DRUG ANTIBODY CONJUGATES

Abstract

Drug conjugates having formula [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-].sub.n-Ab wherein: D is a drug moiety having the following formula (I) or a pharmaceutically acceptable salt or ester thereof, wherein D is covalently attached via a hydroxy group at OR.sub.1, OR.sub.3 or ZH, or a thiol group at ZH to (X).sub.b if any, or (AA).sub.w if any, or to (T).sub.g if any, or (L); that are useful in the treatment of cancer.

##STR00001##

Claims

1. A drug conjugate comprising a drug moiety covalently attached to the rest of the drug conjugate, the drug conjugate having formula [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-].sub.n-Ab wherein: D is a drug moiety having the following formula (I) or a pharmaceutically acceptable salt or ester thereof, ##STR00108## wherein: D is covalently attached via a hydroxy group at OR.sub.1, OR.sub.3 or ZH or via a thiol group at ZH, to (X).sub.b if any, or (AA).sub.w if any, or to (T).sub.g if any, or (L); R.sub.1 and R.sub.2 are each independently selected from hydrogen, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, —C(═O)R.sub.a, —C(═O)OR.sub.b and —C(═O)NR.sub.cR.sub.d; R.sub.3 is selected from hydrogen, —C(═O)R.sub.a, —C(═O)OR.sub.b, and —C(═O)NR.sub.cR.sub.d; Z is selected from —O— and —S—; R.sub.a is selected from hydrogen, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; R.sub.b is selected from substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; R.sub.c and R.sub.d are independently selected from hydrogen, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, substituted or unsubstituted aryl and substituted or unsubstituted heterocyclic group; X and T are extending groups that may be the same or different; each AA is independently an amino acid unit; L is a linker group; w is an integer ranging from 0 to 12; b is an integer of 0 or 1; g is an integer of 0 or 1; Ab is a moiety comprising at least one antigen binding site; and n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] to the moiety comprising at least one antigen binding site and is in the range from 1 to 20.

2. The drug conjugate according to claim 1, wherein D is also a compound of formula (Ia), or a pharmaceutically acceptable salt or ester thereof: ##STR00109## wherein R.sub.1, R.sub.2, R.sub.3 and Z are as defined in formula (I) in claim 1.

3. The drug conjugate according to claim 1, wherein D is a compound of formula: ##STR00110## or a pharmaceutically acceptable salt or ester thereof.

4. The drug conjugate according to claim 2, wherein D is a compound of formula: ##STR00111## or a pharmaceutically acceptable salt or ester thereof or wherein D is a compound of formula: ##STR00112## or a pharmaceutically acceptable salt or ester thereof.

5. (canceled)

6. The drug conjugate according to claim 1 wherein: D is a drug moiety having the following formula (II) or a pharmaceutically acceptable salt or ester thereof: ##STR00113## wherein: the wavy line indicates the point of covalent attachment to (X).sub.b if any, or (AA).sub.w if any, or to (T).sub.gif any, or (L); R.sub.1 and R.sub.2 are each independently selected from hydrogen, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, —C(═O)R.sub.a, —C(═O)OR.sub.b and —C(═O)NR.sub.cR.sub.d; wherein the optional substituents are one or more substituents R.sub.x; R.sub.3 is selected from hydrogen, —C(═O)R.sub.a, —C(═O)OR.sub.b, and —C(═O)NR.sub.cR.sub.d; Z is —O— or —S—; R.sub.a is selected from hydrogen, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; wherein the optional substituents are one or more substituents R.sub.x; R.sub.b is selected from substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; wherein the optional substituents are one or more substituents R.sub.x; R.sub.c and R.sub.d are independently selected from hydrogen, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, substituted or unsubstituted aryl and substituted or unsubstituted heterocyclic group; wherein the optional substituents are one or more substituents R.sub.x; substituents R.sub.x are selected from the group consisting of C.sub.1-C.sub.12 alkyl groups which may be optionally substituted with at least one group R.sub.y, C.sub.2-C.sub.12 alkenyl groups which may be optionally substituted with at least one group R.sub.y, C.sub.2-C.sub.12 alkynyl groups which may be optionally substituted with at least one group R.sub.y, halogen atoms, oxo groups, thio groups, cyano groups, nitro groups, OR.sub.y, OCOR.sub.y, OCOOR.sub.y, COR.sub.y, COOR.sub.y, OCONR.sub.yR.sub.z, CONR.sub.yR.sub.z, SR.sub.y, S(═O)R.sub.y, SO.sub.2R.sub.y, SSR.sub.y, P(O)(R.sub.y)OR.sub.z, NR.sub.yR.sub.z, NR.sub.yCOR.sub.z, NR.sub.yC(═O)NR.sub.yR.sub.z, NR.sub.yC(═NR.sub.y)NR.sub.yR.sub.z, aryl groups having from 6 to 18 carbon atoms in one or more rings which may optionally be substituted with one or more substituents which may be the same or different selected from the group consisting of R.sub.y, OR.sub.y, OCOR.sub.y, OCOOR.sub.y, NR.sub.yR.sub.z, NR.sub.yCOR.sub.z, and NR.sub.yC(═NR.sub.y)NR.sub.yR.sub.z, aralkyl groups comprising an alkyl group having from 1 to 12 carbon atoms substituted with an optionally substituted aryl group as defined above, aralkyloxy groups comprising an alkoxy group having from 1 to 12 carbon atoms substituted with an optionally substituted aryl group as defined above, and a 5- to 14-membered saturated or unsaturated heterocyclic group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said heterocyclic group optionally being substituted with one or more substituents R.sub.y, and where there is more than one optional substituents on any given group the optional substituents R.sub.y may be the same or different; each R.sub.y and R.sub.z is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.12 alkyl groups, C.sub.1-C.sub.12 alkyl groups that are substituted with at least one halogen atom, aralkyl groups comprising a C.sub.1-C.sub.12 alkyl group that is substituted with an aryl group having from 6 to 18 carbon atoms in one or more rings and heterocycloalkyl groups comprising a C.sub.1-C.sub.12 alkyl group that is substituted with a 5- to 14-membered saturated or unsaturated heterocyclic group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s); X and T are extending groups that may be the same or different; each AA is independently an amino acid unit; L is a linker group; w is an integer ranging from 0 to 12; b is an integer of 0 or 1; g is an integer of 0 or 1; where b+g+w is optionally not 0; Ab is a moiety comprising at least one antigen binding site; and n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] to the moiety comprising at least one antigen binding site and is in the range from 1 to 20.

7. The drug conjugate according to claim 6, or a pharmaceutically acceptable salt or ester thereof, wherein D is a drug moiety of formula (IIa): ##STR00114## where the wavy line, R.sub.1, R.sub.2, R.sub.3 and Z are as defined for formula (II).

8.-14. (canceled)

15. The drug conjugate according to am claim 1, wherein the salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate, p-toluenesulfonate, sodium, potassium, calcium, ammonium, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine and basic aminoacids.

16. The drug conjugate according to claim 1, wherein L is a linker group selected from the group consisting of: ##STR00115## wherein the wavy lines indicate the point of covalent attachments to an Ab (the wavy line to the right) and to (T).sub.g if any, or (AA).sub.w if any, or (X).sub.b if any, or to D (the wavy line to the left); R.sub.19 is selected from —C.sub.1-C.sub.12 alkylene-, —C.sub.3-C.sub.8 carbocyclo-, —O—(C.sub.1-C.sub.12 alkylene)-, —C.sub.6-C.sub.18 arylene- in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.1-C.sub.12 alkylene-C.sub.6-C.sub.18 arylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.6-C.sub.18 arylene-C.sub.1-C.sub.12 alkylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.1-C.sub.12 alkylene-(C.sub.3-C.sub.8 carbocyclo)-, —(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.12 alkylene-, —C.sub.5-C.sub.14 heterocyclo- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —C.sub.1-C.sub.12 alkylene-(C.sub.5-C.sub.14 heterocyclo)- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —(C.sub.5-C.sub.14 heterocyclo)-C.sub.1-C.sub.12 alkylene- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —(OCH.sub.2CH.sub.2).sub.r— and —CH.sub.2—(OCH.sub.2CH.sub.2).sub.r—, wherein each of the above alkylene substituents whether alone or attached to another moiety the carbon chain may optionally be substituted by one or more substituents R.sub.x; R.sub.30 is a —C.sub.1-C.sub.6 alkylene- group; M is selected from the group consisting of —C.sub.1-C.sub.6 alkylene-, —C.sub.1-C.sub.6 alkylene-(C.sub.3-C.sub.8 carbocyclo)-, —(CH.sub.2CH.sub.2O).sub.s—, —C.sub.1-C.sub.6 alkylene-(C.sub.3-C.sub.8 carbocyclo)-CON(H or C.sub.1-C.sub.6 alkyl)-C.sub.1-C.sub.6 alkylene-, -phenylene- which may optionally be substituted with one or more substituents R.sub.x, -phenylene-C.sub.1-C.sub.6 alkylene- wherein the phenylene moiety may optionally be substituted with one or more substituents R.sub.x and —C.sub.1-C.sub.6 alkylene-CON(H or C.sub.1-C.sub.6 alkyl)C.sub.1-C.sub.6 alkylene-; Q is selected from the group consisting of —N(H or C.sub.1-C.sub.6 alkyl)phenylene- and —N(H or C.sub.1-C.sub.6 alkyl)-(CH.sub.2).sub.s; r is an integer ranging from 1 to 10; and s is an integer ranging from 1 to 10, or wherein L is a linker group selected from the group consisting of: ##STR00116## wherein: the wavy lines indicate the point of covalent attachments to an Ab (the wavy line to the right) and to (T), if any, or (AA).sub.w if any, or (X).sub.b, if any, or to D (the wavy line to the left); R.sub.19 is selected from —C.sub.1-C.sub.12 alkylene-, —O—(C.sub.1-C.sub.12 alkylene), —C.sub.6-C.sub.12 arylene- in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.1-C.sub.12 alkylene-C.sub.6-C.sub.12 arylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.6-C.sub.12 arylene-C.sub.1-C.sub.12 alkylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.5-C.sub.12 heterocyclo- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —C.sub.1-C.sub.12 alkylene-(C.sub.5-C.sub.12 heterocyclo)- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —(C.sub.5-C.sub.12 heterocyclo)-C.sub.1-C.sub.12 alkylene- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —(OCH.sub.2CH.sub.2).sub.r— and —CH.sub.2—(OCH.sub.2CH.sub.2).sub.r—, wherein each of the above alkylene substituents whether alone or attached to another moiety the carbon chain may optionally be substituted by one or more substituents R.sub.x; R.sub.30 is a —C.sub.1-C.sub.6 alkylene- group; M is selected from the group consisting of —C.sub.1-C.sub.6 alkylene-, —C.sub.1-C.sub.6 alkylene-(C.sub.3-C.sub.8 carbocyclo)- and -phenylene- which may optionally be substituted with one or more substituents R.sub.x; and r is an integer ranging from 1-6.

17. (canceled)

18. The drug conjugate according to claim 1, selected from the formulas (V), (VI) and (VII): ##STR00117## wherein: X and T are extending groups that may be the same or different; each AA is independently an amino acid unit; w is an integer ranging from 0 to 12; b is an integer of 0 or 1; g is an integer of 0 or 1; D is a drug moiety; Ab is a moiety comprising at least one antigen binding site; n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] wherein L is as defined in formula (V), (VI) or (VII) to the moiety comprising at least one antigen binding site and is in the range from 1 to 20; R.sub.19 is selected from —C.sub.1-C.sub.8 alkylene-, —O—(C.sub.1-C.sub.8 alkylene)-, —C.sub.1-C.sub.8 alkylene-C.sub.6-C.sub.12 arylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, and —C.sub.6-C.sub.12 arylene-C.sub.1-C.sub.8 alkylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, wherein each of the above alkylene substituents whether alone or attached to another moiety the carbon chain may optionally be substituted by one or more substituents R.sub.x; R.sub.30 is a —C.sub.2-C.sub.4 alkylene- group; and M is selected from the group consisting of —C.sub.1-C.sub.3 alkylene- and —C.sub.1-C.sub.3 alkylene-(C.sub.5-C.sub.7 carbocyclo)-; or selected from the formulas (V), (VI) and (VII): ##STR00118## wherein: X and T are extending groups that may be the same or different; each AA is independently an amino acid unit; w is an integer ranging from 0 to 12; b is an integer of 0 or 1; g is an integer of 0 or 1; D is a drug moiety; Ab is a moiety comprising at least one antigen binding site; n is the ratio of the group D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] wherein L is as defined in (V), (VI) or (VII) to the moiety comprising at least one antigen binding site and is in the range from 1 to 20; R.sub.19 is selected from —C.sub.1-C.sub.6 alkylene-, -phenylene-C.sub.1-C.sub.6 alkylene- wherein the phenylene group may optionally be substituted with one or more substituents R selected from the group consisting of alkyl groups having from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms, halogen atoms, nitro groups and cyano groups, wherein each of the above alkylene substituents whether alone or attached to another moiety in the carbon chain may optionally be substituted by one or more substituents R.sub.x selected from the group consisting of alkyl groups having from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms, aryl groups having from 6 to 12 carbon atoms, halogen atoms, nitro groups and cyano groups.

19. (canceled)

20. The drug conjugate according to claim 1, wherein (AA).sub.w is of formula (III): ##STR00119## wherein the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or to the drug moiety (the wavy line to the left) and to (T).sub.g if any, or to the linker (the wavy line to the right); and R.sub.21 is, at each occurrence, selected from the group consisting of hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH.sub.2OH, —CH(OH)CH.sub.3, —CH.sub.2CH.sub.2SCH.sub.3, —CH.sub.2CONH.sub.2, —CH.sub.2COOH, —CH.sub.2CH.sub.2CONH.sub.2, —CH.sub.2CH.sub.2COOH, —(CH.sub.2).sub.3NHC(═NH)NH.sub.2, —(CH.sub.2).sub.3NH.sub.2, —(CH.sub.2).sub.3NHCOCH.sub.3, —(CH.sub.2).sub.3NHCHO, —(CH.sub.2).sub.4NHC(═NH)NH.sub.2, —(CH.sub.2).sub.4NH.sub.2, —(CH.sub.2).sub.4NHCOCH.sub.3, —(CH.sub.2).sub.4NHCHO, —(CH.sub.2).sub.3NHCONH.sub.2, —(CH.sub.2).sub.4NHCONH.sub.2, —CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, ##STR00120## and w is an integer ranging from 1 to 12, or wherein (AA).sub.w is of formula (III) wherein: R.sub.21 is selected, at each occurrence, from the group consisting of hydrogen, methyl, isopropyl, sec-butyl, benzyl, indolylmethyl, —(CH.sub.2).sub.3NHCONH.sub.2, —(CH.sub.2).sub.4NH.sub.2, —(CH.sub.2).sub.3NHC(═NH)NH.sub.2 and —(CH.sub.2).sub.4NHC(═NH)NH.sub.2; and w is an integer ranging from 0 to 6, or wherein w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (IV): ##STR00121## wherein: the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or to the drug moiety (the wavy line to the left) and to T), if any, or to the linker (the wavy line to the right); R.sub.22 is selected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl; and R.sub.23 is selected from methyl, —(CH.sub.2)NH.sub.2, —(CH.sub.2).sub.3NHCONH.sub.2 and —(CH.sub.2).sub.3NHC(═NH)NH.sub.2.

21.-22. (canceled)

23. The drug conjugate according to claim 1, wherein X is an extending group selected from: —CONH—(C.sub.1-C.sub.6 alkylene)NH—; —COO—CH.sub.2-(phenylene which may optionally be substituted with one or more substituents R.sub.x)—NH—; —CONH—(C.sub.1-C.sub.6 alkylene)NH—COO—CH.sub.2-(phenylene which may optionally be substituted with one or more substituents R.sub.x)—NH—; —COCH.sub.2NH—COCH.sub.2—NH—; —COCH.sub.2NH—; —CONH—(C.sub.1-C.sub.6 alkylene)S—; —CONH—(C.sub.1-C.sub.6 alkylene)NHCO(C.sub.1-C.sub.6 alkylene)S—; and b is 0 or 1, or wherein X is an extending group selected from the group consisting of: —CONH—(C.sub.1-C.sub.4alkylene)NH—; —COO—CH.sub.2-phenylene-NH—, wherein said phenylene group may optionally be substituted with from one to four substituents R.sub.x selected from the group consisting of alkyl groups having from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms, halogen atoms, nitro groups and cyano groups; —CONH—(C.sub.2-C.sub.4 alkylene)NH—COO—CH.sub.2-(phenylene which may optionally be substituted with from one to four substituents R.sub.x selected from the group consisting of alkyl groups having from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms, halogen atoms, nitro groups and cyano groups)-NH—; —COCH.sub.2NH—COCH.sub.2—NH—; —CONH—(C.sub.2-C.sub.4 alkylene)S—; —CONH—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—; and b is 0 or 1; or wherein X is an extending group selected from the group consisting of: —COO—CH.sub.2-phenylene-NH—; —CONH(CH.sub.2).sub.3NHCOOCH.sub.2-phenylene-NH—; —CONH(CH.sub.2).sub.3NH—; —CONH(CH.sub.2).sub.3—S—; —CONH(CH.sub.2).sub.3NHCO(CH.sub.2).sub.2S—; and b is 0 or 1.

24.-25. (canceled)

26. The drug conjugate according to claim 1, wherein T is an extending group selected from the group consisting of —CO—(C.sub.1-C.sub.6 alkylene)-NH—, —CO—(C.sub.1-C.sub.6 alkylene)-[O—(C.sub.2-C.sub.6 alkylene)].sub.j-NH—, —COO—(C.sub.1-C.sub.6 alkylene)-[O—(C.sub.2-C.sub.6 alkylene)].sub.j-NH—; where j is an integer from 1 to 25, and g is 0 or 1; or wherein T is an extending group selected from the group consisting of —CO—(C.sub.1-C.sub.4 alkylene)NH— —CO—(C.sub.1-C.sub.4 alkylene)-[O—(C.sub.2-C.sub.4 alkylene)].sub.j-NH—, —COO—(C.sub.1-C.sub.4 alkylene)-[O—(C.sub.2-C.sub.4 alkylene)].sub.j-NH—, where j is an integer from 1 to 10; and g is 0 or 1; or wherein T is an extending group selected from the group consisting of —CO—(C.sub.1-C.sub.4 alkylene)NH—, —CO—(C.sub.1-C.sub.4 alkylene)-[O—(C.sub.2-C.sub.4 alkylene)].sub.j-NH—, —COO—(C.sub.1-C.sub.4 alkylene)-[O—(C.sub.2-C.sub.4 alkylene)].sub.j-NH—; where j is an integer from 1 to 5; and g is 0 or 1.

27.-28. (canceled)

29. The drug conjugate according to claim 6, wherein D is a drug moiety of formula (II) or formula (IIa) or a pharmaceutically acceptable salt or ester thereof, wherein: R.sub.1 is hydrogen or substituted or unsubstituted C.sub.1-C.sub.6 alkyl, wherein the optional substituents are one or more substituents R.sub.x; R.sub.2 is hydrogen or —C(═O)R.sub.a, wherein R.sub.a is substituted or unsubstituted C.sub.1-C.sub.6 alkyl, wherein the optional substituents are one or more substituents R.sub.x; R.sub.3 is hydrogen or —C(═O)R.sub.a, wherein R.sub.a is substituted or unsubstituted C.sub.1-C.sub.6 alkyl, wherein the optional substituents are one or more substituents R.sub.x; and Z is —O—; or wherein D is a drug moiety of formula (II) or formula (IIa) or a pharmaceutically acceptable salt or ester thereof, wherein: R.sub.1 is hydrogen or methyl; R.sub.2 is hydrogen; R.sub.3 is hydrogen; and Z is —O—; or wherein D is a drug moiety of formula (II) or formula (IIa), or a pharmaceutically acceptable salt or ester thereof wherein: R.sub.1 is methyl; R.sub.2 is hydrogen; R.sub.3 is hydrogen; and Z is —O—.

30.-31. (canceled)

32. The drug conjugate according to claim 1, wherein D is selected from: ##STR00122## or a pharmaceutically acceptable salt or ester thereof, wherein the wavy line indicates the point of covalent attachment to (X).sub.b if any, or (AA).sub.w if any, or to (T).sub.g if any, or to (L); or wherein D is selected from: ##STR00123## or a pharmaceutically acceptable salt or ester thereof, wherein the wavy line indicates the point of covalent attachment to (X).sub.b if any, or (AA).sub.w if any, or to (T).sub.g if any, or to (L).

33. (canceled)

34. The drug conjugate according to claim 1, wherein the moiety Ab comprising at least one antigen binding site is an antigen-binding peptide; optionally wherein the moiety Ab comprising at least one antigen binding site is an antibody, a single domain antibody or an antigen-binding fragment thereof; optionally wherein the moiety Ab comprising at least one antigen binding site is a monoclonal antibody, polyclonal antibody or bispecific antibody and wherein the antibody or an antigen-binding fragment thereof is derived from any species; optionally wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, an antigen-binding fragment of a human antibody, a humanized antibody, an antigen-binding fragment of a humanized antibody, a chimeric antibody, an antigen-binding fragment of a chimeric antibody, a glycosylated antibody and a glycosylated antigen binding fragment; optionally wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment selected from the group consisting of an Fab fragment, an Fab′ fragment, an F(ab′).sub.2 fragment and an Fv fragment; optionally wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody which immunospecifically binds to cancer cell antigens, viral antigens, antigens of cells that produce autoimmune antibodies associated with autoimmune disease, microbial antigens; optionally wherein the moiety Ab comprising at least one antigen binding site is an antibody selected from the group consisting of Abciximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab, Coltuximab, Daclizumab, Daratumumab, Denintuzumab, Denosumab, Depatuxizumab, Dinutuximab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab, Gemtuzumab, Ibritumomab, Indatuximab, Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab, Lifastuzumab, Lorvotuzumab, Milatuzumab, Mirvetuximab, Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab, Pinatuzumab, Polatuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab, Siltuximab, Sirtratumab, Sofituzumab, Vadastuximab, Vorsetuzumab, an anti-HER2 antibody such as Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, and an anti-CD30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; optionally wherein the moiety Ab comprising at least one antigen binding site is an antibody selected from the group consisting of Abciximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab, Daclizumab, Daratumumab, Denintuzumab, Denosumab, Depatuxizumab, Dinutuximab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab, Gemtuzumab, Ibritumomab, Indatuximab, Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab, Mirvetuximab, Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab, Polatuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab, Siltuximab, Sirtratumab, Vadastuximab, Vorsetuzumab, an anti-HER2 antibody such as Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, and an anti-CD30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; optionally wherein the moiety Ab comprising at least one antigen binding site is an antibody selected from the group consisting of Abciximab, Alemtuzumab, Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab, Daclizumab, Daratumumab, Denosumab, Dinutuximab, Durvalumab, Elotuzumab, Gemtuzumab, Ibritumomab, Inotuzumab, Ipilimumab, Labetuzumab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab, Ramucirumab, Rovalpituzumab, Siltuximab, an anti-HER2 antibody such as Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, and an anti-CD30 antibody, or an antigen-binding fragment or an immunologically active portion thereof.

35.-56. (canceled)

57. The drug conjugate according to claim 1, that is an antibody drug conjugate, selected from the group consisting of: ##STR00124## wherein n is from 2 to 6, and each and is independently selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, an anti-HER2 antibody such as Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, and an anti-CD30 antibody, or an antigen-binding fragment or an immunologically active portion thereof.

58. The drug conjugate according to claim 57, wherein the moiety Ab comprising at least one antigen binding site is an anti-HER2 antibody such as Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof.

59. The drug conjugate according to claim 57, wherein the moiety Ab comprising at least one antigen binding site is selected from Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof.

60. The antibody drug conjugate according to claim 1, in isolated or purified form.

61. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 or of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-H, wherein: L.sub.1 is a linker selected from the group of formulas consisting of: ##STR00125## wherein each of the wavy lines indicates the point of covalent attachment to (T).sub.g if any, or (AA).sub.w if any, or to (X).sub.b if any, or to D; G is selected from halo, —O-mesyl and —O-tosyl; J is selected from halo, hydroxy, —N-succinimidoxy, —O-(4-nitrophenyl), —O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR.sub.20; R.sub.19 is selected from —C.sub.1-C.sub.12 alkylene-, —C.sub.3-C.sub.8 carbocyclo-, —O—(C.sub.1-C.sub.12 alkylene)-, —C.sub.6-C.sub.18 arylene- in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.1-C.sub.12 alkylene-C.sub.6-C.sub.18 arylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.6-C.sub.18 arylene-C.sub.1-C.sub.12 alkylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, —C.sub.1-C.sub.12 alkylene-(C.sub.3-C.sub.8 carbocyclo)-, —(C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.12 alkylene-, —C.sub.5-C.sub.14 heterocyclo- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —C.sub.1-C.sub.12 alkylene-(C.sub.5-C.sub.14 heterocyclo)- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —(C.sub.5-C.sub.14 heterocyclo)-C.sub.1-C.sub.12 alkylene-, wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, —(OCH.sub.2CH.sub.2).sub.r— and —CH.sub.2—(OCH.sub.2CH.sub.2).sub.r—, wherein each of the above alkylene substituents whether alone or attached to another moiety the carbon chain may optionally be substituted by one or more substituents R.sub.x; R.sub.20 is a C.sub.1-C.sub.12 alkyl or an aryl group having from 6 to 18 carbon atoms in one or more aromatic rings, said aryl groups optionally being substituted with one or more substituents R.sub.x; substituents R.sub.x are selected from the group consisting of C.sub.1-C.sub.12 alkyl groups which may be optionally substituted with at least one group R.sub.y, C.sub.2-C.sub.12 alkenyl groups which may be optionally substituted with at least one group R.sub.y, C.sub.2-C.sub.12 alkynyl groups which may be optionally substituted with at least one group R.sub.y, halogen atoms, oxo groups, thio groups, cyano groups, nitro groups, OR.sub.y, OCOR.sub.y, OCOOR.sub.y, COR.sub.y, COOR.sub.y, OCONR.sub.yR.sub.z, CONR.sub.yR.sub.z, SR.sub.y, S(═O)R.sub.y, SO.sub.2R.sub.y, SSR.sub.y, P(O)(R.sub.y)OR.sub.z, NR.sub.yR.sub.z, NR.sub.yCOR.sub.z, NR.sub.yC(═O)NR.sub.yR.sub.z, NR.sub.yC(═NR.sub.y)NR.sub.yR.sub.z, aryl groups having from 6 to 18 carbon atoms in one or more rings which may optionally be substituted with one or more substituents which may be the same or different selected from the group consisting of R.sub.y, OR.sub.y, OCOR.sub.y, OCOOR.sub.y, NR.sub.yR.sub.z, NR.sub.yCOR.sub.z, and NR.sub.yC(═NR.sub.y)NR.sub.yR.sub.z, aralkyl groups comprising an alkyl group having from 1 to 12 carbon atoms substituted with an optionally substituted aryl group as defined above, aralkyloxy groups comprising an alkoxy group having from 1 to 12 carbon atoms substituted with an optionally substituted aryl group as defined above, and a 5- to 14-membered saturated or unsaturated heterocyclic group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said heterocyclic group optionally being substituted with one or more substituents R.sub.y, and where there is more than one optional substituents on any given group the optional substituents R.sub.y may be the same or different; each R.sub.y and R.sub.z is independently selected from the group consisting of hydrogen, C.sub.1-C.sub.12 alkyl groups, C.sub.1-C.sub.12 alkyl groups that are substituted with at least one halogen atom, aralkyl groups comprising a C.sub.1-C.sub.12 alkyl group that is substituted with an aryl group having from 6 to 18 carbon atoms in one or more rings and heterocycloalkyl groups comprising a C.sub.1-C.sub.12 alkyl group that is substituted with a 5- to 14-membered saturated or unsaturated heterocyclic group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s); r is an integer ranging from 1-10; b is an integer of 0 or 1; g is an integer of 0 or 1; w is an integer ranging from 0 to 12; each of D, X, T, and AA is as defined in claim 1; wherein for compounds of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-H, b+w+g is not 0; optionally wherein the compound of formula D-(X).sub.b-(AA).sub.w)-L.sub.1 is selected from ##STR00126##

62.-67. (canceled)

68. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 or of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-H, wherein each of D, X, AA, T, b, g and w are as defined in claim 61; but further wherein if the compound is a compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-H then b+w+g≠0.

69. The drug conjugate according to claim 1, wherein b+g+w is not 0; or wherein b+w is not 0; or wherein when w is not 0, then b is 1.

70.-76. (canceled)

77. A pharmaceutical composition comprising a drug conjugate according to claim 1 and a pharmaceutically acceptable carrier.

78. A method for the prevention or treatment of cancer comprising administering an effective amount of a drug conjugate according to claim 1, to a patient in need thereof.

79. The method for the treatment of cancer according to claim 78, wherein the cancer is selected from lung cancer, including NSCLC, gastric cancer, colorectal cancer, breast cancer, pancreas carcinoma, endometrial cancer, bladder cancer, cervical cancer, esophageal cancer, gallbladder cancer, uterine cancer, salivary duct cancer, ovarian cancer, kidney cancer, leukemia, multiple myeloma, and lymphoma.

80. The method for the treatment of cancer according to claim 79, wherein the cancer is a HER2 positive cancer.

81.-82. (canceled)

83. A kit comprising a therapeutically effective amount of a drug conjugate according to a claim 1 and a pharmaceutically acceptable carrier.

84.-85. (canceled)

86. A process for the preparation of a drug antibody conjugate according to claim 1 comprising conjugating a moiety Ab comprising at least one antigen binding site and a drug D, Ab and D being as defined in claim 1 optionally wherein the preparation of a drug conjugate of formula (G), (G′) or (G″): ##STR00127## comprising the following steps: (i) either: (a) reacting a compound of formula (Ib): ##STR00128## wherein R.sub.1 and R.sub.2 are independently selected from a protecting group for OH, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, —C(═O)R.sub.a, —C(═O)OR.sub.b and —C(═O)NR.sub.cR.sub.d; R.sub.3 is selected from a protecting group for OH, —C(═O)R.sub.a, —C(═O)OR.sub.b, and —C(═O)NR.sub.cR.sub.d; R.sub.1 is selected from hydrogen, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; R.sub.b is selected from substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; R.sub.c and R.sub.d are independently selected from hydrogen, substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, and substituted or unsubstituted aryl and substituted or unsubstituted heterocyclic group; with a compound of formula X.sub.2—C(═O)—X.sub.1 wherein X.sub.1 and X.sub.2 are leaving groups to give a compound of formula (B): ##STR00129## and the point of attachment of the —C(═O)X.sub.1 moiety is the free primary —OH group of the compound of formula (Ib), or (b) reacting said compound of formula (Ib) as defined above with 4-nitro-phenylchloroformate to give a compound of formula (J): ##STR00130## wherein the point of attachment of the (4-nitrophenyl)-O—CO— group is the same as that for the X.sub.1(C═O) moiety in a) above; or (c) reacting a compound of formula (Ib) as defined above with an isocyanate of formula O═C═N—(CH.sub.2).sub.1-6NHProt.sup.NH wherein Prot.sup.NH is a protecting group for amino suitable to be deprotected under basic conditions to give a compound of formula ##STR00131## (ii) either (a) reacting the compound of formula (B) or (J) produced in step (i)(a) or (i)(b) with an amino compound of formula NH.sub.2—(CH.sub.2).sub.1-6NH.sub.2 to give a compound of formula (C): ##STR00132##  or (b) deprotecting the compound of formula ##STR00133## obtained in step (i)(c) to give a compound of formula (C); (iii) reacting the compound of formula (C) with a compound of formula (D′), (E) or E: ##STR00134## wherein R.sub.22 and R.sub.23 are as defined above in the definitions of AA groups; to give a compound of formula (F), (F′) or (F″), respectively: ##STR00135## wherein R.sub.1, R.sub.2 and R.sub.3 are as defined above for the compounds of formula (Ib) and R.sub.22 and R.sub.23 are as defined above in the definitions of AA groups; (iv) if protecting groups for the OH are present in the compounds of formula (F), (F′) or (F″), removing such protecting groups to give compounds of formula (F), (F′) or (F″) wherein R.sub.1, R.sub.2 and R.sub.3 are as defined above for the compounds of formula (II); (v) partial reduction of one or more disulfide bonds in the antibody to be conjugated to give a reduced antibody Ab-SH having free thiol groups: ##STR00136##  and (vi) reacting the partially reduced antibody Ab-SH having free thiol groups with the compound of formula (F), (F′) or (F″) produced in step (iv) to give the desired drug antibody conjugate of formula (G), (G′) or (G″) respectively: ##STR00137##

87.-95. (canceled)

96. A method of inhibiting cancer cell growth, comprising contacting cancer cells with a drug conjugate according to claim 1; optionally wherein the cancer is selected from lung cancer, including NSCLC, gastric cancer, colorectal cancer, breast cancer, pancreas carcinoma, endometrial cancer, bladder cancer, cervical cancer, esophageal cancer, gallbladder cancer, uterine cancer, salivary duct cancer, ovarian cancer, kidney cancer, leukemia, multiple myeloma, and lymphoma; and optionally wherein the cancer is a HER2 positive cancer.

97. The compound according to claim 61, wherein b+g+w is not 0; or wherein b+w is not 0; or wherein when w is not 0, then b is 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0426] The invention is diagrammatically illustrated, by way of example, in the accompanying drawings in which:

[0427] FIG. 1 is a schematic illustration of one process according to the present invention wherein conjugation to the antibody is via free thiol groups;

[0428] FIG. 2 is a schematic illustration of one process according to the present invention wherein conjugation to the antibody is via free amino groups.

[0429] FIG. 3. Tumor volume evaluation of BT-474 tumors in mice treated with placebo, Herceptin, Kadcyla (both at 30 mg/kg) and ADC3 (at 4 mg/kg).

EXAMPLES

[0430] The present invention is further illustrated by way of the following, non-limiting examples. In the examples, the following abbreviations are used:

[0431] CDI, 1,1′-carbonyldiimidazole

[0432] DIPEA, diisopropylethylamine

[0433] Hex, hexane

[0434] EtOAc, ethyl acetate

[0435] DCM, dichloromethane

[0436] NMP, N-methyl-2-pyrrolidone

[0437] DMF, dimethylformamide

[0438] EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

[0439] EDTA, ethylenediaminetetraacetic acid

[0440] MeOH, methanol

[0441] DTT, dithiothreitol

[0442] Py, pyridine

[0443] THF, tetrahydrofuran

[0444] TCEP, Tris[2-carboxyethyl]phosphine hydrochloride

[0445] MC, 6-maleimidocaproyl

[0446] Fmoc, 9-fluorenylmethoxycarbonyl

[0447] Cit, citrulline

[0448] Val, valine

[0449] DMSO, dimethylsulfoxide

[0450] Trt, triphenylmethyl

[0451] HOBt, 1-hydroxybenzotriazole

[0452] DIPCDI, N,N′-diisopropylcarbodiimide

[0453] TFA, trifluoroacetic acid

[0454] PABOH, 4-aminobenzyl alcohol

[0455] bis-PNP, bis(4-nitrophenyl) carbonate

[0456] NAC, N-Acetylcysteine

[0457] SEC, size-exclusion chromatography

[0458] HPLC, high performance liquid chromatography

[0459] ADC, antibody drug conjugate

[0460] ATCC, American Type Culture Collection

[0461] DMEM, Dulbecco's Modified Eagle's Medium

[0462] RPMI, Rosmell Park Memorial Institute medium

[0463] ITS, Insulin-transferrin-sodium selenite media supplement

[0464] FCS, Fetal Calf Serum

[0465] SRB, sulforhodamine B

[0466] PBS, phosphate buffered saline

[0467] DR, dose-response

[0468] UV, ultraviolet

[0469] SMCC, Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

[0470] LAR, Linker to Antibody Ratio

[0471] Synthesis of Intermediates

[0472] Compounds 1 and 2 were obtained following the procedures described in WO2018167270.

[0473] 6-Maleimidohexanoic acid N-hydroxysuccinimide ester was obtained following the procedure described in Org. Biomol. Chem. 2009, 7, 3400-3406.

[0474] Intermediate 7

[0475] a)

##STR00090##

[0476] To a solution of 1 (19.2 mg, 0.039 mmol) and imidazole (10 eq, 0.39 mmol) in CH.sub.2Cl.sub.2 (3 mL) was added chlorotrimethylsilane (6 eq., 0.234 mmol) at 23° C. After being stirred at 23° C. for 1 h, the reaction mixture was concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc, from 90:10 to 70:30) to afford pure 3 (22 mg, 80% yield) as colourless oil.

[0477] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2CO): δ 7.45 (d, J=10.0 Hz, 1H), 5.33 (dd, J=10.0, 6.8 Hz, 1H), 4.77 (t, J=2.2 Hz, 1H), 4.61 (t, J=2.2 Hz, 1H), 4.37 (d, J=0.7 Hz, 1H), 3.90-3.80 (m, 2H), 3.74-3.69 (m, 2H), 3.53 (dd, J=11.3, 5.8 Hz, 1H), 3.36 (s, 3H), 3.35-3.31 (m, 2H), 3.32 (s, 3H), 3.29 (s, 3H), 2.59 (d, J=14.7 Hz, 1H), 2.51 (d, J=14.7 Hz, 1H), 2.23 (qd, J=7.0, 2.5 Hz, 1H), 1.97-1.92 (m, 1H), 1.82-1.77 (m, 1H), 1.71-1.53 (m, 2H), 1.20 (d, J=6.6 Hz, 3H), 1.03 (d, J=7.0, 3H), 0.92 (s, 3H), 0.85 (s, 3H), 0.24 (s, 9H), 0.14 (s, 9H), 0.11 (s, 9H).

[0478] .sup.13C NMR (100 MHz, (CD.sub.3).sub.2CO): δ 170.8, 147.7, 108.7, 99.2, 78.9 (×3), 78.3, 76.0, 72.5, 69.0, 63.5, 55.9, 55.0, 49.6, 41.3, 38.2, 36.3, 30.5, 29.7, 23.7, 19.0, 17.4, 11.4, −0.2, −0.6, −0.9.

[0479] (+)-HRESI-TOFMS m/z: 728.4108 [M+Na].sup.+ (calcd. for C.sub.33H.sub.67NO.sub.9Si.sub.3Na: 728.4021).

[0480] b)

##STR00091##

[0481] To a solution of 3 (20 mg, 0.028 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added SiO.sub.2 (2.5 g) at 23° C. After being stirred at 23° C. for 2.5 h, an extra amount of SiO.sub.2 was added again (1.5 g) and stirred for 3 h. The solid was filtered and washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure and then purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc, from 80:20 to 60:40) to afford pure 4 (15 mg, 85% yield) as colourless oil.

[0482] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2CO): δ 7.50 (d, J=10.0 Hz, 1H), 5.41-5.33 (m, 1H), 4.80-4.74 (m, 1H), 4.61-4.60 (m, 1H), 4.45 (s, 1H), 3.90-3.80 (m, 2H), 3.73 (dd, J=10.1, 4.2 Hz, 1H), 3.67-3.56 (m, 1H), 3.43-3.27 (m, 4H), 3.37 (m, 3H), 3.32 (s, 3H), 3.29 (s, 3H), 2.58-2.48 (m, 2H), 2.27-2.17 (m, 1H), 2.01-1.92 (m, 1H), 1.81-1.61 (m, 2H), 1.51 (ddd, J=14.2, 9.6, 1.8 Hz, 1H), 1.20 (d, J=6.6 Hz, 3H), 1.02 (d, J=7.0 Hz, 3H), 0.91 (s, 3H), 0.84 (s, 3H), 0.24 (s, 9H), 0.14 (s, 9H).

[0483] .sup.13C NMR (100 MHz, (CD.sub.3).sub.2CO): δ 171.4, 147.7, 108.6, 99.2, 79.6, 78.8, 78.1, 75.6, 72.3, 69.1, 63.5, 59.6, 55.7, 55.0, 49.5, 41.3, 38.3, 36.2, 30.4, 23.3, 19.9, 17.4, 13.6, 11.3, −0.3, −0.6.

[0484] (+)-HRESI-TOFMS m/z: 656.3718 [M+Na].sup.+ (calcd. for C.sub.30H.sub.59NO.sub.9Si.sub.2Na: 656.3626).

##STR00092##

[0485] To a solution of 4 (18 mg, 0.028 mmol) in CH.sub.2Cl.sub.2 (3 mL) was added 1,1′-carbonyldiimidazole (6 eq, 0.168 mmol) at 23° C. After being stirred overnight at 23° C., the reaction mixture was concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc, from 80:20 to 50:50) to yield pure 5 (17 mg, 85% yield) as colourless oil.

[0486] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2CO): δ 8.14 (s, 1H), 7.52 (s, 1H), 7.45 (d, J=10.0 Hz, 1H), 7.05 (s, 1H), 5.35 (dd, J=9.9, 6.8 Hz, 1H), 4.74 (d, J=2.4 Hz, 1H), 4.66 (dd, J=11.8, 2.4 Hz, 1H), 4.59-4.57 (m, 1H), 4.37 (ddd, J=11.8, 5.9, 1.3 Hz, 1H), 4.28 (s, 1H), 3.90 (q, J=5.4 Hz, 1H), 3.86-3.78 (m, 1H), 3.76 (dd, J=9.7, 4.1 Hz, 1H), 3.64 (td, J=6.4, 2.9 Hz, 1H), 3.55-3.48 (m, 1H), 3.36 (s, 6H), 3.30 (s, 3H), 2.45 (d, J=14.5 Hz, 1H), 2.33 (d, J=14.7 Hz, 1H), 2.19 (dd, J=10.1, 5.0 Hz, 1H), 2.02-1.88 (m, 2H), 1.79-1.60 (m, 2H), 1.18 (d, J=6.5, 3H), 1.05-0.93 (m, 6H), 0.88 (d, J=1.2 Hz, 3H), 0.23 (s, 9H), 0.14 (s, 9H).

[0487] .sup.13C NMR (100 MHz, (CD.sub.3).sub.2CO): δ 170.9, 148.6, 147.6, 136.8, 130.5, 117.1, 108.7, 99.1, 79.0, 78.0, 76.2 75.5, 72.2, 69.1, 68.2, 59.6, 56.1, 54.9, 49.4, 41.3, 38.2, 35.8, 30.3, 29.7, 23.4, 17.3, 13.6, 11.4, −0.4, −0.6.

[0488] (+)-HRESI-TOFMS m/z: 750.3975 [M+Na].sup.+ (calcd. for C.sub.34H.sub.61N.sub.3O.sub.10Si.sub.2Na: 750.3793).

[0489] d)

##STR00093##

[0490] To a solution of 5 (17 mg, 0.023 mmol) in CH.sub.2Cl.sub.2 (2 mL) was added propane 1,3-diamine (16.6 eq, 32 μL) at 23° C. The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (Si—NH.sub.2, CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:0 to 90:10) to obtain pure 6 (16 mg, 94% yield) as colourless oil.

[0491] .sup.1H NMR (400 MHz, CD.sub.3OD): δ 5.20 (d, J=4.4 Hz, 1H), 4.84-4.77 (m, 1H), 4.65-4.62 (m, 1H), 4.35 (s, 1H), 4.31-4.21 (m, 1H), 4.04 (dd, J=12.0, 5.1 Hz, 1H), 3.96-3.92 (m, 1H), 3.84 (qd, J=6.5, 2.5 Hz, 1H), 3.71 (dd, J=8.2, 3.9 Hz, 1H), 3.55-3.48 (m, 2H), 3.39 (d, J=0.7 Hz, 3H), 3.35 (s, 3H), 3.29 (s, 3H), 3.17 (t, J=6.7 Hz, 2H), 2.76 (t, J=7.1 Hz, 2H), 2.61 (d, J=14.6 Hz, 1H), 2.36 (d, J=14.6 Hz, 1H), 2.25-2.13 (m, 1H), 2.06-2.03 (m, 1H), 2.00-1.86 (m, 2H), 1.73-1.55 (m, 3H), 1.16 (d, J=6.6 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H), 0.94 (s, 3H), 0.85 (s, 3H), 0.20 (s, 9H), 0.13 (s, 9H).

[0492] .sup.13C NMR (100 MHz, CD.sub.3OD): δ 172.4, 157.8, 147.1, 108.6, 99.7, 81.7, 77.2, 76.3, 75.7, 72.8, 70.8, 69.5, 64.1, 55.7, 55.2, 48.6, 41.4, 37.7, 37.6, 37.4, 34.3, 30.8, 30.0, 29.1, 23.9, 16.9, 15.6, 10.9, −1.1, −1.2.

[0493] (+)-HRESI-TOFMS m/z: 734.4526 [M+H].sup.+ (calcd. for C.sub.34H.sub.67N.sub.3O.sub.10Si.sub.2: 733.4365).

[0494] e)

##STR00094##

[0495] To a solution of 6 (16 mg, 0.022 mmol) in MeOH (4 mL) was added 2.4 mL of 0.033 M potassium carbonate solution in methanol (3.65 eq, 0.08 mmol) at 23° C. The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (Si—NH.sub.2, CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:0 to 80:20) to afford pure 7 (12.8 mg, 99% yield) as colourless oil.

[0496] .sup.1H NMR (400 MHz, CD.sub.3OD): δ 5.49 (d, J=1.0 Hz, 1H), 5.30 (d, J=6.7 Hz, 1H), 4.83-4.79 (m, 1H), 4.67-4.63 (m, 1H), 4.28 (d, J=0.8 Hz, 1H), 4.20 (dd, J=11.8, 3.0 Hz, 1H), 4.03 (dd, J=11.8, 5.1 Hz, 1H), 3.94-3.83 (m, 2H), 3.61 (dd, J=10.5, 4.6 Hz, 1H), 3.57-3.51 (m, 1H), 3.39 (s, 3H), 3.33 (s, 3H), 3.28 (d, J=0.8 Hz, 3H), 3.17 (t, J=6.7 Hz, 2H), 2.70 (t, J=7.0 Hz, 2H), 2.47 (d, J=14.3 Hz, 1H), 2.35 (d, J=14.3 Hz, 1H), 2.27-2.13 (m, 1H), 2.04-1.94 (m, 1H), 1.78-1.60 (m, 5H), 1.18 (dd, J=6.6, 0.8 Hz, 3H), 0.98 (dd, J=7.0, 0.8 Hz, 3H), 0.93 (s, 3H), 0.86 (s, 3H).

[0497] .sup.13C NMR (100 MHz, CD.sub.3OD): δ 172.8, 157.8, 146.8, 108.8, 99.8, 80.3, 77.0, 75.7, 72.7, 71.8, 71.1, 69.3, 64.4, 55.7, 55.3, 53.4, 41.5, 38.0, 37.5, 33.5, 31.7, 29.9, 29.0, 22.5, 16.7, 13.0, 12.8, 11.1.

[0498] (+)-HRESI-TOFMS m/z: 590.3687 [M+H].sup.+ (calcd. For C.sub.28H.sub.51N.sub.3O.sub.10: 589.3574).

[0499] Intermediate 12

[0500] a)

##STR00095##

[0501] To a solution of 2 (26 mg, 0.055 mmol) and imidazole (13 eq, 0.715 mmol) in CH.sub.2Cl.sub.2 (3 mL) was added chlorotrimethylsilane (8 eq., 0.44 mmol) at 23° C. After being stirred at 23° C. for 1 h, the reaction mixture was concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc, from 90:10 to 70:30)) to afford pure 8 (26 mg, 60% yield) as colourless oil.

[0502] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2CO): δ 7.46 (d, J=9.9 Hz, 1H), 5.21 (dd, J=9.9, 4.4 Hz, 1H), 4.78-4.76 (m, 1H), 4.61-4.59 (m, 1H), 4.35 (d, J=0.6 Hz, 1H), 4.01-3.92 (m, 2H), 3.88-3.82 (m, 1H), 3.72 (dd, J=7.3, 3.6 Hz, 1H), 3.68-3.61 (m, 1H), 3.56-3.45 (m, 2H), 3.35 (s, 3H), 3.31 (s, 3H), 2.62-2.49 (m, 2H), 2.22 (qd, J=7.0, 2.6 Hz, 1H), 2.10-2.02 (m, 1H), 1.91 (ddd, J=13.5, 7.0, 3.7 Hz, 1H), 1.67 (ddd, J=14.1, 9.9, 2.7 Hz, 1H), 1.59-1.47 (m, 1H), 1.20 (dd, J=6.6, 0.7 Hz, 3H), 1.02 (dd, J=7.0, 0.7 Hz, 3H), 0.97 (s, 3H), 0.85 (s, 3H), 0.23 (s, 9H), 0.13 (s, 9H), 0.12 (s, 9H), 0.11 (s, 9H).

[0503] .sup.13C NMR (100 MHz, (CD.sub.3).sub.2CO): δ 170.7, 147.7, 108.6, 99.3, 80.3, 77.8, 76.6, 72.9, 70.5, 69.1, 65.8, 55.0, 49.4, 41.3, 37.5, 35.9, 33.2, 30.9, 24.9, 17.4, 11.3, −0.2, −0.3, −0.6, −0.9.

[0504] b)

##STR00096##

[0505] To a solution of 8 (40 mg, 0.052 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added SiO.sub.2 (2.5 g) at 23° C. After being stirred at 23° C. for 2.5 h, an extra amount of SiO.sub.2 was added again (1.5 g) and stirred for 3 h. The solid was filtered and washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure and then purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc, from 80:20 to 60:40) to afford pure 9 (16 mg, 44% yield) as colourless oil.

[0506] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2CO): δ 7.52 (d, J=9.9 Hz, 1H), 5.26 (dd, J=9.9, 5.4 Hz, 1H), 4.76-4.78 (m, 1H), 4.59-4.61 (m, 1H), 4.39 (s, 1H), 4.01-3.90 (m, 2H), 3.88-3.82 (m, 1H), 3.71 (dd, J=8.1, 3.8 Hz, 1H), 3.63-3.49 (m, 2H), 3.39-3.28 (m, 2H), 3.36 (s, 3H), 3.31 (s, 3H), 2.62-2.54 (m, 2H), 2.25-2.19 (m, 1H), 1.97-1.91 (m, 2H), 1.69-1.50 (m, 2H), 1.20 (d, J=6.6 Hz, 3H), 1.01 (d, J=7.0 Hz, 3H), 0.96 (s, 3H), 0.85 (s, 3H), 0.23 (s, 9H), 0.14 (s, 9H), 0.11 (s, 9H).

[0507] ESI-MS m/z: 714.4 (M+Na).sup.+.

[0508] c)

##STR00097##

[0509] To a solution of 9 (16 mg, 0.023 mmol) in CH.sub.2Cl.sub.2 (2 mL) was added 1,1′-carbonyldiimidazole (6 eq, 0.138 mmol) at 23° C. After being stirred overnight at 23° C., the reaction mixture was concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc, from 80:20 to 50:50) to yield pure 10 (12.4 mg, 68% yield) as colourless oil.

[0510] .sup.1H NMR (500 MHz, (CD.sub.3).sub.2CO): δ 8.14 (d, J=1.0 Hz, 1H), 7.53 (t, J=1.4 Hz, 1H), 7.48 (d, J=9.8 Hz, 1H), 7.06 (dd, J=1.6, 0.8 Hz, 1H), 5.27 (dd, J=9.8, 4.8 Hz, 1H), 4.76 (m, 1H), 4.61-4.53 (m, 2H), 4.39 (dd, J=11.1, 6.6 Hz, 1H), 4.31 (s, 1H), 4.29 (m, 1H), 4.02 (dt, J=5.6, 3.1 Hz, 1H), 3.84 (qd, J=6.6, 2.6 Hz, 1H), 3.76 (dd, J=7.8, 3.6 Hz, 1H), 3.64 (dd, J=11.5, 2.3 Hz, 1H), 3.36 (s, 3H), 3.31 (s, 3H), 2.46-2.43 (m, 2H), 2.25-2.17 (m, 2H), 2.00-1.95 (m, 1H), 1.80-1.75 (m, 1H), 1.6-1.55 (m, 1H), 1.19 (d, J=6.6 Hz, 3H), 1.02 (s, 3H), 0.98 (d, J=7.0 Hz, 3H), 0.89 (s, 3H), 0.23 (s, 9H), 0.15 (s, 9H), 0.14 (m, 9H).

[0511] .sup.13C NMR (100 MHz, (CD.sub.3).sub.2CO): δ 170.8, 148.6, 147.6, 136.8, 130.5, 117.1, 108.6, 99.3, 80.0, 77.7, 76.0, 72.7, 70.8, 69.2, 67.5, 55.0, 49.4, 41.3, 37.6, 35.6, 33.5, 30.7, 24.5, 17.3, 11.2, −0.4, −0.5, −0.6.

[0512] ESI-MS m/z: 808.4 (M+Na).sup.+.

[0513] d)

##STR00098##

[0514] To a solution of 10 (12.5 mg, 0.016 mmol) in CH.sub.2Cl.sub.2 (2.5 mL) was added propane 1,3-diamine (6 eq, 7 μL) at 23° C. The reaction mixture was stirred at 23° C. overnight, and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (Si—NH.sub.2, CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:0 to 90:10) to obtain pure 11 (5 mg, 39% yield) as colourless oil.

[0515] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2CO): δ 7.47 (d, J=9.7 Hz, 1H), 6.36-6.28 (m, 1H), 5.20 (dd, J=9.8, 4.2 Hz, 1H), 4.77-4.75 (m, 1H), 4.61-4.59 (m, 1H), 4.38 (s, 1H), 4.12-3.95 (m, 4H), 3.89-3.79 (m, 1H), 3.73 (dd, J=7.1, 3.6 Hz, 1H), 3.53 (d, J=10.7 Hz, 1H), 3.36 (s, 3H), 3.30 (s, 3H), 3.26-3.21 (m, 2H), 2.64 (d, J=14.7 Hz, 1H), 2.47 (d, J=14.7 Hz, 1H), 2.26-2.16 (m, 1H), 1.97-1.90 (m, 4H), 1.81-1.65 (m, 5H), 1.50-1.56 (m, 1H), 1.19 (d, J=6.5 Hz, 3H), 1.00 (d, J=7.0 Hz, 3H), 0.97 (s, 3H), 0.85 (s, 3H), 0.23 (s, 9H), 0.13 (s, 9H), 0.12 (s, 9H).

[0516] .sup.13C NMR (100 MHz, (CD.sub.3).sub.2CO): δ 170.8, 156.4, 147.9, 108.5, 99.5, 80.8, 77.2, 76.4, 72.9, 69.7, 69.1, 68.3, 66.9, 55.1, 49.2, 48.9, 41.4, 39.6, 37.4, 35.4, 33.4, 30.8, 30.7, 24.9, 17.4, 11.3, −0.4 (×2), −0.6.

[0517] (+)-HRESI-TOFMS m/z: 792.4403 [M+H].sup.+ (calcd. for C.sub.36H.sub.73N.sub.3O.sub.10Si.sub.3: 791.4604).

[0518] e)

##STR00099##

[0519] To a solution of 11 (6 mg, 0.0076 mmol) in MeOH (4 mL) was added 2.4 mL of 0.033 M potassium carbonate solution in methanol (10.5 eq, 0.08 mmol). The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (Si—NH.sub.2, CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:0 to 70:30) to afford 12 (4.3 mg, 99% yield) as colourless oil.

[0520] .sup.1H NMR (400 MHz, CD.sub.3OD): δ 5.33 (m, 1H), 4.80 (m, 1H), 4.65 (m, 1H), 4.28 (s, 1H), 4.06-3.83 (m, 3H), 3.61-3.46 (m, 3H), 3.47-3.36 (m, 1H), 3.39 (s, 3H), 3.28 (s, 3H), 3.20-3.14 (m, 2H), 2.72-2.65 (m, 2H), 2.53-2.48 (m, 1H), 2.38-2.34 (m, 1H), 2.20 (dt, J=8.1, 4.0 Hz, 1H), 2.01-1.95 (m, 2H), 1.78-1.61 (m, 4H), 1.17 (d, J=6.6 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H), 0.94 (s, 3H), 0.87 (s, 3H).

[0521] (+)-HRESI-TOFMS m/z: 576.3494 [M+H].sup.+ (calcd. for C.sub.27H.sub.49N.sub.3O.sub.10: 575.3418).

[0522] Intermediate 13

[0523] a)

##STR00100##

[0524] To a solution of 9 (80 mg, 0.126 mmol) and Et.sub.3N (6 equiv., 0.756 mmol) in CH.sub.2Cl.sub.2 (5 mL) was dropwise added methanesulfonyl chloride (6 equiv., 0.756 mmol) at 0° C. After being stirred at 0° C. for 30 min., the reaction mixture was concentrated under vacuum. The residue obtained was purified in an automatic system for flash chromatography (SiO.sub.2, Hex:EtOAc, from 90:10 to 70:30) to afford pure 13 (88 mg, 99% yield) as colourless oil.

[0525] .sup.1H NMR (500 MHz, (CD3).sub.2CO): δ 7.50 (d, J=10.0 Hz, 1H), 5.37 (dd, J=10.0, 7.0 Hz, 1H), 4.80-4.75 (m, 1H), 4.63-4.48 (m, 1H), 4.45 (s, 1H), 4.32 (dd, J=11.1, 2.0 Hz, 1H), 4.22 (dd, J=11.1, 6.6 Hz, 1H), 3.92-3.73 (m, 2H), 3.75 (ddd, J=9.7, 4.2, 0.5 Hz, 1H), 3.63-3.55 (m, 1H), 3.47-3.40 (m, 1H), 3.38 (s, 3H), 3.34 (s, 3H), 3.33 (s, 3H), 3.13 (s, 3H), 2.64-2.56 (m, 1H), 2.52 (dt, J=14.5, 0.7 Hz, 1H), 2.26-2.18 (m, 1H), 2.03-1.90 (m, 2H), 1.70-1.60 (m, 2H), 1.20 (d, J=6.6 Hz, 3H), 1.03 (d, J=7.0 Hz, 3H), 0.96 (s, 3H), 0.87 (s, 3H), 0.25 (s, 9H), 0.15 (s, 9H).

[0526] .sup.13C NMR (125 MHz, (CD3).sub.2CO): δ 172.1, 148.7, 109.4, 100.1, 79.7, 78.8, 77.1, 76.2, 73.1, 72.2, 70.0, 56.7, 55.9, 50.3, 42.2, 39.1, 37.2, 36.8, 31.2, 24.3, 18.2, 15.1, 12.1, 0.5, 0.3.

[0527] ESI-MS m/z: 734.3 [M+Na].sup.+.

[0528] Synthesis of Linkers

[0529] Preparation of LIN 1: MC-Val-Cit-PABC-PNP

[0530] Reaction Scheme

##STR00101##

[0531] (a) Preparation of LIN 1-1: MC-Val-Cit-OH

[0532] LIN 1-1

##STR00102##

[0533] CI-TrtCl-resin (20 g, 1.49 mmol/g) (Iris Biotech, Ref.: BR-1065, 2-Chlorotrityl chloride resin (200-400 mesh, 1% DVB, 1.0-1.6 mmol/g), CAS 42074-68-0) was placed in a filter plate. 100 mL of DCM was added to the resin and the mixture was stirred for 1 h. The solvent was eliminated by filtration under vacuum. A solution of Fmoc-Cit-OH (11.83 g, 29.78 mmol) and DIPEA (17.15 mL, 98.45 mmol) in DCM (80 mL) was added and the mixture was stirred for 10 min. After that DIPEA (34.82 mmol, 199.98 mmol) was added and the mixture was stirred for 1 h. The reaction was terminated by addition of MeOH (30 mL) after stirring for 15 minutes. The Fmoc-Cit-O-TrtCl-resin produced as a result was subjected to the following washing/treatments: DCM (5×50 mL×0.5 min), DMF (5×50 mL×0.5 min), piperidine:DMF (1:4, 1×1 min, 2×10 min), DMF (5×50 mL×0.5 min), DCM (5×50 mL×0.5 min). The final piperidine wash gave NH.sub.2—Cit-O-TrtCl-resin. The loading was calculated: 1.15 mmol/g.

[0534] The NH.sub.2—Cit-O-TrtCl-resin produced above was washed with DMF (5×50 mL×0.5 min) and a solution of Fmoc-Val-OH (31.22 g, 91.98 mmol), HOBt (11.23 g, 91.98 mmol) in DMF (100 mL) was added to the NH.sub.2—Cit-O-TrtCl-resin, stirred and DIPCDI (14.24 mL, 91.98 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5×50 mL×0.5 min). The Fmoc-Val-Cit-O-TrtCl-resin thus produced was treated with piperidine:DMF (1:4, 1×1 min, 2×10 min) and washed with DMF (5×50 mL×0.5 min). The final piperidine wash gave NH.sub.2—Val-Cit-O-TrtCl-resin.

[0535] A solution of 6-maleimidocaproic acid (MC-OH) (9.7 g, 45.92 mmol), HOBt (6.21 g, 45.92 mmol) in DMF (100 mL) was added to the NH.sub.2—Val-Cit-O-TrtCl-resin produced above, stirred and DIPCDI (7.12 mL, 45.92 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5×50 mL×0.5 min) and DCM (5×50 mL×0.5 min).

[0536] The peptide was cleaved from the resin by treatments with TFA:DCM (1:99, 5×100 mL). The resin was washed with DCM (7×50 mL×0.5 min). The combined filtrates were evaporated to dryness under reduced pressure and the solid obtained was triturated with Et.sub.2O and filtrated to obtain LIN 1-1 (7.60 g, 71%) as a white solid.

[0537] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 12.47 (s, 1H), 8.13 (d, J=7.3 Hz, 1H), 7.74 (d, J=9.0 Hz, 1H), 6.99 (s, 2H), 5.93 (s, 1H), 5.35 (s, 2H), 4.20 (dd, J=9.0, 6.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.36 (t, J=7.0 Hz, 2H), 3.00-2.88 (m, 2H), 2.21-2.12 (m, 1H), 2.11-2.03 (m, 1H), 1.98-1.86 (m, 1H), 1.74-1.62 (m, 1H), 1.61-1.50 (m, 1H), 1.50-1.31 (m, 6H), 1.21-1.11 (m, 2H), 0.84 (d, J=6.8 Hz, 3H), 0.80 (d, J=6.8 Hz, 3H).

[0538] ESI-MS m/z: 468.3 (M+H).sup.+.

[0539] (b) Preparation of LIN 1-2: MC-Val-Cit-PABOH

##STR00103##

[0540] To a solution of LIN 1-1 (1.6 g, 3.42 mmol) and 4-aminobenzyl alcohol (PABOH) (0.84 g, 6.84 mmol) in DCM (60 mL) was added a solution of HOBt (0.92 g, 6.84 mmol) in DMF (5 mL). DIPCDI (1.05 mL, 6.84 mmol) was added, the reaction mixture was stirred for 2 h at 23° C., Et.sub.2O (150 mL) was added, and the solid obtained was filtrated in a filter plate under vacuum to obtain LIN 1-2 (1.31 g, 67%).

[0541] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 9.88 (s, 1H), 8.03 (d, J=7.6 Hz, 1H), 7.77 (dd, J=12.2, 8.5 Hz, 1H), 7.53 (d, J=8.2 Hz, 2H), 7.21 (d, J=8.2 Hz, 2H), 6.99 (s, 3H), 6.01-5.92 (m, 1H), 5.39 (s, 2H), 5.07 (s, 1H), 4.41 (s, 2H), 4.39-4.31 (m, 1H), 4.23-4.12 (m, 1H), 3.36 (t, J=7.0 Hz, 2H), 3.06-2.97 (m, 1H), 2.96-2.90 (m, 1H), 2.22-2.03 (m, 2H), 2.01-1.88 (m, 1H), 1.76-1.62 (m, 1H), 1.63-1.28 (m, 6H), 1.25-1.11 (m, 2H), 0.84 (d, J=6.9 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H).

[0542] ESI-MS m/z: 573.3 (M+H).sup.+.

[0543] (c) Preparation of LIN 1: MC-Val-Cit-PAB-PNP

[0544] LIN 1

##STR00104##

[0545] To a solution of LIN 1-2 (500 mg, 0.87 mmol) and bis(4-nitrophenyl) carbonate (bis-PNP) (2.64 g, 8.72 mmol) in DCM:DMF (8:2, 25 mL) was added DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred for 20 h at 23° C. and poured onto a silica gel column (DCM:CH.sub.3OH, from 50:1 to 10:1) to afford pure target LIN 1 (364 mg, 57%).

[0546] R.sub.f=0.40 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

[0547] .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): δ 9.45 (s, 1H), 8.23 (d, J=8.3 Hz, 2H), 7.59 (d, J=8.5 Hz, 2H), 7.35 (d, J=8.3 Hz, 2H), 7.34 (d, J=8.5 Hz, 2H), 6.65 (s, 2H), 5.20 (s, 2H), 4.56 (dt, J=10.5, 5.4 Hz, 1H), 4.15 (d, J=7.2 Hz, 1H), 3.46 (dd, J=8.0, 6.4 Hz, 2H), 3.16-2.89 (m, 2H), 2.21 (dd, J=8.3, 6.6 Hz, 2H), 2.06-1.97 (m, 1H), 1.90-1.83 (m, 1H), 1.73-1.46 (m, 7H), 1.34-1.20 (m, 2H), 0.91 (d, J=6.7 Hz, 3H), 0.90 (d, J=6.7 Hz, 3H).

[0548] .sup.13C NMR (125 MHz, CDCl.sub.3/CD.sub.3OD) δ 174.4, 172.4, 171.1, 170.6, 160.5, 155.5, 152.5, 145.3, 138.7, 134.1, 129.9, 129.5, 125.2, 121.8, 120.0, 70.6, 59.0, 53.2, 37.5, 35.8, 30.6, 29.6, 29.3, 28.1, 26.2, 26.2, 25.1, 19.1, 18.1.

[0549] ESI-MS m/z: 738.3 (M+H).sup.+.

Example 1: Synthesis of Compounds of Formula D-X-(AA).SUB.w.-(T).SUB.9.-L.SUB.1

[0550] Preparation of Compound DL-1

##STR00105##

[0551] To a solution of 7 (13 mg, 0.022 mmol) and LIN-1 (24.4 mg, 0.033 mmol) in NMP (3 mL) was added DIPEA (10.5 μL, 0.06 mmol) at 23° C. After 15 h at 23° C. the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by semipreparative HPLC (sunfire C18, 10×150 mm. CH.sub.3CN:H.sub.2O, flow 4 mL/min, UV detection) to obtain pure DL-1 (14.5 mg, 54% yield) as a white solid.

[0552] .sup.1H NMR (500 MHz, CD.sub.3CN:D.sub.2O 2:1): δ 7.53 (d, J=8.5 Hz, 2H), 7.31 (d, J=8.3 Hz, 2H), 6.77 (m, 2H), 5.25 (d, J=7.6 Hz, 1H), 4.99 (s, 2H), 4.81 (t, J=2.1 Hz, 1H), 4.67 (t, J=2.1 Hz, 1H), 4.39 (dd, J=9.1, 5.1 Hz, 1H), 4.24 (s, 1H), 4.11-4.04 (m, 2H), 3.98 (dd, J=11.9, 5.0 Hz, 1H), 3.83 (ddd, J=11.3, 5.5, 3.4 Hz, 2H), 3.57 (dd, J=10.7, 4.5 Hz, 1H), 3.43-3.40 (m, 3H), 3.33 (s, 3H), 3.25 (s, 3H), 3.20 (s, 3H), 3.19-3.18 (m, 1H), 3.10-3.01 (m, 6H), 2.38-2.28 (m, 2H), 2.28-2.17 (m, 3H), 2.06-1.97 (m, 1H), 1.89 (ddd, J=13.7, 4.6, 3.1 Hz, 1H), 1.82 (s, 1H), 1.75-1.44 (m, 10H), 1.30-1.17 (m, 4H), 1.12 (d, J=6.6 Hz, 3H), 0.92 (d, J=7.0 Hz, 3H), 0.89 (d, J=3.0 Hz, 3H), 0.88 (d, J=3.0 Hz, 3H), 0.85 (s, 3H), 0.78 (s, 3H).

[0553] .sup.13C NMR (125 MHz, CD.sub.3CN:D.sub.2O 2:1): δ 175.3, 172.6, 172.5, 172.1, 171.1, 158.3, 158.2, 146.8, 137.7, 134.3, 133.1, 128.6, 120.2, 119.5, 109.7, 99.9, 79.8, 76.8, 75.7, 72.4 (×2), 70.8, 69.6, 65.9, 64.2, 63.9, 59.1, 56.1, 55.9, 53.7, 48.1, 41.1, 38.1, 37.8, 37.4, 35.3, 33.5, 32.1, 30.2, 29.8, 29.4, 28.9, 28.6, 27.8, 25.9, 25.0, 22.6, 21.9, 18.6, 17.6, 17.1, 11.5, 0.9.

[0554] (+)-HRESI-TOFMS m/z: 1210.6301 [M+Na].sup.+ (calcd. for C.sub.57H.sub.89N.sub.9O.sub.18Na: 1210.6223).

[0555] Preparation of Compound DL-2

##STR00106##

[0556] To a solution of 7 (17.8 mg, 0.030 mmol) and 6-Maleimidohexanoic acid N-hydroxysuccinimide ester (0.060 mmol, 19 mg) in CH.sub.2Cl.sub.2 (3 mL), was added DIPEA (16 μL, 0.091 mmol) at 23° C. The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:0 to 80:20) to afford pure DL-2 (17.5 mg, 74% yield).

[0557] .sup.1H NMR (400 MHz, (CD.sub.3).sub.2CO): δ 7.42 (d, J=9.6 Hz, 1H), 7.14-7.12 (m, 1H), 6.86-6.84 (m, 2H), 6.39-6.36 (m, 1H), 5.35 (dd, J=9.6, 7.5 Hz, 1H), 4.82-4.76 (m, 1H), 4.64-4.59 (m, 2H), 4.34-4.32 (m, 1H), 4.15 (dd, J=11.7, 3.3 Hz, 1H), 4.00 (dd, J=11.7, 5.1 Hz, 1H), 3.96-3.87 (m, 1H), 3.90-3.80 (m, 2H), 3.65-3.60 (m, 1H), 3.48-3.44 (m, 3H), 3.36 (s, 3H), 3.34-3.31 (m, 1H), 3.28-3.21 (m, 8H), 3.16-3.11 (m, 2H), 2.42-2.39 (m, 2H), 2.32-2.11 (m, 3H), 2.09-1.94 (m, 1H), 1.79-1.52 (m, 9H), 1.39-1.23 (m, 2H), 1.16 (d, J=6.6 Hz, 3H), 1.01 (d, J=7.0 Hz, 3H), 0.94 (s, 3H), 0.86 (s, 3H).

[0558] .sup.13C NMR (100 MHz, (CD.sub.3).sub.2CO): δ 172.3, 171.7, 170.8, 156.6, 147.2, 134.2, 109.0, 99.7, 79.8, 76.6, 75.5, 73.3, 72.1, 70.7, 69.0, 64.4, 55.9, 55.4, 48.2, 41.4, 38.2, 37.7, 37.2, 35.9, 35.6, 34.2, 30.8, 30.0, 29.8, 28.1, 26.1, 25.0, 23.0, 17.2, 13.2, 11.6.

[0559] (+)-HRESI-TOFMS m/z: 805.4233 [M+Na].sup.+ (calcd. for C.sub.38H.sub.62N.sub.4O.sub.13Na: 805.4211).

[0560] Preparation of Compound DL 3

##STR00107##

[0561] To a solution of 12 (4.5 mg, 0.0078 mmol) and 6-Maleimidohexanoic acid N-hydroxysuccinimide ester (2 eq., 4.8 mg) in CH.sub.2Cl.sub.2 (2 mL), was added DIPEA (4.1 μL, 3 eq.) at 23° C. The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:0 to 80:20) to afford DL-3 (3 mg, 50% yield).

[0562] .sup.1H NMR (500 MHz, (CD.sub.3).sub.2CO): δ 7.58 (d, J=9.7 Hz, 1H), 7.11-7.09 (m, 1H), 6.86 (s, 2H), 6.38-6.34 (m, 1H), 5.45-5.35 (m, 1H), 4.85-4.72 (m, 2H), 4.64-4.62 (m, 1H), 4.35-4.30 (m, 1H), 4.09-3.87 (m, 7H), 3.70-3.58 (m, 2H), 3.51-3.45 (m, 2H), 3.37 (s, 3H), 3.29 (s, 3H), 3.27-3.20 (m, 2H), 3.17-3.12 (m, 2H), 2.55-2.47 (m, 1H), 2.42-2.38 (m, 1H), 2.26-2.19 (m, 1H), 2.18-2.13 (m, 2H), 2.02-1.95 (m, 1H), 1.79-1.54 (m, 11H), 1.15 (d, J=6.6 Hz, 3H), 1.00 (d, J=7.0 Hz, 3H), 0.97 (s, 3H), 0.89 (s, 3H).

[0563] .sup.13C NMR (125 MHz, (CD.sub.3).sub.2CO): δ 173.6, 172.6, 171.4, 157.3, 148.2, 134.9, 109.3, 100.4, 80.2, 79.9, 74.6, 72.3, 71.2, 70.2, 69.5, 68.5, 56.0, 49.0, 42.1, 38.8, 38.3, 37.8, 36.5 (×2), 34.7, 33.3, 30.6, 30.5, 28.7, 26.8, 25.7, 23.8, 17.9, 14.5, 12.1.

[0564] (+)-HRESI-TOFMS m/z: 791.3830 [M+Na].sup.+ (calcd. for C.sub.37H.sub.60N.sub.4O.sub.13Na: 791.4055).

Example 2: Preparation of Antibody-Drug Conjugates (ADCs)

[0565] In this Example, syntheses of antibody-drug conjugates of the present invention are described. It should be noted that these syntheses are exemplary and that the processes described can be applied to all the compounds and antibodies described herein.

[0566] Preparation of Antibody-Drug Conjugate ADC-1 with Trastuzumab and Compound DL-1

[0567] (a) Preparation of Trastuzumab

[0568] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilized powder for the preparation of a concentrated solution for infusion) was dissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM, pH 8.0). Concentration of Trastuzumab (16.1 mg/mL) was determined by measuring the absorbance at 280 nm.

[0569] (b) Reaction of Trastuzumab with 2-Iminothiolane (Traut's Reagent) to Give Thiol-Activated Trastuzumab

[0570] Trastuzumab solution (0.75 mL, 12 mg, 72.7 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8). Traut's reagent was added (72.6 L, 872 nmol, 12 eq.), and the reaction stirred for 2 h at 20° C. The mixture was buffer exchanged using two Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 1.3 mL (9.3 mg/mL). Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.2.

[0571] (c) Preparation of ADC-1

[0572] To the solution of thiol-activated Trastuzumab (200 μL, 1.86 mg, 12.4 nmol), DMA was added (37 L) followed by addition of a freshly prepared solution of DL-1 (10 mM in DMA, 12.3 μL, 123 nmol, 10 eq.). The conjugation reaction was stirred for 2 h at 25° C. and purified by desalting using a Sephadex G25 NAP-5 column into PBS buffer. The final target product ADC-1 was concentrated to a final concentration of 6.32 mg/mL as determined by UV and 210 μL (1.32 mg, 8.8 nmol, 71%) ADC solution was obtained.

[0573] Preparation of Antibody-Drug Conjugate ADC-2 with Trastuzumab and Compound DL-2

[0574] (a) Preparation of Trastuzumab

[0575] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilized powder for the preparation of a concentrated solution for infusion) was dissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM, pH 8.0). Concentration of Trastuzumab (16.1 mg/mL) was determined by measuring the absorbance at 280 nm.

[0576] (b) Reaction of Trastuzumab with 2-Iminothiolane (Traut's Reagent) to Give Thiol-Activated Trastuzumab

[0577] Trastuzumab solution (0.75 mL, 12 mg, 72.7 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8). Traut's reagent was added (72.6 L, 872 nmol, 12 eq.), and the reaction stirred for 2 h at 20° C. The mixture was buffer exchanged using two Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 1.3 mL (9.3 mg/mL). Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.2.

[0578] (c) Preparation of ADC-2

[0579] To the solution of thiol-activated Trastuzumab (200 μL, 1.86 mg, 12.4 nmol), DMA was added (37 μL) followed by addition of a freshly prepared solution of DL-2 (10 mM in DMA, 12.3 μL, 123 nmol, 10 eq.). The conjugation reaction was stirred for 2 h at 25° C. and purified by desalting using a Sephadex G25 NAP-5 column into PBS buffer. The final target product ADC-2 was concentrated to a final concentration of 7.11 mg/mL as determined by UV and 220 μL (1.56 mg, 10.4 nmol, 84%) ADC solution was obtained.

[0580] Preparation of Antibody-Drug Conjugate ADC-3 with Trastuzumab and Compound DL-2

[0581] (a) Preparation of Trastuzumab

[0582] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilized powder for the preparation of a concentrated solution for infusion) was dissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM, pH 8.0). Concentration of Trastuzumab (17.6 mg/mL) was determined by measuring the absorbance at 280 nm.

[0583] (b) Partial reduction of Trastuzumab to give Partially Reduced Trastuzumab

[0584] Trastuzumab solution (0.55 mL, 9.7 mg, 64.5 nmol) was diluted to a concentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partial reduction of the disulfide bonds in the antibody was performed by the addition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP) solution (29.2 μL, 145.8 nmol, 2.2 eq.). The reduction reaction was left to stir for 90 min at 20° C. Immediately after the reduction, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 3.4.

[0585] (c) Preparation of ADC-3

[0586] To the solution of partially reduced Trastuzumab (0.14 mL, 1.78 mg, 11.9 nmol), DMA was added (28.2 μL) followed by addition of a freshly prepared solution of DL-2 (10 mM in DMA, 6.8 μL, 68 nmol, 5.6 eq.). The conjugation reaction was stirred for 30 min at 20° C. and the excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 6.8 μL, 68 nmol) followed by stirring the solution for 20 min. The quenched conjugation reaction was purified by desalting using Sephadex G25 NAP-5 columns into PBS buffer. The final target product ADC-3 was concentrated to a final concentration of 6.73 mg/mL as determined by UV and 188 μL (1.26 mg, 8.4 nmol, 70%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (91%).

[0587] Preparation of Antibody-Drug Conjugate ADC-4 with Trastuzumab and Compound DL-1

[0588] (a) Preparation of Trastuzumab

[0589] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilized powder for the preparation of a concentrated solution for infusion) was dissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM, pH 8.0). Concentration of Trastuzumab (17.1 mg/mL) was determined by measuring the absorbance at 280 nm.

[0590] (b) Partial reduction of Trastuzumab to give Partially Reduced Trastuzumab

[0591] Trastuzumab solution (0.25 mL, 4.27 mg, 28.5 nmol) was diluted to a concentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partial reduction of the disulfide bonds in the antibody was performed by the addition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP) solution (16.5 μL, 81.6 μmol, 3 eq.). The reduction reaction was left to stir for 90 min at 20° C. Immediately after the reduction, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 6.4.

[0592] (c) Preparation of ADC-4

[0593] To the solution of partially reduced Trastuzumab (125 μL, 1.24 mg, 8.3 nmol), DMA was added (19.3 μL) followed by addition of a freshly prepared solution of DL-1 (10 mM in DMA, 7.1 μL, 71 nmol, 8.5 eq.). The conjugation reaction was stirred for 2 h at 25° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 7.1 μL, 71 nmol) followed by stirring the solution for 20 min. The quenched conjugation reaction was purified by desalting using Sephadex G25 NAP-5 columns into PBS buffer. The final target product ADC-4 was concentrated to a final concentration of 4.58 mg/mL as determined by UV and 270 μL (1.24 mg, 8.26 nmol, 99%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (96%).

[0594] Preparation of Antibody-Drug Conjugate ADC-5 with Trastuzumab and Compound DL-3

[0595] (a) Preparation of Trastuzumab

[0596] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilized powder for the preparation of a concentrated solution for infusion) was dissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM, pH 8.0). Concentration of Trastuzumab (16.1 mg/mL) was determined by measuring the absorbance at 280 nm.

[0597] (b) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab

[0598] Trastuzumab solution (0.3 mL, 4.8 mg, 32.2 nmol) was diluted to a concentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partial reduction of the disulfide bonds in the antibody was performed by the addition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP) solution (19.3 μL, 96.6 μmol, 3 eq.). The reduction reaction was left to stir for 90 min at 20° C. Immediately after the reduction, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.3.

[0599] (c) Preparation of ADC-5

[0600] To the solution of partially reduced Trastuzumab (150 μL, 1.5 mg, 10 nmol), DMA was added (29.5 μL) followed by addition of a freshly prepared solution of DL-3 (10 mM in DMA, 8 μL, 80 nmol, 8 eq.). The conjugation reaction was stirred for 30 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 8 μL, 80 nmol) followed by stirring the solution for 20 min. The quenched conjugation reaction was purified by desalting using Sephadex G25 NAP-5 columns into PBS buffer. The final target product ADC-5 was concentrated to a final concentration of 6.1 mg/mL as determined by UV and 180 μL (1.1 mg, 7.3 nmol, 73%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (96%).

Example 3: Demonstrating the Cytotoxicity of the Antibody-Drug Conjugates of the Present Invention

[0601] Bioassays for the detection of antitumor activity

[0602] The aim of the assay was to evaluate the in vitro cytostatic (ability to delay or arrest tumor cell growth) or cytotoxic (ability to kill tumor cells) activity of the samples being tested.

[0603] Cell Lines and Cell Culture

[0604] The following human tumor cell lines were obtained from the American Type Culture Collection (ATCC): SK-BR-3 (ATCC HB-30), HCC-1954 (ATCC CRL-2338) (both being breast cancer, HER2+), MDA-MB-231 (ATCC HTB-26), MCF-7 (ATCC HTB-22) (both being breast cancer, HER2−). Cells were maintained at 37° C., 5% CO.sub.2 and 95% humidity either in Dulbecco's Modified Eagle's Medium (DMEM) (for SK-BR-3, MDA-MB-231 and MCF-7 cells), or RPMI-1640 (for HCC-1954 cell line), all media supplemented with 10% Fetal Calf Serum (FCS), 2 mM L-glutamine and 100 units/mL of penicillin and streptomycin.

[0605] Cytotoxicity Assay

[0606] For SK-BR-3, HCC-1954, MDA-MB-231 and MCF-7 cells, a colorimetric assay using Sulforhodamine B (SRB) was adapted for quantitative measurement of cell growth and cytotoxicity, as described by V. Vichai and K. Kirtikara. In “Sulforhodamine B colorimetric assay for cytotoxicity screening”, Nature Protocols, 2006, 1: 1112-1116. Briefly, cells were seeded in 96-well microtiter plates and allowed to stand for 24 hours in drug-free medium before treatment with vehicle alone or with the tested substances for 72 hours. For quantification, cells were washed twice with phosphate buffered saline (PBS), fixed for 15 min in 1% glutaraldehyde solution, rinsed twice with PBS, stained in 0.4% (w/v) SRB with 1% (v/v) acetic acid solution for 30 min, rinsed several times with 1% acetic acid solution and air-dried. SRB was then extracted in 10 mM Trizma base solution and the optical density measured at 490 nm in a microplate spectrophotometer.

[0607] Cell survival was expressed in all cases as percentage of control, untreated cell survival. All evaluations were performed in triplicate and the resulting data were fitted by nonlinear regression to a four-parameters logistic equation using the Prism 5.0 statistical software (GraphPad, La Jolla, Calif., USA), from which the IC.sub.50 value (the concentration of compound causing 50% effect on cell growth as compared to the untreated control) was calculated. The data presented here correspond to the geometric mean of three independent experiments performed in triplicate.

[0608] The in vitro cytotoxicity of the ADCs along with the parent cytotoxic compounds 1, 2 and DL-2 and Trastuzumab were evaluated against four different human breast cancer cell lines over-expressing or not the HER2 receptor, including SK-BR-3, HCC-1954 (HER2-positive cells) as well as MDA-MB-231 and MCF-7 (HER2-negative cells). Standard dose-response (DR) curves for 72 hours incubation with the tested substances were performed.

[0609] Assays with HER2 Positive/Negative Cell Lines.

[0610] Cytotoxicity of Trastuzumab

[0611] The in vitro cytotoxicty of Trastuzumab was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 50 to 0.01 μg/mL (3.33E-07-8.74E-11 M). Trastuzumab was completely inactive, not reaching the IC.sub.50 in any of the cell lines tested, independently of their HER2 status as shown in Table 3 where results corresponding to the geometric mean of the IC.sub.50 values obtained in three independent experiments are presented.

TABLE-US-00003 TABLE 3 Summary of the in vitro cytotoxicity of Trastuzumab. HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL >50 >50 >50 >50 IC.sub.50, M >3.4E−07 >3.4E−07 >3.4E−07 >3.4E−07

[0612] Cytotoxicity of 1

[0613] The cytotoxicity of the intermediate compound 1 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 to 0.03 ng/mL (2.04E-07-6.12E-11 M).

[0614] As shown in Table 4, where results corresponding to the geometric mean of the IC.sub.50 values obtained in three independent experiments are presented, the cytotoxicity of this compound was similar in all the tumor cell lines regardless of their HER2 expression, with IC.sub.50 values in the nanomolar range.

TABLE-US-00004 TABLE 4 Summary of the in vitro cytotoxicity of 1 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 9.49E−04 6.66E−04 5.96E−04 8.37E−04 IC.sub.50, M 1.94E−09 1.36E−09 1.22E−09 1.71E−09

[0615] Cytotoxicity of 2

[0616] The cytotoxicity of the intermediate compound 2 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 1 μg/mL to 0.3 ng/mL (2.10E-06-6.30E-10 M)

[0617] As shown in Table 5, the cytotoxicity of this compound was similar in all the tumor cell-lines regardless of the HER2 expression, with IC.sub.50 values in the nanomolar range.

TABLE-US-00005 TABLE 5 Summary of the in vitro cytotoxicity of 2 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 1.30E−02 6.18E−03 5.61E−03 1.20E−02 IC.sub.50, M 2.73E−08 1.30E−08 1.18E−08 2.52E−08

[0618] Cytotoxicity of DL-2

[0619] The cytotoxicity of the intermediate compound DL-2 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 10 μg/mL to 2.6 ng/mL (1.28E-05-3.32E-09 M).

[0620] As shown in Table 6, the cytotoxicity of this compound was similar in all the tumor cell lines regardless of their HER2 expression, with IC.sub.50 values in the high nanomolar range.

TABLE-US-00006 TABLE 6 Summary of the in vitro cytotoxicity of DL-2 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 1.08E−01 7.60E−02 6.52E−02 7.59E−02 IC.sub.50, M 1.38E−07 9.71E−08 8.33E−08 9.70E−08

[0621] Cytotoxicity of ADC-3

[0622] The cytotoxicity of ADC-3 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table 7 summarizes the results corresponding to the geometric mean of the IC.sub.50 values obtained. ADC-3 showed a cytotoxicity which is similar to that shown by the parent drug 1 only in HER2-positive cells. However, in HER2-negative cells such toxicity is significantly lower: more than 500-fold lower according to the selectivity ratio obtained by dividing the mean IC.sub.50 value in HER2-negative cells between that in HER2-positive cells. This selectivity leads us to conclude that the conjugate is acting through the interaction of the antibody with the membrane associated HER2 receptor on the tumor cells, followed by intracellular delivery of the cytotoxic drug.

TABLE-US-00007 TABLE 7 Summary of the in vitro cytotoxicity of ADC-3 IC.sub.50 in IC.sub.50 in HER2 negative HER2+ HER2− HER2 positive MDA-MB- (geom. (geom. Selec. SK-BR-3 HCC-1954 231 MCF-7 mean) mean ratio IC.sub.50 1.61E−01 2.10E−01 >1.00E+02 >1.00E+02 1.84E−01 >1.00E+02 >546.7 (μg/mL) IC.sub.50 1.07E−09 1.40E−09 >6.67E−07 >6.67E−07 1.22E−09 >6.67E−07 (M)

[0623] Cytotoxicity of ADC-2

[0624] The cytotoxicity of the ADC-2 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table 8 summarizes the results corresponding to the geometric mean of the IC.sub.50 values obtained. ADC-2 showed a cytotoxicity which is similar to that shown by the parent drug 1 only in HER2-positive cells. However, in HER2-negative cells such toxicity is lower, nearly 7-fold lower according to the selectivity ratio obtained by dividing the mean IC.sub.50 value in HER2-negative cells between that in HER2-positive cells. This selectivity leads us to conclude that the conjugate is acting through the interaction of the antibody with the membrane associated HER2 receptor on the tumor cells, followed by intracellular delivery of the cytotoxic drug.

TABLE-US-00008 TABLE 8 Summary of the in vitro cytotoxicity of ADC-2 IC.sub.50 in IC.sub.50 in HER2 negative HER2+ HER2− HER2 positive MDA-MB- (geom. (geom. Selec. SK-BR-3 HCC-1954 231 MCF-7 mean) mean Ratio IC.sub.50 3.30E−01 5.00E−01 1.91E+00 4.20E+00 4.06E−01 2.83E+00 6.97 (μg/mL) IC.sub.50 2.20E−09 3.33E−09 1.27E−08 2.80E−08 2.71E−09 1.89E−08 (M)

[0625] Cytotoxicity of ADC-1

[0626] The cytotoxicity of the ADC-1 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table 9 summarizes the results corresponding to the geometric mean of the IC.sub.50 values obtained. ADC-1 showed a cytotoxicity which is similar to that shown by the parent drug 1 only in HER2-positive cells. However, in HER2-negative cells such toxicity is lower, more than 400-fold lower according to the selectivity ratio obtained by dividing the mean IC.sub.50 value in HER2-negative cells between that in HER2-positive cells. This selectivity leads us to conclude that the conjugate is acting through the interaction of the antibody with the membrane associated HER2 receptor on the tumor cells, followed by intracellular delivery of the cytotoxic drug.

TABLE-US-00009 TABLE 9 Summary of the in vitro cytotoxicity of ADC-1 IC.sub.50 in IC.sub.50 in HER2 positive HER2 negative HER2+ HER2− HCC- MDA-MB- (geom. (geom. Selec. SK-BR-3 1954 231 MCF-7 mean) mean Ratio IC.sub.50 1.50E−01 3.41E−01 >1.00E+02 >1.00E+02 2.27E−01 >1.00E+02 >440 (μg/mL) IC.sub.50 1.00E−09 2.27E−09 >6.67E−07 >6.67E-07 1.51E−09 >6.67E−07 (M)

[0627] Cytotoxicity of ADC-4

[0628] The cytotoxicity of the ADC-4 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table 10 summarizes the results corresponding to the geometric mean of the IC.sub.50 vales obtained. ADC-4 showed a cytotoxicity which is similar to that shown by the parent drug 1 only in HER2-positive cells. However, in HER2-negative cells such toxicity is lower, more than 1200-fold lower according to the selectivity ratio obtained by dividing the mean IC.sub.50 value in HER2-negative cells between that in HER2-positive cells. This selectivity lead us to conclude that the conjugate is acting through the interaction of the antibody with the membrane associated HER2 receptor on the tumor cells, followed by intracellular delivery of the cytotoxic drug.

TABLE-US-00010 TABLE 10 Summary of the in vitro cytotoxicity of ADC-4 IC.sub.50 in IC.sub.50 in HER2 positive HER2 negative HER2+ HER2− HCC- MDA-MB- (geom. (geom. Selec. SK-BR-3 1954 231 MCF-7 mean) mean Ratio IC.sub.50 <2.60E−02 7.80E−02 >1.00E+02 >1.00E+02 <7.80E−02 >1.00E+02 >1280 (μg/mL) IC.sub.50 <1.73E−10 5.20E−10 >6.67E−07 >6.67E−07 <5.20E−10 >6.67E−07 (M)

[0629] Cytotoxicity of ADC-5

[0630] The cytotoxicity of the ADC-5 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table 11 summarizes the results corresponding to the geometric mean of the IC.sub.50 values obtained. This conjugate selectively impairs growth of HER2-expressing cell lines, with potencies that exceed those shown by the corresponding drug, hence suggesting that the conjugation is favouring cellular uptake of the drug. This remarkable activity in HER2-expressing cell lines is responsible for the large selectivity observed with regard to HER2-negative cells.

TABLE-US-00011 TABLE 11 Summary of the in vitro cytotoxicity of ADC-5 IC.sub.50 in IC.sub.50 in HER2 positive HER2 negative HER2+ HER2- HCC- MDA-MB- (geom. (geom. Selec. SK-BR-3 1954 231 MCF-7 mean) mean Ratio IC.sub.50 >7.61E−02 1.70E−01 >1.00E+02 >1.00E+02 1.13E−01 >1.00E+02 >880 (μg/mL) IC.sub.50 >5.07E−10 1.13E−09 >6.67E−07 >6.67E−07 7.57E−10 >6.67E−07 (M)

[0631] It is also seen from WO 2018/167270 that drug moieties D of the present invention demonstrate efficacy in the following cell lines, the data of which is hereby incorporated by reference.

TABLE-US-00012 Name No ATCC Species Tissue Characteristics A549 CCL-185 human lung lung carcinoma (NSCLC) HT29 HTB-38 human colon colorectal adenocarcinoma MDA-MB-231 HTB-26 human breast breast adenocarcinoma PSN1 CRM-CRL-3211 human pancreas pancreas adenocarcinoma

Example 4. MTD and MTMD of Compound 1

[0632] Maximum Tolerated Dose of 1 in Mice

[0633] CB17/SCID female mice (Envigo) were used for this study. Animals were randomly allocated to dose groups and received a single intravenous administration.

[0634] After the administration, animals were observed for clinical signs at fixed intervals, up to 14 days after dosing. Mortality was recorded daily.

[0635] The Maximum Tolerated Dose (MTD) was defined as the dose level with no mortality recorded. Results are summarized in Table 12.

TABLE-US-00013 TABLE 12 MTD of compound 1 Dose Levels MTD (mg/kg) (mg/kg) 50.0 0.1 25.0 15.0 10.0 5.0 2.5 1.0 0.5 0.25 0.1 Placebo

[0636] Maximum Tolerated Multiple Dose of 1 in Mice

[0637] Athymic and CB17/SCID female mice (Envigo) were used for these studies. Animals were randomly allocated to dose groups and received weekly intravenous administration of 1 for 5 consecutive days (q7dx5).

[0638] After administration, animals were observed fro clinical signs at fixed intervals, up to 14 days after the last dose. Mortality was recorded daily.

[0639] The Maximum Tolerated Multiple Dose (MTMD) was defined as the dose level with no mortality recorded. Results are summarized in Table 13.

TABLE-US-00014 TABLE 13 MTMD of compound 1. Animals Dose Levels (mg/kg) MTMD (mg/kg) Athymmic 2.00  0.1* 1.00 0.10 0.075 0.025 Placebo CB17/SCID 0.10 0.075* 0.075 0.025 Placebo

Example 5. In Vivo Activity of ADC 3

[0640] ADC-3 has been evaluated for in vivo antitumor activity in BT474 cell line, a HER2 positive breast tumor model.

[0641] Experimental Design

[0642] Briefly, 4 to 6 week-old SCID mice were subcutaneously implanted with tumor fragments previously generated in donor mice. Before the initiation of each experiment, a blinded, independent determination of C-erb2 (oncogene related with HER-2 expression) by IHC was performed.

[0643] The animals were implanted as described above and when tumors reached ca. 150-200 mm3, tumor bearing animals (N=10/group) were randomly allocated into treatment groups according to the following experimental design:

TABLE-US-00015 Tumor Group Dose (mg/kg) BT-474 Control 0.0 Herceptin 30.0 Kadcyla 30.0 ADC3 4.0

[0644] Treatments were administered intravenously on days 0.7.14, 21 and 28 (q7dx5).

[0645] Tumor dimensions and body weights were recorded 3 times per week starting from the first day of treatment (Day 0). Treatments producing >20% lethality and/or 20% net body weight loss were considered toxic. Tumor volume was calculated using the equation (a.Math.b.sup.2)/2, where a and b were the longest and shortest diameters, respectively. Animals were euthanized when their tumors reached ca. 2000 mm.sup.3 and/or severe necrosis was seen. Median was calculated for tumor volume on each measurement day. Complete tumor regression (CR) was defined when tumor volume <63 mm.sup.3 for 2 or more consecutive measurements.

[0646] Results

[0647] All the treatments administered to mice bearing BT-474 tumors resulted in antitumor activity (FIG. 3)

[0648] ADC3 administered at 4 mg/kg and Kadcyla (30 mg/kg) induced tumor regressions. ADC3 at 4.0 mg/kg, 7 out of 7 tumor regressions from day 26 to day 190 and Kadcyla at 30.0 mg/kg, 7 out of 7 tumor regressions from day 14 to day 190. Since no tumor regrowth has yet been observed (on Day 190), this experiment is considered to be still on going.

[0649] Herceptin (30 mg/Kg) treatment also induced marked antitumor activity that was statistically significant, p<0.038 from day 12 until the last experimental data registered day, although complete tumor regressions were not achieved.