain may differ from the native human IgG.sub.4 constant domain (Swiss-Prot Accession No. P01861.1) at a position corresponding to position 228 in the EU system and position 241 as determined in the KABAT numbering scheme (See, e.g., Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.)), where the native Ser.sub.108 is replaced with Pro, in order to prevent a potential inter-chain disulfide bond between Cys.sub.106 and Cys.sub.109 (corresponding to positions Cys.sub.226 and Cys.sub.229 in the EU system and positions Cys.sub.239 and Cys.sub.242 in the KABAT system) that could interfere with proper intra-chain disulfide bond formation (See Angal et al. Mol. Imunol. 30:105 (1993)). In other instances, a modified IgG1 constant domain which has been modified to increase half-life or reduce effector function can be used.

(61) In particular aspects, the antibody that has reduced or lacks effector function is an aglycosylated antibody that lacks the N-glycan at position 297 of the heavy chain (as determined using the KABAT Numbering scheme). Aglycosylated antibodies may be produced in a prokaryote expression system, for example, E. coli. The antibody may be encoded by a nucleic acid molecule that introduces an amino acid substitution in any of positions 297-299 of the heavy chain such that the antibody is substantially aglycosylated when the nucleic acid molecule is expressed in a mammalian cell. In IgG.sub.1, the glycosylation site is Asn.sub.297 within the amino acid sequence QYNS (SEQ ID NO:66). In other immunoglobulin isotypes, the glycosylation site corresponds to Asn.sub.297 of IgG1. For example, in IgG.sub.2 and IgG.sub.4, the glycosylation site is the asparagine within the amino acid sequence QFNS (SEQ ID NO:67). Accordingly, a mutation of Asn.sub.297 of IgG.sub.1 removes the glycosylation site in an Fc portion derived from IgG.sub.1. In one embodiment, Asn.sub.297 is replaced with Gln. In other embodiments, the tyrosine within the amino acid sequence QYNS (SEQ ID NO:66) is further mutated to eliminate a potential non-self T-cell epitope resulting from asparagine mutation. As used herein, a T-cell epitope is a polypeptide sequence in a protein that interacts with or binds an MHC class II molecule. For example, the amino acid sequence QYNS (SEQ ID NO:66) within an IgG.sub.1 heavy chain can be replaced with a QAQS (SEQ ID NO:68) amino acid sequence. Similarly, in IgG.sub.2 or IgG.sub.4, a mutation of asparagine within the amino acid sequence QFNS (SEQ ID NO:67) removes the glycosylation site in an Fc portion derived from IgG.sub.2 or IgG.sub.4 heavy chain. In one embodiment, the asparagine is replaced with a glutamine. In other embodiments, the phenylalanine within the amino acid sequence QFNS (SEQ ID NO:67) is further mutated to eliminate a potential non-self T-cell epitope resulting from asparagine mutation. For example, the amino acid sequence QFNS (SEQ ID NO:67) within an IgG.sub.2 or IgG.sub.4 heavy chain can be replaced with a QAQS (SEQ ID NO:68) amino acid sequence.

(62) In particular aspects, the antibody comprises a substitution of one or more of the amino acids at position 318, 320, 322, 234, 235, 236, 237, or 297 of the antibody wherein the antibody with the substitution has a reduced effector function compared to an antibody comprising the native or wild-type amino acid at the position. The effector function may be binding affinity for Clq and/or binding affinity for the Fc receptor. These amino acid substitutions and their effect on reducing effector function have been disclosed in U.S. Pat. No. 5,648,260, which is incorporated herein by reference.

(63) In particular aspects, the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.

(64) Pharmaceutical Formulations and Administration

(65) The conjugates disclosed herein are useful for the manufacture of medicaments for the treatment of diseases or disorders such as an inflammatory disease or cancer. The conjugates disclosed herein may be formulated into pharmaceutical formulations for use in treating diseases or disorders such as an inflammatory disease or cancer.

(66) The present invention provides a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable carrier. The compounds described herein including pharmaceutically acceptable carriers such as addition salts or hydrates thereof, can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections.

(67) In particular embodiments, the conjugates of the invention comprising an antibody or antibody fragment as the targeting moiety are administered parenterally, more preferably intravenously. As used herein, the terms “administering” or “administration” are intended to encompass all means for directly and indirectly delivering a compound to its intended site of action. The compounds described herein, or pharmaceutically acceptable salts and/or hydrates thereof, may be administered singly, in combination with other compounds of the invention, and/or in cocktails combined with other therapeutic agents. The choice of therapeutic agents that can be co-administered with the compounds of the invention will depend, in part, on the condition being treated.

(68) The active compound(s) of the invention are administered per se or in the form of a pharmaceutical composition wherein the active compound(s) is in admixture with one or more pharmaceutically acceptable carriers, excipients or diluents. Pharmaceutical compositions for use in accordance with the present invention are typically formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

(69) For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

(70) For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.

(71) Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxyniethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

(72) Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

(73) Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

(74) For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

(75) For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

(76) The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injection is a preferred method of administration for the compositions of the current invention. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

(77) Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

(78) The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

(79) In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or transcutaneous delivery (e.g., subcutaneously or intramuscularly), intramuscular injection or a transdermal patch. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

(80) The following examples are intended to promote a further understanding of the present invention.

Example 1

(81) The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl(2-((8 S,9R,10S,11S,13 S,14 S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (1-4) was as follows.

(82) ##STR00026##

Step A: 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl dihydrogen phosphate (1-1)

(83) To a stirred solution of dexamethasone (0.40 g, 1.02 mmol) in THF (2.0 mL) at −40° C. was added diphosphoryl chloride (0.31 mL, 2.24 mmol) and the resulting mixture was stirred at −40° C. for 1 hr. The reaction was quenched with water, and treated with saturated sodium bicarbonate solution until pH ˜8. The solution was made acidic using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase washed with brine, dried over sodium sulfate and concentrated to give 1-1 as a solid (497 mg, 103%). LRMS (ES) (M+H).sup.+: observed=473.3, calculated=473.4.

Step B: (9H-fluoren-9-yl)methyl (2-((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)ethyl)carbamate (1-2)

(84) The title compound was prepared from N-(9-fluorenylmethoxycarbonyl)ethanolamine according to the protocol outlined in Example 1-1 to afford 1-2. LRMS (ES) (M+H).sup.+: observed=414.3, calculated=414.4

Step C: (9H-fluoren-9-yl)methyl (2-(((((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)ethyl)carbamate (1-3)

(85) To a stirred solution of 1-2 (0.15 g, 0.41 mmol) in DMF (1.2 mL) was added triethylamine (0.06 mL, 0.41 mmol) and CDI (0.17 g, 1.03 mmol). The resulting solution was stirred at room temperature for 30 minutes. To this mixture was added 1-1 (0.19 g, 0.41 mmol) and ZnCl.sub.2 (0.45 g, 3.31 mmol) and the mixture was allowed to stir at room temperature overnight. The reaction was diluted with 1 N HCl and extracted several times with ethyl acetate. The combined organic layers were concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-35% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 1-3 as a solid (134 mg, 40%). LRMS (ES) (M+H).sup.+: observed=818.6, calculated=818.7.

Step D: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11S,13S14S,14S,6R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (1-4)

(86) To a stirred solution of 1-3 (0.19 g, 0.23 mmol) in DCM (3 mL) was added piperidine (0.15 mL, 1.51 mmol) and the resulting mixture was stirred at room temperature for 3 hrs. The solution was concentrated to dryness and redissolved in DCM (2 mL). In a separate vial a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.045 g, 0.25 mmol) in dichloromethane (1 mL) was added HOAT (0.034 g, 0.25 mmol), EDC (0.056 g, 0.30 mmol) and triethylamine (0.1 mL, 0.68 mmol). The resulting solution was stirred at room temperature for 40 minutes. The two solutions were combined and stirred at room temperature. Additional 2-(cyclooct-2-yn-1-yloxy)acetic acid activated with HOAT/EDC was added as necessary to complete reaction. Upon completion, the mixture was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-30% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 1-4 as a solid (59 mg, 34%). LRMS (ES) (M+H).sup.+: observed=760.6, calculated=760.7. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.64 (br s, 1H), 7.29 (d, J=9.15 Hz, 1H), 6.21 (dd, J=10.05, 1.93 Hz, 1H), 6.00 (s, 1H), 4.57 (d, J=8.3 Hz, 2H), 4.31 (t, J=5.4 Hz, 1H), 4.12 (d, J=11.22 Hz, 1H), 3.92 (dd, J=14.43, 8.59 Hz, 1H), 3.80-3.76 (complex, 3H), 3.30-3.16 (complex, 2H), 3.02-2.91 (complex, 2H), 2.63 (m, 1H), 2.40-2.19 (complex, 3H), 2.17-2.03 (complex, 4H), 1.96-1.82 (m, 3H), 1.80-1.72 (complex, 3H), 1.66-1.52 (complex, 4H), 1.50 (s, 3H), 1.40-1.31 (complex, 2H), 1.07-1.02 (m, 1H), 0.88 (s, 3H), 0.77 (d, J=7.17 Hz, 3H)

Example 2

(87) The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11 S,13 S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) trihydrogen triphosphate (2-7) was as follows.

(88) ##STR00027## ##STR00028##

Step A: 1,3-dioxoisoindolin-2-yl 2-(cyclooct-2-yn-1-yloxy)acetate (2-1)

(89) To a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.20 g, 1.10 mmol) in DCM (4.0 mL) was added N-hydroxyphthalimide (0.36 g, 2.20 mmol) and EDC (0.42 g, 2.20 mmol). The resulting mixture was stirred at room temperature for 45 minutes. The reaction was directly injected onto a silica gel column and flash column separation using a 0-50% ethyl acetate/hexane gradient gave 2-1 as a solid (335 mg, 93%)

Step B: (9H-fluoren-9-yl)methyl dihydrogen phosphate (2-2)

(90) The title compound was prepared from (9H-fluoren-9-yl)methanol according to the protocol outlined in Example 1-1 to afford 2-2. LRMS (ES) (M+H).sup.+: observed=277.1, calculated=276.2

Step C: ((9H-fluoren-9-yl)methyl) (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (2-3)

(91) The title compound was prepared from 2-2 and 1-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 2-3. LRMS (ES) (M+H).sup.+: observed=731.2, calculated=730.6

Step D: 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl trihydrogen diphosphate (2-4)

(92) To a stirred solution of 2-3 (0.29 g, 0.39 mmol) in DCM (2 mL) was added piperidine (0.23 mL, 2.36 mmol) and the resulting mixture was stirred at room temperature for 80 minutes. The solution was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 3-25% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 2-4 as a solid (123 mg, 56%). LRMS (ES) (M+H).sup.+: observed=553.2, calculated=552.4

Step E: (9H-fluoren-9-yl)methyl (2-(((((((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)ethyl)carba mate (2-5)

(93) The title compound was prepared from 2-4 and 1-2 according to the protocol outlined in Example 1 to produce 1-3 to afford 2-5. LRMS (ES) (M+H).sup.+: observed=898.3, calculated=897.7

Step F: 2-amionethyl (2-((8S,9R,10S,1 S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) trihydrogen triphosphate (2-6)

(94) The title compound was prepared from 2-5 according to the protocol outlined in Example 2 to produce 2-4 to afford 2-6. LRMS (ES) (M+H).sup.+: observed=676.2, calculated=675.4

Step G: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11 S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) trihydrogen triphosphate (2-7)

(95) To a stirred solution of 2-6 (0.027 g, 0.04 mmol) in DMF (0.8 mL) was added triethylamine (0.02 mL, 0.16 mmol) and 2-1 and the resulting mixture was stirred at room temperature for 30 minutes. The solution was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-40% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 2-7 as a solid (8 mg, 24%). .sup.1H NMR (499 MHz, DMSO): 0.78 (d, J=7.1 Hz, 3H); 0.88 (s, 3H); 1.10-0.99 (complex, 1H); 1.44-1.28 (complex, 2H); 1.50 (s, 3H); 1.67-1.52 (complex, 4H); 1.80-1.67 (complex, 3H); 1.93-1.83 (m, 3H); 2.17-2.01 (complex, 3H); 2.42-2.17 (complex, 3H); 2.67-2.57 (complex, 1H); 2.81 (d, J=79.7 Hz, 1H); 3.02-2.91 (complex, 1H); 3.17 (s, 1H); 3.26-3.21 (complex, 2H); 3.84-3.74 (complex, 2H); 3.91 (d, J=14.5 Hz, 1H); 4.15 (d, J=11.4 Hz, 1H); 4.31 (t, J=5.1 Hz, 1H); 4.57 (dd, J=18.0, 8.1 Hz, 1H); 4.71 (dd, J=17.9, 7.1 Hz, 1H); 5.99 (s, 1H); 6.20 (d, J=10.1 Hz, 1H); 7.30 (d, J=10.6 Hz, 1H); 8.48 (s, 1H). LRMS (ES) (M+H).sup.+: observed=840.4, calculated=839.6

Example 3

(96) The synthesis of dexamethasone linker 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (2-((8 S,9R,10S,11S,13 S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (3-4) was as follows.

(97) ##STR00029## ##STR00030##

Step A: (9H-fluoren-9-yl)methyl (4-hydroxyphenyl)carbamate (3-1)

(98) To a stirred solution of 4-aminophenol (0.30 g, 2.75 mmol) in DCM (9 mL) was added (9H-fluoren-9-yl)methyl carbonochloridate (0.71 g, 2.75 mmol) and the resulting mixture was stirred at room temperature for 2 hours. The mixture was partitioned between ethyl acetate and 1 N HCl solution. To the organic phase was added methanol until the solution cleared. The organic phase was dried over sodium sulfate and concentrated onto silica gel and flash column separation using a 100% ethyl acetate gave 3-1 as a solid (634 mg, 70%). LRMS (ES) (M+H).sup.+: observed=332.3, calculated=331.3

Step B: (9H-fluoren-9-yl)methyl (4-(phosphonooxy)phenyl)carbamate (3-2)

(99) To a stirred solution of 3-1 (0.31 g, 0.95 mmol) in THF (1.9 mL) at −40° C. was added diphosphoryl chloride (0.31 mL, 2.24 mmol) and triethylamine (1.32 mL, 9.51 mmol) and the resulting mixture was stirred at −40° C. for 3 hr. The reaction was quenched with water, and treated with saturated sodium bicarbonate solution until pH ˜8. The solution was made acidic using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase washed with brine, dried over sodium sulfate and concentrated to give 3-2 as a solid (342 mg, 87%). LRMS (ES) (M+H).sup.+: observed=412.3, calculated=411.3

Step C: (9H-fluoren-9-yl)methyl (4-(((((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)phenyl)carbamate (3-3)

(100) The title compound was prepared from 3-2 and 1-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 3-3. LRMS (ES) (M+H).sup.+: observed=866.5, calculated=865.7

Step D: 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (2-((8S,9R,10S,11S,13S,14S,14S,6R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (3-4)

(101) The title compound was prepared from 3-3 according to the protocol outlined in Example 1 to produce 1-4 to afford 3-4. LRMS (ES) (M+H).sup.+: observed=808.4, calculated=807.7

Example 4

(102) The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8 S,9R,10S,11S,13 S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate (4-3) was as follows.

(103) ##STR00031##

Step A: tert-butyl (2-(((2-((8S,9R,10S,11 S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy)ethyl)carbamate (4-1)

(104) To a stirred solution of dexamethasone (0.10 g, 0.26 mmol) in THF (0.5 mL) at −40° C. was added diphosphoryl chloride (0.12 g, 0.48 mmol) and the resulting mixture was stirred at −40° C. for 1 hr. To this was added tert-butyl N-(2-hydroxyethyl)carbamate (0.12 g, 0.76 mmol) and triethylamine (0.14 mL, 1.0 mmol). The resulting mixture was stirred at −40° C. for 4 hr. The reaction was quenched with water, and treated with saturated sodium bicarbonate solution until pH ˜8. The solution was made acidic using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase was concentrated onto silica gel. Flash column separation using a 0-10% isopropanol/dichloromethane gradient gave 4-1 as a solid (115 mg, 73%). LRMS (ES) (M+H).sup.+: observed=616.5, calculated=616.6.

Step B: 2-aminoethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate, HCl (4-2)

(105) To a stirred solution of 4-1 (0.11 g, 0.18 mmol) in ethyl acetate (1 mL) at 0° C. was bubbled in HCl gas until saturated. The resulting solution was stirred at 0° C. for 1 hr and concentrated to give 4-2 as a solid (99 mg, 100%). LRMS (ES) (M+H).sup.+: observed=516.4, calculated=516.5.

Step C: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate (4-3)

(106) To a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.036 g, 0.20 mmol) in dichloromethane (1 mL) was added HOAT (0.027 g, 0.20 mmol), EDC (0.041 g, 0.22 mmol) and triethylamine (0.05 mL, 0.36 mmol). The resulting solution was stirred at room temperature for 40 minutes. This solution was added to 4-2 (0.10 g, 0.18 mmol) in DCM (1 mL). Additional 2-(cyclooct-2-yn-1-yloxy)acetic acid activated with HOAT/EDC was added as necessary to complete reaction. Upon completion, the mixture was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 4-3 as a solid (45 mg, 37%). LRMS (ES) (M+H).sup.+: observed=680.6, calculated=680.7. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 8.05 (br t, J=5.6 Hz, 1H), 7.30 (d, J=10.15 Hz, 1H), 6.21 (d, J=10.12 Hz, 1H), 6.00 (s, 1H), 5.55 (s, 1H), 5.37 (s, 1H), 4.70 (dd, J=17.2, 6.7 Hz, 1H), 4.29 (br t, J=6.4 Hz, 1H), 4.18-4.10 (complex, 2H), 3.87 (d, J=14.8 Hz, 1H), 3.74 (d, J=14.8 Hz, 1H), 3.69-3.64 (complex, 2H), 3.27-3.18 (complex, 2H), 2.91 (m, 1H), 2.61 (m, 1H), 2.39-2.05 (complex, 7H), 1.97-1.83 (complex, 2H), 1.80-1.69 (complex, 3H), 1.64-1.52 (complex, 3H), 1.48 (s, 3H), 1.47-1.30 (complex, 3H), 1.05 (m, 1H), 0.85 (s, 3H), 0.76 (d, J=7.13 Hz, 3H).

Example 5

(107) The synthesis of dexamethasone linker 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (2-((8 S,9R,10S,11S,13 S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate (5-3) was as follows.

(108) ##STR00032##

Step A: tert-butyl (4-(((2-((8S,9R,10S,11 S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy)phenyl)carbamate (5-1)

(109) To a stirred solution of dexamethasone (0.20 g, 0.51 mmol) in THF (1.0 mL) at −40° C. was added diphosphoryl chloride (0.24 g, 0.97 mmol) and the resulting mixture was stirred at −40° C. for 1 hr 15 min. To this was added N-BOC-4-aminophenol (0.32 g, 1.53 mmol) and triethylamine (0.56 mL, 4.0 mmol). The resulting mixture was stirred at −40° C. for 30 minutes. The reaction was quenched with water, and treated with saturated sodium bicarbonate solution until pH ˜8. The solution was made acidic using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase was concentrated onto silica gel. Flash column separation using a 0-70% isopropanol/dichloromethane gradient gave 5-1 as a solid (370 mg, 88%). LRMS (ES) (M+H).sup.+: observed=664.5, calculated=664.6.

Step B: 4-aminophenyl (2-((8S,9R,10S,11 S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cycl openta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate, HCl (5-2)

(110) The title compound was prepared from 5-1 according to the protocol outlined in Example 4 to produce 4-2 to afford 2-2. LRMS (ES) (M+H).sup.+: observed=564.4, calculated=564.5

Step C: 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate (5-3)

(111) The title compound was prepared from 5-2 according to the protocol outlined in Example 4 to produce 4-3 to afford 5-3. LRMS (ES) (M+H).sup.+: observed=728.6, calculated=728.7 .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.53 (s, 1H), 7.43 (d, J=8.54 Hz, 2H), 7.30 (d, J=10.14 Hz, 1H), 7.04 (d, J=8.54 Hz, 2H), 6.22 (dd, J=10.08, 1.92 Hz, 1H), 6.00 (s, 1H), 5.41 (s, 1H), 5.37 (d, J=4.22 Hz, 1H), 4.78 (dd, J=17.50, 6.20 Hz, 1H), 4.37 (t, J=5.59 Hz, 1H), 4.27 (dd, J=17.47, 9.02, 1H), 4.15-4.12 (m, 1H), 4.06 (d, J=14.53 Hz, 1H), 3.94 (d, J=14.59 Hz, 1H), 3.05 (q, J=7.26 Hz, 1H), 2.92 (m, 1H), 2.61 (m, 1H), 2.40-2.06 (complex, 7H), 1.98 (m, 1H), 1.87 (m, 1H), 1.82-1.73 (complex, 3H), 1.67-1.52 (complex, 3H), 1.48 (s, 3H), 1.43 (d, J=13.54 Hz, 2H), 1.35 (m, 1H), 1.05 (m, 1H), 0.84 (s, 3H), 0.76 (d, J=7.14 Hz, 3H).

Example 6

(112) The synthesis of dexamethasone linker 2-((8 S,9R,10S,11 S,13 S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl hydrogen (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphoramidate (6-2) was as follows.

(113) ##STR00033##

Step A: N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (6-1)

(114) To a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.10 g, 0.55 mmol) in dichloromethane (2 mL) was added HOAT (0.075 g, 0.55 mmol) and EDC (0.126 g, 0.66 mmol). The resulting solution was stirred at room temperature for 20 minutes. This solution was added to 1,2-ethylenediamine (0.49 g, 8.23 mmol) in DCM (1 mL) dropwise. The mixture was concentrated and purified. (Phenomenex Gemini NX C18, 5 um particle size, 21.2 mm i.d. by 5 cm length, 10-50% CH3CN/water w/0.1% NH4OH modifier over 10 min at 40 mL/min) (50 mg, 40%).

Step B: 2-((8S,9R,10S,11 S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl hydrogen (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphoramidate (6-2)

(115) To a stirred solution of 6-1 (0.05 g, 0.22 mmol) and 1-1 (0.035 g, 0.074 mmol) in a solution of t-butanol (1.2 mL) and water (0.25 mL) was added DCC (0.06 g, 0.30 mmol) and the resulting mixture was heated to 100 C for 4 hr. The reaction mixture was allowed to cool and concentrated. The residue was dissolved in a 1:1:1 MeOH:water:MeCN solution and syringe filtered. The mixture was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-45% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 6-2 as a solid (15 mg, 30%). LRMS (ES) (M+H).sup.+: observed=679.5, calculated=678.7.

Example 7

(116) The synthesis of dexamethasone linker 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl (2-((8 S,9R,10S,11S,13 S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (7-1) was as follows.

(117) ##STR00034##

(118) To a stirred solution of 1-3 (0.19 g, 0.23 mmol) in DCM (3 mL) was added piperidine (0.15 mL, 1.51 mmol) and the resulting mixture was stirred at room temperature for 3 hrs. The solution was concentrated to dryness. The crude mixture was taken into a 2:1:1 methanol:acetonitrile:water mixture and filtered. The filtrate was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-35% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min). A portion of the resulting purified amine (0.07 g, 0.11 mmol) was dissolved in DMF (0.8 mL). To this solution was added 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (0.09 mg, 0.34 mmol) and DMAP (0.014 g, 0.11 mmol) and the resulting solution was stirred 20 minutes. The crude reaction mixture was directly purified using reverse phase preparative chromatography (Sunfire Prep C18 OBD 5 um 30×150 mm; 10-35% CH3CN/water w/0.1% TFA modifier over 20 min) gave 7-1 as a solid (13 mg, 15%). LRMS (ES) (M+H).sup.+: observed=747.2, calculated=746.6.

Example 8

(119) The synthesis of dexamethasone linker ((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((2-((8 S,9R,10S,11 S,13 S,14 S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)(hydroxy)phosphoryl)oxy)-4-hydroxytetrahydrofuran-2-yl)methyl (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)carbamate (8-5) was as follows.

(120) ##STR00035## ##STR00036##

Step A: (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-(2H)-yl)tetrahydrofuran-3-yl (2-cyanoethyl) (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) phosphite (8-1)

(121) To a stirred mixture of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (1.20 g, 1.40 mmol) and dexamethasone (0.50 g, 1.27 mmol) in acetonitrile (12 mL) was added 5-(ethylthio)-1H-tetrazole (0.33 g, 2.55 mmol). The resulting mixture was stirred for 20 minutes. To the homogenous solution that resulted was added 5M tert-butyl hydroperoxide (0.51 mL, 2.55 mmol). The reaction was stirred 1 hr at room temperature and concentrated onto silica gel. Flash column separation using a 0-100% ethyl acetate/hexane gradient gave 8-1 as a solid (1.67 g, 100%) LRMS (ES) (M+H).sup.+: observed=1168.3, calculated=1168.3.

Step B: (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl (2-cyanoethyl) (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) phosphate (8-2)

(122) To a stirred solution of 8-1 (1.48 g, 1.26 mmol) in DCM (30 mL) was added TFA (0.3 mL, 3.89 mmol) at room temperature. The reaction was stirred for 30 minutes, washed with saturated bicarbonate solution and the organic phase was concentrated onto silica gel. Flash column separation using a 0-10% isopropanol/DCM gradient gave 8-2 as a solid (0.79 g, 72%) LRMS (ES) (M+H).sup.+: observed=866.5, calculated=865.9.

Step C: ((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3-(((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methyl (4-nitrophenyl) carbonate (8-3)

(123) To a stirred solution of 8-2 (0.30 g, 0.35 mmol) in DCM (5 mL) was added triethylamine (0.15 mL, 1.04 mmol) and 4-nitrophenyl carbonochloridate (0.15 g, 0.76 mmol) and the resulting solution was stirred at room temperature. Additional 4-nitrophenyl carbonochloridate was added until reaction was complete by LCMS. The reaction was directly loaded onto a silica gel column and flash column separation using a 0-50% isopropanol/DCM gradient gave 8-3 as a solid (0.32 g, 93%) LRMS (ES) (M+H).sup.+: observed=978.4, calculated=977.9.

Step D: ((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3-(((2-((8S,9R,10S,1 S,3S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methyl (2-aminoethyl)carbamate (8-4)

(124) To a stirred solution of ethylenediamine (0.28 mL, 4.17 mmol) in DMF (1 mL) was added a solution of 8-3 (0.20 g, 0.20 mmol) in DMF (1 mL) dropwise. The reaction was stirred at room temperature for 10 minutes, then purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 8-4 as a solid (115 mg, 61%). LRMS (ES) (M+H).sup.+: observed=899.5, calculated=898.9.

Step E: ((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)(hydroxy)phosphoryl)oxy)-4-hydroxytetrahydrofuran-2-yl)methyl (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)carbamate (8-5)

(125) The title compound was prepared from 8-4 according to the protocol outlined in Example 4 to produce 4-3 to afford 8-5. LRMS (ES) (M+H).sup.+: observed=949.4, calculated=948.9

Example 9

(126) The synthesis of Budesonide linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((6aR,6bS,7S,8aS,8bS,11 aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (9-4) was as follows.

(127) ##STR00037## ##STR00038##

Step A: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl dihydrogen phosphate (9-1)

(128) The title compound was prepared from budesonide according to the protocol outlined in Example 1 to produce 1-1 to afford 9-1. LRMS (ES) (M+H).sup.+: observed=511.2, calculated=510.5

Step B: (9H-fluoren-9-yl)methyl (2-((hydroxy((hydroxy(2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)phosphoryl)oxy)ethyl)carbamate (9-2)

(129) The title compound was prepared from 9-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 9-2. LRMS (ES) (M+H).sup.+: observed=856.3, calculated=855.8

Step C: 2-aminoethyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (9-3)

(130) The title compound was prepared from 9-2 according to the protocol outlined in Example 2 to produce 2-6 to afford 9-3. LRMS (ES) (M+H).sup.+: observed=634.3, calculated=633.5

Step D: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (9-4)

(131) The title compound was prepared from 9-3 according to the protocol outlined in Example 2-7 to afford 9-4. Mixture of isomers .sup.1H NMR (499 MHz, DMSO): 0.86-0.83 (complex, 6 H); 0.89 (s, 6H); 0.99-0.90 (complex, 2H); 1.09 (t, J=7.0 Hz, 2H); 1.15 (t, J=7.2 Hz, 6H); 1.35-1.25 (complex, 6H); 1.39 (s, 6H); 2.32-1.46 (complex, 32H); 3.02 (q, J=7.0 Hz, 4H); 3.21 (br s, 2H); 3.81-3.73 (complex, 6H); 3.94-3.89 (complex, 2H); 4.33-4.25 (complex, 4H); 4.39 (d, J=18.4 Hz, 2H); 4.56 (t, J=4.3 Hz, 1H); 4.79-4.63 (complex, 3H); 5.01 (d, J=7.2 Hz, 1H); 5.17 (dd, J=4.9, 4.6 Hz, 1H); 5.91 (s, 2H); 6.15 (d, J=10.1 Hz, 2H); 7.31 (d, J=10.3 Hz, 2H); 8.84 (br s, 2H). LRMS (ES) (M+H).sup.+: observed=798.4, calculated=797.7

Example 10

(132) The synthesis of Budesonide linker 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl (2-((6aR,6bS,7S,8aS,8bS,11 aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (10-1) was as follows.

(133) ##STR00039##

(134) The title compound was prepared from 9-2 according to the protocol outlined in Example 7 to produce 7-1 to afford 10-1. LRMS (ES) (M+H).sup.+: observed=785.4, calculated=784.6

Example 11

(135) The synthesis of Budesonide linker 1-(cyclooct-2-yn-1-yloxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl (2-((6aR,6bS,7S,8aS,8bS,11 aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (11-5) was as follows.

(136) ##STR00040## ##STR00041##

Step A: (9H-fluoren-9-yl)methyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (11-1)

(137) To a stirred solution of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol (1.00 g, 5.17 mmol) in DCM (15 mL) was added 9-fluorenylmethyl chloroformate (1.34 g, 5.17 mmol) and triethylamine (1.08 mL, 7.76 mmol). The resulting solution was stirred at room temperature for 10 minutes. The reaction was concentrated onto silica gel and flash column separation using a 0-10% isopropanol/dichloromethane gradient gave 11-1 as an oil (1.43 g, 66%) LRMS (ES) (M+H).sup.+: observed=416.1, calculated=415.4.

Step B: (9H-fluoren-9-yl)methyl (2-(2-(2-(2-(phosphonooxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (11-2)

(138) The title compound was prepared from 11-1 according to the protocol outlined in Example 1 to produce 1-1 to afford 11-2. LRMS (ES) (M+H).sup.+: observed=496.3, calculated=495.4

Step C: (9H-fluoren-9-yl)methyl (2-(2-(2-(2-((hydroxy((hydroxy(2-((6aR,6bS,7S,8aS,8bS,1 aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)phosphoryl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (11-3)

(139) The title compound was prepared from 11-2 and 9-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 11-3. LRMS (ES) (M+H).sup.+: observed=988.6, calculated=987.9

Step D: 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-di][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (11-4)

(140) The title compound was prepared from 11-3 according to the protocol outlined in Example 2 to produce 2-6 to afford 11-4. LRMS (ES) (M+H).sup.+: observed=766.5, calculated=765.7

Step E: 1-(cyclooct-2-yn-1-yloxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (11-5)

(141) The title compound was prepared from 11-4 according to the protocol outlined in Example 2 to produce 2-7 to afford 11-5. LRMS (ES) (M+H).sup.+: observed=930.6, calculated=929.9

Example 12

(142) The synthesis of Budesonide linker 4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((6aR,6b S,7S,8aS,8b S,11aR,12aS,12b S)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) hydrogen phosphate (12-3) was as follows.

(143) ##STR00042## ##STR00043##

Step A: (9H-fluoren-9-yl)methyl ((2S)-1-(((2S)-1-((4-((((2-cyanoethoxy) (2 ((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (12-1)

(144) To a stirred solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (0.20 g, 0.33 mmol) in DMF (4.7 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (0.11 g, 0.37 mmol). To this mixture was added 0.45M tetrazole in acetonitrile (0.81 mL, 0.37 mmol) dropwise and the resulting mixture was stirred for 20 minutes at room temperature. To this was added Budesonide (0.22 g, 0.50 mmol) and 5-(ethylthio)-1H-tetrazole (0.09 g, 0.67 mmol) and allowed to stir to 30 minutes at room temperature. To this was added 6 M tertbutyl hydroperoxide in decane (0.12 mL, 0.73 mmol) and allowed to stir at room temperature for 1 hour. The crude reaction was loaded directly onto silica gel and flash column separation using a 0-100% ethyl acetate/hexane gradient followed by a 0-50% isopropanol/DCM gradient gave 12-1 as a solid. (0.10 g, 27%) LRMS (ES) (M+H).sup.+: observed=1147.7, calculated=1147.2.

Step B: 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2((6aR,6bS,7S,8aS,8bS,11aR,2aS,2bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) hydrogen phosphate (12-2)

(145) To a stirred solution of 12-1 (0.10 g, 0.09 mmol) in DCM (1.8 mL) was added DBU (0.05 mL, 0.35 mmol) and the resulting solution was stirred 20 minutes at room temperature. The reaction was concentrated and dissolved in a 2:1:1 methanol:water:acetonitrile mixture and syringe filtered. The filtrate was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 12-2 as a solid (42 mg, 53%). LRMS (ES) (M+H).sup.+: observed=872.6, calculated=871.9.

Step C: 4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) hydrogen phosphate (12-3)

(146) The title compound was prepared from 12-2 according to the protocol outlined in Example 4 to produce 4-3 to afford 12-3. LRMS (ES) (M+H).sup.+: observed=1036.8, calculated=1036.1

Example 13

(147) The synthesis of Budesonide linker 2-((6aR,6bS,7S,8aS,8bS,11 aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-1) was as follows.

(148) ##STR00044## ##STR00045##

Step A: synthesis 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-1)

(149) To a stirred solution of budesonide (2.00 g, 4.65 mmol) in pyridine (20.0 mL) at room temperature was added acetic anhydride (2.0 mL, 21.20 mmol) and the resulting mixture was stirred for 2.5 hours. The reaction was chilled in an ice bath and quenched with saturated sodium bicarbonate solution (20.0 mL). The solution was extracted several times with ethyl acetate. The combined organic phase washed with brine, dried over sodium sulfate and concentrated to give 13-1 as a solid (2.30 g, 105%). LRMS (ES) (M+H).sup.+: observed=473.4, calculated=472.5.

Step B: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-((hydroxyhydrophosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-2)

(150) To a stirred solution of 13-1 (0.50 g, 1.06 mmol) in THF (10.0 mL) at −78° C. was added phosphorus trichloride (0.18 mL, 2.12 mmol) dissolved in THF (2.0 mL) followed by triethylamine (0.74 mL, 5.29 mmol) dissolved in THF (2.0 mL). The resulting mixture was stirred at −78° C. for 10 minutes and allowed to warm to room temperature for 45 minutes. The reaction was chilled in an ice bath and quenched with water (0.50 mL). The solution was allowed to warm to room temperature and saturated sodium bicarbonate solution was added until pH 9 and stirred for 10 minutes. The mixture was acidified with 1N HCl and was extracted several times with ethyl acetate. The combined organic phase was dried over sodium sulfate and concentrated to give 13-2 as a solid (0.55 g, 97%). LRMS (ES) (M+H).sup.+: observed=537.3, calculated=536.5.

Step C: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-((hydroxy(1H-imidazol-1-yl) phosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-3)

(151) To a stirred solution of 13-2 (0.55 g, 1.03 mmol) and imidazole (0.35 g, 5.13 mmol) in pyridine (8.0 mL) at room temperature was added TMS-Cl (1.31 mL, 10.25 mmol) and the resulting solution was stirred for 10 minutes. To this mixture was added iodine (0.52 g, 2.05 mmol) dissolved in pyridine (2 mL) and stirred room temperature for 50 minutes. The reaction was then cooled in and ice bath and quenched with water (0.5 mL). The reaction was concentrated, dissolved in aqueous acetonitrile and purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 μM 30×100 mm; 10-50% MeCN/water w/0.10% NH.sub.4OH modifier over 20 min) to give 13-3 as a solid (282 mg, 45%). LRMS (ES) (M+H).sup.+: observed=603.4, calculated=602.6.

Step D: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-(((((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-4)

(152) To a stirred solution of 13-3 (0.20 g, 0.33 mmol) and 1-2 (0.12 g, 0.33 mmol) in DMF (1.4 mL) was added ZnCl.sub.2 (0.36 g, 2.66 mmol) and the mixture was allowed to stir at room temperature overnight. The reaction was diluted with 1 N HCl and extracted several times with ethyl acetate. The combined organic layers were concentrated, dissolved in aqueous acetonitrile and purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 μM 30×100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 13-4 as a solid (166 mg, 55%). LRMS (ES) (M+H).sup.+: observed=898.4, calculated=897.8.

Step E: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-(((((2-aminoethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7, 8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1, 3]dioxol-8b-yl)-2-oxoethyl acetate (13-5)

(153) The title compound was prepared from 13-4 according to the protocol outlined in Example 2 to prepare 2-4 to afford 13-5. LRMS (ES) (M+H).sup.+: observed=676.4, calculated=675.6.

Step F: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-(((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-6)

(154) To a stirred solution of 13-5 (0.037 g, 0.055 mmol) and 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.032 g, 0.175 mmol) in DMF (0.8 mL) was added HATU (0.066 g, 0.175 mmol) and triethylamine (0.03 mL, 0.22 mmol). The reaction was stirred at room temperature for 20 minutes, then purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 μM 30×100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 13-6 as a solid (36 mg, 78%). LRMS (ES) (M+H).sup.+: observed=840.5, calculated=839.8.

Step G: 2-((6aR,6bS,7S,8aS,8bS,11 aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-7)

(155) To a stirred solution of 13-6 (0.035 mg, 0.042 mmol) in methanol (0.50 mL) was added 70% perchloric acid (7.2 μL, 0.083 mmol) and the resulting solution was stirred room temperature overnight. The reaction was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 μM 30×100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 13-7 as a solid (16 mg, 47%). LRMS (ES) (M+H).sup.+: observed=798.4, calculated=797.7.

Example 14

(156) The synthesis of Fluticasone linker (6S,8S,9R,10S,11 S,13 S,14S,16R,17R)-11-(((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (14-5) was as follows.

(157) ##STR00046##

Step A: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-((hydroxyhydrophosphoryl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-ylpropionate (14-1)

(158) The title compound was prepared from fluticasone propionate according to the protocol outlined in Example 13 to prepare 13-2 to afford 14-1. LRMS (ES) (M+H).sup.+: observed=565.3, calculated=564.5.

Step B: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (14-2)

(159) The title compound was prepared from 14-1 according to the protocol outlined in Example 13 to prepare 13-3 to afford 14-2. LRMS (ES) (M+H).sup.+: observed=631.3, calculated=630.6.

Step C: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (14-3)

(160) The title compound was prepared from 14-2 according to the protocol outlined in Example 13 to prepare 13-4 to afford 14-3. LRMS (ES) (M+H).sup.+: observed=943.4 (M+H+NH.sub.3), calculated=925.8.

Step D: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-aminoethoxy)(hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (14-4)

(161) The title compound was prepared from 14-3 according to the protocol outlined in Example 2 to prepare 2-4 to afford 14-4. LRMS (ES) (M+H).sup.+: observed=704.3, calculated=703.6.

Step F: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-ylpropionate (14-5)

(162) The title compound was prepared from 14-4 according to the protocol outlined in Example 13 to prepare 13-6 to afford 14-5. LRMS (ES) (M+H).sup.+: observed=868.4, calculated=867.8.

Example 15

(163) The synthesis of Fluticasone linker (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy) phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-7-yl propionate (15-5) was as follows.

(164) ##STR00047##

Step A: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-1-((methylthio)methoxy)-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-1)

(165) To a stirred solution of fluticasone propionate (0.50 g, 1.00 mmol) in MeCN (5.0 mL) at 0° C. was added dimethyl sulfide (0.59 mL, 8.00 mmol) followed by benzoyl peroxide (0.97 g, 4.00 mmol) added in four portions over 20 minutes. The resulting mixture was stirred at 0° C. for 1 hour. The reaction was concentrated, taken up in ethyl acetate and washed with saturated sodium bicarbonate. The combined organic phase was concentrated. The crude was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 40-80% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give to give 15-1 as a solid (0.07 g, 12.7%). LRMS (ES) (M+H).sup.+: observed=561.3, calculated=560.6.

Step B: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-11-((phosphonooxy)methoxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-2)

(166) Phosphoric acid (0.09 g, 0.89 mmol) was heated under nitrogen at 120° C. for 30 minutes. This was allowed to cool and to it was added molecular seives and 15-1 (0.07 g, 0.13 mmol). This mixture was dissolved in THF (1.3 mL) and NIS (0.04 g, 0.19 mmol) was added. The resulting solution was allowed to stir overnight at room temperature. The mixture was filtered and concentrated. The crude was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give to give 15-2 as a solid (0.05 g, 63%). LRMS (ES) (M+H).sup.+: observed=611.3, calculated=610.5.

Step C: (6S,8S,9R,10S,11 S,13S,14S,16R,17R)-11-((((((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-3)

(167) The title compound was prepared from 15-2 according to the protocol outlined in Example 1 to prepare 1-3 to afford 15-3. LRMS (ES) (M+H).sup.+: observed=956.5, calculated=955.8.

Step D: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-aminoethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-4)

(168) The title compound was prepared from 15-3 according to the protocol outlined in Example 12 to prepare 12-2 to afford 15-4. LRMS (ES) (M+H).sup.+: observed=734.5, calculated=733.6.

Step E: (6S,8S,9R,10S,11 S,13S,14S,16R,17R)-1-((((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-5)

(169) The title compound was prepared from 15-4 according to the protocol outlined in Example 13 to prepare 13-6 to afford 15-5. LRMS (ES) (M+H).sup.+: observed=898.4, calculated=897.8.

Example 16

(170) The synthesis of Budesonide linker 1-(cyclooct-2-yn-1-yloxy)-2-oxo-3-aza-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tricosaoxahenheptacont-74-yl (2-((6aR,6b S,7S,8aS,8b S,11aR,12aS,12b S)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (16-5) was as follows.

(171) ##STR00048## ##STR00049##

Step A: (9H-fluoren-9-yl)methyl (71-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontyl)carbamate (16-1)

(172) The title compound was prepared from 71-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontan-1-ol and (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate according to the protocol outlined in Example 11 to produce 11-1 to afford 16-1. LRMS (ES) (M+H).sup.+: observed=1314.1, calculated=1296.5

Step B: (9H-fluoren-9-yl)methyl (71-(phosphonooxy)-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontyl)carbamate (16-2)

(173) The title compound was prepared from 16-1 according to the protocol outlined in Example 1 to produce 1-1 to afford 16-2. LRMS (ES) (M+H).sup.+: observed=1394.0, calculated=1376.5

Step C: (9H-fluoren-9-yl)methyl (71-((hydroxy((hydroxy(2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)phosphoryl)oxy)-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontyl)carbamate (16-3)

(174) The title compound was prepared from 16-2 and 9-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 16-3. LRMS (ES) (M+H).sup.+: observed=1886.7, calculated=1869.0

Step D: 72-amino-3, 6, 9,12,15,18, 21, 24, 27,30, 33, 36, 39, 42, 45, 48, 51, 54, 57,60, 63, 66, 69-tricosaoxahenheptacontanyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (16-4)

(175) The title compound was prepared from 16-3 according to the protocol outlined in Example 12 to produce 12-2 to afford 16-4. LRMS (ES) (M+H).sup.+: observed=1664.0, calculated=1646.7

Step E: 1-(cyclooct-2-yn-1-yloxy)-2-oxo-3-aza-6, 9,12,15,18, 21, 24,27, 30, 33, 36, 39, 42, 45, 48, 51, 54,57, 60, 63, 66, 69,72-tricosaoxahenheptacont-74-yl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (16-5)

(176) The title compound was prepared from 16-4 according to the protocol outlined in Example 13 to produce 13-6 to afford 16-5. LRMS (ES) (M+H).sup.+: observed=1828.7, calculated=1810.9

Example 17

(177) The synthesis of Budesonide linker 4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((6aR,6b S,7S,8aS,8b S,11 aR,12aS,12b S)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (17-2) was as follows.

(178) ##STR00050##

Step A: 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2((6aR,6bS,7S,8aS,8bS,11aR,12aS,2bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (17-1)

(179) To a stirred solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (0.05 g, 0.083 mmol) in DMF (1.2 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (29 uL, 0.09 mmol). To this mixture was added 0.45M tetrazole in acetonitrile (0.20 mL, 0.09 mmol) dropwise and the resulting mixture was stirred for 20 minutes at room temperature. To this was added 9-1 (0.042 g, 0.083 mmol) and 5-(ethylthio)-1H-tetrazole (0.02 g, 0.16 mmol) and allowed to stir to 30 minutes at room temperature. To this was added 6 M tertbutyl hydroperoxide in decane (0.03 mL, 0.18 mmol) and allowed to stir at room temperature for 30 minutes. To this was added DBU (0.12 mL, 0.83 mmol) and the resulting solution was stirred overnight at room temperature. The crude reaction was purified by direct injection using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-40% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 17-1 as a solid (18.5 mg, 23%). LRMS (ES) (M+H).sup.+: observed=952.6, calculated=951.9.

Step B: 4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-H-naphtho[2′,1′: 4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (17-2)

(180) The title compound was prepared from 17-1 according to the protocol outlined in Example 13 to produce 13-6 to afford 17-2. LRMS (ES) (M+H).sup.+: observed=1116.6, calculated=1116.1.

Example 18

(181) The solubility of exemplary drug-linker conjugates in aqueous solutions was evaluated. Linkers utilized in drug conjugates may have aqueous solubility to enable conjugation in aqueous media amenable to protein solubilization. Furthermore, linkers with aqueous solubility are considered hydrophilic, and may confer to the drug conjugate a reduced propensity to aggregate relative to standard hydrophobic linkers in the literature. The following exemplary drug-linkers were tested in an aqueous solution comprising 20% acetonitrile (MeCN/H.sub.2O) for solubility at a concentration of 10 mg/mL. As shown in Table 1, the exemplary linker-drug conjugates displayed high solubility, which may be a result of the contribution of the polarity and charge of the phosphate containing linker to the drug-linker conjugate.

(182) TABLE-US-00001 TABLE 1 Measured Solubility in Drug Linker 20% MeCN/H.sub.2O 1-4 >10 mg/mL 2-7 >10 mg/mL 3-4 >10 mg/mL 4-3 >10 mg/mL 5-3 >10 mg/mL

Example 19

(183) In Vitro Stability Studies of Exemplary Drug-Linkers in Blood and Lysosomal Lysates.

(184) Exemplary dexamethasone-linkers 4-3, 5-3, 1-4, 3-4, and 2-7 were incubated in relevant biomatrices to measure their stability and propensity to release free drug (Tables 2 to 4).

(185) The exemplary dexamethasone-linkers were studied for their stability (Table 1) and propensity to release dexamethasone (Table 2) in human blood. As shown in the tables, all the dexamethasone linkers were stable in blood with little detectable degradation or release of free dexamethasone.

(186) TABLE-US-00002 TABLE 1 Time Course of Calculated Conc. for each compound in human whole blood (nM) Matrix Time Dexamethasone 4-3 5-3 1.4 3-4 2-7 Matrix 2939 2821 n/a 3059 n/a 1059 spiking 0 m 1687 1539 1299 1617 1382 1040 5 m 1251 1325 10 m  15 m  1276 1398 20 m  2409 1861 1390 30 m  1111 1250 1 hr 2224 1619 937 1421 1141 1515 2 hr 1480 1703 3 hr 1502 1677 1899 6 hr 2792 2040 1400  123

(187) TABLE-US-00003 TABLE-2 Time Course of Calculated Conc. for dexamethasone in human whole blood (nM) Matrix Time Dexamethasone 4-3 5-3 1.4 3-4 2-7 Matrix 2939 4 0 2 0 0 Spiking 0 m 1687 0 0 0 0 0 5 m 0 0 10 m  15 m  0 0 20 m  2409 0 0 30 m  0 0 1 hr 2224 0 0 0 0 0 2 hr 0 0 3 hr 1502 1 0 6 hr 2792 3 0 0
General Experimental Procedure
Human Blood Incubation Human blood was collected the morning of the experiment from at least 3 individuals using K2EDTA as the anticoagulant. An equal volume from each individual was combined for use in the experiment. The experiment started no more than 2 hours after the blood collection. All drug-linker conjugates were solubilized in DMSO to form each 10 mM stock solution. Dosing solution for each linker was prepared by serial dilution of each stock solution using 1:3 acetonitrile:water. All solutions were kept on ice during the experiment.

(188) Human blood was pre-warmed in a 37° C. water bath in an appropriate volume to collect samples over a time course from 0 through 6 hours. Incubating blood was mixed well just prior to sampling to give a homogenous mixture. Aliquots of blood were removed at appropriate time points, added to cold stopping solution, which was methanol containing an appropriate internal standard, and mixed rigorously. The samples were centrifuged at 4000 RPM for 10 minutes after which equal volumes of the supernatant fractions were diluted with cold deionized water. The samples were then ready for analysis. A time 0 sample was prepared by spiking blood, which had been pretreated with the same stopping reagent used above with the drug-linker. This sample is referred to in the tables as the matrix spiking.

(189) Representative dexamethasone-linkers 4-3, 5-3, 1-4, 3-4, and 2-7 were studied for their stability (Table 4) and propensity to release dexamethasone (Table 5) in purified rat liver lysosomal lysates. Tables 4 and 5 show that the different dexamethasone-linkers released dexamethasone at different rates depending on the structure of the tuning element. For example, 3-4 was no longer detectable after 10 minutes incubation in the rat lysosomal extract whereas for 4-3 and 5-3 the dexamethasone was more slowly released with 5-3 releasing dexamethasone faster than 4-3.

(190) TABLE-US-00004 TABLE 3 Time Course of Calculated Conc. for each compound in rat lysomal lysate (nM) Dexa- Matrix Time methasone 4-3 5-3 1.4 3-4 2-7 Matrix 2059.0 1590.7 2555.29 2487.3 3128.83 2228 spiking 0 m 1456.4  888.8 1394.08 1148.2 1252 1327 5 m 1851.2 1156.1 1295.86 158.2 501.28  56 10 m  1640.8  983.4 1278.4   19.0 149.18  512 15 m  1666.7 1032.6 1407.73  3.7 N/A 0 30 m  1602.6  980.1 1175.39 N/A N/A  37 1 hr 1576.8  882.4  970.65 N/A N/A 0 2 hr 1681.0  880.1  743.45 N/A N/A 0 3 hr 1689.7  903.8  500.41 N/A N/A 0 6 hr 1689.8  858.9  221.03 N/A N/A 0

(191) TABLE-US-00005 TABLE 4 Time Course of Calculated Conc. for dexamethasone rat lysomeal lysate (nM) Dexa- Matrix Time methasone 4-3 5-3 1.4 3-4 2-7 Matrix 2059.0 N/A N/A N/A N/A 17 Spiking 0 m 1456.4  0.9 N/A 4.7  15.74  44 5 m 1851.2  14.5  10.25  320.7 100.44  415 10 m 1640.8  14.2  21.56  559.4 209.07  682 15 m 1666.7  22.3  31.68  831.7 285.67  808 30 m 1602.6  28.9 78.6 1059.6 425.28 1039 1 hr 1576.8  41.6 162.45 1148.8 526.5  1159 2 hr 1681.0  72.5 290.66  854.5 534.84 1309 3 hr 1689.7  90.2 379.36  841.9 584.61 1207 6 hr 1689.8 141.9 550.43  826.6 561.88 1155
General Experimental Procedure
Rat Lysosome Incubation.

(192) Rat Liver lysosomes were available commercially with a pool of 6 animals. All linker compounds were solubilized in DMSO to form each 10 mM stock solution. Dosing solution for each linker was prepared by serial dilution of each stock solution using 1:3 acetonitrile: water. All solutions were kept on ice during the experiment.

(193) Rat lysosomes were pre-warmed in a 37° C. water bath in an appropriate volume to collect samples over a time course from 0 minutes through 6 hours. Incubating lysosomes were mixed well just prior to sampling to give a homogenous mixture. Aliquots of lysosomes were removed at appropriate time points, added to cold stopping solution, which was methanol containing an appropriate internal standard, and mixed rigorously. The samples were centrifuged at 4000 RPM for 10 minutes after which equal volumes of supernatant were diluted with cold deionized water. The samples were ready for analysis. A time 0 sample was prepared by spiking the drug-linker to lysosomes which had been pretreated with the same stopping reagent as above. This sample is referred to as matrix spiking in the tables.

(194) Liquid Chromatography—Tandem Mass Spectrometry Analysis

(195) A Thermo LX-2 ultra-performance liquid chromatography system coupled with a Sciex API5000 triple quadrupole mass spectrometer was used for the analysis. The payload and drug-linkers were retained and separated by a C18 column and detected by the mass spectrometer. The standard curve for each analyte was prepared to obtain the quantitative results. Samples were kept in cold stack set at 5° C.

Example 20

(196) Synthesis, Purification and Analysis of ADC Using Exemplary Drug-Linker 1-4

(197) To establish the chemical reactivity of this linker design to form a drug conjugate, exemplary drug-linker 1-4 was conjugated to an anti-mouse CD25 antibody (IgG1) (mCD25) to produce antibody-drug conjugate ADC 12-1 or anti-human CD70 antibody (hCD70) to produce ADC 12-2. Specifically, the drug-linker was conjugated to the unnatural amino acid para-azidophenylalanine (pAzF) replacing the alanine at position 1 of CH1 of the antibody using copper-free 3+2 cycloaddition chemistry. Synthesis of antibodies containing an unnatural amino acid has been described in U.S. Pat. No. 7,632,924, incorporated herein by reference, and copper-free 3+2 cycloaddition chemistry has been described in U.S. Pat. No. 7,807,619, incorporated herein by reference. Conjugation, purification, and analysis confirmed synthesis of the Anti-CD25 antibody-drug conjugate ADC 12-1 and the anti-CD70 ADC 12-2.

(198) Experimental for Conjugation of Phosphate Linker 1-4 to Antibodies.

(199) Antibodies were purified over protein A column (NovaSep) followed by SP 650S column (Tosoh Biosciences). The heavy chain and light chain of the anti-CD25 antibody comprise SEQ ID NO:81 and SEQ ID NO:82, respectively, wherein X is pAzF. The heavy chain and light chain of the anti-CD70 antibody comprise SEQ ID NO:89 and SEQ ID NO:80, respectively, wherein X is pAzF.

(200) Site Specific Conjugation Using Click (2+3) Chemistry.

(201) Para-azidophenylalanine containing antibodies were buffer exchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; 0-20% Dimethylamine, pH 6.0 and concentrated to 1-20 mg/mL. 10-15 molar equivalents of cyclooctyne drug-linker were added and reacted for 16-72 hours at 28-30° C. The antibody conjugates were purified over a SP 650S column (Tosoh Biosciences) to remove excess reagents. The conjugates were buffer exchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; pH 6.0, 0.22 μm filtered, and stored at 4° C.

(202) Conjugation Analysis.

(203) Conjugation efficiency and DAR values were determined by reversed phase HPLC. The ADC was run over a Zorbax 300SB-C3 column, 4.6×150 mm (Agilent) at 80° C. and eluted with a linear gradient from 30% B to 90% B (A: water, 0.1% TFA; B: acetonitrile, 0.1% TFA). An Agilent 1200 series HPLC system and Chemstation software were used to resolve and quantify percentage of antibody conjugated with drug-linker.

Example 21

(204) Serum Stability of ADC 12-1

(205) Drug conjugates may be designed with a stable linker to ensure that the attached payload adopts the pharmacokinetic properties of the carrier. In the example of antibody drug conjugates, premature release of the payload will reduce the total payload delivered to a target cell. To establish the potential for circulatory stability of this linker design in the context of a drug conjugate, ADC 12-1 was incubated in mouse serum and monitored for degradation or loss of payload (dexamethasone). As shown in FIG. 2, no measurable loss of dexamethasone was observed over three weeks incubation in serum.

(206) In Vitro Stability Study Design in DBA1 Mice Serum

(207) For the in vitro stability study, the ADC 12-1 was spiked in DBA1 mice serum at 0.1 mg/mL. Samples were sealed under nitrogen, placed at 37° C. in a cell culture incubator and stored at −80° C. until analysis. Time points from 0 min to 21 days of incubation were evaluated.

(208) In Vitro Stability—Free Payload Analysis

(209) Samples were evaluated for free dexamethasone by LC-MS/MS. For the in vitro stability study, 40 μL of serum for each time point underwent protein precipitation with 400 μL acetonitrile containing dexamethasone-d4 (internal standard). Tubes were centrifuged at 14000 RPM (4° C.) for 10 minutes and the supernatant fraction removed and dried in a speed vac. Samples were reconstituted with methanol/water (50/50) and injected in an Acquity/TSQ Vantage triple quad mass spectrometer equipped with an Xbridge C18 column (Waters, Milford). Mobile phases consisted of buffers A (water:acetonitrile:formic acid, 95:5:0.1%) and B (acetonitrile:water:formic acid, 80:20:0.1%) and a linear gradient was performed with buffer B increasing from 30 to 65% in 3 minutes. Flow rate was set at 0.3 mL/minutes. Transitions for acetate adduct of dexamethasone and dexamethasone d-4 were 437>361 and 441>363, respectively. Compounds were detected using negative electrospray ionization. Free dexamethasone was not be detected in the in vitro stability serum samples over the period evaluated.

(210) In Vitro Stability—Immunoprecipitation Coupled to Intact Mass Analysis

(211) ADC 12-1 was pulled down from mice serum using immunoprecipitation (IP) with streptavidin magnetic beads (Dynabeads M-280) coupled to biotinylated CD25 (antigen). Beads were washed 3 times with 100 μL of TBS 1×. Two microliters of 1 mg/mL biotinylated CD25 was added to 30 μL of each sample (serum from each time point) and incubated for 10 minutes at room temperature (RT). Samples were added to the pre-washed beads and incubated for 30 minutes at RT under gentle shaking. The flow through was discarded and beads were washed twice with 0.02% Rapigest in TBS 1×. Elution was performed with 30 μL of TFA 0.1% and 5 μL of each sample was analyzed in an UPLC/Synapt G2-S (Waters, Milford) equipped with a POROS column (ABSciex) using reverse phase gradient.

(212) Spectra deconvolution was performed using MaxEnt1 software (Waters, Milford) and results were compared against the intact mass obtained for the ADC from a stock solution (FIG. 3). FIG. 3 shows a deconvoluted intact mass spectrum for the ADC 12-1 stock solution. G0F, G1F and G2F in the figure refer to the carbohydrate isoforms on the antibody portion of the antibody-drug conjugate.

(213) The deconvoluted spectra of the stock solution (FIG. 3) revealed that the antibody-drug conjugate 12-1 intact mass in its predominant DAR 2 form contained two G0F sugar motifs. Additional peaks at about 162 Da apart showed two other glycoforms containing G0F/G1F (peak at 148701 Da) and 2× G1F (peak at 148863 Da). The glycan profile is typical of that for an IgG.

(214) FIG. 4A-4D shows the deconvoluted spectra of time points 1 hour, 8 hours, 14 days and 21 days from the in vitro stability study. The data analysis showed that no significant mass change occurred for the ADC over the incubation period evaluated in the study (Table 6: intact mass results for the in-vitro stability study).

(215) TABLE-US-00006 TABLE 6 In vitro Stability MW of the ADC 12-1 Incubation (2xG0F form) Time 148539 1 hour 148539 8 hours 148539  2 days 148539  3 days 148540  7 days 148540 14 days 148540 21 days
In Vivo Stability of ADC 12-1

(216) Effective linker designs will not only provide stable tethering to a carrier in drug conjugates, but should also have minimal to no effect on the pharmacokinetic properties of the carrier itself. To establish the potential of this linker design for in vivo circulatory stability and to understand its impact on the pharmacokinetics of the conjugated carrier in the context of a drug conjugate, ADC 12-1 was dosed to DB 1 mice and was monitored for degradation of the mAb, intactness of the antibody-drug conjugate, and loss of payload (dexamethasone). Importantly, the study showed that the inherent pharmacokinetics of naked (non-conjugated) anti-mouse CD25 was not adversely affected by the conjugation of two molecules of drug-linker 1-4 to the antibody. Furthermore, the study showed that no loss of drug linker and no measurable dexamethasone were observed over the course of the 5 day study. The data shows that drug-linker 1-4 was stable in circulation and retained payload on the carrier in this example of an antibody-drug conjugate.

(217) In Vivo Pharmacokinetic (PK)/Stability Study

(218) General Experimental for In Vivo PK Study of ADC 12-1

(219) An in vivo study in DBA1 mice was performed in order to evaluate stability and pharmacokinetics of ADC 12-1. Naked antibody was administered intravenously in a single bolus to all groups. Group 1 was given a dose of 2 mpk; Group 2 and Group 3 were dosed 2 and 4 mpk of ADC 12-1, respectively. Plasma samples were taken from all three groups at 1, 2, 6, 16 hrs and 1, 2, 3, 5 days after dosing. At each of these time points, three animals per group were sacrificed to obtain plasma sample. Samples from group 1 were analyzed for total naked antibody contents, samples from groups 2 and 3 were submitted for total antibody, intact antibody and free-payload analysis. FIG. 5 shows the In vivo stability of ADC 12-1 following IV dosing to DBA1 mice.

(220) In Vivo Pharmacokinetic (PK)/Stability Study—Free Payload Analysis

(221) PK study samples were evaluated for free dexamethasone using the method described above. For increased sensitivity, 100 μL of serum from one mice of each time point was submitted to protein precipitation with acetonitrile. Free dexamethasone was not detected in any PK samples.

(222) In Vivo Pharmacokinetic (PK)/Stability Study—Intact Antibody Drug Conjugate Mass Analysis

(223) Samples from the ADC 12-1 PK study were also evaluated for stability using immunoprecipitation coupled to intact mass analysis. 50 μL of each sample was processed as described above. The results showed no molecular weight change for the antibody-drug conjugate over the study time range (from 1 hour to 5 days) (FIG. 6A-6B, Table 7).

(224) TABLE-US-00007 TABLE 7 In vivo PK/Stability Intact Mass Results MW of the 12-1 PK Time Sample (2xG0F motif) Group Point B1 148548 G2 1 hour C1 148547 G2 1 hour E1 148546 G2  2 hours F1 148548 G2  2 hours A2 148546 G2  6 hours C2 148546 G2 16 hours D2 148547 G2 16 hours F2 148545 G2 1 day G2 148548 G2 1 day A3 148546 G2 2 days D3 148548 G2 3 days G3 148550 G2 5 days H3 148542 G2 5 days B7 148542 G3 1 hour C7 148548 G3 1 hour E7 148547 G3  2 hours F7 148545 G3  2 hours A8 148547 G3  6 hours C8 148546 G3 16 hours D8 148546 G3 16 hours F8 148546 G3 1 day G8 148546 G3 1 day A9 148544 G3 2 days B9 148547 G3 2 days D9 148549 G3 3 days E9 148550 G3 3 days G9 148547 G3 5 days H9 148545 G3 5 days
In Vivo Pharmacokinetic (PK)/Stability Study Naked/Total mAb Analysis

(225) Plasma samples from in vivo PK study were analyzed for naked Antibody/total Antibody ADC 12-1 concentrations using Meso Scale Discovery (MSD) based electro-chemiluminescence method. The capture reagent is recombinant mouse IL-2R alpha (CD25) for both assays. The detection reagent may be goat anti-rat IgG for naked antibody/total Antibody and anti-dex mAb (e.g., Rabbit polyclonal anti-dexamethasone (Abcam Cat # ab35000)) may be used to detect ADC 12-1, respectively. Briefly, 96 well MSD plates were coated with the capture reagent and then washed. Plates were blocked for 1 hour and washed again. Samples were then added and incubated for 2 hours. Following incubation, plates were washed, incubated with the detection antibody for 1 hour, and washed again. The reading buffer was added and the plates were read using MSD plate reader.

Example 22

(226) In this example, anti-CD70 antibody 2H5 was conjugated to exemplary drug-linker 1-4 to produce ADC 12-2 (FIG. 1) as described for making ADC 12-1 in Example 20. In vitro activity and targeted delivery of ADC 12-2, naked antibody, and anti-hexon conjugate control were assessed by transfecting into 786-O (renal cell) and then measuring glucocorticoid-induced leucine zipper (GILZ) mRNA, a widely expressed dexamethasone-induced mRNA transcript. As shown in FIG. 7, ADC 12-2 displayed potent in vitro activity (0.7 ug/ml IP value) in 786-O cells that were confirmed to express CD70. This activity reflects dexamethasone conjugation and targeted delivery as the nonconjugated IgG variant and anti-hexon controls did not induce and observable GILZ in this cell line.

(227) 786-O cells were plated at 30K cells/well overnight at 37° C. in RPMI Media as suggested by ATCC (+10% HI FBS). Cells were stimulated with ADCs for 2, 6, or 24 hours at 37° C. Cells were lysed using RLT and RNA is isolated using RNeasy 96 Kits. PCR was used to measure GAPDH, PER1, or TSC22D3

(228) Quantitation of Glucocorticoid-induced leucine zipper (GILZ) mRNA expression was determined as follows. Cellular quantitation of GILZ mRNA was conducted using the following method. Cells suspension were prepared in HBSS+2% FBS (assay buffer) and plated at 5×10.sup.4 cells per well. Dosing solutions for free drug, ADCs and parental antibodies were prepared by serial dilution of each stock solution using 1:3 in HBSS+2% FBS supplemented with 1% final concentration of (50 mM Histidine, 100 mM NaCl, 5% Trehalose, pH 6.0), and incubated with cells final concentrations ranging from 20 to 0.002 μg/ml and 100 to 0.01 ng dexamethasone/ml for 18 hours. Cell lysis, cDNA synthesis, and real-time PCR were performed according to manufacturer's instructions using TaqMan Gene Expression Cells-to-C.sub.T™ Kit (Invitrogen, Carlsbad, Calif.). Specific primers against human GILZ and GAPDH were purchased from the Life Technologies (Invitrogen, Carlsbad, Calif.). Real-time PCR reactions were performed on the Applied Biosystems 7900 HT Fast Real-Time PCR System. Thermal cycling conditions consisted of an initial UDG incubation hold (50° C., 2 min) denaturing and enzyme activation step (95° C., 2 min) followed by 40 cycles of denaturing (95° C., 15 s), annealing and extending (60° C., 1 min). The mRNA levels were normalized to GAPDH (internal control) using the formula Δ threshold cycle (CT)=CT target−CT reference. The differential expression signal were expressed as delta Ct (ΔCt) by subtracting the Ct values of the un-stimulated samples (containing only assay buffer or DMSO vehicle) from those of the stimulated samples and expressed as relative fold of change using the formula: 2.sup.ΔΔCT.

Example 23

(229) The example shows that conjugates comprising the phosphate-based links have little or no propensity to form aggregates.

(230) Aggregation Assay

(231) An SE-HPLC method was used to conduct the aggregation/% monomer analysis. An isocratic gradient using 0.2 M potassium phosphate, 0.25 M potassium chloride pH 6.0 was used as the mobile phase at a flowrate of 0.5 mL/min. The column used was a Sepax Zenix-C SEC-300, 3 μm, 300 A, 7.8×300 mm (Cat #233300-7830). Detection of signal was monitored at 214 nm (280 for FIO). For a representative run, the analyte load was 10 μg.

(232) The results are shown in Table 8.

(233) TABLE-US-00008 TABLE 8 Sample % High Molecular Weight % Name Drug-Linker (aggregate) Monomer ADC 12-1 Phos-21Dex365 (1-4) 1.4 98.6 ADC 12-2 Phos-21Dex365 (1-4) 0.9 99.1

Example 24

(234) In this example, anti-CD74 antibody LL1 (or negative control IgG1 isotype) was conjugated to exemplary drug-linkers 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 9-4 to produce ADC 12-4, 12-5, 12-6, 12-7, 12-9, 12-10, 12-11, 12-12 (Table 9) as described for making ADC 12-1 in Example 20. The anti-CD74 antibody LL1 comprises a heavy chain (HC) having the amino acid sequence shown in SEQ ID NO:69 and a light chain (LC) having the amino acid sequence shown in SEQ ID NO:73. The amino acid at position 121 is para-azido phenylalanine (pAzF), which was the site for conjugating the exemplary drug-linkers using copper-free 3+2 cycloaddition chemistry to produce the ADCs. The construction of ADC 12-4 is illustrated in FIG. 1.

(235) TABLE-US-00009 TABLE 9 Antibody Linker ADC Anti-hCD74 (LL1) Linker 1-4 ADC 12-4 Anti-hCD74 (LL1) Linker 2-7 ADC 12-5 Anti-hCD74 (LL1) Linker 3-4 ADC 12-6 Anti-hCD74 (LL1) Linker 4-3 ADC 12-7 Anti-hCD74 (LL1) Linker 6-2 ADC 12-9 Anti-hCD74 (LL1) Linker 9-4 ADC 12-10 IgG1 (neg control) Linker 1-4 ADC 12-11 IgG1 (neg control) Linker 9-4 ADC 12-12 Anti-hCD74 (clone11) Linker 9-4 ADC 12-13 Anti-hCD74 (clone11) Linker 15-5 ADC 12-14 Anti-hCD74 (clone11) Linker 16-5 ADC 12-15

(236) In vitro activity and targeted delivery of ADC 12-4, 12-5, 12-6, 12-7, 12-9, 12-10, the isotype-matched negative controls 12-11, 12-12, and naked anti-hCD74 (LL1) control were assessed in HUT-78 an SUDHL-6 cells (CD74 positive T and B lymphoma cell lines respectively) as well as 786-O cells (a CD74 negative renal carcinoma cell line). The cells were treated with individual ADC, anti-hCD74 antibody, or budesonide (Bud) for 18 hours and then glucocorticoid-induced leucine zipper (GILZ) mRNA, a glucocorticoid-regulated gene, was measured by RT-PCR. As shown in FIGS. 8 and 9, the tested ADCs displayed a range of potency in up-regulating GILZ mRNA in both HUT-78 and SUDHL-6 cells, with ADC 12-10 most active with an EC50 value of about 0.2 μg/mL in both cell lines (see Table 10). In contrast, the isotype-matched negative control ADCs did not induce measurable GILZ mRNA in both cell lines. Furthermore, all the ADCs did not induce measurable GILZ mRNA except at the highest concentration (30 μg/mL) in 786-O cells (FIG. 10). (The activity at the highest concentration in 786-O cells may be due to impurity of the sample or possible low CD74 expression in cells.). The overall activity profile of the ADC is consistent with targeted delivery of a payload (e.g. dexamethasone or budesonide) that is dependent on the binding and internalization of the antibody with its antigen on the cell surface, and on the efficiency of linker cleavage inside the cells.

(237) TABLE-US-00010 TABLE 10 The Max effects and the EC.sub.50 values of ADC and budesonide on human cell lines HUT-78 cells SUDHL-6 cells 786-0 cells (CD74(—)) ADC or EC.sub.50 EC.sub.50 EC.sub.50 Antibody Emax (μg/mL) Emax (μg/mL) Emax (μg/mL) ADC 12-4 3.95 1.08 3.44 0.99 1.71 >30 (LL1) ADC 12-5 4.13 1.32 3.4 0.4 1.34 >30 (LL1) ADC 12-6 4.61 1.87 3.33 0.47 2.26 >30 (LL1) ADC 12-7 1.93 >30 1.52 14.3 1.73 >30 (LL1) ADC 12-9 2.55 >30 6.48 >30 4.02 >30 (LL1) ADC 12-10 6.47 0.2 5.89 0.21 3.52 >30 (LL1) ADC 12-11 0.99 N/A 1.13 >30 1.14 >30 (IgG1) ADC 12-12 0.99 N/A 1.28 >30 2.66 >30 (IgG1) ADC12-13 4.94 0.2 6.77 0.63 9.19 >30 (Clone 11) CD74 Ab 1.34 N/A 1.02 N/A 1.02 N/A (LL1) ADC 12-14 8.36 0.055 14.2 0.023 18.78 4.45 (Clone 11) ADC 12-15 8.34 0.26 7.53 0.67 5.26 >30 (Clone11) Budesonide 9.03 0.65 (nM) 14.33 2.21 (nM) 25.68 0.58 (nM) Fluticasone 10.27 0.45 (nM) 15.78 0.52 (nM) 25.38 0.46 (nM) Propionate N/A: EC.sub.50 value can't be generated.
Materials and Methods

(238) The HUT-78 (ATCC TIB-161), SUDHL-6 (ATCC CRL-2959) and 786-O (ATCC CRL-1932) cells were purchased from ATCC and maintained in culture medium as suggested by ATCC, in which Iscove's MDM/20% HI FBS for HUT-78 cells and RPMI-1640/10% FBS for SUDHL-6 and 786-O cells.

(239) The quantitation of Glucocorticoid-induced leucine zipper (GILZ) mRNA expression was determined by real time-PCR. In brief, actively growing cells were harvested and then resuspended in the assay buffer (HBSS plus 2% FBS) at concentration of 1.1×10.sup.6/ml. 5×10.sup.4 cells/well in 45 μl volume were plated to Greiner 384 well v-bottom reagent plates (Ref #781280). Dosing solutions for free drug, ADCs and parental antibodies were prepared in the v-bottom Greiner reagent plates at 10-fold over the final concentration by serial dilution of each stock solution using 1:3 in HBSS+2% FBS supplemented with 1% final concentration of (50 mM Histidine, 100 mM NaCl, 5% Trehalose, pH 6.0), and 5 μl of the 10-fold solutions were added to each well to reach final concentrations ranging from 30 to 0.0005 μg/ml for ADC/parental antibody and 100 to 0.002 nM for dexamethasone or budesonide (11 concentrations). After 18 hour incubation, the cells were lysed, and the lysates were used for cDNA synthesis and real-time PCR, according to manufacturer's instructions in TaqMan Gene Expression Cells-to-C.sub.T™ Kit (Invitrogen, Carlsbad, Calif.). Specific primers against human GILZ and GAPDH were purchased from the Life Technologies (Invitrogen, Carlsbad, Calif.). Real-time PCR reactions were performed on the Applied Biosystems 7900 HT Fast Real-Time PCR System. Thermal cycling conditions consisted of an initial UDG incubation hold (50° C., 2 min) denaturing and enzyme activation step (95° C., 2 min) followed by 40 cycles of denaturing (95° C., 15 s), annealing and extending (60° C., 1 min). The mRNA levels were normalized to GAPDH (internal control) using the formula Δ threshold cycle (CT)=CT target−CT reference. The differential expression signal were expressed as delta Ct (ΔCt) by subtracting the Ct values of the un-stimulated samples (containing only assay buffer or DMSO vehicle) from those of the stimulated samples and expressed as relative fold of change using the formula: 2.sup.ΔΔCT. The graphs were generated in GraphPad Prism and the EC.sub.50 values were calculated with non-linear regression curve fit of the data in GraphPad Prism.

Example 25

(240) The anti-CD74 antibody LL1 (or negative control isotype) was conjugated to exemplary drug-linkers 15-5, 16-5, or 17-2 as described for making ADC 12-1 to make ADC 12-13, ADC 12-14, and ADC 12-15, respectively. The anti-CD74 antibody LL1 comprises a heavy chain (HC) having the amino acid sequence shown in SEQ ID NO:69 and a light chain (LC) having the amino acid sequence shown in SEQ ID NO:73. The amino acid at position 121 is para-azido phenylalanine (pAzF) and may serve as the site for conjugating the exemplary drug-linkers using copper-free 3+2 cycloaddition chemistry to produce the ADCs.

(241) TABLE-US-00011 Table of Sequences SEQ ID NO: Description Amino Acid Sequence  1 Anti-CD25 LC CDR1 RASQSVSSSYLA  2 Anti-CD25 LC CDR2 GASSRAT  3 Anti-CD25 LC CDR3 QQYSSSPLT  4 Anti-CD25 HC CDR1 RYIIN  5 Anti-CD25 HC CDR2 RIIPILGVENYAQKFQG  6 Anti-CD25 HC CDR3 KDWFDY  7 Anti-CD25 HC CDR1 RYPIN  8 Anti-CD25 HC CDR2 RIIPILGIADYAQRFQG  9 Anti-CD25 HC CDR3 RDWGDY 10 Anti-CD25 LC CDR3 QQYGSSPIT 11 Anti-CD25 HC CDR1 RYAIN 12 Anti-CD25 HC CDR2 RIIPILDIADYAQKFQD 13 Anti-CD25 HC CDR3 KDWFDP 14 Anti-CD25 HC CDR1 RYPIN 15 Anti-CD70 LC CDR1 RASQSVSSYLA 16 Anti-CD70 LC CDR2 YDASNRAT 17 Anti-CD70 LC CDR3 QQRTNWPLT 18 Anti-CD70 HC CDR1 SYIMH 19 Anti-CD70 HC CDR2 VISYDGRNKYYADSVK 20 Anti-CD70 HC CDR3 DTDGYDFDY 21 Anti-CD70 LC CDR1 RASQGISSALA 22 Anti-CD70 LC CDR2 DASSLES 23 Anti-CD70 LC CDR3 QQFNSYPFT 24 Anti-CD70 HC CDR1 YYAMH 25 Anti-CD70 HC CDR2 VISYDGSIKYYADSVK 26 Anti-CD70 HC CDR3 EGPYSNYLDY 27 Anti-CD70 LC CDR1 RASQGISSWLA 28 Anti-CD70 LC CDR2 AASSLQS 29 Anti-CD70 LC CDR3 QQYNSYPLT 30 Anti-CD70 HC CDR1 DYGMH 31 Anti-CD70 HC CDR2 VIWYDGSNKYYADSVK 32 Anti-CD70 HC CDR3 DSIVMVRGDY 33 Anti-CD70 LC CDR1 RASQGISSWLA 34 Anti-CD70 LC CDR2 AASSLQS 35 Anti-CD70 LC CDR3 QQYNSYPLT 36 Anti-CD70 HC CDR1 DHGMH 37 Anti-CD70 HC CDR2 VIWYDGSNKYYADSVK 38 Anti-CD70 HC CDR3 DSIMVRGDY 39 Anti-CD70 LC CDR2 DASNRAT 40 Anti-CD70 LC CDR3 QQRSNWPLT 41 Anti-CD70 HC CDR1 SDYYYWS 42 Anti-CD70 HC CDR2 YIYYSGSTNYDPSLKS 43 Anti-CD70 HC CDR3 GDGDYGGNCFDY 44 Anti-CD74 LC CDR1 RSSQSLVHRNGNTYLH 45 Anti-CD74 LC CDR2 TVSNRFS 46 Anti-CD74 LC CDR3 SQSSHVPPT 47 Anti-CD74 HC CDR1 NYGVN 48 Anti-CD74 HC CDR2 WINPNTGEPTFDDDFKG 49 Anti-CD74 HC CDR3 SRGKNEAWFAY 50 Anti-CD74 LC CDR1 QGISSW 51 Anti-CD74 LC CDR3 QQYNSYPLT 52 Anti-CD74 HC CDR1 GFTFSSYA 53 Anti-CD74 HC CDR2 ISYDGSNK 54 Anti-CD74 HC CDR3 ASGRYYGSGSYSSYFD 55 Anti-CD74 HC CDR2 ISYDGSIK 56 Anti-CD74 HC CDR3 ARGREYTSQNIVILLD 57 Anti-CD74 HC CDR3 ARGREITSQNIVILLD 58 Anti-CD74 HC CDR2 IWYDGSNK 59 Anti-CD74 HC CDR3 ARGGTLVRGAMYGTDV 60 Anti-CD163 LC CDR1 ASQSVSSDV 61 Anti-CD163 LC CDR3 QDYTSPRT 62 Anti-CD163 HC CDR1 GYSITSDY 63 Anti-CD163 HC CDR3 CVSGTYYFDYWG 64 Anti-CD163 LC CDR1 ASQSVSHDV 65 Anti-CD163 LC CDR3 QDYSSPRT 66 Glycosylation site QYNS atN297 of IgG1 67 Glycosylation site QFNS atN297 of IgG4 68 Mutated  QAQS glycosylation site of IgG1 or IgG4 69 Anti-CD74 IgG1; QVQLQQSGSELKKPGASVKVSCKAS X at position 121 GYTFTNYGVNWIKQAPGQGLQWMGW is para-azido- INPNTGEPTFDDDFKGRFAFSLDTS phenylalanine  VSTAYLQISSLKADDTAVYFCSRSR (pAzF) (CDRs bold GKNEAWFAYWGQGSLVTVSSXSTKG type; Fc  PSVFPLAPSSKSTSGGTAALGCLVK underlined) DYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 70 Anti-CD74 IgG4; QVQLQQSGSELKKPGASVKVSCKAS X at position 121 GYTFTNYGVNWIKQAPGQGLQWMGW is para-azido- INPNTGEPTFDDDFKGRFAFSLDTS phenylalanine  VSTAYLQISSLKADDTAVYFCSRSR (pAzF) (CDRs bold GKNEAWFAYWGQGSLVTVSSXSTKG type; Fc PSVFPLAPCSRSTSESTAALGCLVK underlined) DYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK 71 Anti-CD74 IgG1 QVQLQQSGSELKKPGASVKVSCKAS (CDRs bold type; GYTFTNYGVNWIKQAPGQGLQWMGW Fc underlined) INPNTGEPTFDDDFKGRFAFSLDTS VSTAYLQISSLKADDTAVYFCSRSR GKNEAWFAYWGQGSLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 72 Anti-CD74 IgG4 QVQLQQSGSELKKPGASVKVSCKAS (CDRs bold type; GYTFTNYGVNWIKQAPGQGLQWMGW Fc underlined) INPNTGEPTFDDDFKGRFAFSLDTS VSTAYLQISSLKADDTAVYFCSRSR GKNEAWFAYWGQGSLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK 73 Anti-CD74 LC DIQLTQSPLSLPVTLGQPASISCRS (CDRs bold type) SQSLVHRNGNTYLHWFQQRPGQSPR LLIYTVSNRFSGVPDRFSGSGSGTD FTLKISRVEAEDVGVYFCSQSSHVP PTFGAGTRLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 74 Anti-CD74 IgG1; QVQLVESGGGVVQPGRSLRLSCAAS X at position 126 GFTFSSYAMHWVRQAPGKGLEWVAV is para-azido- ISYDGSIKYYADSVKGRFTISRDNS phenylalanine  KNTLYLQMNSLRVEDTAVFYCARGR (pAzF)(CDRs bold EEITSQNIVILLDYWGQGTLVTVTS type; Fc  XSTKGPSVFPLAPSSKSTSGGTAAL underlined) GCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLS LSPGK 75 Anti-CD74 IgG4; QVQLVESGGGVVQPGRSLRLSCAAS X at position 126 GFTFSSYAMHWVRQAPGKGLEWVAV is para-azido- ISYDGSIKYYADSVKGRFTISRDNS phenylalanine  KNTLYLQMNSLRVEDTAVFYCARGR (pAzF) (CDRs bold EEITSQNIVILLDYWGQGTLVTVTS type; Fc  XSTKGPSVFPLAPCSRSTSESTAAL underlined) GCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSS LGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSL GK 76 Anti-CD74 IgG1 QVQLVESGGGVVQPGRSLRLSCAAS (CDRs bold type; GFTFSSYAMHWVRQAPGKGLEWVAV Fc underlined) ISYDGSIKYYADSVKGRFTISRDNS KNTLYLQMNSLRVEDTAVFYCARGR EEITSQNIVILLDYWGQGTLVTVTS ASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLS LSPGK 77 Anti-CD74 IgG4 QVQLVESGGGVVQPGRSLRLSCAAS (CDRs bold type; GFTFSSYAMHWVRQAPGKGLEWVAV Fc underlined) ISYDGSIKYYADSVKGRFTISRDNS KNTLYLQMNSLRVEDTAVFYCARGR EEITSQNIVILLDYWGQGTLVTVTS ASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSS LGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQED PEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSL GK 78 Anti-CD74 LC DIQMTQSPSSLSASVGDRVTITCRA (CDRs bold type) SQGISSWLAWYQQKPEKAPKSLIYA ASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQYNSYPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 79 Anti-CD70 2H5 IgG1 QVQLVESGGGVVQPGRSLRLSCAAS X at position 119 GFTFSSYIMHWVRQAPGKGLEWVAV is para-azido- ISYDGRNKYYADSVKGRFTISRDNS phenylalanine  KNTLYLQMNSLRAEDTAVYYCARDT (pAF) (CDRs bold DGYDFDYWGQGTLVTVSSXSTKGPS type; Fc VFPLAPSSKSTSGGTAALGCLVKDY underlined) FPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG 80 Anti-CD70 Kappa  EIVLTQSPATLSLSPGERATLSCRA light chain  SQSVSSYLAWYQQKPGQAPRLLIYD (CDRs bold type) ASNRATGIPARFSGSGSGTDFTLTI SSLEPEDFAVYYCQQRTNWPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 81 Anti-murine CD25 QVKLLQSGAALVKPGASVKMSCKAS muIgG1 D265A GYSFPDSWVTWVKQSHGKSLEWIGD X at position 115 IFPNSGATNFNEKFKGKATLTVDKS is para-azido- TSTAYMELSRLTSEDSAIYYCTRLD phenylalanine  YGYWGQGVMVTVSSXKTTPPSVYPL (pAF) APGSAAQTNSMVTLGCLVKGYFPEP VTVTWNSGSLSSGVHTFPAVLQSDL YTLSSSVTVPSSTWPSETVTCNVAH PASSTKVDKKIVPRDCGCKPCICTV PEVSSVFIFPPKPKDVLTITLTPKV TCVVVAISKDDPEVQFSWFVDDVEV HTAQTQPREEQFNSTFRSVSELPIM HQDWLNGKEFKCRVNSAAFPAPIEK TISKTKGRPKAPQVYTIPPPKEQMA KDKVSLTCMITDFFPEDITVEWQWN GQPAENYKNTQPIMDTDGSYFVYSK LNVQKSNWEAGNTFTCSVLHEGLHN HHTEKSLSHSPGK 82 Anti-murine CD25 DVVLTQTPPTLSATIGQSVSISCRS muKappa SQSLLHSNGNTYLNWLLQRPGQPPQ LLIYLASRLESGVPNRFSGSGSGTD FTLKISGVEAEDLGVYYCVQSSHFP NTEGVGTKLELKRADAAPTVSIFPP SSEQLTSGGASVVCFLNNFYPKDIN VKWKIDGSERQNGVLNSWTDQDSKD STYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC

(242) While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.