CHEMOSELECTIVE THIOL-CONJUGATION WITH ALKENE OR ALKYNE-PHOSPHONAMIDATES

Abstract

Disclosed are novel conjugates and processes for the preparation thereof. A process for the preparation of alkene- or alkyne-phosphonamidates comprises the steps of (I) reacting a compound of formula (III), with an azide of formula (IV), to prepare a compound of formula (V), reacting a compound of formula (V) with a thiol-containing molecule of formula (VI), resulting in a compound of formula (VII).

##STR00001##

Claims

1. Process for the preparation of alkene- or alkyne-phosphonamidates comprising the steps of (I) Reacting a compound of formula (III) ##STR00212## wherein custom-character represents a double or triple bond; X represents R.sub.3C when is a triple bond; or X represents (R.sub.3 R.sub.4)C when is a double bond; R.sub.1 independently represents an optionally substituted aliphatic or aromatic residue, such as phenyl; optionally, R.sub.1 represents C.sub.1-C.sub.8-alkyl optionally substituted with at least one of (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, Br, I, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2, N(C.sub.1-C.sub.8-alkyl).sub.2, ?O, C.sub.3-C.sub.8-cycloalkyl, SS(C.sub.1-C.sub.8-alkyl), hydroxy-(C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, C.sub.2-C.sub.8-alkynyl or optionally substituted phenyl such as ##STR00213## wherein # represents the position of O in formula (III); or optionally, R.sub.1 represents phenyl optionally independently substituted with at least one of C.sub.1-C.sub.8-alkyl, (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, I, Br, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2 or N(C.sub.1-C.sub.8-alkyl).sub.2; or optionally, R.sub.1 represents a 5- or 6-membered heteroaromatic system such as pyridyl; preferably, R.sub.1 represents C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkyl substituted with SS(C.sub.1-C.sub.8-alkyl), C.sub.1-C.sub.8-alkyl substituted with (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, C.sub.1-C.sub.8-alkyl substituted with optionally substituted phenyl, phenyl or phenyl substituted with NO.sub.2; R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; and R.sub.4 represents H or C.sub.1-C.sub.8-alkyl; with an azide of formula (IV)
?-N.sub.3 (IV) wherein ? represents an aliphatic or aromatic residue; to prepare a compound of formula (V) ##STR00214## wherein ?, custom-character , R.sub.1, and X are as defined above; (II) Reacting a compound of formula (V) with a thiol-containing molecule of formula (VI)
-SH (VI) wherein represents an optionally substituted C.sub.1-C.sub.8-alkyl, an optionally substituted Phenyl, an optionally substituted aromatic 5- or 6-membered heterocyclic system, an amino acid, a peptide, a protein, an antibody, a saccharide, a polysaccharide, a nucleotide, a oligonucleotide or a polymer; resulting in a compound of formula (VII) ##STR00215## wherein custom-character represents a bond if in a compound of formula (V) represents a double bond; or represents a double bond if in a compound of formula (V) represents a triple bond; and , ?, R.sub.1 and X are as defined above.

2. The process according to claim 1, comprising step a) prior to step (I) a) Reacting a compound of formula (I) ##STR00216## wherein R.sub.1 is defined as above; Hal represents a halogen selected from the group consisting of Cl, Br, I, preferably Cl, with an alpha unsaturated compound of formula (II) comprising a double or triple bond in alpha-position ##STR00217## wherein custom-character represents a double or triple bond; X represents R.sub.3C when is a triple bond; or X represents (R.sub.3 R.sub.4)C when is a double bond; R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; and R.sub.4 represents H or C.sub.1-C.sub.8-alkyl; to form a compound of formula (III) ##STR00218## wherein custom-character , X and R.sub.1 are as defined above; alternatively, reacting a compound of formula (I) ##STR00219## wherein R.sub.5 independently represents C.sub.1-C.sub.8-alkyl; Hal represents a halogen selected from the group consisting of Cl, Br, I, preferably Cl. with an alpha unsaturated compound of formula (II) comprising a double or triple bond in alpha-position ##STR00220## wherein custom-character represents a double or triple bond; X represents R.sub.3C when is a triple bond; or X represents (R.sub.3 R.sub.4)C when is a double bond; R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; and R.sub.4 represents H or C.sub.1-C.sub.8-alkyl. to form a compound of formula (III) ##STR00221## and reacting said compound of formula (III) with R.sub.1OH to form a compound of formula (III) ##STR00222## wherein custom-character and X are defined as above and R.sub.1 is as defined above but not individually selected.

3. Process for the preparation of alkene-phosphonamidates comprising the steps of: (I) Reacting a compound of formula (III) ##STR00223## wherein V represents C.sub.1-C.sub.8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl; R.sub.1 independently represents an optionally substituted aliphatic or aromatic residue, such as phenyl; optionally, R.sub.1 represents C.sub.1-C.sub.8-alkyl optionally substituted with at least one of (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, Br, I, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2, N(C.sub.1-C.sub.8-alkyl).sub.2, ?O, C.sub.3-C.sub.8-cycloalkyl, SS(C.sub.1-C.sub.8-alkyl), hydroxy-(C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, C.sub.2-C.sub.8-alkynyl or optionally substituted phenyl such as ##STR00224## wherein # represents the position of O in formula (III*); or optionally, R.sub.1 represents phenyl optionally independently substituted with at least one of C.sub.1-C.sub.8-alkyl, (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, I, Br, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2 or N(C.sub.1-C.sub.8-alkyl).sub.2; or optionally, R.sub.1 represents a 5- or 6-membered heteroaromatic system such as pyridyl; preferably, R.sub.1 represents C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkyl substituted with SS(C.sub.1-C.sub.8-alkyl), C.sub.1-C.sub.8-alkyl substituted with (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, C.sub.1-C.sub.8-alkyl substituted with optionally substituted phenyl, phenyl or phenyl substituted with NO.sub.2; R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; and R.sub.4 represents H or C.sub.1-C.sub.8-alkyl; with an azide of formula (IV) ##STR00225## wherein ? represents an aliphatic or aromatic residue; to prepare a compound of formula (V*) ##STR00226## wherein ?, V, and R.sub.1 are as defined above; X is (R.sub.3 R.sub.4)C; and R.sub.3 and R.sub.4 are as defined above; (II) Reacting a compound of formula (V*) with a thiol-containing molecule of formula (VI)
custom-character -SH (VI) wherein represents an optionally substituted C.sub.1-C.sub.8-alkyl, an optionally substituted Phenyl, an optionally substituted aromatic 5- or 6-membered heterocyclic system, an amino acid, a peptide, a protein, an antibody, a saccharide, a polysaccharide, a nucleotide, a oligonucleotide or a polymer; resulting in a compound of formula (VII*) ##STR00227## wherein custom-character , ?, V, R.sub.1 and X are as defined above.

4. The process according to claim 1, wherein R.sub.1 independently represent methyl, ethyl, propyl or butyl, more preferably methyl or ethyl.

5. The process according to claim 1, wherein R.sub.1 represents ##STR00228## wherein R.sub.10 and R.sub.11 independently represent hydrogen or C.sub.1-C.sub.8-alkyl; and # represents the position of O.

6. The process according to claim 1, wherein R.sub.1 represents C.sub.1-C.sub.8-alkyl substituted with phenyl, said phenyl being further substituted with ##STR00229## wherein Z is O or NH, preferably 0, and wherein # represents the position of said phenyl.

7. The process according to claim 1, wherein R.sub.1 represents C.sub.1-C.sub.8-alkyl substituted with phenyl, said phenyl being further substituted with ##STR00230## and wherein # represents the position of said phenyl.

8. The process according to claim 1, wherein R.sub.1 represents hydroxyethyl or homopropargyl.

9. The process according to claim 1, wherein custom-character represents a double bond, X represents (R.sub.3 R.sub.4)C, R.sub.3 and R.sub.4 independently represent H or C.sub.1-C.sub.8-alkyl and represents a bond.

10. The process according to claim 1, wherein custom-character represents a triple bond, X represents R.sub.3C, R.sub.3 represents H or C.sub.1-C.sub.8-alkyl and represents a double bond.

11. The process according to claim 1, wherein ? represents an optionally substituted C.sub.1-C.sub.8-alkyl, preferably ##STR00231## an optionally substituted phenyl, preferably ##STR00232## a radioactive or non-radioactive nuclide, biotin, a reporter enzyme, a nucleotide, an oligonucleotide, a fluorophore such as CY.sub.5 or EDANS, an amino acid, a peptide, an optionally substituted 5- or 6-membered heteroaromatic system.

12. The process according to claim 10, wherein ? represents a cyclic RGD peptide of structure (VIII) (c(RGDfK) ##STR00233## wherein * represents the position of the N.sub.3 group; Biotin; CY.sub.5 or EDANS; phenyl, optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkoxy, halogen, CN, NO.sub.2, NH.sub.2, N(C.sub.1-C.sub.8-alkyl), N(C.sub.1-C.sub.8-alkyl).sub.2-COOH, COO(C.sub.1-C.sub.8-alkyl), OC(O)(C.sub.1-C.sub.8-alkyl), C(O)N(C.sub.1-C.sub.8-alkyl), N(H)C(O)(C.sub.1-C.sub.8-alkyl) preferably optionally substituted with one substituent selected from the group consisting of C.sub.1-C.sub.8-alkoxy, COOH, COO(C.sub.1-C.sub.8-alkyl and NO.sub.2. C.sub.1-C.sub.8-alkyl optionally substituted with at least one substituent selected from the group consisting of C.sub.3-C.sub.8-cycloalkyl; heterocyclyl with 3 to 8 ring members wherein the heteroatom(s) are selected from N, O, S; C.sub.1-C.sub.8-alkoxy; halogen; CN; NO.sub.2; NH.sub.2; N(C.sub.1-C.sub.8-alkyl); N(C.sub.1-C.sub.8-alkyl).sub.2; COOH; COO(C.sub.1-C.sub.8-alkyl); OC(O)(C.sub.1-C.sub.8-alkyl); CONH.sub.2; C(O)N(C.sub.1-C.sub.8-alkyl).sub.2; C(O)NH(C.sub.1-C.sub.8-alkyl); N(H)C(O)(C.sub.1-C.sub.8-alkyl), preferably C.sub.1-C.sub.8-alkoxy, COOH, COO(C.sub.1-C.sub.8-alkyl and NO.sub.2, phenyl or a heteroaromatic system, a monosaccharide, a polysaccharide, a peptide, a nucleotide, an oligonucleotide, a polymer, an amino acid, a fluorophor, a protein tag (substituent 1.sup.st generation), wherein a substituent 1.sup.st generation may again optionally be substituted with C.sub.3-C.sub.8-cycloalkyl; heterocyclyl with 3 to 8 ring members wherein the heteroatom(s) are selected from N, O, S; C.sub.1-C.sub.8-alkoxy; halogen; CN; NO.sub.2; NH.sub.2; N(C.sub.1-C.sub.8-alkyl); N(C.sub.1-C.sub.8-alkyl).sub.2; COOH; COO(C.sub.1-C.sub.8-alkyl); OC(O)(C.sub.1-C.sub.8-alkyl); CONH.sub.2; C(O)N(C.sub.1-C.sub.8-alkyl).sub.2; C(O)NH(C.sub.1-C.sub.8-alkyl); N(H)C(O)(C.sub.1-C.sub.8-alkyl), preferably C.sub.1-C.sub.8-alkoxy, COOH, COO(C.sub.1-C.sub.8-alkyl and NO.sub.2, phenyl or a heteroaromatic system (substituents 2.sup.nd generation) and wherein a substituent 2.sup.nd generation may be substituted again by at least one substituent selected from the same group and wherein such substitution may go until generation 3, 4, 5, 6, 7, 8, 9 or 10.

13. The process according to claim 1, wherein ? represents an optionally substituted C.sub.1-C.sub.8-alkyl such as a linker, a drug, or a linker-drug conjugate; or wherein ? represents an optionally substituted phenyl such as a linker, a drug, or a linker-drug conjugate.

14. (canceled)

15. The process according to claim 1, wherein custom-character represents an antibody, preferably a IgG-antibody, more preferably a Cetuximab or a Trastuzumab; a peptide, preferably GFP protein or eGFP-protein, a tripeptide, more preferably a peptide of formula (IX) ##STR00234## wherein # represents the position of S; Optionally substituted C.sub.1-C.sub.8-alkyl, preferably the substituted C.sub.1-C.sub.8-alkyl ##STR00235## wherein # markes the S-position.

16. The process according to claim 1, wherein the ?-N.sub.3 and the custom-character -SH are in the same molecule.

17. A compound of formula (V) or formula (V*) ##STR00236## wherein ?, custom-character , R1 and X are as defined in claim 1; and V represents C.sub.1-C.sub.8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl.

18. (canceled)

19. A compound of formula (VII) or formula (VII*) ##STR00237## wherein custom-character represents a bond ; or represents a double bond; , ?, R.sub.1 and X are as defined in claim 1; and V represents C.sub.1-C.sub.8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl.

20. (canceled)

21. The compound according to claim 19, wherein custom-character represents an antibody and ? represents a protein tag or a fluorophore such as CY.sub.5 or EDANS, or a protein.

22. The compound according to claim 19, wherein custom-character represents a protein and ? represents a protein tag or a fluorophore such as CY.sub.5 or EDANS, an antibody, or a protein.

23. The compound according to claim 19, wherein custom-character represents a protein and ? represents a protein.

24. The compound of according to claim 19, wherein custom-character represents an antibody and ? represents a linker, a drug, or a linker-drug conjugate.

25. (canceled)

26. (canceled)

Description

DETAILED DESCRIPTION

[0060] The invention provides a new chemoselective reaction of Cys residues in (unprotected) peptides, proteins, such as enzymes and co-enzymes (e.g. coenzyme A), antibodies or other thiol-comprising compounds with alkene- or alkyne-phosphonamidates. In one embodiment, the peptides, proteins, antibodies or other thiol-comprising compounds are unprotected. In another embodiment, the alkene- or alkyne-phosphonamidates are electron deficient alkene- or alkyne-phosphonamidates. The resulting conjugates have not been described in the literature previously.

[0061] Scheme 1 describes the general strategy for a synthesis according to the present invention at the example of ethenyl or ethynyl phosphonites. R1 represents an optionally substituted aliphatic or aromatic residue:

##STR00003##

[0062] Scheme 2 shows the difference between a process known in the art (e.g., 15) and a process according to the present invention A) Sequential azide-azide couplings using alkyne phosphonites; B) Staudinger-induced thiol-addition (the thiol addition may be also denoted as Michael addition, as e.g. in Scheme 2B) for the modification of Cys residues according to the invention. Merely as examples, ethenyl and ethynyl (diethyl)phosphonite were used:

##STR00004##

[0063] It is submitted that the processes described herein allow to combine a huge amount of different organic compounds in position R.sub.1 custom-character and ?.

[0064] Furthermore, the invention refers to a method for bioconjugation of two complex molecules: a chemoselective reaction, which induces a second chemoselective reaction for the conjugation to proteins. This concept is based on the unique reactivity of an azide-building block with an unprotected alkyne or alkene phosphonite via the Staudinger-phosphonite reaction (SPhR) resulting in the generation of a, preferably, electron-deficient double or triple bond (Scheme 1 and 2B). The resulting electrophilic system can subsequently be employed for the reaction with thiol-containing proteins and antibodies or further thiol-comprising compounds to deliver functional conjugates such as antibody or protein conjugates.

[0065] It is demonstrated with the attached results: [0066] The synthesis of different alkene and alkyne phosphonites [0067] (Chemoselective) Staudinger reactions with alkene and alkyne phosphonites [0068] Conjugation reactions of alkene- or alkyne-phosphonamidates with thiol-containing molecules, including small molecules, peptides, proteins and antibodies [0069] Thiol addition to alkyne-phosphonamidates in aqueous systems showed a high diastereoselectivity for the formation of the Z-Product [0070] Stability of these conjugates under physiologically relevant conditions [0071] Synthesis of conjugates comprising a cleavable group

[0072] This invention features several innovative aspects, which further ease the accessibility of conjugates such as antibody or protein conjugates, in particular with complex payloads and labels containing several functional groups, with novel conjugation chemistry: [0073] A new reaction for modifying thiols in small molecules, polymers, proteins and antibodies, therefore [0074] Unprecedented chemical structure at Cys-moiety [0075] Two complex molecule (e.g. peptide and proteins or peptide and antibody) can be connected by straightforward step-wise chemoselective conjugations [0076] No need of final protecting group manipulations after installation of chemoselective handle (i.e., preferably electron-deficient, alkene or alkyne-phosphonamidate) or after the chemoselective conjugation [0077] Linker with great variability (P-substituents can be varied, various O-substituents at the phosphorus center, O-substituents comprising a cleavable group) [0078] High stability of conjugates as opposed to usual Maleimide reagents; fast conjugation reactions [0079] High stereoselectivity of the thiol addition to alkyne-phosphonamidates

[0080] Generally, the process according to the present invention can be carried out to conjugate different compounds such as small molecules (e.g. optionally substituted alkyl, phenyl or heterocycles), proteins, antibodies, oligonucleotides or polysaccharides with tags, proteins oligonucleotides etc. To achieve this coupling, the present invention refers in a first aspect to a process for the preparation of conjugates of formula (VII) comprising the steps of [0081] (I) Reacting a compound of formula (III)

##STR00005## [0082] wherein [0083] custom-character represents a double or triple bond; [0084] X represents R.sub.3C when is a triple bond [0085] (thus, the structure is

##STR00006## [0086] X represents (R.sub.3 R.sub.4)C when custom-character is a double bond [0087] (thus, the structure is

##STR00007## [0088] R.sub.1 independently represents an optionally substituted aliphatic or aromatic residue, such as phenyl; with (C.sub.1-C.sub.8-alkoxy).sub.n, wherein n is 1, 2, 3, 4, 5 or 6 with F, with Cl, with Br, with I, with NO.sub.2, with N(C.sub.1-C.sub.8-alkyl)H, with NH.sub.2, with N(C.sub.1-C.sub.8-alkyl).sub.2, with ?O, with C.sub.3-C.sub.8-cycloalky, with optionally substituted phenyl substituted C.sub.1-C.sub.8-alkyl such as

##STR00008##

or optionally independently with C.sub.1-C.sub.8-alkyl, (C.sub.1-C.sub.8-alkoxy).sub.n, F, Cl, I, Br, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2, N(C.sub.1-C.sub.8-alkyl).sub.2, substituted phenyl; or 5- or 6-membered heteroaromatic system such as pyridyl; preferably C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkyl substituted with (C.sub.1-C.sub.8-alkoxy).sub.n, phenyl or phenyl substituted with NO.sub.2; [0089] or [0090] which may be again substituted at one of the Nitrogen-ring-atoms with biotin or any other peptide, protein, such as an enzyme or co-enzyme (e.g. coenzyme A), antibody, protein tag, fluorophore, oligonucleotide, or polysaccharide and wherein # represents the position of O; [0091] R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; [0092] R.sub.4 represents H or C.sub.1-C.sub.8-alkyl; and [0093] with an azide of formula (IV)

?-N.sub.3 (IV) [0094] wherein [0095] ? represents an aliphatic or aromatic residue; [0096] to prepare a compound of formula (V)

##STR00009## [0097] wherein ?, custom-character , R.sub.1, and X are as defined above. [0098] (II) Reacting a compound of formula (V) with a thiol-containing molecule of formula (VI)

custom-character -SH (VI) [0099] wherein represents an optionally substituted C.sub.1-C.sub.8-alkyl, an optionally substituted Phenyl, an optionally substituted aromatic 5- or 6-membered heterocyclic system, an amino acid, a peptide, a protein, an antibody, a saccharide, a polysaccharide, a nucleotide, a oligonucleotide or a polymer; resulting in a compound of formula (VII)

##STR00010## [0100] wherein [0101] custom-character represents a bond if in a compound of formula (V) represents a double bond; or [0102] represents a double bond if in a compound of formula (V) represents a triple bond; and [0103] , ?, R.sub.1 and X are as defined above.

[0104] The invention also refers to a process comprising a step (a) prior to step (I) of the process described above. Thus, such a process comprises the steps of [0105] a) Reacting a compound of formula (I)

##STR00011## [0106] wherein R.sub.1 and Hal are defined as above; [0107] with an alpha unsaturated compound of formula (II) comprising a double or triple bond in alpha-position

##STR00012## [0108] wherein [0109] custom-character represents a double or triple bond; [0110] X represents R.sub.3C when is a triple bond; or [0111] X represents (R.sub.3 R.sub.4)C when is a double bond; [0112] R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; and [0113] R.sub.4 represents H or C.sub.1-C.sub.8-alkyl; [0114] to form a compound of formula (III)

##STR00013## [0115] wherein [0116] custom-character , X and R.sub.1 are as defined above; [0117] alternatively, reacting a compound of formula (I)

##STR00014## [0118] wherein [0119] R.sub.5 independently represents C.sub.1-C.sub.8-alkyl; [0120] Hal represents a halogen selected from the group consisting of Cl, Br, I, preferably Cl; [0121] with an alpha unsaturated compound of formula (II) comprising a double or triple bond in alpha-position

##STR00015## [0122] wherein [0123] custom-character represents a double or triple bond; [0124] X represents R.sub.3C when is a triple bond; or [0125] X represents (R.sub.3 R.sub.4)C when is a double bond; [0126] R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; and [0127] R.sub.4 represents H or C.sub.1-C.sub.8-alkyl; [0128] to form a compound of formula (III)

##STR00016## [0129] and reacting said compound of formula (III) with R.sub.1OH [0130] to form a compound of formula (III)

##STR00017## [0131] wherein [0132] custom-character and X are defined as above and R.sub.1 is as defined above but not individually selected; [0133] (I) Reacting a compound of formula (III) with an azide of formula (IV)

?-N.sub.3 (IV) [0134] wherein [0135] ? represents an aliphatic or aromatic residue; [0136] to prepare a compound of formula (V)

##STR00018## [0137] wherein [0138] ?, custom-character , R.sub.1 and X are as defined above; [0139] (II) Reacting a compound of formula (V) with a thiol-containing molecule of formula (VI)

custom-character -SH (VI) [0140] wherein represents an optionally substituted C.sub.1-C.sub.8-alkyl, an optionally substituted Phenyl, an optionally substituted aromatic 5- or 6-membered heterocyclic system, an amino acid, a peptide, a protein, an antibody, a saccharide, a polysaccharide, a nucleotide, a oligonucleotide or a polymer; [0141] resulting in a compound of formula (VII)

##STR00019## [0142] wherein [0143] custom-character represents a bond if in a compound of formula (V) represents a double bond; or [0144] represents a double bond if in a compound of formula (V) represents a triple bond; and [0145] , ?, R.sub.1 and X are as defined above.

[0146] Preferably, in this process custom-character represents a triple bond.

[0147] In one embodiment, the P-atom of compounds of formula (III), preferably wherein custom-character represents a double bond, can be protected by BH.sub.3 prior to the Staudinger reaction (e.g. for purification purposes) and can easily be deprotected before the Staudinger reaction: [0148] b) reacting a compound of formula (III) [0149] to form a compound of formula (III)

##STR00020## [0150] wherein X and R.sub.1 are as defined above; [0151] with BH.sub.3 to form a compound of formula (III)

##STR00021## [0152] wherein X and R.sub.1 are as defined above.

[0153] Deprotection of boran protected phosphonites of formula (III) to form the reactive P(III) species can be easily achieved by the addition of a weak base such as DABCO (1,4-Diazabicyclo[2.2.2]octan=Triethylendiamin (TEDA)).

[0154] Compounds of formula (III) can also be synthesized starting from PCl.sub.3:

##STR00022##

wherein R, is as defined herein.

[0155] The processes described herein can also be carried out with a compound of formula (III*) instead of a compound (III)

##STR00023##

[0156] Wherein V represents C.sub.1-C.sub.8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl; and R.sub.1, R.sub.2 and R.sub.3 are as defined for compound (III) above. For the preparation of compounds of formula (III*), compounds of formula (II*) can be used

##STR00024##

wherein V, R.sub.3 and R.sub.4 are defined herein.

[0157] A process according to the invention with compound (III*) results in compounds of formula (V*)

##STR00025##

wherein V represents C.sub.1-C.sub.8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl; [0158] and R.sub.1 are as defined for compound (V);
and compounds of formula (VII*)

##STR00026##

wherein V represents C.sub.1-C.sub.8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl; ?, custom-character and R.sub.1 are as defined for compound (VII). All steps for the processes described herein for compounds of formula (V) and (VII) can be performed analogously for compounds of formula (V*) and (VII*).

[0159] Accordingly, the present invention also relates to a process for the preparation of alkene-phosphonamidates comprising the steps of:

(I) Reacting a compound of formula (III)

##STR00027## [0160] wherein [0161] V represents C.sub.1-C.sub.8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl; [0162] R.sub.1 independently represents an optionally substituted aliphatic or aromatic residue, such as phenyl; with (C.sub.1-C.sub.8-alkoxy).sub.n, wherein n is 1, 2, 3, 4, 5 or 6, with F, with Cl, with Br, with I, with NO.sub.2, with N(C.sub.1-C.sub.8-alkyl)H, with NH.sub.2, with N(C.sub.1-C.sub.8-alkyl).sub.2, with ?O, with C.sub.3-C.sub.8-cycloalkyl, with optionally substituted phenyl substituted C.sub.1-C.sub.8-alkyl such as

##STR00028##

or optionally independently with C.sub.1-C.sub.8-alkyl, (C.sub.1-C.sub.8-alkoxy).sub.n, F, Cl, I, Br, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2, N(C.sub.1-C.sub.8-alkyl).sub.2, substituted phenyl; or 5- or 6-membered heteroaromatic system such as pyridyl; preferably C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkyl substituted with (C.sub.1-C.sub.8-alkoxy).sub.n, phenyl or phenyl substituted with NO.sub.2; [0163] or [0164] which may be again substituted at one of the Nitrogen-ring-atoms with biotin or any other peptide, protein, such as an enzyme or co-enzyme (e.g. coenzyme A), antibody, protein tag, fluorophore, oligonucleotide, or polysaccharide and wherein # represents the position of O; [0165] R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; [0166] R.sub.4 represents H or C.sub.1-C.sub.8-alkyl; and [0167] with an azide of formula (IV)

?-N.sub.3 (IV) [0168] wherein [0169] ? represents an aliphatic or aromatic residue; [0170] to prepare a compound of formula (V*)

##STR00029## [0171] Wherein ?, V, and R.sub.1 are as defined above; [0172] X is (R.sub.3 R.sub.4)C; and [0173] R.sub.3 and R.sub.4 are as defined above;
(II) Reacting a compound of formula (V*) with a thiol-containing molecule of formula (VI)

custom-character -SH (VI) [0174] wherein represents an optionally substituted C.sub.1-C.sub.8-alkyl, an optionally substituted Phenyl, an optionally substituted aromatic 5- or 6-membered heterocyclic system, an amino acid, a peptide, a protein, an antibody, a saccharide, a polysaccharide, a nucleotide, a oligonucleotide or a polymer; [0175] resulting in a compound of formula (VII*)

##STR00030## [0176] wherein [0177] custom-character , ?, V, R.sub.1 and X are as defined above.

[0178] One embodiment of the present invention also refers to compounds of formula (V*) and (VII*).

[0179] In the processes of the invention described herein it is not required that the compound (V) or (V*) obtained in step (I) has exactly the same structure as the compound (V) or (V*) used for reacting with the thiol-containing molecule of formula (VI) in step (II). In this respect, the ?, R1 and/or X moieties of the compound (V) or (V*) may be modified before the compound (V) or (V*) is used for reacting with the thiol-containing molecule of formula (VI) in step (II). Such modification may be carried out as long as the ?, R1 and/or X moieties after modification are still covered by the definitions disclosed herein above. As a merely illustrative example, as shown in the following reaction scheme, the ? moiety of a compound A of formula (V) obtained in step (I) may be modified to give the compound B of formula (V), which is then used for reacting with the thiol-containing molecule of formula (VI) in step (II):

##STR00031##

wherein TFA.sup.? is trifluoroacetate, Cy5 is the fluorescence dye Cy5, HATU is (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate), DIPEA is N,N-diisopropylethylamine and DMF is N,N-dimethylformamide.

[0180] In one preferred embodiment of a process according to the invention, R.sub.1 independently represents methyl, ethyl, propyl, butyl, phenyl, nitro-substituted phenyl, (C.sub.1-C.sub.2-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, more preferably 2-(2-methoxyethoxy)ethyl, phenyl, benzyl or nitro-substituted benzyl, methyl or ethyl, even more preferably methyl or ethyl. In an even more preferred embodiment R.sub.1 is the same.

[0181] In another preferred embodiment, R.sub.1 can even be modified after the thiol-addition (step (II), for example, the substituent R.sub.1 can comprise a triple bond as in

##STR00032##

[0182] Which can be reacted with any desired organic compound-N.sub.3 (such as peptide-N.sub.3, protein-N.sub.3, such as an enzyme-N.sub.3 or co-enzyme-N.sub.3 (e.g. coenzyme A-N.sub.3), antibody-N.sub.3, protein tag-N.sub.3, fluorophore-N.sub.3, oligonucleotide-N.sub.3, or polysaccharide-N.sub.3 e.g. Biotin-N.sub.3) to form a triazole-bridged complex, for example

##STR00033##

[0183] Accordingly, R.sub.1 may represent

##STR00034##

wherein compound may represent a peptide, a protein, an enzyme, a co-enzyme (e.g. co-enzyme A), an antibody, a protein tag, a fluorophore, an oligonucleotide, a polysaccharide, or biotin; wherein # represents the position of O.

[0184] In another preferred embodiment, R.sub.1 is an optionally substituted aliphatic or aromatic residue, such as phenyl; with (C.sub.1-C.sub.8-alkoxy).sub.n, wherein n is 1, 2, 3, 4, 5 or 6 with F, with Cl, with Br, with I, with NO.sub.2, with N(C.sub.1-C.sub.8-alkyl)H, with NH.sub.2, with N(C.sub.1-C.sub.8-alkyl).sub.2, with ?O, with C.sub.3-C.sub.8-cycloalky, with optionally substituted phenyl substituted C.sub.1-C.sub.8-alkyl such as

##STR00035##

[0185] Accordingly, R.sub.1 may independently represent an optionally substituted aliphatic or aromatic residue, such as phenyl. Optionally substituted in the optionally substituted aliphatic or aromatic residue refers to optional substitution of the aliphatic or aromatic residue independently with any possible residue.

[0186] R.sub.1 may represent C.sub.1-C.sub.8-alkyl optionally substituted with at least one of (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, Br, I, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2, N(C.sub.1-C.sub.8-alkyl).sub.2, ?O, C.sub.3-C.sub.8-cycloalkyl, SS(C.sub.1-C.sub.8-alkyl), hydroxy-(C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, C.sub.2-C.sub.8-alkynyl or optionally substituted phenyl such as

##STR00036##

wherein # represents the position of O in formula (III) or formula (III*).

[0187] R.sub.1 may represent phenyl optionally independently substituted with at least one of C.sub.1-C.sub.8-alkyl, (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, F, Cl, 1, Br, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2 or N(C.sub.1-C.sub.8-alkyl).sub.2.

[0188] R.sub.1 may represent a 5- or 6-membered heteroaromatic system such as pyridyl.

[0189] R.sub.1 may represent C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkyl substituted with SS(C.sub.1-C.sub.8-alkyl), C.sub.1-C.sub.8-alkyl substituted with (C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6, C.sub.1-C.sub.8-alkyl substituted with optionally substituted phenyl, phenyl or phenyl substituted with NO.sub.2.

[0190] In some embodiments R.sub.1 represents an aliphatic or aromatic residue which is optionally substituted with SS(C.sub.1-C.sub.8-alkyl). In a preferred embodiment, R.sub.1 represents

##STR00037##

wherein R.sub.10 and R.sub.11 independently represent hydrogen or C.sub.1-C.sub.8-alkyl; and # represents the position of O. In a more preferred embodiment R.sub.10 and R.sub.11 independently represent hydrogen, methyl or ethyl. In a still more preferred embodiment, R.sub.1 represents

##STR00038##

wherein R.sub.10 and R.sub.11 independently represent hydrogen, methyl or ethyl; and # represents the position of O. In some of these embodiments R.sub.10 and R.sub.11 are both hydrogen. In some of these embodiments R.sub.10 is hydrogen and R.sub.11 is C.sub.1-C.sub.6-alkyl. In some of these embodiments R.sub.10 is hydrogen and R.sub.11 is methyl or ethyl. Preferably, in these embodiments both R.sub.1 are the same.

[0191] In some embodiments R.sub.1 represents C.sub.1-C.sub.8-alkyl substituted with phenyl, said phenyl being further substituted with

##STR00039##

wherein Z is O or NH, and wherein # represents the position of said phenyl. In some embodiments Z is O. In some embodiments Z is NH. The C.sub.1-C.sub.8-alkyl in the

##STR00040##

may be, for example, methyl, ethyl, propyl or butyl; preferably methyl, ethyl or propyl; more preferably methyl or ethyl; most preferably methyl. In a preferred embodiment R.sub.1 represents

##STR00041##

wherein the C.sub.1-C.sub.8-alkyl may be, for example, methyl, ethyl, propyl or butyl; preferably methyl, ethyl or propyl; more preferably methyl or ethyl; most preferably methyl; wherein Z is O or NH, and wherein # represents the position of O. In another preferred embodiment R.sub.1 represents

##STR00042##

[0192] In some embodiments R.sub.1 represents C.sub.1-C.sub.8-alkyl substituted with phenyl, said phenyl being further substituted with

##STR00043##

and wherein # represents the position of said phenyl. In some embodiments R.sub.1 represents C.sub.1-C.sub.8-alkyl substituted with phenyl, said phenyl being further substituted with

##STR00044##

wherein # represents the position of said phenyl. In a preferred embodiment R.sub.1 represents

##STR00045##

wherein # represents the position of O. In another preferred embodiment R.sub.1 represents

##STR00046##

wherein # represents the position of O. Preferably, in these embodiments both R.sub.1 are the same.

[0193] In some embodiments R.sub.1 represents an aliphatic or aromatic residue which is optionally substituted with hydroxy-(C.sub.1-C.sub.8-alkoxy).sub.n wherein n is 1, 2, 3, 4, 5 or 6. In a preferred embodiment R.sub.1 is hydroxyethoxyethyl, more preferably (CH.sub.2).sub.2O(CH.sub.2).sub.2OH.

[0194] In some embodiments R.sub.1 represents an aliphatic or aromatic residue which is optionally substituted with C.sub.2-C.sub.8-alkynyl. In a preferred embodiment R.sub.1 is homopropargyl.

[0195] In another preferred embodiment of a process according to the invention, custom-character represents a double bond, X represents (R.sub.3 R.sub.4)C, R.sub.3 and R.sub.4 independently represent H or C.sub.1-C.sub.8-alkyl and represents a bond. In another preferred embodiment, R.sub.3 and R.sub.4 each represent H.

[0196] In another preferred embodiment of a process according to the invention custom-character represents a triple bond, X represents R.sub.3C, R.sub.3 represents H or C.sub.1-C.sub.8-alkyl, more preferably H, and represents a double bond.

[0197] In another preferred embodiment of a process according to the invention ? represents an optionally substituted C.sub.1-C.sub.8-alkyl such as

##STR00047##

##STR00048##

wherein # represents the position of the N.sub.3 group of compounds of formula (IV), a radioactive or non-radioactive nuclide, biotin, a nucleotide, an oligonucleotide, a polymer, a carbohydrate, an amino acid, a peptide, an optionally substituted 5- or 6-membered heteroaromatic system, a protein tag, or a fluorophore such as CY.sub.5 or EDANS.

[0198] In another preferred embodiment of a process according to the invention ? represents [0199] a cyclic RGD peptide of structure (VIII) (c(RGDfK)

##STR00049## [0200] wherein [0201] * represents the position of the N.sub.3 group; [0202] Biotin; [0203] CY.sub.5 or EDANS; [0204] phenyl, optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkoxy, halogen, CN, NO.sub.2, NH.sub.2, N(C.sub.1-C.sub.8-alkyl), N(C.sub.1-C.sub.8-alkyl).sub.2-COOH, CO(C.sub.1-C.sub.8-alkyl), OC(O)(C.sub.1-C.sub.8-alkyl), C(O)N(C.sub.1-C.sub.8-alkyl), N(H)C(O)(C.sub.1-C.sub.8-alkyl) preferably optionally substituted with one substituent selected from the group consisting of C.sub.1-C.sub.8-alkoxy, COOH, COO(C.sub.1-C.sub.8-alkyl and NO.sub.2. [0205] C.sub.1-C.sub.8-alkyl optionally substituted with at least one substituent selected from the group consisting of C.sub.3-C.sub.8-cycloalkyl; heterocyclyl with 3 to 8 ring members wherein the heteroatom(s) are selected from N, O, S; C.sub.1-C.sub.8-alkoxy; halogen; CN; NO.sub.2; NH.sub.2; N(C.sub.1-C.sub.8-alkyl); N(C.sub.1-C.sub.8-alkyl).sub.2; COOH; COO(C.sub.1-C.sub.8-alkyl); OC(O)(C.sub.1-C.sub.8-alkyl); CONH.sub.2; C(O)N(C.sub.1-C.sub.8-alkyl).sub.2; C(O)NH(C.sub.1-C.sub.8-alkyl); N(H)C(O)(C.sub.1-C.sub.8-alkyl), preferably C.sub.1-C.sub.8-alkoxy, COOH, COO(C.sub.1-C.sub.8-alkyl and NO.sub.2, phenyl or a heteroaromatic system, a monosaccharide, a polysaccharide, a peptide, a nucleotide, an oligonucleotide, a polymer, an amino acid, a fluorophor, a protein tag (substituent 1.sup.st generation), wherein a substituent 1.sup.st generation may again optionally be substituted with C.sub.3-C.sub.8-cycloalkyl; heterocyclyl with 3 to 8 ring members wherein the heteroatom(s) are selected from N, O, S; C.sub.1-C.sub.8-alkoxy; halogen; CN; NO.sub.2; NH.sub.2; N(C.sub.1-C.sub.8-alkyl); N(C.sub.1-C.sub.8-alkyl).sub.2; COOH; COO(C.sub.1-C.sub.8-alkyl); OC(O)(C.sub.1-C.sub.8-alkyl); CONH.sub.2; C(O)N(C.sub.1-C.sub.8-alkyl).sub.2; C(O)NH(C.sub.1-C.sub.8-alkyl); N(H)C(O)(C.sub.1-C.sub.8-alkyl), preferably C.sub.1-C.sub.8-alkoxy, COOH, COO(C.sub.1-C.sub.8-alkyl and NO.sub.2, phenyl or a heteroaromatic system (substituents 2.sup.nd generation) and wherein a substituent 2.sup.nd generation may be substituted again by at least one substituent selected from the same group and wherein such substitution may go until generation 3, 4, 5, 6, 7, 8, 9 or 10.

[0206] In another preferred embodiment of a process according to the invention ? represents [0207] a cyclic RGD peptide of structure (VIII) (c(RGDfK)

##STR00050## [0208] wherein [0209] * represents the position of the N.sub.3 group; [0210] Biotin; [0211] CY.sub.5 or EDANS; [0212] phenyl, optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkoxy, halogen, CN, NO.sub.2, NH.sub.2, N(C.sub.1-C.sub.8-alkyl), N(C.sub.1-C.sub.8-alkyl).sub.2-COOH, CO(C.sub.1-C.sub.8-alkyl), OC(O)(C.sub.1-C.sub.8-alkyl), C(O)N(C.sub.1-C.sub.8-alkyl), N(H)C(O)(C.sub.1-C.sub.8-alkyl) preferably optionally substituted with one substituent selected from the group consisting of C.sub.1-C.sub.8-alkoxy, COOH, COO(C.sub.1-C.sub.8-alkyl and NO.sub.2. [0213] C.sub.1-C.sub.8-alkyl optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of phenyl which may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkoxy, halogen, CN, NO.sub.2, NH.sub.2, N(C.sub.1-C.sub.8-alkyl), N(C.sub.1-C.sub.8-alkyl).sub.2-COOH, COO(C.sub.1-C.sub.8-alkyl), OC(O)(C.sub.1-C.sub.8-alkyl), C(O)N(C.sub.1-C.sub.8-alkyl), N(H)C(O)(C.sub.1-C.sub.8-alkyl), preferably optionally substituted with one substituent selected from the group consisting of C.sub.1-C.sub.8-alkoxy, COOH, CO(C.sub.1-C.sub.8-alkyl and NO.sub.2; C.sub.1-C.sub.8-alkoxy; halogen; CN; NO.sub.2; NH.sub.2; N(C.sub.1-C.sub.8-alkyl); N(C.sub.1-C.sub.8-alkyl).sub.2; COOH; COO(C.sub.1-C.sub.8-alkyl); OC(O)(C.sub.1-C.sub.8-alkyl); C(O)N(C.sub.1-C.sub.8-alkyl); N(H)C(O)(C.sub.1-C.sub.8-alkyl), preferably C.sub.1-C.sub.8-alkoxy, COOH, COO(C.sub.1-C.sub.8-alkyl, NO.sub.2;

##STR00051##

wherein # represents the N-position.

[0214] In another preferred embodiment of a process according to the invention ? represents an optionally substituted phenyl such as

##STR00052##

wherein # represents the position of the N.sub.3 group. TFA.sup.? is trifluoroacetate.

[0215] In another preferred embodiment of a process according to the invention ? represents an optionally substituted C.sub.1-C.sub.8-alkyl such as a linker, a drug, or a linker-drug conjugate.

[0216] In another preferred embodiment of a process according to the invention ? represents an optionally substituted phenyl such as a linker, a drug, or a linker-drug conjugate.

[0217] In another preferred embodiment of a process according to the invention custom-character represents an antibody, preferably a IgG-antibody, such as Cetuximab or Trastuzumab; a peptide, such as GFP protein, eGFP-protein, a tripeptide, e.g., a peptide of formula (IX)

##STR00053## [0218] Wherein # represents the position of S; or [0219] optionally substituted C.sub.1-C.sub.8-alkyl such as

##STR00054## [0220] Wherein # markes the S-position.

[0221] In another preferred embodiment of a process according to the invention custom-character represents

##STR00055##

wherein # represents the position of S.

[0222] In an embodiment of a process according to the ?-N.sub.3 invention the and the custom-character -SH are in the same molecule.

[0223] Accordingly, the present invention also relates to a process wherein a compound of formula (XX)

##STR00056##

wherein ?-N.sub.3 the and the custom-character -SH are in the same molecule as indicated by the arc connecting the ? and the .
is reacted with a compound of formula (III) as defined herein to give a compound of formula (VIIa):

##STR00057## [0224] wherein custom-character represents a bond if in a compound of formula (III) represents a double bond; or [0225] represents a double bond if in a compound of formula (III) represents a triple bond; and [0226] , ?, R.sub.1 and X are as defined herein.

[0227] Accordingly, the present invention also relates to a process wherein a compound of formula (XX)

##STR00058##

wherein the ?-N.sub.3 and the custom-character -SH are in the same molecule as indicated by the arc connecting the ? and the .
is reacted with a compound of formula (III*) as defined herein to give a compound of formula (VII*a):

##STR00059## [0228] wherein custom-character , ?, V, R.sub.1 and X are as defined herein.

[0229] In some embodiments the compound (XX) having the ?-N.sub.3 and the custom-character -SH in the same molecule is a peptide, such as for example the BCL9 peptide. Accordingly, the compound of formula (VIIa) or (VII*a) obtained by the process may be a cyclic peptide, such as for example a cyclic peptide derived from the BCL9 peptide.

[0230] All steps for the processes described herein for compounds of formula (V), (V*), (VII) and (VII*) can be performed analogously for compounds of formula (VIIa) and (VII*a).

[0231] The incorporation of both an azide and a thiol into the same molecule provides for an intramolecular Staudinger-induced thiol addition that can realize an intramolecular cyclization as exemplarily shown in the following scheme:

##STR00060##

[0232] Without wishing to be bound by any theory, it is assumed that first the azide is reacting with the electron-rich alkyne/alkene-phosphonite upon which the phosphonamidate is formed and an electron-poor alkyne/alkene-phosphonamidate is formed that undergoes a fast intramolecular thiol addition with the SH moiety.

[0233] One embodiment of the present invention also refers to compounds of formula (VIIa) and (VII*a).

Compounds

[0234] The invention also refers to compounds of formula (V)

##STR00061##

[0235] Wherein R.sub.1 and X and ? are as defined above.

[0236] The invention also refers to compounds of formula (V*)

##STR00062## [0237] wherein ?, V, R.sub.1, and X are as defined above.

[0238] Preferably, in the compounds of formula (V) or (V*) ? represents an optionally substituted C.sub.1-C.sub.8-alkyl such as

##STR00063##

an optionally substituted phenyl such as

##STR00064##

wherein # represents the N-position; a radioactive or non-radioactive nuclide, biotin, a nucleotide, an oligonucleotide, a polymer, a carbohydrate, an amino acid, a peptide, an optionally substituted phenyl, an optionally substituted 5- or 6-membered heteroaromatic system, an optionally substituted C.sub.1-C.sub.8-alkyl, a protein tag or a fluorophore such as CY.sub.5.

[0239] Preferably, in the compounds of formula (V) or (V*) ? represents an optionally substituted phenyl such as

##STR00065##

wherein # represents the position of N. TFA.sup.? is trifluoroacetate.

[0240] Preferably, in the compounds of formula (V) or (V*) ? represents an optionally substituted C.sub.1-C.sub.8-alkyl such as a linker, a drug, or a linker-drug conjugate.

[0241] Preferably, in the compounds of formula (V) or (V*) ? represents an optionally substituted phenyl such as a linker, a drug, or a linker-drug conjugate.

[0242] The invention also refers to compounds of formula (VII)

##STR00066## [0243] wherein [0244] custom-character represents a bond and X represents (R.sub.3 R.sub.4)C; or [0245] represents a double bond and X represents R.sub.3C; [0246] R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; [0247] R.sub.4 represents H or C.sub.1-C.sub.8-alkyl; [0248] represents an optionally substituted C.sub.1-C.sub.8-alkyl, an optionally substituted Phenyl, an optionally substituted aromatic 5- or 6-membered heterocyclic system, an amino acid, a peptide, a protein, an antibody, a saccharide, a polysaccharide, a nucleotide, a oligonucleotide or a polymer; [0249] ? represents an aliphatic or aromatic residue; [0250] R.sub.1 independently represents an optionally substituted aliphatic or aromatic residue, such as phenyl; with (C.sub.1-C.sub.8-alkoxy).sub.n, wherein n is 1, 2, 3, 4, 5 or 6 with F, with Cl, with Br, with I, with NO.sub.2, with N(C.sub.1-C.sub.8-alkyl)H, with NH.sub.2, with N(C.sub.1-C.sub.8-alkyl).sub.2, with ?O, with C.sub.3-C.sub.8-cycloalky, with optionally substituted phenyl substituted C.sub.1-C.sub.8-alkyl such as

##STR00067##

[0251] The invention also refers to compounds of formula (VII*)

##STR00068## [0252] wherein [0253] custom-character represents an optionally substituted C.sub.1-C.sub.8-alkyl, an optionally substituted Phenyl, an optionally substituted aromatic 5- or 6-membered heterocyclic system, an amino acid, a peptide, a protein, an antibody, a saccharide, a polysaccharide, a nucleotide, a oligonucleotide or a polymer; [0254] ? represents an aliphatic or aromatic residue; [0255] R.sub.1 independently represents an optionally substituted aliphatic or aromatic residue, such as phenyl; with (C.sub.1-C.sub.8-alkoxy).sub.n, wherein n is 1, 2, 3, 4, 5 or 6 with F, with Cl, with Br, with I, with NO.sub.2, with N(C.sub.1-C.sub.8-alkyl)H, with NH.sub.2, with N(C.sub.1-C.sub.8-alkyl).sub.2, with ?O, with C.sub.3-C.sub.8-cycloalky, with optionally substituted phenyl substituted C.sub.1-C.sub.8-alkyl such as

##STR00069##

or optionally independently with C.sub.1-C.sub.8-alkyl, (C.sub.1-C.sub.8-alkoxy).sub.n, F, Cl, I, Br, NO.sub.2, N(C.sub.1-C.sub.8-alkyl)H, NH.sub.2, N(C.sub.1-C.sub.8-alkyl).sub.2, substituted phenyl; or 5- or 6-membered heteroaromatic system such as pyridyl; preferably C.sub.1-C.sub.8-alkyl, C.sub.1-C.sub.8-alkyl substituted with (C.sub.1-C.sub.8-alkoxy).sub.n, phenyl or phenyl substituted with NO.sub.2; [0256] V represents C.sub.1-C.sub.8-alkyl, preferably methyl, ethyl or propyl, more preferably methyl; [0257] X represents (R.sub.3 R.sub.4)C [0258] R.sub.3 represents H or C.sub.1-C.sub.8-alkyl; and [0259] R.sub.4 represents H or C.sub.1-C.sub.8-alkyl.

[0260] Preferably, in the compounds of formula (VII) or (VII*) ? represents an optionally substituted C.sub.1-C.sub.8-alkyl such as

##STR00070##

an optionally substituted phenyl such as

##STR00071##

a radioactive or non-radioactive nuclide, biotin, a nucleotide, an oligonucleotide, a polymer, a carbohydrate, an amino acid, a peptide, an optionally substituted phenyl, an optionally substituted 5- or 6-membered heteroaromatic system, an optionally substituted C.sub.1-C.sub.8-alkyl, a protein tag or a fluorophore such as CY.sub.5 or EDANS.

[0261] Preferably, in the compounds of formula (VII) or (VII*) ? represents an optionally substituted phenyl such as

##STR00072##

wherein # represents the position of N. TFA.sup.? is trifluoroacetate.

[0262] Preferably, in the compounds of formula (VII) or (VII*) ? represents an optionally substituted C.sub.1-C.sub.8-alkyl such as a linker, a drug, or a linker-drug conjugate.

[0263] Preferably, in the compounds of formula (VII) or (VII*) ? represents an optionally substituted phenyl such as a linker, a drug, or a linker-drug conjugate.

[0264] Preferably, in the compounds of formula (VII) or (VII*) custom-character represents an antibody, preferably a IgG-antibody, more preferably a Cetuximab or a Trastuzumab; a peptide, preferably GFP protein or eGFP-protein or a tripeptide, more preferably a peptide of formula (IX) or C.sub.1-C.sub.8-alkyl.

[0265] Preferably, in the compounds of formula (VII) or (VII*) custom-character represents

##STR00073##

wherein # represents the position of S.

[0266] Preferred conjugates of formula (VII) or formula (VII*) are conjugates wherein

custom-character represents an antibody and
? represents a protein tag or a fluorophore such as CY.sub.5 or EDANS, or a protein.

[0267] Further preferred conjugates of formula (VII) are conjugates wherein

custom-character represents a protein and
? represents a protein tag or a fluorophore such as CY.sub.5 or EDANS, an antibody or a protein.

[0268] One preferred embodiment are conjugates of formula (VII) wherein

custom-character represents a protein and
? represents a protein.

[0269] Further, preferred conjugates of formula (VII) or formula (VII*) are conjugates wherein

custom-character represents an antibody and
? represents a linker, a drug, or a linker-drug conjugate.

[0270] The invention also refers to compounds of formula (VIIa)

##STR00074##

wherein ? and custom-character are in the same molecule as indicated by the arc connecting the ? and the , and wherein ?, , , X and R.sub.1 are as defined herein, in particular as defined with regard to compound (VII). Preferably, the compound (VIIa) is a cyclic peptide, such as for example a cyclic peptide derived from the BCL9 peptide.

[0271] The invention also refers to compounds of formula (VII*a)

##STR00075##

wherein ? and custom-character are in the same molecule as indicated by the arc connecting the ? and the , and wherein ?, , V, X and R.sub.1 are as defined herein, in particular as defined with regard to compound (VII*). Preferably, the compound (VII*a) is a cyclic peptide, such as for example a cyclic peptide derived from the BCL9 peptide.

[0272] The following compounds of formula (VII) are also preferred:

wherein the protein on the left side is GFP-protein, preferably eGFP-protein;

##STR00076##

[0273] A fluorescently labeled ASGP-R addressing Cy.sub.5 conjugate of formula (X) which can be produced via the modular addition to vinyl phosphonamidates:

##STR00077## ##STR00078## ##STR00079##

[0274] The following compounds of formula (VII) are also preferred:

##STR00080##

##STR00081##

wherein LD represents a linker drug conjugate having the
structure

##STR00082##

and # represents the position of the N; and

##STR00083##

[0275] Moreover, also compounds provided herein as examples in the example section for compounds of formula (VII) are preferred.

[0276] The skilled person understands that embodiments according to the invention can be combined with each other with the proviso that a combination which would contravene any natural law is excluded.

Synthesis of Phosphonamidate of Formula (V)

Step a)

[0277] General procedure for the preparation of alkenyl or alkynyl phosphonamidates by Staudinger phosphonite reaction requires the reaction of an alkenyl- or alkynylmagnesiumbromide of formula (II) with a dialkyl halogenchlorophosphite of formula (I), preferably a chlorophosphonite, below ?20? C., e.g. between ?100? C. and ?40? C., preferably between ?90? C. and ?50? C. (e.g. around 87? C.). Preferably, the reaction is carried out under inert gas such as argon. Inert in this situation refers to a gas which will not react with any of the educts or products of this reaction under the given reaction conditions. Of course, the reaction time depends on the reaction volume and amount of substance. However, as a guideline, the reaction time should be in a range from 2 min to 4 h. The amounts of compound of formula (I) and (II) should be in a range from 5:1 to 1:5 such as 2:1 to 1:2, e.g., around 1:1.

step (I)

[0278] The reaction of a compound of formula (III) with an azide of formula (IV) can be performed at room temperature, i.e. around 25? C. However, the reaction can also be carried out at temperatures in a range from 0? C. to 50? C. The reaction time depends on the reaction volume and the amount of substance. However, as a guideline, the reaction should be carried out in a time frame from 1 h to 72 h. The amounts of compound of formula (III) and (IV) should be in a range from 5:1 to 1:5 such as 2:1 to 1:2, e.g., around 1:1.

[0279] Preferred solvents for step (I) described herein is carried out in a polar aprotic solvent system such as tetrahydrofurane (THF), dimethylformamide (DMF), acetonitrile (MeCN), acetone, dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), N-methylpyrrolidone or mixtures thereof, preferably THF, DMF, MeCN, THF/DMF, THF/MeCN; or a mixture of a polar unprotic solvent and a non-polar solvent such as hexane, toluene, benzene, 1,4-dioxane, chloroform, diethylether or dichloromethane (DCM), preferably THF/toluene. Step (I) may be also carried out in an aqueous medium, for example in water or in an aqueous buffer, such as for example phosphate-buffered saline (PBS), tris(hydroxymethyl)-aminomethane (TRIS) or bicarbonate.

Procedure for Base Mediated Hydrothiolations of Electron-Deficient Phosphonamidate Alkynes

Step (II)

[0280] Phosphonamidate of formula (V) and a base (and additive where required) can be suspended in a respective solvent. Then a thiol of formula (VI) can be added, e.g., via a microliter syringe and the mixture is allowed to react at room temperature, i.e. around 25? C. However, the reaction can also be carried out at temperatures in a range from 0? C. to 50? C. The reaction time depends on the reaction volume and the amount of substance. However, as a guideline, the reaction should be carried out in a time frame from 0.1 h (hours) to 10 h, e.g., in a time frame from 0.1 h to 3 h or even within a time frame between 0.1 h and 1 h.

[0281] In a preferred embodiment, step (II) described herein is carried out in the presence of a weak base. Preferred weak bases are carbonates such as ammonium (NH.sub.4).sub.2 CO.sub.3, Na.sub.2 CO.sub.3, Rb.sub.2 CO.sub.3, K.sub.2 CO.sub.3, or Cs.sub.2 CO.sub.3 or correlating hydrogencarbonates thereof (e.g. NaHCO.sub.3 etc.); and weak Nitrogen containing bases such as triethylamine Et.sub.3N (pK.sub.a 10,76 at 25? C.). Preferably, a base with a pK.sub.a value within the range of 7.5 to 11.5 is used.

[0282] The solvent (system) can be chosen from a wide range of solvents. The solvent can be a polar aprotic solvent system such as tetrahydrofurane (THF), dimethylformamide (DMF), acetonitrile (MeCN), acetone, dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), N-methylpyrrolidone or mixtures thereof, preferably THF, DMF, DMSO; non-polar solvents such as hexane, toluene, benzene, 1,4-dioxane, chloroform, diethylether or dichloromethane (DCM), preferably DCM; polar protic solvents suc as water, ethanol, isopropanol, methanol, n-butanol, preferably erthanol; or mixtures thereof, e.g., DMF/water. The solvent may be also an aqueous medium, such as for example water or an aqueous buffer, such as for example phosphate-buffered saline (PBS), tris(hydroxymethyl)-aminomethane (TRIS) or bicarbonate.

EXAMPLES

General Procedure for the Preparation of Alkynyl Phosphonamidates

[0283] In a flame dried Schlenk flask under an atmosphere of argon a solution of ethynyl magnesiumbromide (0.5 M in THF, 2 mL, 1 mmol) was cooled to ?78? C. in a bath of dry ice/acetone. The diethyl chlorophosphite (157 mg, 144 ?L, 1 mmol) was added dropwise via a syringe. The solution was stirred at ?78? C. for 30 minutes, then warmed up to room temperature and subsequently stirred for another 1.5 hours. Afterwards 3 mL of dry THF and azide (1 mmol) was added and the solution was stirred at room temperature for 24 hours. Then H.sub.2O (5 mL) was added and the solution was stirred for another 24 hours open to air. After removal of the solvent under reduced pressure the crude mixture was analyzed by .sup.31P NMR.

Synthesis of Vinyl Phosphonites.

[0284] General Procedure a for the Synthesis of Vinyl Phosphonites from Phosphorous Trichloride.

[0285] A flame-dried Schlenkflask was charged with 1.50 mmol (1.0 eq.) of phosphorous trichloride in 20 ml of dry toluene and cooled to ?78? C. 3.3 mmol of pyridine (2.2 eq.) and a solution of 3.3 mmol of the alcohol (2.2 eq.) in 5 ml Et.sub.2O were added drop wise. The resulting suspension was allowed to warm to room temperature, stirred for another 30 min and cooled again to ?78? C. 1.65 mmol (1.1 eq.) of Vinylgrignard (1.0 M in THF) was added and the reaction was stirred at room temperature for two hours. Finally 2.25 mmol (1.5 eq.) of borane (1.0 M in THF) were added at 0? C. and stirred for another hour. The crude product was dry packed on a silica column for purification.

General Procedure B for the Synthesis of Vinyl Phosphonites from Bis(Diisopropylamino)Chlorophosphine

[0286] A flame-dried Schlenkflask was charged with 1.5 mmol (1.0 eq.) Bis(diisopropylamino)chlorophosphine, dissolved in 200 ?l of dry THF and cooled to ?78? C. 1.65 mmol (1.1 eq.) of Vinylgrignard (1.0 M in THF) was added and the reaction was stirred at room temperature for 30 minutes. A solution of 3.3 mmol vacuum dried alcohol (2.2 eq.) in 1 ml of dry THF or MeCN and 3.3 mmol (2.2 eq.) Tetrazole (0.45 M in MeCN) was added. The resulting suspension was stirred at room temperature overnight. Finally 2.25 mmol (1.5 eq.) of borane (1.0 M in THF) were added at 0? C. and stirred for another hour. The crude product was dry packed on a silica column for purification.

General Procedure C for the Synthesis of Vinyl Phosphonamidates from Diethylchloro Phosphite, Vinyl Grignard Reagent and Different Azides

[0287] A 25-ml Schlenk flask was charged with 1.71 ml vinylmagnesium bromide (0.7 M in THF, 1.20 mmol, 1.2 eq.) under an argon atmosphere, cooled to ?78? C. and 140 ?l diethyl chlorophosphite (1.00 mmol, 1.0 eq.) were added drop wise. The yellowish solution was allowed to warm to 0? C., stirred for another two hours and 1.00 mmol of azide (1.0 eq.) dissolved in 3.2 ml of THF was added and stirred over night at room temperature. 5 ml of water were added and stirred for another 24 h. The solvents were removed under reduced pressure and the crude product was purified by flash column chromatography on silica gel.

Ethyl-N-phenyl-P-ethynyl-phosphonamidate

[0288] ##STR00084##

[0289] Ethyl N-phenyl-P-ethynyl-phosphonamidate was prepared after general procedure for the preparation of alkenyl or alkynyl phosphonamidates on 5 mmol scale from phenyl azide (595 mg, 5 mmol). The crude mixture was purified by silica gel column chromatography eluting with hexane/ethyl acetate. The product was obtained as colourless solid in a yield of 430 mg (2.1 mmol, 42%). .sup.1H NMR (300 MHz, Chloroform-d): ?=7.28 (t, J=7.7 Hz, 2H, ArH), 7.11 (d, J=8.0 Hz, 2H, ArH), 7.02 (t, J=7.3 Hz, 1H, ArH), 6.74 (d, J=7.6 Hz, 1H, NH), 4.55-3.93 (m, 2H, CH.sub.2), 2.89 (d, J=12.8 Hz, 1H, CH), 1.39 (t, J=7.0 Hz, 3H, CH.sub.3) ppm. .sup.13C NMR (75 MHz, Chloroform-d): ?=138.99, 129.39, 122.42, 118.23, 118.13, 88.10, 87.45, 76.34 (d, J=273.3 Hz), 62.26 (d, J=5.2 Hz), 16.23 (d, J=7.4 Hz) ppm. .sup.31P NMR (122 MHz, Chloroform-d): ?=?9.17 ppm. HRMS ESI-TOF m/z [M+H].sup.+=210.0678 (calcd.); 210.0687 (found).

Ethyl-N-benzyl-P-ethynyl-phosphonamidate

[0290] ##STR00085##

[0291] Ethyl N-benzyl-P-ethynyl-phosphonamidate was prepared after general procedure for the preparation of alkenyl or alkynyl phosphonamidates from benzyl azide (133 mg, 125 ?L, 1 mmol). The crude mixture was purified by silica gel column chromatography eluting with hexane/ethyl acetate. The product was obtained as colourless solid in a yield of 37 mg (0.17 mmol, 17%). .sup.1H NMR (300 MHz, Chloroform-d): ?=7.51-7.18 (m, 5H, ArH), 4.26-4.04 (m, 4H, 2?CH.sub.2), 3.34 (s, 1H, CH), 2.91 (d, J=12.7 Hz, 1H, NH), 1.34 (t, J=7.1 Hz, 3H, CH.sub.3) ppm. .sup.13C NMR (75 MHz, Chloroform-d): ?=138.99, 138.90, 128.71, 127.62, 127.54, 87.77, 87.16, 76.83 (d, J=260.0 Hz), 62.03 (d, J=5.1 Hz), 44.86, 16.25 (d, J=7.3 Hz) ppm. .sup.31P NMR (122 MHz, Chloroform-d): ?=?2.76 ppm. HRMS ESI-TOF m/z [M+H].sup.+=224.0835 (calcd.); 224.0835 (found).

Ethyl-N-phenyl-P-vinyl-phosphonamidate

[0292] ##STR00086##

[0293] The compound was synthesized according to the general procedure C from 1.15 ml diethyl chlorophosphite (8 mmol). The pure phosphonamidate was purified by flash column chromatography (EtOAc) and obtained as a white solid. (675 mg, 3.20 mmol, 40.0%)

[0294] .sup.1H NMR (600 MHz, Chloroform-d) ?=7.24 (dd, J=8.5, 7.3, 2H), 7.05-7.01 (m, 2H), 6.99 (d, J=5.8, 1H), 6.94 (tt, J=7.3, 1.1, 1H), 6.33-6.23 (m, 2H), 6.10 (ddd, J=50.3, 9.6, 5.1, 1H), 4.29-4.04 (m, 2H), 1.35 (t, J=7.1, 3H). .sup.13C NMR (151 MHz, Chloroform-d) ?=140.43, 134.44, 129.28, 127.51 (d, J=172.7), 121.26, 117.31 (d, J=6.6), 60.44 (d, J=6.2), 16.22 (d, J=7.0). .sup.31P NMR (122 MHz, Chloroform-d) ?=15.68. HRMS for C.sub.10H.sub.15NO.sub.2P.sup.+ [M+H].sup.+ calcd: 212.0835, found: 212.0839.

Ethyl-N-(4-carboxy-phenyl)-P-vinyl-phosphonamidate

[0295] ##STR00087##

[0296] The compound was synthesized according to the general procedure C from 288 ?l diethyl chlorophosphite (2 mmol). The pure phosphonamidate was purified by flash column chromatography (CH.sub.2Cl.sub.2/MeOH, 9:1 to 4:1) and obtained as a white solid. (173 mg, 0.68 mmol, 34.0%)

[0297] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.37 (d, J=7.9, 1H), 7.80 (d, J=8.7, 2H), 7.12 (d, J=8.7, 2H), 6.42-6.04 (m, 3H), 4.11-3.94 (m, 2H), 1.26 (t, J=7.0, 3H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) ?=167.56, 146.36, 135.00, 131.21, 129.04 (d, J=165.8), 123.06, 117.00 (d, J=6.9), 60.84 (d, J=5.7), 16.61 (d, J=6.3). .sup.31P NMR (122 MHz, DMSO-d.sub.6) ?=14.36. HRMS for C.sub.11H.sub.15NO.sub.4P.sup.+ [M+H].sup.+ calcd: 256.0733, found: 256.0723.

Ethyl-N-benzyl-P-vinyl-phosphonamidate

[0298] ##STR00088##

[0299] The compound was synthesized according to the general procedure C from 290 ?l diethyl chlorophosphite (2 mmol). The pure phosphonamidate was purified by flash column chromatography (EtOAc) and obtained as a colourless oil. (155 mg, 0.69 mmol, 34.3%)

[0300] .sup.1H NMR (300 MHz, Chloroform-d) ?=7.36-7.21 (m, 5H), 6.33-5.88 (m, 3H), 4.16-3.90 (m, 4H), 3.21 (d, J=8.5, 1H), 1.28 (t, J=7.1, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=139.65 (d, J=5.9), 133.21 (d, J=1.5), 129.45, 128.46, 127.20, 127.17, 60.11 (d, J=5.7), 44.58, 16.27 (d, J=6.7). .sup.31P NMR (122 MHz, Chloroform-d) ?=20.52. HRMS for C.sub.11H.sub.17NO.sub.2P calcd: 226.0991, found: 226.1003

Ethyl-N-(2-nitro-Benzyl)-P-vinyl-phosphonamidate

[0301] ##STR00089##

[0302] The compound was synthesized according to the general procedure C from 120 ?l diethyl chlorophosphite (0.83 mmol). The pure phosphonamidate was purified by flash column chromatography (2% MeOH in CH.sub.2Cl.sub.2) and obtained as a brown oil. (125 mg, 0.46 mmol, 55.4%)

[0303] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.03 (d, J=8.1, 1H), 7.73-7.57 (m, 2H), 7.45 (t, J=7.6, 1H), 6.31-5.83 (m, 3H), 4.39 (dd, J=11.2, 7.7, 2H), 4.12-3.85 (m, 2H), 3.65 (q, J=8.6, 1H), 1.26 (t, J=7.1, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=148.09, 135.45 (d, J=4.2), 133.83, 133.52 (d, J=1.6), 131.10, 128.41, 128.26 (d, J=169.7), 124.95, 60.35 (d, J=5.7), 42.42 (d, J=1.3), 16.22 (d, J=6.7). .sup.31P NMR (122 MHz, Chloroform-d) ?=20.63. HRMS for C.sub.11H.sub.16N.sub.2O.sub.4P calcd: 271.0842, found: 271.0851.

Ethyl-N-(3-phenyl-propyl)-P-vinyl-phosphonamidate

[0304] ##STR00090##

[0305] The compound was synthesized according to the general procedure C from 290 ?l diethyl chlorophosphite (2 mmol). The pure phosphonamidate was purified by flash column chromatography (EtOAc) and obtained as a colourless oil. (165 mg, 0.65 mmol, 32.5%)

[0306] .sup.1H NMR (300 MHz, Chloroform-d) ?=7.28 (dd, J=8.1, 6.2, 2H), 7.23-7.11 (m, 3H), 6.28-5.89 (m, 3H), 4.04 (qt, J=10.2, 7.2, 2H), 2.92 (dq, J=9.1, 7.0, 2H), 2.84-2.70 (m, 1H), 2.70-2.60 (m, 2H), 1.82 (p, J=7.3, 2H), 1.31 (t, J=7.1, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=141.28, 132.98 (d, J=1.5), 128.42 (d, J=169.0), 128.34, 128.24, 125.88, 59.95 (d, J=5.7), 40.23, 33.53 (d, J=5.6), 32.86, 16.32 (d, J=6.7). .sup.31P NMR (122 MHz, Chloroform-d) ?=20.82. HRMS for C.sub.11H.sub.16N.sub.2O.sub.4P.sup.+ calcd: 271.0842, found: 271.0851. HRMS for C.sub.13H.sub.21NO.sub.2P.sup.+ calcd: 254.1304, found: 254.1312.

Ethyl-N-cyclohexyl-P-vinyl-phosphonamidate

[0307] ##STR00091##

[0308] The compound was synthesized according to the general procedure C from 140 ?l diethyl chlorophosphite (1 mmol). The pure phosphonamidate was purified by flash column chromatography (1.5% MeOH in CH.sub.2Cl.sub.2) and obtained as a colourless oil. (70 mg, 0.32 mmol, 32.2%)

[0309] .sup.1H NMR (600 MHz, Chloroform-d) ?=6.25-5.93 (m, 3H), 4.14-3.97 (m, 2H), 2.96 (dqd, J=13.8, 9.6, 8.1, 4.2, 1H), 2.51 (t, J=9.6, 2H), 1.97-1.84 (m, 2H), 1.74-1.65 (m, 1H), 1.57 (dt, J=13.0, 3.9, 1H), 1.32 (t, J=7.1, 3H), 1.30-1.09 (m, 5H). .sup.13C NMR (75 MHz, Chloroform-d) ?=132.56 (d, J=1.8), 129.30 (d, J=168.8), 59.80 (d, J=5.9), 49.71, 36.03, 25.24, 24.96, 16.32 (d, J=6.8). .sup.31P NMR (122 MHz, Chloroform-d) ?=19.34. HRMS for C.sub.10H.sub.21NO.sub.2P.sup.+ calcd: 218.1304, found: 218.1302.

Staudinger-Induced Thiol-Additions with Alkynyl-Phosphonites:
Synthesis of Diethyl-Alkynyl-Phosphonite and Reaction with Different Azides (Step b)

[0310] Diethyl-alkynyl-phosphonite was synthesized according to published protocols (13) and reacted with different aliphatic and aromatic azides (Scheme 3). The formation of the desired alkynyl-phosphonamidates was monitored by .sup.31P-NMR (see Table 1 for conversions for different azide substrates).

##STR00092##

TABLE-US-00001 TABLE 1 Substrate scope for the Staudinger phosphonite reaction of diethyl-alkynyl- phosphonite (values in %) n. d. = not detected); determined by .sup.31P-NMR. Entry R = [00093] embedded image [00094] [00095] 1 [00096] 94 n. d. n. d. 2 [00097] 91 n. d. n. d. 3 [00098] 70 n. d. 25 4 [00099] 44 n. d. n. d. 5 [00100] 70 4 25 6 [00101] 73 10 14 7 [00102] 76 5 18 8 [00103] 45 17 28

[0311] N-phenyl- and N-benzyl-phosphonamidates were isolated by column chromatography in yields of 41% and 17% respectively. The highest conversions were obtained in THF (Table 2).

TABLE-US-00002 TABLE 2 Influence of the solvent on the Staudinger phosphonite reaction between diethyl-alkynyl-phosphonite and phenylazide. Entry Solvent Conversion 1 THF 94 2 THF/DMF 86 3 THF/Acetonitrile 91 4 THF/Toluene 88

General Procedure for Base Mediated Hydrothiolations of Phosphonamidate Alkynes or Alkenes

[0312] To a capped vial Ethyl N-phenyl-P-ethynyl-phosphonamidate (10 mg, 0.05 mmol) and the respective base (and additive where required) was added. The mixture was suspended in 200 ?L of respective solvent. Then ethanethiol (3.1 mg, 3.6 ?L, 0.05 mmol) was added via a microliter syringe and the mixture was stirred at room temperature for 3 hours. Afterwards the mixture was diluted with CH.sub.2Cl.sub.2 (5 mL) and H.sub.2O (5 mL) was added. After extraction the phases were separated and the aqueous layer was extracted three times with CH.sub.2Cl.sub.2 (5 mL). The combined organic layers were washed two times with H.sub.2O (5 mL) and with brine (5 mL). After removal of the solvent the crude mixture was analyzed by .sup.1H NMR and .sup.31P NMR. The preparation of alkene phosphonamidates is analogous to the preparation of alkyne phosphonamidates.

Ethyl-N-phenyl-P-(2-ethylsulfanyl)-ethenyl-phosphonamidate

[0313] ##STR00104##

[0314] To a capped vial Ethyl N-phenyl-P-ethynyl-phosphonamidate (10 mg, 0.05 mmol) and potassium carbonate (2.8 mg, 0.02 mmol) was added. The mixture was suspended in a 1 to 1 mixture of DMF/H.sub.2O (200 ?L). Then ethanethiol (3.1 mg, 3.6 ?L, 0.05 mmol) was added via a microliter syringe and the mixture was stirred at room temperature for 3 hours. Afterwards the mixture was diluted with CH.sub.2Cl.sub.2 (5 mL) and H.sub.2O (5 mL) was added. After extraction the phases were separated and the aqueous layer was extracted three times with CH.sub.2Cl.sub.2 (5 mL). The combined organic layers were washed two times with H.sub.2O (5 mL) and with brine (5 mL).

[0315] After removal of the solvent under reduced pressure the product was obtained in a yield of 12 mg (0.044 mmol, 89%). .sup.1H NMR (300 MHz, Chloroform-d): ?=7.45 (dd, J=21.7, 16.7 Hz, 1H, PCH, E), 7.30-7.17 (m, 3H, ArH), 7.06 (d, J=12.5 Hz, 1H, SCH, Z), 7.01-6.90 (m, 2H, ArH), 5.75 (dd, J=16.7, 12.5 Hz, 1H, PCH, Z), 4.35-4.00 (m, 2H, OCH.sub.2), 2.75 (q, J=7.5 Hz, 2H, SCH.sub.2), 1.36 (t, J=7.0 Hz, 3H, OCH.sub.2CH.sub.3), 1.28 (t, J=7.5 Hz, 3H, SCH.sub.2CH.sub.3) ppm. .sup.13C NMR (75 MHz, Chloroform-d): ?=150.40, 140.11, 129.31, 121.50, 117.47 (d, J=6.4 Hz), 60.61 (d, J=6.0 Hz), 29.59, 25.90, 16.42 (d, J=6.9 Hz), 15.53, 13.75 ppm. .sup.31P NMR (122 MHz, Chloroform-d) ? 15.13, 14.35 ppm. HRMS ESI-TOF m/z [M+H].sup.+=272.0869 (calcd.); 272.0855 (found).

(Ethyl-N-phenyl-P-ethenyl-phosphonamidate)-S-glutathion conjugate

[0316] ##STR00105##

[0317] To a capped vial Ethyl N-phenyl-P-ethynyl-phosphonamidate (31 mg, 0.15 mmol) and potassium carbonate (7 mg, 0.05 mmol) was added. The mixture was suspended in a 1 to 1 mixture of DMF/H.sub.2O (500 ?L). Then (2S)-2-amino-4-{[(1R)-1-[(carboxymethyl)carbamoyl]-2-sulfanylethyl]carbamoyl}-butanoic acid (31 mg, 0.1 mmol) was added and the mixture was stirred at room temperature for 3 hours. Afterwards the mixture was diluted with H.sub.2O (5 mL) and CH.sub.2Cl.sub.2 (5 mL) was added. After extraction the phases were separated and the organic layer was extracted three times with H.sub.2O (5 mL). The aqueous layers were washed three times with CH.sub.2Cl.sub.2 (5 mL). Afterwards the solvent was removed under reduced pressure. The crude mixture was purified by preparative HPLC eluting with acetonitrile and ammonium acetate buffer. The product was obtained as ammonium acetate salt in a yield of 35.5 mg (0.061 mmol, 61%). .sup.1H NMR (300 MHz, Deuterium Oxide): ?=7.37 (d, J=12.6 Hz, 1H, SCH, Z), 7.21 (t, J=7.9 Hz, 2H, ArH), 6.94 (dd, J=7.8, 5.3 Hz, 3H, ArH), 5.77 (dd, J=17.5, 12.2 Hz, 1H, PCH, Z), 4.45 (ddd, J=12.8, 8.3, 5.2 Hz, 1H), 4.01 (q, J=7.4 Hz, 2H, CH.sub.2), 3.84-3.52 (m, 2H, CH.sub.2), 3.21 (dd, J=14.7, 5.1 Hz, 1H, CH), 3.02 (dd, J=14.6, 8.5 Hz, 1H, CH), 2.45-2.20 (m, 2H, CH.sub.2), 1.96 (q, J=7.2 Hz, 2H, CH.sub.2), 1.19 (t, J=7.1 Hz, 3H, CH.sub.3) ppm. .sup.13C NMR: (75 MHz, Deuterium Oxide) ?=174.66, 174.45, 173.59, 171.25, 171.19, 151.73, 139.17, 129.38, 122.06, 117.75 (d, J=6.8 Hz), 86.89, 86.83, 86.72, 62.14, 53.76 (d, J=12.1 Hz), 42.35, 35.94, 31.18, 25.92, 15.41 ppm. .sup.31P NMR: (122 MHz, Deuterium Oxide) ?=18.96, 18.11 (d, J=4.2 Hz) ppm. HRMS ESI-TOF m/z [M+H].sup.+=517.1516 (calcd.); 517.1526 (found).

Thiol-Addition of Ethanethiol and Glutathione to Alkynyl-Phosphonamidate

[0318] Ethanethiol was chosen as aliphatic model substrate. All experiments were conducted for 3 hours at room temperature in 0.1 mmol scale using 400 ?L of the solvent. Conversions and diastereoselectivities were determined by .sup.31P-NMR and .sup.1H-NMR (Scheme 4).

##STR00106##

[0319] First experiments confirmed the formation of both the E- and the Z-conformational isomer. The vicinal HH coupling constant of 12.5 Hz of the major diastereomer and 21.7 Hz of the minor diastereomer in the .sup.1H NMR of the diastereomeric mixture indicates that the Z-isomer is the major product for all the reaction conditions (see Table 3).

TABLE-US-00003 TABLE 3 Screening of solvents for the base mediated hydrothiolation of electron-deficient alkynyl phosphonamidates. Entry Solvent Base Conversion E/Z 1 CH.sub.2Cl.sub.2 K.sub.2CO.sub.3 100% 1:99 2 EtOH K.sub.2CO.sub.3 100% 2:98 3 DMF/H.sub.2O K.sub.2CO.sub.3 100% 2:98 (1:1) 4 THF K.sub.2CO.sub.3 100% 2:98 5 DMF K.sub.2CO.sub.3 100% 5:95 6 DMSO K.sub.2CO.sub.3 100% 12:88

[0320] The influence of the solvent to the thiol-addition was then further investigated revealing quantitative formation of the thiol adduct for every tested solvent. Full conversion was achieved in all of the tested solvents. DMSO showed the lowest diastereoselectivity (12% E-product). Therefore the influence of the base was than further investigated in DMSO and in DMF/H.sub.2O (1:1) (Table 4).

TABLE-US-00004 TABLE 4 Screening of bases for the hydrothiolation of electron-deficient alkynyl phosphonamidates in DMSO and DMF/H.sub.2O (1:1). DMSO DMF/H.sub.2O Entry Base Conversion E/Z Conversion E/Z 1 Et.sub.3N 5% / 36% 3:97 2 (NH.sub.4).sub.2CO.sub.3 100% 4:96 100% 2:98 3 Na.sub.2CO.sub.3 100% 6:94 100% 1:99 4 Rb.sub.2CO.sub.3 100% 8:92 100% 2:98 5 K.sub.2CO.sub.3 100% 12:88 100% 2:98 6 Cs.sub.2CO.sub.3 100% 17:83 100% 2:98

[0321] It turned out that the diastereoselectivity of the reaction in DMSO is dependent of the applied base. In contrast to this, reactions in aqueous systems always delivered the Z-alkene as the major product.

[0322] In conclusion it was possible to optimize the reaction conditions of the model reactions. The reaction can be applied in aqueous solvent systems and quantitative conversions can be achieved at room temperature after 3 hours using mild carbonate bases. No side reactions were observed.

[0323] In the next step these optimized reaction conditions were now applied to synthesize a water soluble glutathion phosphonamidate conjugate (Scheme 5).

##STR00107##

[0324] The conjugate could be isolated by semipreparative HPLC under basic conditions as a diastereomeric mixture in a yield of 61%. Having reasonable quantities of this water soluble phosphonamidate-conjugate in hand, studies could be performed in order to determine the hydrolytic properties of the phosphorus-nitrogen bond. For these studies a 3 ?M solution of conjugate and the standard tetramethylphosphonium bromide (1.2 ?M) in aqueous buffer was prepared and the hydrolysis of the phosphonamidate was characterized by monitoring the decay of the conjugate against the standard by means of .sup.31P NMR over 24 hours. The results are shown in FIG. 1 which shows the hydrolytic decay of the GSH-phosphonamidate conjugate under acidic conditions.

[0325] Under strong acidic conditions (1 M HCL, pH 0.36) the phosphonamidate showed rapid decomposition, which is represented by the lower curve (circles). For slight acidic conditions (150 mM NH.sub.4OAc-buffer, pH=4.76), as depicted in the blue curve the compound was stable over the duration of the measurement (squares).

[0326] The rate of the reaction was determined by HPLC. Glutathione was added to a solution of ethyl-N-phenyl alkynyl phosphonamidate in aqueous buffer at slightly basic pH. The reaction was stopped after several time points by the addition of an acidic buffer and analyzed by HPLC-UV, referring to inosine as an internal standard. FIG. 2 refers to the consumption of ethyl-N-phenyl alkynyl phosphonamidate in the reaction with glutathione at pH 8.5. HPLC UV traces were taken at different time points. Experiment was performed in triplicate.

[0327] As FIG. 2 shows, we achieve a very fast conversion of more than 95% of the alkynyl phosphonamidate starting material after 15 min at pH 8.5.

Staudinger-Induced Thiol-Addition of RGD Peptides to GFP

[0328] In a next proof of principle study we synthesized an azido-containing cyclic RGD peptide (c(RGDfK)), which is known to bind to overexpressed integrins in cancer cells. This cyclic azido-peptide was reacted with the bisethoxyalkyne-phosphonite to form the highly reactive phosphonamidate in 53% isolated yield after HPLC with no observed by-product formation (Scheme 6).

##STR00108##

Synthesis of c(RGDfK)-azide

[0329] ##STR00109##

[0330] The cyclic RGDfK-azido peptide was synthesized manually on a NovaSynTGT alcohol resin with a loading of 0.26 mmol/g. First the resin was activated by stirring 480.7 mg resin in 2.5 ml toluene and 480 ?l acetylchloride at 60? C. for three hours. Double coupling of Fmoc-Asp(OAII)-OH (123.56 mg, 0.3125 mmol, 2.5 eq) was performed in DCM using DIPEA (212.6 ?l, 1.25 mmol, 10 eq.) as activating base each for one hour. Further amino acid couplings were performed by mixing amino acid (0.25 mmol, 2 eq.), HATU (0.25 mmol, 2 eq.) and DIPEA (0.5 mmol, 4 eq.) in DMF and coupling once for 30 minutes and once for one hour. Fmoc deprotection was accomplished with 20% piperidine in DMF. After the final amino acid coupling the alloc deprotection was achieved by treating the resin with Pd(P(Ph.sub.3).sub.4) (433 mg, 0.375 mmol, 3 eq.) in chloroform/acetic acid/NMM (37:2:1; v:v:v) for two hours in an argon atmosphere, followed by Fmoc deprotection and cyclisation with HATU (0.25 mmol, 2 eq.) and DIPEA (0.5 mmol, 4 eq.) in DMF for 16 hours. To be abled to install the aromatic azide on the lysine residue Fmoc-Lys(dde)-OH was used in the solid phase synthesis and was orthogonally deprotected on resin using 2% hydrazine in DMF three times for three minutes, followed by coupling of 4-azidobenzoic acid (81.65 mg, 0.5 mmol, 4 eq.) with HATU (190 mg, 0.5 mmol, 4 eq.) and DIPEA (1 mmol, 8 eq.) in DMF for two hours. Cleavage from the resin was performed using TFA/DCM (75:25; v:v) for 2.5 hours. Precipitation was carried out in cold and dry ether. The crude was analyzed by UPLC-MS and either used as crude in the following staudinger reaction or purified by preparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)). The product was gained as white powder (8.0 mg, 11.0 ?mol, 8.5% yield) and was analyzed by analytical UPLC (5 to 95% of acetonitrile in water containing 0.1% TFA on a RP-C18 column). The UPLC chromatogram of the c(RGDfK)-azide is shown in FIG. 7. LRMS: m/z: 749.67 [M+H].sup.+ (calcd. m/z: 749.3485).

Synthesis of c(RGDfK)-Phosphonamidate Alkyne

Bisethoxyalkyne-Phosphonite Synthesis

[0331] ##STR00110##

[0332] Ethynyl magnesium bromide in THF (5 M, 2 ml, 1 mmol, 1 eq.) was cooled to ?78? C. in a flame dried schlenk flask and diethylchlorophosphite (0.143 ml, 1 mmol, 1 eq.) was added. The solution was stirred for 10 minutes at ?78? C. and let warm to room temperature and stirred for another 90 minutes. The full consumption of starting material was checked by .sup.31P-NMR (product at 126.73 ppm; see FIG. 6: Crude Bisethoxyalkyne-phosphonite synthesis to FIG. 9) and used as crude in the following staudinger reaction with azido-c(RGDfK).

Staudinger Reaction on c(RGDfK)-Azide

##STR00111##

[0333] When crude peptide was used it (66 mg, 88.2 ?mol, 1 eq.) was dissolved in DMSO (4 ml, 22 mM) and dried in a flame dried flask for one hour prior to adding bisethoxyalkyne-phosphonite (volume according to percentage of product determined by NMR, 132.3 ?mol, 1.5 eq.). After the reaction mixture was stirred over night at room temperature 4 ml of water were added and stirred for 6 hours, before lyophilization. The crude product was purified by semi-preparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)) and gave the product as a white powder (6.2 mg, 6.64 ?mol, 5.3% overall yield).

[0334] Using the purified c(RGDfK)-azido peptide (6.9 mg, 9.14 ?mol, 1 eq.) it was dissolved in DMSO (1.5 ml, 6 mM) and dried in a flame dried flask for one hour prior to adding bisethoxyalkyne-phosphonite (volume according to percentage of product determined by NMR, 36.56 ?mol, 4 eq.). After the reaction mixture was stirred over night at room temperature 1.5 ml water was added and stirred again for six hours before lyophilization. The crude product was purified by semi-preparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)) and gave the product as a white powder (4.1 mg, 4.89 ?mol, 53.5% yield).

[0335] The final product was analyzed by LC-UV: rt. 5.0 min (0-1 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 1-16.5 min 5/95, water (0.1% TFA)/MeCN (0.1% TFA) on RP-C18 column) and mass. The chromatogram of the c(RGDfK)-alkyne is shown in FIG. 8. HRMS: m/z: 839.3636 [M+H].sup.+ (calcd. m/z: 839.3606)

Hydrothiolations of Electron-Deficient c(RGDfK)-Phosphonamidate Alkyne
Modelreaction with Glutathione

##STR00112##

[0336] Glutathione (1 mg, 3.25 ?mol, 1 eq.) and c(RGDfK)-phosphonamidate alkyne (1.24 mg, 3.25 ?mol, 1 eq.) were mixed in 135 ?l 10 mM ammoniumbicarbonate buffer pH 9.2 and 15 ?l acetonitrile (c=21.6 mM). After 10 minutes of shaking quantitative conversion to the addition product was observed by LC-UV/MS.

[0337] The final product was analyzed by LC-UV: rt. 4.3/4.4 min (0-1 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 1-16.5 min 5/95, water (0.1% TFA)/MeCN (0.1% TFA) on RP-C18 column)

[0338] HRMS: m/z: 1146.4451 [M+H].sup.+ (calcd. m/z: 1146.4444), 573.7321 [M+2H].sup.2+ (calcd. m/z: 573.7262)

[0339] As a first test substrate for the reaction with thiols, we used glutathione (GSH) and found a fast and high yielding addition of the thiol to the phosphonamidate-alkyne in nearly quantitative conversions after 10 minutes under slightly basic conditions at pH 8.8 at room temperature (FIG. 3: Staudinger-induced thiol-addition of cyclic azido-RGD-peptides to GSH).

[0340] On this model addition product we conducted stability studies at different pH and under the addition of thiols like MesNa and DTT at neutral and basic pH. It could have been shown that the formed product is stable in a broad pH range from pH 2.3 until pH 9.0 (FIG. 4: pH stability of c(RGDfK)-Glutathion).

[0341] Also the product is stable towards high concentration (0.2 M, 100 eq.) of DTT and MesNa at physiological pH (PBS buffer; pH 7.4). At pH 9.0 MesNa is slowly adding to the formed double bond (10% addition product formed after four days). In contrast to that DTT is rapidly forming an addition product with (42% after 30 hours) and followed by degradation over time.

Staudinger-Induced Thiol-Addition of GFP Protein

[0342] In the next step we probed the Staudinger-induced conjugation reaction with a Cys-containing model protein. Here we used a mutated eGFP bearing only one addressable cysteine for the thiol conjugation to the cyclic RGD-phosphonamidate.

[0343] GFP C70M S147C (3.13 nmol, 1 eq) was rebuffered to 100 ?l 10 mM Ammoniumbicarbonte pH 8.4 and c(RGDfK)-phosphonamidate alkyne (0.08 mg, 93.9 nmol, 30 eq.) was added. The reaction mixture was shaken at 37? C. and 800 rpm for three hours. Finally the mixture was spin filtrated using Amicon Spin filters with a 10 kDa MWCO. After spinfiltrating the sample ten times at 14000 rpm for 5 minutes and adding fresh 10 mM Ammoniumbicarbonate buffer MALDI-TOF analysis was conducted and verified total conversion of GFP C70M S147C to the desired product.

[0344] MALDI TOF: expected (in Da): 28605.31 (M+H.sup.+), 14303.16 (M+2H.sup.+); found (in Da): 28608.46 (M+H.sup.+), 14294.46 (M+2H.sup.+)

[0345] With this approach we could validate the feasibility of this reaction on the protein level at a concentration of 31 ?M, in which the conjugate was formed again in virtually quantitative conversions, as verified by MALDI-MS analysis and MS/MS analysis of the digested protein conjugate (FIG. 5: Staudinger-induced thiol-addition to thiol-containing eGFP.).

Stability Studies for c(RGDfK)-Glutathion

[0346] c(RGDfK)-glutathion was solved at a concentration of 2 mM in different solvents (0.1 M HCl at pH 1; 30% acetonitrile in water containing 0.1% TFA with a pH of 2.3; PBS buffer at pH 7.4; ammonium acetate buffer at pH 9.0; 0.05 M NaOH at pH 12) and 0.5 mM of Inosine was added as internal standard. The stability of the starting material was then monitored over three days.

[0347] The stability studies in presence of a competing thiol c(RGDfK)-glutathion was solved in either PBS or 1 M Tris HCl pH 9.0 at a concentration of 2 mM and 10 eq. DTT or MesNa was added. The mixture was monitored over several days.

Antibody Conjugation with Alkyne Phosphonamidates

[0348] First experiments were conducted with Cetuximab, a monoclonal IgG1 antibody against human epidermal growth factor. The antibody was modified with a biotin phosphonamidate and analyzed by SDS-PAGE under non reducing conditions, followed by anti-biotin western blotting (Scheme 7).

##STR00113##

[0349] The intact antibody was reduced by incubation with DTT in 50 mM borate containing PBS (pH 8.0) at 37? C. Excess of DTT was removed after the reaction by size exclusion columns and the reduced antibody fragments were incubated with a biotin phosphonamidate (1.1 equivalents per thiol) in 50 mM ammonium bicarbonate buffer (pH 8.5). EDTA (1 mM) was added to the reaction mixture to complex heavy metal ions that promote disulfide formation.

[0350] Western blot analysis confirmed modification of the antibody fragments, even though the intact antibody is not formed back by reoxidation of the remaining cysteins. This could be explained by a high degree of modification. No modification could be detected without prior reduction of the disulfide bonds. Thus further confirming the high selectivity of these compounds for free cysteine residues. Further experiments will include the determination of the degree of modification and experiments that prove the functionality of the modified antibody (see FIG. 10: Western blot analysis after non reducing SDS-PAGE. SM: Cetuximab starting material. 1: 5 min, 2: 1 h, 3: 2 h, 4: 20 h incubation with a biotin modified phosphonamidate. Reaction with (left) and without (right) prior reduction of the disulfides).

[0351] Cysteine selective modification was further confirmed by tryptic digest of the cetuximab phosphonamidate conjugates, followed by MS/MS analysis. To simplify the MS/MS spectra, the modification was conducted under previously described conditions with the structurally simpler ethyl-N-phenyl alkynyl phosphonamidate. Modification of Cys 263 of the heavy chain and Cys 214 of the light chain could be confirmed by MS/MS (HCD fragmentation) while no modification was detected without prior reduction of the disulfide bonds.

Staudinger-Induced Thiol-Additions with Vinyl Phosphonites:

a) Synthesis of Various Borane Protected Vinyl Phosphonites

[0352] Diethyl vinylphosphonite was synthesized based on previously published protocols by alkylation of diethyl chlorophosphite with vinylmagnesium bromide and subsequent borane addition (13) (Scheme 8). The desired phosphonite was isolated in 37% yield.

##STR00114##

[0353] Vinylphosphonites with different O-substituents were synthesized starting from phosphorous trichloride by substitution of two chlorides with the corresponding alcohols in the presence of pyridine. The formed mono chloro phosphite was reacted with the vinyl Grignard reagent and protected with borane. All these steps were performed in a one-pot strategy.

##STR00115##

[0354] As some alcohols are not compatible with subsequent addition of the Grignard reagent we applied an alternative route to the synthesis of phosphonites derived from these alcohols, starting from bis(diisopropylamino)chlorophosphine. alkylation to bis(diisopropylamino)vinylphosphine in the first step enabled tetrazole mediated addition of the alcohol in more polar solvents like acetonitrile in the second step. All phosphonites were treated with borane in situ and isolated by flash chromatography.

##STR00116##

Experimental Part for IIa)

Diethyl Vinylphosphonite Borane

[0355] ##STR00117##

[0356] A 25-ml Schlenk flask was charged with 2.14 ml vinylmagnesium bromide (0.7 M in THF, 1.50 mmol, 1.5 eq.) under an argon atmosphere, cooled to ?78? C. and 140 ?l diethyl chlorophosphite (1.00 mmol, 1.0 eq.) were added drop wise. The yellowish solution was allowed to warm to 0? C., stirred for another two hours and 1.00 ml of Borane (1.0 M in THF, 1.00 mmol, 1.0 eq.) were added and stirred for one more hour at 0? C. The organic solvents were removed under reduced pressure and the crude product was purified by flash column chromatography on silica gel (Hexane/EtOAc, 9:1) to yield the desired compound as colourless oil. (60 mg, 0.37 mmol, 37.0%)

[0357] .sup.1H NMR (300 MHz, Chloroform-d) ?=6.36-6.03 (m, 3H), 4.19-3.96 (m, 4H), 1.33 (t, J=7.1, 6H), 0.55 (ddd, J=190.3, 94.1, 16.6, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=134.62 (d, J=8.7), 130.12 (d, J=75.0), 63.16 (d, J=4.8), 16.59 (d, J=5.6). .sup.31P NMR (122 MHz, Chloroform-d) ?=129.58 (dd, J=167.1, 82.6).

[0358] NMR data is in accordance with those reported in the literature. .sup.18

Di(2-nitrobenzyl) vinylphosphonite borane

[0359] ##STR00118##

[0360] The compound was synthesized according to the general procedure A from PCl.sub.3 (260 ?l, 3.00 mmol). The pure borane protected phosphonite was purified by flash column chromatography (Hexane/EtOAc, 4:1) and obtained as a yellowish solid. (555 mg, 1.48 mmol, 49.2%)

[0361] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.10 (d, J=8.2, 2H), 7.77-7.63 (m, 4H), 7.57-7.44 (m, 2H), 6.51-6.18 (m, 3H), 5.45 (qd, J=14.8, 7.5, 4H), 1.42-?0.02 (m, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=146.80, 136.84 (d, J=10.2), 132.52 (d, J=6.8), 129.10, 129.05, 128.67, 128.61 (d, J=74.3), 125.09, 65.56 (d, J=3.6). .sup.31P NMR (122 MHz, Chloroform-d) ?=136.23 (dd, J=151.3, 56.0). HRMS for C.sub.16H.sub.18BN.sub.2NaO.sub.6P.sup.+ [M+Na].sup.+ calcd: 399.0888, found: 399.0885

Di(2-(2-methoxyethoxy)ethyl) vinylphosphonite borane

[0362] ##STR00119##

[0363] The compound was synthesized according to the general procedure A from PCl.sub.3 (130 ?l, 1.50 mmol). The pure borane protected phosphonite was purified by flash column chromatography (CH.sub.2Cl.sub.2/MeOH, 19:1 to 9:1) and obtained as a colourless oil. (34 mg, 0.11 mmol, 7.3%)

[0364] .sup.1H NMR (300 MHz, Chloroform-d) ?=6.37-6.00 (m, 3H), 4.16 (dq, J=7.6, 4.9, 4H), 3.70 (t, J=4.8, 4H), 3.64 (dd, J=5.8, 3.3, 4H), 3.54 (dd, J=5.9, 3.3, 4H), 3.38 (s, 6H), 1.14-?0.12 (m, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=135.00 (d, J=8.9), 129.51 (d, J=75.7), 71.79, 70.47, 70.21 (d, J=6.0), 65.92 (d, J=5.2), 58.95. .sup.31P NMR (122 MHz, Chloroform-d) ?=133.77-130.56 (m).

Diphenyl Vinylphosphonite Borane

[0365] ##STR00120##

[0366] The compound was synthesized according to the general procedure A from PCl.sub.3 (393 ?l, 4.50 mmol). The pure borane protected phosphonite was purified by flash column chromatography (Hexane/EtOAc, 4:1) and obtained as a colourless oil. (700 mg, 2.71 mmol, 60.3%)

[0367] .sup.1H NMR (300 MHz, Chloroform-d) ?=7.39 (td, J=7.7, 5.5, 4H), 7.30-7.17 (m, 6H), 6.67-6.18 (m, 3H), 1.48-0.01 (m, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=151.27 (d, J=8.7), 137.01 (d, J=12.5), 129.70 (d, J=1.0), 129.05 (d, J=71.1), 125.35 (d, J=1.3), 120.90 (d, J=4.2). .sup.31P NMR (122 MHz, Chloroform-d) ?=134.08-130.87 (m). HRMS for C.sub.14H.sub.16BNaO.sub.2P.sup.+ [M+Na].sup.+ calcd: 281.0873, found: 281.0873.

Bis(4-(2-nitro-5-(oxypropargyl)benzyloxy)phenyl) vinyl phosphonite borane

[0368] ##STR00121##

[0369] The compound was synthesized according to the general procedure B from Bis(diisopropylamino)chlorophosphine (71 mg, 0.27 mmol). The pure borane protected phosphonite was purified by flash column chromatography (Hexane/CH.sub.2Cl.sub.2, 1:1) and obtained as a yellowish solid. (75 mg, 0.11 mmol, 41.9%)

[0370] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.25 (d, J=9.1, 2H), 7.47 (d, J=2.8, 2H), 7.17-6.87 (m, 10H), 6.62-6.14 (m, 3H), 5.48 (s, 4H), 4.78 (d, J=2.4, 4H), 2.55 (t, J=2.4, 2H), 1.51-?0.24 (m, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=161.89, 155.27 (d, J=1.3), 145.34 (d, J=8.4), 140.04, 137.08 (d, J=12.6), 136.97, 129.06 (d, J=71.3), 127.77, 121.93 (d, J=4.0), 115.74 (d, J=1.1), 113.85, 113.79, 76.89, 76.89, 67.37, 56.24. .sup.31P NMR (122 MHz, Chloroform-d) ?=136.36-131.69 (m). HRMS for C.sub.34H.sub.30BN.sub.2NaO.sub.10P.sup.+ [M+Na].sup.+ calcd: 691.1623, found: 691.1629.

Bis(2-nitro-5-(oxypropargyl)benzyl) vinyl phosphonite borane

[0371] ##STR00122##

[0372] The compound was synthesized according to the general procedure B from Bis(diisopropylamino)chlorophosphine (513 mg, 1.92 mmol). The pure borane protected phosphonite was purified by flash column chromatography (Hexane/CH.sub.2Cl.sub.2, 4:1) and obtained as a yellowish solid. (704 mg, 1.45 mmol, 75.6%)

[0373] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.19 (d, J=9.1, 2H), 7.31 (d, J=2.8, 2H), 7.00 (dd, J=9.2, 2.8, 2H), 6.58-6.22 (m, 3H), 5.49 (qd, J=15.5, 7.4, 4H), 4.81 (d, J=2.4, 4H), 2.62 (t, J=2.4, 2H), 1.31-0.12 (m, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=161.86, 139.86, 136.92 (d, J=10.4), 135.78 (d, J=6.8), 128.43 (d, J=73.2), 127.79, 114.10, 113.92, 76.95, 76.89, 65.54 (d, J=3.6), 56.31. .sup.31P NMR (122 MHz, Chloroform-d) ?=136.32 (d, J=107.0). HRMS for C.sub.22H.sub.22BN.sub.2NaO.sub.8P.sup.+ calcd: 507.1099, found: 507.1111.

Bis(2,2,2-trifluoroethyl) vinyl phosphonite borane

[0374] ##STR00123##

[0375] The compound was synthesized according to the general procedure B from Bis(diisopropylamino)chlorophosphine (266 mg, 1.00 mmol). The pure borane protected phosphonite was purified by flash column chromatography (Hexane/CH.sub.2Cl.sub.2, 9:1 to 4:1) and obtained as a colourless liquid. (87 mg, 0.32 mmol, 32.2%)

[0376] .sup.1H NMR (300 MHz, Chloroform-d) ?=6.52-6.11 (m, 3H), 4.36 (p, J=8.1, 4H), 0.62 (ddd, J=203.0, 103.5, 15.0, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=137.57, 127.48 (d, J=79.1), 122.37 (qd, J=276.0, 7.5), 63.60 (qd, J=37.7, 2.5). .sup.19F NMR (282 MHz, Chloroform-d) b=2.13. .sup.31P NMR (122 MHz, Chloroform-d) ?=145.49 (dd, J=135.6, 65.1).

Bis-(4-Hydroxyphenyl) vinyl phosphonite borane

[0377] ##STR00124##

[0378] The compound was synthesized according to the general procedure B from Bis(diisopropylamino)chlorophosphine (534 mg, 2.00 mmol) and Hydrochinon (2.20 g, 10 eq.). The pure borane protected phosphonite was purified by flash column chromatography (Hexane/EtOAc, 4:1 to 1:1) and obtained as a colourless solid. (280 mg, 0.96 mmol, 48.3%)

[0379] .sup.1H NMR (300 MHz, DMSO-d.sub.6) ?=9.49 (s, 2H), 6.96 (d, J=8.5, 4H), 6.74 (d, J=8.9, 4H), 6.63-6.22 (m, 3H), 1.20-?0.12 (m, 3H). .sup.31P NMR (122 MHz, DMSO-d.sub.6) ?=131.80. HRMS for C.sub.14H.sub.16BNaO.sub.4P.sup.+ calcd: 313.0771, found: 313.0774.

Di(4-nitrobenzyl) vinylphosphonite borane

[0380] ##STR00125##

[0381] The compound was synthesized according to the general procedure B from Bis(diisopropylamino)chlorophosphine (533 mg, 2.00 mmol). The pure borane protected phosphonite was purified by flash column chromatography (Hexane/CH.sub.2Cl.sub.2, 9:1 to 4:1) and obtained as a white solid. (540 mg, 1.44 mmol, 71.8%)

[0382] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.17 (d, J=8.6, 4H), 7.49 (d, J=8.5, 4H), 6.47-6.16 (m, 3H), 5.12 (qd, J=13.2, 8.3, 4H), 1.40-?0.00 (m, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=147.66, 143.09 (d, J=6.1), 136.50 (d, J=10.2), 128.80 (d, J=76.0), 127.78, 123.73, 67.30 (d, J=3.8). .sup.31P NMR (122 MHz, Chloroform-d) ?=137.95 (d, J=95.6).

2-nitro-5-(oxypropargyl)benzyl alcohol

[0383] ##STR00126##

[0384] A 5 ml-microwave tube was charged with 200 mg of 5-Hydroxy-2-nitrobenzyl alcohol (1.18 mmol, 1.0 eq.), 245 mg K.sub.2CO.sub.3 (1.77 mmol, 1.5 eq.), 132 ?l Propargyl bromide (80 wt. % solution in Toluene) and 4 ml DMF. The resulting suspension was irradiated for 1 h at 100? C. After cooling to room temperature, 5 ml of water were added. The resulting precipitate was filtered, washed three times with water and vacuum dried to give 179 mg of light brown solid. (0.87 mmol, 73.2%)

[0385] NMR data is in accordance with those reported in the literature. .sup.19

4-(2-nitro-5-(oxypropargyl)benzyloxy)phenol

[0386] ##STR00127##

[0387] A flame dried Schlenk-tube, 400 mg of 2-nitro-5-(oxypropargyl)benzyl alcohol (1.93 mmol, 1.0 eq.), together with 850 mg hydrochinon (7.72 mmol, 4.0 eq.) and 750 mg of triphenylphosphine (2.90 mmol, 1.5 eq.) were dissolved in 10 ml of dry THF. The solution was cooled to 0? C. and 1.33 ml of diethyl azodicarboxylate (40% solution in Toluene) (2.90 mmol, 1.5 eq.) were added dropwise and the reaction was allowed to warm to room temperature overnight. The crude product was dry packed on a silica column for purification, eluting with Hexan/EtOAc (7:3 to 3:2), yielding 505 mg of a yellow solid. (1.68 mmon, 87.5%)

[0388] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.26 (d, J=9.1, 1H), 7.51 (d, J=2.8, 1H), 7.00 (dd, J=9.2, 2.9, 1H), 6.89 (d, J=9.0, 2H), 6.80 (d, J=9.0, 2H), 5.46 (s, 2H), 4.79 (d, J=2.4, 2H), 2.54 (t, J=2.4, 1H). .sup.13C NMR (75 MHz, Chloroform-d) ?=161.86, 152.11, 150.03, 140.10, 137.71, 127.69, 116.11, 116.03, 113.88, 113.69, 76.95, 76.68, 67.66, 56.20. HRMS for C.sub.16H.sub.14NO.sub.5.sup.+ [M+H].sup.+ calcd: 300.0866, found: 300.0871.

Small Molecule Studies: Reaction of Unprotected Alkene Phosphonites with Different Azides

[0389] The Staudinger phosphonite reaction with vinyl phosphonites was first investigated with rather simple diethyl derivatives. Those were synthesized by alkylation of commercial available diethyl chlorophosphite and reacted with different aliphatic and aromatic azides in situ. The desired phosphonamidates were isolated by column chromatography, after Hydrolysis of the Phosphonimidates.

##STR00128## ##STR00129##

[0390] Varying the O-substituents of the Phosphonamidates allows the fine tuning of the reactivity in the thiol-addition as well as the installation of a third functionality to the system. Phosphonamidates with substituents other than ethyl were synthesized by staudinger phosphonite reaction of the respective phosphonites. Isolated borane protected phosphonites were treated with DABCO to form the reactive P(III) species and reacted with an azide in situ to form the phosphonimidate. Subsequent Hydrolysis by water addition formed the desired phosphonamidates in moderate yields.

##STR00130##

[0391] The 2-nitro-benzyl group is widely known as a photolabile substituent and has been shown to release attached molecules upon UV-irradiation..sup.20 From our expertise in phosphonamidate chemistry, we knew that the PN-Bond of the phosphonamidate the very labile once the Phosphonamidate ester is cleaved. Therefore we wanted to synthesize 2-nitro-benzyl substituted Phosphonamidates that enable the controlled light mediated release of an amine from the thiol addition conjugates.

##STR00131##

[0392] Several 2-nitro-benzyl substituted phosphonamidates could be synthesized, including a photo cleavable biotin as well as a Cy5-dye and a DABCYL quencher variant. An additional alkyne at the 2-nitro-benzyl group enables the installation of a third functionality to the system wire copper catalyzed click chemistry, which can be cleaved of again by photo irradiation.

##STR00132##

[0393] Further fine tuning of the reactivity of the subsequent thiol-addition was achieved by changing the electronic properties of the phosphonamidates. Therefore different phenyl- as well as trifuloroethyl derivatives were synthesized.

##STR00133## ##STR00134##

[0394] Some phosphonites could not be isolated with a borane protection group, as their corresponding alcohols are not compatible with borane addition. We were able to show that these phosphonites can be used in an in situ synthesis with an azide without isolation of the phosphonite as depicted in Scheme 16.

##STR00135##

[0395] Stability of a phosphonamidate to different pHs was proven by .sup.31P-NMR in aqueous buffers at room temperature. In a first experiment, Ethyl-N-phenyl-P-vinyl-phosphonamidate was chosen to measure stability. It turned out that the compound is stable over a broad pH range. PN-bond cleavage occurred under strong acidic conditions (FIG. 11: Stability of Ethyl-N-phenyl-P-vinyl-phosphonamidate to different pHs over time).

Thiol-Addition of Small Molecule Thiols and Glutathione to Vinyl Phosphonamidates

[0396] In a first study, vinyl phosphonamidates were reacted with different small molecule thiols under reaction conditions that previously worked well for alkynyl phosphonamidates. Full conversion of the Phosphonamidate starting material could be observed after 3 h treatment with one equivalent of a thiol in presence of potassium carbonate.

##STR00136##

[0397] In the next step these reaction conditions were now applied to synthesize water soluble glutathione phosphonamidate conjugates. The reaction proceeded in case of the water soluble 4-carboxyphenyl-phosphonamidate without the addition of any organic solvent. The highly polar products were isolated by semi preparative HPLC with a slightly basic gradient.

##STR00137##

[0398] Stability of a thiol-adduct to different pHs was proven by .sup.31P-NMR in aqueous buffers at room temperature. The conjugates showed excellent stability over a broad pH range. PN-bond cleavage occurred under strong acidic conditions. An elimination of the thiol referred to as retro thiol-addition was not observed (FIG. 12: Stability of a glutathione phosphonamidate adduct to different pHs over time.).

[0399] The effect of the O-substituent on the reaction rate was investigated by the addition of glutathione to a solution of various N-phenyl alkynyl phosphonamidates in ammonium bicarbonate buffer at pH 8.5. Conversion of different phosphonamidates over time is shown in FIG. 11.

[0400] We found out that that vinyl phosphonamidates are much slower in the reaction with thiols than there corresponding alkynyl derivatives. We assumed that exchanging the electron donating ethyl group of the phosphonamidates to more electron withdrawing substituents should further increase the electrophilicity and therefore raise the rate of the thiol addition. Exchanging the ethyl to a phenyl group already reduces the half-life time t.sub.1/2 of the staring material in the reaction from ten hours to one hour. Trifluoroethyl further reduces t.sub.1/2 to thirty minutes while 2-nitro benzyl reacts to fifty percent in two hours (FIG. 13: Consumption of various N-phenyl vinyl phosphonamidates in the reaction with glutathione at pH 8.5. HPLC UV traces were taken at different time points. Experiments were performed in triplicates).

Thiol-Addition to Vinyl Phosphonamidates on Protein Level

[0401] First experiments with alkene phosphonamidates on protein level were conducted with the water soluble Ethyl-N-(4-carboxy-phenyl)-P-vinyl-phosphonamidate. As previous studies indicated that carbonate bases work very well in the promotion of the thiol-addition, ammonium bicarbonate buffer at pH 9.0 was chosen for the first experiments. A mutated eGFP variant bearing only one addressable cysteine was selected for the study.

[0402] The protein was incubated with 50 equivalents of the phosphonamidate at 37? C. Even though MALDI/MS analysis of the reaction mixture after 16 hours showed still unreacted protein, we were very pleased to observe formation of the desired protein conjugate.

[0403] Further eGFP conjugation experiments were conducted with a fluorescent Cy5-Phosphonamidate and observed by in-gel fluorescence measurements of the Cy5-channel.

[0404] The selectivity of the reaction for cysteine residues could be confirmed in this experiment. Neither an eGFP variant without any accessible cysteine incubated with the phosphonamidate nor addition of a Cy5 azide to the Cys containing eGFP showed fluorescent labeling. Addition of 5% DMSO (line 1) or acetonitrile (line 3) to the reaction mixture were both sufficient in solubilizing the dye without influencing the reaction itself.

Light Cleavable Triple Conjugation

[0405] We mentioned earlier that we were able to synthesize phosphonamidates with o-nitro benzyl substituents bearing an additional alkyne handle for CuAAC. One possible application for these compounds is the installation of a biotin to the alkyne to purify protein conjugates. We envision that the biotin binds to streptavidin beads, unbound material can be washed away and pure protein can be eluted by light irradiation.

[0406] In a first experiment on protein level, a simple N-phenyl Phosphonamidate with an O-substituted light cleavable Alkyne was reacted first with our single cysteine containing eGFP under previously optimized conditions. After the step, an azido modified biotin was attached to the contruct via CuACC and the conjugates were analyzed by anti-biotin western blotting.

[0407] Western blot analysis confirmed successful conjugation of the biotin to the eGFP construct. When eGFP without attached phosphonamidate was used in the CuACC reaction no biotin was detected. The same is true in the absence of Copper.

[0408] Further immobilization experiments on Streptavidin beads were conducted with a phosphonamidate, Synthesized from an azido containing peptide and a single cystein containing Ubiquitin. The high molecular weight of the peptide induces a shift of the protein in the SDS gel, allowing the estimation of the conjugation yield.

Scheme 22:

[0409] Photocleavable alkyne labeling of ubiquitin with one addressable cysteine with subsequent biotin labeling via CuACC. Western blot analysis after immobilization on streptavidin beads. 1: Ubiquitin starting material, 2: reaction mixture after CuACC, 3: Supernatant after incubation of the reaction mixture with streptavidin agarose, 4: flow through after wash of streptavidin agarose, 5: boiled beads, 6: Irradiated beads

[0410] The conjugation yield of the peptide to the protein could be estimated to 60%. The final construct was successfully immobilized on streptavidin beads. The constructs can be released by either boiling the beads in SDS buffer, which releases the intact protein peptide conjugate or irradiation by UV light. The latter method cleaves the phosphonamidate ester, leading to instability of the PN-Bond and therefore release of the unconjugated protein. Further experiments will be conducted with phosphonamidates that form intact esters upon light irradiation to release the conjugated construct upon light irradiation.

b) Antibody Conjugation with Vinyl Phosphonamidates

[0411] Vinyl phosphonamidates were also applied to the modification of monoclonal antibodies. As we found out that 2-nitro-benzyl substituted vinyl phosphonamidates react faster in the thiol addition, we chose those phosphonamidate with a biotin modification.

##STR00138##

[0412] We chose the same reaction conditions for the reduction-alkylation procedure as described previously with the exception of 4? C. for the thiol-addition, because we found out that lower temperatures slow down disulfide formation and therefore lead to higher conjugation yield.

[0413] Western blot analysis confirmed cysteine selective modification of the antibody. High selectivity for free cysteine residues could be observed by the absence of a Signal in the anti-biotin western blot without prior reduction of the disulfide bonds. In contrast to the labeling experiments with alkynyl phosphonamidates, this time reformation of the antibody fragments could be observed (FIG. 14: Western blot analysis after non reducing SDS-PAGE. SM: Cetuximab starting material. 1: 5 min, 2: 1 h, 3: 2 h, 4: 4 h, 5: 20 h incubation with a biotin modified phosphonamidate. Reaction with (left) and without (right) prior reduction of the disulfides).

[0414] Cysteine selective modification was further confirmed by tryptic digest of the cetuximab phosphonamidate conjugates, followed by MS/MS analysis. To simplify the MS/MS spectra, the modification was conducted under previously described conditions with the structurally simpler phenyl-N-phenyl alkynyl phosphonamidate. Modification of Cys 264 and Cys 146 of the heavy chain could be confirmed by MS/MS (HCD fragmentation) while no modification was detected without prior reduction of the disulfide bonds.

Alkene Phosphonites in the Synthesis ASGP-R Addressing Drug Conjugates

[0415] We further want to apply our modular conjugation approach to the synthesis of targeted drug conjugates. Khorev et al described previously the synthesis of an ASGP-R addressing trivalent ligand with a terminal amino modification. Based on this route, we synthesized the same ligand with a terminal thiol modification (20).

##STR00139##

[0416] Having the thiol modified, fully deprotected ligand in hand we successfully conjugated the construct to our fluorescent Cy5-phosphonamidate. With this conjugate, we can now monitor the sufficient uptake into hepatocytes by FACS analysis and fluorescent microscopy.

[0417] FIG. 15 shows the sequences mentioned throughout this description.

Introduction of the Alkyne-Phosphonamidate Moiety by Generic Building Blocks Via an Amide Bond

[0418] Generic building blocks as the amino-modified derivative N2 or the NHS-ester N1 shown in Scheme 25 can introduce an alkyne-phosphonamidate moiety into functional molecules via amide bond forming reactions.

Scheme 25:

[0419] Alkyne-phosphonamidates for the chemoselective modification of Cys-residues. Introduction via chemoselective Staudinger-phosphonite reaction or amide coupling with the generic building blocks N1 and N2.

[0420] This approach can be advantageous as one does not have to handle labile P(III) compounds. Furthermore, it has been shown that high yields can be achieved by using those generic building blocks, which is of a particular interest for expensive starting materials.

##STR00140##

Procedures for the Introduction of the Alkyne-Phosphonamidate Moiety by Generic Building Blocks Via an Amide Bond

Preperative HPLC

[0421] Preperative HPLC was performed on a Gilson PLC 2020 system (Gilson Inc, WI, Middleton, USA) using a VP 250/32 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany). The following gradients were used throughout all sections of this disclosure: Method C: (A=H.sub.2O+0.1% TFA (trifluoroacetic acid), B=MeCN (acetonitrile)++0.1% TFA, flow rate 30 ml/min, 5% B 0-5 min, 5-90% B 5-60 min, 90% B 60-65 min. Method D: (A=H.sub.2O+0.1% TFA, B=MeCN++0.1% TFA), flow rate 30 ml/min, 5% B 0-5 min, 5-25% B 5-10 min, 25%-45% B 10-50 min, 45-90% 50-60 min, 90% B 60-65 min.

Semi-Preperative HPLC

[0422] Semi-preperative HPLC was performed on a Shimadzu prominence HPLC system (Shimadzu Corp., Japan) with a CBM20A communication bus module, a FRC-10A fraction collector, 2 pumps LC-20AP, and a SPD-20A UV/VIS detector, using a VP250/21 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany). The following gradients were used throughout all sections of this disclosure: Method E: (A=H.sub.2O+0.1% TFA, B=MeCN++0.1% TFA), flow rate 10 ml/min, 5% B 0-5 min, 5-99% B 5-65 min, 99% B 65-75 min.

General Procedure 1 for the Synthesis of Aromatic Azides

[0423] A 500-ml round-bottom flask was charged with 10 mmol aromatic amine, suspended in 15 ml water and cooled to 0? C. 5 ml of concentrated aqueous HCl were added, followed by drop-wise addition of 1.27 g sodium nitrite (15.00 mmol, 1.50 eq.) solution in 10 ml Water. The mixture was stirred for 20 min at 0? C., 100 ml EtOAc (ethyl acetate) were added and a solution of 0.98 g sodium azide (15.00 mmol, 1.5 eq.) in 5 ml water was added drop-wise. The solution was allowed to warm to room temperature and stirred for one more hour. Phases were separated, the aqueous phase was extracted two times with EtOAc, combined organic fractions were washed two times with water, dried (MgSO.sub.4) and all volatiles were removed under reduced pressure.

General Procedure 2 for the Synthesis of O-Ethyl-Alkynyl Phosphonamidates from Diethyl Chlorophosphite

[0424] A 25-ml Schlenk flask was charged with 173 ?l diethyl chlorophosphite (1.20 mmol, 1.2 eq.) under an argon atmosphere, cooled to ?78? C. and 2.40 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.20 mmol, 1.2 eq.) was added drop wise. The yellowish solution was allowed to warm to room temperature and 1.00 mmol of azide (1.0 eq.) dissolved in 3.0 ml of THF or DMF was added and stirred over night at room temperature. 5 ml of water were added and stirred for another 2 h. The reaction mixture was extracted with EtOAc, the combined organic fractions dried (MgSO.sub.4) and solvents were removed under reduced pressure. The crude product was purified by flash column chromatography on silica gel or preperative reversed phase HPLC.

4-azidobenzoic acid

[0425] ##STR00141##

[0426] The compound was synthesized according to the general procedure 1 from 2.00 g 4-aminobenzoic acid (14.58 mmol) and obtained as a yellowish solid. (2.00 g, 12.26 mmol, 84.1%)

[0427] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.11 (d, J=8.4, 2H), 7.11 (d, J=8.4, 2H). NMR data was in accordance with literature values (23).

4-azidobenzoic-acid-N-hydroxysuccinimide ester

[0428] ##STR00142##

[0429] In a 50-ml round-bottom-flask, 500 mg 4-azidobenzoic acid (3.056 mmol, 1.00 eq.), 705 mg N-hydroxysuccinimide (6.112 mmol, 2.00 eq.) and 20 mg 4-Dimethylaminopyridine (0.164 mmol, 0.05 eq.) were suspended in 10 ml of dry CH.sub.2Cl.sub.2. 1.172 g EDC*HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 6.112 mmol, 2.00 eq.) were added slowly at 0? C. and the reaction mixture was allowed to stir at room temperature for two hours. The solvent was removed under reduced pressure and the crude product purified by column chromatography on silicagel (50% EtOAc in hexane) and obtained as white solid (763 mg, 2.934 mmol, 96.0%)

[0430] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.14 (d, J=8.6, 2H), 7.15 (d, J=8.6, 2H), 2.92 (s, 4H). .sup.13C NMR (75 MHz, CDCl.sub.3) ?=169.15, 160.97, 146.85, 132.42, 121.19, 119.21, 25.59. NMR data was in accordance with literature values (24).

Ethyl-N-(4-(2, 5-dioxo-1-pyrrolidinyl)oxy-carbonyl-phenyl)-P-ethynyl phosphonamidate

[0431] ##STR00143##

[0432] The compound was synthesized according to the general procedure 2 from 173 ?l diethyl chlorophosphite (1.20 mmol, 1.20 eq.), 2.40 ml ethynylmagnesium bromide solution (0.5 M in THF (tetrahydrofuran), 1.20 mmol, 1.20 eq.) and 260 mg 4-azidobenzoic-acid-N-hydroxysuccinimide ester (1.00 mmol, 1.00 eq.). The crude phosphonamidate was purified by flash column chromatography on silicagel (100% EtOAc) and obtained as a yellowish solid. (225 mg, 0.643 mmol, 64.3%)

[0433] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.05 (d, J=8.6, 2H), 7.37 (d, J=7.4, 1H), 7.16 (d, J=8.6, 2H), 4.38-4.13 (m, 2H), 2.96 (d, J=13.2, 1H), 2.90 (s, 4H), 1.40 (t, J=7.1, 3H). .sup.13C NMR (75 MHz, Chloroform-d) ?=169.59, 161.51, 145.64, 132.55, 118.38, 117.59 (d, J=8.0), 88.69 (d, J=50.2), 62.93 (d, J=5.2), 25.82, 16.24 (d, J=7.3). .sup.31P NMR (122 MHz, Chloroform-d) ?=?10.65. HR-MS for C.sub.15H.sub.16N.sub.2O.sub.6P.sup.+ [M+H].sup.+ calcd: 351.0740, found 351.0749.

2-(4-Azidophenyl)-ethanol

[0434] ##STR00144##

[0435] The compound was synthesized according to the general procedure 1 from 1.00 g of 2-(4-Aminophenyl)-ethanol (7.21 mmol) and obtained as brown oil (0.50 g, 3.06 mmol, 42.5%).

[0436] .sup.1H NMR (300 MHz, Chloroform-d) ?=7.21 (d, J=8.3, 2H), 6.97 (d, J=8.3, 2H), 3.83 (t, J=6.5, 2H), 2.84 (t, J=6.5, 2H), 1.81 (s, 1H). .sup.13C NMR (75 MHz, CDCl.sub.3) ?=138.26, 135.41, 130.42, 119.18, 63.55, 38.50. NMR data was in accordance with literature values (25).

2-(4-Azidophenyl)-ethyl-4-toluenesulfonate

[0437] ##STR00145##

[0438] A 50-ml round-bottom flask was charged with 455 mg of 2-(4-Azidophenyl)-ethanol (2.79 mmol, 1.00 eq.), dissolved in 8 ml pyridine and cooled to 0? C. 787 mg of solid tosyl chloride (4.18 mmol, 1.50 mmol) was added portion-wise and the mixture was stirred for 4 h at room temperature, 10 ml of saturate NaCl-solution and 10 ml water were added and the yellow solution was extracted three times with EtOAc, combined organic fractions were washed two times with 1N HCl, twice with saturate NaHCO.sub.3-solution and once with water. The organic layer was dried (MgSO.sub.4) and all volatiles were removed under reduced pressure. Product was obtained as yellow oil (0.72 g, 2.44 mmol, 87.4%).

[0439] .sup.1H NMR (300 MHz, Chloroform-d) ?=7.69 (d, J=8.2, 2H), 7.30 (d, J=8.2, 2H), 7.10 (d, J=8.3, 2H), 6.91 (d, J=8.3, 2H), 4.21 (t, J=6.8, 2H), 2.94 (t, J=6.8, 2H), 2.45 (s, 3H). .sup.13C NMR (75 MHz, CDCl.sub.3) ?=144.66, 138.60, 132.96, 132.76, 130.19, 129.69, 127.72, 119.05, 70.34, 34.60, 21.55. NMR data was in accordance with literature values (26).

2-(4-Azidophenyl)-ethyl phtalimide

[0440] ##STR00146##

[0441] A 50-ml round-bottom flask was charged with 4.11 g of 2-(4-Azidophenyl)-ethyl-4-toluenesulfonate (12.95 mmol, 1.00 eq.), together with 3.60 g potassium phtalimide (19.42 mmol, 1.50 eq.) and dissolved in 60 ml DMF (N,N-dimethylformamide). The brown solution was stirred over night at 100? C. All volatiles were removed under reduced pressure, 50 ml of water were added extracted three times with EtOAc, the combined organic fractions were washed two times with water, the organic layer was dried (MgSO.sub.4) and all volatiles were removed under reduced pressure. The product was used in the next step without further purification. Pure product was obtained by flash column chromatography on silicagel (10% to 20% EtOAc in n-hexan) as a yellow solid (1.75 g, 5.99 mmol, 46.2%). .sup.1H NMR (600 MHz, Chloroform-d) ?=7.85 (dd, J=5.4, 3.1, 2H), 7.73 (dd, J=5.4, 3.1, 2H), 7.25 (d, J=8.4, 2H), 6.96 (d, J=8.4, 2H), 3.93 (dd, J=8.3, 6.8, 2H), 3.00 (dd, J=8.3, 6.8, 2H). .sup.13C NMR (151 MHz, CDCl.sub.3) ?=168.12, 138.43, 134.76, 133.96, 132.00, 130.22, 123.26, 119.17, 39.14, 33.92.

2-(4-Azidophenyl)-ethylamine hydrochloride

[0442] ##STR00147##

[0443] A 100-ml round-bottom flask was charged with 722 mg of 2-(4-Azidophenyl)-ethyl phtalimide (2.47 mmol, 1.00 eq.), 144 ?l hydrazine hydrate (2.96 mmol, 1.20 eq.), dissolved in 20 ml of dry ethanol under argon atmosphere and the solution was refluxed for 4 h. Most of the solvent was removed under reduced pressure, 50 ml water was added and the suspension was basified with 1N NaOH. It was extracted three times with EtOAc, the combined organic fractions were washed two times with water, the organic layer was dried (MgSO.sub.4) and all volatiles were removed under reduced pressure. Pure product was obtained by flash column chromatography on silicagel (10% MeOH (methanol) in DCM (dichloromethane)+0.5% N,N-ethyldimethylamine) and lyohilisation from HCl as yellowish solid (224 mg, 1.14 mmol, 46.2% over two steps). .sup.1H NMR (600 MHz, Deuterium Oxide) ?=7.29 (d, J=7.6, 2H), 7.05 (d, J=7.6, 2H), 3.22 (t, J=7.2, 2H), 2.94 (t, J=7.2, 2H). .sup.13C NMR (151 MHz, D.sub.2O) ?=138.81, 133.24, 130.32, 119.40, 40.51, 32.13. NMR data was in accordance with literature values (27).

Ethyl-N-(4-(2-aminoethyl)phenyl)-P-ethynyl phosphonamidate TFA salt

[0444] ##STR00148##

[0445] The compound was synthesized according to the general procedure 2 from 181 ?l diethyl chlorophosphite (1.26 mmol, 1.20 eq.), 2.52 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.26 mmol, 1.20 eq.) and 322 mg 2-(4-azidophenyl)ethyl amine hydrochloride (1.05 mmol, 1.00 eq.). The crude phosphonamidate was purified by preparative RP-HPLC (Method C described above) and obtained as brown oil. (209 mg, 0.57 mmol, 54.5%)

[0446] .sup.1H NMR (300 MHz, Acetonitrile-d.sub.3) ?=7.58 (s, 3H), 7.20-7.01 (m, 4H), 6.96 (d, J=8.5, 1H), 4.26-4.05 (m, 2H), 3.42 (d, J=12.8, 1H), 3.08 (d, J=7.8, 2H), 2.88 (dd, J=9.0, 6.4, 2H), 1.31 (t, J=7.1, 3H). .sup.13C NMR (75 MHz, Acetonitrile-d.sub.3) ?=161.38 (q, J=34.7), 139.20 (d, J=1.3), 131.75, 130.66, 119.63 (d, J=7.3), 90.09 (d, J=47.2), 77.02 (d, J=265.0), 63.54 (d, J=5.3), 41.92, 33.19, 16.41 (d, J=7.3). .sup.31P NMR (122 MHz, Acetonitrile-d.sub.3) ?=?9.71. HR-MS for C12H18N.sub.2O2P.sup.+ [M+H].sup.+ calcd: 253.1100, found 253.1095.

5-((2-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino) naphthalene-1-sulfonic acid

[0447] ##STR00149##

[0448] The reaction was carried out in DMF. 265 ?l of a 100 mM solution of Ethyl-N-(4-(2,5-dioxo-1-pyrrolidinyl)oxy-carbonyl-phenyl)-P-ethynyl phosphonamidate (0.0265 mmol, 1.00 eq.) and 1.06 ml of a 50 mM solution of 5-((2-Aminoethyl)aminonaphthalene-1-sulfonate (0.0530 mmol, 2.00 eq.) together with 795 ?l DMF was premixed and 530 ?l of a solution of 200 mM DIPEA (0.1060 mmol, 4.00 eq.) was added. The mixture was shaken for 2 hours at room-temperature, all volatiles were removed under reduced pressure, the crude mixture was purified by preperative HPLC using method C described above, and the desired compound obtained as a white solid after lyophilisation. (9.30 mg, 0.0186 mmol, 70.0%)

[0449] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.78 (d, J=8.5, 1H), 8.57 (t, J=5.7, 1H), 8.36 (d, J=8.6, 1H), 8.11 (d, J=8.4, 1H), 7.99 (d, J=7.0, 1H), 7.80 (d, J=8.7, 2H), 7.43 (dd, J=8.5, 7.1, 1H), 7.38 (t, J=8.1, 1H), 7.14 (d, J=8.7, 2H), 6.92 (d, J=7.5, 1H), 4.43 (d, J=12.7, 1H), 4.21-4.05 (m, 2H), 3.62 (q, J=6.3, 2H), 3.46 (t, J=6.6, 2H), 1.31 (t, J=7.0, 3H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) ?=167.03, 144.64, 143.48, 141.01, 130.59, 128.98, 127.65, 126.47, 125.13, 124.62, 123.86, 123.13, 119.62, 117.34 (d, J=7.8), 107.91, 91.69 (d, J=45.5), 77.26 (d, J=260.8), 62.31 (d, J=5.0), 45.51, 38.15, 16.42 (d, J=6.9). .sup.31P NMR (243 MHz, DMSO) ?=?10.35. HR-MS for C23H25N.sub.3O6PS.sup.+ [M+H].sup.+ calcd: 502.1196, found 502.1195.

Cy5-O-ethyl-P-alkynyl-phosphonamidate

[0450] ##STR00150##

[0451] The Cy5-COOH was synthesized according to a procedure, previously published by our lab (28). A 5-ml-round bottom flask was charged with 33.2 mg Cy5-COOH (0.0628 mmol, 1.00 eq.), 35.8 mg HATU ((1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate), 0.0942 mmol, 1.5 eq.) and 200 ?l DMF. The deep blue solution was cooled to 0? C. and 32 ?l DIPEA (N,N-diisopropylethylamine, 0.1884 mmol, 3.0 eq.) were added. After 5 minutes a solution of 23 mg Ethyl-N-(4-(2-aminoethyl)phenyl)-P-ethynyl phosphonamidate TFA salt (0.0628 mmol, 1.00 eq.) in 300 ?l DMF were added drop-wise. The solution was allowed to warm to room-temperature and stirred for 2 hours. All volatiles were removed under reduced pressure and the crude product was purified by flash column chromatography on silicagel (0% to 5% MeOH in DCM) and obtained as blue solid. (45 mg, 0.0590 mmol, 93.9%).

[0452] .sup.1H NMR (600 MHz, Chloroform-d) ?=7.88 (td, J=13.0, 4.9, 2H), 7.43-7.33 (m, 4H), 7.23 (t, J=7.4, 2H), 7.15-7.07 (m, 4H), 7.01 (d, J=8.4, 2H), 6.72 (t, J=12.5, 1H), 6.46 (bs, 1H), 6.18 (dd, J=13.6, 8.5, 2H), 6.11 (q, J=7.6, 1H), 4.27-4.09 (m, 2H), 3.98 (t, J=7.6, 2H), 3.56 (s, 3H), 3.43 (q, J=6.9, 2H), 2.97 (d, J=12.8, 1H), 2.75 (t, J=7.5, 2H), 2.25 (t, J=7.3, 2H), 1.81 (p, J=8.0, 2H), 1.73-1.67 (m, 2H), 1.70 (s, 6H), 1.69 (s, 6H), 1.55-1.42 (m, 2H), 1.35 (t, J=7.1, 3H). .sup.13C NMR (151 MHz, CDCl3) ?=173.64, 173.19, 173.11, 153.34, 152.99, 142.72, 141.90, 141.17, 140.89, 136.88, 133.32, 129.69, 128.78, 128.66, 126.32, 126.22, 125.34, 125.15, 122.21, 122.13, 118.60, 118.53, 110.83, 110.36, 103.77, 103.64, 88.54, 88.23, 75.27, 62.46, 49.40, 49.17, 44.22, 41.03, 35.94, 34.78, 27.96, 27.90, 27.84, 27.09, 26.32, 25.24, 16.17, 16.11, 16.04. .sup.31P NMR (243 MHz, CDCl3)=?9.08.

Staudinger-Induced Thiol Addition with Alkynyl-Phosphonites for the Generation of Antibody Drug Conjugates (ADCs)

[0453] As set our herein above, we were able to show that a modification of full length igG antibodies with alkyne- and alkene-phosphonamidates is possible. In the above examples we used Cetuximab as a model antibody and modified the interchain-disulfides with a biotinylated phorsphonamidate via the reduction and alkylation protocol, previously described by Senter and coworkers (29). This concept was further developed towards a feasible system for the generation of ADCs by phosphonamidate mediated conjugation the highly potent tubulin binding cytotoxin MMAE and the Her2 binding antibody Trastuzumab.

[0454] Similar to our above studies with Cetuximab, we reduced the inter-chain disulfide bonds of Trastuzumab with dithiothreitol (DTT) and carried out Cys-conjugation reactions with different electrophilic biotin derivatives, including maleimide, iodoacetamide and alkyne-phopshonamidate (phosphonamidate-labelling), to have a direct comparison to state-of-the art techniques. The latter was synthesized by the Staudinger phosphonite reaction protocol in 72% overall yield. The antibody-labelling reactions were carried out with and without prior reduction of the disulfide bonds to probe the chemoselectivity of the Cys-conjugation reactions (FIG. 16). Westernblot analysis revealed sufficient labelling for all of the tested biotin derivatives with reduced trastuzumab. Most strikingly, we observed high reactivity of maleimides with non-reduced trastuzumab, which was further confirmed by trypsin digestion and MS/MS analysis. In contrast, phosphonamidate-labelling demonstrated outstanding selectivity for Cys-residues (FIG. 16).

[0455] FIG. 16 shows: A: Trastuzumab modification with three different Cys-reactive bitotin derivatives. Disulfide reduction was carried out with 1000 eq. DTT in 50 mM borate containing PBS for 30 minutes at 37? C. Excess DTT was removed by size exclusion chromatography. Labelling was conducted with 35 eq. biotin derivative with a final DMSO content of 1% in a Buffer containing 50 mM NH.sub.4HCO.sub.3 and 1 mM EDTA, pH 8.5 for the amidate and PBS containing 1 mM EDTA, pH 7.4 for the other two compounds. B: Western blot analysis. Lane 1 and 5: untreated antibody. Lane 2-4: reactions with prior DTT treatment. Lane 6-8: Control reactions without prior DTT treatment.

[0456] Phosphonamidate-linked ADCs were generated from the very efficient antimitotic toxin MMAF and the FDA approved Her2-addressing antiproliferative antibody trastuzumab (FIG. 17). To investigate release of the toxic payload, ADCs with a cathepsin B cleavage side (Valine-Citruline linker VC) were prepared between the antibody and the toxin. Amidate-VC-PAB-MMAF constructs were synthesized based on a previously described procedure, as depicted in Scheme 27.

##STR00151##

[0457] Conjugation to Trastuzumab was carried out in 50 mM ammoniumbicarbonate buffer at pH 8.5 for 16 hours at 14? C., after reduction of the interchain-disulfide bonds with DTT and removal of the excess reducing agent by Zeba? Spin desalting columns.

[0458] FIG. 17 shows: Trastuzumab modification with phosphonamidate modified, cathepsin cleavable MMAF (Amidate-VC-PAB-MMAF). A: Reaction scheme reduction and alkylation of interchain disulfides. B: SDS-PAGE analysis of the reaction. C. Deconvolutet MS spectra of the antibody fragments after deglaycosylation with PNGase F and reduction with DTT. LC: Light chain; HC: Heavy chain; mod: Amidate-VC-PAB-MMAF.

[0459] An average loading of 4.6 drug molecules per antibody was determined by ESI-MS after deglycosylation and reduction. We approximated the drug-to-antibody ratio (DAR) with the mass signal intensities of the heavy- and light-chain species bearing different degrees of modification.

[0460] The obtained Phosphonamidate-ADC conjugates were evaluated in a previously established Her2 based proliferation assay with two different Her2-overexpressing cell lines BT474 and SKBR3 (30). The Her2-non overexpressing cell line MDAMB468 was used as a control to proof Her2 selectivity. Phosphonamidate-linked conjugates were compared to a maleimide-linked cathepsin B-cleavable trastumzumab MMAF conjugate. These experiments clearly demonstrate that phosphonamidate-labelled MMAF-ADCs enable sufficient and selective killing of Her2 overexpressing cells. The measured IC.sub.50-values values were at least as good as the compared maleimide controls (FIG. 18). It is important to note, that it is not to be expected that the advantages of phosphonamidate-labelling have a positive effect on in vitro cell killing efficiency when compared to maleimide chemistry.

[0461] FIG. 18 shows: Increased antiproliferative potency of MMAF linked trastuzumab on two different Her2 overexpressing cell lines (BT474 and SKBR3) and one control (MDAMB468). Plots depict the number of proliferating cells after 4 days of antibody treatment in dependency of the antibody concentration. Trastuzumab alone (pink), trastuzumab-phosphonamidate-MMAF (blue) and trastuzumab-maleimide-MMAF (green).

Procedures for the Staudinger-Induced Thiol Addition with Alkynyl-Phosphonites for the Generation of Antibody Drug Conjugates (ADCs)

N-(4-azidobenzoyl)-L-valine

[0462] ##STR00152##

[0463] A 50-ml Schlenk-flask was charged with 1.00 g of 4-azidobenzoic acid (6.13 mmol, 1.00 eq.) and suspended in 8.5 ml of dry DCM (dichloromethane) together with a drop of DMF (N,N-dimethylformamide) under argon. 630 ?l of oxalylchloride were added drop-wise at 0? C. and the reaction mixture was stirred at room temperature for 2 h until the solution became clear. All volatiles were removed under reduced pressure and the corresponding solid was redissolved in 4 ml of DMF. The corresponding solution was added drop-wise at 0? C. to a solution of 720 mg L-valin (6.13 mmol, 1.00 eq.) and 612 mg sodium hydroxide (15.33 mmol, 2.50 eq.) in 8 ml water and stirred for 2 more hours. The solution was acidified with 1 N HCl and extracted three times with diethylether. The organic fractions were pooled, dried (MgSO.sub.4) and the solvents were removed under reduced pressure. Pure product was obtained by flash column chromatography on silicagel (30% EtOAc, 0.5% formic acid in n-hexane) as colourless fume. (954 mg, 4.96 mmol, 80.9%)

[0464] .sup.1H NMR (600 MHz, Chloroform-d) ?=10.12 (s, 1H), 7.79 (d, J=8.6, 2H), 7.05 (d, J=8.6, 2H), 6.79 (d, J=8.5, 1H), 4.76 (dd, J=8.5, 4.9, 1H), 2.33 (pd, J=6.9, 4.9, 1H), 1.03 (d, J=6.9, 3H), 1.01 (d, J=6.9, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) ?=175.82, 167.28, 144.03, 130.17, 129.13, 119.20, 77.16, 57.79, 31.40, 19.16, 17.99. HR-MS for C12H15N.sub.4O3.sup.+ [M+H].sup.+ calcd: 263.1139, found 263.1151.

N-(4-azidobenzoyl)-L-valine-anhydride

[0465] ##STR00153##

[0466] In a 100-ml round-bottom flask, 954 mg N-(4-azidobenzoyl)-L-valine (3.64 mmol, 1.00 eq.), 750 mg dicyclohexylcarbodiimide (3.64 mmol, 1.00 eq.), 418 mg N-hydroxysuccinimide (3.64 mmol, 1.00 eq.) and 9 mg 4-(dimethylamino)-pyridine (0.07 mmol, 0.02 eq.) were dissolved in 25 ml of THF and stirred over night at room temperature. The reaction mixture was filtered, the solids were washed several times with THF, the solvent was removed under reduced pressure and the crude product was purified by flash column chromatography on silicagel (20 to 40% EtOAc in n-hexane). The compound was isolated as white powder (513 mg, 1.01 mmol, 55.7%)

[0467] .sup.1H NMR (600 MHz, Chloroform-d) ?=8.01 (d, J=8.7, 2H), 7.13 (d, J=8.7, 2H), 4.29 (d, J=4.6, 1H), 2.39 (heptd, J=6.9, 4.6, 1H), 1.16 (d, J=6.9, 3H), 1.03 (d, J=6.9, 3H). .sup.13C NMR (151 MHz, CDCl.sub.3) ?=177.52, 160.90, 144.51, 129.60, 122.43, 119.30, 70.68, 31.28, 18.76, 17.57.

N-(4-azidobenzoyl)-L-valine-L-citrulline

[0468] ##STR00154##

[0469] In a 50-ml round-bottom flask, 380 mg N-(4-azidobenzoyl)-L-valine-anhydride (0.75 mmol, 1.00 eq.) were dissolved in 2 ml of 1,2-Dimethoxyethane and cooled to 0? C. A solution of 351 mg L-citrulline (1.50 mmol, 2.00 eq.) and 144 mg sodium hydrogencarbonate (2.25 mmol, 3.00 eq.) in 4 ml H.sub.2O and 2 ml THF (tetrahydrofuran) was added dropwise and stirred over night at room temperature. All volatiles were removed under reduced pressure and the crude product was purified by flash column chromatography on silicagel (10% MeOH, 0.5% formic acid in CH.sub.2Cl.sub.2). The compound was isolated as colourless oil (312 mg, 0.74 mmol, 99.0%).

[0470] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.31 (d, J=8.8, 1H), 8.27-8.21 (m, 1H), 7.96 (d, J=8.6, 2H), 7.20 (d, J=8.6, 2H), 6.05 (t, J=5.5, 1H), 5.47 (s, 2H), 4.37 (t, J=8.3, 1H), 4.18 (td, J=8.1, 5.1, 1H), 2.98 (q, J=6.4, 2H), 2.15 (dq, J=13.6, 6.8, 1H), 1.78-1.68 (m, 1H), 1.68-1.56 (m, 1H), 1.51-1.35 (m, 2H), 0.96 (d, J=6.8, 3H), 0.94 (d, J=6.8, 3H). .sup.13C NMR (151 MHz, DMSO) ?=174.09, 171.54, 165.99, 159.40, 142.77, 131.36, 129.93, 119.23, 59.31, 52.57, 49.07, 30.77, 29.01, 27.07, 19.75, 19.28. HR-MS for C18H26N.sub.7O5.sup.+ [M+H].sup.+ calcd: 420.1990, found 420.1990.

N-(4-azidobenzoyl)-L-valine-L-citrulline-4-aminobenzyl alcohol

[0471] ##STR00155##

[0472] In a 50-ml round-bottom flask, 330 mg N-(4-azidobenzoyl)-L-vaine-L-citrulline (0.787 mmol, 1.0 eq.) and 107 mg 4-aminobenzyl alcohol (0.866 mmol, 1.10 eq.) were dissolved in 8 ml CH.sub.2Cl.sub.2 and 4 ml MeOH (methanol) under an argon atmosphere and cooled to 0? C. 390 mg N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.574 mmol, 2.00 eq.) were added portion-wise and the resulting solution was allowed to warm to room temperature overnight. All volatiles were removed under reduced pressure and the crude product was isolated by flash column chromatography on silicagel (10% to 15% MeOH in CH.sub.2Cl.sub.2) and obtained as white solid (164 mg, 0.313 mmol, 39.8%). Enantiomeric pure compound was isolated by preperative HPLC (Method D described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond) and obtained as a white solid after lyophilisation.

[0473] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.93 (s, 1H), 8.32 (d, J=8.4, 1H), 8.21 (d, J=7.6, 1H), 7.96 (d, J=8.6, 2H), 7.55 (d, J=8.6, 2H), 7.24 (d, J=8.6, 2H), 7.21 (d, J=8.6, 2H), 6.12 (bs, 2H), 4.44 (s, 2H), 4.46-4.40 (m, 1H), 4.36 (t, J=8.1, 1H), 3.09-2.93 (m, 2H), 2.24-2.04 (m, J=6.7, 1H), 1.84-1.58 (m, 2H), 1.55-1.34 (m, 2H), 0.95 (d, J=6.7, 3H), 0.94 (d, J=6.7, 3H). .sup.13C NMR (151 MHz, DMSO) ?=171.62, 170.79, 166.15, 159.46, 142.83, 137.95, 137.91, 131.29, 129.96, 127.38, 119.34, 119.26, 63.07, 59.56, 53.64, 39.20, 30.61, 29.88, 27.16, 19.79, 19.37. HR-MS for C.sub.25H.sub.33N.sub.8O.sub.5.sup.+ [M+H].sup.+ calcd: 525.2568, found 525.2563. [?].sub.D.sup.24=?49.6 (c=0.81; MeOH)

N-(4-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)-L-valine-L-citrulline-4-aminobenzyl alcohol

[0474] ##STR00156##

[0475] The compound was synthesized according to the general procedure from 230 ?l diethyl chlorophosphite (0.925 mmol, 5.0 eq.), 1.85 ml ethynylmagnesium bromide solution (0.5 M in THF, 0.925 mmol, 5.0 eq.) and 97 mg N-(4-azidobenzoyl)-L-valine-L-citrulline-4-aminobenzyl alcohol (0.185 mmol, 1.0 eq.). The crude phosphonamidate was purified by preperative HPLC (method C described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond) and obtained as a white solid after lyophilisation. (60 mg, 0.098 mmol, 52.9%).

[0476] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.96 (s, 1H), 8.80 (d, J=8.5, 1H), 8.22 (d, J=7.7, 1H), 8.11 (dd, J=8.6, 1.9, 1H), 7.82 (d, J=8.7, 2H), 7.56 (d, J=8.4, 2H), 7.24 (d, J=8.4, 2H), 7.14 (d, J=8.7, 2H), 4.39-4.46 (m, 4H), 4.33 (t, J=8.1, 1H), 4.20-4.03 (m, 2H), 3.02 (ddt, J=38.3, 13.4, 6.8, 2H), 2.20-2.09 (m, J=6.9, 1H), 1.79-1.57 (m, 2H), 1.53-1.35 (m, 2H), 1.31 (t, J=7.1, 3H), 0.95 (d, J=6.9, 3H), 0.93 (d, J=6.9, 3H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) ?=171.74, 170.81, 166.59, 159.53, 143.51, 137.93 (d, J=9.0), 129.31, 127.57, 127.38, 119.34, 117.24 (d, J=7.7), 91.68 (d, J=45.5), 77.25 (d, J=261.1), 63.06, 62.29 (d, J=5.0), 59.45, 53.61, 39.27, 30.70, 29.82, 27.03, 19.82, 19.32, 16.42 (d, J=6.9). .sup.31P NMR (243 MHz, DMSO-d.sub.6) ?=?13.28, ?13.32. HR-MS for C.sub.29H.sub.40N.sub.6O.sub.7P.sup.+ [M+H].sup.+ calcd: 615.2691, found 615.2716.

N-(4-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)-L-valine-L-citrulline-4-aminobenzyl-4-nitrophenyl carbonate

[0477] ##STR00157##

[0478] A 5-ml round-bottom flask was charged with 31 mg N-(4-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)-L-valine-L-citrulline-4-aminobenzyl alcohol (0.050 mmol, 1.00 eq.) and 31 mg Bis(4-nitrophenyl) carbonate (0.101 mmol, 2.00 eq.). The solids were dissolved in 140 ?l of DMF (N,N-dimethylformamide) and 17.4 ?l DIPEA (N,N-diisopropylethylamine, 0.101 mmol, 2.00 eq.) were added. The yellow solution was stirred for 1 h at room temperature and the solution was added to 30 ml of ice-cold diethyl ether. The precipitate was collected by centrifugation, redissolved in DMF and again precipitated with ether. The procedure was conducted three times in total and finally the solid was dried under high vacuum conditions. The compound was isolated in quantitative yields and sufficiently pure for the next step. Analytical pure material was purified by preperative HPLC using method C described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond.

[0479] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=10.10 (s, 1H), 8.79 (d, J=8.5, 1H), 8.32 (d, J=9.1, 1H), 8.23 (d, J=7.4, 1H), 8.07 (dd, J=8.5, 2.2, 1H), 7.81 (d, J=8.7, 2H), 7.66 (d, J=8.5, 2H), 7.57 (d, J=9.1, 1H), 7.42 (d, J=8.5, 2H), 7.13 (d, J=8.7, 2H), 5.25 (s, 2H), 4.47-4.40 (m, 2H), 4.34 (t, J=8.0, 1H), 4.20-4.05 (m, 2H), 3.01 (ddt, J=47.1, 13.4, 6.8, 2H), 2.20-2.09 (m, J=6.8, 1H), 1.80-1.59 (m, 2H), 1.55-1.35 (m, 2H), 1.30 (t, J=7.0, 3H), 0.95 (d, J=6.7, 3H), 0.93 (d, J=6.7, 3H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) ?=171.79, 171.17, 166.58, 159.44, 155.75, 152.42, 145.63, 143.50, 139.83, 129.95, 129.77, 129.30, 127.59, 125.86, 123.08, 119.51, 117.24 (d, J=7.8), 91.67 (d, J=45.6), 77.26 (d, J=261.0), 70.71, 62.26 (d, J=5.0), 59.31, 53.68, 39.14, 30.71, 29.76, 27.19, 19.80, 19.30, 16.41 (d, J=6.9). .sup.31P NMR (243 MHz, DMSO) ?=?10.39, ?10.44. HR-MS for C.sub.36H.sub.43N.sub.7O.sub.11P.sup.+ [M+H].sup.+ calcd: 780.2753, found 780.2744.

Amidate-Val-Cit-Pab-MMAF

[0480] ##STR00158##

[0481] A 15-mL falcon-tube was charged with 14.35 mg N-(4-(O-Ethyl-P-ethynyl-phosphonamidato-N-benzoyl)-L-valine-L-citrulline-4-aminobenzyl-4-nitrophenyl carbonate (0.0184 mmol, 1.00 eq.), 0.50 mg 1-Hydroxybenzotriazol (0.0037 mmol, 0.20 eq.) and 13.15 mg MMAF (monomethylauristatin F, 0.0184 mmol, 1.00 eq.). The solids were dissolved in 250 ml dry DMF and 25 ml pyridine and heated to 60? C. over-night. All volatiles were removed under reduced pressure, the crude product was purified by semi-preperative HPLC using method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond, and the desired compound obtained as a white solid after lyophilisation. (4.84 mg, 0.0035 mmol, 19.2%). HR-MS for C.sub.69H.sub.104N.sub.11O.sub.16P.sup.2+ [M+2H].sup.2+ calcd: 686.8695, found 686.8694.

[0482] FIG. 19 shows the UPLC-UV purity of phosphonamidate-Val-Cit-Pab-MMAF.

Trastuzumab Production

[0483] Trastuzumab expression and purification was executed as previously published with an additional final purification by gel filtration on a Superdex 200 Increase 10/300 from GE with phosphate-buffered saline (PBS) anf flow rate of 0.75 ml/min (30).

General Procedure for the Modification of Trastuzumab Via the Reduction/Alkylation Protocol

[0484] ##STR00159##

[0485] Trastuzumab modification was carried out by incubating freshly expressed antibody (c=0.55 mg/ml) in a buffer containing 50 mM sodium borate and 4 mM DTT in PBS (pH 8.0) with a total volumn of 80 ?l at 37? C. for 40 min. Excess DTT removal and buffer exchange to a solution containing 50 mM NH.sub.4HCO.sub.3 and 1 mM EDTA (pH 8.5) was conducted afterwards using 0.5 mL Zeba? Spin Desalting Columns with 7K MWCO (Thermo Fisher Scientific, Waltham, United States). 1.60 ?l of a solution containing 13 mM amidate in DMSO was added quickly. And the mixture was shaken at 800 rpm and 14? C. for 16 hours. Excess amidate was again removed by buffer exchange to sterile PBS using 0.5 mL Zeba? Spin Desalting Columns with 7K MWCO.

Cell Based Antiproliferation Assays

[0486] Antiproliferation assays were conducted as previously reported (30) with the following minor changes: [0487] For MDAMB468 cells, a reduced amount of 2*10.sup.3 cells were seeded in each well of a 96-well optical cell culture plate supplemented with 100 ?L culture media. [0488] Images were acquired with an Operetta High-Content Imaging system (PerkinElmer, Waltham, Mass., USA) equipped with a 20? high NA objective. [0489] Cell counts were calculated from duplicates
Staudinger-Induced Thiol Addition with Alkynyl-Phosphonites for the Generation of Antibody Fluorophore Conjugates (AFCs)

[0490] In a similar manner, as described above under Staudinger-induced thiol addition with alkynyl-phosphonites for the generation of Antibody Drug Conjugates (ADCs) the fluorescent dye Cy5 was conjugated to Trastuzumab to generate an antibody-fluorophore conjugate. Synthesis of Cy5-O-ethyl-P-alkynyl-phosphonamidate was conducted as described above under Introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond. The obtained Phosphonamidate-AFC conjugates were evaluated by immunostaining of two different Her2-overexpressing cell lines BT474 and SKBR3. The Her2-non overexpressing cell line MDAMB468 was used as a control to proof Her2 selectivity. Sufficient membrane staining after cell fixation was observed the two Her2-expressing cell lines, while the Her2-non expressing cell lines did not show increased fluorescence.

[0491] FIG. 20: Depicted are immunostainings of fixed cells over expressing the cell surface receptorHer2 (BT474 and SKBR3) or exhibiting low Her2 expression levels (MDAMB468). The AFC Trastuzumab-Amidate-Cy5 shows clear localization to the plasma membrane for Her2+ cell lines and no staining of Her2-cells. The merged images show the DAPI signal in blue and the Tras-phosphonamidate-Cy5 signal in red. Scale bar represents 10 ?m.

Procedures for the Staudinger-Induced Thiol Addition with Alkynyl-Phosphonites for the Generation of Antibody Fluorophore Conjugates (AFCs)

##STR00160##

[0492] Trastuzumab-Cy5 conjugates were synthesized according to the general procedure, described above under Staudinger-induced thiol addition with alkynyl-phosphonites for the generation of Antibody Drug Conjugates (ADCs) with the following slight modifications: the amidate equivalents were raised to 130, and the DMSO (dimethylsulfoxide) content was raised to 5% (more precisely, from 2% to 5%) to solubilize the Cy5.

AFC Imaging Procedure

[0493] BT474, SKBR3 and MDAMB468 were seeded on sterile cover slips and incubated ON at 37? C., 5% CO2 for cell attachment. Cells were washed three times with 1?PBS prior to fixation for 10 min in 1?PBS/4% PFA (formaldehyde). Fixation was stopped by the addition of an equal volume 1?PBST (PBS+0.05% Tween20) followed by two more washes with PBST. AFCs were added to a final concentration of 5 ?g/mL and incubated for 1 h at RT. Unbound AFC was removed by three washes with PBS.

[0494] Images were acquired on a Leica SP5 confocal microscopy system equipped with a 63?1.40 Oil immersion objective. Laserlines 405 nm and 594 nm were used in combination with standard DAPI and Cy5 filter settings. Image processing was carried out with ImageJ 1.5.1 h software extended by the Fiji processing package.

Stability Studies of the Phosphonamidate Linkage

[0495] To study the stability of the phosphonamidate bond in complex systems as cell lysate or serum, a dye-quencher pair was synthesized which generates a fluorescent signal upon cleavage of the phosphonamidate bond. Conjugates consist of the fluorescent dye EDANS, the quencher DABCYL and an attached peptide to ensure water solubility of the conjugates (FIG. 21). A maleimide linked conjugate was synthesized for comparison experiments (FIG. 21B).

[0496] FIG. 21 shows: A: Structure of the phosphonamidate linked FRET conjugate. B: Structure of the maleimide linked FRET conjugate C: Principle of the fluorescence-quencher based readout. Conjugates were incubated at room temperature at a concentration of 10 ?M. Measurements were performed at least in triplicates in a 96-well plate. D: Fluorescence increase was monitored over time. HCl samples were neutralized before the measurements. Lysate was freshly prepared from HeLa-cells, lysed in PBS. Serum originated from human blood. E: Comparison of a phosphonamidate- and a maleimide-linked dye-quencher pair during exposure to 1000 eq. glutathion in PBS.

[0497] As shown in FIG. 21, the phosphonamidate adducts show a high stability in PBS, HeLA cell lysate and human serum, whereas only strong acidic conditions (1N HCl) lead to phosphonamidate bond cleavage. The FRET-conjugates were also exposed to a high excess of glutathione. After 2 days of incubation with 1000 eq. of glutathione at physiological pH, 15% of the maleimide linkage was cleaved, while 99% of the phosphonamidate adducts were still intact (FIG. 21C).

[0498] In the next experiment, we probed whether the modification element of phoshonamidate-labelled or maleimide-labelled ADCs is transferred to serum proteins in the presence of thiols, as the stability of ADCs for several days is crucial during circulation in the blood stream. Trastuzumab modified with different biotin derivatives (FIG. 22) was exposed to serum-like albumin concentrations of 0.5 mM, incubated at 37? C., and transfer of the modification from the antibody to the serum protein was monitored by western blotting (FIG. 22B). A significant transfer of the biotin to BSA (bovine serum albumin) at serum concentrations was observed for maleimide linkage while the phoshonamidate-linkage was stable under the tested conditions. Taken together, these stability experiments experiment clearly point to superior stability of the phoshonamidate-labelled ADCs as compared to maleimide-labelled ADCs potentially leading to a reduction of off-target toxicity when compared to conventional maleimide-linked conjugates.

[0499] FIG. 22 shows: Transfer of the antibody modification to serum proteins. A: Tratuzumab-biotin conjugates were incubated at a concentration of 3 ?M with 500 ?M BSA in PBS at 37? C. B: Biotin transfer to albumin was monitored by western blot analysis. Lane 1: Untreated maleimide conjugate. Lane 2-5: Anlaysis of the BSA exposed maleimide adduct after 0, 1, 2 and 5 days. Lane 6: Untreated amidate conjugate. Lane 6-10: Analysis of the BSA exposed amidate adduct after 0, 1, 2 and 5 days.

Procedures for the Stability Studies of the Phosphonamidate Linkage

DABCYI-Cys Peptide

[0500] ##STR00161##

[0501] DABCYI-Cys peptide was synthesized by standard Fmoc-based chemistry in a linear synthesis by manual coupling. 0.1 mmol of Rink amide resin (subst: 0.4 mmol/g) was added to a reaction vessel and synthesis was performed with five-fold amino acid excess. Fmoc de-blocking was achieved by resin treatment with 20% piperidine in DMF twice for 5 minutes. Coupling was achieved by addition of HOBt/HBTU/DIPEA (5 eq./5 eq./10 eq) in DMF for 45 min. After the final Cys coupling, 5 eq. of the DABCYL acid was coupled with 5 eq. HATU and 10 eq. DIPEA in DMF for 45 min. The peptide was cleaved of the resin by addition of TFA/DTT/TIS (95/2.5/2.5, w,w,w) within 3 h. Subsequently, the peptide was precipitated by the addition of ice-cold diethyl ether. The precipitate was collected by centrifugation, dried and purified by preperative HPLC (method C described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). The peptide was obtained as a red solid in a yield of 35.8% (38.2 mg, 35.8 ?mol). ESI-MS for C48H66N.sub.12O14S.sup.+ [M+2H].sup.+ calcd: 533.23, found 533.34.

DABCYI-Cys Peptid Phosphonamidate EDANS Adduct

[0502] ##STR00162##

[0503] A 1.5-ml Eppendorf tube was charged with 263 ?l of a solution of DABCYI-Cys peptide (20 mM) in 50 mM NH.sub.4HCO.sub.3 at a pH of 8.5. 158 ?l 50 mM NH.sub.4HCO.sub.3 at a pH of 8.5 and 105 ?l of a solution of EDANS amidate (100 mM) in DMF was added to give a final concentration of 20 mM peptide and 10 mM amidate in 20% DMF/Buffer. The tube was shaken at 800 rpm at room temperature for 3 h. All volatiles were removed under reduced pressure and the crude product purified by semi-preperative HPLC (method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). The peptide was obtained as a red solid ESI-MS for C.sub.71H.sub.90N.sub.15O.sub.20PS.sub.2.sup.+ [M+2H].sup.+ calcd: 783.78, found 784.47.

DABCYI-Cys Peptid Maleimide EDANS Adduct

[0504] ##STR00163##

[0505] A 1.5-ml Eppendorf tube was charged with 188 ?l of a solution of DABCYI-Cys peptide (20 mM) in PBS. 188 ?l of a solution of EDANS maleimide (40 mM) in DMF was added to give a final concentration of 10 mM peptide and 20 mM maleimide in 50% DMF/Buffer. The tube was shaken at 800 rpm at room temperature for 3 h. All volatiles were removed under reduced pressure and the crude product purified by semi-preperative HPLC (method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). The peptide was obtained as a red solid. ESI-MS for C.sub.66H.sub.83N.sub.15O.sub.20S.sub.2.sup.+ [M+2H].sup.+ calcd 734.77: found. 734.79

Stability Studies of the Dabcyl-EDANS Adducts

[0506] Stabilities studies were conducted in 96-well plate (Corning 3615, black with clear, flat bottom) at least in triplicates. 5 ?l of a 200 ?M Stock solution of the Dabcyl-EDANS adducts and 95 ?l of the respective test solutions were added to each well.

[0507] HeLa cell lysate was generated from approximately 1*10.sup.7 cells, lysed in 400 ?l PBS by sonification. Cells were grown on a 75 cm.sup.2 cell culture plate, washed twice with PBS and harvested with a cell scraper. Human serum was purchased from Sigma Aldrich. Glutathione was dissolved at a concentration of 10 mM in PBS and the pH was adjusted to 7.4. 1N HCl studies were conducted at 200 ?M, neutralized to pH 7 and diluted to 10 ?M before fluorescence measurements.

[0508] Fluorescence was measured on a Tecan Safire plate reader. Excitation: 336 nm, emission: 490 nm, bandwith: 5 nm at 20? C.

Incubation of Trastuzumab-Biotin Conjugates with BSA

[0509] Trstuzumab-Biotin conjugates were incubated at a consentration of 3 ?M in PBS with a final concentration of 0.5 mM BSA at 37? C. Samples were drawn after 0, 1, 2 and 5 days, deep frozen in liquid Nitrogen and finally subjected to SDS/Page and westernblot analysis.

Further Kinetic Investigations of the Thiol Addition

[0510] To study the kinetics of the thiol addition to alkyne-phosphonamidates at low concentrations, a fluorescent EDANS-based phosphonamidate was synthesized as described in chapter 1.1. Addition of glutathione as a model substrate was probed over time by fluorescence HPLC. Peak integration and normalization to unconjugated EDANS as an internal standard was applied to determine the second order rate constant of the reaction. A second order rate constant of 37.32?0.41 I/mol*s was measured.

[0511] FIG. 23 shows: Determination of the second order rate constant of the thiol addition. A: Reaction of the EDANS phosphonamidate with glutathione. B: Fluorescence HPLC trace after 30 min reaction time. C: Monitoring of the phosphonamidate decrease over time. D: Plot of the inverse concentration against reaction time. Error bars represent the mean of three replicates (n=3).

Procedures for the Further Kinetic Investigations of the Thiol Addition

[0512] Glutathione addition to the EDANS-phosphonamidate was conducted at a final concentration of 0.1 mM amidate, 0.1 mM glutathione and 0.02 mM EDANS as an internal standard in 50 mM NH.sub.4HCO.sub.3-buffer containing 1 mM EDTA at pH 8.5 with 1% DMF. 2.5 ?l of a 20 mM stock solution of EDANS-phosphonamidate in DMF was premixed with 488 ?l buffer and 5 ?l of a 2 mM stock solution of EDANS in a 1:1 mixture of DMF and buffer. The reaction was started by the addition of 5 ?l of a 10 mM solution of glutathione in buffer. 10 ?l samples were drawn at 0, 15, 30, 60, 120, 240 and 480 minutes and acidified with 190 ?l 10 mM NaOAc-Buffer (pH 5.0) and subjected to fluorescence HPLC analysis.

Synthesis of Further Phosphonites

[0513] Further, the O-substituent oft he alkyne phosphonites was varied as shown in Scheme E2, and electron-rich phosphonites E1 to E5 were synthesized:

##STR00164##

Procedures for the Synthesis of Further Phosphonites E1 to E5

General Procedure for the Synthesis of O-Substituted Alkynyl Phosphonamidates from Bis(Diisopropylamino)Chlorophosphine

[0514] ##STR00165##

[0515] A 25-ml Schlenk flask was charged with 267 mg bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.) under an argon atmosphere, cooled to 0? C. and 2.20 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.) was added drop wise. The yellowish solution was allowed to warm to room temperature and stirred for further 30 minutes. The respective alcohol, dissolved in 5.56 ml 1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol) was added and the white suspension was stirred over night at room temperature. The reaction mixture was directly placed on a silica gel flash column.

Di-(2-(2-Hydroxyethoxy)ethyl) ethynylphosphonite (compound E1)

[0516] ##STR00166##

[0517] The compound was synthesized according to the above General procedure for the synthesis of O-substituted alkynyl phosphonamidates from bis(diisopropylamino)chlorophosphine from 267 mg bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.), 2.20 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.), 1.06 g 2-(2-Hydroxyethoxy)ethan-1-ol (10.00 mmol, 10.00 eq.), 5.56 ml 1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol) and purified by flash column chromatography on silicagel (5% MeOH in CH.sub.2Cl.sub.2). The compound was obtained as a yellowish oil. (112 mg, 0.421 mmol, 42.1%).

[0518] .sup.1H NMR (300 MHz, Chloroform-d) ?=4.14-3.98 (m, 4H), 3.65-3.59 (m, 4H), 3.58-3.49 (m, 8H), 3.15 (d, J=2.4, 1H). .sup.13C NMR (75 MHz, Chloroform-d) ?=92.52, 92.50, 84.61, 83.98, 72.60, 70.72 (d, J=4.0), 67.20 (d, J=6.0), 61.44. .sup.13C NMR (75 MHz, Chloroform-d) ?=92.51 (d, J=1.4), 84.30 (d, J=46.8), 72.60, 70.72 (d, J=4.0), 67.20 (d, J=6.0), 61.44. .sup.31P NMR (122 MHz, CDCl.sub.3) ?=131.97.

Di-(3-Butinyl) ethynyiphosphonite (compound E2)

[0519] ##STR00167##

[0520] The compound was synthesized according to the above General procedure for the synthesis of O-substituted alkynyl phosphonamidates from bis(diisopropylamino)chlorophosphine from 267 mg bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.), 2.20 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.), 189 ?l 3-Butyn-1-ol (2.50 mmol, 2.50 eq.), 5.56 ml 1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol) and purified by flash column chromatography on silicagel (10% EtOAC in n-hexane). The compound was obtained as a colourless oil. (152 mg, 0.774 mmol, 77.4%).

[0521] .sup.1H NMR (300 MHz, Chloroform-d) ?=4.07 (dtd, J=8.1, 7.0, 1.5, 4H), 3.14 (d, J=2.3, 1H), 2.56 (tdd, J=7.0, 2.7, 0.6, 4H), 2.03 (t, J=2.7, 2H). .sup.13C NMR (75 MHz, Chloroform-d) ?=92.42 (d, J=1.3), 83.92 (d, J=47.1), 80.24, 70.02, 65.81 (d, J=6.5), 21.28 (d, J=4.7). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=130.15.

[0522] The procedures for the synthesis of compounds E3, E4 and E5 are provided herein below under Procedures for the synthesis of compounds having a cleavable group on the 0-substituent.

Staudinger Phosphonite Reaction with Phosphonite E1 and E2

[0523] The highly stable nature of electron rich phosphonites was further exploited by performing the Staudinger phosphonite reaction with alkyne-phosphonites in aqueous solvents. As depicted in FIG. 24, formation of the desired product from alkyne phosphonite E1 was observed in a pure aqueous system.

[0524] FIG. 24 shows: Reaction of an azido modified peptide with the water soluble phosphonite E1 in Tris buffer. A: Reaction scheme. B: HPLC-trace; orange: starting material; blue: reaction after 2 h.

Procedures for the Staudinger Phosphonite Reaction with Phosphonites E1 and E2

Peptide E9

[0525] Peptide E9 was synthesized by standard Fmoc-based chemistry in a linear synthesis by manual coupling. 0.1 mmol of Rink amide resin (subst: 0.4 mmol/g) was added to a reaction vessel and synthesis was performed with five-fold amino acid excess. Fmoc de-blocking was achieved by resin treatment with 20% piperidine in DMF twice for 5 minutes. Coupling was achieved by addition of HOBt/HBTU/DIPEA (5 eq./5 eq./10 eq) in DMF for 45 min. After the final Gly coupling, 5 eq. of the 4-azido benzoic acid was coupled with 5 eq. HATU and 10 eq. DIPEA in DMF for 45 min. The peptide was cleaved of the resin by addition of TFA/TIS/H.sub.2O (95/2.5/2.5, w,w,w) within 3 h. Subsequently, the peptide was precipitated by the addition of ice-cold diethyl ether. The precipitate was collected by centrifugation, dried and purified by preperative HPLC (method C described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). ESI-MS for C37H50N.sub.11O13.sup.+ [M+H].sup.+ calcd: 856.36, found 856.36.

Staudinger Phosphonite Reaction of Peptide E9 with Amidate E1 in Basic Tris-Buffer

[0526] 10 ?l of a 50 mM stock solution of peptide E9 in 100 mM Tris buffer (pH 9.0) was added to 80 ?l of 100 mM Tris buffer (pH 9.0). 10 ?l of a solution of 500 mM phosphonite E1 in the same buffer was added and shaken at 37? C. for 2 hours at 800 RPM. A sample of 10 ?l was drawn, diluted with 90 ?l 1% TFA in H.sub.2O and subjected to UPLC-MS-analysis.

Synthesis of E6

[0527] ##STR00168##

[0528] General procedure: 1.00 mmol of an organic azide (1.00 eq.) was stirred together with 1.00 mmol of an alkynyl phosphonite (1.00 eq.) in 5 ml DMF overnight. The organic solvent was removed under educed pressure and the residue purified by column chromatographie on silica. Following this general procedure, 37 mg Di-(3-Butinyl) ethynylphosphonite (compound E2) (0.192 mmol, 1.00 eq.) and 50 mg 4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.162 mmol, 1.00 eq.) were mixed in 1 ml of DMF and purified by flash column chromatography on silicagel (70% EtOAc in hexane). The compound was obtained as colourless oil. (55 mg, 0.147 mmol, 76.6%).

[0529] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.33-7.93 (m, 3H), 7.21 (d, J=8.8, 2H), 4.47-3.94 (m, 2H), 3.06 (d, J=13.2, 1H), 2.89 (s, 4H), 2.65 (td, J=6.7, 2.7, 2H), 2.07 (t, J=2.7, 1H). .sup.13C NMR (75 MHz, Chloroform-d) ? 169.61, 161.42, 145.52, 132.30, 118.19, 117.72 (d, J=8.1 Hz), 89.38 (d, J=50.0 Hz), 79.09, 70.94, 63.92 (d, J=5.0 Hz), 31.48, 25.69, 20.57 (d, J=8.2 Hz). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=?9.74.

Synthesis of Compounds Having a Cleavable Group on the O-Substituent and Cleavage Experiments

Introduction of a Cleavable Group on the O-Substituent

[0530] It has been described previously that cleavable disulfides can be used to liberate a specific payload under reducing conditions. For example, this approach has been applied to the specific release of a cytotoxic payload from an Antibody Drug Conjugate (ADC) within a cellular environment (31). In this context, it could be shown that disulfides that carry a leaving group in the beta position undergo cyclisation to a thiirane after disulfide cleavage and liberate a given payload (32).

[0531] For the purpose of the present invention, the synthesis of a conjugate having a cleavable disulfide-comprising O-substituent was envisaged as shown in Scheme 29.

##STR00169##

[0532] The following compounds having a cleavable group R on the O-substituent were synthesized and subjected to cleavage experiments:

##STR00170##

wherein R is

##STR00171##

Procedures for the Synthesis of Compounds Having a Cleavable Group on the O-Substituent

General Procedure 1 for the Synthesis of 2-Hydoxyethyl Disulfides

[0533] ##STR00172##

[0534] A 250 ml-round bottom flask was charged with 10 mmol (1.00 eq.) of the respective thiol, 10 mmol 2-mercaptoethanol (1.00 eq.), 0.1 mmol sodium iodide and 20 ml EtOAc. The mixture was rapidly stirred and 10 mmol of a solution of 30% H.sub.2O.sub.2 in water was added drop-wise. The mixture was stirred at room temperature for 1 h, volatiles were removed under reduced pressure and the disulfide was isolated by column chromatographie.

2-Hydroxyethyl ethyldisulfide

[0535] ##STR00173##

[0536] The compound was synthesized according to the above General procedure 1 for the synthesis of 2-hydoxyethyl disulfides from 2.00 ml Ethanethiol (27.74 mmol, 1.00 eq.), 1.96 ml 2-mercaptoethanol (27.74 mmol, 1.00 eq.), 41 mg sodium iodide (0.28 mmol, 0.01 eq.) and 3.14 ml hydrogen peroxide solution (aqueous, 30%) (27.74 mmol, 1.00 eq.). The disulfide was isolated by column chromatographie on silica (20% EtOAc in hexane) as colourless oil. Yield: 2.15 g (15.53 mmol, 56.0%).

[0537] .sup.1H NMR (300 MHz, Chloroform-d) ?=3.91 (dd, J=5.7, 2H), 2.87 (t, J=5.7, 2H), 2.74 (q, J=7.3, 2H), 2.08 (s, 1H), 1.35 (t, J=7.3, 3H).

2-Hydroxyethyl isopropyldisulfide

[0538] ##STR00174##

[0539] The compound was synthesized according to the above General procedure 1 for the synthesis of 2-hydoxyethyl disulfides from 2.00 ml isopropylthiol (21.53 mmol, 1.00 eq.), 1.52 ml 2-mercaptoethanol (21.53 mmol, 1.00 eq.), 32 mg sodium iodide (0.21 mmol, 0.01 eq.) and 2.44 ml hydrogen peroxide solution (aqueous, 30%) (21.53 mmol, 1.00 eq.). The disulfide was isolated by column chromatographie on silica (10% EtOAc in hexane) as colourless oil. Yield: 1.10 g (7.22 mmol, 33.6%)

[0540] .sup.1H NMR (300 MHz, Chloroform-d) ?=3.88 (m, 2H), 3.02 (hept, J=6.7, 1H), 2.85 (t, J=5.9, 2H), 2.37 (m, 1H), 1.32 (d, J=6.7, 6H). .sup.13C NMR (75 MHz, CDCl.sub.3) ?=60.48, 41.94, 41.14, 22.54.

2-Hydroxyethyl tert-butyldisulfide

[0541] ##STR00175##

[0542] The compound was synthesized according to the above General procedure 1 for the synthesis of 2-hydoxyethyl disulfides from 2.00 ml tert-Butylthiol (17.74 mmol, 1.00 eq.), 1.24 ml 2-mercaptoethanol (17.74 mmol, 1.00 eq.), 26 mg sodium iodide (0.18 mmol, 0.01 eq.) and 2.04 ml hydrogen peroxide solution (aqueous, 30%) (17.74 mmol, 1.00 eq.). The disulfide was isolated by column chromatographie on silica (10% EtOAc in hexane) as colourless oil. Yield: 0.90 g (5.41 mmol, 30.5%).

[0543] .sup.1H NMR (300 MHz, Chloroform-d) ?=3.87 (t, J=5.9, 2H), 2.86 (t, J=5.9, 2H), 2.33 (bs, 1H), 1.35 (s, 9H). NMR Data was in accordance with literature values (33).

2-(3-Hydroxypropyl) isopropyl disulfide

[0544] ##STR00176##

[0545] A 500-ml round-bottom flask was charged with 2.00 ml thiolactic acid (23.55 mmol, 1.00 eq.) and 150 ml dry THF. At 0? C., 1.60 g Lithium alluminium hydride (47.10, 2.0 eq.) were added portion-wise. The mixture was stirred at room temperature for 1 h, cooled again to 0? C. and quenched carefully with 6 N HCl. The aqueous phase was extracted with twice with 100 ml EtOAc, the organic fractions pooled, dried (MgSO.sub.4) and all volatiles were removed under reduced pressure. The resulting colourless oil was redissolved in 20 ml EtOH and 2.18 ml isobutyl thiol (23.55 mmol, 1.00 eq.), 55 mg sodium iodide (0.24 mmol, 0.01 eq.) and 2.70 ml hydrogen peroxide solution (aqueous, 30%) (23.55 mmol, 1.00 eq.) were added. The yellowish solution was stirred for another hour. Volatiles were removed under reduced pressure and the above stated disulfide isolated by column chromatographie on silica (20% EtOAc in hexane) as colouless oil. Yield: 1.15 g (6.928 mmol, 29.4%).

[0546] .sup.1H NMR (300 MHz, Chloroform-d) ?=3.69 (dd, J=5.8, 3.2, 2H), 3.08-2.83 (m, 2H), 1.37-1.25 (m, 9H). .sup.13C NMR (75 MHz, CDCl3) ?=65.49, 48.70, 41.66, 22.60, 22.51, 16.89.

1-(4-(hydroxymethyl)phenyl)-2-phenyldiazene

[0547] ##STR00177##

[0548] The compound was synthesized according to previously published procedure and isolated as orange solid (34).

[0549] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.03-7.86 (m, 4H), 7.68-7.42 (m, 5H), 4.81 (s, 2H). NMR Data was in accordance with literature values (34).

General Procedure 2 for the Synthesis of O-Substituted Alkynyl Phosphonites from Bis(Diisopropylamino) Chlorophosphine

[0550] ##STR00178##

[0551] A 25-ml Schlenk flask was charged with 267 mg bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.) under an argon atmosphere, cooled to 0? C. and 2.20 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.) was added drop wise. The yellowish solution was allowed to warm to room temperature and stirred for further 30 minutes. The respective alcohol, dissolved in 5.56 ml 1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol) was added and the white suspension was stirred over night at room temperature. The reaction mixture was directly placed on a silica gel flash column.

Di-(ethyl disulfido)ethyl) ethynylphosphonite

[0552] ##STR00179##

[0553] The compound was synthesized according to the above General procedure 2 for the synthesis of O-substituted alkynyl phosphonites from bis(diisopropylamino)chlorophosphine from 116 mg bis(diisopropylamino)chlorophosphine (0.44 mmol, 1.00 eq.), 0.96 ml ethynylmagnesium bromide solution (0.5 M in THF, 0.48 mmol, 1.10 eq.), 150 mg 2-Hydroxyethyl ethyldisulfide (1.10 mmol, 2.50 eq.), 2.42 ml 1H-tetrazole solution (0.45 M in MeCN, 1.10 mmol, 2.50 eq.) and purified by flash column chromatography on silicagel (10% to 20% EtOAc in hexane). The compound was obtained as yellowish oil. (112 mg, 0.34 mmol, 77.0%).

[0554] .sup.1H NMR (300 MHz, Chloroform-d) ?=4.22 (dt, J=7.6, 6.8, 4H), 3.15 (d, J=2.3, 1H), 2.95 (t, J=6.8, 4H), 2.75 (q, J=7.3, 4H), 1.35 (t, J=7.3, 6H). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=130.46.

Di-(2-isopropyl disulfido)ethyl) ethynylphosphonite

[0555] ##STR00180##

[0556] The compound was synthesized according to the above General procedure 2 for the synthesis of O-substituted alkynyl phosphonites from bis(diisopropylamino)chlorophosphine from 213 mg bis(diisopropylamino)chlorophosphine (0.80 mmol, 1.00 eq.), 1.76 ml ethynylmagnesium bromide solution (0.5 M in THF, 0.88 mmol, 1.10 eq.), 370 mg 2-Hydroxyethyl isopropyldisulfide (2.00 mmol, 2.50 eq.), 4.44 ml 1H-tetrazole solution (0.45 M in MeCN, 2.00 mmol, 2.50 eq.) and purified by flash column chromatography on silicagel (10% EtOAc in hexane). The compound was obtained as yellowish oil. (183 mg, 0.51 mmol, 63.9%).

[0557] .sup.1H NMR (300 MHz, Chloroform-d) ?=4.21 (dt, J=8.0, 6.8, 4H), 3.15 (d, J=2.3, 1H), 3.04 (p, J=6.7, 2H), 2.94 (t, J=6.8, 4H), 1.33 (d, J=6.7, 12H). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=130.40.

Di-(2-tert-butyl disulfido)ethyl) ethynylphosphonite

[0558] ##STR00181##

[0559] The compound was synthesized according to the above General procedure 2 for the synthesis of O-substituted alkynyl phosphonites from bis(diisopropylamino)chlorophosphine from 167 mg bis(diisopropylamino)chlorophosphine (0.63 mmol, 1.00 eq.), 1.38 ml ethynylmagnesium bromide solution (0.5 M in THF, 0.69 mmol, 1.10 eq.), 260 mg 2-Hydroxyethyl tert-butyldisulfide (1.57 mmol, 2.50 eq.), 3.48 ml 1H-tetrazole solution (0.45 M in MeCN, 1.57 mmol, 2.50 eq.) and purified by flash column chromatography on silicagel (10% EtOAc in hexane). The compound was obtained as yellowish oil. (190 mg, 0.49 mmol, 78.5%).

[0560] .sup.1H NMR (300 MHz, Chloroform-d) ?=4.20 (dt, J=7.9, 6.9, 4H), 3.14 (d, J=2.2, 1H), 2.95 (t, J=6.9, 4H), 1.36 (s, 18H). .sup.13C NMR (75 MHz, Chloroform-d) ?=92.35 (d, J=1.0), 84.21 (d, J=47.8), 66.37 (d, J=6.1), 47.98, 40.77 (d, J=4.3), 29.89. .sup.31P NMR (122 MHz, CDCl.sub.3) ?=130.28.

Di-((2-isopropyl disulfido)-3-propyl) ethynyiphosphonite

[0561] ##STR00182##

[0562] The compound was synthesized according to the above General procedure 2 for the synthesis of O-substituted alkynyl phosphonites from bis(diisopropylamino)chlorophosphine from 267 mg bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.), 2.20 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.), 415 mg 2-(3-Hydroxypropyl) isopropyl disulfide (2.50 mmol, 2.50 eq.), 5.55 ml 1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol, 2.50 eq.) and purified by flash column chromatography on silicagel (0-10% EtOAc in hexane). The compound was obtained as a diastereomeric mixture as yellowish oil. (91 mg, 0.235 mmol, 23.5%).

[0563] .sup.1H NMR (300 MHz, Chloroform-d) ?=4.28-4.06 (m, 2H), 3.99-3.81 (m, 2H), 3.21-3.10 (m, 1H), 3.07-2.95 (m, 4H), 1.37-1.28 (m, 18H). .sup.13C NMR (75 MHz, Chloroform-d) ?=92.39 (d, J=3.6), 84.32 (d, J=49.3), 71.18 (d, J=4.9), 48.18-44.79 (m), 41.65, 22.55 (d, J=6.5), 17.12. .sup.31P NMR (122 MHz, CDCl.sub.3) ?=130.56, 130.32, 130.10.

Di-(4-acetoxy benzyl) ethynylphosphonite

[0564] ##STR00183##

[0565] The compound was synthesized according to the above General procedure 2 for the synthesis of O-substituted alkynyl phosphonites from bis(diisopropylamino)chlorophosphine from 267 mg bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 eq.), 2.20 ml ethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 eq.), 415 mg 2-(3-Hydroxypropyl) isopropyl disulfide (2.50 mmol, 2.50 eq.), 5.55 ml 1H-tetrazole solution (0.45 M in MeCN, 2.50 mmol, 2.50 eq.) and purified by flash column chromatography on silicagel (30% EtOAc in hexane). The compound was obtained as a as colourless oil. (118 mg, 0.306 mmol, 30.6%).

[0566] .sup.1H NMR (300 MHz, Chloroform-d) ?=7.34 (d, J=8.5, 4H), 7.08 (d, J=8.5, 4H), 4.95 (dd, J=8.4, 1.7, 4H), 3.20 (d, J=2.3, 1H), 2.32 (s, 6H). .sup.13C NMR (75 MHz, Chloroform-d) b=169.44, 150.37, 135.31 (d, J=4.3), 128.89, 121.68, 92.68, 84.40 (d, J=47.6), 69.35 (d, J=6.8), 21.16. .sup.31P NMR (122 MHz, CDCl.sub.3) ?=131.09.

Di (4-(diazophenyl)-benzyl) ethynyiphosphonite

[0567] ##STR00184##

[0568] The compound was synthesized according to the above General procedure 2 for the synthesis of O-substituted alkynyl phosphonites from bis(diisopropylamino)chlorophosphine from 98 mg bis(diisopropylamino)chlorophosphine (0.37 mmol, 1.00 eq.), 0.80 ml ethynylmagnesium bromide solution (0.5 M in THF, 0.4 mmol, 1.10 eq.), 195 mg 1-(4-(hydroxymethyl)phenyl)-2-phenyldiazene (0.93 mmol, 2.50 eq.), 2.00 ml 1H-tetrazole solution (0.45 M in MeCN, 0.93 mmol, 2.50 eq.) and purified by flash column chromatography on silicagel (0-10% EtOAc in hexane). The compound was obtained as orange solid. (82 mg, 0.171 mmol, 46.3%).

[0569] .sup.1H NMR (300 MHz, Chloroform-d) ?=7.98-7.86 (m, 8H), 7.59-7.44 (m, 10H), 5.08 (d, J=8.5, 4H), 3.24 (d, J=2.3, 1H). .sup.13C NMR (75 MHz, Chloroform-d) ?=152.60, 152.27, 140.55 (d, J=4.3), 131.07, 129.09, 128.24, 123.03, 122.90, 92.89, 84.35 (d, J=47.2), 69.54 (d, J=6.9). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=131.77.

General Procedure 3 for the Synthesis of O-Substituted Alkynyl Phosphonamidates from Alkynyl Phosphonites and Azides

[0570] ##STR00185##

[0571] 1.00 mmol of an organic azide (1.00 eq.) was stirred together with 1.00 mmol of an alkynyl phosphonite (1.00 eq.) in 5 ml DMF overnight. The organic solvent was removed under educed pressure and the residue purified by column chromatographie on silica.

2-Isopropyl-disulfido-ethyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate

[0572] ##STR00186##

[0573] The compound was synthesized according to the above General procedure 3 for the synthesis of O-substituted alkynyl phosphonamidates from alkynyl phosphonites and azides from 147 mg Di-(2-isopropyl disulfido)ethyl) ethynylphosphonite (0.411 mmol, 1.00 eq.) and 106 mg 4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.411 mmol, 1.00 eq.) and purified by flash column chromatography on silicagel (60% EtOAc in hexane). The compound was obtained as colourless oil. (80 mg, 0.175 mmol, 42.6%).

[0574] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.08 (d, J=8.7, 2H), 7.20 (d, J=8.8, 2H), 7.13 (d, J=7.5, 1H), 4.63-4.18 (m, 2H), 3.23-2.76 (m, 8H), 1.31 (dd, J=6.7, 1.0, 6H). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=?10.16.

2-tert-butyl-disulfido-ethyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate

[0575] ##STR00187##

[0576] The compound was synthesized according to the above General procedure 3 for the synthesis of O-substituted alkynyl phosphonamidates from alkynyl phosphonites and azides from 50 mg Di-(2-tert-butyl disulfido)ethyl) ethynylphosphonite (0.129 mmol, 1.00 eq.) and 33 mg 4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.129 mmol, 1.00 eq.) and purified by flash column chromatography on silicagel (70% EtOAc in hexane). The compound was obtained as colourless solid. (29 mg, 0.0638 mmol, 47.4%).

[0577] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.06 (d, J=8.7, 2H), 7.49 (d, J=7.5, 1H), 7.21 (d, J=8.7, 2H), 4.58-4.26 (m, 2H), 3.09-2.81 (m, 7H), 1.33 (s, 9H). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=?9.98.

2-isopropyl disulfido-3-propyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate

[0578] ##STR00188##

[0579] The compound was synthesized according to the above General procedure 3 for the synthesis of O-substituted alkynyl phosphonamidates from alkynyl phosphonites and azides from 61 mg Di-((2-isopropyl disulfido)-3-propyl) ethynylphosphonite (0.158 mmol, 1.00 eq.) and 40 mg 4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.158 mmol, 1.00 eq.) and purified by flash column chromatography on silicagel (70% EtOAc in hexane). The compound was obtained as mixture of diastereomers as colourless oil. (32 mg, 0.068 mmol, 43.0%).

[0580] .sup.1H NMR (300 MHz, Chloroform-d) ?=8.05 (d, J=8.7, 2H), 7.84-7.74 (m, 1H), 7.21 (d, J=8.8, 2H), 4.57-4.27 (m, 2H), 3.20-2.66 (m, 7H), 1.46-1.21 (m, 9H). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=?9.87.

4-acetoxy-benzyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate

[0581] ##STR00189##

[0582] The compound was synthesized according to the above General procedure 3 for the synthesis of O-substituted alkynyl phosphonamidates from alkynyl phosphonites and azides from 103 mg Di-(4-acetoxy benzyl) ethynylphosphonite (0.267 mmol, 1.00 eq.) and 69 mg 4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.267 mmol, 1.00 eq.) and purified by flash column chromatography on silicagel (70% EtOAc in hexane). The compound was obtained as colourless oil. (36 mg, 0.077 mmol, 28.7%).

[0583] .sup.1H NMR (300 MHz, Chloroform-d) ?=7.53-7.34 (m, 2H), 7.20-6.99 (m, 7H), 5.14 (d, J=8.8, 2H), 3.01 (d, J=13.3, 1H), 2.91 (s, 4H), 2.32 (s, 3H). .sup.31P NMR (122 MHz, CDCl.sub.3) ?=?10.33.

4-Diazophenyl-benzyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate

[0584] ##STR00190##

[0585] The compound was synthesized according to the above General procedure 3 for the synthesis of O-substituted alkynyl phosphonamidates from alkynyl phosphonites and azides from 71 mg Di (4-(diazophenyl)-benzyl) ethynylphosphonite (0.148 mmol, 1.00 eq.) and 39 mg 4-azidobenzoic-acid-N-hydroxysuccinimide ester (0.148 mmol, 1.00 eq.) and purified by flash column chromatography on silicagel (50% EtOAc in hexane). The compound was obtained as orange solid. (58 mg, 0.112 mmol, 75.8%).

[0586] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=9.44 (d, J=8.6, 1H), 8.02 (d, J=8.8, 2H), 7.96-7.89 (m, 4H), 7.67 (d, J=8.5, 2H), 7.64-7.57 (m, 3H), 7.33 (d, J=8.8, 2H), 5.28 (ddd, J=45.1, 12.5, 8.7, 2H), 4.61 (d, J=13.0, 1H), 2.88 (s, 4H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) ?=170.90, 161.75, 152.36, 152.23, 147.36, 139.31 (d, J=7.6), 132.33, 132.17, 129.97, 129.41, 123.14, 123.08, 118.17 (d, J=8.1), 117.25, 93.06 (d, J=46.9), 76.49 (d, J=265.4), 66.88, 25.98. .sup.31P NMR (243 MHz, DMSO) ?=?10.42.

General Procedure 4 for the Amide Bond Formation Between Phosphonamidate-NHS Esters and EDANS

[0587] ##STR00191##

[0588] 0.1 mmol NHS-phosphonamidate (1.00 eq.) and 0.12 mmol 5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (1.20 eq.) were dissolved in 10 mL DMF. 0.40 mmol of DIPEA (4.0 eq.) was added and the mixture stirred for 3 hours at room-temperature. All volatiles were removed under reduced pressure and the crude mixture was purified by preperative HPLC using method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond.

5-((2-(O-(2-Isopropyl-disulfido-ethyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino) naphthalene-1-sulfonic acid

[0589] ##STR00192##

[0590] The compound was synthesized according to the above General procedure 4 for the amide bond formation between phosphonamidate-NHS esters and EDANS from 72 mg 2-Isopropyl-disulfido-ethyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate (0.157 mmol, 1.00 eq.), 54 mg 5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.188 mmol, 1.20 eq.) and 109 ?l DIPEA (0.628 mmol, 4.0 eq.) and purified by semi-preperative HPLC (method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). The compound was obtained as white solid. (62 mg, 0.102 mmol, 64.9%).

[0591] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.90 (d, J=8.8, 1H), 8.61 (t, J=5.6, 1H), 8.56 (d, J=8.6, 1H), 8.13 (d, J=8.3, 1H), 8.04 (dd, J=7.2, 1.1, 1H), 7.82 (d, J=8.7, 2H), 7.60-7.31 (m, 2H), 7.17 (d, J=8.8, 2H), 4.51 (d, J=12.8, 1H), 4.37-4.17 (m, 2H), 3.65 (q, J=6.3, 2H), 3.52 (t, J=6.5, 2H), 3.07 (p, J=6.7, 1H), 3.03 (t, J=6.3, 2H), 1.24 (dd, J=6.7, 2.8, 6H). 13C NMR (151 MHz, DMSO-d.sub.6) ?=167.03, 144.53, 143.31, 130.53, 129.02, 127.63, 126.33, 125.44, 125.15, 124.70, 123.21, 117.55, 117.50, 92.38 (d, J=45.7), 76.79 (d, J=262.5), 63.85 (d, J=4.7), 46.85, 40.77, 39.01 (d, J=7.7), 37.65, 22.72. .sup.31P NMR (243 MHz, DMSO) ?=?9.84.

5-((2-(O-(2-tert-butyl-disulfido-ethyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino) naphthalene-1-sulfonic acid

[0592] ##STR00193##

[0593] The compound was synthesized according to the above General procedure 4 for the amide bond formation between phosphonamidate-NHS esters and EDANS from 10 mg 2-tert-butyl-disulfido-ethyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate (0.021 mmol, 1.00 eq.), 7 mg 5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.025 mmol, 1.20 eq.) and 15 ?l DIPEA (0.084 mmol, 4.0 eq.) and purified by semi-preperative HPLC (method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). The compound was obtained as white solid. (8 mg, 0.013 mmol, 62.3%).

[0594] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.87 (d, J=8.7, 1H), 8.57 (t, J=5.8, 1H), 8.21 (d, J=8.6, 1H), 8.10 (d, J=8.5, 1H), 7.94 (dd, J=7.1, 1.2, 1H), 7.80 (d, J=8.7, 2H), 7.36 (dd, J=8.5, 7.1, 1H), 7.31 (dd, J=8.7, 7.5, 1H), 7.14 (d, J=8.8, 2H), 6.74 (d, J=7.6, 1H), 4.49 (d, J=12.8, 1H), 4.37-4.11 (m, 2H), 3.60 (q, J=6.4, 2H), 3.40 (t, J=6.5, 2H), 3.03 (t, J=6.5, 2H), 1.29 (s, 9H). .sup.31P NMR (243 MHz, DMSO) ?=?9.87.

5-((2-(O-2-isopropyl disulfido-3-propyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonic acid

[0595] ##STR00194##

[0596] The compound was synthesized according to the above General procedure 4 for the amide bond formation between phosphonamidate-NHS esters and EDANS from 29 mg 2-isopropyl disulfido-3-propyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate (0.061 mmol, 1.00 eq.), 21 mg 5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.073 mmol, 1.20 eq.) and 42 ?l DIPEA (0.244 mmol, 4.0 eq.) and purified by semi-preperative HPLC (method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). The compound was obtained as a mixture of diastereomers as white solid. (15 mg, 0.024 mmol, 39.5%).

[0597] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.88 (d, J=8.8, 1H), 8.58 (t, J=5.7, 1H), 8.35 (d, J=8.6, 1H), 8.10 (dt, J=8.6, 1.1, 1H), 7.98 (dd, J=7.1, 1.1, 1H), 7.81 (d, J=8.7, 2H), 7.39 (ddd, J=31.3, 8.6, 7.3, 2H), 7.15 (d, J=8.8, 2H), 6.91 (d, J=7.5, 1H), 4.51 (dd, J=12.9, 1.8, 1H), 4.25-4.13 (m, 1H), 4.13-3.98 (m, 1H), 3.61 (q, J=6.4, 2H), 3.45 (t, J=6.6, 2H), 3.18 (dtd, J=10.6, 6.8, 5.2, 1H), 3.03 (h, J=6.7, 1H), 1.30-1.19 (m, 9H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) ?=166.92, 144.74, 143.21, 130.61, 128.96, 127.79, 126.43, 125.10, 124.61, 123.82, 123.09, 117.50 (d, J=7.5), 92.58 (d, J=9.5), 92.28 (d, J=9.4), 76.76 (d, J=262.3), 68.10 (d, J=4.9), 45.48, 41.32 (d, J=8.6), 38.15, 22.74, 17.14 (d, J=4.1). .sup.31P NMR (243 MHz, DMSO) ?=?9.76, ?9.79.

5-((2-(O-(4-acetoxy benzyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonic acid

[0598] ##STR00195##

[0599] The compound was synthesized according to the above General procedure 4 for the amide bond formation between phosphonamidate-NHS esters and EDANS from 36 mg 4-acetoxy-benzyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate (0.076 mmol, 1.00 eq.), 22 mg 5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.095 mmol, 1.20 eq.) and 53 ?l DIPEA (0.284 mmol, 4.0 eq.) and purified by semi-preperative HPLC (method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). The compound was obtained as white solid. (14 mg, 0.023 mmol, 30.4%).

[0600] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.92 (d, J=8.6, 1H), 8.56 (t, J=5.7, 1H), 8.32 (d, J=8.7, 1H), 8.10 (d, J=8.5, 1H), 8.03-7.92 (m, 1H), 7.80 (d, J=8.6, 2H), 7.47 (d, J=8.5, 2H), 7.41 (dd, J=8.5, 7.2, 1H), 7.36 (t, J=8.1, 1H), 7.17 (d, J=6.7, 2H), 7.15 (d, J=6.7, 2H), 6.88 (d, J=7.5, 1H), 5.25-5.05 (m, 2H), 4.49 (d, J=12.8, 1H), 3.61 (q, J=6.3, 2H), 3.44 (t, J=6.6, 2H), 2.28 (s, 3H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) ?=169.61, 166.95, 150.96, 144.73, 143.29, 133.66 (d, J=7.7), 130.61, 129.81, 128.99, 127.82, 126.47, 125.06, 124.53, 123.68, 123.10, 122.42, 117.44 (d, J=7.9), 92.28 (d, J=45.6), 77.01 (d, J=261.8), 66.59 (d, J=4.4), 45.26, 38.24, 21.31. .sup.31P NMR (243 MHz, DMSO) ?=?9.87.

5-((2-(O-(4-Diazophenyl-benzyl)-P-ethynyl-phosphonamidato-N-benzoyl)ethyl)amino)naphthalene-1-sulfonic acid

[0601] ##STR00196##

[0602] The compound was synthesized according to the above General procedure 4 for the amide bond formation between phosphonamidate-NHS esters and EDANS from 27 mg 4-Diazophenyl-benzyl-N-(4-benzoic-acid-N-hydroxysuccinimide ester)-P-ethynyl phosphonamidate (0.053 mmol, 1.00 eq.), 15 mg 5-((2-Aminoethyl)aminonaphthalene-1-sulfonate sodium salt (0.064 mmol, 1.20 eq.) and 37 ?l DIPEA (0.212 mmol, 4.0 eq.) and purified by semi-preperative HPLC (method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). The compound was obtained as orange solid. (18 mg, 0.027 mmol, 50.9%).

[0603] .sup.1H NMR (600 MHz, DMSO-d.sub.6) ?=8.98 (d, J=8.7, 1H), 8.57 (t, J=5.7, 1H), 8.36 (d, J=8.6, 1H), 8.11 (d, J=8.5, 1H), 7.98 (d, J=7.1, 1H), 7.97-7.88 (m, 4H), 7.81 (d, J=8.8, 2H), 7.66 (d, J=8.5, 2H), 7.64-7.52 (m, 4H), 7.42 (dd, J=8.5, 7.1, 1H), 7.37 (t, J=8.1, 1H), 7.18 (d, J=8.7, 2H), 6.92 (d, J=7.5, 1H), 5.37-5.04 (m, 2H), 4.53 (d, J=12.8, 1H), 3.61 (q, J=6.4, 2H), 3.45 (t, J=6.6, 2H). .sup.13C NMR (151 MHz, DMSO-d.sub.6) ?=166.95, 152.36, 152.18, 144.75, 143.27, 139.56, 139.51, 132.15, 130.61, 129.97, 129.32, 129.01, 127.84, 126.44, 125.11, 124.62, 123.83, 123.13, 123.07, 117.49 (d, J=8.0), 92.47 (d, J=45.9), 76.95 (d, J=262.7), 66.56 (d, J=4.4), 45.47, 38.15. .sup.31P NMR (243 MHz, DMSO) ?=?9.68.

General Procedure 5 for the Addition of a Cys-Model Peptide to Different O-Substituted EDANS Phosphonamidates

[0604] ##STR00197##

[0605] Equal volumes of a 5 mM solution of the respective EDANS-phosphonamidate in DMF and a 5 mM solution of the above stated DABCYL-Modified Cys-peptide in 100 mM NH.sub.4HCO.sub.3-Buffer (pH8.5) were freshly prepared, mixed and shaken at room temperature for 1 h. All volatiles were removed under reduced pressure and the thiol adducts isolated by semi-preperative HPLC (method E described above under Procedures for the introduction of the alkyne-phosphonamidate moiety by generic building blocks via an amide bond). Isolated conjugates were analyzed by HPLC-MS as set out in the following Table 5 and FIG. 25:

TABLE-US-00005 TABLE 5 Isolated R HPLC trace Mass analysis Yield [00198] embedded image see FIG 25A C.sub.74H.sub.96N.sub.15O.sub.20PS.sub.4.sup.2+ [M + 2H].sup.2+ calcd: 836.78, found: 836.94 39.4% [00199] see FIG 25B C.sub.75H.sub.98N.sub.15O.sub.20PS.sub.4.sup.2+ [M + 2H].sup.2+ calcd: 843.77, found: 844.10 44.7% [00200] see FIG 25C C.sub.75H.sub.98N.sub.15O.sub.20PS.sub.4.sup.2+ [M + 2H].sup.2+ calcd: 843.77, found: 844.05 24.5% [00201] embedded image see FIG 25D C.sub.78H.sub.94N.sub.15O.sub.22PS.sub.2.sup.2+ [M + 2H].sup.2+ calcd: 843.79, found: 844.13 26.1% [00202] see FIG 25E C.sub.82H.sub.96N.sub.17O.sub.20PS.sub.2.sup.2+ [M + 2H].sup.2+ calcd: 866.81, found: 867.12 Product not isolated

Procedure for the Cleavage of the Disulfide Containing Amidate-Adducts with TCEP

[0606] ##STR00203##

[0607] 10 ?l of a 1 mM stock solution of the respective peptide (SM1-3) in phosphate buffered saline (PBS) was premixed with 80 ?l of PBS. 10 ?l of a 10 mM stock solution of Tris-(2-carboxyethyl)-phosphin (TCEP) in PBS was added and the solutions were shaken at 37? C. for one hour. 15 ?l samples were drawn afterwards, diluted with 15 ?l of 2% trifuloroacetic acid (TFA) solution in water and subjected to UPLC-MS analysis. The UPLC-MS analysis is depicted in FIG. 26A-C. Red line shows incubation with TCEP, black with PBS only. Peaks were identified by MS. The results show that the disulfide-containing O-substituents are cleaved and the EDANS-containing part is liberated from the starting materials.

Procedure for the Cleavage of the Ester-Containing Amidate-Adducts with Cell Lysate

[0608] ##STR00204##

[0609] 10 ?l of a 1 mM stock solution of the peptide SM4 in PBS was premixed with 90 ?l of freshly prepared HeLa-lysate in PBS. The solutions was shaken at 37? C. for one hour. A 15 ?l sample was drawn afterwards, diluted with 15 ?l of 2% TFA solution in water and subjected to UPLC-MS analysis. The UPLC-MS analysis is depicted in FIG. 27. Red line shows incubation with cell lysate, black with PBS only. Peaks were identified by MS. The results show that the O-substituent on the phosphorus comprising an ester moiety is cleaved and the EDANS-containing part is liberated from the starting material.

Procedure for the Diazo-Containing Amidate-Adducts with Sodium Dithionite

[0610] ##STR00205##

[0611] 10 ?l of a 1 mM stock solution of the peptide SM5 in PBS was premixed with 80 ?l of PBS. 10 ?l of a 200 mM stock solution of TCEP in PBS was added and the solutions were shaken at 37? C. for one hour. 15 ?l samples were drawn afterwards, diluted with 15 ?l of 2% TFA solution in water and subjected to UPLC-MS analysis. The UPLC-MS analysis is depicted in FIG. 28. Red line shows incubation with TCEP, black with PBS only. Peaks were identified by MS. The results show that the O-substituent on the phosphorus comprising a diazo moiety is cleaved and the EDANS-containing part is liberated from the starting material.

[0612] Thus, it has been demonstrated that a cleavage of the amidates having various cleavable groups as O substituent on the phosphorus is possible.

[0613] Without wishing to be bound by any theory, for a disulfide-containing group on the phosphorus it is believed that the mechanism of the cleavage proceeds as exemplarily depicted in Scheme 31, i.e. through reductive cleavage of the disulfide, cyclisation to a thiirane to generate a free phosphonamidic acid which undergoes PN-hydrolysis to liberate the payload as a free amine.

##STR00206##

Disulfide Substituted Phosphonites for Protein Conjugation

[0614] The cyclic cell-penetrating peptide c(Tat) was conjugated to eGFP via the Staudinger induced thiol addition with a disulfide substituted phosphonite.

[0615] First, we synthesized the cyclic Tat-peptide via solid phase peptide synthesis (SPPS) (see Scheme 32). By capping the N-terminus with 4-azidobenzoic acid we obtained compound E11 having an azide moiety. After purification by preparative HPLC the Staudinger phosphonite reaction of E11 with the disulfide containing alkyne phosphonites was carried out in DMF to give compounds E12 and E13, which were purified again by preparative H PLC.

##STR00207##

[0616] With the alkyne functionalized peptides in hand we further tested the thiol addition towards a cysteine containing eGFP as shown in FIG. 29. The eGFP C70M S147C is a mutant, which exhibits only two cysteines of which only one is addressable.

[0617] For the tert-butyl-disulfide substituent (E13) the thiol addition reaction went to completion after incubating eGFP with 6 equivalents phosphonite in PBS at 37? C. for 16 hours at a proteinconcentration of 63 ?M. When applying the same reaction conditions with the isopropyl-disulfide substituent (E12) the product was obtained in about 50% conversion according to MALDI analysis as shown in FIG. 29.

[0618] FIG. 29 shows: Thiol addition of alkyne-c(Tat) to eGFP C70M S147C.

Procedures for the Disulfide Substituted Phosphonites for Protein Conjugation

Synthesis of c(Tat)-Azide

[0619] The c(Tat) was synthesized in a 0.1 mmol scale on a Rink Amide Resin with a loading of 0.78 mm/g. The synthesis was carried out on a PTI synthesizer with single couplings of each amino acid (10 eq. amino acid for 40 min) in DMF. After the final building block coupling the peptide, still Fmoc protected, was treated with Pd(PPh.sub.3).sub.4 (24 mg, 20 ?mol, 20 mol %) and Phenylsilane (308 ?l, 2.5 mmol, 2.5 eq.) in 4 ml dry DCM for 1 hour in order to cleave the alloc and allyl protecting groups in one step. After confirmation of full deprotection by test cleavage, cyclization with 2 eq. HATU 4 eq. DIPEA was carried out over night in DMF.

[0620] The peptide was then Fmoc-deprotected using 20% Piperidine in DMF and the 4-azidobenzoic acid (81.6 mg, 0.5 mmol, 5 eq.) was coupled to the N-terminus with HATU (190.1 mg, 0.5 mmol, 5 eq.) and DIPEA (170 ?l, 1.0 mmol, 10 eq.) for 1 hour. Finally the peptide was cleaved from the resin by treatment with 4 ml of a TFA:TIS:H.sub.2O (95:2.5:2.5) for 3 hours and precipitated in cold diethylether. The crude peptide was purified by preparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)). The product was gained as white powder (30.0 mg, 11.4 ?mol, 11.4% yield) and was analyzed by analytical UPLC (5 to 95% of acetonitrile in water containing 0.1% TFA on a RP-C18 column). LRMS: m/z: 648.49 [M+3H].sup.3+ (calcd. m/z: 648.0569).

Synthesis of c(Tat)-Phosphonamidate Alkyne: Staudinger Reaction on c(Tat)-Azide

[0621] The purified c(Tat)-azido peptide (5 mg, 1.9 ?mol, 1 eq.) was reacted with both disulfide substituted phosphonites according to the general protocol. The crude peptide was purified by preparative reverse phase C18 HPLC. The product was gained as white powder and was analyzed by MALDI-TOF.

Hydrothiolation of Electron-Deficient c(Tat)-Phosphonamidate Alkyne

[0622] eGFP C70M S147C (2.7 nmol, 1 eq) in PBS was concentrated to 40 ?l and c(Tat)-phosphonamidate alkyne (0.05 mg, 16.2 nmol, 6 eq.) was added. After the reaction mixture was shaken at 37? C. and 800 rpm over night it was purified by ZebaSpin filters with a MWCO of 7 kDa. The product was analyzed by MALDI-TOF. For the conjugation of peptide E12 an approximately 50% conversion to the product was observed, while in contrast the conjugation of peptide E13 gave a full conversion.

[0623] MALDI TOF for E14: expected Product (in Da): 29919 (M+H.sup.+), 14960 (M+2H.sup.+); found (in Da): 29933 (M+H.sup.+), 14967 (M+2H.sup.+)

[0624] MALDI TOF for E15: expected Product (in Da): 29933 (M+H.sup.+), 14967 (M+2H.sup.+); found (in Da): 29940 (M+H.sup.+), 14965 (M+2H.sup.+)

Intramolecular Staudinger Induced Thiol Addition for Peptide Cyclization

[0625] The incorporation of an azide as well as a thiol into a complex molecule, e.g. a peptide, leads the way for the intramolecular staudinger induced thiol addition, that can realize an intramolecular cyclization as shown in the following scheme:

##STR00208##

[0626] Without wishing to be bound by any theory, it is assumed that first the azide is reacting with the electron-rich alkyne/alkene-phosphonite upon which the phosphonamidate is formed and an electron-poor alkyne/alkene-phosphonamidate is formed that undergoes a fast intramolecular thiol addition with the cysteine in the peptide structure.

[0627] First we synthesized a peptide taken from the protein sequence of BCL-9 and we incorporated an azidohomoalanine and a cysteine distanced by three amino acids into the peptide by standard solid phase peptide synthesis. After cleavage from the solid phase and purification by preparative HPLC we gained the peptide. With this in hand we could probe the intramolecular cyclization by staudinger induced thiol addition.

[0628] We reacted the in dry DMSO solubilized peptide with either diethyl-ethynylphosphonite or diethyl-vinylphosphonite for 24 hours. After preparative HPLC the cyclized peptide was gained, which was confirmed by Ellman's test.

Procedures for the Intramolecular Staudinger Induced Thiol Addition for Peptide Cyclization

Synthesis of BCL9-Azide

[0629] ##STR00209##

[0630] The BCL9-azide was synthesized in a 0.1 mmol scale on a Rink Amide Resin with a loading of 0.78 mm/g. The synthesis was carried out on a PTI synthesizer with single couplings of each amino acid (5 eq. amino acid for 40 min) in DMF. Finally the peptide was cleaved from the resin by treatment with 4 ml of a TFA:TIS:H.sub.2O (95:2.5:2.5) for 2 hours and precipitated in cold diethylether. The crude peptide was purified by preparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)). The product was gained as white powder (35.0 mg, 11.5 ?mol, 11.5% yield) and was analyzed by analytical UPLC (5 to 95% of acetonitrile in water containing 0.1% TFA on a RP-C18 column). LRMS: m/z: [M+3H].sup.3+ 759.86 (calcd. m/z: 759.6590).

Intramolecular Staudinger Induced Thiol Addition

Alkyne-Phosphonamidate

[0631] ##STR00210##

Staudinger Reaction on BCL9-Azide

[0632] The peptide 1 (20 mg, 6.55 ?mol, 1 eq.) was dissolved in dry DMSO (1.5 ml, 4.4 mM). After drying under high vacuum in a previously flame dried flask the Bisethoxyalkyne-phosphonite was given to the reaction mixture (volume according to percentage of product determined by NMR, 39.3 ?mol, 6 eq.). The reaction mixture was heated to 50? C. and stirred for 24 hours. After addition of water, the reaction mixture was purified via basic (10 mM ammonium acetate buffer pH 9.0/MeCN) semi-preparative reverse phase C18 Nucleodur HPLC (0-5 min 95/5, Buffer/MeCN; 5-70 min 10/90, Buffer/MeCN) and gave the cyclized product as a white powder (3.82 mg, 1.22 ?mol, 18.7% overall yield). The product was further analyzed with an Ellman's test which showed that 97% of the cysteine was reacted. The final product 2 was analyzed by LC-UV: rt. 5.0 min (0-1 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 1-16.5 min 5/95, water (0.1% TFA)/MeCN (0.1% TFA) on RP-C18 column) and mass. LRMS: m/z: [M+3H].sup.3+ 1049.19 (calcd. m/z: 1048.5349).

Alkene-Phosphonamidate

[0633] ##STR00211##

Staudinger Reaction on BCL9-Azide

[0634] The peptide 3 (34 mg, 11.55 ?mol, 1 eq.) was dissolved in dry DMSO (4 ml, 2.9 mM). After drying under high vacuum in a previously flame dried flask the Bisethoxyvinyl-phosphonite was given to the reaction mixture (volume according to percentage of product determined by NMR, 39.3 ?mol, 6 eq.). The reaction mixture stirred for 24 hours at room temperature. After addition of water, the reaction mixture was purified by preparative reverse phase C18 HPLC (0-5 min 95/5, water (0.1% TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water (0.1% TFA)/MeCN (0.1% TFA)). The product was gained as white powder (14.9 mg, 4.8 ?mol, 41.3% yield) and was analyzed by analytical UPLC (5 to 95% of acetonitrile in water containing 0.1% TFA on a RP-C18 column). LRMS: m/z: [M+4H].sup.4+ 782.89 (calcd. m/z: 782.6660). The product 4 was further analyzed with an Ellman's test which showed that 99% of the cysteine was reacted.

REFERENCES

[0635] (1) Hackenberger, C. P. R.; Schwarzer, D. Angew. ChemieInt. Ed. 2008, 47 (52), 10030. [0636] (2) Spicer, C. D.; Davis, B. G. Nat. Commun. 2014, 5, 4740. [0637] (3) Sletten, E. M.; Bertozzi, C. R. Angew. ChemieInt. Ed. 2009, 48 (38), 6974. [0638] (4) Patterson, D. M.; Nazarova, L. A.; Prescher, J. A. ACS Chem. Biol. 2014, 9 (3), 592. [0639] (5) Lang, K.; Chin, J. W. ACS Chem. Biol. 2014, 9 (1), 16. [0640] (6) Nikic, I.; Plass, T.; Schraidt, O.; Szymaski, J.; Briggs, J. A. G.; Schultz, C.; Lemke, E. A. Angew. ChemieInt. Ed. 2014, 53 (8), 2245. [0641] (7) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126 (46), 15046. [0642] (8) Chalker, J. M.; Bernardes, G. J. L.; Lin, Y. A.; Davis, B. G. Chem.An Asian J. 2009, 4 (5), 630. [0643] (9) Hoesl, M. G.; Budisa, N. Angew. ChemieInt. Ed. 2011, 50 (13), 2896. [0644] (10) Artner, L. M.; Merkel, L.; Bohlke, N.; Beceren-Braun, F.; Weise, C.; Dernedde, J.; Budisa, N.; Hackenberger, C. P. R. Chem. Commun. 2012, 48 (4), 522. [0645] (11) van Kasteren, S. I.; Kramer, H. B.; Jensen, H. H.; Campbell, S. J.; Kirkpatrick, J.; Oldham, N. J.; Anthony, D. C.; Davis, B. G. Nature 2007, 446 (7139), 1105. [0646] (12) Witte, C.; Martos, V.; Rose, H. M.; Reinke, S.; Klippel, S.; Schr?der, L.; Hackenberger, C. P. R. Angew. ChemieInt. Ed. 2015, 54 (9), 2806. [0647] (13) Vall?e, M. R. J.; Artner, L. M.; Dernedde, J.; Hackenberger, C. P. R. Angew. ChemieInt. Ed. 2013, 52 (36), 9504. [0648] (14) Vall?e, M. R. J.; Majkut, P.; Wilkening, I.; Weise, C.; Miller, G.; Hackenberger, C. P. R. Org. Lett. 2011, 13 (20), 5440. [0649] (15) Vall?e, M. R. J.; Majkut, P.; Krause, D.; Gerrits, M.; Hackenberger, C. P. R. Chemistry 2015, 21 (3), 970. [0650] (16) Pfaff, M.; Tangemann, K.; Miller, B.; Gurrath, M.; Miller, G.; Kessler, H.; Timpl, R.; Engel, J. J. Biol. Chem. 1994, 269 (32), 20233. [0651] (17) Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. a; Cerveny, C. G.; Chace, D. F.; DeBlanc, R. L.; Gearing, R. P.; Bovee, T. D.; Siegall, C. B.; Francisco, J. a; Wahl, A. F.; Meyer, D. L.; Senter, P. D. Nat. Biotech. 2003, 21 (7), 778. [0652] (18) Ortial, S.; Montchamp, J. L. Org. Lett. 2011, 13 (12), 3134. [0653] (19) Park, C.; Lee, K.; Kim, C. Angew. ChemieInt. Ed. 2009, 48 (7), 1275. [0654] (20) Barltrop, J. A.; Plant, P. J.; Schofield, P. Chem. Commun. (London) 1966, 822. [0655] (21) Khorev, O.; Stokmaier, D.; Schwardt, O.; Cutting, B.; Ernst, B. Bioorganic Med. Chem. 2008, 16 (9), 5216 [0656] (22) Gunnoo, S. B.; Madder, A.; ChemBioChem. 2016, 17, 529-553 [0657] (23) T. K. Claus, S. Telitel, A. Welle, M. Bastmeyer, A. P. Vogt, G. Delaittre, C. Barner-Kowollik, Chem. Commun. (Cambridge, U. K.) 2017, 53, 1599-1602. [0658] (24) M. M. Dcona, D. Mitra, R. W. Goehe, D. A. Gewirtz, D. A. Lebman, M. C. T. Hartman, Chem. Commun. (Cambridge, U. K.) 2012, 48, 4755-4757. [0659] (25) Y. Chen, A. S. Kamlet, J. B. Steinman, D. R. Liu, Nat Chem 2011, 3, 146-153. [0660] (26) J. I. Degraw, J. S. Engstrom, Journal of Labelled Compounds 1975, 11, 233-239. [0661] (27) A. J. Perez, F. Wesche, H. Adihou, H. B. Bode, ChemistryA European Journal 2016, 22, 639-645. [0662] (28) H. D. Herce, D. Schumacher, A. F. L. Schneider, A. K. Ludwig, F. A. Mann, M. Fillies, M.-A. Kasper, S. Reinke, E. Krause, H. Leonhardt, M. C. Cardoso, C. P. R. Hackenberger, Nat Chem 2017, advance online publication. [0663] (29) S. O. Doronina, B. E. Toki, M. Y. Torgov, B. A. Mendelsohn, C. G. Cerveny, D. F. Chace, R. L. DeBlanc, R. P. Gearing, T. D. Bovee, C. B. Siegall, J. A. Francisco, A. F. Wahl, D. L. Meyer, P. D. Senter, Nat Biotech 2003, 21, 778-784. [0664] (30) A. Stengl, D. Horl, H. Leonhardt, J. Helma, Slas Discovery 2017, 22, 309-315. [0665] (31) a) T. H. Pillow, J. D. Sadowsky, D. Zhang, S.-F. Yu, G. Del Rosario, K. Xu, J. He, S. Bhakta, R. Ohri, K. R. Kozak, E. Ha, J. R. Junutula, J. A. Flygare, Chemical Science 2017, 8, 366-370; b) L. R. Staben, S. G. Koenig, S. M. Lehar, R. Vandlen, D. Zhang, J. Chuh, S.-F. Yu, C. Ng, J. Guo, Y. Liu, A. Fourie-O'Donohue, M. Go, X. Linghu, N. L. Segraves, T. Wang, J. Chen, B. Wei, G. D. L. Phillips, K. Xu, K. R. Kozak, S. Mariathasan, J. A. Flygare, T. H. Pillow, Nat Chem 2016, 8, 1112-1119. [0666] (32) a) A. Satyam, Bioorg. Med. Chem. Lett. 2008, 18, 3196-3199; b) L. W. C. Miles, L. N. Owen, Journal of the Chemical Society (Resumed) 1952, 817-826. [0667] (33) G.-M. Fang, J.-X. Wang, L. Liu, Angew. Chem., Int. Ed. 2012, 51, 10347-10350. [0668] (34) P. Fat?s, E. Longo, F. Rastrelli, M. Crisma, C. Toniolo, A. I. Jimenez, C. Cativiela, A. Moretto, ChemistryA European Journal 2011, 17, 12606-12611.

CHEMOSELECTIVE THIOL-CONJUGATION WITH ALKENE OR ALKYNE-PHOSPHONAMIDATES

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Abstract

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