SULFOMALEIMIDE-BASED LINKERS AND CORRESPONDING CONJUGATES

Abstract

The present invention relates to a linker of the following formula (I) or a salt thereof: (I). The present invention relates to a linker-drug conjugate of the following formula (II) or a salt thereof: (II). The present invention relates also to a binding unit-drug corrugate, such as an antibody-drug conjugate, of the following formula (III) or (IV) or a salt thereof: (III), (IV), as well as a pharmaceutical composition comprising such a binding unit-drug corrugate and its use in the treatment of cancer.

##STR00001##

Claims

1. A linker of the following formula (I): ##STR00161## or a salt thereof, wherein; X.sub.1 and X.sub.2 represent, independently of each other, H, a halogen atom, a (C.sub.1-C.sub.6)alkoxy, an optionally substituted aryloxy, or —O—(CH.sub.2CH.sub.2O).sub.rH, provided that X.sub.1 and X.sub.2 do not represent H at the same time; L.sub.1 represents a group of formula L.sub.1′-(CO—Z′).sub.z′ with L.sub.1′ being —(CH.sub.2).sub.n—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2—, arylene, heteroarylene, cycloalkanediyl, —(CH.sub.2).sub.n-arylene-, —(CH.sub.2).sub.n-heteroarylene-, —(CH.sub.2).sub.n-cycloalkanediyl-, -arylene-(CH.sub.2).sub.p—, -heteroarylene-(CH.sub.2).sub.p—, -cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-arylene-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-heteroarylene-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-arylene-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-heteroarylene-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-arylene-CH.sub.2—CH.sub.2—(OCH.sub.2CH.sub.2).sub.m—, —(CH.sub.2).sub.n-heteroarylene-CH.sub.2—CH.sub.2—(OCH.sub.2CH.sub.2).sub.m—, or —(CH.sub.2).sub.n-cycloalkanediyl-CH.sub.2—CH.sub.2—(OCH.sub.2CH.sub.2).sub.m—, each W independently represents an amino acid unit; Y is PAB-CO—(Z).sub.z—, with PAB being ##STR00162## the oxygen of the PAB unit being linked to CO—(Z).sub.z; Z is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—, —NR.sub.4—(CH.sub.2).sub.n—NR.sub.5—CO—, —NR.sub.4—(CH.sub.2).sub.n—NR.sub.5—CO—(CH.sub.2).sub.v—, or —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—(CH.sub.2).sub.v—CO—, the NR.sub.4 group being linked to the CO group of PAB-CO; Z′ is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5— or —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—(CH.sub.2).sub.v—, the NR.sub.4 group being linked to the CO group of CO—Z′; R.sub.4 and R.sub.5 are independently H or a (C.sub.1-C.sub.6)alkyl group, c is 0 or 1; m is an integer from 1 to 15; n is an integer from 1 to 6; p is an integer from 1 to 6; q is 0, 1 or 2; r is an integer from 1 to 21; u is an integer from 1 to 6; v is an integer from 1 to 6; w is an integer from 0 to 5; y is 0 or 1; z is 0 or 1; z′ is 0 or 1; and X.sub.3 represents H when y=z=1 and Z is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5— or when c=w=y=0, z′=1 and Z′ is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5— and in the other cases, X.sub.3 represents OH, NH.sub.2 or a leaving group, wherein the leaving group is a halogen atom, a sulfonate of formula —OSO.sub.2—R.sub.LG, N-succinimidyloxy, 4-nitro-phenyloxy, pentafluorophenoxy or N-benzotriazoloxy, R.sub.LG representing a (C.sub.1-C.sub.6)alkyl, aryl, aryl-(C.sub.1-C.sub.6)alkyl or (C.sub.1-C.sub.6)alkyl-aryl group, the said group being optionally substituted with one or several halogen atoms such as fluorine atoms, with the proviso that it is not a compound of formula (I) for which, X.sub.1 is Cl, X.sub.2 is H, q is 0, L.sub.1 is ##STR00163## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is Cl, q is 0, L.sub.1 is ##STR00164## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is H, q is 0, L.sub.1 is ##STR00165## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is H, q is 0, L.sub.1 is ##STR00166## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is H, q is 0, L.sub.1 is ##STR00167## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is H, q is 0, L.sub.1 is ##STR00168## c is 0, w is 0, y is 0 and X.sub.3 is Br; X.sub.1 is Cl, X.sub.2 is H, q is 0, L.sub.1 is ##STR00169## c is 0, w is 0, y is 0 and X.sub.3 is I; X.sub.1 is H, X.sub.2 is Cl, q is 0, L.sub.1 is ##STR00170## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is H, X.sub.2 is Br, q is 0, L.sub.1 is ##STR00171## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is H, X.sub.2 is Br, q is 0, L.sub.1 is ##STR00172## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is Cl, q is 0, L.sub.1 is ##STR00173## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is Cl, q is 0, L.sub.1 is ##STR00174## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is Cl, q is 0, L.sub.1 is ##STR00175## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is Br, q is 0, L.sub.1 is ##STR00176## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is Br, q is 0, L.sub.1 is ##STR00177## c is 0, w is 0, y is 0 and X.sub.3 is Cl; X.sub.1 is Cl, X.sub.2 is Br, q is 0, L.sub.1 is ##STR00178## c is 0, w is 0, y is 0 and X.sub.3 is Cl; or X is Br, X.sub.2 is Br, q is 0, L.sub.1 is ##STR00179## c is 0, w is 0, y is 0 and X.sub.3 is Cl; wherein the dashed line indicates the point of attachment of L.sub.1 to the nitrogen atom of ##STR00180## and the wavy line indicates the point of attachment of L.sub.1 to X.sub.3.

2. The linker according to claim 1, wherein it has the following formula (Ia): ##STR00181## or a salt thereof, wherein: y is 0 when w is 0 and y is 0 or 1 when w is an integer from 1 to 5.

3. The linker according to claim 2, wherein at least X.sub.1 or X.sub.2 represents a halogen atom.

4. The linker according to claim 3, wherein at least X.sub.1 or X.sub.2 represents Br or Cl.

5. The linker according to claim 4, wherein one of X.sub.1 and X.sub.2 represents Br or Cl and the other group represents H, Cl or Br.

6. The linker according to claim 1, wherein L.sub.1′ represents —(CH.sub.2).sub.n—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2—, arylene, -cycloalkanediyl-, —(CH.sub.2).sub.n-arylene-, - arylene-(CH.sub.2).sub.n—, —(CH.sub.2).sub.n-cycloalkanediyl-, -cycloalkanediyl-(CH.sub.2).sub.n—, ##STR00182##

7. The linker according to claim 6, wherein L.sub.1′ is —(CH.sub.2).sub.n— or —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2.

8. The linker according to claim 1, wherein each W is selected from alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, lysine, lysine protected with acetyl or formyl, arginine, arginine protected with tosyl or nitro group(s), histidine, ornithine, ornithine protected with acetyl or formyl, and citrulline.

9. The linker according to claim 1, wherein: w=0 and (W).sub.w is a bond, or w=2 and (W).sub.w is Val-Cit or Val-Ala, preferably Val Cit.

10. The linker according to claim 1, wherein: X.sub.1 and X.sub.2 are identical and are selected from Cl, Br, (C.sub.1-C.sub.6)alkoxy and an aryloxy optionally substituted with one or several groups selected from halogen, CN, NO.sub.2 and an aryloxy optionally substituted with one or several halogen atoms, or one of X.sub.1 and X.sub.1 is H and the other is selected from Cl, Br, (C.sub.1-C.sub.6)alkoxy and an aryloxy optionally substituted with one or several groups selected from halogen, CN, NO.sub.2 and an aryloxy optionally substituted with one or several halogen atoms.

11. The linker according to claim 1, wherein X.sub.3 is H when y=z=1 and Z is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5— or when c=w=y=0, z′=1 and Z′ is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5— and in the other cases, X.sub.3 is OH, Cl or N-succinimidyloxy.

12. A linker-drug conjugate of the following formula (II): ##STR00183## or a salt thereof, wherein: X.sub.1 and X.sub.2 represent, independently of each other, H, a halogen atom, a (C.sub.1-C.sub.6)alkoxy, an optionally substituted aryloxy, or —O—(CH.sub.2CH.sub.2O).sub.rH, provided that X.sub.1 and X.sub.2 do not represent H at the same time; L.sub.1 represents a group of formula L.sub.1′-(CO—Z′).sub.z′ with L.sub.1′ being —(CH.sub.2).sub.n—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2—, arylene, heteroarylene, cycloalkanediyl, —(CH.sub.2).sub.n-arylene-, —(CH.sub.2).sub.n-heteroarylene-, —(CH.sub.2).sub.n-cycloalkanediyl-, -arylene-(CH.sub.2).sub.p—, -heteroarylene-(CH.sub.2).sub.p—, -cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2).sub.D-arylene-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-heteroarylene-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-arylene-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-heteroarylene-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-arylene-CH.sub.2—CH.sub.2—(OCH.sub.2CH.sub.2).sub.m—, —(CH.sub.2).sub.n-heteroarylene-CH.sub.2—CH.sub.2—(OCH.sub.2CH.sub.2).sub.m—, or —(CH.sub.2).sub.n-cycloalkanediyl-CH.sub.2—CH.sub.2—(OCH.sub.2CH.sub.2).sub.m—, each W independently represents an amino acid unit; Y is PAB-CO—(Z).sub.z—, with PAB being ##STR00184## the oxygen of the PAB unit being linked to CO—(Z).sub.z, Z is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—, —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—, —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—(CH.sub.2).sub.v—, or —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—(CH.sub.2).sub.v—CO—, the NR.sub.4 group being linked to the CO group of PAB-CO; Z′ is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5— or —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—(CH.sub.2).sub.v—, the NR.sub.4 group being linked to the CO group of CO—Z′; R.sub.4 and R.sub.5 are independently H or a (C.sub.1-C.sub.6)alkyl group; Q represents a drug moiety; c is 0 or 1; m is an integer from 1 to 15; n is an integer from 1 to 6; p is an integer from 1 to 6; q is 0, 1 or 2; r is an integer from 1 to 21; u is an integer from 1 to 6; v is an integer from 1 to 6; w is an integer from 0 to 5; y is 0 or 1; z is 0 or 1; and z′ is 0 or 1.

13. The linker-drug conjugate according to claim 12, wherein it has the following formula (IIa): ##STR00185## or a salt thereof, wherein: y is 0 when w is 0 and y is 0 or 1 when w is an integer from 1 to 5.

14. (canceled)

15. The linker-drug conjugate according to claim 12, wherein Q is a residue of an auristatin, an anthracycline, camptothecin, SN-38, a tubulysin, a calicheamicin, a maytansinoid, a duocarmycin, an amanitine, a pyrrolobenzodiazepine, or an activator of immune check point.

16. The linker-drug conjugate according to claim 12, wherein Q is: a residue of monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), or monomethyl dolastatin-10 or a residue of a derivative thereof having the following formula (C): ##STR00186## wherein: R.sub.1 is H or OH, R.sub.2 is a (C.sub.1-C.sub.6)alkyl, COOH, COO—((C.sub.1-C.sub.6)alkyl) or a thiazolyl, R.sub.3 is H or a (C t-C.sub.6)alkyl, X.sub.4 is O or NR.sub.9, R.sub.9 is H or (C.sub.1-C.sub.6)alkyl, and t is an integer from 1 and 8; a residue of daunorubicine, doxorubicine, epirubicine, idarubicine, 2-pyrrolinodoxorubicine, pro-2-pyrrolinodoxorubicine, or PNU-159682 or a residue of following formula (A) or (B): ##STR00187## a residue of camptothecin or SN-38; a residue of tubulysin A, tubulysin B, tubulysin C or tubulysin D; a residue of esperamicin, calicheamicin yl, or N-acetyl dimethyl hydrazide calicheamicin; a residue of maytansine, DM1 or DM4; a residue of duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D duocarmycin SA, or CC-1065, a residue of α-amanitine, β-amanitine, γ-amanitine or ε-amanitine; a residue of anthramycin or SGD-1882, a residue of following formula (D): ##STR00188## wherein: X.sub.1 i and X.sub.21 are independently O or S, X.sub.12 and X.sub.22 are independently OH, SH, O or S, A.sub.11 and A.sub.21 are independently a group of formula: ##STR00189## Z.sub.1 is OR.sub.11, NR.sub.11R.sub.12, O or NR.sub.11, with R.sub.11 and R.sub.12 being independently H, R.sub.13 or COR.sub.13, with R.sub.13 being (C.sub.1-C.sub.6)alkyl, aryl or aryl(C.sub.1-C.sub.6)alkyl, Z.sub.2 is H, NR.sub.21R.sub.22 or NR.sub.21, with R.sub.21 and R.sub.22 being independently H, R.sub.23 or COR.sub.23, with R.sub.23 being (C.sub.1-C.sub.6)alkyl, aryl or aryl(C.sub.1-C.sub.6)alkyl, Z.sub.3 is N or CR.sub.33, with R.sub.33 being H or a halogen atom, and Z.sub.4 is H or a (C.sub.1-C.sub.6)alkyl, A.sub.12 and A.sub.22 are independently H, OH or F, and A.sub.2 is H or A.sub.2 and A.sub.22 are linked together with A.sub.2 being CH.sub.2 and A.sub.22 being O, wherein: when X.sub.11 is O or S, then X.sub.22 is not O and is not S, Z.sub.1 is not O and is not NR.sub.11, Z.sub.2 is not NR.sub.21, and the residue of the STING agonist is linked to the rest of the molecule by X.sub.11; when X.sub.22 is O or S, then X.sub.12 is not O and is not S, X.sub.22 is not O and is not NR.sub.11, Z.sub.2 is not NR.sub.21, and the residue of the STING agonist is linked to the rest of the molecule by X.sub.22; when Z.sub.1 is O or NR.sub.11, then X.sub.12 is not O and is not S, X.sub.22 is not O and is not S, Z.sub.2 is not NR.sub.21, and the residue of the STING agonist is linked to the rest of the molecule by Z.sub.1; when Z.sub.2 is NR.sub.21, then X.sub.12 is not O and is not S, X.sub.22 is not O and is not S, Z.sub.1 is not O and is not NR.sub.11, and the residue of the STING agonist is linked to the rest of the molecule by Z.sub.2.

17. The linker-drug conjugate according to claim 12, wherein Q has: the following formula (A): ##STR00190## the following formula (B): ##STR00191## the following formula (C): ##STR00192## wherein: R.sub.1 is H or OH, R.sub.2 is a (C.sub.1-C.sub.6)alkyl, COOH, COO—((C.sub.1-C.sub.6)alkyl) or a thiazolyl, R.sub.3 is H or a (C.sub.1-C.sub.6)alkyl, X.sub.4 is O or NR.sub.9, R.sub.9 is H or (C.sub.1-C.sub.6)alkyl, and t is an integer from 1 and 8; or the following formula (D): ##STR00193## wherein: X.sub.11 and X.sub.21 are independently O or S, preferably Q, X.sub.12 and X.sub.11 are independently OH, SH, O or S, A.sub.11 and A.sub.21 are independently a group of formula: ##STR00194## Z.sub.1 is OR.sub.11, NR.sub.11R.sub.12, O or NR.sub.11, with R.sub.11 and R.sub.12 being independently H, R.sub.13 or COR.sub.13, with R.sub.13 being (C.sub.1-C.sub.6)alkyl, aryl or aryl(C.sub.1-C.sub.6)alkyl, Z.sub.2 is H, NR.sub.21R.sub.22 or NR.sub.21, with R.sub.21 and R.sub.22 being independently H, R.sub.23 or COR.sub.23, with R.sub.23 being (C.sub.1-C.sub.6)alkyl, aryl or aryl(C.sub.1-C.sub.6)alkyl, Z.sub.3 is N or CR.sub.33, with R.sub.33 being H or a halogen atom, and Z.sub.4 is H or a (C.sub.1-C.sub.6)alkyl, A.sub.12 and A.sub.22 are independently H, OH or F, and A.sub.2 is H or A.sub.2 and A.sub.22 are linked together with A.sub.2 being CH.sub.2 and Au being O, wherein: when X.sub.12 is O or S, then X.sub.22 is not O and is not S, Z.sub.1 is not O and is not NR.sub.11, Z.sub.2 is not NR.sub.21, and the residue of the STING agonist is linked to the rest of the molecule by X.sub.12; when X.sub.22 is O or S, then X.sub.12 is not O and is not S, Z.sub.1 is not O and is not NR.sub.11, Z.sub.2 is not NR.sub.21, and the residue of the STING agonist is linked to the rest of the molecule by X.sub.22, when Z.sub.1 is O or NR.sub.11, then X.sub.12 is not O and is not S, X.sub.22 is not O and is not S, Z.sub.2 is not NR.sub.21, and the residue of the STING agonist is linked to the rest of the molecule by Z.sub.1; when Z.sub.2 is NR.sub.21, then X.sub.12 is not O and is not S, X.sub.22 is not O and is not S, Z.sub.1 is not O and is not NR.sub.11, and the residue of the STING agonist is linked to the rest of the molecule by Z.sub.2.

18-19. (canceled)

20. A binding unit-drug conjugate of the following formula (III) or (IV): ##STR00195## or a salt thereof, wherein: the binding unit is a peptide, a protein, an antibody, or an antigen binding fragment thereof, L.sub.1 represents a group of formula L.sub.1′-(CO—Z′).sub.z′ with L.sub.1′ being —(CH.sub.2).sub.n—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2—, arylene, heteroarylene, cycloalkanediyl, —(CH.sub.2).sub.n-arylene-, —(CH.sub.2).sub.n-heteroarylene-, —(CH.sub.2).sub.n-cycloalkanediyl-, -arylene-(CH.sub.2).sub.p—, -heteroarylene-(CH.sub.2).sub.p—, -cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2).sub.D-arylene-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-heteroarylene-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-arylene-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-heteroarylene-(CH.sub.2).sub.p—, —(CH.sub.2CH.sub.2O).sub.m—CH.sub.2—CH.sub.2-cycloalkanediyl-(CH.sub.2).sub.p—, —(CH.sub.2).sub.n-arylene-CH.sub.2—CH.sub.2—(CH.sub.2CH.sub.2O).sub.m—, —(CH.sub.2).sub.n-heteroarylene-CH.sub.2—CH.sub.2—(CH.sub.2CH.sub.2O).sub.m—, or —(CH.sub.2).sub.n-cycloalkanediyl-CH.sub.2—CH.sub.2—(CH.sub.2CH.sub.2O).sub.m—, each W independently represents an amino acid unit; Y is PAB-CO—(Z).sub.z—, with PAB being ##STR00196## the oxygen of the PAB unit being linked to CO—(Z).sub.z, Z is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—, —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—, —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—(CH.sub.2).sub.v—, or —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—(CH.sub.2).sub.v—CO—, the NR.sub.4 group being linked to the CO group of PAB-CO; Z′ is —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5— or —NR.sub.4—(CH.sub.2).sub.u—NR.sub.5—CO—(CH.sub.2).sub.v—, the NR.sub.4 group being linked to the CO group of CO—Z′; R.sub.4 and R.sub.5 are independently H or a (C.sub.1-C.sub.6)alkyl group; Q represents a drug moiety; c is 0 or 1; m is an integer from 1 to 15; n is an integer from 1 to 6; p is an integer from 1 to 6; s is an integer from 1 to 8, u is an integer from 1 to 6; v is an integer from 1 to 6; w is an integer from 0 to 5; y is 0 or 1; z is 0 or 1; and z′ is 0 or 1.

21. (canceled)

22. The binding unit-drug conjugate according to claim 20, wherein the binding unit is an antibody, or an antigen binding fragment thereof.

23. The binding unit-drug conjugate according to claim 22, wherein the antibody is an IGF-1R antibody or a HER2 antibody.

24. A pharmaceutical composition comprising a binding unit-drug conjugate according to claim 20 and at least one pharmaceutically acceptable excipient.

25. (canceled)

26. The linker-drug conjugate according to claim 15, wherein the activator of immune check point is a residue of a stimulator of interferon genes (STING) agonist or a residue of an indoleamine 2,3-dioxygenase (IDO) inhibitor.

27. A method for covalently linking a drug to a binding unit by a linker according to claim 1, wherein the binding unit is selected from a peptide, a protein, an antibody and an antigen binding fragment thereof.

28. The method according to claim 27, wherein q is 2.

29. A method for covalently linking a drug to a binding unit by reacting a linker-drug conjugate according to claim 12 with the binding unit, wherein the binding unit is selected from a peptide, a protein, an antibody and an antigen binding fragment thereof.

30. The method according to claim 29, wherein q is 2.

31. A method for treating cancer comprising the administration to a person in need thereof of an effective amount of a binding unit-drug conjugate according to claim 20.

32. A method for treating cancer comprising the administration to a person in need thereof of an effective amount of a pharmaceutical composition according to claim 24.

Description

FIGURES

[0488] FIGS. 1A, 2, 3A, 4A, 5A, 6A, 7A, 21 and 22A represent mass spectra of drug-linker conjugates according to the invention.

[0489] FIGS. 1B, 3B, 4B, 5B, 6B, 7B, 8 and 9 represent .sup.1H-NMR spectra of drug-linker conjugates according to the invention.

[0490] FIGS. 10 and 11 represent mass spectra of drug-somatostatin conjugates according to the invention.

[0491] FIG. 12 represents the SDS-PAGE analysis of the Ab I antibody (I) and purified ADCs according to the invention (ADC1-A (2), ADC1-B (3), ADC1-C (4), ADC1-D (5). ADC1-E (6). ADC 1-F (7) and ADC 1-G (8)) under reducing and non-reducing conditions. The bands observed on the gels correspond to completely bridged antibody (i.e. LHHL); partially bridged (i.e. HHL, HH, HL) and no bridging (i.e. H and L).

[0492] FIG. 13 represents the SEC analysis of the Abl antibody and ADCs according to the invention (ADC1-A, ADC1-B, ADC1-C, ADC1-D, ADC1-E, ADC1-F and ADC1-G).

[0493] FIGS. 14A, 14B, 14C and 14D represent ADC m/z spectra before deconvolution of ADCs according to the invention: (A) ADC1-A, (B) ADC1-B, (C) ADC1-C and (D) ADC-1D respectively.

[0494] FIGS. 15A, 15B and 15C represent DAR distribution after Maxent deconvolution for (A) ADC1-A, (B) ADC1-B and (C) ADC1-D respectively.

[0495] FIGS. 16A and 16B represent an analysis by native mass spectrometry of ADCs: (A) a reference ADC Ref-A and (B) ADC1-C according to the invention.

[0496] FIGS. 17A, 17B and 17C represent the results of the in vitro stability study by presenting the percentage of total antibody (100%) and ADC at each timepoint (D0, D3. D7 and D14) in (1) human, (2) cynomolgus, (3) mouse and (4) rat sera for (A) a reference ADC Ref-B, (B) ADC1-C and (C) ADC1-E respectively.

[0497] FIGS. 18A and 18B represent the in vitro cell cytotoxicity evaluation of different ADCs in NCI-H2122 (A) and MCF-7 (B) cells respectively.

[0498] FIGS. 19 and 20 represent the in vivo activity of ADC1-C and reference ADC Ref-A in an ovarian cancer model.

[0499] FIG. 22B represents a TOF-MS spectrum of a drug-linker conjugate according to the invention.

[0500] FIG. 23A represents the SEC analysis of the Abl antibody and ADCs according to the invention which are synthesized with the PNU-159682 derivatives.

[0501] FIG. 23B represents the SEC analysis of the Ab2 antibody and ADCs according to the invention which are synthesized with the PNU-159682 derivatives.

[0502] FIGS. 24: 24A, 24B, 24C and 24D represent ADC m/z spectra before deconvolution of the ADCs according to the invention: (A) hz208F2-F562524, (B) c9G4-F562524, (C) hz208F2-F562616 and (D) c9G4-F562646 respectively.

[0503] FIGS. 25: 25A, 25B, 25C and 25D represent DAR distribution, after Maxent deconvolution, for ADCs according to the invention: (A) hz208F2-F562524, (B) c9G4-F562524. (C) hz208F2-F562616 and (D) c9G4-F562646 respectively.

[0504] FIGS. 26: 26A and 26B represent the in vitro cell cytotoxicity evaluation of the ADC hz208F2-F562524, and the corresponding control ADC c9G4-F562524, in NCI-H2122 (A) and MCF-7 (B) cells respectively.

[0505] FIGS. 27: 27A and 27B represent the in vitro cell cytotoxicity evaluation of the ADC hz208F2-F562646, and the corresponding control ADC c9G4-F562646, in NCI-H2122 (A) and MCF-7 (B) cells respectively.

[0506] FIG. 28 represents the in vivo activity of the ADC hz208F2-F562524, and the corresponding control ADC c9G4-F562524, in an ovarian cancer model.

EXAMPLES

Abbreviations

[0507] ACN: Acetonitrile [0508] ADC: Antibody-Drug Conjugate [0509] aq: aqueous [0510] BBO: Broadband Observe [0511] BCA: Bicinchoninic acid [0512] CDR: Complementarity Determining Region [0513] DAR: Drug-to-Antibody Ratio [0514] DCM: Dichloromethane [0515] DIPEA: N,N-Diisopropylethylamine [0516] DMF: Dimethylformamide [0517] DMSO: Dimethylsulfoxide [0518] EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [0519] EDTA: Ethylenediaminetetraacetic acid [0520] eq: equivalent [0521] ES: Electrospray [0522] ESI: Electrospray ionisation [0523] HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate [0524] HOBt: 1-Hydroxybenzotriazole [0525] HIC: Hydrophobic Interaction Chromatography [0526] HPLC: High Performance Liquid Chromatography [0527] HRMS: High Resolution Mass Spectrometry [0528] LBA: Ligand Binding Assay [0529] LC: Liquid Chromatography [0530] LCMS: Liquid Chromatography-Mass Spectrometry [0531] mCPBA: meta-Chloroperoxybenzoic acid [0532] Ms: Mesyl [0533] MS: Mass Spectrum [0534] NMR: Nuclear Magnetic Resonance [0535] PBS: Phosphate buffered saline [0536] Q-TOF: Quadrupole-time-of-flight [0537] Rf: Retardation factor [0538] rt: Room Temperature [0539] sat.: saturated [0540] SDS-PAGE: Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis [0541] SEC: Size Exclusion Chromatography [0542] TBME: Tert-butyl methyl ether [0543] TCEP: Tris(2-carboxyethyl)phosphine [0544] TEA: Triethylamine [0545] TFA: Trifluoroacetic acid [0546] THF: Tetrahydrofuran [0547] TLC: Thin Layer Chromatography [0548] TOF: Time of Flight [0549] Ts: Tosyl [0550] UV: Ultraviolet

EXPERIMENTAL PROCEDURES

[0551] All reactions requiring anhydrous conditions were conducted in oven-dried apparatus under an atmosphere of nitrogen. Anhydrous solvents were received in sealed bottles under inert atmosphere. All reagents were used as received. Column chromatography was carried out on puriFlash® Columns with silica gel (50 μm) on an Interchim puriFlash®430 and a Grace Reveleris® X.sub.2. TLC was performed on aluminum sheets pre-coated with silica (Merck silica gel 60 F.sub.254) which were visualized with an UV-Lamp 254 nm. Proton (.sup.1H) and carbon (.sup.13C) NMR spectra were recorded in CDCl.sub.3 and DMSO at room temperature with Bruker 500 MHz Ascend™ equipped with a BBO Prodigy probe (5 mm). Spectra were interpreted using Topspin™ 3.2 software. Chemical shifts (δ.sub.H and δ.sub.C) are reported in parts per million (ppm) and are referenced relative to either CDCl.sub.3 (.sup.1H NMR 7.26, .sup.13C NMR 77.0, central signal of triplet) or DMSO (.sup.1H NMR 2.50, .sup.13C NMR 37.9, central signal of septuplet). Assignments were aided by COSY and HSQC experiments. Coupling constants (J: vicinal protons, J.sub.cis: vicinal protons in cis position, J.sub.o: proton in ortho position, J.sub.m: proton in meta position) are given in Hertz to the nearest ±0.1 Hz. Multiplicities are given as singular (s), doublet (d), triplet (t), quartet (q), triplet of triplet (t. of t.), multiplet (m) and broad (b) where applicable. Mass spectra (w/z) were recorded on a Waters® ZQ Mass Detector spectrometer using the technique of electrospray ionization (ES+), source temperature: 120° C., dessolvatation temperature: 350° C., capillary voltage: 3.20 kV, cone voltage: 25 V, extractor voltage: 5 V, Rf lens voltage: 0.5 V, MS Scan range: 100-2000. HPLC analysis were performed using a Waters® X-Bridge Shield RP18 3.5 μm (3.0 mm×30 mm) column and a Waters® X-Bridge Shield RP18 3.5 μm (3.0 mm×20 mm) pre-column on a HPLC Waters® Alliance 2695 with MassLynx 4.1 software and a Waters 2996 PDA Detector UV/vis at the appropriate wavelength for the sample under analysis. Retention times (R.sub.t) are given in 10 minutes to the nearest 0.01 min. Two methods of elution were used:

TABLE-US-00008 TABLE 7 Method 1 of elution for LC Solvent A (Water + 0.1% Solvent B (ACN + 0.1% Time (min.) formic acid) % formic acid) % 0.00 97.0 3.0 2.25 0.0 100.0 2.50 0.0 100.0 2.60 97.0 3.0 3.00 97.0 3.0

TABLE-US-00009 TABLE 8 Method 2 of elution for LC Solvent A (Water + 0.1% Solvent B (ACN + 0.1% Time (min.) formic acid) % formic acid) % 0.00 50.0 50.0 2.10 25.0 75.0 2.25 0.0 100.0 2.50 0.0 100.0 2.75 97.0 3.0 3.00 97.0 3.0

[0552] Retention times given by Method 1 are indicated by R.sub.t i and ones given by Method 2 by R.sub.t,2.

1. SYNTHESIS OF THE LINKERS

Example of a Synthetic Path for Oxoisothiazolones

[0553] ##STR00077##

I.1. 3,3′-disulfanediyldipropanoyl Chloride

[0554] ##STR00078##

[0555] I.2. 3,3′-disulfanediyldipropionic acid (4 g, 0.019 mol, 1 eq.) was suspended in anhydrous DCM (100 mL) and anhydrous DMF (300 μL) was added, followed by oxalyl chloride (7.24 g, 0.057 mol, 3 eq.) at 0° C., under inert atmosphere. The solution clarified. The mixture was left for 3 h at rt until it clarified and no gas formation was longer observed. The crude was evaporated and kept under reduced pressure for another 30 min to remove remnants of oxalyl chloride. A yellow oil (4.70 g, 100%) was obtained. The crude was used without further purification; R.sub.t,1 (in MeOH): 2.13; MS ES+ m/z: 206.86.

I.3. Dibenzyl 6,6′-((3,3′-disulfanediylbis(propanoyl))bis(azanediyl))dihexanoate

[0556] ##STR00079##

[0557] 6-(benzyloxy)-6-oxohexan-1-aminium 4-methylbenzenesulfonate (12.20 g, 0.031 mol, 2.2 eq.) was suspended under vigorous stirring in anhydrous DCM (75 mL) in an ice bath at 0° C., under inert atmosphere. TEA (15.72 mL, 0.113, 8 eq.) was added to the solution. A solution of freshly prepared 3,3′-disulfanediyldipropanoyl chloride (3.88 g, 0.014 mol, 1 eq.) in DCM (25 mL) was slowly dropped into the solution maintained at 0° C. Stirring was continued for 24 hours while the solution was let to come to rt. Water was added (50 mL) and the mixture transferred to a separatory funnel. The organic layer was separated and washed with brine (1×100 mL) then washed with HCl 1M (1×100 mL), saturated solution of NaHCO.sub.3 salt (2×100 mL) and brine (1×100 mL) again. The combined aqueous layers were extracted with DCM (2×100 mL). The organic layers were dried with MgSO.sub.4, filtered and evaporated to dryness affording a yellow solid which was triturated in MeOH to give Dibenzyl 6,6′-((3,3′-disulfanediylbis(propanoyl)) bis(azanediyl))dihexanoate as a light yellow powder (6.0 g, 66%). .sup.1H NMR (500 MHz, CDCl.sub.3), δ 7.35 (m, 10H), 6.0 (s, 2H), 5.11 (s, 4H), 3.25 (q, J=5.9 Hz, 4H), 3.00 (t, J=7.0 Hz, 4H), 2.55 (t, J=7.10 Hz, 4H), 2.36 (t, J=7.30 Hz, 4H), 1.66 (t of t., J=8.10 Hz, 4H), 1.52 (t of t., J=8.10 Hz, 4H), 1.35 (t of t, J=8.52 Hz, 4H); .sup.13C NMR (500 MHz, CDCl.sub.3), δ 173.5 (2-NH—C═O), 170.9 (—O—C═O), 136.0 (2 C.sub.quat from aromatic cycles), 128.6 (2H—C.sub.aromatic), 128.3 (H—C.sub.aromatic), 128.2 (2H—C.sub.aromatic), 66.2 (2-CH.sub.2—O—), 39.4 (2-CH.sub.2—N), 35.8 (2-CH.sub.2—COO—), 34.3 (2-CH—S—), 34.1 (2-CH.sub.2—CNO—), 29.1 (2C), 26.3 (2C), 24.4 (2C); R.sub.t,1 (in MeOH): 2.50; MS ES+ M/Z: 617.00.

I.4. Benzyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate

[0558] ##STR00080##

[0559] Dibenzyl 6,6′-((3,3′-disulfanediylbis(propanoyl))bis(azanediyl))dihexanoate (2.50 g, 4.05 mmol, 1 eq.) was dissolved in anhydrous DCM (20.3 mL). SO.sub.2Cl.sub.2 (pur. 97%, 2.96 mL, 0.036 mol, 9 eq.) was added dropwise to the solution, and the mixture was stirred at rt for 5 h under inert atmosphere. The solution clarified and became pale yellow. Subsequently, the solution was washed with water (2×100 mL) and brine (1×100 mL). The combined aqueous layers were extracted with DCM (2×100 mL). The organic layers were dried with MgSO.sub.4 and filtered, then concentrated under reduced pressure and purified using a chromatography column. Benzyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate (0.824 g, 29.9%) was obtained as a light yellow oil along with benzyl 6-(3-oxoisothiazol-2(3H)-yl)hexanoate. .sup.1H NMR (CDCl.sub.3), δ 7.35 (m, 5H), 6.25 (s, 1H), 5.11 (s, 2H), 3.72 (t, J=7.34 Hz, 2H), 2.37 (t, J=7.39 Hz, 2H), 1.69 (m, 4H), 1.38 (m, 2H); .sup.13C NMR (500 MHz, CDCl.sub.3), δ 173.2 (—O—C═O), 166.9 (—N—C═O), 145.6 (Cl—HC═CH—), 136.0 (C.sub.quat from aromatic cycle), 128.6-128.3 (5H—C.sub.aromatic), 114.8 (Cl—HC═CH—), 66.2 (—CH.sub.2—O—), 43.5 (—CH.sub.2—N—), 34.0 (—CH.sub.2—C═O), 29.4, 25.9, 24.4; R.sub.t,1 (in ACN): 2.35; MS ES+ m/z: 339.84.

I.5. Benzyl 6-(5-chloro-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0560] ##STR00081##

[0561] Benzyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate (802 g, 3.58 mmol) was diluted in anhydrous DCM (25 mL), 3-chlorobenzoperoxoic acid (1.2 eq.) is added. The solution was stirred for 48 h at rt under inert atmosphere. The solution was then diluted with DCM and treated by 10% aq. Na.sub.2S.sub.2O.sub.3. The organic phase was then extracted successively by a saturated solution of NaHCO.sub.3 salt (2×100 mL), followed by brine (1×100 mL). The combined aqueous layers were extracted with DCM (2×100 mL). The organic layers were dried over MgSO.sub.4, filtered, then concentrated under reduced pressure and purified using a chromatography column. Benzyl 6-(5-chloro-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate was then obtained (753 mg) as a colorless oil.

I.6. Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0562] ##STR00082##

[0563] Standard Procedure for the Oxidation of Mono-Chloride Compounds:

[0564] Benzyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate (1.11 g, 0.0036 mol, 1 eq.) was diluted in anhydrous DCM (7 mL), 3-chlorobenzoperoxoic acid (2.69 g, 0.0109 mol, 3 eq.) is added. The solution was stirred for 48 h at rt under inert atmosphere. The solution was then diluted with DCM and treated by 10% aq. Na.sub.2S.sub.2O.sub.3. The organic phase was then extracted successively by a saturated solution of NaHCO.sub.3 salt (2×100 mL), followed by brine (1×100 mL). The combined aqueous layers were extracted with DCM (2×100 mL). The organic layers were dried over MgSO.sub.4, filtered, then concentrated under reduced pressure and purified using a chromatography column. Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate was obtained as a light yellow oil (0.760 g, 64.3%). .sup.1H NMR (CDCl.sub.3), δ 7.36 (m, 5H), 6.68 (s, 1H), 5.11 (s, 2H), 3.67 (t, J=7.68 Hz, 2H), 2.37 (t, J=7.27 Hz, 2H), 1.78 (t of t, J=7.73 Hz, 2H), 1.70 (t of t, J=7.38 Hz, 2H), 1.40 (m, 2H); .sup.13C NMR (500 MHz, CDCl.sub.3), δ 173.2 (—O—CO), 157.0 (—N—CO), 144.6 (Cl—HC═CH—), 136.0 (C.sub.quat from aromatic cycle), 128.6-128.2 (H—C.sub.aromatic), 123.6 (Cl—HC═CH—), 66.2 (—CH.sub.2—O), 40.3 (—CH.sub.2—N—), 34.0 (—CH.sub.2—C═O), 27.9, 26.0, 24.2; R.sub.t,1 (in ACN): 2.49; MS ES+ M/Z: 371.83.

Example 1. 6-(5-Chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic Acid

[0565] ##STR00083##

[0566] 6-(5-Chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid was obtained as a light white powder following the standard procedure of deprotection of benzyl esters starting from Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (0.445 g, 77%). .sup.1H NMR (CDCl.sub.3), δ 10.89 (br, s, 1H), 6.70 (s, 1H), 3.70 (t, J=7.43 Hz, 2H), 2.38 (t, J=7.38 Hz, 2H), 1.81 (t of t., J=7.60 Hz, 2H), 1.69 (t of t, J=7.71 Hz, 2H), 1.43 (splitted t of t, J=7.75 Hz, J=3.31 Hz, 2H); .sup.13C NMR (500 MHz, CDCl.sub.3), δ 178.7 (O═C—OH), 157.1 (—N—C═O), 144.7 (Cl—HC═CH—), 123.6 (Cl—HC═CH—), 40.2 (—CH.sub.2—N), 33.5 (—CH.sub.2—C═O), 27.9, 25.9, 23.9; R.sub.t,1 (in ACN): 1.98; MS ES+ m/z: 349.89.

I.7. Benzyl 6-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)hexanoate

[0567] ##STR00084##

[0568] Dibenzyl 6,6′-((3,3′-disulfanediylbis(propanoyl))bis(azanediyl))dihexanoate (3.21 g, 0.0052 mol, 1 eq.) was dissolved in anhydrous DCM (26 mL). SO.sub.2Cl.sub.2 (pur. 97%, 3.80 mL, 0.047 mol, 9 eq.) was added dropwise to the solution, and the mixture was stirred at rt for 24 h under inert atmosphere. The solution clarified and became pale yellow. Subsequently, the mixture was washed with water (2×100 mL) and brine (1×100 mL). The combined aqueous layers were extracted with DCM (2×100 mL). The organic layers were dried with MgSO.sub.4 and filtered then concentrated under reduced pressure and purified using a chromatography column. Benzyl 6-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)hexanoate was obtained as a light yellow oil (1.391 g, 35.7%). .sup.1H NMR (CDCl.sub.3), δ 7.35 (m, 5H), 5.11 (s, 2H), 3.79 (t, J=7.18 Hz, 2H), 2.37 (t, J=7.33 Hz, 2H), 1.70 (m, 4H), 1.38 (m, 2H); .sup.13C NMR (500 MHz, CDCl.sub.3), δ 173.2 (—O—C═O), 161.9 (—N—C═O), 138 (—CH.sub.2—O—), 0.3 (Cl—C—S—), 135.6 (C.sub.quat from aromatic cycle), 128.6-128.3 (5H—C.sub.aromatic), 115.1 (Cl—C—C═O), 66.2, 44.9 (—CH.sub.2—N—), 33.9 (—CH.sub.2—C═O), 29.1, 25.9, 24.3; R.sub.t,1 (in ACN): 2.50; MS ES+ m/z: 373.75.

I.8. Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0569] ##STR00085##

[0570] Standard Procedure for the Oxidation of Di-Chloride Compounds:

[0571] Ruthenium trichloride monohydrated (16 mg, 0.07 mmol, 0.013 eq.) was added in one portion to a stirred solution of Benzyl 6-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)hexanoate (1.94 g, 0.0052 mol, 1 eq.) in water:DCM:ACN (2:1:1, 1 ml). Sodium periodate (3.33 g, 0.0156 mol, 3 eq.) was then added over 5 min and the resulting mixture stirred at rt for 90 minutes under inert atmosphere. The solids were filtered and the filtrate was diluted with water (50 mL), extracted with EtOAc (2×100 mL), dried with MgSO.sub.4, filtered, and concentrated under reduced pressure. The grey solid obtained was then purified using a chromatography column and Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate was obtained as a pale yellow oil (0.930 g, 44.2%). .sup.1H NMR (CDCl.sub.3), δ 7.36 (m, 5H), 5.12 (s, 2H), 3.72 (t, J=7.53 Hz, 2H), 2.73 (t, J=7.50 Hz, 2H), 1.80 (t of t, J=7.53 Hz, 2H), 1.70 (t of t, J=7.70 Hz), 1.41 (m, 2H); .sup.13C NMR (500 MHz, CDCl.sub.3), δ 173.1 (—O—C═O—), 154.1 (N—C═O), 138.0 (Cl—C—SO.sub.2—), 136.0 (C.sub.quat from aromatic cycle), 130.7 1 (Cl—C—C═O), 128.6-128.3 (5H—C.sub.aromatic), 66.2 (—CH.sub.2—O—), 41.1 (—CH.sub.2—N—), 33.9 (—CH.sub.2—C═O), 27.9, 26.0, 24.2; R.sub.t,1 (in ACN): 2.59; MS ES+ m/z: 405.76.

Example 2. 6-(4,5-Dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic Acid

[0572] ##STR00086##

[0573] Standard Procedure of Deprotection of Benzyl Esters:

[0574] Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (0.930 g, 2.23 mmol, 1 eq.) was diluted in anhydrous DCM (11.5 mL). Methanesulfonic acid (1.5 mL, 0.023 mol, 10 eq.) was added. The solution was stirred for 24 h at rt under inert atmosphere. The solution was then diluted with DCM, and treated with water (50 mL). The organic layer was extracted with water (2×100 mL) then brine (1×100 mL). The combined aqueous layers were extracted with DCM (2×100 mL). The organic layers were dried over MgSO.sub.4 and concentrated under reduced pressure, then purified using a chromatography column. 6-(4,5-Dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid was obtained as a light white powder (0.563 g, 78%). .sup.1H NMR (500 MHz, CDCl.sub.3), δ=3.76 (t, J=7.34 Hz, 2H), 2.38 (t, J=7.32 Hz, 2H), 1.83 (t of t, J=7.65 Hz, 2H), 1.70 (t of t, J=1.11 Hz, 2H), 1.45 (t of t, J=7.54 Hz, J=3.31 Hz, 2H); .sup.13C NMR (500 MHz, CDCl.sub.3), δ 178.2 (O═C—OH), 154.2 (N—C═O), 138.1 (Cl—C—SO.sub.2—), 130.9 (Cl—C—C═O), 41.1 (—CH.sub.2—N—), 33.4 (—CH.sub.2—C═O), 27.9, 25.9, 23.9: R.sub.t,1 (in ACN): 2.09; MS ES+m/z: 315.76.

I.9. Benzyl 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0575] ##STR00087##

[0576] To a solution of benzyl 6-(1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (obtained following the standard procedure for the oxidation of mono-chloride compounds starting from benzyl 6-(3-oxoisothiazol-2(3H)-yl)hexanoate) (3 g, 8.89 mmol, 1.00 equiv) in CCl.sub.4 (40 mL) was added Br: (1.2 mL, 19.58 mmol, 2.2 equiv) dropwise with stirring at ambient temperature over 30 min and stirred overnight at 75° C. The reaction mixture was concentrated under vacuum and diluted with CHCl.sub.3 (40 mL), which was followed by the addition of pyridine (0.9 g). The resulting solution was stirred for 30 min at ambient temperature and then quenched by the addition of 50 ml saturated NaHCO.sub.3 solution. The resulting mixture was washed with saturated sodium carbonate (2×50 mL) and 50 mL of brine. The resulting mixture was concentrated under vacuum and the residue was purified by a silica gel column with ethyl acetate/petroleum ether (1:5) to afford 0.4 g (10.8%) of benzyl 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate as a light yellow oil. LC-MS (ES, m/z): 416 [M+H].sup.+, 433 [M+NH.sub.4].sup.+; .sup.1H-NMR (400 MHz, Chloroform-d) δ 7.42-7.28 (m, 2H), 5.12 (s, 1H), 3.69 (t, J=7.4 Hz, 1H), 2.38 (t, J=7.4 Hz, 1H), 1.86-1.64 (m, 2H), 1.47-1.34 (m, 1H).

Example 3. 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic Acid

[0577] ##STR00088##

[0578] To a solution of benzyl 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (1 g, 2.40 mmol, 1.00 equiv) in dioxane (10 mL) was added 4N HCl (10 mL) dropwise with stirring at 0′C. The resulting solution was stirred for 2 days at room temperature. The resulting mixture was concentrated under vacuum and extracted with dichloromethane (3×50 mL). The combined organic layer was washed with brine (2×100 mL), dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified via a silica gel column with DCM/MeOH (10:1) to afford 100 mg (13%) of 6-(4-bromo-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid as a white solid. LC-MS (ES, m/z): 308 [M+NH.sub.4].sup.+, 326/328 [M+H].sup.+; .sup.1H-NMR (300 MHz, Chloroform-d) δ 7.58 (s, 1H), 3.75 (t, J=7.4 Hz, 2H), 2.41 (t, J=7.4 Hz, 2H), 1.78 (dq, J=34.6, 7.4 Hz, 4H), 1.47 (t, 0.7=7.7 Hz, 2H).

Example of a Synthetic Path for the Synthesis of a Linker with X.SUB.1.=OR

[0579] ##STR00089##

I.10. Benzyl 6-(5-(4-cyanophenoxy)-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0580] ##STR00090##

[0581] A solution of 4-hydroxybenzenecarbonitrile (76 mg, 0639 mmol) in THF (2 mL) was added at 0° C., to a mixture of NaH (0.639 mmol) in THF (2 mL). After 30 minutes under stirring, benzyl 6-(5-chloro-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate (250 mg, 0,703 mmol) in THF (2 mL) was added. The reaction mixture was then stirred at room temperature for 18 h. The reaction mixture was diluted with AcOEt and NH.sub.4Cl (10% aqueous) was added. The organic phase was then washed with brine and dried over MgSO4, filtered and concentrated. The crude product was purified over silica gel column using DCM/MeOH mixture (80/20) to afford Benzyl 6-(5-(4-cyanophenoxy)-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate (210 mg, 75% yield) as a colorless oil. LC-MS (ES, m/z): 439.0 [M+H]+; .sup.1H-NMR (300 MHz, Chloroform-d) δ 7.79 (m, 2H), 7.35 (m, 7H), 5.59 (s, 1H), 5.12 (s, 2H), 3.69 (m, 2H), 2.37 (m, 2H), 1.71 (m, 4H), 1.39 (m, 2H).

I.11. Benzyl 6-(5-(4-cyanophenoxy)-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0582] ##STR00091##

[0583] Obtained as a colorless oil following the standard procedure for the oxidation of mono-chloride compounds (136 mg, 48% yield) starting from Benzyl 6-(5-(4-cyanophenoxy)-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate. LC-MS (ES, m/z): 455.0 [M+H]+; .sup.1H-NMR (300 MHz, Chloroform-d) δ 7.82 (m, 2H), 7.40 (m, 2H), 7.35 (m, 5H), 5.63 (s, 1H), 5.12 (s, 2H), 3.65 (m, 2H), 2.38 (m, 2H), 1.79 (m, 2H), 1.70 (m, 2H), 1.41 (m, 2H).

Example 4. 6-(5-(4-cyanophenoxy)-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic Acid

[0584] ##STR00092##

[0585] Obtained as a white solid following the standard procedure for deprotection of benzyl esters (38 mg, 35% yield) starting from Benzyl 6-(5-(4-cyanophenoxy)-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate. LC-MS (ES, m/z): 365.0 [M+H]+; .sup.1H-MR (300 MHz, Chloroform-d) 7.82 (m, 2H), 7.41 (m, 2H), 5.65 (s, 1H), 3.67 (m, 2H), 2.38 (m, 2H), 1.80 (m, 2H), 1.69 (m, 2H), 1.44 (m, 2H).

I.12. Benzyl 6-(5-methoxy-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0586] ##STR00093##

[0587] A mixture of benzyl 6-(5-chloro-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate (270 mg, 0,759 mmol) in methanol (5 mL) and triethylamine (113 μl, 0,835 mmol) was stirred at room temperature for 18 h. Volatiles were then removed under vacuum and the residue purified over a silica column cyclohexane/AcOEt (1/1) to afford Benzyl 6-(5-methoxy-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate (107 mg, 40%) as a yellow oil. LC-MS (ES, m/z): 352.0 [M+H]+; .sup.1H-NMR (300 MHz, Chloroform-d) δ 7.36 (m, 5H), 5.57 (s, 1H), 5.11 (s, 2H), 4.04 (s, 3H), 3.60 (m, 2H), 2.37 (m, 2H), 1.75 (m, 2H), 1.69 (m, 2H), 1.39 (m, 2H).

I.13. Benzyl 6-(5-methoxy-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0588] ##STR00094##

[0589] Obtained as a white solid following the standard procedure for the oxidation of mono-chloride compounds (62 mg, 36% yield) starting from benzyl 6-(5-methoxy-1-oxido-3-oxoisothiazol-2(3H)-yl)hexanoate. LC-MS (ES, m/z): 368.0 [M+H]+; .sup.1H-NMR (300 MHz, Chloroform-d) δ 7.36 (m, 5H), 5.57 (s, 1H), 5.11 (s, 2H), 4.04 (s, 3H), 3.60 (m, 2H), 2.37 (m, 2H), 1.75 (m, 2H), 1.69 (m, 2H), 1.39 (m, 2H).

Example 5. 6-(5-Methoxy-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic Acid

[0590] ##STR00095##

[0591] Obtained as a white solid following the standard procedure for deprotection of benzyl esters (37 mg, 67% yield) starting from benzyl 6-(5-methoxy-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate. HRMS (ES, m/z): [M+H] found 278.0691 for 278.0698 calculated; .sup.1H-NMR (300 MHz, Chloroform-d) δ 5.60 (s, 1H), 4.05 (s, 3H). 3.62 (m, 2H), 2.36 (m, 2H), 1.77 (m, 2H), 1.68 (m, 2H), 1.42 (m, 2H).

I.14. Benzyl 4-(aminomethyl)cyclohexane-1-carboxylate 4-methylbenzeaesulfonate

[0592] ##STR00096##

[0593] A mixture of 4-(aminomethyl)cyclohexanecarboxylic acid (10 g, 63.61 mmol, 1 eq), phenylmethanol (55.03 g, 508.87 mmol, 52.91 mL, 8 eq) and TsOH.H.sub.2O (12.70 g, 66.79 mmol, 1.05 eq) in toluene (50 mL) was stirred at 140° C., for 16 hours using a Dean and Stark apparatus to collect the water of condensation and from the toluene sulphonic acid monohydrate. The reaction turned to clear after refluxing for several hours. The clear reaction mixture was poured into TBME (500 mL) and the resultant white solid Altered off, washed with TBME (200 mL) and dried in vacuum. Benzyl 4-(aminomethyl)cyclohexanecarboxylate; 4-methylbenzenesulfonic acid (26.6 g, 63.40 mmol, 99.68% yield) was obtained as a white solid; .sup.1H NMR (400 MHz, METHANOL-d4) δ ppm 7.71 (d, J=8.16 Hz, 2H) 7.28-7.40 (m, 5H) 7.23 (d, J=7.94 Hz, 2H) 5.11 (s, 2H) 2.77 (d, J=7.06 Hz, 2H) 2.29-2.39 (m, 4H) 1.99-2.08 (m, 2H) 1.85 (br d, J=11.25 Hz, 2H) 1.53-1.65 (m, I H) 1.43 (qd, J=12.97, 3.20 Hz, 2H) 1.06 (qd, J=12.75, 3.20 Hz, 2H).

I.15. Dibenzyl 4,4′-(((3,3′-disulfanediylbis(propanoyl))bis(azanediyl))bis(methylene))bis (cyclohexane-1-carboxylate)

[0594] ##STR00097##

[0595] To a solution of 3-(2-carboxyethyldisulfanyl)propanoic acid (6.64 g, 31.58 mmol, I eq), HOBt (9.39 g, 69.48 mmol, 2.2 eq) and TEA (12.78 g, 126.33 mmol, 17.58 mL, 4 eq) in DCM (300 mL) was added EDCI (13.32 g, 69.48 mmol, 2.2 eq) at 0° C. Then benzyl 4-(aminomethyl)cyclohexanecarboxylate; 4-methylbenzenesulfonic acid (26.5 g, 63.17 mmol, 2 eq) was added at this temperature. The mixture was stirred at 0-20° C., for 4 hrs. TLC (Petroleum ether:Ethyl acetate=2:1. R.sub.f=0.5) indicated the reaction was completed. The mixture was poured into sat. NaHCO.sub.3 (100 mL) and H.sub.2O (100 mL) and the organic layer was separated. The aqueous layer was extracted with DCM (200 mL). The combined organic layers were washed with H.sub.2O (100 mL), brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was dissolved into DCM (50 mL), added petroleum ether very slowly until the white precipitate was formed. Filtered and washed with petroleum ether, dried over vacuum. Benzyl 4-[[3-[[3-[(4-benzyloxycarbonylcyclohexyl)methylamino]-3-oxo-propyl]disulfanyl]propanoylamino]methyl]cyclohexanecarboxylate (19 g, 28.40 mmol, 89.94% yield) was obtained as a white solid; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.27-7.41 (m, 10H) 6.05 (br s, 2H) 5.11 (d, J=1.54 Hz, 4H) 3.10-3.17 (m, 4H) 2.96-3.02 (m, 4H) 2.54-2.63 (m, 4H) 2.30 (td, J=12.24, 1.76 Hz, 2H) 2.04 (br d, J=12.57 Hz, 4H) 1.85 (br d, J=12.79 Hz, 4H) 1.38-1.54 (m, 6H) 0.99 (q, J=12.72 Hz, 4H).

I.16. Benzyl 4-((5-chloro-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylate

[0596] ##STR00098##

I.17. Benzyl 4-((4,5-dichloro-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylate

[0597] ##STR00099##

[0598] To a solution of benzyl 4-[[3-[[3-[(4-benzyloxycarbonylcyclohexyl)methylamino]-3-oxo-propyl] disulfanyl]propanoylamino]methyl]cyclohexanecarboxylate (10 g, 14.95 mmol, 1 eq) in DCM (100 mL) was added dropwise sulfuryl chloride (10.09 g, 74.75 mmol, 7.47 mL, 5 eq) at 0° C. The mixture was stirred at 0-20° C., for 12 hrs. The clear solution was obtained after the addition of the sulfuryl chloride. TLC (Petroleum ether:Ethyl acetate=2:1, Rf. (major)=0.5) indicated the reaction was completed. The mixture was poured into H.sub.2O (30 mL), extracted with DCM (50 mL*2). The combined organic layers were washed with H.sub.2O (50 mL), brine (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum. Purification over silica gel column afforded Benzyl 4-[(3-oxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (2.1 g, 3.42 mmol, 11.44% yield, S4% purity) obtained as a brown solid. Benzyl 4-[(5-chloro-3-oxo-isothiazol-2-yl)methyl]cyclohexanecarboxylate (7.1 g, 18.82 mmol, 62.96% yield, 97% purity) obtained as an off-white solid and Benzyl 4-[(4,5-dichloro-3-oxo-isothiazol-2-yl)methyl]cyclohexanecarboxylate (0.4 g, 869.31 umol, 2.91% yield, 87% purity) obtained as a brown oil.

I.18. Benzyl 4-((5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylate

[0599] ##STR00100##

[0600] To a mixture of benzyl 4-[(5-chloro-3-oxo-isothiazol-2-yl)methyl]cyclohexanecarboxylate (2 g, 5.01 mmol, 1 eq) in H.sub.2O (20 mL), ACN (10 mL) and DCM (10 mL) was added RuCl.sub.3.H.sub.2O (22.58 mg, 100.14 umol, 0.02 eq) and NaIO.sub.4 (6.43 g, 30.04 mmol, 1.66 mL, 6 eq) in one portion at 0° C., under N.sub.2. The mixture then heated to 20° C., and stirred for 16 hours. TLC showed the reaction was completed (Petroleum ether:Ethyl acetate=2:1, Rf-pl=0.6). The mixture was filtered, the filtrate was concentrated by nitrogen flow, and the solid that appeared again was filtered, the filtrate was concentrated by nitrogen. The residue was purified by preparative TLC (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel. Petroleum ether:Ethyl acetate=2:1) to afford benzyl 4-[(5-chloro-1,1,3-trioxo-isothiazol-2-yl)methyl]cyclohexanecarboxylate (1.4 g, 3.17 mmol, 63.25% yield, 90% purity) as white solid.

Example 6. 4-((5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylic Acid

[0601] ##STR00101##

[0602] To a mixture of benzyl 4-[(5-chloro-1,1,3-trioxo-isothiazol-2-yl)methyl]cyclohexanecarboxylate (0.1 g, 226.20 umol, 1 eq) in DCM (S mL) was added MsOH (217.39 mg, 2.26 mmol, 161.03 uL, 10 eq) in one portion at 30° C., under N.sub.2. The mixture was stirred at 30° C., for 16 hours. TLC showed the reaction was completed. LCMS (ET17992-54-PI A) showed desired MS detected. The mixture was poured into ice-water (5 mL) and stirred for 5 min. The aqueous phase was extracted with DCM (3 mL*2). The combined organic phase was washed with brine (3 mL), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by preparative TLC (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether/Ethyl acetate=2:1) to afford 4-[(5-chloro-1,1,3-trioxo-isothiazol-2-yl)methyl]cyclohexanecarboxylic acid (0.02 g, 64.99 umol, 28.73% yield) as colorless oil; LC-MS (ES, m/z): 306.0 [M−H].sup.−; .sup.1H-NMR (300 MHz, DMSO-D6) δ 12.01 (bs, 1H), 7.62 (s, 1H), 3.45 (m, 2H), 2.11 (m, 1H), 1.90 (m, 2H), 1.76 (m, 2H), 1.73 (m, 1H), 1.23 (m, 2H), 0.96 (m, 2H).

I.19 Benzyl 4-((4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylate

[0603] ##STR00102##

[0604] To a mixture of benzyl 4-[(4,5-dichloro-3-oxo-isothiazol-2-yl)methyl]cyclohexanecarboxylate (1.2 g, 2.70 mmol, 1 eq) in H.sub.2O (20 mL), DCM (10 mL) and ACN (10 mL) was added RuCl.sub.3.H.sub.2O (12.16 mg, 53.96 umol, 0.02 eq) and NaIO.sub.4 (3.46 g, 16.19 mmol, 896.96 uL, 6 eq) in one portion at 0° C., under N.sub.2. The mixture was stirred at 20° C., for 2 hours. TLC showed the reaction was completed (Petroleum ether:Ethyl acetate=2:1, Rf-pl=0.7). The mixture was poured into ice-water (30 mL) and stirred for 5 min. The aqueous phase was extracted with ethyl acetate (20 mL*2). The combined organic phases were washed with brine (20 mL), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether:Ethyl acetate=5:1 to 3:1) to afford benzyl 4-[(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)methyl]cyclohexanecarboxylate (0.8 g, 1.67 mmol, 61.73% yield, 90% purity) as white solid.

Example 7. 4-((4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)methyl)cyclohexane-1-carboxylic Acid

[0605] ##STR00103##

[0606] To a mixture of benzyl 4-[(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)methyl]cyclohexanecarboxylate (0.8 g, 1.67 mmol, 1 eq) in DCM (20 mL) was added MsOH (1.60 g, 16.65 mmol, 1.19 mL, 10 eq) in one portion at 30° C., under N.sub.2. The mixture was stirred at 30° C., for 16 hours. LCMS showed the reaction was completed. The mixture was poured into ice-water (5 mL) and concentrated under reduced pressure then a solid appeared. The solution was filtered and trituration by EtOAc (2 mL*3) and the filter cake was dried in vacuum to afford 4-[(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)methyl]cyclohexanecarboxylic acid (0.170 g, 486.86 umol, 29.23% yield, 98% purity) as white solid; LC-MS (ES, m/z): 364.0 [M+Na].sup.+; .sup.1H-NMR (300 MHz, DMSO-D6) δ 11.99 (bs, 1H), 3.49 (m, 2H), 2.11 (m, 1H), 1.88 (m, 2H), 1.79 (m, 2H), 1.66 (m, 1H), 1.23 (m, 2H), 0.96 (m, 2H).

I.20 Dibenzyl 3,3′-(((3,3′-disulfanediylbis(propanoyl))bis(azanediyl))bis(4,1-phenylene))dipropionate

[0607] ##STR00104##

[0608] To a solution of 3-(2-carboxyethyldisulfanyl)propanoic acid (651.56 mg, 3.10 mmol, 1 eq), HOBt (921.15 mg, 6.82 mmol, 2.2 eq) and TEA (1.25 g, 12.39 mmol, 1.73 mL, 4 eq) in DCM (50 mL) was added EDCI (1.31 g, 6.82 mmol, 2.2 eq) at 0° C. Then benzyl 4-(aminomethyl)cyclohexanecarboxylate; 4-methylbenzenesulfonic acid (2.6 g, 6.20 mmol, 2 eq) was added at this temperature. The mixture was stirred at 0-20° C., for 12 hrs. TLC (Petroleum ether:Ethyl acetate=2:1, Rf=0.5) indicated the reaction was completed. The mixture was poured into sat. NaHCO.sub.3 (20 mL) and H.sub.2O (20 mL) and the organic layer was separated. The aqueous layer was extracted with DCM (50 mL). The combined organic layers were washed with H.sub.2O (50 mL), brine (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum. Benzyl 4-[[3-[[3-[(4-benzyloxycarbonylcyclohexyl)methylamino]-3-oxo-propyl]disulfanyl]propanoyl amino]methyl]cyclohexanecarboxylate (1.1 g, 1.64 mmol, S3.07% yield) was obtained as a white solid; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.28-7.41 (m, 10H) 5.96 (br s, 2H) 5.10 (s, 4H) 3.13 (t, J=6.39 Hz, 4H) 2.98 (t, J=6.84 Hz, 4H) 2.57 (t, J=6.95 Hz, 4H) 2.23-2.34 (m, 2H) 2.03 (br d, J=12.79 Hz, 4H) 1.84 (br d, J=12.13 Hz, 4H) 1.37-1.63 (m, 6H) 0.91-1.05 (m, 4H).

I.21. Benzyl 3-(4-(5-chloro-3-oxoisothiazol-2(3H)-yl)phenyl)propanoate

[0609] ##STR00105##

[0610] To a solution of benzyl 3-[4-[3-[[3-[4-(3-benzyloxy-3-oxo-propyl)amino]-3-oxo-propyl]disulfanyl]propanoylamino]phenyl]propanoate (10 g, 14.60 mmol, 1 eq) in DCM (200 mL) was added sulfuryl chloride (5.91 g, 43.80 mmol, 4.38 mL, 3 eq) dropwise at 25° C., under N.sub.2. The solution was stirred at 25° C., for 8 hours. The color of solution changed from colorless to black when sulfuryl chloride was added and then changed to yellow after 1 hour. TLC (Petroleum ether:Ethyl acetate=2:1, Rf=0.60) showed starting material was consumed and two new main spots were generated. The residue was poured into ice-water (200 ml) and then concentrated in vacuum to remove DCM. After concentration, the aqueous phase was extracted with ethyl acetate (200 mL*3) and then the combined organic phase was washed with water (200 mL*l), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate=5:1 to 1:1) to get benzyl 3-[4-(3-oxoisothiazol-2-yl)phenyl]propanoate (2.5 g, 7.37 mmol, 50.47% yield) .sup.1H NMR (400 MHz, METHANOL-d4) δ=8.56 (d, J=6.2 Hz, 1H), 7.44-7.39 (m, 2H), 7.37-7.26 (m, 7H), 6.30 (d, J=6.4 Hz, 1H), 5.09 (s, 2H), 2.98 (t, J=7.4 Hz, 2H), 2.78-2.66 (m, 2H); and benzyl 3-[4-(5-chloro-3-oxo-isothiazol-2-yl)phenyl]propanoate (5 g, 13.37 mmol, 91.60% yield) was a yellow solid; .sup.1H NMR (ET17992-22-P2A) confirmed ET17992-22-P2. .sup.1H NMR (400 MHz, METHANOL-d4) δ=7.42-7.36 (m, 2H), 7.35-7.25 (m, 7H), 6.49-6.45 (m, 1H), 5.08 (s, 2H), 2.99-2.91 (m, 2H), 2.69 (t. J=7.5 Hz, 2H).

I.22. Benzyl 3-(4-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)phenyl)propanoate

[0611] ##STR00106##

[0612] To a mixture of benzyl 3-[4-(5-chloro-3-oxo-isothiazol-2-yl)phenyl]propanoate (1.4 g, 3.74 mmol, 1 eq) in H2O (12 mL), DCM (6 mL) and ACN (6 mL) was added NaIO.sub.4 (4.81 g, 22.47 mmol, 1.25 mL, 6 eq) in one portion at 25° C., and then the mixture was purged with N.sub.2 three times. Then, RuCl.sub.3.H.sub.2O (42.21 mg, 187.24 umol, 0.05 eq) was added under N.sub.2. The mixture was stirred at 25° C., for 12 hrs. The mixture turned turbidity and the color become to gray. The residue was poured into Ethyl acetate (100 ml) and then filtered. The filtrate was concentrated in vacuum. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether:Ethyl acetate=10:1 to 4:1) to give benzyl 3-[4-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)phenyl]propanoate (460 mg, 1.08 mmol, 28.75% yield, 95% purity) as a yellow solid; .sup.1H NMR (ET17992-63-P1A) confirmed ET17992-63-P1. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.41-7.30 (m, 7H), 6.84 (s, 1H), 5.13 (s, 2H), 3.04 (t, J=7.6 Hz, 2H), 2.72 (t, J=7.7 Hz, 2H).

Example 8. 3-(4-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)phenyl)propanoic Acid

[0613] ##STR00107##

[0614] A solution of benzyl 3-[4-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)phenyl]propanoate (460 mg, 1.13 mmol, 1 eq) in DCM (15 mL) was added methanesulfonic acid (1.09 g, 11.33 mmol, 806.87 uL, 10 eq) dropwise at 10° C. Then, the solution was heated to 35° C., and stirred for 12 hrs. The residue was washed with water (15 ml*3), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was poured into water (20 ml) and then filtered. The filter cake was dissolved in DCM (S ml) and then petroleum ether (30 ml) was poured into the residue, the solution was stirred at 10° C., for 2 min, and then filtered, the filter cake was dried in vacuum to give 3-[4-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)phenyl]propanoic acid (162 mg, 501.81 umol, 44.27% yield, 97.8% purity) as a white solid; LC-MS (ES, m/z): 313.9 .sup.1H-NMR (300 MHz, DMSO-D6) δ 12.18 (bs, 1H), 7.81 (s, 1H), 7.45 (m, 2H), 7.37 (m, 2H), 2.89 (m, 2H), 2.58 (m, 2H).

I.23. Benzyl 3-(4-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)phenyl)propanoate

[0615] ##STR00108##

[0616] To a mixture of benzyl 4-[(3-oxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (1.2 g, 3.32 mmol, 1 eq) in H.sub.2O (20 mL). ACN (10 mL) and DCM (10 mL) was added RuCl.sub.3.H.sub.2O (14.95 mg, 66.33 umol, 0.02 eq) and NaIO.sub.4 (4.26 g, 19.90 mmol, 1.10 mL, 6 eq) in one portion at 0° C., under N.sub.2. The mixture then heated to 20° C., and stirred for 16 hours. The mixture was filtered, and the filtrate was concentrated by nitrogen flow, and a solid appeared again and filtered, the filtrate was concentrated by nitrogen. The residue was purified by prep-TLC (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, Petroleum ether:Ethyl acetate=2:1) to afford benzyl 4-[(1,1,3-trioxoisothiazol-2-yl)methyl]cyclohexanecarboxylate (0.45 g, 1.11 mmol, 33.60% yield, 90% purity) as a colorless oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.53 (dd, J=3.2, 8.7 Hz, 2H), 7.17 (ddd, J=3.2, 7.6, 9.0 Hz, 2H), 6.97-6.89 (m, 2H), 5.56 (br s, 1H), 4.12-4.06 (m, 4H), 3.92 (s, 5H), 3.55 (q, J=5.1 Hz, 5H).

I.24. Benzyl 3-(4-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)phenyl)propanoate

[0617] ##STR00109##

[0618] To a mixture of benzyl 3-[4-(4,5-dichloro-3-oxo-isothiazol-2-yl)phenyl]propanoate (650 mg, 1.59 mmol, 1 eq) and NaIO.sub.4 (1.36 g, 6.37 mmol, 352.86 uL, 4 eq) in H.sub.2O (10 mL), DCM (5 mL), ACN (5 mL) was added RuCl.sub.3.H.sub.2O (7.18 mg, 31.84 umol, 0.02 eq) in one portion at 0° C., under N.sub.2. The mixture was stirred at 20° C., for 2 hrs. The residue was extracted with ethyl acetate (30 mL*2). The combined organic phase was dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether:Ethyl acetate=30:1 to 5:1) to give benzyl 3-[4-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)phenyl]propanoate (180 mg, 25.68% yield) as a yellow solid; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.43-7.29 (m, 9H), 5.13 (s, 2H), 3.05 (t, J=7.6 Hz, 2H), 2.73 (t, J=7.6 Hz, 2H).

Example 9. 3-(4-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)phenyl)propanoic Acid

[0619] ##STR00110##

[0620] A solution of benzyl 3-[4-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)phenyl]propanoate (180 mg, 408.81 umol, 1 eq) in DCM (5 mL) was added methanesulfonic acid (392.89 mg, 4.09 mmol, 291.03 uL, 10 eq) dropwise at 10° C. The solution was stirred at 35° C., for 12 hrs. The residue was poured into water (5 ml) and then filtered. The filter cake was washed with water (10 ml*3) and then dried in vacuum. The residue was dissolved into Dichloromethane:Methanol (5.5 ml, v/v=10:1) and then purified by prep-TLC (Ethyl acetate, Rf=0.26) to give 3-[4-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)phenyl]propanoic acid (S7.64 mg, 156.83 umol, 38.36% yield, 95.278% purity) as a white solid; LC-MS (ES, m/z): 347.9 [M−H]−; .sup.1H NMR (400 MHz, DMSO-d6) δ=12.22 (br s, 1H), 7.51-7.40 (m, 4H), 2.90 (br t, J=7.6 Hz, 2H), 2.60 (br t, J=7.6 Hz, 2H).

I.25. Dibenzyl 4,4′-((3,3′-disulfanediylbis(propanoyl))bis(azanediyl))dibenzoate

[0621] ##STR00111##

[0622] To a solution of 3-(2-carboxyethyldisulfanyl)propanoic add (6.01 g, 28.60 mmol, 1 eq) and pyridine (14.93 g, 188.77 mmol, 15.24 mL, 6.6 eq) in DMF (120 mL) was added EDO (12.06 g, 62.92 mmol, 2.2 eq) and benzyl 4-aminobenzoate (13 g, 57.20 mmol, 2 eq) at 10° C. Then, the mixture was stirred at 50° C., for 12 hrs. The residue was poured into ice-water (200 mL) and stirred for 20 min. The aqueous phase was extracted with ethyl acetate (200 mL*3). The combined organic phase was washed with sat. NaCl (200 mL*3), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. Then, the residue was recrystallized from Petroleum ether:DCM=50:1 to get the solid. The solid was washed petroleum three times (150 ml*3), and then dried in vacuum to give benzyl 4-[3-[[3-(4-benzyloxycarbonylamino)-3-oxo-propyl]disulfanyl]propanoylamino]benzoate (14 g, crude) as a white solid; .sup.1H NMR (400 MHz, DMSO-d6) 5=10.37 (s, 2H), 8.02-7.83 (m, 4H), 7.72 (d, J=8.8 Hz, 4H), 7.48-7.32 (m, 10H), 5.31 (s, 4H), 3.05-2.98 (m, 4H), 2.81-2.75 (m, 4H).

I.26. Benzyl 4-(5-chloro-3-oxoisothiazol-2(3H)-yl)benzoate

[0623] ##STR00112##

I.27, Benzyl 4-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)benzoate

[0624] ##STR00113##

[0625] To a solution of benzyl 4-[3-[[3-(4-benzyloxycarbonylamino)-3-oxo-propyl]disulfanyl]propanoylamino]benzoate (9.4 g, 14.95 mmol, 1 eq) in DCM (120 mL) was added dropwise sulfuryl chloride (10.09 g, 74.75 mmol, 7.47 mL, 5 eq) at 0° C. The mixture was stirred at 0-20° C., for 12 hrs. The mixture turned to clear after stirring for several minutes. TLC (Petroleum ether:Ethyl acetate=2:1) indicated the reaction was completed. The mixture was poured into H.sub.2O (200 mL), extracted with DCM (200 mL*2). The combined organic layers were washed with H.sub.2O (200 mL*2), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography on silica gel (Petroleum ether:Ethyl acetate=5:1 to 1:1) to give benzyl 4-(3-oxoisothiazol-2-yl)benzoate (1.2 g, 3.43 mmol, 11.47% yield, 89% purity) was obtained as an off-white solid; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.09-8.27 (m, 3H) 7.63-7.82 (m, 2H) 7.32-7.52 (m, 5H) 6.34 (br d, J=6.36 Hz, 1H) 5.39 (s, 2H); Benzyl 4-(5-chloro-3-oxo-isothiazol-2-yl)benzoate (3.5 g, 10.01 mmol, 33.49% yield, 98.92% purity) was obtained as an off-white solid; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.15 (d, J=8.60 Hz, 2H) 7.69 (d, J=8.82 Hz, 2H) 7.33-7.49 (m, 4H) 6.38 (s, 1H) 5.38 (s, 2H) and Benzyl 4-(4,5-dichloro-3-oxo-isothiazol-2-yl)benzoate (2.8 g, 7.19 mmol, 24.06% yield, 97.68% purity) was obtained as an off-white solid; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.18 (d, J=8.60 Hz, 2H) 7.71 (d, J=8.60 Hz, 2H) 7.33-7.50 (m, 4H) 5.39 (s, 2H).

I.28. Benzyl 4-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)benzoate

[0626] ##STR00114##

[0627] To a mixture of benzyl 4-(5-chloro-3-oxo-isothiazol-2-yl)benzoate (1 g, 2.89 mmol, 1 eq) in H.sub.2O (10 mL), ACN (5 mL) and DCM (5 mL) was added RuCl.sub.3.H.sub.2O (13.04 mg, 57.84 umol, 0.02 eq) and NaIO.sub.4 (2.47 g, 11.57 mmol, 640.97 uL, 4 eq) in one portion at 0° C., under N.sub.2. The mixture then heated to 20° C., and stirred for 2 hours. The residue was filtered and the filtrate was poured into water (40 ml). The aqueous phase was extracted with ethyl acetate (50 mL*l). The organic phase was washed with sat. NaCl (30 mL*3), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether:Ethyl acetate=15:1 to 5:1) to give benzyl 4-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)benzoate (500 mg, 1.32 mmol, 45.77% yield) as a yellow oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=8.27-8.21 (m, 2H), 7.61-7.55 (m, 2H), 7.49-7.34 (m, 5H), 6.87 (s, 1H), 5.40 (s, 2H).

Example 10. 4-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)benzoic Acid

[0628] ##STR00115##

[0629] A solution of benzyl 4-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)benzoate (450 mg, 1.19 mmol, 1 eq) in DCM (20 mL) was added methanesulfonic acid (1.14 g, 11.91 mmol, 847.94 uL, 10 eq) dropwise at 10° C. The solution was stirred at 35° C., for 10 hrs. The residue was concentrated in vacuum to remove DCM. Then, the residue was dissolved into Ethyl acetate (10 ml) and then the organic phase was washed with water (20 mL*5), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was dissolved into methanol (3 ml) and then petroleum ether (30 ml) was poured into the residue, the solution was stirred at 10° C., for 2 min, and then filtered, the filter cake was dried in vacuum to give 4-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)benzoic acid (112.56 mg, 379.48 umol, 31.86% yield, 96.986% purity) as a white solid; LC-MS (ES, m/z): 285.9 [M−H]−; .sup.1H NMR (400 MHz, DMSO-d6) δ=13.33 (br s, 1H), 8.17-8.11 (m, 2H), 7.87 (d, J=1.5 Hz, 1H), 7.67-7.61 (m, 2H).

I.29. Benzyl 4-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)benzoate

[0630] ##STR00116##

[0631] To a mixture of benzyl 4-(4,5-dichloro-3-oxo-isothiazol-2-yl)benzoate (I g, 2.63 mmol, 1 eq) in H.sub.2O (10 mL), ACN (5 mL) and DCM (5 mL) was added RuCl.sub.3.H.sub.2O (11.86 mg, 52.60 umol, 0.02 eq) and NaIO.sub.4 (2.25 g, 10.52 mmol, 582.91 uL, 4 eq) in one portion at 0° C., under N.sub.2. The mixture then heated to 20° C., and stirred for 2 hours. The residue was filtered and the filtrate was concentrated in vacuum. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether:Ethyl acetate=20:1 to 5:1) to give benzyl 4-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)benzoate (130 mg, 315.35 umol, 11.99% yield) as a white solid; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=8.25 (d, J=8.6 Hz, 2H), 7.58 (d, J=8.8 Hz, 2H), 7.49-7.34 (m, 5H), 5.41 (s, 2H).

Example 11. 4-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)benzoic Acid

[0632] ##STR00117##

[0633] A solution of benzyl 4-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)benzoate (130 mg, 315.35 umol, 1 eq) in DCM (5 mL) was added methanesulfonic acid (303.07 mg, 3.15 mmol, 224.49 uL, 10 eq) dropwise at 10° C. The solution was stirred at 10° C., for 5 min, then the solution was heated to 35° C., and stirred for 10 hours. The color of solution changed from colorless to yellow. The reaction solution was poured into water (20 ml) and then filtered. The filter cake was washed with water (10 ml*3) and DCM (10 ml*3) three times respectively. Then the filter cake was dried in vacuum. The residue was dispersed with DCM (10 ml) and then petroleum ether (30 ml) was poured into the residue, the mixture was stirred at 10° C., for 2 min, and then filtered, the filter cake was dried in vacuum to give 4-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)benzoic acid (93.4 mg, 282.97 umol, 89.73% yield, 97.590% purity) as a white solid; LC-MS (ES, m/z): 319.9 [M−H]−; .sup.1H NMR (400 MHz, DMSO-d6) δ=8.16 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.4 Hz, 2H).

I.30. Benzyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate 4-methylbenzenesulfonate

[0634] ##STR00118##

[0635] A mixture of 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoic acid (4 g, 18.08 mmol, 1 eq), phenylmethanol (15.64 g, 144.63 mmol, 15.04 mL, 8 eq) and TsOH.H.sub.2O (3.61 g, 18.98 mmol, 1.05 eq) in toluene (30 mL) was stirred at 140° C., for 8 hours using a Dean and Stark apparatus to collect the water of condensation. The reaction turned to clear after refluxing for several hours. TLC (Dichloromethane:Methanol=10:1, Rf=0.3) indicated the reaction was complete. The clear reaction mixture was poured into TBME:Petroleum ether (1:1, 50 mL) and removed the clear solution. The residue was washed with TBME:Petroleum ether (1:1, 50 mL) for 2 times and dried in vacuum. The crude product Benzyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoate; 4-methylbenzenesulfonic acid (8.9 g, crude) was obtained as a yellow oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.76 (br d, J=8.07 Hz, 2H) 7.33-7.38 (m, 5H) 7.15 (d, J=7.95 Hz, 2H) 5.11 (s, 2H) 3.72 (q, J=6.11 Hz, 4H) 3.53-3.64 (m, 8H) 3.11-3.24 (m, 2H) 2.53-2.69 (m, 2H) 2.30-2.41 (m, 1H) 2.34 (s, 3H).

I.31. Benzyl 3-[2-[2-[2-[3-[[3-[2-[2-[2-(3-benzyloxy-3-oxo-propoxy)ethoxy]ethoxy]ethyl amino]-3-oxopropyl]disulfanyl]propanoylamino]ethoxy]ethoxy]ethoxy]propanoate

[0636] ##STR00119##

[0637] To a mixture of 3-(2-carboxyethyldisulfanyl)propanoic acid (1.90 g, 9.04 mmol, 1 eq), HOBt (2.69 g, 19.88 mmol, 2.2 eq) and TEA (4.57 g, 45.18 mmol, 6.29 mL, 5 eq) in DCM (100 mL) was added EDCI (3.81 g, 19.88 mmol, 2.2 eq) at 20° C. Then benzyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propanoate; 4-methylbenzenesulfonic acid (8.74 g, 18.07 mmol, 2 eq) was added to the above solution. The mixture was stirred at 20° C., for 12 hours. TLC (Petroleum ether:Ethyl acetate=2:1, Rf=0.25) indicated that the reaction was complete. MeOH was added and the solution was concentrated under reduced pressure and dried over vacuum. The residue was poured into H.sub.2O (20 mL), extracted with EtOAc (50 mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The crude product was purified by prep-TLC (Petroleum ether:Ethyl acetate=1:1, Rf=0.5) to give Benzyl 3-[2-[2-[2-[3-[[3-[2-[2-[2-(3-benzyloxy-3-oxo-propoxy)ethoxy]ethoxy]ethylamino]-3-oxo-propyl]disulfanyl]propanoyl amino]ethoxy]ethoxy]ethoxy]propanoate (6.52 g, 7.94 mmol, 87.81% yield, 97% purity) as a yellow oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.29-7.42 (m, 10H) 6.42 (br s, 2H) 5.15 (s, 4H) 3.79 (t, J=6.42 Hz, 4H) 3.53-3.69 (m, 18H) 2.92-3.00 (m, 4H) 2.62-2.71 (m, 4H) 2.53-2.62 (m, 4H).

I.32. Benzyl 3-(2-(2-(2-(5-chloro-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoate

[0638] ##STR00120##

[0639] To a solution of benzyl 3-[2-[2-[2-[3-[[3-[2-[2-[2-(3-benzyloxy-3-oxo-propoxy)ethoxy]ethoxy]ethylamino]-3-oxopropyl]disulfanyl]propanoylamino]ethoxy]ethoxy]ethoxy]propanoate (6.4 g, 8.03 mmol, 1 eq) in DCM (50 mL) was added dropwise the solution of sulfuryl chloride (4.34 g, 32.12 mmol, 3.21 mL, 4 eq) in DCM (10 mL) at 0° C. The mixture was stirred at 0-10° C., for 12 hrs. TLC (Petroleum ether:Ethyl acetate=0:1, Rf=0.15, 0.35) indicated the reaction was complete. The mixture was poured into ice/water (100 mL), extracted with DCM (200 mL*2). The combined organic layers were washed with H.sub.2O (100 mL*2), brine (100 mL), dried over Na.sub.2SO.sub.4. Filtration and concentrated in vacuum. The residue was purified by column chromatography on silica gel (Petroleum ether:Ethyl acetate=1:1 to 0:1) to give Benzyl 3-[2-[2-[2-(3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (820 mg, 1.66 mmol, 10.33% yield, 80% purity) as a brown oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.06 (d, J=6.17 Hz, 1H) 7.30-7.41 (m, 5H) 6.24 (d, J=6.39 Hz, 1H) 5.15 (s, 2H) 3.95-4.03 (m, 2H) 3.79 (t, J=6.39 Hz, 2H) 3.68-3.75 (m, 2H) 3.59-3.68 (m, 8H) 2.63-2.70 (m, 2H) and Benzyl 3-[2-[2-[2-(5-chloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (2.5 g, 4.30 mmol, 26.79% yield, 74% purity) as a colorless oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.28-7.43 (m, 5H) 6.25 (s, 1H) 5.15 (s, 2H) 3.92-3.98 (m, 2H) 3.77-3.81 (m, 2H) 3.59-3.71 (m, 10H) 2.66 (t, J=6.39 Hz, 2H).

I.33. Benzyl 3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoate

[0640] ##STR00121##

[0641] To a mixture of benzyl 3-[2-[2-[2-(5-chloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (I g, 2.33 mmol, 1 eq) and NaIO.sub.4 (1.99 g, 9.30 mmol, 515.57 uL, 4 eq) in H.sub.2O (20 mL), DCM (10 mL), ACN (10 mL) was added RuCl.sub.3.H.sub.2O (26.22 mg, 116.30 umol, 0.05 eq) in one portion at 20° C., under N.sub.2. Then, the mixture was stirred at 20° C., for 1 hr. The residue was extracted with ethyl acetate (20 mL*2). The combined organic phase was dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether:Ethyl acetate=5:1 to 1:1) to give benzyl 3-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (560 mg, 1.21 mmol, 52.12% yield, 100% purity) as a purple oil: .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.42-7.29 (m, 5H), 6.70 (s, 1H), 5.15 (s, 2H), 3.92-3.86 (m, 2H), 3.77 (td, J=6.0, 11.9 Hz, 4H), 3.68-3.58 (m, 8H), 2.67 (t, J=6.5 Hz, 2H).

Example 12. 3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoic Acid

[0642] ##STR00122##

[0643] A solution of benzyl 3-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (560 mg, 1.21 mmol, 1 eq) in DCM (30 mL) was added methanesulfonic acid (1.17 g, 12.12 mmol, 863.06 uL, 10 eq) dropwise at 10° C. The mixture was heated to 35° C., and stirred for 10 hrs. The color of solution turned to yellow. The residue was washed with water (30 ml*3) and then the organic phase was dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by prep-TLC (Ethyl acetate:Ethyl acetate:Methanol:Acetic acid=40:8:1, Rf=0.77) to give 3-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy] propanoic acid (95.61 mg, 240.16 umol, 19.81% yield, 93.389% purity) as a colorless oil; LC-MS (ES, m/z): 372.1 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d6) 5=12.13 (br s, 1H), 7.64 (s, 1H), 3.86-3.74 (m, 2H), 3.61 (td, J=6.0, 16.9 Hz, 4H), 3.54-3.46 (m, 8H), 2.43 (t, J=6.4 Hz, 2H).

I.34. Benzyl 3-(2-(2-(2-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoate

[0644] ##STR00123##

[0645] To a solution of benzyl 3-[2-[2-[2-(5-chloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy] propanoate (1.6 g, 3.72 mmol, 1 eq) in DCM (30 mL) was added dropwise sulfuryl chloride (1.00 g, 7.44 mmol, 744.17 uL, 2 eq) at 0° C. The mixture was stirred at 0-10° C., for 12 hrs. A clear pale yellow solution was obtained after the addition of sulfuryl chloride. TLC (Ethyl acetate:Petroleum ether=2:1, Rf=0.5) indicated the reaction was complete. The mixture was concentrated in vacuum to give a crude product. The residue was poured into H.sub.2O (50 mL), extracted with DCM (50 mL*2). The combined organic layers were washed with H.sub.2O (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography on silica gel (Ethyl acetate:Petroleum ether=1:2 to 2:1) to give Benzyl 3-[2-[2-[2-(4,5-dichloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy] propanoate (1.06 g, 1.76 mmol, 47.17% yield, 76.9% purity) as a colorless oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.28-7.40 (m, 4H) 5.13 (s, 2H) 3.98-4.04 (m, 2H) 3.77 (t, J=6.39 Hz, 2H) 3.67-3.72 (m, 2H) 3.57-3.67 (m, 8H) 2.65 (t, J=6.39 Hz, 2H).

I.35. Benzyl 3-(2-(2-(2-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy) ethoxy)propanoate

[0646] ##STR00124##

[0647] To a mixture of benzyl 3-[2-[2-[2-(4,5-dichloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy] propanoate (1 g, 2.15 mmol, 1 eq) and NaIO.sub.4 (1.84 g, 8.61 mmol, 477.32 uL, 4 eq) in H.sub.2O (20 mL), CH.sub.3CN (10 mL) and DCM (10 mL) was added RuCl.sub.3.H.sub.2O (7.28 mg, 32.30 umol, 0.015 eq) under N.sub.2 at 0° C. The mixture was stirred at 0-10 for 2 hrs. TLC indicated the reaction was complete. The mixture was diluted with EtOAc (50 mL), filtered to remove the unsoluble solid. The organic layer was separated and concentrated in vacuum. The residue was purified by prep-TLC (Petroleum ether:Ethyl acetate=1:1, Rf=0.6) to give Benzyl 3-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (830 mg, 1.61 mmol, 74.96% yield, 96.533% purity) as a colorless oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.28-7.41 (m, 5H) 5.15 (s, 2H) 3.91-3.97 (m, 2H) 3.75-3.83 (m, 4H) 3.59-3.68 (m, 8H) 2.66 (t, J=6.50 Hz, 2H).

Example 13. 3-(2-(2-(2-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanoic Acid

[0648] ##STR00125##

[0649] A solution of benzyl 3-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoate (820.00 mg, 1.65 mmol, 1 eq) in DCM (10 mL) was added methanesulfonic acid (1.59 g, 16.52 mmol, 1.18 mL, 10 eq) dropwise at 10° C. Then, the solution was heated at 35° C., and stirred for 10 hrs. The residue was diluted by DCM (20 ml) and then the solution was washed with water (15 ml*3), the organic phase was dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by prep-TLC (Ethyl acetate:Acetic acid=250:1, Rf=0.55) to 3-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]propanoic acid (249.4 mg, 602.76 umol, 36.49% yield, 98.180% purity) as a yellow oil; LC-MS (ES, m/z): 406.0 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d6) δ=12.16 (br s, 1H), 3.85 (t, J=5.5 Hz, 2H), 3.65 (br t, J=5.4 Hz, 2H), 3.61-3.45 (m, 10H), 2.43 (t, J=6.3 Hz, 2H).

I.36. Benzyl 1-amino-3,6,9,12,15,18-hexaoxahenicosan-21-oate 4-methylbenzenesulfonate

[0650] ##STR00126##

[0651] A mixture of phenylmethanol (2.45 g, 22.64 mmol, 2.35 mL, 8 eq), 3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (1 g, 2.83 mmol, 1 eq) and TsOH.H.sub.2O (565.16 mg, 2.97 mmol, 1.05 eq) in toluene (30 mL) was stirred at 140° C., with a Dean-Stark trap for 14 hrs. The mixture was changed from turbidity to clearly several hours later. The residue was concentrated in vacuum to remove toluene, and then TBME (50 ml) was poured into the residue and stirred for 1 min. Then, supernatant was remove and dried to give benzyl 3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate; 4-methylbenzene sulfonic acid (1.45 g, crude) as a yellow oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.80 (d, J=8.1 Hz, 2H), 7.67-7.46 (m, 2H), 7.39-7.31 (m, 5H), 7.15 (d, J=7.8 Hz, 2H), 5.13 (s, 2H), 3.96-3.83 (m, 2H), 3.75-3.50 (m, 22H), 3.24-3.14 (m, 2H), 2.63 (t, J=6.2 Hz, 2H), 2.34 (s, 3H).

I.37. Dibenzyl 23,30-dioxo-4,7,10,13,16,19,34,37,40,43,46,49-dodecaoxa-26,27-dithia-22,31-diazadopentacontanedioate

[0652] ##STR00127##

[0653] To a mixture of 3-(2-carboxyethyldisulfanyl)propanoic acid (47.41 mg, 225.47 umol, 1 eq) and TEA (91.26 mg, 901.86 umol, 125.53 uL, 4 eq), HOBt (91.40 mg, 676.40 umol, 3 eq), EDCI (129.67 mg, 676.40 umol, 3 eq) in DCM (5 mL) was added benzyl 3-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate; 4-methyl benzenesulfonic acid (277.65 mg, 450.93 umol, 2 eq) dropwise at 25° C. After addition, the mixture was stirred at 25° C., for 8 hours. TLC (Ethyl acetate:Methanol=3:1, Rf=0.33) showed starting material was consumed and a new main spot was generated. The residue was poured into sat. NaCl (10 ml) and stirred for 2 min. Then, the aqueous phase was extracted with DCM (5 mL*3). The combined organic phase was washed with sat. NaCl (10 mL*2), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 5u; mobile phase: [water (10 mM NH4HCO3)-ACN]; B %: 32%-62%, 12 min) to give benzyl 3-(2-[2-[2-[2-[2-[2-[3-[[3-[2-[2-[2-[2-[2-[2-(3-benzyloxy-3-oxo-propoxy)ethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-3-oxo-propyl]disulfanyl]propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] propanoate (70 mg, 65.96 umol, 29.25% yield) as a colorless oil: .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.41-7.29 (m, 10H), 6.50 (br s, 2H), 5.14 (s, 4H), 3.78 (t, J=6.5 Hz, 4H), 3.67-3.61 (m, 40H), 3.59-3.55 (m, 4H), 3.45 (q, J=5.1 Hz, 4H), 2.97 (t, J=7.2 Hz, 4H), 2.66 (t, J=6.5 Hz, 4H), 2.60 (t, J=7.1 Hz, 4H).

I.38. Benzyl 1-(5-chloro-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oate

[0654] ##STR00128##

[0655] To a solution of benzyl 3-[2-[2-[2-[2-[2-[2-[3-[[3-[2-[2-[2-[2-[2-[2-(3-benzyloxy-3-oxo-propoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethylamino]-3-oxo-propyl]disulfanyl]propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-propanoate (5.5 g, 5.18 mmol, 1 eq) in DCM (60 mL) was added dropwise su I fury I chloride (3.50 g, 25.91 mmol, 2.59 mL, 5 eq) at 0° C. The mixture was stirred at 0-20° C., for 12 hrs. TLC (Ethyl acetate:Methanol=10:1, Rf=0.3, 0.5) indicated the reaction was completed. The mixture was poured into ice/water (10 mL), extracted with DCM (20 mL*2). The combined organic layers were washed with H.sub.2O (20 mL*2), brine (20 mL), dried over Na.sub.2SO.sub.4. Filtration and concentrated in vacuum. The residue was purified by column chromatography on silica gel (Petroleum ether:Ethyl acetate=1:1 to 0:1) to give Benzyl 3-[2-[2-[2-[2-[2-[2-(3-oxoisothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1.4 g, 2.29 mmol, 22.05% yield, 86.148% purity) as a brown oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.02 (d, J=6.17 Hz, 1H) 7.22-7.32 (m, 5H) 6.17 (br d, J=6.17 Hz, 1H) 5.07 (s, 2H) 3.92 (br t, J=4.30 Hz, 2H) 3.51-3.73 (m, 24H) 2.58 (t, J=6.39 Hz, 2H) and Benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (3.1 g, 4.44 mmol, 42.85% yield, 80.532% purity) as a colorless oil: .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.30-7.40 (m, 5H) 6.26 (s, 1H) 5.15 (s, 2H) 3.93-3.99 (m, 2H) 3.78 (t, J=6.50 Hz, 2H) 3.60-3.72 (m, 23H) 2.66 (t, J=6.50 Hz, 2H).

I.39 Benzyl 1-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oate

[0656] ##STR00129##

[0657] To a mixture of benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1 g, 1.78 mmol, 1 eq) and NaIO.sub.4 (1.52 g, 7.12 mmol, 394.34 uL, 4 eq) in H.sub.2O (20 mL), DCM (10 mL), ACN (10 mL) was added RuCl.sub.3.H.sub.2O (20.05 mg, 88.96 umol, 0.05 eq) in one portion at 20° C., under N.sub.2. Then, the mixture was stirred at 20° C., for 1 hr. The residue was extracted with ethyl acetate (20 mL*2). The combined organic phase was dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether:Ethyl acetate=1:1 to Ethyl acetate:Methanol=10:1) to give benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (S20 mg, 849.06 umol, 47.72% yield, 97% purity) as a yellow oil: .sup.1H NMR (400 MHz, CHLOROFORM-d) 5=7.44-7.29 (m, 5H), 6.72 (s, 1H), 5.14 (s, 2H), 3.92-3.86 (m, 2H), 3.80-3.73 (m, 4H), 3.69-3.59 (m, 20H), 2.66 (t, J=6.4 Hz, 2H).

Example 14, 1-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oic Acid

[0658] ##STR00130##

[0659] A solution of benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (520 mg, 875.32 umol, 1 eq) in DCM (30 mL) was added methanesulfonic acid (841.23 mg, 8.75 mmol, 623.13 uL, 10 eq) dropwise at 10° C. The solution was heated to 35° C., and stirred for 10 hrs. The residue was washed with water (30 ml*3) and then the organic phase was dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by prep-TLC (Ethyl acetate:Methanol:Acetic acid=40:8:1, Rf=0.58). The residue was purified again by prep-HPLC (column: Nano-micro Kromasil C18 100*30 mm 5 μm; mobile phase: [water (0.05% HCl)-ACN]; B %: 1%-30%, 10 min) to give 3-[2-[2-[2-[2-[2-[2-(5-chloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (55.93 mg, 106.24 umol, 12.14% yield, 95.726% purity) as a colorless oil; LC-MS (ES, m/z): 504.2 [M+H].sup.+; .sup.1H NMR (400 MHz, DMSO-d6) δ=12.30-11.96 (m, 1H), 7.64 (s, 1H), 3.83-3.76 (m, 2H), 3.65-3.58 (m, 4H), 3.54-3.52 (m, 2H), 3.52-3.48 (m, 18H), 2.44-2.42 (m, 2H).

I.40 Benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy] ethoxy]ethoxy]ethoxy]propanoate

[0660] ##STR00131##

[0661] A solution of benzyl 3-[2-[2-[2-[2-[2-[2-(5-chloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (1.3 g, 2.31 mmol, 1 eq) in DCM (30 mL) was added sulfuryl chloride (624.34 mg, 4.63 mmol, 462.47 uL, 2 eq) dropwise at 20° C. The solution was stirred at 20° C., for 2 hrs. The solution turned to yellow. The residue was poured into ice-water (30 ml) and stirred for 30 min. The DCM phase was washed with water (50 mL*6), dried with anhydrous Na2SO4, filtered and concentrated in vacuum. The residue was purified by prep-TLC (Ethyl acetate:Methanol=10:1, Rf=0.50) to give benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (800 mg, 1.21 mmol, 52.19% yield, 90% purity) as a yellow oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) 6=7.40-7.29 (m, 5H), 5.15 (s, 2H), 4.04 (t, J=4.7 Hz, 2H), 3.78 (t, J=6.4 Hz, 2H), 3.72 (t, J=4.7 Hz, 2H), 3.69-3.63 (m, 16H), 3.62 (s, 4H), 2.66 (t, J=6.5 Hz, 2H).

I.41 Benzyl 1-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oate

[0662] ##STR00132##

[0663] To a mixture of benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-3-oxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (600 mg, 1.01 mmol, 1 eq) and NaIO.sub.4 (860.56 mg, 4.02 mmol, 222.94 uL, 4 eq) in H.sub.2O (20 mL), DCM (10 mL), ACN (10 mL) was added RuCl.sub.3.H.sub.2O (11.34 mg, 50.29 umol, 0.05 eq) in one portion at 0° C., under N.sub.2. The mixture was stirred at 0° C., for 2 min, then heated to 25° C., and stirred for 1 hour. The residue was poured into Ethyl acetate (30 ml), and then filtered. The filtrate was extracted with ethyl acetate (30 mL*3). The combined organic phase was concentrated in vacuum. The residue was purified by prep-TLC (Ethyl acetate, Rf=0.50) to give benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (220 mg, 350.03 umol, 34.80% yield, 100% purity) as a colorless oil; .sup.1H NMR (400 MHz, CHLOROFORM-d) δ=7.40-7.27 (m, 4H), 5.15 (s, 2H), 3.99-3.90 (m, 2H), 3.78 (t, J=6.2 Hz, 4H), 3.70-3.58 (m, 20H), 2.66 (t, J=6.4 Hz, 2H).

Example 15, 1-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-3,6,9,12,15,18-hexaoxahenicosan-21-oic Acid

[0664] ##STR00133##

[0665] A solution of benzyl 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoate (220 mg, 350.03 umol, 1 eq) in DCM (5 mL) was added methanesulfonic acid (504.60 mg, 5.25 mmol, 373.78 uL, eq) dropwise at 10° C. Then, the solution was heated to 40° C., and stirred for 20 hrs. The residue was diluted by DCM (20 ml) and then the solution was washed with water (15 ml*3), the organic phase was dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (column: Nano-micro Kromasil C18 100*30 mm 5 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 25%-55%, 10 min) to give 3-[2-[2-[2-[2-[2-[2-(4,5-dichloro-1,1,3-trioxo-isothiazol-2-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid (53.79 mg, 98.63 umol, 28.18% yield, 98.724% purity) as a yellow oil: LC-MS (ES, m/z): 538.2 [M+H].sup.+; 1H NMR (400 MHz, DMSO-d6) δ=12.13 (br s, 1H), 3.89-3.81 (m, 2H), 3.65 (t, J=5.5 Hz, 2H), 3.59 (t, J=6.4 Hz, 2H), 3.56-3.48 (m, 20H), 2.43 (t, J=6.4 Hz, 2H).

Example 16

[0666] ##STR00134##

[0667] In a flask under argon was added product 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl) hexanoic acid (12.58 mg, 0.045 mmol), DCM (2 mL) and DMF (10 μl). The mixture was cooled to 0° C., then oxalyl dichloride (11.65 μl, 0.136 mmol) was added dropwise. The mixture was warmed up to rt and was stirred until complete conversion was observed by LCMS (follow-up by LCMS by adding to the aliquot dry MeOH to form the methyl ester). The crude was evaporated under vacuo. The residue was taken in DCM and dried again under vacuo to give a yellow solid. The crude material was used without further purification for the next step.

2. SYNTHESIS OF THE DRUG-LINKER CONJUGATES

Example A. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-<(4-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-N-methylhexanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0668] ##STR00135##

Standard Procedure for the Synthesis of Drug-Linkers:

[0669] In a flask under nitrogen, were added at rt 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (205 mg, 0.73 mmol) (Example 1), dichloromethane (10 mL) and DMF (100 μl). The mixture was cooled to 0° C., using an ice bath, then oxalyl chloride was added (190.4 μl, 2.18 mmol). The mixture was warmed up to rt and was stirred for 2 h. The reaction mixture was evaporated under vacuum. The residue was taken in CH.sub.2Cl.sub.2 and dried again under vacuum to give 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoyl chloride as a yellow solid. In a vial under N.sub.2 at rt were introduced (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid, (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methy)amino)phenethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid compound with 2,2,2-trifluoroacetic acid (1:1) (111 mg, 0.102 mmol) and dichloromethane (3.7 mL). The mixture was cooled to 0° C., and DIPEA (70.9 μl, 0.406 mmol) was added. The reaction mixture was stirred for 10 min at 0° C., then 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoyl chloride (36.6 mg, 0.122 mmol) was added dropwise as a solution in DCM (248 mg of acid chloride in 2 mL of DCM). The mixture was stirred at 0° C., for 1 h 15. The reaction was stopped by adding trifluoroacetic acid (32.9 μl, 0,426 mmol), acetonitrile (2.1 mL) and water (0.3 mL) into the mixture at 0° C. The crude material was concentrated in vacuum and the residue purified by preparative HPLC (Column X-Bridge C18 (100*30) using a gradient of ACN and water with 0.1% TFA as a mobile phase) to give ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-N-methylhexanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine. The mass spectrum and the .sup.1H-NMR spectrum of this drug-linker conjugate are represented respectively on FIGS. 1A and 1B.

Example B. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-(6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)-N-methylhexanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0670] ##STR00136##

[0671] It was synthesized following the standard procedure for the synthesis of drug-linkers using 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 2) and ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)—N,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine compound with 2,2,2-trifluoroacetic acid (1:1) as starting materials.

[0672] The mass spectrum of this drug-linker conjugate is represented on FIG. 2.

Example C. ((2R,3R)-3-((S)-1-<(3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)beazyl)oxy)carbonyl)(metliyl)amino)pheaethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0673] ##STR00137##

[0674] It was obtained following the standard procedure for drug-linker synthesis using 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 1) and ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl) (methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine as starting materials.

[0675] The mass spectrum and the .sup.1H-NMR spectrum of this drug-linker conjugate are represented respectively on FIGS. 3A and 3B.

Example D. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine

[0676] ##STR00138##

[0677] It was obtained following the standard procedure for drug-linker synthesis using 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 2) and ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine as starting materials.

[0678] The mass spectrum and the .sup.1H-NMR spectrum of this drug-linker conjugate are represented respectively on FIGS. 4A and 4B.

Example E. ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-(3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine 2,2,2-trifluoroacetic Acid Salt

[0679] ##STR00139##

[0680] Synthesized following the standard procedure for the synthesis of drug-linkers using 3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy) propanoic acid (Example 12) and (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((4-((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5 ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3-methylbutanamido)-N3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid as starting materials.

[0681] The mass spectrum and the .sup.1H-NMR spectrum of this drug-linker conjugate are represented respectively on FIGS. 5A and 5B.

Example F. ((2R,3R)-3-(1-((3R,4R,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-(3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy)propanamido)-3-methylbutanamido)-N-methylpropanamido)plienethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-D-phenylalanine

[0682] ##STR00140##

[0683] Synthesized following the standard procedure for the synthesis of drug-linkers using 3-(2-(2-(2-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)ethoxy)ethoxy)ethoxy) propanoic acid (Example 12) and ((2R,3R)-3-(1-((3R,4R,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-amino-3-methylbutanamido)-N-methylpropanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-D-phenylalanine as starting materials.

[0684] The mass spectrum and the .sup.1H-NMR spectrum of this drug-linker conjugate are represented respectively on FIGS. 6A and 6B.

Example G. ((2R,3R)-3-(1-((3R,4R,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-N-methylpropanamido)phenethyl)(methyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-D-phenylalanine

[0685] ##STR00141##

[0686] Synthesized following the standard procedure for the synthesis of drug-linkers using 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (Example 1) and ((2R,3R)-3-(1-((3R,4R,5S)-4-((S)-2-((S)-2-((4-((S)-2-((S)-2-amino-3-methylbutanainido)-N-methylpropaiiamido)phenethyl)(niethyl)amino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-D-phenylalanine as starting materials.

[0687] The mass spectrum and the .sup.1H-NMR spectrum of this drug-linker conjugate are represented respectively on FIGS. 7A and 7B.

Example H. 4-((S)-2-((S)-2-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamido)ethyl)carbamate

[0688] ##STR00142##

[0689] Example H has been synthesized according to the following synthetic path:

##STR00143## ##STR00144##

I.42. (2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxylic Acid

[0690] In a flask was added PNU-159682 (52 mg, 0.081 mmol) in a mixture of methanol (15 mL) and water (10 mL). A solution of NaIO.sub.4 (34.7 mg, 0.162 mmol) in water (5 mL) was added. The reaction mixture was stirred at rt until complete conversion was observed by LCMS. The solvents were removed under vacuo to give 1.42 as a red solid which was used directly in the next step.

I.43 (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-((((2-aminoethyl)carbamoyl)oxy) methyl)phenyl) amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate

[0691] In a flask under argon were added (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (1 g, 1.662 mmol) and bis(4-nitrophenyl) carbonate (1.011 g, 3.32 mmol) in DMF (0.443 mol/L). Then, the mixture was cooled at 0° C., and DIPEA (639 μL, 3.66 mmol) was added dropwise. The reaction mixture was warmed up to rt and stirred for 18 h. The crude mixture was concentrated under vacuo. The crude product was taken in 1:1 mixture of Et.sub.2O/EtOAc and filtered. The precipitate was washed with Et.sub.2O, citric acid 5%, H.sub.2O then Et.sub.2O again to obtain a yellow solid. This solid was purified by automatic column chromatography silica gel (100 DCM:0 MeOH to 80 DCM:20 MeOH) to give 345 mg of (9H-fluoren-9-yl)methyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate (white solid), 27.1% yield.

[0692] To a solution of the previous product (150 mg, 0.196 mmol) in DMF (6 mL) were added HOBt (34.4 mg, 0.254 mmol) and pyridine (63.3 μl, 0.782 mmol) at 0° C. After 5 min, tert-butyl (2-aminoethyl)carbamate 1-2 (40.7 mg, 0.254 mmol) in DMF (1.5 mL) was added to the mixture, followed by DIPEA (102 μl, 0.587 mmol). The mixture was warmed to rt and stirred for 2 h. The crude was concentrated under vacuo to give a white solid which was purified by automatic column chromatography silica gel (100 DCM:0 MeOH to 80 DCM:20 MeOH) to give 129 mg of 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl tert-butyl ethane-1,2-diyldicarbamate (white solid), 84% yield.

[0693] In a flask was placed the previous product (154 mg, 0.195 mmol) in DCM (6 mL). The mixture was cooled at 0° C., and TFA (753 μL, 9.77 mmol) was added and the mixture was stirred at 0° C., until complete conversion was observed by LCMS. The crude mixture was concentrated in vacuo to give 1.43 as a white solid (quantitative yield).

I.44 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-carboxamido)ethyl)carbamate

[0694] In a flask were added 1.42 (50.9 mg, 0.081 mmol), 1.43 (78.0 mg, 0.097 mmol) and DMF (8 mL) followed by HATU (30.8 mg, 0.081 mmol) and DIPEA (56.7 μl, 0.324 mmol). The reaction mixture was stirred at rt for 18 h. To this mixture was then added piperidine (80 μl, 0.811 mmol). The reaction mixture was stirred for 1 h (until complete conversion was observed by LCMS). The mixture was concentrated under vacuo. The crude product obtained was immediately purified by automatic column chromatography silica gel (100 DCM:0 MeOH/NH.sub.3 aq to 85 DCM:25 MeOH/NH.sub.3aq) to give 20 mg of 1.44 (red oil), 23% yield.

Example H

[0695] In a flask under N.sub.2 was added 6-(5-Chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (example 1) (7.86 mg, 0.028 mmol) in DCM (1 mL) and DMF (10 μl). The mixture was cooled at 0° C., then oxalyl chloride (7.28 μl, 0.085 mmol) was added dropwise. The mixture was warmed up to rt and was stirred until complete conversion was observed by LCMS (follow-up by LCMS by adding to the aliquot dry MeOH to form the methyl ester). The crude mixture was evaporated under vacuo. The residue was taken in DCM and dried again under vacuo to give 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoyl chloride as a yellow solid (yield quantitative). The crude material was used without further purification for the next step.

[0696] In a flask under N.sub.2 were introduced at rt 1.44 (20 mg, 0.019 mmol) in DCM (2 mL). The mixture was cooled to 0° C., and DIPEA (12.96 μl, 0.074 mmol) was added. The mixture was stirred at 0° C., for 10 min then the product of previous step (8.40 mg, 0.028 mmol) diluted in DCM (1 mL) was added. The mixture was then stirred at 0° C., for 2 h (until complete conversion was observed by LCMS). The crude mixture was concentrated under vacuo and purified by automatic column chromatography, silica gel (100 DCM:0 MeOH to 85 DCM: 15 MeOH) to give 6.85 mg of example H (also named compound F562524) as a red solid, 27% yield.

[0697] The .sup.1H-NMR spectrum of this drug-linker conjugate is represented on FIG. 8.

Example I. 4-((S)-2-((S)-2-(6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl) ethane-1,2-diylbis(methylcarbamate)

[0698] ##STR00145##

[0699] Example I has been synthesized according to the following synthetic path:

##STR00146## ##STR00147##

I.45 2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl (Perfluorophenyl) Carbonate

[0700] In a flask under argon were added PNU-159682 (12 mg, 0.0180 mmol) and DMF (1.5 mL). The mixture was cooled at 0° C., and bis(perfluorophenyl) carbonate (36.9 mg, 0.094 mmol) was added. Then a solution of DIPEA (9.80 μl, 0.056 mmol) in DMF (0.5 mL) was slowly added over a period of 5 min. The mixture was finally stirred for 3 h at 0° C. (conversion observed by LCMS). The crude mixture was concentrated in vacuo and purified by automatic column chromatography, silica gel (100 DCM:0 [80 DCM:20 MeOH] to 50 DCM:50 [80 DCM:2 0 MeOH]) to give 5.52 mg of 1.45 as a red oil, 36% yield.

I.46 (9H-fluoren-9-yl)methyl ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl) oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate 2,2,2-trifluoroacetate

[0701] In a flask under argon were added (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (I g, 1.662 mmol) and bis(4-nitrophenyl) carbonate (1.011 g, 3.32 mmol) in DMF (0.443 mol/L). Then, the mixture was cooled at 0° C., and DIPEA (639 μL, 3.66 mmol) was added dropwise. The reaction mixture was warmed up to rt and stirred for 18 h. The crude mixture was concentrated under vacuo, taken in Et:0/EtOAc (1/1) and filtered. The precipitate was washed with Et:0, citric acid 5%, H.sub.2O then Et.sub.2O again to obtain a yellow solid. This solid was purified by automatic column chromatography, silica gel (100 DCM:0 MeOH to 80 DCM:20 MeOH) to give 345 mg of a white solid, 27.1% yield. To a solution of this compound (118 mg, 0.154 mmol) in DMF (6 mL) were added HOBt (27.0 mg, 0.200 mmol) and pyridine (49.8 μl, 0.616 mmol) at 0° C. After 5 min, tert-butyl methyl(2-(methylamino)ethyl)carbamate (37.7 mg, 0.200 mmol) in DMF (1.5 mL) was added to the mixture, followed by DIPEA (81.0 μl, 0.462 mmol). The mixture was then warmed to rt and stirred for 2 h (until complete conversion was observed by LCMS). The crude mixture was concentrated under vacuo to give a yellow oil which was purified by automatic column chromatography, silica gel (100 DCM:0 MeOH to 80 DCM:20 MeOH) to give 103 mg of a white solid, 82% yield.

[0702] In a flask was placed this product (198 mg, 0.243 mmol) in DCM (12 mL). The mixture was cooled to 0° C., and TFA (935 μl, 12.13 mmol) was added and the mixture was stirred for 4 h at 0° C. (until complete conversion was observed by LCMS). The crude mixture was concentrated under vacuo to give 220 mg of 1.46 as a clear yellow solid (quantitative yield).

I.47 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl) ethane-1,2-diylbis(methylcarbamate)

[0703] To a solution of product I.4S (19 mg, 0.022 mmol) in DMF (1 mL) was added at rt a solution of product 1.46 (22.2 mg, 0.027 mmol) and DIPEA (15.59 μl, 0.089 mmol) in DMF (I mL). The reaction mixture was stirred at rt for 3 h (until complete conversion was observed by LCMS). Then, to the mixture was added piperidine (22.09 μl, 0.223 mmol). The reaction mixture was stirred for 1 h (complete conversion observed by LCMS). The crude mixture was concentrated under vacuo and purified by automatic column chromatography, silica gel (100 DCM:0 MeOH/NH.sub.3 (9/1) to 75 DCM:25 MeOH/NH.sub.3 (9/1)) to give 10 mg of 1.47 as a red oil, 39% yield.

Example I

[0704] In a flask under N.sub.2 was added 6-(5-Chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoic acid (example 1) (7.86 mg, 0.028 mmol) in DCM (1mL) and DMF (10 μl). The mixture was cooled at 0° C., then oxalyl chloride (7.28 μl, 0.085 mmol) was added dropwise. The mixture was warmed up to rt and was stirred until complete conversion was observed by LCMS (follow-up by LCMS by adding to the aliquot dry MeOH to form the methyl ester). The crude mixture was evaporated under vacuo. The residue was taken in DCM and dried again under vacuo to give 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoyl chloride as a yellow solid (yield quantitative). The crude material was used without further purification for the next step.

[0705] In a flask under N.sub.2 at rt was introduced product 1.47 (10 mg, 0.0086 mmol) in DCM (2 mL). The mixture was cooled at 0° C., and DIPEA (6.0 μl, 0.034 mmol) was added. The mixture was stirred at 0° C., for 10 min then addition of previous product (3.90 mg, 0.013 mmol) diluted in DCM (1 mL). The mixture was then stirred at 0° C., for 2 h (until complete conversion was observed by LCMS). The crude was concentrated in vacuo and purified by automatic column chromatography, silica gel (100 DCM:0 MeOH to 85 DCM:15 MeOH) to give 2.45 mg of example I as a red solid, 19% yield.

[0706] The .sup.1H-NMR spectrum of this drug-linker conjugate is represented on FIG. 9.

Example J

[0707] ##STR00148##

[0708] Example J has been synthesized according to the following synthetic path:

##STR00149## ##STR00150##

[0709] Compound I.50 has been prepared according to the following synthetic path:

##STR00151##

Compound I.48

[0710] In a flask under argon were added 2-([1,1′-biphenyl]-4-yl) propan-2-ol (1 g, 4.71 mmol) and pyridine (0.465 ml, 5.75 mmol) in DCM (5 mL). Then, the mixture was cooled to 0° C., and phenyl chloroformate (0.662 ml, 5.28 mmol) in DCM dry (2.4 mL) were added dropwise. The reaction mixture was warmed up to rt and stirred for 18 h (check by LCMS). The crude was concentrated in vacuo. The solid mixture was dissolved in DCM and washed with brine 3 times. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give the desired compound I.48, Yield 880 mg, 56% as white solid. LCMS (ESI): 333.40 (MH+).

Compound I.49

[0711] In a flask under argon containing N,N′-dimethyl-1,2-ethanediamine (2791 μl, 26.2 mmol). N-ethyl-N-isopropylpropan-2-amine (305 μl, 1.748 mmol) and DMF (4 mL), a solution of 2-([1,1′-biphenyl]-4-yl)propan-2-yl phenyl carbonate (581 mg, 1.748 mmol) in DMF (1.5 mL) was added at 0° C. The reaction mixture was warmed up to rt and stirred for 24 h (check by LCMS). The crude was concentrated in vacuo and the residue was purified by automatic column chromatography (Interchim, solid deposit): DCM/MeOH: 9/1. The desired fractions were concentrated in vacuo to give the desired compound I.49, Yield 433 mg, 76% as yellow oil. LCMS (ESI): 327.43 (MH+).

Compound I.50

[0712] In a flask under argon containing bis(trichloromethyl)carbonate (157 mg, 0.531 mmol) and toluene (4.3 mL), a solution of 2-([1,1′-biphenyl]-4-yl)propan-2-yl methyl(2-(methylamino)ethyl)carbamate (433 mg, 1,326 mmol) and triethyl amine (368 μl, 2.65 mmol) in toluene (2.9 mL) was added at 0° C. The reaction mixture was warmed up to rt and stirred for 1 h (check by LCMS). The solution was filtered, and the solvent was concentrated in vacuo and the residue was purified by automatic column chromatography (Interchim, solid deposit): cyclohexane/ethyl acetate: 7/3. The desired fractions were concentrated in vacuo to give the desired compound I.50, Yield 166 mg, 33% as white solid. LCMS (ESI): 405.60 (MH+).

[0713] Compound I.52 has been prepared according to the following synthetic path:

##STR00152##

Compound I.51

[0714] In a flask under argon containing (8S,10S)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-10-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-7,8,9,10-tetrahydrotetracene-5,12-dione (50 mg, 0.078 mmol), 4-dimethylaminopyridine (47.6 mg, 0,390 mmol), molecular sieves 0.4 nm (33 mg) and DCM (1 mL), a solution of 2-([1,1′-biphenyl]-4-yl)propan-2-yl (2-((chlorocarbonyl)(methyl)amino)ethyl)(methyl)carbamate (91 mg, 0.234 mmol)) in DCM (0.5 mL) were added. This mixture was stirred in the dark at 25° C., for 5 days. The solution was filtered, and the solvent was concentrated in vacuo and the residue was used without further purification in the next step.

Compound I.52

[0715] To a solution of product 1.51 in DCM (1 ml) in ice bath, a solution of dichloroacetic acid (96 μl, 1.169 mmol) in 0.5 mL of DCM was added. The solution was stirred at rt for 2 h.

[0716] The solvent was concentrated in vacuo and the residue was purified by automatic column chromatography (Interchim, solid deposit): DCM/MeOH: 9/1. The desired fractions were concentrated in vacuo to give the desired compound I.52, Yield 13 mg, 22% as red solid. LCMS (ESI): 756.76 (MH+).

Compound I.53

[0717] In a flask under argon were added (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (250 mg, 0.485 mmol), bis(perfluorophenyl) carbonate (382 mg, 0.970 mmol) and DMF (4 mL). Then, the mixture was cooled to 0° C., and N-ethyl-N-isopropylpropan-2-amine (127 μl, 0.727 mmol) was added dropwise. The reaction mixture was warmed up to rt and stirred for 2 h (check by LCMS). The crude was concentrated in vacuo. The crude was purified by automatic column chromatography (Interchim, solid deposit): DCM/MeOH: 9/1. The desired fractions were concentrated in vacuo to give the desired compound I.53, Yield 281 mg, 80% as yellow oil. LCMS (ESI): 726.65 (MH+).

Compound I.54

[0718] In a flask under argon were added 2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl methyl(2-(methylamino)ethyl)carbamate (78 mg, 0.103 mmol), 1-hydroxybenzotriazole (27.9 mg, 0.206 mmol), N,N′-diisopropylethylamine (35.1 μl, 0.206 mmol) and DMF (2 mL). Then, the mixture was cooled to 0° C., and (9H-fluoren-9-yl)methyl ((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-((((perfluorophenoxy)carbonyl)oxy)methyl)phenyl)amino)propan-2-yl)amino)butan-2-yl)carbamate (112 mg, 0.155 mmol) was added dropwise. The reaction mixture was warmed up to rt and stirred for 2 h (check by LCMS). The crude was concentrated in vacuo. The crude was purified by automatic column chromatography (Interchim, solid deposit): DCM/MeOH: 9/1. The desired fractions were concentrated in vacuo to give the desired compound I.54, Yield 77 mg, 58% as red oil. LCMS (ESI): 1298.0 (MH+).

Compound I.55

[0719] In a flask under argon were added 4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl) ethane-1,2-diylbis(methylcarbamate) (77.7 mg, 0.060 mmol) and DMF (2 mL). Then, the mixture was cooled to 0° C., and morpholine (259 μl, 2.99 mmol) was added dropwise. The reaction mixture was warmed up to rt and stirred for 2 h (check by LCMS). The crude was concentrated in vacuo. The crude was purified by automatic column chromatography (Interchim, solid deposit): DCM/MeOH: 9/1. The desired fractions were concentrated in vacuo to give the desired compound I.55, Yield 32 mg, 50% as red oil. LCMS (ESI): 1075.80 (MH+).

Example J

[0720] In a flask under argon at 25° C., were introduced 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido) benzyl(2-oxo-2-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[4′,3′:4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-2-yl)ethyl) ethane-1,2-diylbis(methylcarbamate) (32 mg, 0.030 mmol, 1 eq) in dichloromethane (2 mL). The mixture was cooled to 0° C., and N-ethyl-N-isopropylpropan-2-amine (20.74 μl, 0.119 mmol) was added. The mixture was stirred at 0° C., for 10 min then addition of Example 16 diluted in dichloromethane (2 mL). The mixture was then stirred at 0° C., for 2 h (until complete conversion was observed by LCMS). The crude was concentrated in vacuo and purified by automatic column chromatography (Interchim, 12 g, solid deposit): DCM/MeOH: 9/1. The desired fractions were concentrated in vacuo to give the desired Example J (also named compound F562646), Yield 17.4 mg, 40% as red oil. LCMS (ESI): 1338.41 (MH+).

[0721] The mass spectrum of this drug-linker conjugate is represented on FIG. 21.

Example K

[0722] ##STR00153##

[0723] Example K has been synthesized according to the following synthetic path:

##STR00154## ##STR00155##

Compound I.56

[0724] In a flask under argon was added starting material SMI (100 mg, 0.135 mmol) and DMF (I ml). The mixture was cooled to 0° C., then a solution of 3-bromopropanoic acid (22.79 mg, 0.149 mmol) and 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a] azepine (40.5 μl, 0.271 mmol) in DMF (0.5 mL) was added dropwise. Then The mixture was warmed up to rt and was stirred until complete conversion was observed by LCMS. The crude was concentrated in vacuo and purified by automatic column chromatography (Interchim, 12 g, solid deposit): DCM/MeOH: 80/20. The desired fractions were concentrated in vacuo to give the desired compound I.56, Yield 108 mg, 98% as white solid. LCMS (ESI): 811.35 (MH+).

Compound I.57

[0725] In a flask under argon were added compound I.56 (87.0 mg, 0.107 mmol) and DCM (2 mL). Then, the mixture was cooled to 0° C., N-hydroxysuccinimide (13.59 mg, 0.118 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (30.9 mg, 0.161 mmol) were added. The reaction mixture was warmed up to rt and stirred for 2 h (CHECK LCMS). The crude was concentrated in vacuo. The crude material was used without further purification for the next step.

Compound I.58

[0726] In a flask under argon was added compound I.57 (97 mg, 0.107 mmol) and DCM (I ml), then a solution of compound I.49 in 1 mL of DCM and N, N′-diisopropylethylamine (37.3 μl, 0.214 mmol) was added dropwise. The mixture was stirred until complete conversion was observed by LCMS. The crude was concentrated in vacuo and used without further purification for the next step.

Compound I.59

[0727] In a flask under argon was added compound I.58 (58 mg, 0.052 mmol) and DCM (1 ml). The mixture was cooled to 0° C., then dichloroacetic acid (86 μl, 1.037 mmol) was added dropwise. Then the mixture was warmed up to rt and was stirred until complete conversion was observed by LCMS. The crude was concentrated in vacuo and purified by automatic column chromatography (Interchim, 12 g, solid deposit): DCM/MeOH: 80/20. The desired fractions were concentrated in vacuo to give the desired compound 1.59, Yield 39 mg, 86% as white solid. LCMS (ESI): 882.6 (MH+).

Example K

[0728] In a flask under argon at 25° C., were introduced compound I.59 in dichloromethane (1 mL). The mixture was cooled to 0° C., and N-ethyl-N-isopropylpropan-2-amine (15.83 μl, 0.091 mmol) was added. The mixture was stirred at 0° C., for 10 min then addition of Example 16 diluted in dichloromethane (2 mL). The mixture was then stirred at 0° C., for 2 h (until complete conversion was observed by LCMS). The crude was concentrated in vacuo and purified by preparative HPLC (HCOOH conditions) to give the desired Example K, Yield 6 mg, 22% as white solid. LCMS (ESI): 1165.37 (M+Na).sup.+.

[0729] The mass spectrum and the TOF-MS spectrum of this drug-linker conjugate are represented respectively on FIGS. 22A and 22B.

3. CONJUGATION WITH SOMATOSTATIN

VI1. Reaction of Somatostatin with Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0730] ##STR00156##

[0731] 1 mg of lyophilized somatostatin (m.sub.exact=1636.72) was solubilized in 4 mL of buffer (57.5% NaH.sub.2PO.sub.4 20 mM, pH 6.5, 40% ACN, 2.5% DMF) to yield a concentration of 153 μM (0.25 mg/mL), 33.5 mg of TCEP were dissolved in 4 mL of buffer (57.5% NaH.sub.2PO.sub.4 20 mM, pH 6.5, 40% ACN, 2.5% DMF). To 300 μL of somatostatin solution (1 eq.) were added 3 μL of TCEP solution (1.1 eq.). The solution is stirred at 37° C., for 1 h. Commercial somatostatin: R.sub.t,1 (in ACN): 1.57; MS ES+: M.sup.+3/3=546.4, M.sup.+2/2=819.2. Reduced disulfide bond somatostatin: R.sub.t,1 (in ACN): 1.50; MS ES+: M.sup.+3/3=547.2, M.sup.+2/2=820.3, 5 mg of Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (I.6) were solubilized in 800 μL of ACN. 3 μL of Benzyl 6-(5-chloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (1.1 eq.) in solution were added to the somatostatin solution. The solution was stirred at 37° C. R.sub.t,1 (in ACN): 1.66; MS ES+: M.sup.+3/3=637.6, M.sup.+2/2=956.0.

[0732] The same reaction was performed in buffer pH 8 (57.5% NaH.sub.2PO.sub.4 20 mM, pH 8, 40% ACN, 2.5% DMF).

[0733] The mass spectrum of the obtained conjugate is represented on FIG. 10.

VI2. Reaction of Somatostatin with Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate

[0734] ##STR00157##

[0735] 1 mg of lyophilized somatostatin (m.sub.exact=1636.72) was solubilized in 4 mL of buffer (57.5% NaH.sub.2PO.sub.4 20 mM, pH 6.5, 40% ACN, 2.5% DMF) to yield a concentration of 153 μM (0.25 mg/mL), 33.5 mg of TCEP were dissolved in 4 mL of buffer (57.5% NaH.sub.2PO.sub.4 20 mM, pH 6.5, 40% ACN, 2.5% DMF). To 300 μL of somatostatin solution (1 eq.) were added 3 μL of TCEP solution (1.1 eq.). The solution is stirred at 37° C., for 1 h. Commercial somatostatin: R.sub.t,1 (in ACN): 1.57; MS ES+: M.sup.+3/3=546.4, M.sup.+2/2=819.2. Reduced disulfide bond somatostatin: R.sub.t,1 (in ACN): 1.50; MS ES+: M.sup.+3/3=547.2, M.sup.+2/2=820.3, 5.4 mg of Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (1.7) were solubilized in 800 μL of ACN. 3 μL of Benzyl 6-(4,5-dichloro-1,1-dioxido-3-oxoisothiazol-2(3H)-yl)hexanoate (1.1 eq.) in solution were added to the somatostatin solution. The solution was stirred at 37° C. R.sub.t,1 (in ACN): 1.65; MS ES+: M.sup.+3/3=637.3, M.sup.+2/2=955.5.

[0736] The same reaction was performed in buffer pH 8 (57.5% NaH.sub.2PO.sub.4 20 mM, pH 8, 40% ACN, 2.5% DMF).

[0737] The mass spectrum of the obtained conjugate is represented on FIG. 11.

4. CONJUGATION WITH MONOCLONAL ANTIBODIES

4.1. ADC Synthesis, Purification and Characterization

[0738] The procedure described below applies to chimeric, humanized and human IgG1 forms. It must be understood that for any other forms, such as IgG2, IgG4, etc., the person skilled in the art would be capable of adapting this procedure using the general knowledge.

[0739] Ab1 antibody is an anti-IGF1R IgG1 monoclonal antibody. This antibody corresponds to antibody 208F2 of WO2015162291 (see table 3, page 36) for which the three light chain CDRs have sequences SEQ ID Nos. 9, 5 and 11; the three heavy chain CDRs have sequences SEQ ID Nos. 7, 2 and 3; the light chain variable domain has sequence SEQ ID No. 18; and the heavy chain variable domain has sequence SEQ ID No. 13.

[0740] Ab2 antibody is an irrelevant chimeric (IgG1) antibody directed at a bacterial protein, which is the outer membrane protein A from E. coli, and called c9G4 (Haeuw J. F, and Beck A. Proteomics for development of immunotherapies, In Proteomics: Biomedical and Pharmaceutical Applications, Kluwer Academic Publishers, Ed. Hondermarck H., 2014, pages 243-278; WO2015162291).

[0741] Antibodies (1-5 mg/ml) were partially reduced with TCEP hydrochloride in 10 mM borate buffer pH 8.4 containing 150 mM NaCl and 2 mM EDTA for 2-4 hours at 37° C. Typically, 6-20 molar equivalents of TCEP were used to target a DAR of around 4. The partial antibody reduction was confirmed by SDS-PAGE analysis under non-reducing conditions. The antibody concentration was then adjusted to 1 mg/ml with 10 mM borate buffer pH 8.4 containing 150 mM NaCl, 2 mM EDTA, 6% sucrose and a 5-20 molar excess of drug-linker conjugate to antibody was added from a 10 mM solution in DMSO. Seven examples of drug-linker conjugate according to the invention were coupled to Abl: [0742] Example A and Example B giving respectively ADC1-A and ADC1-B (non-cleavable linkers); [0743] Example C, Example D, Example E, Example F and Example G giving respectively ADC1-C, ADC1-D, ADC1-E, ADC1-F and ADC1-G (cleavable linkers).

[0744] The final DMSO concentration was adjusted to 10% to maintain the solubility of the drug in the aqueous medium during coupling. The reaction was carried out for 1-4 h at room temperature or 37° C. The drug excess was quenched by addition of 2.5 moles of N-acetylcysteine per mole of drug and incubation for 1 h at room temperature.

[0745] After dialysis against 25 mM His buffer pH 6.5 containing 150 mM NaCl and 6% sucrose overnight at 4° C., the re-bridged antibody-drug conjugates were purified by using methods known to persons skilled in the art with commercial chromatography columns and ultrafiltration units. The purified ADCs were stored at 4° C., after sterile filtration on 0.2 μm filter.

[0746] They were further analyzed by SDS-PAGE under reducing and non-reducing conditions to confirm drug conjugation and by SEC on analytical TSK G3000 SWXL column to determine the content of monomers and aggregated forms. The content of aggregated forms deduced from the SEC chromatograms (FIG. 13) was lower than 5% as shown in Table 9.

TABLE-US-00010 TABLE 9 Content of aggregated forms Ab/ADC % monomer Ab1 99.6 ADC1-A 99.0 ADC1-B 99.3 ADC1-C 98.1 ADC1-D 99.5 ADC1-E 99.4 ADC1-F 99.6 ADC1-G 95.2

[0747] SDS-PAGE analyses confirm formation of fully bridged antibody H2L2 (FIG. 12). However other species (H2L, H2 and HL), corresponding to partially bridged antibody, were also detected. It's important to note that these species were visible when samples were heat-treated in reducing conditions before the run to ensure full dissociation of heavy and light chains (H and L), not connected by an intact interchain bridge.

[0748] The protein concentrations were determined by using the BCA assay with IgG as standard. The DAR was estimated for each purified ADC by HIC using a TSK-Butyl-NPR column. It was comprised between 3.5 and 4.3 (Table 10). HIC profiles revealed that no DAR0 and a major peak of DAR 4 were observed for most of the ADCs synthesised. Indeed, only ADC1-C and E show trace of DAR0. Moreover, for ADC1-A, B. C and G only DAR3, DAR4 and DAR5 were observed. Excepted for ADC1-D, the major peak is a DAR4. Compare to a second-generation ADC, these ADCs are more homogeneous as shown in Table 10.

TABLE-US-00011 TABLE 10 DAR distribution estimated by HIC using a TSK-Butyl-NPR column DAR % DAR 0 DAR 1 DAR 2 DAR 3 DAR 4 DAR 5 DAR 6 DAR 7 DAR 8 Adcetris 6.0 1.34 25.2 3.7 32.9 0 22.5 1.5 6.9 ADC1-A 0 0 0 0 64.9 35.1 0 0 0 ADC1-B 0 0 0 39.8 47.6 12.5 0 0 0 ADC1-C 0.6 1.0 0.8 8.2 63.1 26.3 0 0 0 ADC1-D 0 0 0 44.6 39.5 10.6 3.8 1.5 0 ADC1-E 2.1 4.5 4.0 11.9 56.3 21.1 0 0 0 ADC1-F 0 0.5 3.2 22.6 73.7 0 0 0 0 ADC1-G 0.1 0.4 2.6 18.6 68.1 10.2 0 0 0

[0749] Adcetris® (brentuximab vedotin) has been used as a reference since it uses a second generation maleimide linker conjugated to the cysteines of the antibody. It is the best representative example of second generation ADC; the technology described in this demand being can be considered as the third generation.

4.2. ADC ANALYSIS BY NATIVE MASS SPECTROMETRY

[0750] All chemicals were purchased from Sigma-Aldrich: ammonium acetate (A1542), caesium iodide (21004), 2-propanol (19516). IgGZERO (A0-IZ1-010) enzyme was obtained from Genovis. Aqueous solutions were prepared using an ultra-pure water system (Sartorius, Göttingen, Germany).

[0751] ADC1-A to ADC1-G were deglycosylated prior to native MS experiments. This was performed by incubating one unit of IgGZERO per microgram of ADC for 30 min at 37° C. Then, ADCs were buffer exchanged against a 150 mM ammonium acetate solution (pH 6.9) using six cycles of concentration/dilution using a microconcentrator (Vivaspin, 10-kD cutoff, Sartorius, Göttingen, Germany). Protein concentration was determined by UV absorbance using a NanoDrop spectrophotometer (Thermo Fisher Scientific, France). Non-denaturing (native) mass spectrometry of ADCs was performed on a Q-TOF (Synapt G2 HDMS, Waters, Manchester, UK) mass spectrometer operating in the positive ion mode both coupled to an automated chip-based nanoelectrospray device (Triversa Nanomate, Advion, Ithaca, USA). Analyses were performed in the m/z 1000-10 000 range. Samples were diluted in 150 mM NH.sub.4OAc at pH 6.9 and infused at 10 μM. External calibration was performed using singly charged ions produced by a 2 g/L solution of caesium iodide in 2-propanol/water (S0/S0 v/v).

[0752] The voltage of the nanoelectrospray was set at 1.75 kV and nitrogen nanoflow at 0.75 psi. The cone voltage was set to 180 volts and the backing pressure to 6 mbar.

[0753] FIG. 14 presents examples of non-deconvoluted MS spectrum.

[0754] The DAR distribution (FIG. 15) was determined after deconvolution using MaxEnt™ algorithm from Mass Lynx 4.1 (Waters, Manchester, UK). The parameters of the software were optimized for each spectrum.

[0755] Average DAR values (FIG. 15) were calculated by using the following equation (where j is the maximum number of drug load).

[00001] $DAR = \frac{({.Math.}_{i = 0}^{j} i * intensity Di)}{{.Math.}_{i = 0}^{j} intensity Di}$

[0756] The results were derived from the relative peak intensities of each charge states in the raw spectra and are presented in Table 11 below.

TABLE-US-00012 TABLE 11 DAR distribution calculated using MaxEnt ™ algorithm from Mass Lynx 4.1 DAR distribution (%) Average ADC DAR0 DAR1 DAR2 DAR3 DAR4 DAR5 DAR6 DAR7 DAR8 DAR ADC1-A 0 0 0 0 64 36 0 0 0 4.4 ± 0.1 ADC1-B 0 0 0 42 39 19 0 0 0 3.8 ± 0.1 ADC1-D 0 0 0 47 40 13 0 0 0 3.7 ± 0.1

[0757] FIG. 16 compares the DAR distribution, determined from raw spectra after mass deconvolution, for 2 different ADCs, i.e.: [0758] ADC1-C according to the invention prepared from Abl antibody and the drug-linker conjugate C (FIG. 16B) and [0759] a reference ADC Ref-A which is a comparative ADC synthesized from the same antibody (Abl) and from a drug-linker conjugate corresponding to the drug-linker conjugate C in which the sulfomaleimide moiety

##STR00158## has been replaced by a maleimide moiety

##STR00159## (FIG. 16A).

[0760] A heterogeneous distribution from DAR 0 to DAR 8 is observed for the ADC synthesized by using the classical maleimide chemistry to link the drug to the antibody (FIG. 16A), whereas the ADC generated by using the sulfomaleimide chemistry according to the invention is highly homogeneous with 75% of DAR 4 and no DAR 0/2 and 6/8 species (FIG. 16B). These results are summarized in Table 12 below.

TABLE-US-00013 TABLE 12 DAR distribution calculated using MaxEnt ™ algorithm from Mass Lynx 4.1 DAR distribution (%) Average ADC DAR0 DAR1 DAR2 DAR3 DAR4 DAR5 DAR6 DAR7 DAR8 DAR ADC Ref-A 6 4 29 0 33 0 18 0 10 3.8 ADC1-C 0 0 0 5 76 19 0 0 0 4.1

4.3. In Vitro Stability Study of ADCs in 4 Mammalian Sera Using a Ligand Binding Assay Method

[0761] To establish the gain of stability, an in vitro stability study was conducted. It consists in the incubation of the ADCs at 37° C., for a period of 14 days. Samples were collected at day 0, 3, 7 and 14. The various samples (DO, D3, D7 and D14) were then analyzed by LBA to determine the concentration of total antibody versus the concentration of ADC. In practice, a solution of each ADC is prepared at 100 μg/ml in 4 sera (human, cynomolgus, mouse and rat) and incubated at 37° C., for a maximum of 14 days. Then aliquots are collected at DO, D3, D7 and D14, and stored at −80° C., until dosage. For total Ab and ADC quantification, the plates are thawed at room temperature with shaking and both LBA assays are run in parallel. Briefly, standard microtiter plates (MSD, Gaithersburg, USA) are coated using 30 μl of an anti-His antibody solution at 2 μg/ml prepared in PBS 1×. After an overnight incubation at 4° C., assay plates are treated with blocking buffer (3% MSD Blocker A (MSD, Gaithersburg, USA)) for I hour at 37° C. Then the recombinant His-taged antigen is added for 1 hour at 37° C., at the concentration of 2.5 μg/ml in assay buffer. After a washing step, samples are analyzed as duplicates at the 1/5000° dilution and incubated for 1 hour at 37° C., while standard ADCs are loaded in duplicate onto the assay plate. The detection step is done using either a goat anti-human Ig Kappa sulfo-tag solution at 1 μg/ml for the detection of total Ab or a mouse monoclonal anti-Drug antibody labelled with sulfotag for ADC detection. After a 1-hour incubation period at 37° C., the detection is realized using 150 μL of a 2×MSD-read T buffer containing surfactant (MSD, Gaithersburg, USA) just before reading using MSD Sector Imager.

[0762] The total antibody and ADC concentrations are determined at each timepoint and transformed in percentage, taking 100% as the quantity of total ADCs or antibody at each timepoint.

[0763] Data are illustrated in FIGS. 17A, 17B and 17C for 3 ADCs: ADC1-C (FIG. 17B) and ADC1-E (FIG. 17C) in which the drug has been linked to the Abl antibody using the sulfomaleimide chemistry according to the invention (by means of the drug-linker conjugate C or E respectively), in comparison to a reference ADC Ref-B in which the drug has been linked to the antibody using a classical maleimide chemistry (FIG. 17A).

##STR00160##

Drug-Linker Moiety Used to Prepare Reference ADC Ref-B

[0764] As a comparator, a drug-linker using the same payload and a non-cleavable linker was chosen (drug-linker of ADC Ref-B). It was conjugated to the same antibody using a maleimide chemistry. The choice of this comparator limits “the instability” of the reference ADC in the sera by deconjugation from the antibody through a retro-Michael reaction. Compared to our constructs based on a cleavable linker, this comparator is thus favoured which makes the stability improvement of our drug-linkers even more spectacular.

[0765] As expected a decrease in ADC concentration is observed for the ADC synthesized using classical maleimide chemistry (ADC Ref-B), whereas the ADCs generated using sulfomaleimide chemistry according to the invention (ADC1-C & ADC1-E) surprisingly are much more stable over the 14-day period.

4.4. In Vitro Cytotoxicity of ADCs

[0766] The in vitro cytotoxicity of ADC according to the invention was evaluated. In order to evaluate the non-specific cytotoxicity, the compounds were also coupled to an irrelevant chimeric antibody (Ab2), called c9G4, at the same DAR and using the same drug-linker conjugates to give ADC2-C with Example C, ADC2-E with Example E and ADC2-F with Example F.

[0767] MCF-7 and NCI-H2122 cells were plated on 96 well plates (2500 cells per well) in complete growth media. The day after, serial dilutions of the tested ADCs were added to the corresponding wells and incubated at 37° C., for 6 days. Six days after the addition of the ADCs, a Cell Titer Glo assay (PROMEGA) was performed on the plates to check the viability of the cells.

[0768] The results obtained, expressed in percentage of viability, are shown in FIGS. 18A and 18B. As expected, the ADCs synthesized with the irrelevant antibody showed no or modest cytotoxic activity on both MCF-7 and NCI-H2122 cells. On the opposite, the ADCs of the invention: ADC1-C, ADC1-E and ADC1-F decreased dramatically cell viability. EC.sub.50 values of 7.61.Math.10.sup.−11, 7.16.Math.10.sup.−11 and 3.64.Math.10.sup.−11 M were obtained for ADC1-C, ADC1-E and ADC1-F respectively on NCI-H2122 and EC.sub.50 values of 1.04.Math.10.sup.−11, 1.33.Math.10.sup.−11 and 7.39.Math.10.sup.−11 M were obtained for ADC1-C, ADC1-E and ADC1-F respectively on MCF7, indicating potent cytotoxic activity.

4.5. In Vivo

[0769] All experimental protocols were approved by Pierre Fabre's Institutional Animal Care and use Committee.

[0770] For ovarian cancer model, 7 weeks old female SCID mice (Charles RIVER Laboratories) were engrafted subcutaneously with 10.Math.10.sup.6 CaoV3 cells (6 animals per groups).

[0771] Treatment by intravenous administration of ADC1-C according to the invention, reference ADC Ref-A or ADC vehicle was initiated when tumors reached approximately 150 mm.sup.3. The animals were either treated by one injection (Q1d1) or by 3 injections (once weekly) (Q7d3). Tumor volume (Length×width×height×0.52) was measured by electronic caliper at least twice weekly during approximatively 25 days after the first injection. The results are presented on FIG. 19 (animals treated at Q1d1) and FIG. 20 (animals treated at Q7d3).

[0772] As can be seen on FIGS. 19 and 20, the ADCs according to the invention have a great efficacy with a complete tumor regression even after a single injection.

5. CONJUGATION OF PNU-159682 DERIVATIVES TO MONOCLONAL ANTIBODIES

5.1. ADC Synthesis, Purification and Characterization

[0773] Two PNU-159682 derivatives, namely F562524 (example J) and F562646 (example H), were coupled to the antibodies 208F2 (Abl) and c9G4 (Ab2), under the conditions previously described in example 4, 208F2 (Abl) and c9G4 (Ab2) are as disclosed in example 4. Briefly, antibodies (1-5 mg/ml) were partially reduced with 6-20 equivalents of TCEP hydrochloride in 10 mM borate buffer pH 8.4 containing 150 mM NaCl and 2 mM EDTA for 2-4 hours at 37° C. The antibody concentration was then adjusted to 1 mg/ml with 10 mM borate buffer pH 8.4 containing 150 mM NaCl, 2 mM EDTA, 6% sucrose and a 5-20 molar excess of drug-linker conjugate to antibody was added from a 10 mM solution in DMSO. The reaction was carried out for 1-4 h at room temperature or 37° C., in the presence of 10% DMSO. The drug excess was quenched by addition of 2.5 moles of N-acetylcysteine per mole of drug and incubation for 1 h at room temperature. After dialysis against 25 mM His buffer pH 6.5 containing 150 mM NaCl and 6% sucrose overnight at 4° C., the ADCs were purified by chromatography or ultrafiltration. The ADC concentrations were determined by using the BCA assay with IgG as standard and the purified ADCs were stored at 4° C., after sterile filtration on 0.2 μm filter.

[0774] ADCs were further analyzed by SDS-PAGE and SEC (TSK G3000 SWXL column), as previously described in example 4, to confirm drug conjugation and rebridging, and to determine the content of monomers and aggregates. The content of monomers was around 95% (FIG. 23 and table 13).

TABLE-US-00014 TABLE 13 Content of monomers ADC % of monomers Ab1 (208F2) 99.8 Ab1- F562524 (208F2-F562524) 94.1 Ab1- F562646 (208F2-F562646) 94.8 Ab2 (c9G4) 99.7 Ab2- F562524 (c9G4-F562524) 94.4 Ab2- F562646 (c9G4-F562646) 94.7

5.2. ADC Analysis by Native LC-MS

[0775] ADCs were analyzed by native liquid chromatography-mass spectrometry on a UPLC Acquity H Class Bio system coupled to a Synapt G2Si mass spectrometer (Waters). LC separation was performed on 2 Polyhydroxyethyl A columns (Poly-LC, 150×1 mm, 300 A, 5 μm) connected in series. Samples were diluted to 0.2 mg/ml with the eluant buffer (150 mM ammonium acetate). Four μg of sample were injected and eluted at a flow rate of 40 μL/min. The mass spectrometer was operated in positive mode with a capillary voltage of 2.9 kV. The sample cone was set at 150 V. Analyses were performed in the range of m/z 1000-8000 with a 1 sec scan time. FIG. 24 shows the m/z spectra before deconvolution. The DAR distribution was determined after deconvolution of MS spectra using MaxEnt™ algorithm from Mass Lynx software (Waters) (FIG. 25). Average DAR values were calculated by using the following equation (where j is the maximum number of drug load):

[00002] $DAR = \frac{({.Math.}_{i = 0}^{j} i * intensity Di)}{{.Math.}_{i = 0}^{j} intensity Di}$

[0776] The results are presented in the Table 14 below.

TABLE-US-00015 TABLE 14 ADC analysis by native LC-MS analysis: DAR distribution and average DAR DAR distribution (%) Average ADC DAR0 DAR1 DAR2 DAR3 DAR4 DAR5 DAR6 DAR7 DAR8 DAR hz208F2- 0 0 0 11 68 21 0 0 0 4.1 F562524 hz208F2- 0 0 0 0 79 21 0 0 0 4.2 F562646 c9G4- 0 0 0 11 67 23 0 0 0 4.1 F562524 c9G4- 0 0 0 0 82 18 0 0 0 4.2 F562646

5.3. In Vitro Stability

[0777] The ADC hz208F2-F562524 was incubated at 200 μg/ml in cynomolgus serum at 37° C., for a period of 14 days. Samples were collected at day 0, 3, 7 and 14, and stored at −80° C., until LC-MS analysis to determine the average DAR.

[0778] Before LC-MS analysis, the samples were immunopurified by using Streptavidin magnetic beads (M-280, Invitrogen) coated with a Capture Select anti-human IgG-Biotin conjugate (Life Technologies, 8 μg antibody/200 μL beads). Samples were incubated with the anti-IgG-coated beads for 2 h at room temperature (100 μL sample/200 μL beads) before acidic elution with 40 μL of 0.4% trifluoroacetic acid. The pH was increased by adding 4 μL of a 3 M Tris/HCL pH 8.8 solution. The immunopurified samples were incubated with 2 μL of IgGZero for 30 minutes at 37° C., before LC-MS analysis in native conditions as described above. The DAR distribution was determined after deconvolution of MS spectra using MaxEnt™ algorithm from Mass Lynx software (Waters), and average DAR values were calculated by using the following equation (where j is the maximum number of drug load):

[00003] $DAR = \frac{({.Math.}_{i = 0}^{j} i * intensity Di)}{{.Math.}_{i = 0}^{j} intensity Di}$

[0779] The results are presented in the Table 15 below. The ADC hz208F2-F562524 was shown to be highly stable up to 14 days after in vitro incubation in cynomolgus serum.

TABLE-US-00016 TABLE 15 In vitro stability study of hz208F2-F562524: DAR distribution and average DAR DAR distribution (%) Average Day DAR0 DAR1 DAR2 DAR3 DAR4 DAR5 DAR6 DAR7 DAR8 DAR 0 0 0 0 18 57 25 0 0 0 4.1 3 0 0 0 19 54 27 0 0 0 4.1 7 0 0 0 18 50 32 0 0 0 4.1 10 0 0 0 19 54 28 0 0 0 4.1

5.4. In Vitro Cytotoxicity

[0780] The cytotoxicity of the ADCs was evaluated in MCF-7 and NCI-H2122 cells. Cells were plated on 96 well plates (2500 cells per well) in complete growth media. The day after, serial dilutions of the tested ADCs were added to the corresponding wells and incubated at 37° C., for 6 days. Cell viability was determined by measuring ATP using the cell Titer Glo kit (Promega). Luminescence was read using the plate reader Mithras from Berthold Company. The results obtained, expressed in percentage of viability, are shown in FIGS. 26 and 27. The viability in the non-treated wells was considered as 100%.

[0781] As expected, the ADCs hz208F2-F562524 and hz208F2-F562646 decreased dramatically cell viability. EC.sub.50 values of 2.48.Math.10.sup.−11 and 1.92.Math.10.sup.−12 M were determined on NCI-H2122 cells for hz208F2-F562524 and hz208F2-F562646, respectively, and EC.sub.50 values of 5.86.Math.10.sup.−12 and 9.45.Math.10.sup.−13 M were obtained on MCF-7 cells for hz208F2-F562524 and hz208F2-F562646, respectively, indicating potent cytotoxic activity. On the opposite, the corresponding ADCs synthesized with the irrelevant antibody showed a modest cytotoxic activity on both MCF-7 and NCI-H2122 cells.

5.5. In Vivo Anti-Tumoral Activity

[0782] Seven weeks old female SCID mice (Charles River Laboratories) were engrafted subcutaneously with 10.106 Caov3 cells (6 animals per groups). Treatment by intravenous administration (Q7d2) of the ADC hz208F2-F562524 (0.3 mg/kg), the corresponding control ADC c9G4-F562524 (0.3 mg/kg), or the vehicle was initiated when tumors reached approximately 150 mm3. Tumor volume (Length×Width×Height×0.52) was measured by electronic caliper twice weekly during approximatively 50 days after the first injection. The results are presented on FIG. 28. A complete tumor regression can be observed after 2 injections of the ADC hz208F2-F562524 whereas no anti-tumoral effect was observed with the control ADC or the vehicle.

5.6. Conclusion

[0783] The ADCs synthesized with PNU-159682 derivatives by using the sulfomaleimide-linker technology are highly homogeneous and stable in serum. Their efficacy was demonstrated in different in vitro and in vivo models.

6. OVERALL CONCLUSIONS

[0784] Overall, the sulfomaleimide-based linker technology described in this invention give a better stability in the plasma of different species and a better efficacy in in vitro models compared to usual maleimide-based linkers used for compounds in the market such as Adcetris. These properties have translated in a clear improvement of in vivo efficacy in different cell lines and more notably for cell lines with a lower expression of the antigen (CAOV3).

[0785] A better tolerability is also expected since the ADCs according to the invention are more stable in the circulation associated with an improved efficacy and safety margin in human treatment.

SULFOMALEIMIDE-BASED LINKERS AND CORRESPONDING CONJUGATES

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C07K5/06026

CHEMISTRY; METALLURGY

Classification Explorer

A61K47/6849

HUMAN NECESSITIES

Classification Explorer

A61K38/12

HUMAN NECESSITIES

Classification Explorer

A61P35/00

HUMAN NECESSITIES

International classification

Classification Explorer

A61K47/68

HUMAN NECESSITIES

Classification Explorer

A61K38/07

HUMAN NECESSITIES

Classification Explorer

A61K38/12

HUMAN NECESSITIES