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ANTIBODY DRUG CONJUGATES COMPRISING ECTEINASCIDIN DERIVATIVES

20230012681 · 2023-01-19

    Inventors

    Cpc classification

    International classification

    Abstract

    Drug conjugates having formula [D-(X).sub.6-(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, ester, solvate, tautomer or stereoisomer thereof, (I) wherein D is covalently attached via a hydroxy or amine group 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.

    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, ester, solvate, tautomer or stereoisomer thereof, ##STR00256## wherein: D is covalently attached via a hydroxy or amine group to (X).sub.b if any, or (AA).sub.w if any, or to (T).sub.g if any, or (L); Y is —NH— or —O—; R.sub.1 is —OH or —CN; R.sub.2 is a —C(═O)R.sub.a group; R.sub.3 is hydrogen or a —OR.sub.b group; R.sub.4 is selected from —CH.sub.2O— and —CH.sub.2NH—; 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, and substituted or unsubstituted C.sub.2-C.sub.12 alkynyl; 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, and substituted or unsubstituted C.sub.2-C.sub.12 alkynyl; 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; wherein the drug moiety D is conjugated via the R.sub.4 position.

    2. The drug conjugate according to claim 1, wherein D is selected from formula Ia or Ib, or a pharmaceutically acceptable salt or ester thereof: ##STR00257## wherein: Y is —NH— or —O—; R.sub.1 is —OH or —CN; R.sub.2 is a —C(═O) R.sub.a group; R.sub.3 is hydrogen or a —OR.sub.b group; R.sub.4 is selected from —CH.sub.2O— and —CH.sub.2NH—; 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, and substituted or unsubstituted C.sub.2-C.sub.12 alkynyl; and 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, and substituted or unsubstituted C.sub.2-C.sub.12 alkynyl.

    3. The drug conjugate according to claim 1 or claim 2, wherein R.sub.4 is —CH.sub.2O-.

    4. The drug conjugate according to claim 1 or claim 2, wherein R.sub.4 is —CH.sub.2NH—.

    5. The drug conjugate according to claim 1, wherein D is a compound of formula: ##STR00258## ##STR00259## 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 (L); preferably of formula: ##STR00260## ##STR00261## ##STR00262## ##STR00263## or a pharmaceutically acceptable salt or ester thereof; preferably of formula: ##STR00264## ##STR00265## or a pharmaceutically acceptable salt or ester thereof.

    6. The drug conjugate according to claim 1, wherein D is a compound of formula: ##STR00266## 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 (L).

    7. The drug conjugate according to claim 1, wherein D is a compound of formula: ##STR00267## 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 (L).

    8. The drug conjugate according to claim 1, wherein D is a compound of formula: ##STR00268## 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 (L).

    9. The drug conjugate according to claim 1, wherein D is a compound of formula: ##STR00269## 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 (L).

    10. The drug conjugate according to claim 1, wherein D is a compound of formula: ##STR00270## 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 (L).

    11. 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 (IH) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof: ##STR00271## 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 (L); each Y and Z is independently selected from —NH— and —O—; R.sub.1 is —OH or —CN; R.sub.2 is a —C(═O)R.sub.a group; R.sub.3 is hydrogen or a —OR.sub.b group; 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, and substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, wherein the optional substituents are one or more substituents R.sub.x; and 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, and substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, 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, S(O)R.sub.y, SO.sub.2R.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; 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.

    12. A drug conjugate according to claim 11, or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein D is a drug moiety selected from formulas (IHa) and (IHb): ##STR00272## where the wavy lines, R.sub.1, R.sub.2, R.sub.3, Y, and Z are as defined for formula (IH).

    13. The drug conjugate according to any one of claims 1 to 4, 11 or 12, wherein Y is —NH—.

    14. The drug conjugate according to any one of claims 1 to 4, 11 or 12, wherein Y is —O—.

    15. The drug conjugate according to any one of claims 1 to 4, or 11 to 14, wherein R.sub.1 is —OH.

    16. The drug conjugate according to any one of claims 1 to 4, or 11 to 14, wherein R.sub.1 is —CN.

    17. The drug conjugate according to any one of claims 1 to 4, or 11 to 16, wherein R.sub.2 is a —C(═O)R.sub.a group where R.sub.a is substituted or unsubstituted C.sub.1-C.sub.6 alkyl; preferably wherein R.sub.a is selected from substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted isopropyl, substituted or unsubstituted n-butyl, substituted or unsubstituted isobutyl, substituted or unsubstituted sec-butyl and substituted or unsubstituted tert-butyl.

    18. The drug conjugate according to claim 17 wherein R.sub.2 is acetyl.

    19. The drug conjugate according to any one of claims 1 to 4, or 11 to 18, wherein R.sub.3 is hydrogen or —OR.sub.b wherein R.sub.b is substituted or unsubstituted C.sub.1-C.sub.6 alkyl; preferably wherein R.sub.b is selected from substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted isopropyl, substituted or unsubstituted n-butyl, substituted or unsubstituted isobutyl, substituted or unsubstituted sec-butyl and substituted or unsubstituted tert-butyl.

    20. The drug conjugate according to claim 19 wherein R.sub.3 is hydrogen.

    21. The drug conjugate according to claim 19 wherein R.sub.3 is —OR.sub.b wherein R.sub.b is substituted or unsubstituted C.sub.1-C.sub.6 alkyl; preferably wherein R.sub.b is selected from substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted isopropyl, substituted or unsubstituted n-butyl, substituted or unsubstituted isobutyl, substituted or unsubstituted sec-butyl and substituted or unsubstituted tert-butyl.

    22. The drug conjugate according to claim 21 wherein R.sub.3 is methoxy.

    23. The drug conjugate according to any one of claims 1 to 22, 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.

    24. A drug conjugate according to any one of the preceding claims, wherein L is a linker group selected from the group consisting of: ##STR00273## 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 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.8carbocyclo)-, —(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.6alkyl)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.

    25. A drug conjugate according to any of claims 1 to 24, wherein L is a linker group selected from the group consisting of: ##STR00274## 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 to (X).sub.b (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.8carbocyclo)- and phenylene which may optionally be substituted with one or more substituents R.sub.x; and r is an integer ranging from 1-6.

    26. A drug conjugate according to any one of claims 1 to 25, selected from the formulas (IV), (V) and (VI): ##STR00275## 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 (IV), (V) or (VI) 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)-.

    27. A drug conjugate according to claim 26 selected from the formulas (IV), (V) and (VI): ##STR00276## 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 (IV), (V) or (VI) 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.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, 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, and preferably R.sub.19 is a —C.sub.1-C.sub.6 alkylene group; R.sub.30 is a —C.sub.2-C.sub.4 alkylene- group; and M is —C.sub.1-C.sub.3 alkylene-(C.sub.5-C.sub.7carbocyclo)-.

    28. A drug conjugate according to any one of claims 1 to 27, wherein (AA).sub.w is of formula (II): ##STR00277## 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, ##STR00278## and w is an integer ranging from 0 to 12.

    29. A drug conjugate according to claim 28, wherein (AA).sub.w is of formula (II) 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.

    30. A drug conjugate according to any one of claims 1 to 28, wherein w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00279## 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); R.sub.22 is selected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl; and R.sub.23 is selected from methyl, —(CH.sub.2).sub.4NH.sub.2, —(CH.sub.2).sub.3NHCONH.sub.2 and —(CH.sub.2).sub.3NHC(═NH)NH.sub.2.

    31. A drug conjugate according to any one of claims 1 to 30, wherein X is an extending group selected from: where D is covalently attached via an amine group (for example where Z is —NH—): —COO—(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—; —COO—(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—; —COO—(C.sub.1-C.sub.6 alkylene)S—; —COO—(C.sub.1-C.sub.6 alkylene)NHCO(C.sub.1-C.sub.6 alkylene)S—; and where D is covalently attached via an hydroxy group (for example where Z is —O—): —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, preferably 1.

    32. A drug conjugate according to any one of claims 1 to 31, wherein X is an extending group selected from the group consisting of: where D is covalently attached via an amine group (for example where Z is —NH—): —COO—(C.sub.2-C.sub.4 alkylene)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; —COO—(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—; —COO—(C.sub.2-C.sub.4 alkylene)S—; —COO—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—; or where D is covalently attached via an hydroxy group (for example where Z is —O—): —CONH—(C.sub.2-C.sub.4 alkylene)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, preferably 1.

    33. A drug conjugate according to claim 32, wherein X is an extending group selected from the group consisting of: where D is covalently attached via an amine group (for example where Z is —NH—): —COO-CH.sub.2-phenylene-NH— —COO(CH.sub.2).sub.3NHCOOCH.sub.2-phenylene-NH—; —COO(CH.sub.2).sub.3NH—; —COO(CH.sub.2).sub.3—S—; —COO(CH.sub.2).sub.3NHCO(CH.sub.2).sub.2S—; or where D is covalently attached via an hydroxy group (for example where Z is —O—): —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, preferably 1.

    34. A drug conjugate according to any one of claims 1 to 33, 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.6alkylene)].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.

    35. A drug conjugate according to claim 34, 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.

    36. A drug conjugate according to claim 35, 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

    37. A drug conjugate according to any one of claims 11 to 36, wherein D is a drug moiety of formula (IHa) or formula (IHb) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein: R.sub.1 is CN or OH; R.sub.2 is C(═O)R.sub.a, wherein R.sub.a is selected from hydrogen and 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 a —OR.sub.b group wherein R.sub.b is a substituted or unsubstituted C.sub.1-C.sub.6 alkyl group, wherein the optional substituents are one or more substituents R.sub.x, Y is —NH— or —O—; and Z is —NH— or —O—.

    38. A drug conjugate according to claim 37, wherein D is a drug moiety of formula (IHa) or formula (IHb) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein: R.sub.1 is CN or OH; R.sub.2 is acetyl; R.sub.3 is hydrogen or methoxy, more preferably hydrogen; Y is —NH— or —O—; and Z is —NH— or —O—.

    39. A drug conjugate according to claim 37 or claim 38 wherein D is a drug moiety of formula (IHa) or formula (IHb), or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof wherein: R.sub.1 is CN; R.sub.2 is acetyl: R.sub.3 is hydrogen; Y is —NH— or —O—; and Z is —NH—.

    40. A drug conjugate according to any one of claims 1, 3, 4, 11, or 13 to 39, wherein D is selected from: ##STR00280## or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer 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).

    41. A drug conjugate according to any one of claims 1 to 4 or 11 to 40, wherein D is ##STR00281## or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer 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).

    42. A drug conjugate according to any one of claims 1 to 41, wherein the moiety Ab comprising at least one antigen binding site is an antigen-binding peptide.

    43. A drug conjugate according to claim 42, wherein the moiety Ab comprising at least one antigen binding site is an antibody, a single domain antibody or an antigen-binding fragment thereof.

    44. A drug conjugate according to claim 42 or 43, 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, preferably a human, mouse or rabbit.

    45. A drug conjugate according to claim 43 or 44, 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.

    46. A drug conjugate according to any one of claims 43 to 45, 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.

    47. A drug conjugate according to any one of claims 43 to 46, 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, and preferably a monoclonal antibody which immunospecifically binds to cancer cell antigens.

    48. A drug conjugate according to any one of claims 1 to 47, 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, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof.

    49. A drug conjugate according to claim 48, 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, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof.

    50. A drug conjugate according to claim 48, 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, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof, more preferably Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologicallly active portion thereof, preferably Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof, more preferably Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof.

    51. A drug conjugate according to claim 11 or claim 12, wherein: L is a linker group selected from the group consisting of: ##STR00282## 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 to (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.8carbocyclo)- and phenylene which may optionally be substituted with one or more substituents R.sub.x; r is an integer ranging from 1-6; (AA).sub.w is of formula (II): ##STR00283## 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); 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, ##STR00284## w is an integer ranging from 0 to 12; wherein X is an extending group selected from where Z is —NH—: —COO—(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—, —COO—(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.2—NH—, —COO—(C.sub.1-C.sub.6 alkylene)S—, —COO—(C.sub.1-C.sub.8 alkylene)NHCO(C.sub.1-C.sub.6 alkylene)S—; or where Z is —O-: —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—; b is 0 or 1, preferably 1; wherein T is an extending group selected from —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—, and —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; g is 0 or 1; D is a drug moiety of formula (IH), formula (IHa) or formula (IHb), or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof wherein: R.sub.1 CN or OH; R.sub.2 is C(═O)R.sub.a, wherein R.sub.a is selected from hydrogen and 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 a —OR.sub.b group wherein R.sub.b is a substituted or unsubstituted C.sub.1-C.sub.6 alkyl group, wherein the optional substituents are one or more substituents R.sub.x; Y is —NH— or —O—; Z is —NH— or —O—; the moiety Ab comprising at least one antigen binding site is an antibody or an antigen-binding fragment thereof and it 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; and n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] to the moiety Ab comprising at least one antigen binding site and is in the range from 1 to 12.

    52. A drug conjugate according to claim 11 or claim 12, selected from the formulas (IV), (V), and (VI): ##STR00285## wherein: 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; 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)-; (AA).sub.w is of formula (II) ##STR00286## 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); R.sub.21 is, at each occurrence, selected 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; w is an integer from 0 to 6; X is an extending group selected from the group consisting of where Z is —NH—: —COO—(C.sub.2-C.sub.4 alkylene)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, —COO—(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—, —COO—(C.sub.2-C.sub.4 alkylene)S—, and —COO—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—; or where Z is —O-: —CONH—(C.sub.2-C.sub.4 alkylene)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—, and —CONH—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—; b is 0 or 1, preferably 1; wherein T is an extending group selected from —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—, and —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; g is 0 or 1; D is a drug moiety of formula (IH), formula (IHa) or formula (IHb), or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof wherein: R.sub.1 is CN or OH; R.sub.2 is acetyl; R.sub.3 is hydrogen or methoxy, more preferably hydrogen; Y is —NH— or —O—; Z is —NH— or —O—; the moiety Ab comprising at least one antigen binding site is an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment 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, and preferably a monoclonal antibody which immunospecifically binds to cancer cell antigens; and n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] wherein L is as defined in formulas (IV), (V) or (VI) to the moiety Ab comprising at least one antigen binding site and is in the range from 3 to 8.

    53. A drug conjugate according to claim 11 or claim 12, selected from the formulas (IV), (V) and (VI): ##STR00287## wherein: 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.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, 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, and preferably R.sub.19 is a C.sub.1-C.sub.6 alkylene group; R.sub.30 is a —C.sub.2-C.sub.4 alkylene- group; M is —C.sub.1-C.sub.3 alkylene-(C.sub.5-C.sub.7carbocyclo)-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00288## 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); R.sub.22 is selected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl; R.sub.23 is selected from methyl, —(CH.sub.2).sub.4NH.sub.2, —(CH.sub.2).sub.3NHCONH.sub.2 and —(CH.sub.2).sub.3NHC(═NH)NH.sub.2; X is an extending group selected from the group consisting of —COO—(C.sub.2-C.sub.4 alkylene)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, —COO—(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 or cyano groups)-NH—, —COCH.sub.2NH—COCH.sub.2—NH—, —COO—(C.sub.2-C.sub.4 alkylene)S—, and —COO—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—; b is 0 or 1, preferably 1; wherein T is an extending group selected from —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—, and —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; g is 0 or 1; D is a drug moiety of formula (IHa) or formula (IHb), or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof wherein: R.sub.1 is CN; R.sub.2 is acetyl: R.sub.3 is hydrogen; Y is —NH— or —O—; Z is —NH—; the moiety Ab comprising at least one antigen binding site is a monoclonal 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, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; preferably 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, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof, more preferably Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof, preferably Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof, more preferably Trastuzumab or an antigen-binding fragment or an immunologicallly active portion thereof; and n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] wherein L is as defined in formulas (IV), (V) or (VI) to the moiety Ab comprising at least one antigen binding site and is in the range from 3 to 5.

    54. A drug conjugate according to claim 53, 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, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof.

    55. A drug conjugate according to claim 53, 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, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof, more preferably Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof, preferably Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof, more preferably Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof.

    56. A drug conjugate according to claim 11, selected from the formulas (IV), (V) and (VI): ##STR00289## wherein: R.sub.19 is —C.sub.2-C.sub.6 alkylene-; R.sub.30 is a —C.sub.2-C.sub.4 alkylene-; M is —C.sub.1-C.sub.3 alkylene-(C.sub.5-C.sub.7carbocyclo)-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00290## wherein R.sub.22 is isopropyl, R.sub.23 is selected from methyl and —(CH.sub.2).sub.3NHCONH.sub.2, 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); X is an extending group selected from the group consisting of —COO—(C.sub.2-C.sub.4 alkylene)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, —COO—(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—, —COO—(C.sub.2-C.sub.4 alkylene)S—, and —COO—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S; b is 0 or 1, preferably 1; wherein T is an extending group selected from —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—, and —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; g is 0 or 1; D is a drug moiety selected from: ##STR00291## or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof; wherein the wavy lines indicate 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 linker; the moiety Ab comprising at least one antigen binding site is selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; and n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] wherein L is as defined in formulas (IV), (V) or (VI) to the moiety Ab comprising at least one antigen binding site and is in the range from 3 to 5.

    57. A drug conjugate according to claim 56, wherein the moiety Ab comprising at least one antigen binding site is selected from Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof.

    58. A drug conjugate according to claim 56, wherein the moiety Ab comprising at least one antigen binding site is Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof.

    59. A drug conjugate according to claim 11 or claim 12, selected from the formulas (IV), (V), and (VI): ##STR00292## wherein: R.sub.19 is —C.sub.2-C.sub.6 alkylene-; R.sub.30 is —C.sub.2-C.sub.4 alkylene-; M is —C.sub.1-C.sub.3 alkylene-(C.sub.5-C.sub.7carbocyclo)-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00293## wherein R.sub.22 is isopropyl, R.sub.23 is selected from methyl and —(CH.sub.2).sub.3NHCONH.sub.2, and the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or 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); X is an extending group selected from the group consisting of —COO—(C.sub.2-C.sub.4 alkylene)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, —COO—(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—, —COO—(C.sub.2-C.sub.4 alkylene)S—, and —COO—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—; b is 0 or 1, preferably 1; wherein T is an extending group selected from —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—, and —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; g is 0 or 1; D is a drug moiety selected from: ##STR00294## or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof; wherein the wavy lines indicate 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 linker; the moiety Ab comprising at least one antigen binding site is selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; and n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-] wherein L is as defined in formulas (IV), (V) or (VI) to the moiety comprising at least one antigen binding site and is in the range from 3 to 5.

    60. A drug conjugate according to claim 59, wherein the moiety Ab comprising at least one antigen binding site is selected from Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof.

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

    62. A drug conjugate according to any one of claims 1 to 4, or 11 to 49, of formula (IV): ##STR00295## wherein: R.sub.19 is C.sub.2-C.sub.5 alkylene-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00296## wherein R.sub.22 is isopropyl, R.sub.23 is selected from methyl and —(CH.sub.2).sub.3NHCONH.sub.2, and 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); X is a —COOCH.sub.2-phenylene-NH— group; b is 1; T is an extending group of formula —CO—(C.sub.1-C.sub.4 alkylene)-[O—(C.sub.2-C.sub.4 alkylene)].sub.4—NH—; g is 0 or 1; or of formula (V) ##STR00297## wherein M is -methyl-cyclohexylene-; b is 1; w is 0; X is an extending group selected from —(CH.sub.2).sub.3S- and —(CH.sub.2).sub.3NHCO(CH.sub.2).sub.2S— g is 0; or of formula (VI) ##STR00298## wherein R.sub.19 is —C.sub.2-C.sub.5 alkylene-; R.sub.30 is —C.sub.3 alkylene-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00299## wherein R.sub.22 is isopropyl, R.sub.23 is selected from methyl and —(CH.sub.2).sub.3NHCONH.sub.2, and 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 X is a —COOCH.sub.2-phenylene-NH group; b is 1; T is an extending group of formula —CO—(C.sub.1-C.sub.4 alkylene)-[O—(C.sub.2-C.sub.4 alkylene)].sub.4—NH—; g is 0 or 1; D is a drug moiety selected from: ##STR00300## or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer 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 the linker; the moiety Ab comprising at least one antigen binding site is is selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; and 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 (IV) to the moiety comprising at least one antigen binding site and is in the range from 3 to 5, and preferably 4.

    63. A drug conjugate according to claim 62, wherein the moiety Ab comprising at least one antigen binding site is selected from Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof.

    64. A drug conjugate according to claim 62, wherein the moiety Ab comprising at least one antigen binding site is Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof.

    65. An antibody drug conjugate according to claim 1, selected from the group consisting of: ##STR00301## ##STR00302## wherein n is from 2 to 6, more preferably 3, 4, or 5 and each custom-character and custom-character is independently selected from is independently selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof, and more preferably its is selected from Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof, particularly Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof.

    66. A drug conjugate according to claim 65, wherein the moiety Ab comprising at least one antigen binding site is selected from Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof.

    67. A drug conjugate according to claim 65, 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.

    68. A drug conjugate according to claim 1, selected from the group consisting of: ##STR00303## ##STR00304## ##STR00305## wherein n is from 2 to 6, more preferably 3, 4, or 5 and each custom-character and custom-character is independently selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof, and more preferably its is selected from Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof, particularly Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof; or a drug conjugate of formula ##STR00306## wherein n is from 2 to 6, more preferably 3, 4, or 5 and custom-character is an anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof.

    69. A drug conjugate according to claim 68, wherein the moiety Ab comprising at least one antigen binding site is selected from Trastuzumab and anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof.

    70. A drug conjugate according to claim 68, 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.

    71. An antibody drug conjugate according to any one of claims 1 to 70 in isolated or purified form.

    72. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1, wherein: L.sub.1 is a linker selected from the group of formulas consisting of: ##STR00307## wherein each of the 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; 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, R.sub.x, X, T, and AA is as defined in any one of claims 1 to 71.

    73. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 according to claim 72, wherein: L.sub.1 is a linker of formula: ##STR00308## wherein: the wavy line 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; 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 is an integer ranging from 1-6; 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, R.sub.x, X, AA, and T is as defined in any one of claims 1 to 71.

    74. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 according to claim 72 wherein: L.sub.1 is linker of formula: ##STR00309## wherein: the wavy line 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; 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; (AA).sub.w is of formula (II): ##STR00310## wherein the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or D (the wavy line to the left) and to (T).sub.g if any, or L.sub.1 or to a hydrogen atom (the wavy line to the right); 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 from 0 to 6; X is an extending group selected from the group consisting of where D is conjugated via an amine group (for example where Z is —NH—): —COO—(C.sub.2-C.sub.4 alkylene)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, —COO—(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—, —COO—(C.sub.2-C.sub.4 alkylene)S—, and —COO—(C.sub.2-C.sub.4 alkylene)-NHCO(C.sub.1-C.sub.3 alkylene)S—, or where D is conjugated via an hydroxy group (for example where Z is —O—): —CONH—(C.sub.2-C.sub.4 alkylene)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—, and —CONH—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—; T is an extending group selected from —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— and —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; b is 0 or 1; g is 0 or 1; and D is a drug moiety of formula (Ia) or a formula (Ib), or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof: ##STR00311## or D is a drug moiety of formula (IHa) or a formula (IHb), or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof ##STR00312## wherein the wavy lines of (IHa) and (IHb) indicate 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.sub.1; R.sub.1 is —OH or —CN; R.sub.2 is a —C(═O)R.sub.a group, wherein R.sub.a is selected from hydrogen and 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 a —OR.sub.b group wherein R.sub.b is a substituted or unsubstituted C.sub.1-C.sub.6 alkyl group, wherein the optional substituents are one or more substituents R.sub.x; Y is —NH— or —O—; R.sub.4 (if present) is —CH.sub.2OH, or —CH.sub.2NH.sub.2; and Z (if present) is —NH— or —O—.

    75. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 according to claim 72 wherein: L.sub.1 is a group of formula: ##STR00313## wherein: the wavy line 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; 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.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, 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, and preferably R.sub.19 is a C.sub.1-C.sub.6 alkylene group; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00314## wherein the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or D (the wavy line to the left) and to (T).sub.g if any, or L.sub.1 or to a hydrogen atom (the wavy line to the right); R.sub.22 is selected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl; R.sub.23 is selected from methyl, —(CH.sub.2).sub.4NH.sub.2, —(CH.sub.2).sub.3NHCONH.sub.2 and —(CH.sub.2).sub.3NHC(═NH)NH.sub.2; X is an extending group selected from where D is conjugated via an amine group (for example where Z is —NH—): —COO—CH.sub.2-phenylene-NH, —COO(CH.sub.2).sub.3)NHCOO—CH.sub.2-phenylene-NH, —COO—(CH.sub.2).sub.3NH—, —COO(CH.sub.2).sub.3-S-, and —COO—(CH.sub.2).sub.3NHCO—(CH.sub.2).sub.2S—; or where D is conjugated via an hydroxy group (for example where Z is —O—): —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—, and —CONH(CH.sub.2).sub.3NHCO(CH.sub.2).sub.2S—; wherein T is an extending group selected from —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—, and —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; b is an integer of 0 or 1; g is 0 or 1; and D is a drug moiety of formula (Ia) or a formula (Ib), or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof: ##STR00315## or D is a drug moiety of formula (IHa) or a formula (IHb), or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof: ##STR00316## wherein the wavy lines of (IHa) and (IHb) indicate 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.sub.1; R.sub.1 is CN or OH; R.sub.2 is acetyl; R.sub.3 is hydrogen or methoxy, preferably hydrogen; Y is —NH— or —O—; R.sub.4 (if present) is —CH.sub.2OH, or —CH.sub.2NH.sub.2; and Z (if present) is —NH— or —O—.

    76. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 according to claim 72 wherein: L.sub.1 is a linker of formula: ##STR00317## wherein: the wavy line 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); R.sub.19 is —C.sub.2-C.sub.6 alkylene-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00318## R.sub.22 is isopropyl, R.sub.23 is selected to methyl and —(CH.sub.2).sub.3NHCONH.sub.2, wherein the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or D (the wavy line to the left) and to (T).sub.g if any, or L.sub.1 or to a hydrogen atom (the wavy line to the right); X is an extending group selected from —COO—CH.sub.2-phenylene-NH—, —COO(CH.sub.2).sub.3NHCOO—CH.sub.2-phenylene-NH, —COO—(CH.sub.2).sub.3)NH—, —COO(CH.sub.2).sub.3—S—, and —COO—(CH.sub.2).sub.3NHCO—(CH.sub.2).sub.2S—; wherein T is an extending group selected from —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—, and —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; b is 0 or 1; g is 0 or 1; and D is a drug selected from: ##STR00319## or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer 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.sub.1.

    77. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 according to claim 72 wherein: L.sub.1 is a group of formula: ##STR00320## wherein: the wavy line 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; R.sub.19 is-C.sub.2-C.sub.5 alkylene-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00321## wherein R.sub.22 is isopropyl, R.sub.23 is selected from methyl and —(CH.sub.2).sub.3NHCONH.sub.2, wherein the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or to D (the wavy line to the left) and to (T).sub.g if any, or L.sub.1 or to a hydrogen atom (the wavy line to the right); X is a —COO—CH.sub.2-phenylene-NH— group; T is a —CO—(CH.sub.2).sub.2-[O—(CH.sub.2).sub.2].sub.4—NH— group; b is an integer of 0 or 1; g is 0 or 1; and D is a drug moiety selected from: ##STR00322## or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer 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.sub.1.

    78. A compound of formula D-X-(AA).sub.w-(T).sub.g-L.sub.1 according to any one of claims 72 to 77 selected from: ##STR00323## ##STR00324##

    79. A compound of formula D-X-(AA).sub.w-(T).sub.g-L.sub.1 according to any one of claims 72 to 77 selected from: ##STR00325## ##STR00326##

    80. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1, wherein each of D, X, AA, T, L.sub.1, b, g and w are as defined in any one of claims 1 to 70.

    81. A drug conjugate according to any one of claims 1 to 71, or a compound according to any one of claims 72 to 80, wherein b+g+w is not 0.

    82. A drug conjugate according to any one of claims 1 to 71 or 81, or a compound according to any one of claims 72 to 81, wherein b+w is not 0.

    83. A drug conjugate according to any one of claims 1 to 71, 81 or 82, or a compound according to any one of claims 72 to 82, wherein when w is not 0, then b is 1.

    84. Use of a drug moiety as described in any one of claims 1 to 23, as a payload in an antibody drug conjugate.

    85. Use of a drug moiety as described in any one of claims 1 to 23, in the manufacture of a antibody drug conjugate.

    86. A drug conjugate according to any one of claims 1 to 71, for use as a medicament.

    87. A drug conjugate according to any one of claims 1 to 71 for use in the treatment of cancer, and more preferably a cancer selected from lung cancer, colorectal cancer, breast cancer, pancreas carcinoma, kidney cancer, leukaemia, multiple myeloma, lymphoma, gastric and ovarian cancer.

    88. A pharmaceutical composition comprising a drug conjugate according to any one of claims 1 to 71 and a pharmaceutically acceptable carrier.

    89. A method for the prevention or treatment of cancer comprising administering an effective amount of a drug conjugate according to any one of claims 1 to 71 to a patient in need thereof.

    90. A method for the treatment of cancer according to claim 89, wherein the cancer is selected from lung cancer, colorectal cancer, breast cancer, pancreas carcinoma, kidney cancer, leukaemia, multiple myeloma, lymphoma, gastric and ovarian cancer.

    91. Use of a drug conjugate according to any one of claims 1 to 71 in the preparation of a medicament for the treatment of cancer, preferably a cancer selected from lung cancer, colorectal cancer, breast cancer, pancreas carcinoma, kidney cancer, leukaemia, multiple myeloma, lymphoma, gastric and ovarian cancer.

    92. A kit comprising a therapeutically effective amount of a drug conjugate according to any one of claims 1 to 71 and a pharmaceutically acceptable carrier.

    93. The kit according to claim 92 for use in the treatment of cancer, and more preferably a cancer selected from lung cancer, colorectal cancer, breast cancer, pancreas carcinoma, kidney cancer, leukaemia, multiple myeloma, lymphoma, gastric and ovarian cancer.

    94. The drug conjugate according to any one of claims 1 to 50, wherein n is in the range of from 1-12, 1-8, 3-8, 3-6, 3-5, 1, 2, 3, 4, 5 or 6; preferably 3, 4 or 5 or more preferably 4.

    95. A process for the preparation of a drug antibody conjugate according to any one of claims 1 to 71 comprising conjugating a moiety Ab comprising at least one antigen binding site and a drug D, Ab and D being as defined in any one of claims 1 to 71.

    96. A process according to claim 95 for the preparation of a drug antibody conjugate of formula (G) or (G′): ##STR00327## said process comprising the following steps: (i) reacting a drug D-H of formula (IH)-H: ##STR00328## wherein the substituents in the definitions of (IH)-H are as defined in any one of claims 1 to 71, with a compound of formula (D′) or (E): ##STR00329## to give a compound of formula (F) or (F′),respectively: ##STR00330## (ii) 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: ##STR00331## and (iii) reaction of the partially reduced antibody Ab-SH having free thiol groups with the compound of formula (F) or (F′) produced in step (i) to give the desired drug antibody conjugate of formula (G) or (G′) respectively: ##STR00332##

    97. A process according to claim 96, wherein the antibody is selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof which is partially reduced in step (ii) using tris[2-carboxyethyl]phosphine hydrochloride, preferably the antibody is selected from Trastuzumab and an anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof which is partially reduced in step (ii) using tris[2-carboxyethyl]phosphine hydrochloride and most preferably it is Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof which is partially reduced in step (ii) using tris[2-carboxyethyl]phosphine hydrochloride.

    98. A process according to claim 95 for the preparation of a drug antibody conjugate of formula (W) or (W′): ##STR00333## said process comprising the following steps: (i) reacting the antibody with 2-iminothiolane hydrochloride (Traut's reagent) to give a thiol-activated antibody: ##STR00334## (ii) reacting the thiol-activated antibody with the compound of formula (F) or (F′), to give the desired drug antibody conjugate of formula (W) or (W′), respectively. ##STR00335##

    99. A process according to claim 98, wherein the antibody is selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-binding fragment or an immunologically active portion thereof, preferably the antibody is selected from Trastuzumab and an anti-CD13 antibody or an antigen-binding fragment or an immunologically active portion thereof and most preferably it is Trastuzumab or an antigen-binding fragment or an immunologically active portion thereof.

    100. A process according to claim 95 for the preparation of a drug antibody conjugate of formula (O) or (P): ##STR00336## said process comprising the following steps: (i) either: (a) reacting a drug (D-H) of formula formula (IH)-H: ##STR00337## wherein the substituents in the definitions of (IH)-H are as defined in any one of claims 1 to 71, 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): ##STR00338## and the point of attachment of the —(C═O)X.sub.1 moiety is the free —NH.sub.2 group of the compound of formula D-H, or (b) reacting said drug (D-H) of formula (IH)-H as defined above with 4-nitro-phenylchloroformate to give a compound of formula (J): ##STR00339## and the point of attachment of the (4-nitrophenyl)-O—CO— group is the same as that for the X.sub.1(CO) moiety in (a) above; (ii) either: (c) reacting the compound of formula (B) produced in step (i) with a hydroxy compound of formula HO—(CH.sub.2).sub.1-6NHProt.sup.NH and removing the Prot.sup.NH group from the coupled compound to give a compound of formula (C): ##STR00340## and then reacting the resulting compound of formula (C) with a compound of formula Me-S-S—(CH.sub.2).sub.1-3—CO.sub.2H to give a compound of formula (K) ##STR00341## or (d) reacting the compound (J) produced in step (i) with a compound of formula HO—(CH.sub.2).sub.1-3SProt.sup.SH and removing the Prot.sup.SH group from the coupled compound to give a compound of formula (L): ##STR00342## (iii) reacting (K) or (L) produced in step (ii) with dithiothreitol under disulfide reducing conditions to give compounds of formula (M) and (N) respectively: ##STR00343## (iv) reacting the antibody to be conjugated with succininimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate to derivatise said antibody at one or more lysine groups with a succininimidyl-4-(N-maleimidomethyl)cyclohexane-1-carbonyl group: ##STR00344## (v) reacting the derivatised antibody produced in step (iv) with either (M) or (N) produced in step (iii) to give the desired drug antibody conjugate of formula (O) or (P): ##STR00345##

    101. A process according to claim 100, wherein the compound of formula X.sub.2-C(O)-X.sub.1 is 1,1′-carbonyldiimidazole.

    102. A process according to claim 100 or claim 101, wherein the hydroxy compound reacted with the compound of formula (B) is preferably HO—(CH.sub.2).sub.2-4—NHProtNH, and preferably HO—(CH.sub.2).sub.3—NHProt.sup.NH.

    103. A process according to claim 100 or claim 101, wherein the compound reacted with the compound of formula (C) to give the compound of formula (K) is 3-(methyldisulfanyl)propanoic acid.

    104. A process according to claim 100, wherein the compound of formula HO—(CH.sub.2).sub.1-3SProt.sup.SH that is reacted with a compound of formula (J) to give a compound of formula (L) is HO—(CH.sub.2).sub.3SProt.sup.SH.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0704] The invention is diagrammatically illustrated, by way of example, in the accompanying drawings in which:

    [0705] FIG. 1 is a schematic illustration of one process according to the present invention wherein conjugation to the antibody is via free thiol groups;

    [0706] FIG. 2 is a schematic illustration of one process according to the present invention wherein conjugation to the antibody is via free amino groups.

    [0707] FIG. 3. Dose-response curves corresponding to one representative experiment conducted to evaluate the antiproliferative potential of the ADC 1 in HER2-positive (black symbols) or HER2-negative (hollow symbols) cell lines. Dots correspond to the average of triplicates with error bars denoting SD, drawing lines correspond to the best fitting by nonlinear regression of the experimental points to a four-parameters logistic curve used to obtain the IC.sub.50 values reported in Table 13.

    [0708] FIG. 4. Histograms showing the effect on cell survival of either Trastuzumab at 20 μg/mL or the ADC 1 at 16 or 2.5 μg/mL in several cell lines, either HER2-positive (SK-BR-3 and HCC-1954) or HER2-negative (MDA-MB-231 and MCF-7). Bars correspond to the average of three determinations with error bars denoting SD. Statistical significance was measured using an unpaired two-tailed t-test, p values are summarized as follows:***, p<0.001; *, p<0.01; *, p<0.05.

    [0709] FIG. 5. Dose-response curves showing the antiproliferative potential of the ADC 2 in HER2-positive (black symbols) or HER2-negative (hollow symbols) cell lines. Dots correspond to the average of triplicates with error bars denoting SD, drawing lines correspond to the best fitting by nonlinear regression of the experimental points to a four-parameters logistic curve used to obtain the IC.sub.50 values reported in Table 15.

    [0710] FIG. 6. Histograms showing the effect on cell survival of either Trastuzumab at 20 μg/mL or the ADC 2 at 16 or 2.5 μg/mL in several cell lines, either HER2-positive (SK-BR-3 and HCC-1954) or HER2-negative (MDA-MB-231 and MCF-7). Bars correspond to the average of three determinations with error bars denoting SD. Statistical significance was measured using an unpaired two-tailed t-test, p values are summarized as follows: ***, p<0.001; *, p<0.01; *, p<0.05.

    [0711] FIG. 7. Dose-response curves showing the antiproliferative potential of the ADC 3 in HER2-positive (black symbols) or HER2-negative (hollow symbols) cell lines. Dots correspond to the average of triplicates with error bars denoting SD, drawing lines correspond to the best fitting by nonlinear regression of the experimental points to a four-parameters logistic curve used to obtain the IC.sub.50 values reported in Table 17.

    [0712] FIG. 8. Histograms showing the effect on cell survival of either Trastuzumab at 20 μg/mL or the ADC 3 at 20 or 3 μg/mL in several cell lines, either HER2-positive (SK-BR-3 and HCC-1954) or HER2-negative (MDA-MB-231 and MCF-7). Bars correspond to the average of three determinations with error bars denoting SD. Statistical significance was measured using an unpaired two-tailed t-test, p values are summarized as follows: ***, p<0.001; *, p<0.01; *, p<0.05.

    [0713] FIG. 9. Dose-response curves showing the antiproliferative potential of the ADC 4 in HER2-positive (black symbols) or HER2-negative (hollow symbols) cell lines. Dots correspond to the average of triplicates with error bars denoting SD, drawing lines correspond to the best fitting by nonlinear regression of the experimental points to a four-parameters logistic curve used to obtain the IC50 values reported in Table 18.

    [0714] FIG. 10. Histograms showing the effect on cell survival of either Trastuzumab at 3 μg/mL or the ADC 4 at 3 or 0.2 μg/mL in several cell lines, either HER2-positive (SK-BR-3 and HCC-1954) or HER2-negative (MDA-MB-231 and MCF-7). Bars correspond to the average of three determinations with error bars denoting SD. Statistical significance was measured using an unpaired two-tailed t-test, p values are summarized as follows: ***, p<0.001; *, p<0.01; *, p<0.05.

    [0715] FIG. 11. Dose-response curves showing the antiproliferative potential of the ADC 6 in HER2-positive (black symbols) or HER2 negative (hollow symbols) cell lines. Dots correspond to the average of triplicates with error bars denoting SD, drawing lines correspond to the best fitting by nonlinear regression of the experimental points to a four-parameters logistic curve used to obtain the IC.sub.50 values reported in Table 21.

    [0716] FIG. 12. Tumor volume evaluation of BT-474 tumors in mice treated with placebo, 11-R (at 5 mg/kg), ADC 1 (2.2 TCEP) (at 1.6 and 6.5 mg/kg) and ADC 1 (at 2.24 mg/kg).

    [0717] FIG. 13. Tumor volume evaluation of JIMT-1 tumors in mice treated with placebo, 11-R (at 5.0 mg/kg) and ADC 1 (at 5, 10 and 30 mg/kg).

    [0718] FIG. 14. Tumor volume evaluation of SKOV3 tumors in mice treated with placebo, 11-R (at 5.0 mg/kg) and ADC 1 (at 5, 10 and 30 mg/kg).

    [0719] FIG. 15. Tumor volume evaluation of N87 tumors in mice treated with placebo, 11-R (at 5 mg/kg) and ADC 1 (2.2 TCEP) (at 5, 10 and 30 mg/kg)—

    [0720] FIG. 16 Tumor volume evaluation of Gastric-008 (PDX) tumors in mice treated with placebo, 11-R (at 5 mg/kg) and ADC-1 (at 5, 10 and 30 mg/kg).

    EXAMPLES

    [0721] The present invention is further illustrated by way of the following, non-limiting examples. In the examples, the following abbreviations are used: [0722] CDI, 1,1′-Carbonyldiimidazole [0723] DIPEA, N,N-Diisopropylethylamine [0724] Hex, Hexane [0725] EtOAc, Ethyl acetate [0726] DCM, Dichloromethane [0727] NMP, N-Methyl-2-pyrrolidone [0728] DMF, Dimethylformamide [0729] EDC, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride [0730] EDTA, Ethylenediaminetetraacetic acid [0731] MeOH, Methanol [0732] DTT, Dithiothreitol [0733] Py, Pyridine [0734] THF, Tetrahydrofuran [0735] TCEP, Tris[2-carboxyethyl]phosphine hydrochloride [0736] MC, 6-Maleimidocaproyl [0737] Fmoc, 9-Fluorenylmethoxycarbonyl [0738] Cit, Citrulline [0739] Val, Valine [0740] DMSO, Dimethylsulfoxide [0741] Trt, Triphenylmethyl [0742] HOBt, 1-Hydroxybenzotriazole [0743] DIPCDl, N,N′-Diisopropylcarbodiimide [0744] TFA, Trifluoroacetic acid [0745] PABOH, 4-Aminobenzyl alcohol [0746] bis-PNP, bis(4-Nitrophenyl) carbonate [0747] NAC, N-Acetylcysteine [0748] SEC, Size-Exclusion Chromatography [0749] HPLC, High Performance Liquid Chromatography [0750] ADC, Antibody Drug Conjugate [0751] ATCC, American Type Culture Collection [0752] DMEM, Dulbecco's Modified Eagle's Medium [0753] RPMI, Rosmell Park Memorial Institute Medium [0754] ITS, Insulin-transferrin-sodium selenite media supplement [0755] FCS, Fetal Calf Serum [0756] SRB, Sulforhodamine B [0757] PBS, Phosphate Buffered Saline [0758] DR, Dose-Response [0759] UV, Ultraviolet [0760] SMCC, Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [0761] LAR, Linker to Antibody Ratio

    Synthesis of Compounds

    [0762] Compound 1 was prepared as described in Example 20 of WO 01/87895.

    Example 0-1

    A)

    [0763] ##STR00150##

    [0764] To a solution of 1 (0.5 g, 0.80 mmol) in acetic acid (20 mL, 0.04 M) was added L-tryptophanol (2-S) (533 mg, 3.0 mmol, Sigma-Aldrich). The reaction mixture was stirred at 23° C. for 16 h and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) gave compounds 3-S (616 mg, 97%) and 3a-S (12 mg, 2%).

    3-S

    [0765] R.sub.f=0.50 (Hexane:EtOAc, 1:1).

    [0766] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.71 (s, 1H), 7.36 (dd, J=7.9, 1.0 Hz, 1H), 7.27 (dd, J=8.2, 0.9 Hz, 1H), 7.13 (ddd, J=8.3, 7.0, 1.2 Hz, 1H), 7.03 (ddd, J=8.0, 7.0, 1.0 Hz, 1H), 6.62 (s, 1H), 6.26 (d, J=1.4 Hz, 1H), 6.04 (d, J=1.3 Hz, 1H), 5.75 (s, 1H), 5.14 (dd, J=11.7, 1.2 Hz, 1H), 4.60 (s, 1H), 4.41 (s, 1H), 4.36-4.24 (m, 2H), 4.21 (d, J=2.7 Hz, 1H), 3.82 (s, 3H), 3.52 (s, 1H), 3.50-3.47 (m, 1H), 3.45 (dq, J=8.4, 2.2 Hz, 1H), 3.35 (t, J=10.1 Hz, 1H), 3.01-2.78 (m, 5H), 2.62 (dd, J=15.3, 4.7 Hz, 1H), 2.41 (s, 1H), 2.38 (s, 3H), 2.37-2.31 (m, 1H), 2.28 (s, 3H), 2.17 (s, 3H), 2.06 (s, 3H).

    [0767] ESI-MS m/z: 794.2 (M+H).sup.+.

    3a-S

    [0768] R.sub.f=0.70 (Hexane:EtOAc, 1:1).

    [0769] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.83 (s, 1H), 7.38 (dt, J=7.9, 0.9 Hz, 1H), 7.25 (dt, J=8.3, 0.9 Hz, 1H), 7.11 (ddd, J=8.2, 7.1, 1.2 Hz, 1H), 7.02 (ddd, J=8.0, 7.0, 1.0 Hz, 1H), 6.62 (s, 1H), 6.24 (d, J=1.4 Hz, 1H), 6.03 (d, J=1.3 Hz, 1H), 5.79 (s, 1H), 5.13 (d, J=11.7 Hz, 1H), 4.60 (s, 1H), 4.39 (s, 1H), 4.36-4.22 (m, 3H), 4.17-4.09 (m, 1H), 3.91 (dd, J=10.5, 8.6 Hz, 1H), 3.83 (s, 3H), 3.51-3.41 (m, 2H), 3.04-2.92 (m, 3H), 2.72 (dd, J=15.1, 4.0 Hz, 1H), 2.54-2.41 (m, 2H), 2.38 (s, 3H), 2.35-2.30 (m, 1H), 2.29 (s, 3H), 2.21-2.16 (m, 1H), 2.18 (s, 3H), 2.12 (s, 3H); 2.05 (s, 3H).

    [0770] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 171.2, 170.7, 168.6, 147.5, 145.8, 143.0, 141.1, 140.4, 135.6, 130.1, 129.5, 126.7, 122.2, 121.2, 120.9, 119.4, 118.4, 118.2, 118.2, 113.6, 113.5, 110.9, 110.0, 109.1, 102.1, 91.4, 67.2, 63.4, 61.3, 60.4, 59.7, 59.1, 54.8, 54.6, 47.7, 42.0, 41.6, 31.6, 24.0, 22.6, 21.0, 15.9, 14.2, 9.7.

    [0771] ESI-MS m/z: 836.2 (M+H).sup.+.

    B)

    [0772] ##STR00151##

    [0773] To a solution of 3-S (616 mg, 0.77 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 51 mL, 0.015 M) was added AgNO.sub.3 (3.40 g, 23.3 mmol). After 3 h at 23° C., the reaction mixture was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give 4-S (471 mg, 78%).

    [0774] R.sub.f=0.50 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0775] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.71 (s, 1H), 7.36 (dd, J=7.8, 1.1 Hz, 1H), 7.26 (dd, J=7.8, 1.1 Hz, 1H), 7.12 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 7.03 (ddd, J=8.0, 7.1, 1.0 Hz, 1H), 6.64 (s, 1H), 6.23 (d, J=1.3 Hz, 1H), 6.01 (d, J=1.4 Hz, 1H), 5.75 (s, 1H), 5.25 (d, J=11.4 Hz, 1H), 4.92 (s, 1H), 4.52 (br s, 3H), 4.22 (dd, J=11.4, 2.2 Hz, 1H), 4.19 (s, 1H), 3.83 (s, 3H), 3.54 (br s, 2H), 3.35 (t, J=10.2 Hz, 1H), 3.26 (s, 1H), 3.01-2.93 (m, 3H), 2.88 (br s, 3H), 2.63 (dd, J=15.2, 4.8 Hz, 1H), 2.38 (s, 3H), 2.36-2.31 (m, 2H), 2.28 (s, 3H), 2.05 (s, 3H).

    [0776] .sup.13C NMR (126 MHz, CDCl.sub.3): δ 171.9, 168.6, 147.5, 145.4, 142.9, 141.2, 140.7, 135.5, 130.4, 126.8, 122.3, 122.0, 121.3, 119.4, 118.4, 115.2, 112.8, 111.0, 110.0, 109.6, 101.8, 81.9, 76.8, 65.2, 62.8, 62.5, 60.4, 58.1, 57.9, 55.9, 55.1, 53.4, 51.6, 41.8, 41.3, 39.6, 24.1, 23.8, 20.5, 15.8, 9.7.

    [0777] ESI-MS m/z: 767.3 (M−H.sub.2O+H).sup.+.

    [0778] (+)-HR-ESI-TOF-MS m/z 767.2788 [M−H.sub.2O+H].sup.+ (Calcd. for C41H.sub.43N.sub.4O.sub.9S: 767.2745).

    B′)

    [0779] ##STR00152##

    [0780] To a solution of 3a-S (30 mg, 0.035 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 2.4 mL, 0.015 M) was added AgNO.sub.3 (180 mg, 1.07 mmol). After 3 h at 23° C., the reaction mixture was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give 4a-S (24 mg, 83%).

    [0781] R.sub.f=0.60 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0782] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.81 (s, 1H), 7.37 (d, J=7.8 Hz, 1H), 7.30-7.21 (m, 1H), 7.06 (dddt, J=34.7, 8.0, 7.1, 1.1 Hz, 2H), 6.63 (s, 1H), 6.22 (d, J=1.3 Hz, 1H), 6.02 (dd, J=12.9, 1.4 Hz, 1H), 5.74 (s, 1H), 5.25-5.21 (m, 1H), 4.89 (d, J=8.7 Hz, 1H), 4.55-4.45 (m, 2H), 4.30-4.18 (m, 1H), 4.14 (dd, J=10.5, 4.2 Hz, 1H), 4.00-3.88 (m, 2H), 3.82 (s, 3H), 3.56-3.44 (m, 2H), 3.23 (d, J=9.0 Hz, 1H), 2.95 (d, J=15.7 Hz, 2H), 2.87-2.78 (m, 2H), 2.71 (dd, J=15.0, 3.9 Hz, 1H), 2.48 (dd, J=15.1, 9.6 Hz, 1H), 2.37 (s, 3H), 2.35-2.29 (m, 1H), 2.28 (s, 3H), 2.22-2.16 (m, 1H), 2.15 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H).

    [0783] ESI-MS m/z: 809.2 (M−H.sub.2O+H).sup.+.

    Example 0-2

    A)

    [0784] ##STR00153##

    [0785] To a solution of 1 (0.5 g, 0.80 mmol) in acetic acid (20 mL, 0.04 M) was added D-tryptophanol (2-R) (533 mg, 3.0 mmol, Sigma-Aldrich). The reaction mixture was stirred at 23° C. for 16 h and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) gave compound 3-R (479 mg, 75%).

    [0786] R.sub.f=0.44 (Hexane:EtOAc, 1:1).

    [0787] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.61 (s, 1H), 7.39 (d, J=7.8 Hz, 1H), 7.29 (d, J=9.6 Hz, 1H), 7.12 (t, J=7.3 Hz, 1H), 7.03 (t, J=7.3 Hz, 1H), 6.60 (s, 1H), 6.25 (s, 1H), 6.03 (s, 1H), 5.75 (s, 1H), 5.04 (d, J=11.7 Hz, 1H), 4.62 (s, 1H), 4.37 (s, 1H), 4.32-4.25 (m, 1H), 4.22 (d, J=2.7 Hz, 1H), 4.19-4.09 (m, 1H), 3.82 (s, 3H), 3.77 (s, 1H), 3.64 (d, J=9.0 Hz, 1H), 3.49-3.41 (m, 2H), 3.02-2.90 (m, 2H), 2.60-2.52 (m, 2H), 2.45 (d, J=14.7 Hz, 2H), 2.40 (s, 3H), 2.28 (s, 3H), 2.22-2.14 (m, 2H), 2.18 (s, 3H), 2.10 (m, 3H).

    [0788] ESI-MS m/z: 794.3 (M+H).sup.+.

    B)

    [0789] ##STR00154##

    [0790] To a solution of 3-R (479 mg, 0.60 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 40 mL, 0.015 M) was added AgNO.sub.3 (3.03 g, 18.1 mmol). After 3 h at 23° C., the reaction mixture was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford 4-R (428 mg, 91%).

    [0791] R.sub.f=0.45 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0792] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.62 (s, 1H), 7.39 (d, J=8.1 Hz, 1H), 7.28 (d, J=8.1 Hz, 1H), 7.11 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 7.02 (ddd, J=7.9, 7.1, 1.0 Hz, 1H), 6.61 (s, 1H), 6.22 (d, J=1.3 Hz, 1H), 5.99 (d, J=1.3 Hz, 1H), 5.73 (s, 1H), 5.17 (dd, J=11.5, 1.2 Hz, 1H), 4.86 (s, 1H), 4.56-4.47 (m, 2H), 4.17 (dd, J=5.1, 1.6 Hz, 1H), 4.08 (dd, J=11.5, 2.1 Hz, 1H), 3.81 (s, 3H), 3.78 (d, J=3.8 Hz, 1H), 3.64 (dd, J=10.8, 3.8 Hz, 2H), 3.51 (d, J=5.1 Hz, 1H), 3.48-3.43 (m, 2H), 3.24 (d, J=8.6 Hz, 1H), 3.00-2.80 (m, 2H), 2.57 (s, 1H), 2.55-2.43 (m, 1H), 2.40 (s, 3H), 2.27 (s, 3H), 2.19-2.12 (m, 1H), 2.16 (s, 3H), 2.08 (s, 3H).

    [0793] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 171.8, 168.6, 147.6, 145.4, 143.0, 141.3, 140.7, 136.0, 131.1, 130.0, 129.6, 126.6, 122.1, 121.6, 121.2, 119.4, 118.4, 115.6, 112.9, 111.1, 110.6, 101.8, 81.7, 65.8, 62.7, 61.8, 60.4, 60.3, 57.9, 57.8, 56.1, 55.0, 52.1, 42.2, 41.3, 41.1, 23.8, 23.4, 20.5, 15.7, 9.8.

    [0794] ESI-MS m/z: 767.6 (M−H.sub.2O+H).sup.+.

    [0795] (+)-HR-ESI-TOF-MS m/z: 767.2799 [M−H.sub.2O+H].sup.+ (Calcd. for C41H.sub.43N.sub.4O.sub.9S: 767.2745).

    Example 0-3. Synthesis of allyl N—[(R)-(2-amino-3-(1H-indol-3-yl)propyl)]carbamate (9-R)

    [0796] ##STR00155##

    A)

    [0797] ##STR00156##

    [0798] To a solution of D-tryptophanol (2-R) (2.0 g, 10.4 mmol) in CH.sub.3CN (42 mL, 4 mL/mmol) was added di-tert-butyl dicarbonate (4.6 g, 20.8 mmol). The reaction mixture was stirred at 23° C. for 3 h and concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH from 99:1 to 85:15) to afford 5-R (2.2 g, 73%).

    [0799] R.sub.f=0.5 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0800] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.13 (s, 1H), 7.67 (dd, J=7.8, 1.1 Hz, 1H), 7.38 (dd, J=8.1, 1.3 Hz, 1H), 7.29-7.10 (m, 2H), 7.06 (s, 1H), 4.82 (s, 1H), 4.00 (s, 1H), 3.71 (dd, J=11.0, 3.8 Hz, 1H), 3.62 (dd, J=11.0, 5.5 Hz, 1H), 3.01 (d, J=6.7 Hz, 2H), 2.14 (s, 1H), 1.44 (s, 9H).

    B)

    [0801] ##STR00157##

    [0802] To a solution of 5-R (2.4 g, 8.2 mmol) in CH.sub.2Cl.sub.2 (50 mL, 6 mL/mmol) was added phthalimide (2.7 g, 18.2 mmol), triphenylphosphine (4.8 g, 18.2 mmol) and the mixture was cooled at 0° C. A solution of diethyl azodicarboxylate (DEAD) solution in CH.sub.2Cl.sub.2 (25 mL, 3 mL/mmol) was added for 15 min. The reaction was stirred at 23° C. for 16 h, concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford 6-R (3.3 g, 96%).

    [0803] R.sub.f=0.7 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0804] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.50 (s, 1H), 7.81 (dd, J=5.5, 3.1 Hz, 2H), 7.66 (dd, J=5.6, 3.2 Hz, 2H), 7.60 (d, J=7.8 Hz, 1H), 7.35 (d, J=8.0 Hz, 1H), 7.19-7.04 (m, 3H), 4.81 (s, 1H), 4.40 (s, 1H), 3.83 (dd, J=13.9, 3.7 Hz, 1H), 3.72 (dd, J=13.9, 9.9 Hz, 1H), 3.08-3.01 (m, 2H), 1.23 (s, 9H).

    C)

    [0805] ##STR00158##

    [0806] To a solution of 6-R (3.25 g, 7.74 mmol) in ethanol (231 mL, 30 mL/mmol) was added hydrazine monohydrate (37 mL, 774 mmol). The reaction mixture was stirred at 80° C. in sealed tube for 2.25 h, concentrated under vacuum. Flash chromatography (EtOAc:CH.sub.3OH, from 100:1 to 50:50) afforded 7-R (2.15 g, 96%).

    [0807] R.sub.f=0.2 (EtOAc:CH.sub.3OH, 6:4).

    [0808] .sup.1H NMR (400 MHz, CD3OD): δ 7.60 (d, J=7.9 Hz, 1H), 7.33 (d, J=8.1 Hz, 1H), 7.13-7.04 (m, 2H), 7.05-6.96 (m, 1H), 4.02-3.94 (m, 1H), 2.99-2.87 (m, 3H), 2.78 (dd, J=13.1, 9.7 Hz, 1H), 1.39 (s, 9H).

    [0809] ESI-MS m/z: 290.2 (M+H).sup.+.

    D)

    [0810] ##STR00159##

    [0811] To a solution of 7-R (2.15 g, 7.4 mmol) in CH.sub.3CN (74 mL, 10 mL/mmol) and DMF (7.4 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (1.06 mL, 5.9 mmol) and allyl chloroformate (7.9 mL, 74 mmol). The reaction was stirred at 23° C. for 16 h. The mixture was diluted with EtOAc, NH.sub.4Cl was added and the mixture was 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 flash chromatography (Hexane:EtOAc, from 100:1 to 1:100) to afford 8-R (1.69 g, 61%).

    [0812] R.sub.f=0.4 (Hexane:EtOAc, 1:1).

    [0813] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.25 (s, 1H), 7.62 (d, J=7.8 Hz, 1H), 7.35 (dd, J=8.1, 0.9 Hz, 1H), 7.16 (dddd, J=27.8, 8.0, 7.0, 1.1 Hz, 2H), 7.04 (d, J=2.4 Hz, 1H), 5.90 (ddt, J=17.3, 10.7, 5.6 Hz, 1H), 5.34-5.22 (m, 1H), 5.20 (dt, J=10.5, 1.4 Hz, 1H), 5.12 (s, 1H), 4.82 (s, 1H), 4.55 (dq, J=5.4, 1.7 Hz, 2H), 4.02 (s, 1H), 3.35 (dt, J=10.0, 4.7 Hz, 1H), 3.21 (s, 1H), 2.95 (ddd, J=21.6, 15.4, 9.1 Hz, 2H), 1.42 (s, 9H).

    [0814] ESI-MS m/z: 274.3 (M−Boc+H).sup.+.

    ##STR00160##

    [0815] To a solution of 8-R (1.30 g, 3.50 mmol) in CH.sub.2Cl.sub.2 (58 mL, 16.6 mL/mmol) was added trifluoroacetic acid (30 mL, 8.3 mL/mmol). The reaction mixture was stirred at 23° C. for 1.5 h, concentrated under vacuum to give crude 9-R which was used in the next steps without further purification.

    [0816] R.sub.f=0.2 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0817] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.95 (s, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.36 (d, J=8.1 Hz, 1H), 7.17 (s, 1H), 7.09 (t, J=7.5 Hz, 1H), 7.03 (t, J=7.5 Hz, 1H), 5.87 (ddt, J=16.4, 10.8, 5.6 Hz, 1H), 5.34-5.13 (m, 2H), 4.50 (d, J=5.5 Hz, 2H), 3.62 (bs, 1H), 3.42 (dd, J=14.9, 3.9 Hz, 1H), 3.36-3.20 (m, 1H), 3.11-3.00 (m, 2H).

    [0818] ESI-MS m/z: 274.3 (M+H).sup.+.

    Example 0-4. Synthesis of allyl N—[(S)-(2-amino-3-(1H-indol-3-yl)propyl)]carbamate (9-S)

    [0819] ##STR00161##

    A)

    [0820] To a solution of L-tryptophanol (2-S) (2.0 g, 10.4 mmol) in CH.sub.3CN (42 mL, 4 mL/mmol) was added Di-tert-butyl dicarbonate (4.6 g, 20.8 mmol). The reaction mixture was stirred at 23° C. for 3 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford 5-S (2.24 g, 73%).

    [0821] R.sub.f=0.5 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0822] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.10 (s, 1H), 7.65 (dd, J=7.8, 1.1 Hz, 1H), 7.37 (dd, J=8.1, 1.3 Hz, 1H), 7.23-7.11 (m, 2H), 7.06 (s, 1H), 4.81 (s, 1H), 3.99 (s, 1H), 3.70 (dd, J=11.0, 3.8 Hz, 1H), 3.61 (dd, J=11.0, 5.5 Hz, 1H), 3.00 (d, J=6.7 Hz, 2H), 2.01 (s, 1H), 1.42 (s, 9H).

    B)

    [0823] ##STR00162##

    [0824] To a solution of 5-S (1.2 g, 4.13 mmol) in CH.sub.2Cl.sub.2 (24.8 mL, 6 mL/mmol) was added phthalimide (1.33 g, 9.1 mmol), triphenylphosphine (2.4 g, 9.1 mmol) and the mixture was cooled at 0° C. A solution of diethyl azodicarboxylate (DEAD) solution (3 mL, 10.32 mmol) in CH.sub.2Cl.sub.2 (12.4 mL, 3 mL/mmol) was added for 15 min. The reaction was stirred at 23° C. for 16 h, concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford 6-S (2.8 g, >100%).

    [0825] R.sub.f=0.7 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0826] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.49 (s, 1H), 7.80 (dd, J=5.4, 3.1 Hz, 2H), 7.66 (dd, J=5.6, 3.2 Hz, 2H), 7.60 (d, J=7.8 Hz, 1H), 7.34 (d, J=8.0 Hz, 1H), 7.21-7.04 (m, 3H), 4.74 (s, 1H), 4.42 (s, 1H), 3.83 (dd, J=13.9, 3.7 Hz, 1H), 3.72 (dd, J=13.9, 9.9 Hz, 1H), 3.10-3.01 (m, 2H), 1.23 (s, 9H).

    C)

    [0827] ##STR00163##

    [0828] To a solution of 6-S (0.86 g, 2.07 mmol) in ethanol (72 mL, 36 mL/mmol) was added hydrazine monohydrate (10 mL, 207 mmol). The reaction mixture was stirred at 80° C. in sealed tube for 2.25 h, concentrated under vacuum. Flash chromatography (EtOAc:CH.sub.3OH, from 100:1 to 50:50) to afford 7-S (1.0 g, 84%).

    [0829] R.sub.f=0.2 (EtOAc:CH.sub.3OH, 6:4).

    [0830] .sup.1H NMR (400 MHz, CD3OD): δ 7.61 (d, J=7.9 Hz, 1H), 7.35 (d, J=8.1 Hz, 1H), 7.13-6.97 (m, 2H), 7.09 (s, 1H), 4.06-3.96 (m, 1H), 3.01-2.76 (m, 4H), 1.38 (s, 9H).

    [0831] ESI-MS m/z: 290.3 (M+H).sup.+.

    D)

    [0832] ##STR00164##

    [0833] To a solution of 7-S (0.95 g, 3.3 mmol) in CH.sub.3CN (33 mL, 10 mL/mmol) and DMF (3.3 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (0.5 mL, 2.6 mmol) and allyl chloroformate (3.5 mL, 33 mmol). The reaction was stirred at 23° C. for 20 h. The mixture was diluted with EtOAc, NH.sub.4Cl was added and the mixture was 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 flash chromatography (Hexane:EtOAc, from 100:1 to 1:100) to afford 8-S (0.88 g, 73%).

    [0834] R.sub.f=0.5 (Hexane:EtOAc, 1:1).

    [0835] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.17 (s, 1H), 7.63 (d, J=7.8 Hz, 1H), 7.20 (dd, J=8.1, 0.9 Hz, 1H), 7.13 (dddd, J=27.8, 8.0, 7.0, 1.1 Hz, 2H), 7.06 (d, J=2.4 Hz, 1H), 5.90 (ddt, J=17.3, 10.7, 5.6 Hz, 1H), 5.31-5.18 (m, 2H), 5.09 (s, 1H), 4.80 (s, 1H), 4.59-4.52 (m, 2H), 4.03 (s, 1H), 3.37 (dt, J=10.0, 4.7 Hz, 1H), 3.21 (s, 1H), 3.05-2.87 (m, 2H), 1.42 (s, 9H).

    [0836] ESI-MS m/z: 274.3 (M−Boc+H).sup.+.

    E)

    [0837] ##STR00165##

    [0838] To a solution of 8-S (0.875 g, 2.3 mmol) in CH.sub.2Cl.sub.2 (38 mL, 16.6 mL/mmol) was added trifluoroacetic acid (19 mL, 8.3 mL/mmol). The reaction mixture was stirred at 23° C. for 2 h, concentrated under vacuum to give crude 9-S which was used in the next steps without further purification.

    [0839] R.sub.f=0.2 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0840] .sup.1H NMR (400 MHz, CD3OD): δ 7.56 (d, J=7.8 Hz, 1H), 7.37 (d, J=8.1 Hz, 1H), 7.21 (s, 1H), 7.13 (t, J=7.5 Hz, 1H), 7.05 (t, J=7.5 Hz, 1H), 5.94 (ddt, J=16.4, 10.8, 5.6 Hz, 1H), 5.34-5.16 (m, 2H), 4.56 (d, J=5.5 Hz, 2H), 3.60 (bs, 1H), 3.43 (dd, J=14.9, 3.9 Hz, 1H), 3.37-3.31 (m, 1H), 3.14-2.99 (m, 2H).

    [0841] ESI-MS m/z: 274.3 (M+H).sup.+.

    Example 0-5

    A)

    [0842] ##STR00166##

    [0843] To a solution of 1 (1.45 g, 2.33 mmol) in acetic acid (58 mL, 0.08 M) was added 9-R (0.95 g, 3.50 mmol). The reaction mixture was stirred a 50° C. for 18 h and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4. Flash chromatography (Hexane:EtOAc, 1:1) gives compound 10-R (1.3 g, 64%).

    [0844] R.sub.f=0.5 (Hexane:EtOAc, 1:1).

    [0845] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.66 (s, 1H), 7.36 (d, J=7.9 Hz, 1H), 7.27 (d, J=7.9 Hz, 1H), 7.10 (ddd, J=8.3, 7.0, 1.3 Hz, 1H), 7.01 (td, J=7.5, 7.0, 1.0 Hz, 1H), 6.62 (s, 1H), 6.23 (d, J=1.4 Hz, 1H), 6.01 (d, J=1.4 Hz, 1H), 5.99-5.89 (m, 1H), 5.79 (s, 1H), 5.44-5.21 (m, 2H), 5.14-4.99 (m, 2H), 4.63 (ddd, J=7.3, 4.4, 1.5 Hz, 2H), 4.36 (s, 1H), 4.33-4.24 (m, 1H), 4.29-4.26 (m, 1H), 4.21 (d, J=2.7 Hz, 1H), 4.19-4.13 (m, 3H), 3.80 (s, 3H), 3.56 (s, 1H), 3.48-3.43 (m, 3H), 3.27 (dt, J=13.2, 4.0 Hz, 1H), 3.04-2.88 (m, 2H), 2.56 (dd, J=15.2, 3.8 Hz, 1H), 2.49-2.35 (m, 2H), 2.31 (s, 3H), 2.28 (s, 3H), 2.17 (s, 3H), 2.07 (s, 3H).

    [0846] ESI-MS m/z: 877.3 (M+H).sup.+.

    B)

    [0847] ##STR00167##

    [0848] To a solution of 10-R (600 mg, 0.68 mmol) in CH.sub.2Cl.sub.2 (12 mL, 18 mL/mmol) was added bis(triphenylphosphine)palladium(II) dichloride (77 mg, 0.1 mmol) and acetic acid (0.4 mL, 6.8 mmol). Tributyltin hydride (1.1 mL, 4.08 mmol) was added at 0° C., the reaction mixture was stirred at 0° C. for 0.5 h and concentrated under vacuum. The crude obtained was diluted with EtOAc, saturated aqueous solution of NH.sub.4Cl was added, and the mixture was extracted with EtOAc. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH.sub.3OH, from 100:1 to 1:100) to afford 11-R (440 mg, 82%).

    [0849] R.sub.f=0.5 (CH.sub.2Cl.sub.2:CH.sub.3OH, 1:1).

    [0850] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.64 (s, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.29 (d, J=8.1 Hz, 1H), 7.11 (ddt, J=8.3, 7.0, 1.4 Hz, 1H), 7.03 (ddt, J=8.3, 7.0, 1.4 Hz, 1H), 6.58 (s, 1H), 6.24 (d, J=1.5 Hz, 1H), 6.02 (d, J=1.5 Hz, 1H), 5.02 (d, J=11.8 Hz, 1H), 4.63 (s, 1H), 4.36 (s, 1H), 4.28 (d, J=5.1 Hz, 1H), 4.21 (d, J=2.2 Hz, 1H), 4.16 (s, 1H), 3.80 (s, 3H), 3.51-3.39 (m, 4H), 3.32-3.13 (m, 3H), 2.95 (d, J=8.9 Hz, 2H), 2.89-2.76 (m, 2H), 2.73-2.57 (m, 1H), 2.42 (d, J=14.8 Hz, 1H), 2.36 (s, 3H), 2.25 (s, 3H), 2.16 (s, 3H), 2.09 (s, 3H).

    [0851] ESI-MS m/z: 793.2 (M+H).sup.+.

    C)

    [0852] ##STR00168##

    [0853] To a solution of 11-R (850 mg, 1.07 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 70 mL, 0.015 M) was added AgNO.sub.3 (3.64 g, 21.4 mmol). After 17 h at 23° C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give 12-R (553 mg, 66%).

    [0854] R.sub.f=0.3 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0855] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.60 (s, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.28 (d, J=7.9 Hz, 1H), 7.11 (ddt, J=8.3, 7.1, 1.2 Hz, 1H), 7.02 (ddt, J=8.3, 7.1, 1.2 Hz, 1H), 6.58 (s, 1H), 6.22 (s, 1H), 6.00 (s, 1H), 5.16 (d, J=11.5 Hz, 1H), 4.87 (s, 1H), 4.54 (s, 1H), 4.51 (d, J=3.3 Hz, 1H), 4.17 (d, J=5.4 Hz, 1H), 4.07 (dd, J=11.3, 2.2 Hz, 1H), 3.81 (s, 3H), 3.52 (d, J=5.1 Hz, 1H), 3.24 (d, J=8.8 Hz, 2H), 2.99-2.78 (m, 4H), 2.66 (dd, J=14.9, 3.5 Hz, 1H), 2.49-2.39 (m, 2H), 2.38 (s, 3H), 2.28 (m, 2H), 2.25 (s, 3H), 2.21-2.16 (m, 2H), 2.15 (s, 3H), 2.08 (s, 3H).

    [0856] .sup.13C NMR (101 MHz, CD3OD): δ 171.7, 169.4, 148.7, 145.9, 143.7, 141.4, 140.9, 136.9, 130.8, 130.0, 129.7, 126.0, 121.4, 121.0, 119.7, 119.1, 118.4, 117.5, 114.9, 110.8, 107.5, 106.4, 102.1, 91.3, 63.2, 60.0, 59.0, 58.6, 55.3, 54.6, 52.7, 52.4, 48.4, 45.8, 42.5, 40.2, 24.5, 23.2, 19.2, 15.0, 8.2.

    [0857] ESI-MS m/z: 766.2 (M−H.sub.2O+H).sup.+.

    [0858] (+)-HR-ESI-TOF-MS m/z: 766.2972 [M−H.sub.2O+H].sup.+ (Calcd. for C.sub.41H.sub.44N.sub.5O.sub.8S+: 766.2905).

    C′)

    [0859] ##STR00169##

    [0860] To a solution of 10-R (700 mg, 0.8 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 52.5 mL, 0.015 M) was added AgNO.sub.3 (2.66 g, 16 mmol). After 20 h at 23° C., the reaction mixture was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give 13-R (438 mg, 63%).

    [0861] R.sub.f=0.40 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0862] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.64 (s, 1H), 7.37 (d, J=7.9 Hz, 1H), 7.32-7.20 (m, 1H), 7.11 (t, J=7.7 Hz, 1H), 7.01 (t, J=7.4 Hz, 1H), 6.62 (s, 1H), 6.21 (s, 1H), 6.05-5.90 (m, 1H), 5.99 (s, 1H), 5.75 (d, J=6.0 Hz, 1H), 5.40-5.07 (m, 4H), 4.88 (d, J=14.7 Hz, 1H), 4.68-4.50 (m, 3H), 4.28-4.13 (m, 1H), 4.08 (dt, J=11.4, 2.4 Hz, 1H), 3.83 (s, 3H), 3.68-3.40 (m, 4H), 3.37-3.19 (m, 2H), 2.98-2.79 (m, 2H), 2.59-2.36 (m, 3H), 2.29 (s, 3H), 2.27 (s, 3H), 2.14 (s, 3H), 2.10-2.16 (m, 1H), 2.08 (s, 3H).

    [0863] ESI-MS m/z: 850.3 (M−H.sub.2O+H).sup.+.

    Example 0-6

    A)

    [0864] ##STR00170##

    [0865] To a solution of 1 (955 mg, 1.5 mmol) in acetic acid (37.5 mL, 0.08 M) was added 9-S (627 mg, 2.29 mmol). The reaction mixture was stirred a 50° C. for 18 h and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4. Flash chromatography (Hexane:EtOAc, 1:1) gives compound 10-S (756 mg, 58%).

    [0866] R.sub.f=0.4 (Hexane:EtOAc, 1:1).

    [0867] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.78 (s, 1H), 7.36 (d, J=7.9 Hz, 1H), 7.24 (d, J=7.9 Hz, 1H), 7.10 (ddd, J=8.3, 7.0, 1.3 Hz, 1H), 7.01 (td, J=7.5, 7.0, 1.0 Hz, 1H), 6.68 (s, 1H), 6.23 (d, J=1.4 Hz, 1H), 6.01 (d, J=1.4 Hz, 1H), 6.07-5.93 (m, 1H), 5.82 (s, 1H), 5.41-5.19 (m, 2H), 5.1 (d, J=11.7 Hz, 1H), 4.66 (dt, J=5.9, 1.3 Hz, 1H), 4.57 (s, 1H), 4.37 (s, 1H), 4.33-4.20 (m, 3H), 3.81 (s, 3H), 3.46 (d, J=4.2 Hz, 2H), 3.22-3.13 (m, 1H), 3.11-2.88 (m, 4H), 2.66 (dd, J=15.2, 4.2 Hz, 1H), 2.51 (dd, J=15.3, 6.0 Hz, 1H), 2.43-2.32 (m, 2H), 2.31 (s, 3H), 2.26 (s, 3H), 2.19 (s, 3H), 2.04 (s, 3H).

    [0868] ESI-MS m/z: 877.3 (M+H).sup.+.

    B)

    [0869] ##STR00171##

    [0870] To a solution of 10-S (650 mg, 0.72 mmol) in CH.sub.2Cl.sub.2 (13.3 mL, 18 mL/mmol) was added bis(triphenylphosphine)palladium(II) dichloride (83 mg, 0.11 mmol) and acetic acid (0.42 mL, 7.4 mmol). Tributyltin hydride (1.2 mL, 4.4 mmol) was added at 0° C., the reaction mixture was stirred at 23° C. for 0.5 h, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH.sub.3OH, from 100:1 to 1:100) to afford 11-S (445 mg, 78%).

    [0871] R.sub.f=0.5 (CH.sub.2Cl.sub.2:CH.sub.3OH, 1:1).

    [0872] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.74 (s, 1H), 7.36 (d, J=7.9 Hz, 1H), 7.26 (d, J=8.1 Hz, 1H), 7.12 (ddt, J=8.3, 7.0, 1.4 Hz, 1H), 7.02 (ddt, J=8.3, 7.0, 1.4 Hz, 1H), 6.62 (s, 1H), 6.26 (d, J=1.5 Hz, 1H), 6.04 (d, J=1.5 Hz, 1H), 5.12 (d, J=11.8 Hz, 1H), 4.59 (s, 1H), 4.42 (s, 1H), 4.36-4.17 (m, 3H), 3.81 (s, 3H), 3.51-3.39 (m, 3H), 2.98-2.75 (m, 4H), 2.69-2.60 (m, 2H), 2.47 (d, J=16.1 Hz, 1H), 2.38 (s, 3H), 2.35-2.17 (m, 2H), 2.28 (s, 3H), 2.13 (s, 3H), 2.04 (s, 3H).

    [0873] ESI-MS m/z: 793.3 (M+H).sup.+.

    C)

    [0874] ##STR00172##

    [0875] To a solution of 11-S (435 mg, 0.55 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 38.5 mL, 0.015 M) was added AgNO.sub.3 (1.84 g, 11 mmol). After 24 h at 23° C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give 12-S (152 mg, 35%).

    [0876] R.sub.f=0.2 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0877] .sup.1H NMR (500 MHz, CD3OD): δ 7.34 (dd, J=7.7, 1.5 Hz, 1H), 7.28 (dd, J=7.7, 1.5 Hz, 1H), 7.04 (ddt, J=8.2, 7.0, 1.1 Hz, 1H), 6.95 (ddt, J=8.2, 7.0, 1.2 Hz, 1H), 6.55 (s, 1H), 6.31-6.25 (m, 1H), 6.15-6.05 (m, 1H), 5.31 (d, J=11.4 Hz, 1H), 4.91 (s, 1H), 4.64 (s, 1H), 4.40-4.19 (m, 3H), 3.76 (s, 3H), 3.64 (d, J=5.2 Hz, 1H), 3.44 (d, J=9.0 Hz, 1H), 3.03-2.85 (m, 4H), 2.85-2.65 (m, 2H), 2.59 (d, J=15.6 Hz, 1H), 2.52-2.39 (m, 2H), 2.37 (s, 3H), 2.27 (s, 3H), 2.09 (s, 3H), 2.00 (s, 3H).

    [0878] .sup.13C NMR (126 MHz, CD3OD): δ 171.4, 169.3, 148.6, 145.8, 143.5, 141.2, 140.8, 136.5, 131.2, 130.3, 129.5, 126.3, 121.6, 121.2, 119.8, 119.4, 118.6, 117.5, 114.9, 111.0, 107.5, 107.4, 102.2, 91.1, 63.5, 60.5, 59.2, 58.5, 55.3, 54.7, 53.4, 52.7, 48.6, 44.7, 42.7, 39.9, 24.3, 23.4, 19.2, 15.1, 8.2.

    [0879] ESI-MS m/z: 766.2 (M−H.sub.2O+H).sup.+.

    [0880] (+)-HR-ESI-TOF-MS m/z: 766.2958 [M−H.sub.2O+H].sup.+ (Calcd. for C41H.sub.44N.sub.5O.sub.8S: 766.2905).

    C′)

    [0881] ##STR00173##

    [0882] To a solution of 10-S (5 mg, 0.006 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 0.5 mL, 0.015 M) was added AgNO.sub.3 (29 mg, 0.17 mmol). After 20 h at 23° C., the reaction mixture was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give 13-S (5 mg, 100%).

    [0883] R.sub.f=0.40 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0884] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.75 (s, 1H), 7.37 (d, J=7.9 Hz, 1H), 7.32-7.20 (m, 1H), 7.12 (t, J=7.7 Hz, 1H), 7.02 (t, J=7.4 Hz, 1H), 6.84 (s, 1H), 6.24 (s, 1H), 6.08-5.97 (m, 1H), 6.01 (s, 1H), 5.87 (s, 1H), 5.42-5.19 (m, 4H), 4.88 (s, 1H), 4.69-4.65 (m, 2H), 4.58 (s, 1H), 4.28-4.13 (m, 2H), 3.84 (s, 3H), 3.68-3.40 (m, 2H), 3.24-3.15 (m, 2H), 3.08-2.90 (m, 2H), 2.73-2.57 (m, 2H), 2.53-2.37 (m, 3H), 2.34 (s, 3H), 2.25 (s, 3H), 2.14 (s, 3H), 2.10-2.16 (m, 1H), 2.03 (s, 3H).

    [0885] ESI-MS m/z: 850.3 (M−H.sub.2O+H).sup.+.

    Example 0-7

    A) Synthesis of (S)-5-methoxy-tryptophanol (17-S)

    [0886] ##STR00174##

    [0887] To a solution of LiAlH.sub.4 (23.4 mL, 1.0 M in THF, 23.4 mmol) at −40° C. was added carefully H.sub.2SO.sub.4 (0.31 mL, 5.57 mmol) and a suspension of 5-methoxy-L-tryptophan (16-S) (1.0 g, 4.26 mmol, Chem-Impex) in THF (13.4 mL, 0.3 M). The reaction mixture was left evolution at 23° C., heated for 3 h at 80° C. and 18 h at 23° C. Cool at −21° C. the reaction mixture was quenched carefully with NaOH 2N until basic pH. EtOAc was added and the mixture filtered through Celite® and washed with CH.sub.3OH. The crude was concentrated under vacuum to give 17-S as a crude which was used in the next step without further purification.

    [0888] R.sub.f=0.2 (CH.sub.2Cl.sub.2:CH.sub.3OH, 4:1).

    [0889] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.19 (dt, J=8.8, 0.7 Hz, 1H), 7.06-7.00 (m, 2H), 6.72 (dd, J=8.8, 2.4 Hz, 1H), 3.77 (s, 3H), 3.63-3.48 (m, 1H), 3.42-3.33 (m, 1H), 3.17-3.06 (m, 1H), 2.86 (ddt, J=14.3, 6.1, 0.8 Hz, 1H), 2.66 (dd, J=14.3, 7.5 Hz, 1H).

    [0890] ESI-MS m/z: 221.4 (M+H).sup.+.

    B) Synthesis of (R)-5-methoxy-tryptophanol (17-R)

    [0891] ##STR00175##

    [0892] To a solution of LiAlH.sub.4 (11.7 mL, 1.0 M in THF, 11.7 mmol) at −40° C. was added carefully H.sub.2SO.sub.4 (0.31 mL, 5.75 mmol) and a suspension of 5-methoxy-D-tryptophan (16-R) (0.5 g, 2.13 mmol, Aldrich) in THF (6.7 mL, 0.3 M). The reaction mixture was left evolution at 23° C., heated for 3.5 h at 80° C. and 18 h at 23° C. Cool at −21° C. the reaction mixture was quenched carefully with NaOH 2N until basic pH. EtOAc was added and the mixture filtered through Celite® and washed with CH.sub.3OH. The crude was concentrated under vacuum to give 17-R as a crude which was used in the next step without further purification.

    [0893] R.sub.f=0.2 (CH.sub.2Cl.sub.2:CH.sub.3OH, 4:1).

    [0894] .sup.1H NMR (400 MHz, CD3OD): δ 7.20 (d, J=8.9 Hz, 1H), 7.06-6.96 (m, 2H), 6.71 (dd, J=8.8, 2.5 Hz, 1H), 3.75 (s, 3H), 3.62-3.52 (m, 1H), 3.37 (dd, J=10.8, 7.0 Hz, 1H), 3.09 (br s, 1H), 2.82 (dd, J=14.3, 5.9 Hz, 1H), 2.62 (dd, J=14.4, 7.6 Hz, 1H).

    [0895] ESI-MS m/z: 221.6 (M+H).sup.+.

    Example 0-8

    A)

    [0896] ##STR00176##

    [0897] To a solution of 1 (530 mg, 0.85 mmol) in acetic acid (10.6 mL, 0.08 M) was added 17-S (469 mg, 2.13 mmol). The reaction mixture was stirred at 50° C. for 18 h and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) gave compound 18-S (420 mg, 60%).

    [0898] R.sub.f=0.3 (Hexane:EtOAc, 1:1).

    [0899] .sup.1H NMR (400 MHz, CD3OD): δ 7.13 (d, J=8.8 Hz, 1H), 6.80 (d, J=2.4 Hz, 1H), 6.66 (dd, J=8.8, 2.5 Hz, 1H), 6.51 (s, 1H), 6.27 (s, 1H), 6.11 (s, 1H), 5.21 (d, J=11.7 Hz, 1H), 4.67 (s, 1H), 4.49-4.29 (m, 4H), 3.75 (s, 3H), 3.73 (s, 3H), 3.47 (t, J=5.8 Hz, 3H), 3.37 (d, J=5.1 Hz, 1H), 3.01-2.81 (m, 2H), 2.75 (d, J=7.4 Hz, 1H), 2.66 (dd, J=15.1, 4.1 Hz, 1H), 2.55-2.35 (m, 4H), 2.34 (s, 3H), 2.28 (s, 3H), 2.11 (s, 3H), 1.99 (s, 3H).

    [0900] ESI-MS m/z: 824.3 (M+H).sup.+.

    B)

    [0901] ##STR00177##

    [0902] To a solution of 18-S (420 mg, 0.519 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 36 mL, 0.015 M) was added AgNO.sub.3 (2.60 g, 15.3 mmol). After 3 h at 23° C., the reaction mixture was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to obtain 19-S (250 mg, 60%).

    [0903] R.sub.f=0.45 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0904] .sup.1H NMR (500 MHz, CD3OD): δ 7.15 (dd, J=8.9, 0.6 Hz, 1H), 6.82 (dd, J=2.4, 0.6 Hz, 1H), 6.68 (dd, J=8.8, 2.5 Hz, 1H), 6.54 (s, 1H), 6.27 (d, J=1.3 Hz, 1H), 6.08 (d, J=1.3 Hz, 1H), 5.30 (d, J=11.5 Hz, 1H), 4.62 (s, 1H), 4.34 (dd, J=11.4, 2.0 Hz, 1H), 4.31-4.27 (m, 2H), 3.76 (s, 3H), 3.75 (s, 3H), 3.66-3.58 (m, 1H), 3.55-3.45 (m, 2H), 3.42 (d, J=7.8 Hz, 1H), 2.93-2.73 (m, 3H), 2.68 (dd, J=15.1, 4.2 Hz, 1H), 2.54 (d, J=15.4 Hz, 1H), 2.42 (dd, J=15.1, 10.1 Hz, 2H), 2.35 (s, 3H), 2.29 (s, 3H), 2.09 (s, 3H), 2.00 (s, 3H).

    [0905] .sup.13C NMR (126 MHz, CD3OD): δ 172.7, 170.8, 155.1, 149.9, 147.2, 145.0, 142.6, 142.2, 133.1, 132.4, 132.1, 131.3, 128.1, 122.5, 121.6, 120.3, 116.4, 113.0, 112.9, 111.4, 109.0, 103.6, 100.8, 92.5, 66.6, 65.0, 61.7, 60.4, 59.9, 56.7, 56.1, 54.8, 54.1, 51.7, 44.1, 41.3, 30.7, 25.4, 24.7, 20.6, 16.3, 9.5.

    [0906] ESI-MS m/z: 798.1 (M−H.sub.2O+H).sup.+.

    [0907] (+)-HR-ESI-TOF-MS m/z: 797.2899 [M−H.sub.2O+H].sup.+ (Calcd. for C.sub.42H.sub.45N.sub.4O.sub.10S 797.2851).

    Example 0-9

    A)

    [0908] ##STR00178##

    [0909] To a solution of 1 (311 mg, 0.50 mmol) in acetic acid (6.25 mL, 0.08 M) was added 17-R (220 mg, 1.0 mmol). The reaction mixture was stirred at 50° C. for 18 h and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) gave compound 18-R (280 mg, 68%).

    [0910] R.sub.f=0.3 (Hexane:EtOAc, 1:1).

    [0911] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.53 (s, 1H), 7.18 (d, J=8.7 Hz, 1H), 6.82 (d, J=2.4 Hz, 1H), 6.78 (dd, J=8.6, 2.3 Hz, 1H), 6.60 (s, 1H), 6.23 (s, 1H), 6.02 (s, 1H), 5.76 (s, 1H), 5.04 (d, J=11.7 Hz, 1H), 4.62 (s, 1H), 4.36 (s, 1H), 4.28 (d, J=5.0 Hz, 1H), 4.24-4.09 (m, 3H), 3.81 (s, 3H), 3.79 (s, 3H), 3.64 (s, 1H), 3.47-3.40 (m, 3H), 3.01-2.90 (m, 2H), 2.53 (d, J=6.9 Hz, 2H), 2.45-2.41 (m, 1H), 2.40 (s, 3H), 2.27 (s, 3H), 2.22-2.14 (m, 1H), 2.18 (s, 3H), 2.06 (s, 3H).

    [0912] ESI-MS m/z: 824.3 (M+H).sup.+.

    B)

    [0913] ##STR00179##

    [0914] To a solution of 18-R (330 mg, 0.40 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 28 mL, 0.015 M) was added AgNO.sub.3 (2.04 g, 12.0 mmol). After 3 h at 23° C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to obtain 19-R (224 mg, 69%).

    [0915] R.sub.f=0.44 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0916] .sup.1H NMR (500 MHz, CD3OD): δ 7.14 (dd, J=8.8, 0.5 Hz, 1H), 6.83 (d, J=2.5 Hz, 1H), 6.68 (dd, J=8.8, 2.5 Hz, 1H), 6.59 (s, 1H), 6.26 (d, J=1.4 Hz, 1H), 6.07 (d, J=1.4 Hz, 1H), 5.21 (d, J=11.5 Hz, 1H), 4.68-4.55 (m, 1H), 4.32-4.25 (m, 2H), 4.12 (dd, J=11.5, 2.1 Hz, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.60 (d, J=5.2 Hz, 1H), 3.57-3.45 (m, 3H), 3.41 (d, J=8.8 Hz, 1H), 2.97-2.83 (m, 3H), 2.73 (dd, J=15.0, 3.4 Hz, 1H), 2.69 (d, J=14.9 Hz, 1H), 2.34 (s, 3H), 2.30 (s, 3H), 2.20 (dd, J=15.1, 10.4 Hz, 1H), 2.12 (s, 3H), 2.11-2.08 (m, 1H), 2.05 (s, 3H).

    [0917] .sup.13C NMR (126 MHz, CD3OD): δ 173.0, 170.8, 155.0, 149.8, 147.3, 145.0, 142.8, 142.3, 133.5, 133.1, 132.2, 132.1, 131.1, 130.5, 127.8, 122.5, 121.7, 120.0, 116.4, 113.5, 112.9, 111.4, 110.2, 103.5, 100.9, 92.6, 66.8, 64.5, 61.3, 60.4, 60.0, 56.8, 56.1, 55.9, 54.1, 44.1, 41.3, 25.6, 24.5, 20.6, 16.2, 9.6.

    [0918] ESI-MS m/z: 797.4 (M−H.sub.2O+H).sup.+.

    [0919] (+)-HR-ESI-TOF-MS m/z: 797.2896 [M−H.sub.2O+H].sup.+ (Calcd. for C42H.sub.45N.sub.4O.sub.10S 797.2851).

    Example 0-10. Synthesis of allyl N—[(S)-2-amino-3-(5-methoxy-1H-indol-3-yl)propyl)]carbamate (24-S)

    [0920] ##STR00180##

    A)

    [0921] ##STR00181##

    [0922] To a solution of 17-S (6.9 g, 31.4 mmol) in CH.sub.3CN (126 mL, 4 mL/mmol) was added di-tert-butyl dicarbonate (13.7 g, 62.8 mmol). The reaction mixture was stirred at 23° C. for 5.5 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) gives 20-S (4.5 g, 45%).

    [0923] R.sub.f=0.6 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0924] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.04 (s, 1H), 7.25 (d, J=8.4 Hz, 1H), 7.10 (d, J=2.4 Hz, 1H), 7.03 (s, 1H), 6.87 (dd, J=8.8, 2.5 Hz, 1H), 4.83 (s, 1H), 3.98 (s, 1H), 3.87 (s, 3H), 3.73-3.58 (m, 2H), 2.96 (d, J=6.6 Hz, 2H), 1.42 (s, 9H).

    B)

    [0925] ##STR00182##

    [0926] To a solution of 20-S (4.5 g, 14 mmol) in CH.sub.2Cl.sub.2 (84 mL, 6 mL/mmol) was added phthalimide (4.5 g, 30.9 mmol), triphenylphosphine (8.1 g, 30.9 mmol) and the mixture was cooled at 0° C. A solution of 40% of diethyl azodicarboxylate (DEAD) in CH.sub.2Cl.sub.2 (10.4 mL, 35 mmol) was added for 15 min. The reaction was stirred at 23° C. for 18 h, concentrated under vacuum. The residue obtained was purified by flash chromatography (Hexane:EtOAc, from 99:1 to 85:15) to yield 21-S (5.8 g, 92%).

    [0927] R.sub.f=0.55 (Hexane:EtOAc, 1:1).

    [0928] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.48 (s, 1H), 7.78 (dd, J=5.5, 3.1 Hz, 2H), 7.69-7.61 (m, 2H), 7.21 (d, J=8.8 Hz, 1H), 7.06 (dd, J=18.5, 2.4 Hz, 2H), 6.81 (dd, J=8.8, 2.4 Hz, 1H), 4.87 (s, 1H); 4.39 (s, 1H), 3.87 (s, 3H), 3.83-3.66 (m, 2H), 2.98 (d, J=6.1 Hz, 2H), 1.20 (s, 9H).

    C)

    [0929] ##STR00183##

    [0930] To a solution of 21-S (6.29 g, 14 mmol) in ethanol (420 mL, 30 mL/mmol) was added hydrazine monohydrate (61.1 mL, 1260 mmol). The reaction mixture was stirred at 80° C. in sealed tube for 2 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:1 to 50:50) affords 22-S (4.2 g, 95%).

    [0931] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 8:2).

    [0932] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.22 (d, J=8.8 Hz, 1H), 7.12 (d, J=2.4 Hz, 1H), 7.06 (s, 1H), 6.76 (dd, J=8.8, 2.4 Hz, 1H), 4.06-3.97 (m, 1H), 3.82 (s, 3H), 3.06-2.82 (m, 4H), 1.37 (s, 9H).

    D)

    [0933] ##STR00184##

    [0934] To a solution of 22-S (4.0 g, 12.52 mmol) in CH.sub.3CN (125 mL, 10 mL/mmol) and DMF (12 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (1.8 mL, 10 mmol) and allyl chloroformate (13.3 mL, 125 mmol). The reaction was stirred at 23° C. for 5 h. The mixture was diluted with EtOAc and NH.sub.4Cl was added and the mixture was 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 flash chromatography (Hexane:EtOAc, from 100:1 to 1:100) to obtain 23-S (2.65 g, 52%).

    [0935] R.sub.f=0.5 (Hexane:EtOAc, 1:1).

    [0936] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.11 (s, 1H), 7.28-7.20 (m, 1H), 7.04 (d, J=13.1 Hz, 2H), 6.85 (dd, J=8.9, 2.4 Hz, 1H), 5.97-5.82 (m, 1H), 5.33-5.24 (m, 1H), 5.19 (dt, J=10.4, 1.3 Hz, 1H), 5.11 (s, 1H), 4.82 (s, 1H), 4.55 (d, J=5.6 Hz, 2H), 4.01 (s, 1H), 3.86 (s, 3H), 3.37 (d, J=13.7 Hz, 1H), 3.21 (s, 1H), 2.89 (dd, J=14.5, 7.0 Hz, 1H), 1.41 (s, 9H).

    E)

    [0937] ##STR00185##

    [0938] To a solution of 23-S (2.60 g, 6.44 mmol) in CH.sub.2Cl.sub.2 (106 mL, 16.6 mL/mmol) was added trifluoroacetic acid (54 mL, 8.3 mL/mmol). The reaction mixture was stirred at 23° C. for 1.5 h, concentrated under vacuum to afford 24-S (3.9 g, 100%).

    [0939] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0940] .sup.1H NMR (400 MHz, CD3OD): δ 8.27 (s, 1H), 7.25 (dd, J=9.0, 2.4 Hz, 1H), 7.10 (s, 1H), 6.96 (d, J=2.3 Hz, 1H), 6.87 (dd, J=9.0, 2.4 Hz, 1H), 5.81 (ddt, J=16.3, 10.9, 5.7 Hz, 1H), 5.23 (dd, J=19.3, 13.6 Hz, 2H), 4.49 (d, J=5.9 Hz, 2H), 3.82 (s, 3H), 3.81-3.55 (m, 1H), 3.62-3.39 (m, 2H), 3.08 (qd, J=15.1, 7.3 Hz, 2H).

    Example 0-11

    A)

    [0941] ##STR00186##

    [0942] To a solution of 1 (120 mg, 0.19 mmol) in acetic acid (6 mL, 0.08 M) was added 24-S (117 mg, 0.35 mmol). The reaction mixture was stirred at 23° C. for 18 h and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) gives compound 25-S (95 mg, 54%).

    [0943] R.sub.f=0.4 (Hexane:EtOAc, 1:1).

    [0944] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.64 (s, 1H), 7.14 (d, J=8.8 Hz, 1H), 6.80 (s, 1H), 6.77 (d, J=8.8 Hz, 1H), 6.68 (s, 1H), 6.24 (s, 1H), 6.03 (s, 1H), 6.02-5.93 (m, 1H), 5.76 (s, 1H), 5.38 (d, J=10.5 Hz, 1H), 5.26 (d, J=10.5 Hz, 1H), 5.11 (d, J=11.7 Hz, 1H), 4.66 (d, J=5.6 Hz, 2H), 4.57 (s, 1H), 4.37 (s, 1H), 4.33-4.19 (m, 3H), 3.82 (s, 3H), 3.79 (s, 3H), 3.46 (s, 2H), 3.17 (s, 1H), 3.10-2.90 (m, 3H), 2.68-2.45 (m, 2H), 2.38-2.33 (m, 1H), 2.32 (s, 3H), 2.27 (s, 3H), 2.16 (s, 3H), 2.04 (s, 2H).

    [0945] ESI-MS m/z: 907.1 (M+H).sup.+.

    B)

    [0946] ##STR00187##

    [0947] To a solution of 25-S (90 mg, 0.1 mmol) in CH.sub.2Cl.sub.2 (2 mL, 18 mL/mmol) was added bis(triphenylphosphine)palladium(II)dichloride (12 mg, 0.1 mmol) and acetic acid (0.056 mL, 0.99 mmol). Tributyltin hydride (0.16 mL, 0.60 mmol) was added at 0° C., the reaction mixture was stirred at 0° C. for 0.5 h, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH.sub.3OH, from 100:1 to 1:100) to afford 26-S (75 mg, 92%).

    [0948] R.sub.f=0.25 (CH.sub.2Cl.sub.2:CH.sub.3OH, 1:1).

    [0949] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.62 (s, 1H), 7.15 (d, J=9.3 Hz, 1H), 6.81-6.76 (m, 2H), 6.72 (s, 1H), 6.25 (d, J=1.2 Hz, 1H), 6.03 (d, J=1.2 Hz, 1H), 5.12 (d, J=11.7 Hz, 1H), 4.57 (s, 1H), 4.41 (s, 1H), 4.36-4.24 (m, 2H), 4.20 (d, J=11.7 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H), 3.44 (dd, J=22.0, 7.1 Hz, 2H), 3.08-2.78 (m, 4H), 2.73-2.64 (m, 2H), 2.41-2.22 (m, 3H), 2.28 (s, 3H), 2.25-2.15 (m, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H).

    [0950] ESI-MS m/z: 823.3 (M+H).sup.+.

    C)

    [0951] ##STR00188##

    [0952] To a solution of 26-S (70 mg, 0.085 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 6 mL, 0.015 M) was added AgNO.sub.3 (335 mg, 1.7 mmol). After 18 h at 23° C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give 27-S (23 mg, 33%).

    [0953] R.sub.f=0.2 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0954] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.62 (s, 1H), 7.15 (d, J=7.8 Hz, 1H), 6.78 (s, 1H), 6.75 (d, J=7.8 Hz, 1H), 6.21 (d, J=1.5 Hz, 1H), 6.01 (d, J=1.5 Hz, 1H), 5.78 (s, 1H), 5.22 (d, J=11.5 Hz, 1H), 4.90 (s, 1H), 4.58-4.42 (m, 3H), 4.29-4.10 (m, 2H), 3.84-3.80 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.53-3.48 (m, 2H), 3.22 (d, J=8.7 Hz, 1H), 3.12 (s, 1H), 3.02 (d, J=12.8 Hz, 1H), 2.89-2.64 (m, 3H), 2.46 (s, 3H), 2.42-2.34 (m, 2H), 2.27 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H).

    [0955] .sup.13C NMR (126 MHz, CDCl.sub.3): δ 172.1, 168.7, 154.0, 147.6, 145.6, 143.0, 141.2, 140.8, 131.6, 130.6, 129.6, 127.1, 121.8, 120.9, 118.4, 115.2, 112.5, 111.8, 101.8, 100.2, 81.5, 62.6, 60.6, 58.0, 57.8, 56.0, 55.8, 55.0, 42.3, 41.4, 31.9, 29.7, 27.8, 26.9, 25.6, 24.0, 22.7, 20.5, 16.0, 14.1, 13.6, 9.7.

    [0956] ESI-MS m/z: 796.3 (M−H.sub.2O+H).sup.+.

    [0957] (+)-HR-ESI-TOF-MS m/z: 796.3062 [M−H.sub.2O+H].sup.+ (Calcd. for C42H.sub.46N.sub.5O.sub.9S 796.3011).

    Example 0-12. Synthesis of allyl N—[(R)-2-amino-3-(5-methoxy-1H-indol-3-yl)propyl)]carbamate (24-R)

    [0958] ##STR00189##

    A)

    [0959] ##STR00190##

    [0960] To a solution of 17-R (2.35 g, 10.7 mmol) in CH.sub.3CN (43 mL, 4 mL/mmol) was added di-tert-butyl dicarbonate (4.67 g, 21.4 mmol). The reaction mixture was stirred at 23° C. for 2.5 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) afforded 20-R (1.7 g, 50%).

    [0961] R.sub.f=0.6 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0962] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.05 (s, 1H), 7.25 (d, J=8.9 Hz, 1H), 7.09 (d, J=2.4 Hz, 1H), 7.02 (d, J=2.4 Hz, 1H), 6.86 (dd, J=8.8, 2.4 Hz, 1H), 4.83 (s, 1H), 3.98 (s, 1H), 3.87 (s, 3H), 3.69 (td, J=9.2, 7.5, 5.3 Hz, 1H), 3.61 (dd, J=10.9, 5.6 Hz, 1H), 2.95 (d, J=6.8 Hz, 2H), 1.42 (s, 9H).

    B)

    [0963] ##STR00191##

    [0964] To a solution of 20-R (1.7 g, 5.3 mmol) in CH.sub.2Cl.sub.2 (32 mL, 6 mL/mmol) was added phthalimide (1.72 g, 11.7 mmol), triphenylphosphine (3.06 g, 11.7 mmol) and the mixture was cooled at 0° C. A solution of 40% of diethyl azodicarboxylate (DEAD) in CH.sub.2Cl.sub.2 (4.0 mL, 13.2 mmol) was added for 15 min. The reaction was stirred at 23° C. for 16 h, concentrated under vacuum. The residue obtained was purified by flash chromatography (Hexane:EtOAc, from 99:1 to 85:15) to afford 21-R (2.0 g, 84%).

    [0965] R.sub.f=0.45 (Hexane:EtOAc, 1:1).

    [0966] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.31 (s, 1H), 7.80 (dd, J=5.4, 3.0 Hz, 2H), 7.67 (dd, J=5.4, 3.0 Hz, 2H), 7.30-7.12 (m, 2H), 7.08 (dd, J=15.2, 2.4 Hz, 1H), 6.84 (dd, J=8.8, 2.4 Hz, 1H), 4.85 (d, J=9.2 Hz, 1H), 4.43 (q, J=5.3 Hz, 1H), 3.86 (s, 3H), 3.83-3.68 (m, 2H), 3.01 (d, J=5.4 Hz, 2H), 1.22 (s, 9H).

    C)

    [0967] ##STR00192##

    [0968] To a solution of 21-R (2.0 g, 4.45 mmol) in ethanol (133 mL, 30 mL/mmol) was added hydrazine monohydrate (21.6 mL, 445 mmol). The reaction mixture was stirred at 80° C. in sealed tube for 2 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:1 to 50:50) to afford 22-R (1.15 g, 81%).

    [0969] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 8:2).

    [0970] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.21 (d, J=8.8 Hz, 1H), 7.12 (s, 1H), 7.05 (s, 1H), 6.75 (dd, J=8.8, 2.4 Hz, 1H), 3.95 (ddd, J=10.7, 8.7, 5.4 Hz, 1H), 3.82 (s, 3H), 2.98-2.79 (m, 3H), 2.75 (dd, J=13.1, 9.4 Hz, 1H), 1.37 (s, 9H).

    D)

    [0971] ##STR00193##

    [0972] To a solution of 22-R (1.1 g, 3.4 mmol) in CH.sub.3CN (34 mL, 10 mL/mmol) and DMF (3.4 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (0.5 mL, 2.7 mmol) and allyl chloroformate (3.7 mL, 34 mmol). The reaction was stirred at 23° C. for 19 h. The mixture was diluted with EtOAc and NH.sub.4Cl was added and the mixture was 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 flash chromatography (Hexane:EtOAc, from 100:1 to 1:100) to afford 23-R (0.95 g, 69%).

    [0973] R.sub.f=0.5 (Hexane:EtOAc, 1:1).

    [0974] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.55 (s, 1H), 7.20 (d, J=8.8 Hz, 1H), 7.05 (s, 1H), 6.98-6.87 (m, 1H), 6.82 (dt, J=8.8, 1.8 Hz, 1H), 5.96-5.81 (m, 1H), 5.37-5.22 (m, 2H), 5.22-5.14 (m, 1H), 5.02-4.97 (m, 1H), 4.60-4.47 (m, 2H), 4.00 (s, 1H), 3.84 (s, 3H), 3.31 (s, 1H), 3.19 (s, 1H), 2.88 (td, J=14.5, 13.3, 5.9 Hz, 2H), 1.40 (s, 9H).

    E)

    [0975] ##STR00194##

    [0976] To a solution of 23-R (0.94 g, 2.3 mmol) in CH.sub.2Cl.sub.2 (39 mL, 16.6 mL/mmol) was added trifluoroacetic acid (19 mL, 8.3 mL/mmol). The reaction mixture was stirred at 23° C. for 1.5 h, concentrated under vacuum to afford 24-R (0.72 g, 100%).

    [0977] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0978] .sup.1H NMR (400 MHz, CD3OD): δ 7.27 (d, J=8.8, 1H), 7.18 (s, 1H), 7.04 (d, J=2.4 Hz, 1H), 6.80 (ddd, J=8.8, 2.4, 0.9 Hz, 1H), 5.95 (ddt, J=16.4, 10.8, 5.5 Hz, 1H), 5.32 (d, J=17.1 Hz, 1H), 5.20 (d, J=10.5 Hz, 1H), 4.60-4.53 (m, 2H), 3.83 (s, 3H), 3.59 (dt, J=11.4, 5.5 Hz, 1H), 3.47-3.30 (m, 2H), 3.13-2.94 (m, 2H).

    Example 0-13

    A)

    [0979] ##STR00195##

    [0980] To a solution of 1 (0.71 g, 1.14 mmol) in acetic acid (45 mL, 0.08 M) was added 24-R (0.54 mg, 1.8 mmol). The reaction mixture was stirred at 23° C. for 7 h and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) gives compound 25-R (670 mg, 65%).

    [0981] R.sub.f=0.4 (Hexane:EtOAc, 1:1).

    [0982] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.52 (s, 1H), 7.17 (d, J=8.8 Hz, 1H), 6.83-6.73 (m, 2H), 6.61 (s, 1H), 6.23 (d, J=1.0 Hz, 1H), 6.02 (d, J=1.0 Hz, 1H), 6.05-5.89 (m, 1H), 5.75 (s, 1H), 5.44-5.30 (m, 1H), 5.25 (d, J=10.4 Hz, 1H), 5.13-4.99 (m, 2H), 4.71-4.59 (m, 2H), 4.36 (s, 1H), 4.30-4.07 (m, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.61-3.53 (m, 1H); 3.48-3.41 (m, 3H), 3.26 (dt, J=13.3, 3.8 Hz, 1H), 3.04-2.88 (m, 2H), 2.52 (dd, J=14.9, 3.7 Hz, 1H), 2.46-2.35 (m, 2H), 2.31 (s, 3H), 2.29 (s, 3H), 2.16 (s, 3H), 2.12-2.02 (m, 1H), 2.09 (s, 3H).

    [0983] ESI-MS m/z: 907.3 (M+H).sup.+.

    B)

    [0984] ##STR00196##

    [0985] To a solution of 25-R (745 mg, 0.82 mmol) in CH.sub.2Cl.sub.2 (15 mL, 18 mL/mmol) was added bis(triphenylphosphine)palladium(II) dichloride (92 mg, 0.1 mmol) and acetic acid (0.47 mL, 8.2 mmol). Tributyltin hydride (1.33 mL, 4.9 mmol) was added at 0° C., the reaction mixture was stirred at 0° C. for 0.75 h and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH.sub.3OH, from 100:1 to 1:100) to afford 26-R (680 mg, >100%).

    [0986] R.sub.f=0.25 (CH.sub.2Cl.sub.2:CH.sub.3OH, 1:1).

    [0987] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.57 (s, 1H), 7.16 (d, J=8.8 Hz, 1H), 6.85-6.72 (m, 2H), 6.57 (s, 1H), 6.21 (d, J=1.4 Hz, 1H), 6.00 (d, J=1.3 Hz, 1H), 5.05-4.97 (m, 1H), 4.63 (s, 1H), 4.35 (s, 1H), 4.31-4.09 (m, 4H), 3.80 (s, 3H), 3.78 (s, 3H), 3.50-3.40 (m, 3H), 3.24 (dq, J=9.9, 5.3 Hz, 1H), 2.95 (s, 1H), 2.91-2.75 (m, 2H), 2.62 (dd, J=14.8, 3.6 Hz, 1H), 2.43-2.28 (m, 2H), 2.36 (s, 3H), 2.25 (s, 3H), 2.22-2.14 (m, 1H), 2.15 (s, 3H), 2.08 (s, 3H).

    [0988] ESI-MS m/z: 823.3 (M+H).sup.+.

    C)

    [0989] ##STR00197##

    [0990] To a solution of 26-R (660 mg, 0.80 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 56 mL, 0.015 M) was added AgNO.sub.3 (2.70 g, 16.0 mmol). After 16.5 h at 23° C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give 27-R (271 mg, 42%).

    [0991] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [0992] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.46 (s, 1H), 7.16 (d, J=8.9 Hz, 1H), 6.83 (s, 1H), 6.72 (d, J=8.9 Hz, 1H), 6.58 (s, 1H), 6.20 (d, J=1.8 Hz, 1H), 5.99 (d, J=1.8 Hz, 1H), 5.76 (s, 1H), 5.15 (d, J=11.4 Hz, 1H), 4.86 (s, 1H), 4.52 (m, 2H), 4.17 (d, J=5.3 Hz, 1H), 4.07 (d, J=11.4 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.55-3.43 (m, 2H), 3.32-3.20 (m, 2H), 3.01-2.82 (m, 4H), 2.68-2.59 (m, 1H), 2.44-2.31 (m, 1H), 2.38 (s, 3H), 2.30-2.19 (m, 1H), 2.26 (s, 3H), 2.15 (s, 3H), 2.07 (s, 3H).

    [0993] .sup.13C NMR (101 MHz, CD3OD): δ 171.7, 171.3, 153.8, 153.3, 148.0, 147.6, 145.4, 145.4, 143.1, 141.3, 140.7, 131.6, 131.4, 131.2, 129.3, 126.8, 121.6, 120.9, 118.3, 115.6, 112.2, 111.8, 101.8, 100.2, 81.7, 63.5, 63.1, 61.7, 58.0, 57.8, 56.1, 55.8, 55.0, 42.2, 42.1, 41.4, 41.0, 25.1, 23.8, 20.5, 16.0, 9.7.

    [0994] ESI-MS m/z: 796.3 (M−H.sub.2O+H).sup.+.

    [0995] (+)-HR-ESI-TOF-MS m/z: 796.3045 [M−H.sub.2O+H].sup.+ (Calcd. for C.sub.42H.sub.46N.sub.5O.sub.9S 796.3011).

    Example 0-14

    A)

    [0996] ##STR00198##

    [0997] To a solution of compound 1 (2.0 g, 3.21 mmol) in acetonitrile (200 mL, 0.01 M) was added 2-benzofuran-3-yl-ethylamine hydrochloride (30) (1.90 g, 9.65 mmol, Sigma Aldrich) and cyanuric chloride (TCT) (200 mg, 10%). The reaction mixture was stirred at 85° C. for 24 h and then aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 9:1 to 1:9) gives compound 31 (1.95 g, 79%).

    [0998] R.sub.f=0.5 (Hexane:EtOAc, 1:1).

    [0999] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.38-7.36 (m, 2H), 7.19-7.10 (m, 2H), 6.64 (s, 1H), 6.20 (d, J=1.5 Hz, 1H), 6.05 (d, J=1.5 Hz, 1H), 5.76 (s, 1H), 5.05 (d, J=11.7 Hz, 1H), 4.54 (s, 1H), 4.33-4.24 (m, 2H), 4.23-4.16 (m, 2H), 3.81 (s, 3H), 3.49-3.38 (m, 2H), 3.28-3.21 (m, 1H), 3.06-2.78 (m, 5H), 2.57-2.50 (m, 2H), 2.37 (s, 3H), 2.27 (s, 3H), 2.21 (m, 3H), 2.08 (s, 3H).

    [1000] ESI-MS m/z: 765.3 (M+H).sup.+.

    B)

    [1001] ##STR00199##

    [1002] To a solution of compound 31 (380 mg, 0.49 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 25 mL, 0.015 M) was added AgNO.sub.3 (1.30 g, 7.45 mmol). After 5 h at 23° C., a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3 was added, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford compound 32 (175 mg, 47%).

    [1003] R.sub.f=0.40 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1004] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.35 (ddd, J=10.7, 7.6, 1.1 Hz, 2H), 7.14 (dtd, J=19.7, 7.3, 1.3 Hz, 2H), 6.65 (s, 1H), 6.16 (d, J=1.5 Hz, 1H), 6.01 (d, J=1.5 Hz, 1H), 5.75 (s, 1H), 5.15 (dd, J=11.5, 1.2 Hz, 1H), 4.80 (s, 1H), 4.48 (d, J=3.2 Hz, 1H), 4.44 (s, 1H), 4.20-4.06 (m, 2H), 3.81 (s, 1H), 3.50 (d, J=18.8 Hz, 1H), 3.30 (ddd, J=12.6, 7.9, 5.1 Hz, 1H), 3.22 (d, J=9.1 Hz, 1H), 2.99 (d, J=17.9 Hz, 1H), 2.84 (dd, J=19.2, 12.0 Hz, 3H), 2.59-2.49 (m, 2H), 2.36 (s, 3H), 2.27 (s, 3H), 2.21-2.14 (m, 1H), 2.18 (s, 3H), 2.06 (s, 3H).

    [1005] .sup.13C NMR (101 MHz, CDCl.sub.3): δ 171.2, 168.7, 154.4, 150.0, 147.9, 145.5, 142.9, 140.9, 140.8, 131.3, 129.0, 127.7, 123.7, 122.2, 121.2, 120.8, 118.9, 118.3, 115.5, 113.5, 111.7, 101.7, 82.1, 62.7, 61.7, 60.3, 57.8, 57.4, 55.9, 55.0, 42.2, 41.3, 39.7, 38.2, 29.7, 23.7, 21.3, 20.6, 15.9, 9.7.

    [1006] ESI-MS m/z: 738.6 (M−H.sub.2O+H).sup.+.

    [1007] (+)-HR-ESI-TOF-MS m/z: 756.2654 [M+H].sup.+ (Calcd. for C.sub.40H.sub.42N.sub.3O.sub.10S 756.2585).

    Example 0-15

    A)

    [1008] ##STR00200##

    [1009] To a solution of 1 (500 mg, 0.80 mmol) in acetic acid (10 mL, 0.08 M) was added 2-(5-methoxybenzofuran-3-yl)-ethylamine hydrochloride (33) (Diverchim, ref: DW04590) (444 mg, 1.60 mmol). The reaction mixture was stirred at 50° C. for 6 days and then acetic acid was evaporated. An aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) affords 34 (270 mg, 43%).

    [1010] R.sub.f=0.3 (Hexane:EtOAc, 1:1).

    [1011] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.25 (d, J=9.1 Hz, 1H), 6.80-6.73 (m, 2H), 6.63 (s, 1H), 6.18 (d, J=1.4 Hz, 1H), 6.03 (d, J=1.4 Hz, 1H), 5.78 (s, 1H), 5.03 (dd, J=11.5, 1.3 Hz, 1H), 4.52 (s, 1H), 4.29 (s, 1H), 4.26 (dd, J=4.7, 1.5 Hz, 1H), 4.23-4.16 (m, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 3.46-3.43 (m, 1H), 3.43-3.37 (m, 1H), 3.24 (s, 1H), 3.03 (d, J=18.0 Hz, 1H), 2.91 (dd, J=17.9, 9.2 Hz, 1H), 2.87-2.72 (m, 2H), 2.53-2.47 (m, 2H), 2.36 (s, 3H), 2.27 (s, 3H), 2.20 (s, 3H), 2.06 (s, 3H).

    [1012] ESI-MS m/z: 795.8 (M+H).sup.+.

    B)

    [1013] ##STR00201##

    [1014] To a solution of 34 (345 mg, 0.43 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 30 mL, 0.015 M) was added AgNO.sub.3 (2.20 g, 13.0 mmol). After 3 h at 23° C., a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3 was added, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to obtain 35 (175 mg, 51%).

    [1015] R.sub.f=0.35 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1016] .sup.1H NMR (500 MHz, CD3OD): δ 7.27 (d, J=9.0 Hz, 1H), 6.90 (d, J=2.6 Hz, 1H), 6.80 (dd, J=9.0, 2.6 Hz, 1H), 6.57 (s, 1H), 6.23 (d, J=1.2 Hz, 1H), 6.05 (d, J=1.2 Hz, 1H), 5.23 (d, J=11.5 Hz, 1H), 4.27-4.08 (m, 4H), 3.77 (s, 3H), 3.75 (s, 3H), 3.63 (d, J=14.1 Hz, 2H), 3.40-3.34 (m, 2H), 2.93-2.87 (m, 5H), 2.80 (d, J=15.5 Hz, 1H), 2.57-2.54 (m, 2H), 2.34 (s, 3H), 2.30 (s, 3H), 2.14 (s, 3H), 2.05 (s, 3H).

    [1017] .sup.13C NMR (126 MHz, CD3OD): δ 171.9, 170.6, 157.5, 147.0, 145.0, 142.3, 141.0, 132.2, 131.1, 129.1, 122.2, 120.9, 120.2, 116.3, 115.1, 114.0, 112.7, 111.4, 103.5, 102.7, 92.9, 62.0, 60.3, 59.8, 59.4, 56.5, 56.2, 56.0, 54.0, 43.8, 41.2, 40.7, 30.8, 30.3, 28.7, 24.5, 21.6, 20.6, 16.2, 9.6.

    [1018] ESI-MS m/z: 768.6 (M−H.sub.2O+H).sup.+.

    [1019] (+)-HR-ESI-TOF-MS m/z: 768.2630 [M−H.sub.2O+H].sup.+ (Calcd. for C41H.sub.42N.sub.3O.sub.10S 768.2585).

    Example 0-16

    [1020] ##STR00202##

    [1021] To a solution of LiAlH.sub.4 (148 mL, 1.0 M in THF, 148 mmol) at −40° C. was added carefully H.sub.2SO.sub.4 (7.14 mL, 72.9 mmol) and a suspension of (S)-2-amino-3-(benzofuran-3-yl)propanoic acid (36-S) (prepared as described in Tetrahedron Asymmetry 2008, 19, 500-511) (5.54 g, 26.9 mmol) in THE (85 mL, 0.003 M). The reaction mixture was left evolution at 23° C., heated at 80° C. for 3 h and 18 h at 23° C. Cool at −21° C. the reaction mixture was quenched carefully with NaOH 2N until basic pH. EtOAc was added and the mixture filtered through Celite® and washed with CH.sub.3OH. The crude was concentrated under vacuum to afford compound 37-S (3.93 g, >100%).

    [1022] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 4:1).

    [1023] .sup.1H NMR (400 MHz, CD3OD): δ 7.67-7.62 (m, 1H), 7.61 (s, 1H), 7.51-7.41 (m, 1H), 7.34-7.18 (m, 2H), 3.69-3.48 (m, 1H), 3.44 (dd, J=10.8, 6.6 Hz, 1H), 3.18 (dtd, J=7.4, 6.4, 4.6 Hz, 1H), 2.88 (ddd, J=14.4, 6.1, 1.0 Hz, 1H), 2.68 (ddd, J=14.4, 7.5, 0.9 Hz, 1H).

    Example 0-17

    [1024] ##STR00203##

    [1025] To a solution of LiAlH.sub.4 (118 mL, 1.0 M in THF, 118 mmol) at −40° C. was added carefully H.sub.2SO.sub.4 (3.1 mL, 57.8 mmol) and a suspension of (R)-2-amino-3-(benzofuran-3-yl)propanoic acid (36-R) (prepared as described in Tetrahedron Asymmetry 2008, 19, 500-511) (4.4 g, 21.4 mmol) in THE (67.4 mL, 0.003 M). The reaction mixture was left evolution at 23° C., heated at 80° C. for 3 h and 18 h at 23° C. Cool at −21° C. the reaction mixture was quenched carefully with NaOH 2N until basic pH. EtOAc was added and the mixture filtered through Celite® and washed with CH.sub.3OH. The crude was concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15, Silice amine) to afford compound 37-R (2.77 g, 68%).

    [1026] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 4:1).

    [1027] .sup.1H NMR (400 MHz, CD3OD): δ 7.63-7.52 (m, 1H), 7.56 (s, 1H), 7.46-7.33 (m, 1H), 7.21 (dtd, J=19.9, 7.3, 1.3 Hz, 2H), 3.57 (dd, J=10.7, 4.6 Hz, 1H), 3.42 (dd, J=10.8, 6.6 Hz, 1H), 3.15 (dtd, J=7.6, 6.3, 4.6 Hz, 1H), 2.84 (ddd, J=14.4, 6.0, 1.0 Hz, 1H), 2.64 (ddd, J=14.4, 7.5, 0.9 Hz, 1H).

    Example 0-18

    A)

    [1028] ##STR00204##

    [1029] To a solution of compound 1 (850 mg, 1.36 mmol) in CH.sub.3CN (136 mL, 0.01 M) was added (S)-2-amino-3-(benzofuran-3-yl)propan-1-ol (37-S) (1.30 g, 6.83 mmol and cyanuric chloride (TCT) (170 mg, 20%). The reaction mixture was stirred at 85° C. for 24 h and then aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 9:1 to 1:9) gives compound 38-S (750 mg, 69%).

    [1030] R.sub.f=0.25 (Hexane:EtOAc, 1:1).

    [1031] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.39-7.33 (m, 1H), 7.33-7.29 (m, 1H), 7.20 (ddd, J=8.3, 7.2, 1.4 Hz, 1H), 7.14 (td, J=7.4, 1.0 Hz, 1H), 6.61 (s, 1H), 6.21 (d, J=1.4 Hz, 1H), 6.06 (d, J=1.4 Hz, 1H), 5.74 (s, 1H), 5.08 (d, J=11.2 Hz, 1H), 4.58 (s, 1H), 4.37 (s, 1H), 4.32-4.23 (m, 2H), 4.19 (d, J=2.7 Hz, 1H), 3.81 (s, 3H), 3.52-3.41 (m, 3H), 3.36-3.29 (m, 1H), 3.13 (d, J=9.8 Hz, 1H), 3.00-2.81 (m, 3H), 2.57 (dd, J=15.7, 4.9 Hz, 1H), 2.50 (d, J=15.2 Hz, 1H), 2.37 (s, 3H), 2.31-2.25 (m, 1H), 2.29 (s, 3H), 2.16 (s, 3H), 2.10 (d, J=7.2 Hz, 1H), 2.05 (s, 3H).

    [1032] ESI-MS m/z: 795.2 (M).sup.+.

    B)

    [1033] ##STR00205##

    [1034] To a solution of compound 38-S (890 mg, 1.12 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 75 mL, 0.015 M) was added AgNO.sub.3 (4.70 g, 28.0 mmol). After 18 h at 23° C., a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3 was added, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford compound 39-S (500 mg, 57%).

    [1035] R.sub.f=0.30 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1036] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.38-7.33 (m, 1H), 7.33-7.28 (m, 1H), 7.23-7.16 (m, 1H), 7.16-7.09 (m, 1H), 6.62 (s, 1H), 6.18 (d, J=1.4 Hz, 1H), 6.03 (d, J=1.4 Hz, 1H), 5.71 (s, 1H), 5.19 (d, J=11.2 Hz, 1H), 4.85 (s, 1H), 4.49 (s, 2H), 4.24-4.10 (m, 3H), 3.81 (s, 3H), 3.54 (d, J=4.9 Hz, 1H), 3.49 (d, J=2.3 Hz, 3H), 3.33 (t, J=10.1 Hz, 2H), 3.22 (s, 1H), 2.98 (s, 1H), 2.84 (d, J=7.6 Hz, 2H), 2.62-2.53 (m, 2H), 2.37 (s, 3H), 2.30-2.24 (m, 1H), 2.28 (s, 3H), 2.14 (s, 3H), 2.04 (s, 3H).

    [1037] .sup.13C NMR (126 MHz, CDCl.sub.3): δ 172.0, 170.7, 156.1, 150.6, 149.9, 147.1, 145.0, 142.4, 142.2, 132.0, 131.4, 128.7, 125.5, 123.8, 122.6, 121.6, 120.1, 116.5, 114.4, 112.3, 103.5, 92.6, 66.0, 65.1, 62.2, 60.4, 59.7, 56.6, 56.1, 54.8, 54.1, 51.6, 44.0, 41.3, 38.3, 30.8, 24.8, 20.6, 16.3, 9.6.

    [1038] ESI-MS m/z: 768.2 (M−H.sub.2O+H).sup.+.

    [1039] (+)-HR-ESI-TOF-MS m/z: 768.2652 [M−H.sub.2O+H].sup.+ (Calcd. for C41H.sub.42N.sub.3O.sub.10S 768.2585)

    Example 0-19

    A)

    [1040] ##STR00206##

    [1041] To a solution of compound 1 (100 mg, 0.16 mmol) in CH.sub.3CN (16 mL, 0.01 M) was added (R)-2-amino-3-(benzofuran-3-yl)propan-1-ol (37-R) (307 mg, 1.6 mmol) and cyanuric chloride (TCT) (40 mg, 40%). The reaction mixture was stirred at 85° C. for 44 h and then aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 9:1 to 1:9) gives compound 38-R (95 mg, 75%).

    [1042] R.sub.f=0.3 (Hexane:EtOAc, 1:1).

    [1043] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.42-7.27 (m, 2H), 7.28-7.09 (m, 2H), 6.58 (s, 1H), 6.20 (d, J=1.4 Hz, 1H), 6.05 (d, J=1.4 Hz, 1H), 5.79 (s, 1H), 5.00 (d, J=11.4 Hz, 1H), 4.59 (s, 1H), 4.34 (s, 1H), 4.31-4.16 (m, 4H), 3.80 (s, 3H), 3.79-3.76 (m, 1H), 3.63 (s, 1H), 3.54-3.40 (m, 4H), 2.99-2.87 (m, 2H), 2.68 (d, J=15.0 Hz, 1H), 2.56-2.47 (m, 1H), 2.38 (s, 3H), 2.27 (s, 3H), 2.17 (s, 3H), 2.07 (s, 3H).

    [1044] ESI-MS m/z: 795.2 (M+H).sup.+.

    B)

    [1045] ##STR00207##

    [1046] To a solution of compound 38-R (95 mg, 0.11 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 11 mL, 0.015 M) was added AgNO.sub.3 (601 mg, 3.58 mmol). After 18 h at 23° C., a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3 was added, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford compound 39-R (66 mg, 70%).

    [1047] R.sub.f=0.3 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1048] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.39-7.31 (m, 2H), 7.23-7.07 (m, 2H), 6.59 (s, 1H), 6.17 (d, J=1.4 Hz, 1H), 6.01 (d, J=1.4 Hz, 1H), 5.75 (s, 1H), 5.12 (dd, J=11.3, 1.2 Hz, 1H), 4.84 (s, 1H), 4.56-4.43 (m, 2H), 4.19-4.07 (m, 3H), 3.79 (s, 3H), 3.83-3.74 (m, 1H), 3.66-3.51 (m, 3H), 3.24 (s, 1H), 2.99-2.79 (m, 2H), 2.75-2.64 (m, 1H), 2.59-2.43 (m, 2H), 2.38 (s, 3H), 2.27 (s, 3H), 2.16 (s, 3H), 2.07 (s, 3H).

    [1049] .sup.13C NMR (101 MHz, CD3OD): δ 170.5, 169.1, 154.9, 148.9, 148.5, 145.7, 143.6, 141.1, 140.8, 130.6, 129.9, 127.1, 124.1, 122.4, 122.4, 121.2, 120.3, 118.7, 118.2, 115.1, 113.6, 110.9, 102.1, 91.1, 65.0, 63.3, 60.2, 59.0, 58.4, 55.4, 54.5, 52.7, 52.3, 42.5, 38.7, 29.4, 23.5, 23.2, 19.1, 14.8, 8.3.

    [1050] ESI-MS m/z: 768.2 (M−H.sub.2O+H).sup.+.

    [1051] (+)-HR-ESI-TOF-MS m/z: 767.2628 [M−H.sub.2O+H].sup.+ (Calcd. for C41H.sub.42N.sub.3O.sub.10S 768.2585).

    Example 0-20. Synthesis of allyl —N—[(S)-2-amino-3-(benzofuran-3-yl)propyl]carbamate (44-S)

    [1052] ##STR00208##

    A)

    [1053] ##STR00209##

    [1054] To a solution of compound 37-S (1.0 g, 5.22 mmol) in CH.sub.3CN (21 mL, 4 mL/mmol) was added di-tert-butyl dicarbonate (2.28 g, 10.4 mmol). The reaction mixture was stirred at 23° C. for 2 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford compound 40-S (0.5 g, 33%).

    [1055] R.sub.f=0.7 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1056] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.64 (d, J=7.6 Hz, 1H), 7.49 (s, 1H), 7.46 (d, J=7.6 Hz, 1H), 7.36-7.19 (m, 2H), 4.94 (s, 1H), 3.98 (s, 1H), 3.71-3.56 (m, 2H), 2.93 (d, J=6.9 Hz, 2H), 1.41 (s, 9H).

    B)

    [1057] ##STR00210##

    [1058] To a solution of compound 40-S (0.5 g, 1.71 mmol) in CH.sub.2Cl.sub.2 (11 mL, 6 mL/mmol) was added phthalimide (0.55 g, 3.77 mmol), triphenylphosphine (0.99 g, 3.77 mmol) and the mixture was cooled at 0° C. A solution of 40% of diethyl azodicarboxylate (DEAD) in CH.sub.2Cl.sub.2 (1.26 mL, 4.29 mmol) was added for 15 min. The reaction was stirred at 23° C. for 18 h, concentrated under vacuum. The residue obtained was purified by flash chromatography (Hexane:EtOAc, from 99:1 to 40:60) to afford compound 41-S (0.68 g, 94%).

    [1059] R.sub.f=0.8 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1060] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.89-7.79 (m, 2H), 7.83-7.62 (m, 2H), 7.65-7.55 (m, 2H), 7.49-7.42 (m, 1H), 7.33-7.20 (m, 2H), 4.83 (d, J=9.0 Hz, 1H), 4.39 (ddt, J=12.1, 6.3, 2.9 Hz, 1H), 3.88-3.70 (m, 2H), 2.96 (d, J=6.4 Hz, 2H), 1.24 (s, 9H).

    C)

    [1061] ##STR00211##

    [1062] To a solution of compound 41-S (345 mg, 0.82 mmol) in ethanol (25 mL, 30 mL/mmol) was added hydrazine monohydrate (3.6 mL, 73.8 mmol). The reaction mixture was stirred at 80° C. in sealed tube for 2 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:1 to 50:50) to afford compound 42-S (233 mg, 98%).

    [1063] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 8:2).

    [1064] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.62 (d, J=7.5 Hz, 1H), 7.49-7.42 (m, 2H), 7.33-7.18 (m, 2H), 4.85 (d, J=8.8 Hz, 1H), 3.91 (s, 1H), 2.91-2.76 (m, 3H), 2.67 (dd, J=13.1, 6.8 Hz, 1H), 1.25 (s, 9H).

    D)

    [1065] ##STR00212##

    [1066] To a solution of compound 42-S (280 mg, 0.96 mmol) in CH.sub.3CN (10 mL, 10 mL/mmol) and DMF (16 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (0.14 mL, 0.77 mmol) and allyl chloroformate (1.02 mL, 9.64 mmol). The reaction was stirred at 23° C. for 2 h. The mixture was diluted with EtOAc and NH.sub.4Cl was added and the mixture was 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 flash chromatography (Hexane:EtOAc, from 100:1 to 1:100) to afford compound 43-S (445 mg, >100%).

    [1067] R.sub.f=0.5 (Hexane:EtOAc, 1:1).

    [1068] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.6 Hz, 1H), 7.52-7.43 (m, 2H), 7.34-7.20 (m, 2H), 5.90 (ddt, J=16.4, 10.8, 5.6 Hz, 1H), 5.32-5.17 (m, 2H), 4.93-4.86 (m, 1H), 4.56 (d, J=5.6 Hz, 2H), 4.08-3.98 (m, 1H), 3.40-3.21 (m, 2H), 2.88 (m, 2H), 1.25 (s, 9H).

    E)

    [1069] ##STR00213##

    [1070] To a solution of compound 43-S (160 mg, 0.43 mmol) in CH.sub.2Cl.sub.2 (8 mL, 16.6 mL/mmol) was added trifluoroacetic acid (4 mL, 8.3 mL/mmol). The reaction mixture was stirred at 23° C. for 1.5 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:1 to 50:50) to afford compound 44-S (175 mg, >100%).

    [1071] R.sub.f=0.2 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1072] .sup.1H NMR (400 MHz, CD3OD): δ 7.72 (s, 1H), 7.64 (dt, J=8.4, 0.9 Hz, 1H), 7.49 (dt, J=8.4, 0.9 Hz, 1H), 7.37-7.22 (m, 2H), 5.94 (ddt, J=16.3, 10.7, 5.5 Hz, 1H), 5.32 (dq, J=17.3, 1.7 Hz, 1H), 5.19 (dq, J=10.6, 1.5 Hz, 1H), 4.56 (dt, J=5.7, 1.5 Hz, 2H), 3.56 (qd, J=7.0, 4.4 Hz, 1H), 3.46-3.32 (m, 1H), 3.32-3.24 (m, 1H), 3.03 (dd, J=14.8, 6.9 Hz, 1H), 2.91 (ddd, J=14.8, 7.1, 0.9 Hz, 1H).

    Example 0-21. Synthesis of allyl —N—[(R)-2-amino-3-(benzofuran-3-yl)propyl]carbamate (44-R)

    [1073] ##STR00214##

    A)

    [1074] ##STR00215##

    [1075] To a solution of compound 37-R (2.75 g, 14.4 mmol) in CH.sub.3CN (58 mL, 4 mL/mmol) was added di-tert-butyl dicarbonate (6.27 g, 28.76 mmol). The reaction mixture was stirred at 23° C. for 2.5 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford compound 40-R (3.7 g, 88%).

    [1076] R.sub.f=0.6 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1077] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.64 (d, J=7.6 Hz, 1H), 7.52-7.43 (m, 2H), 7.35-7.20 (m, 2H), 4.85 (d, J=8.2 Hz, 1H), 4.00 (bs, 1H), 3.69 (dd, J=11.0, 4.0 Hz, 1H), 3.62 (dd, J=10.9, 5.1 Hz, 1H), 2.94 (d, J=6.9 Hz, 2H), 1.42 (s, 9H).

    B)

    [1078] ##STR00216##

    [1079] To a solution of compound 40-R (3.7 g, 12.7 mmol) in CH.sub.2Cl.sub.2 (76 mL, 6 mL/mmol) was added phthalimide (4.1 g, 28 mmol), triphenylphosphine (7.3 g, 28 mmol) and the mixture was cooled at 0° C. A solution of 40% of diethyl azodicarboxylate (DEAD) in CH.sub.2Cl.sub.2 (9.4 mL, 31.7 mmol) was added for 15 min. The reaction was stirred at 23° C. for 16 h, concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to afford compound 41-R (4.05 g, 76%).

    [1080] R.sub.f=0.8 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1081] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.67-7.68 (m, 4H), 7.61 (d, J=7.5 Hz, 1H), 7.58 (s, 1H), 7.46 (d, J=7.5 Hz, 1H), 7.27 (dtd, J=17.2, 7.3, 1.4 Hz, 2H), 4.84 (d, J=9.0 Hz, 1H), 4.46-4.30 (m, 1H), 3.89-3.66 (m, 2H), 2.97 (d, J=6.4 Hz, 2H), 1.24 (s, 9H).

    C)

    [1082] ##STR00217##

    [1083] To a solution of compound 41-R (4.0 g, 9.5 mmol) in ethanol (285 mL, 30 mL/mmol) was added hydrazine monohydrate (41.5 mL, 856 mmol). The reaction mixture was stirred at 80° C. in sealed tube for 2 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:1 to 50:50) to afford compound 42-R (2.2 g, 80%).

    [1084] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 8:2).

    [1085] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.5 Hz, 1H), 7.45 (s, 1H), 7.44 (d, J=7.1 Hz, 1H), 7.25 (dtd, J=18.8, 7.3, 1.3 Hz, 2H), 4.94 (d, J=8.8 Hz, 1H), 3.98-3.78 (m, 1H), 2.90-2.77 (m, 2H), 2.65 (dd, J=13.1, 7.0 Hz, 1H), 1.40 (s, 9H).

    D)

    [1086] ##STR00218##

    [1087] To a solution of compound 42-R (2.2 g, 7.6 mmol) in CH.sub.3CN (76 mL, 10 mL/mmol) and DMF (7.6 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (1.1 mL, 6.08 mmol) and allyl chloroformate (8.05 mL, 76 mmol). The reaction was stirred at 23° C. for 7 h. The mixture was diluted with EtOAc and NH.sub.4Cl was added and the mixture was 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 flash chromatography (Hexane:EtOAc, from 100:1 to 1:100) to afford compound 43-R (2.3 g, 81%).

    [1088] R.sub.f=0.7 (Hexane:EtOAc, 1:1).

    [1089] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.60 (d, J=7.5 Hz, 1H), 7.52-7.43 (m, 2H), 7.34-7.20 (m, 2H), 5.90 (ddt, J=17.3, 10.8, 5.6 Hz, 1H), 5.29 (d, J=17.2, 1H), 5.20 (d, J=10.4, 1H), 5.10 (t, J=6.2 Hz, 1H), 4.86 (d, J=8.4 Hz, 1H), 4.56 (d, J=5.4, 2H), 4.08-3.97 (m, 1H), 3.36 (dt, J=10.7, 4.7 Hz, 1H), 3.30-3.23 (m, 1H), 2.87 (td, J=14.8, 6.5 Hz, 2H), 1.41 (s, 9H).

    E)

    [1090] ##STR00219##

    [1091] To a solution of compound 43-R (1.32 g, 3.52 mmol) in CH.sub.2Cl.sub.2 (60 mL, 16.6 mL/mmol) was added Trifluoroacetic acid (30 mL, 8.3 mL/mmol). The reaction mixture was stirred at 23° C. for 1.5 h, concentrated under vacuum. Flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:1 to 50:50) to afford compound 44-R (0.90 g, 94%).

    [1092] R.sub.f=0.2 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1093] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.75 (s, 1H), 7.69-7.61 (m, 1H), 7.54-7.46 (m, 1H), 7.39-7.24 (m, 2H), 5.95 (ddt, J=16.3, 10.8, 5.5 Hz, 1H), 5.32 (dd, J=17.3, 1.8 Hz, 1H), 5.24-5.16 (m, 1H), 4.57 (dt, J=5.7, 1.5 Hz, 2H), 3.68 (qd, J=7.1, 4.2 Hz, 1H), 3.48 (dd, J=14.8, 4.2 Hz, 1H), 3.42-3.30 (m, 1H), 3.14-2.95 (m, 2H).

    Example 0-22

    A)

    [1094] ##STR00220##

    [1095] To a solution of compound 1 (750 mg, 1.2 mmol) in CH.sub.3CN (120 mL, 0.01 M) was added compound 44-S (1370 mg, 6 mmol) and cyanuric chloride (TCT) (184 mg, 20%). The reaction mixture was stirred at 85° C. for 23 h and then aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 9:1 to 1:9) gives compound 45-S (755 mg, 72%).

    [1096] R.sub.f=0.36 (Hexane:EtOAc, 1:1).

    [1097] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.38-7.28 (m, 2H), 7.23-7.08 (m, 2H), 6.67 (s, 1H), 6.19 (d, J=1.4 Hz, 1H), 6.09-5.95 (m, 1H), 6.04 (d, J=1.4 Hz, 1H), 5.92 (s, 1H), 5.80 (s, 1H), 5.44-5.34 (m, 1H), 5.26 (dq, J=10.4, 1.3 Hz, 1H), 5.08 (dd, J=11.4, 1.1 Hz, 1H), 4.70-4.63 (m, 2H), 4.56 (s, 1H), 4.34 (s, 1H), 4.31-4.18 (m, 3H), 3.80 (s, 3H), 3.50-3.39 (m, 2H), 3.24-3.15 (m, 1H), 3.00 (dt, J=12.2, 6.0 Hz, 2H), 2.95 (d, J=5.2 Hz, 2H), 2.60 (dd, J=15.4, 4.5 Hz, 2H), 2.44 (dd, J=15.6, 5.2 Hz, 1H), 2.29 (s, 3H), 2.27 (s, 3H), 2.25-2.20 (m, 1H), 2.18 (s, 3H), 2.12 (s, 1H), 2.04 (s, 3H).

    [1098] ESI-MS m/z: 878.2 (M+H).sup.+.

    B)

    [1099] ##STR00221##

    [1100] To a solution of compound 45-S (750 mg, 0.85 mmol) in CH.sub.2Cl.sub.2 (15.3 mL, 18 mL/mmol) was added bis(triphenylphosphine)palladium(II) dichloride (96 mg, 0.14 mmol) and acetic acid (0.5 mL, 8.5 mmol). Tributyltin hydride (1.4 mL, 5.1 mmol) was added at 0° C., and the reaction mixture was stirred at 0° C. for 30 minutes, and was concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 100:1 to 1:100 and CH.sub.2Cl.sub.2:CH.sub.3OH, from 100:1 to 1:100) to afford compound 46-S (430 mg, 64%).

    [1101] R.sub.f=0.3 (CH.sub.2Cl.sub.2:CH.sub.3OH, 1:1).

    [1102] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.37-7.29 (m, 2H), 7.22-7.11 (m, 2H), 6.57 (s, 1H), 6.21 (d, J=1.5 Hz, 1H), 6.06 (d, J=1.5 Hz, 1H), 5.07 (d, J=11.5 Hz, 1H), 4.57 (s, 1H), 4.37 (s, 1H), 4.29-4.23 (m, 2H), 4.14 (s, 1H), 3.79 (s, 3H), 3.50-3.47 (m, 2H), 3.38 (d, J=8.7 Hz, 1H), 2.95-2.71 (m, 4H), 2.68-2.52 (m, 2H), 2.51-2.38 (m, 1H), 2.35 (s, 3H), 2.33-2.26 (m, 1H), 2.29 (s, 3H), 2.17-2.08 (m, 1H), 2.10 (s, 3H), 2.04 (s, 3H).

    [1103] ESI-MS m/z: 794.3 (M+H).sup.+.

    C)

    [1104] ##STR00222##

    [1105] To a solution of compound 46-S (550 mg, 0.7 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 49 mL, 0.015 M) was added AgNO.sub.3 (2.4 g, 14 mmol). After 16 h at 23° C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give compound 47-S (53 mg, 10%).

    [1106] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1107] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.36 (d, 7.9 Hz, 1H), 7.33 (d, 7.4 Hz, 1H), 7.23 (t, J=7.4 Hz, 1H), 7.16 (t, J=7.4 Hz, 1H), 6.77 (s, 1H), 6.20 (s, 1H), 6.04 (s, 1H), 5.92 (s, 1H), 5.20 (d, J=11.1 Hz, 1H), 4.90 (s, 1H), 4.50 (s, 1H), 4.46-4.39 (m, 1H), 4.25 (d, J=11.1 Hz, 1H), 4.20 (s, 1H), 3.84 (s, 3H), 3.81 (d, J=4.2 Hz, 1H), 3.58 (s, 1H), 3.40-3.14 (m, 3H), 2.90 (t, J=13.0 Hz, 1H), 2.76 (m, 3H), 2.50 (s, 3H), 2.46-2.37 (m, 1H), 2.32-2.26 (m, 2H), 2.30 (s, 3H), 2.15 (s, 3H), 2.04 (s, 3H).

    [1108] .sup.13C NMR (126 MHz, CD3OD): δ 170.5, 169.2, 154.6, 149.1, 148.7, 145.7, 143.5, 141.0, 140.9, 131.2, 129.6, 126.9, 124.4, 122.5, 121.4, 119.7, 118.7, 115.0, 112.7, 111.0, 110.7, 102.1, 91.2, 63.5, 61.2, 59.2, 58.5, 55.3, 54.7, 53.4, 52.7, 43.3, 42.5, 39.9, 36.9, 29.3, 24.1, 23.6, 19.1, 15.0, 8.2.

    [1109] ESI-MS m/z: 767.2 (M−H.sub.2O+H).sup.+.

    [1110] (+)-HR-ESI-TOF-MS m/z: 767.2794 [M−H.sub.2O+H].sup.+ (Calcd. for C41H.sub.43N.sub.4O.sub.9S 767.2745).

    Example 0-23

    A)

    [1111] ##STR00223##

    [1112] To a solution of compound 1 (621 mg, 1 mmol) in CH.sub.3CN (100 mL, 0.01 M) was added compound 44-R (825 mg, 3 mmol) and cyanuric chloride (TCT) (248 mg, 40%). The reaction mixture was stirred at 85° C. for 66 h and then aqueous saturated solution of NaHCO.sub.3 was added and the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 9:1 to 1:9) gives compound 45-R (530 mg, 58%).

    [1113] R.sub.f=0.4 (Hexane:EtOAc, 1:1).

    [1114] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.42-7.28 (m, 2H), 7.23-7.08 (m, 2H), 6.60 (s, 1H), 6.20 (d, J=1.4 Hz, 1H), 6.04 (d, J=1.4 Hz, 1H), 6.01-5.92 (m, 1H), 5.77 (s, 1H), 5.44-5.20 (m, 2H), 5.09 (s, 1H), 5.04-4.96 (m, 1H), 4.71-4.55 (m, 2H), 4.34 (s, 1H), 4.30-4.18 (m, 3H), 3.79 (s, 3H), 3.53 (dd, J=10.2, 4.4 Hz, 1H), 3.46 (m, 2H), 3.50-3.40 (m, 1H), 3.03-2.87 (m, 2H), 2.67 (d, J=15.0 Hz, 1H), 2.47 (dd, J=15.6, 3.7 Hz, 1H), 2.40-2.32 (m, 2H), 2.30 (s, 3H), 2.29 (s, 3H), 2.19-2.12 (m, 2H), 2.16 (s, 3H), 2.09 (s, 3H).

    [1115] ESI-MS m/z: 878.3 (M+H).sup.+.

    B)

    [1116] ##STR00224##

    [1117] To a solution of compound 45-R (552 mg, 0.63 mmol) in CH.sub.2Cl.sub.2 (11.3 mL, 18 mL/mmol) was added bis(triphenylphosphine)palladium(II) dichloride (70.7 mg, 0.1 mmol) and acetic acid (0.36 mL, 6.3 mmol). Tributyltin hydride (1.02 mL, 3.8 mmol) was added at 0° C. and the reaction mixture was stirred at 0° C. for 0.5 h, and concentrated under vacuum The crude obtained was diluted with EtOAc, saturated aqueous solution of NH.sub.4Cl was added and the mixture was extracted with EtOAc. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH.sub.3OH, from 100:1 to 1:100) to afford compound 46-R (423 mg, 85%).

    [1118] R.sub.f=0.3 (CH.sub.2Cl.sub.2:CH.sub.3OH, 1:1).

    [1119] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.45-7.28 (m, 2H), 7.23-7.08 (m, 2H), 6.56 (s, 1H), 6.19 (d, J=1.4 Hz, 1H), 6.05 (d, J=1.4 Hz, 1H), 4.98 (d, J=11.5 Hz, 1H), 4.59 (s, 1H), 4.34 (s, 1H), 4.27 (dd, J=5.1, 1.7 Hz, 1H), 4.22-4.16 (m, 2H), 3.80 (s, 3H), 3.49-3.39 (m, 2H), 3.31 (dq, J=9.8, 5.5, 4.5 Hz, 2H), 2.95 (s, 1H), 2.83 (d, J=5.6 Hz, 2H), 2.74-2.51 (m, 3H), 2.35 (s, 3H), 2.32-2.21 (m, 2H), 2.26 (s, 3H); 2.16 (s, 3H), 2.06 (s, 3H).

    [1120] ESI-MS m/z: 794.3 (M+H).sup.+.

    C)

    [1121] ##STR00225##

    [1122] To a solution of compound 46-R (412 mg, 0.52 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 36 mL, 0.015 M) was added AgNO.sub.3 (1.76 g, 10.4 mmol). After 22 h at 23° C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2Cl.sub.2, stirred for 5 min, and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 85:15) to give compound 47-R (175 mg, 43%).

    [1123] R.sub.f=0.1 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1124] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.34 (dd, J=11.1, 7.9 Hz, 2H), 7.22-7.07 (m, 2H), 6.57 (s, 1H), 6.17 (d, J=1.2 Hz, 1H), 6.01 (d, J=1.2 Hz, 1H), 5.11 (d, J=11.2 Hz, 1H), 4.84 (s, 1H), 4.53-4.47 (m, 2H), 4.21-4.07 (m, 2H), 3.80 (s, 3H), 3.56 (d, J=5.1 Hz, 1H), 3.43 (s, 1H), 3.24 (d, J=9.1 Hz, 1H), 2.98-2.78 (m, 4H), 2.72-2.58 (m, 2H), 2.38 (s, 3H), 2.35-2.27 (m, 2H), 2.28 (s, 3H), 2.14 (s, 3H), 2.08 (s, 3H).

    [1125] .sup.13C NMR (101 MHz, CD3OD): δ 170.6, 169.1, 155.0, 148.8, 145.6, 143.7, 141.1, 140.8, 130.9, 129.7, 126.9, 124.2, 122.4, 121.1, 119.6, 118.9, 118.7, 115.0, 113.2, 112.5, 111.0, 102.1, 91.3, 63.3, 60.4, 59.0, 58.4, 55.3, 54.6, 52.6, 51.1, 44.9, 42.4, 39.8, 38.7, 29.4, 24.0, 23.2, 19.1, 15.0, 8.3.

    [1126] ESI-MS m/z: 767.2 (M−H.sub.2O+H).sup.+.

    [1127] (+)-HR-ESI-TOF-MS m/z: 767.2806 [M−H.sub.2O+H].sup.+ (Calcd. for C41H.sub.43N.sub.4O.sub.9S 767.2745).

    Bioactivity Example of the Payloads

    [1128] The aim of this assay is 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.

    Cell Lines

    [1129]

    TABLE-US-00003 Name No ATCC Species Tissue Characteristics A549 CCL-185 human Lung lung carcinoma (NSCLC) HT29 HTB-38 human Colon colorectal adenocarcinoma MDA-MB- HTB-26 human Breast breast adenocarcinoma 231 PSN1 CRM-CRL- human Pancreas pancreas 3211 adenocarcinoma PC-3 CRL-1435 human Prostate prostate adenocarcinoma 22Rv1 CRL-2505 human Prostate prostate carcinoma

    Evaluation of Cytotoxic Activity Using the SRB and the MTT Colorimetric Assays

    [1130] A colorimetric assay, using Sulforhodamine B (SRB) reaction has been adapted to provide a quantitative measurement of cell growth and viability (following the technique described by Skehan et al. J. Natl. Cancer Inst. 1990, 82, 1107-1112). Another colorimetric assay based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to a purple formazan has been also used to assess the antiproliferative activity (following the technique described by Mosmann et al. J. Immunol. Meth. 1983, 65, 55-63).

    [1131] These forms of assays employ 96-well cell culture microplates following the standards of the American National Standards Institute and the Society for Laboratory Automation and Screening (ANSI SLAS January 2004 (R2012) Oct. 12, 2011. All the cell lines used in this study were obtained from the American Type Culture Collection (ATCC) and derive from different types of human cancer.

    [1132] A549, HT29, MDA-MB-231 and PSN1 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) while PC-3 and 22Rv1 cells were maintained in Roswell Park Memorial Institute Medium (RPMI). All cell lines were supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin at 37° C., 5% CO2 and 98% humidity. For the experiments, cells were harvested from subconfluent cultures using trypsinization and resuspended in fresh medium before counting and plating.

    [1133] A549, HT29, MDA-MB-231 and PSN1 cells were seeded in 96 well microtiter plates, at 5000 cells per well in aliquots of 150 μL, and allowed to attach to the plate surface for 18 hours (overnight) in drug free medium. After that, one control (untreated) plate of each cell line was fixed (as described below) and used for time zero reference value. Culture plates were then treated with test compounds (50 μL aliquots of 4× stock solutions in complete culture medium plus 4% DMSO) using ten 2/5 serial dilutions (concentrations ranging from 10 to 0.003 μg/mL) and triplicate cultures (1% final concentration in DMSO). After 72 hours treatment, the antitumor effect was measured by using the SRB methodology: Briefly, cells were washed twice with PBS, fixed for 15 min in 1% glutaraldehyde solution at room temperature, rinsed twice in PBS, and stained in 0.4% SRB solution for 30 min at room temperature. Cells were then rinsed several times with 1% acetic acid solution and air-dried at room temperature. SRB was then extracted in 10 mM trizma base solution and the absorbance measured in an automated spectrophotometric plate reader at 490 nm.

    [1134] An appropriate number of PC-3 and 22Rv1 cells, to reach a final cell density in the assay ranging from 5,000 to 15,000 cells per well depending on the cell line, were seeded in 96-well plates and allowed to stand in culture medium for 24 h at 37° C. under 5% CO2 and 98% humidity. Then, compounds or DMSO in culture medium were added to reach a final volume of 200 μL and the intended compound concentration in a range covering ten serial 2/5 dilutions starting from 0.1 μg/mL in 1% (v/v) DMSO. At this point a set of “time zero control plates” treated with 1% (v/v) DMSO were processed with MTT as described below. The rest of the plates were incubated during 72 h under the aforementioned environmental conditions. Afterwards 50 μL of a 1 mg/mL MTT solution in culture medium were added to the wells and incubated for 6-8 hours at 37° C. to allow formazan crystals generation. Culture medium was then removed and 100 μL of neat DMSO added to each well to dissolve the formazan product into a coloured solution whose absorbance at 540 nm was finally measured in a PolarStar Omega microplate multilabel reader (BMG Labtech, Ortenberg, Germany).

    [1135] Effects on cell growth and survival were estimated by applying the NCl algorithm (Boyd M R and Paull K D. Drug Dev. Res. 1995, 34, 91-104). The values obtained in triplicate cultures were fitted by nonlinear regression to a four-parameters logistic curve by nonlinear regression analysis. Three reference parameters were calculated (according to the aforementioned NCl algorithm) by automatic interpolation of the curves obtained by such fitting: GI.sub.50=compound concentration that produces 50% cell growth inhibition, as compared to control cultures; TGI=total cell growth inhibition (cytostatic effect), as compared to control cultures, and LC.sub.50=compound concentration that produces 50% net cell killing cytotoxic effect).

    [1136] Tables 3-9 illustrate data on the biological activity of compounds of the present invention.

    TABLE-US-00004 TABLE 3 Biological activity (Molar) Compound [00226]embedded image 3-S R.sub.1 = CN, R.sub.4 = —CH.sub.2OH 3a-S R.sub.1 = CN, R.sub.4 = —CH.sub.2OAc 10-S R.sub.1 = ON, R.sub.4 = —CH.sub.2NHAlloc 11-S R.sub.1 = CN, R.sub.4 = —CH.sub.2NH.sub.2 4-S R.sub.1 = OH, R.sub.4 = —CH.sub.2OH 4a-S R.sub.1 = OH, R.sub.4 = —CH.sub.2OAc 12-S R.sub.1 = OH, R.sub.4 = —CH.sub.2NH.sub.2 13-S R.sub.1 = OH, R.sub.1 = —CH.sub.2NHAlloc MDA-MB- A549 HT29 231 PSN1 PC-3 22Rv1 GI.sub.50 3-S 4.03E-10 2.77E-10 4.91E-10 9.95E-10 TGI 6.17E-10 >1.26E-07 5.29E-10 1.64E-09 LC.sub.50 >1.26E-07 >1.26E-07 6.17E-10 >1.26E-07 GI.sub.50 3a-S 3.11E-09 2.99E-09 2.87E-09 2.15E-09 TGI 3.23E-09 3.23E-09 3.59E-09 3.59E-09 LC.sub.50 >1.20E-07 >1.20E-07 4.90E-09 1.20E-08 GI50 10-S 2.05E-08 1.14E-08 4.79E-09 7.64E-09 TGI 3.08E-08 1.25E-08 8.44E-09 1.25.Math.E-08 LC.sub.50 7.53E-08 >1.14E-06 1.60E-08 2.39E-08 GI.sub.50 11-S 8.45E-09 3.41E-09 2.27E-09 3.28E-09 TGI 2.65E-08 >1.26E-07 3.41E-09 4.54E-09 LC.sub.50 >1.26E-07 >1.26E-07 6.43E-09 8.07E-09 GI.sub.50 4-S 1.27E-09 1.27E-09 1.22E-09 1.78E-09 8.08E-10 3.58E-10 TGI 1.40E-09 1.40E-09 2.55E-09 2.29E-09 LC.sub.50 >1.27E-07 >1.27E-07 6.50E-09 3.44E-09 GI.sub.50 4a-S 3.99E-09 3.14E-09 3.39E-09 3.02E-09 TGI 6.17E-09 3.39E-09 5.44E-09 3.27E-09 LC.sub.50 >1.21E-07 >1.21E-07 1.00E-08 3.51E-09 GI.sub.50 12-S 2.04E-08 4.85E-09 5.23E-09 3.44E-09 TGI 5.61E-08 8.42E-09 8.42E-09 5.49E-09 LC.sub.50 >1.28E-07 >1.28E-07 1.53E-08 1.21E-08 GI.sub.50 13-S 1.15E-08 1.15E-08 1.15E-08 1.96E-08 TGI 1.61E-08 1.27E-08 1.27E-08 2.88E-08 LC.sub.50 2.42E-08 >1.15E-06 1.38E-08 4.61E-08

    TABLE-US-00005 TABLE 4 Biological activity (Molar) Compound [00227]embedded image 3-R R.sub.1 = CN, R.sub.4 = —CH.sub.2OH 10-R R.sub.1 = CN, R.sub.4 = —CH.sub.2NHAlloc 11-R R.sub.1 = CN, R.sub.4 = —CH.sub.2NH.sub.2 4-R R.sub.1 = OH, R.sub.4 = —CH.sub.2OH 12-R R.sub.1 = OH, R.sub.4 = —CH.sub.2NH.sub.2 13-R R.sub.1 = OH, R.sub.4 = —CH.sub.2NHAlloc A549 HT29 MDA-MB-231 PSN1 GI.sub.50 3-R 4.03E-10 2.77E-10 2.77E-10 3.90E-10 TGI 5.79E-10 >1.26E-07 5.04E-10 6.05E-10 LC.sub.50 >1.26E-07 >1.26E-07 1.25E-09 >1.26E-07 GI.sub.50 10-R 3.76E-09 3.08E-09 2.85E-09 2.62E-09 TGI 5.93E-09 >1.14E-07 4.33E-09 3.88E-09 LC.sub.50 >1.14E-07 >1.14E-07 7.18E-09 6.61E-09 GI.sub.50 11-R 1.77E-09 1.39E-09 1.01E-09 1.39 E-09 TGI 4.54E-09 >1.26E-07 1.51E-09 1.89E-09 LC.sub.50 >1.26E-07 >1.26E-07 2.65E-09 >1.26E-07 GI.sub.50 4-R 1.27E-09 1.26E-09 1.27E-09 4.59E-10 TGI 1.40E-09 1.40E-09 1.40E-09 8.54E-10 LC.sub.50 >1.27E-07 >1.27E-07 1.53E-09 2.55E-09 GI.sub.50 12-R 1.40E-09 5.74E-10 3.19E-10 4.98E-10 TGI 2.93E-09 1.10E-09 6.76E-10 1.22E-09 LC.sub.50 1.22E-08 2.93E-09 1.40E-09 >1.28E-07 GI.sub.50 13-R 7.26E-09 6.91E-09 4.95E-09 2.88E-09 TGI 7.72E-09 7.60E-09 7.95E-09 3.11E-09 LC.sub.50 >1.15E-07 >1.15E-07 1.38E-08 3.46E-09

    TABLE-US-00006 TABLE 5 Biological activity (Molar) Compound [00228]embedded image 38-S R.sub.1 = CN, R.sub.4 = —CH.sub.2OH 45-S R.sub.1 = CN, R.sub.4 = —CH.sub.2NHAlloc 46-S R.sub.1 = CN, R.sub.4 = —CH.sub.2NH.sub.2 39-S R.sub.1 = OH, R.sub.4 = —CH.sub.2OH 47-S R.sub.1 = OH, R.sub.4 = —CH.sub.2NH.sub.2 MDA-MB- A549 HT29 231 PSN1 PC-3 22Rv1 GI.sub.50 38-S 8.05E-09 4.53E-09 2.52E-09 5.03E-09 TGI 8.55E-09 7.05E-09 4.28E-09 8.18E-09 LC.sub.50 9.44E-09 >1.26E-07 7.80E-09 1.51E-08 GI.sub.50 45-S 1.82E-08 1.82E-08 1.71E-08 1.94E-08 TGI 1.94E-08 1.94E-08 2.16E-08 2.62E-08 LC.sub.50 2.16E-08 >1.14E-07 2.96E-08 3.64E-08 GI.sub.50 46-S 8.19E-09 2.77E-09 3.65E-09 3.15E-09 TGI 2.14E-08 6.17E-09 6.80E-09 4.79E-09 LC.sub.50 >1.26E-07 >1.26E-07 1.26E-08 9.20E-09 GI.sub.50 39-S 4.84E-09 3.94E-09 3.44E-09 8.02E-09 2.78E-09 4.81E-10 TGI 8.27E-09 6.74E-09 7.13E-09 1.02E-08 LC.sub.50 1.65E-08 >1.27E-07 1.78E-08 1.27E-08 GI.sub.50 47-S 1.40E-08 4.33E-09 6.24E-09 5.99E-09 TGI 2.80E-08 6.75E-09 9.68E-09 8.54E-09 LC.sub.50 >1.27E-07 >1.27E-07 1.66E-08 1.27E-08

    TABLE-US-00007 TABLE 6 Biological (Molar) activity Compound [00229]embedded image 38-R R.sub.1 = CN, R.sub.4 = —CH.sub.2OH 45-R R.sub.1 = CN, R.sub.4 = —CH.sub.2NHAlloc 46-R R.sub.1 = CN, R.sub.4 = —CH.sub.2NH.sub.2 39-R R.sub.1 = OH, R.sub.4 = —CH.sub.2OH 47-R R.sub.1 = OH, R.sub.4 = —CH.sub.2NH.sub.2 A549 HT29 MDA-MB-231 PSN1 GI.sub.50 38-R 6.54E-10 5.41E-10 4.53E-10 6.54E-10 TGI 1.04E-09 5.91E-10 8.43E-10 9.94E-10 LC.sub.50 >1.26E-07 >1.26E-07 2.01E-09 1.76E-09 GI.sub.50 45-R 1.82E-08 1.25E-08 9.57E-09 1.06E-08 TGI 1.94E-08 2.28E-08 1.94E-08 1.94E-08 LC.sub.50 2.39E-08 >1.14E-07 4.33E-08 3.76E-08 GI.sub.50 46-R 1.51E-09 1.21E-09 1.23E-09 9.95E-10 TGI 2.77E-09 1.39E-09 1.39E-09 1.51E-09 LC.sub.50 >1.26E-07 >1.26E-07 1.51E-09 2.65E-09 GI.sub.50 39-R 2.67E-10 2.93E-10 2.04E-10 3.65E-10 TGI 4.33E-10 6.24E-10 5.98E-10 5.73E-10 LC.sub.50 >1.27E-07 >1.27E-07 2.80E-09 1.06E-09 GI.sub.50 47-R 2.04E-09 8.03E-10 5.99E-10 1.40E-09 TGI 3.82E-09 1.40E-09 1.17E-09 2.04E-09 LC.sub.50 1.40E-08 >1.27E-07 2.55E-09 3.31E-09

    TABLE-US-00008 TABLE 7 Biological activity (Molar) Compound [00230]embedded image 18-S R.sub.1 = CN, R.sub.4 = —CH.sub.2OH 25-S R.sub.1 = CN, R.sub.4 = —CH.sub.2NHAlloc 26-S R.sub.1 = CN, R.sub.4 = —CH.sub.2NH.sub.2 19-S R.sub.1 = OH, R.sub.4 = —CH.sub.2OH 27-S R.sub.1 = OH, R.sub.4 = —CH.sub.2NH.sub.2 A549 HT29 MDA-MB-231 PSN1 GI.sub.50 18-S 1.70E-09 1.21E-09 1.21E-09 9.59E-10 TGI 3.03E-09 1.34E-09 1.34E-09 1.34E-09 LC.sub.50 >1.21E-07 >1.21E-07 1.58E-09 >1.21E-07 GI.sub.50 25-S 7.17E-09 7.17E-09 5.84E-09 6.84E-09 TGI 7.61E-09 7.72E-09 9.04E-09 9.26E-09 LC.sub.50 >1.10E-07 >1.10E-07 1.54E-08 1.43E-08 GI.sub.50 26-S 1.12E-08 2.79E-09 1.34E-09 3.04E-09 TGI 2.19E-08 3.16E-09 1.94E-09 3.28E-09 LC.sub.50 >1.22E-07 >1.22E-07 3.89E-09 3.52E-09 GI.sub.50 19-S 3.07E-09 1.35E-09 1.96E-09 2.95E-09 TGI 3.31E-09 1.60E-09 3.31E-09 3.19E-09 LC.sub.50 >1.23E-07 >1.23E-07 1.10E-08 >1.23E-07 GI.sub.50 27-S 6.02E-09 1.23E-09 1.19E-09 1.97E-09 TGI 1.12E-08 1.35E-09 1.23E-09 2.83E-09 LC.sub.50 >1.23E-07 >1.23E-07 1.35E-09 4.55E-09

    TABLE-US-00009 TABLE 8 Biological activity (Molar) Compound [00231]embedded image 18-R R.sub.1 = CN, R.sub.4 = —CH.sub.2OH 25-R R.sub.1 = CN, R.sub.4 = —CH.sub.2NHAlloc 26-R R.sub.1 = CN, R.sub.4 = —CH.sub.2NH.sub.2 19-R R.sub.1 = OH, R.sub.4 = —CH.sub.2OH 27-R R.sub.1 = OH, R.sub.4 = —CH.sub.2NH.sub.2 MDA-MB- A549 HT29 231 PSN1 GI.sub.50 18-R 1.21E-09 1.21E-09 1.21E-09 5.70E-10 TGI 1.34E-09 1.34E-09 1.34E-09 1.06E-09 LC.sub.50 >1.21E-07 >1.21E-07 1.46E-09 >1.21E-07 GI.sub.50 25-R 1.32E-09 1.54E-09 1.21E-09 1.21E-09 TGI 2.43E-09 2.76E-09 2.54E-09 2.32E-09 LC.sub.50 9.92E-09 >1.10E-07 8.38E-09 6.73E-09 GI.sub.50 26-R 1.94E-09 7.29E-10 1.17E-09 9.72E-10 TGI 3.40E-09 1.58E-09 1.22E-09 1.70E-09 LC.sub.50 >1.22E-07 >1.22E-07 1.46E-09 3.52E-09 GI.sub.50 19-R 1.47E-09 1.72E-09 1.23E-09 1.23E-09 TGI 3.56E-09 1.72E-09 1.35E-09 1.35E-09 LC.sub.50 >1.23E-07 >1.23E-07 1.23E-07 1.47E-09 GI.sub.50 27-R 2.09E-09 5.04E-10 3.07E-10 6.39E-10 TGI 3.93E-09 5.53E-10 5.41E-10 1.17E-09 LC.sub.50 1.01E-08 >1.23E-07 8.60E-10 2.46E-09

    TABLE-US-00010 TABLE 9 Biological activity (Molar) Compound [00232]embedded image 31 R.sub.1 = CN, R.sub.3 = H 32 R.sub.1 = OH, R.sub.3 = H 34 R.sub.1 = CN, R.sub.3 = OMe 35 R.sub.1 = OH, R.sub.3 = OMe A549 HT29 MDA-MB-231 PSN1 GI.sub.50 31 1.96E-08 1.05E-08 8.89E-09 6.80E-09 TGI 2.09E-08 1.57E-08 1.70E-08 1.57E-08 LC.sub.50 2.35E-08 >1.31E-07 3.53E-08 4.31E-08 GI.sub.50 32 6.88E-09 6.88E-09 4.76E-09 6.09E-09 TGI >1.32E-08 >1.32E-08 1.05E-08 8.34E-09 LC.sub.50 >1.32E-08 >1.32E-08 >1.32E-08 1.20E-08 GI.sub.50 34 5.91E-08 5.41E-08 4.53E-08 5.41E-08 TGI 8.05E-08 8.55E-08 7.67E-08 5.91E-08 LC.sub.50 >1.26E-07 1.25E-07 1.12E-07 >1.26E-07 GI.sub.50 35 8.14E-09 7.89E-09 4.58E-09 6.24E-09 TGI 8.78E-09 8.65E-09 8.27E-09 9.03E-09 LC.sub.50 >1.27E-07 >1.27E-07 1.65E-08 1.40E-08
    This data demonstrates that the payloads employed in the present invention have high potency in vitro.

    Synthesis of Linkers

    Preparation of LIN 1: MC-Val-Cit-PABC-PNP

    [1137] ##STR00233##

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

    [1138] ##STR00234##

    [1139] Cl-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.

    [1140] 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.

    [1141] 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).

    [1142] 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.

    [1143] .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).

    [1144] ESI-MS m/z: Calcd. for C.sub.21H.sub.33N.sub.5O.sub.7: 467.2. Found: 468.3 (M+H).sup.+.

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

    [1145] ##STR00235##

    [1146] 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%).

    [1147] .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).

    [1148] ESI-MS m/z: Calcd. for C.sub.28H.sub.40N.sub.6O.sub.7: 572.3. Found: 573.3 (M+H).sup.+.

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

    [1149] ##STR00236##

    [1150] 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%).

    [1151] R.sub.f=0.40 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    [1152] .sup.1H NMR (400 MHz, CDCl.sub.3/CD3OD): 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).

    [1153] .sup.13C NMR (125 MHz, CDCl.sub.3/CD3OD) 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.

    [1154] ESI-MS m/z: Calcd. for C35H.sub.43N7011: 737.3. Found: 738.3 (M+H).sup.+.

    Preparation of LIN-2: MC2-PEG4-Val-Cit-PABC-PNP

    [1155] ##STR00237##

    a) Preparation of LIN 2-1: MC2-PEG4-Val-Cit-OH

    [1156] ##STR00238##

    [1157] Cl-TrtCl-resin (5 g, 1.49 mmol/g) was placed in a filter plate. To the resin was added CH.sub.2Cl.sub.2 (25 mL) and the mixture was stirred for 1 h at 23° C. The solvent was eliminated by filtration over vacuum. A solution of Fmoc-Cit-OH (2.95 g, 7.44 mmol) and DIPEA (4.29 mL, 24.61 mmol) in CH.sub.2Cl.sub.2 (20 mL) was added and the mixture was stirred for 10 min at 23° C. DIPEA (8.70 mL, 49.99 mmol) was additionally added and the mixture was stirred for 1 h at 23° C. The reaction was stopped by addition of MeOH (10 mL) and stirred 15 min at 23° C. The Fmoc-Cit-O-TrtCl-resin was subjected to the following washing/treatments: CH.sub.2Cl.sub.2 (5×15 mL×0.5 min), DMF (5×15 mL×0.5 min), piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min), DMF (5×15 mL×0.5 min), CH.sub.2Cl.sub.2 (5×15 mL×0.5 min). The loading was calculated: 1.17 mmol/g.

    [1158] The NH.sub.2—Cit-O-TrtCl-resin was washed with DMF (5×15 mL×0.5 min) and a solution of Fmoc-Val-OH (7.80 g, 22.99 mmol) and HOBt (2.80 g, 24.5 mmol) in DMF (25 mL) was added to the NH.sub.2-Cit-O-TrtCl-resin followed by addition of DIPCDI (3.56 mL, 24.5 mmol) at 23° C. The reaction mixture was stirred for 1.5 h at 23° C. The reaction was stopped by washing with DMF (5×15 mL×0.5 min). The Fmoc-Val-Cit-O-TrtCl-resin was treated with piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min) and washed with DMF (5×15 mL×0.5 min).

    [1159] A solution of 15-(9-Fluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoic acid (Fmoc-NH-PEG4-OH) (4.27 g, 8.75 mmol) and HOBt (1.18 g, 8.72 mmol) in DMF (30 mL) was added to the NH.sub.2-Val-Cit-O-TrtCl-resin followed by addition of DIPCDI (1.35 mL, 8.72 mmol) at 23° C. The reaction mixture was stirred for 24 h at 23° C. The reaction was stopped by washing with DMF (5×15 mL×0.5 min). The Fmoc-NH-PEG4-Val-Cit-O-TrtCl-resin was treated with piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min) and washed with DMF (5×15 mL×0.5 min).

    [1160] A solution of 3-(Maleimido)propionic acid (MC2-OH) (3.95 g, 23.35 mmol) and HOBt (3.16 g, 23.37 mmol) in DMF (30 mL) was added to the NH.sub.2-PEG4-Val-Cit-O-TrtCl-resin followed by addition of DIPCDI (3.62 mL, 23.37 mmol) at 23° C. The reaction mixture was stirred for 2 h at 23° C. The reaction was stopped by washing with DMF (5×15 mL×0.5 min) and CH.sub.2Cl.sub.2 (5 x 15 mL×0.5 min).

    [1161] The peptide was cleaved from the resin by treatments with TFA:CH.sub.2Cl.sub.2 (1:99, 5×50 mL). The resin was washed with CH.sub.2Cl.sub.2 (7×50 mL×0.5 min). The combined filtrates were evaporated to dryness under reduced pressure, the solid obtained was triturated with Et.sub.2O and filtrated to obtain LIN 2-1 (4.59 g, 87% yield) as a white solid.

    [1162] .sup.1H NMR (300 MHz, CDCl.sub.3): δ 7.67-7.57 (m, 1H), 7.44 (d, J=8.3 Hz, 1H), 7.11 (t, J=5.4 Hz, 1H), 6.73 (s, 2H), 4.49 (d, J=7.2 Hz, 1H), 4.35 (t, J=7.7 Hz, 1H), 3.82 (t, J=7.0 Hz, 2H), 3.74 (t, J=6.2 Hz, 2H), 3.68-3.56 (m, 13H), 3.56-3.45 (m, 2H), 3.39 (q, J=5.4 Hz, 2H), 3.17 (s, 2H), 2.55 (q, J=7.0, 6.0 Hz, 4H), 2.16-1.99 (m, 1H), 1.91 (s, 1H), 1.75 (s, 1H), 1.43 (s, 2H), 0.94 (d, =9.7 Hz, 3H), 0.93 (d, =9.7 Hz, 3H).

    [1163] ESI-MS m/z: 673.3 (M+H).sup.+.

    (b) Preparation of LIN 2-2: MC2-PEG4-Val-Cit-PABOH

    [1164] ##STR00239##

    [1165] To a solution of LIN 2-1 (1.5 g, 2.22 mmol) and 4-aminobenzyl alcohol (PABOH) (0.55 g, 4.45 mmol) in CH.sub.2Cl.sub.2 (60 mL) was added a solution of HOBt (0.60 g, 4.45 mmol) in DMF (5 mL) followed by addition of DIPCDI (0.69 mL, 4.45 mmol) at 23° C. The reaction mixture was stirred for 5 h at 23° C., Et.sub.2O (150 mL) was added, and the solid obtained was filtrated under vacuum to obtain crude LIN 2-2 (2.37 g, >100% yield) which was used in the next step without further purification.

    [1166] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 7.57 (d, J=8.6 Hz, 2H), 7.30 (d, J=8.6 Hz, 2H), 6.81 (s, 2H), 4.58 (s, 1H), 4.56 (s, 2H), 4.50 (dd, J=9.1, 5.1 Hz, 1H), 4.21 (d, J=7.0 Hz, 1H), 3.80-3.68 (m, 4H), 3.65-3.59 (m, 12H), 3.55-3.47 (m, 1H), 3.20 (dd, J=13.6, 6.9 Hz, 1H), 3.12 (dt, J=13.5, 6.7 Hz, 1H), 2.55 (td, J=6.1, 2.1 Hz, 2H), 2.46 (t, J=6.9 Hz, 2H), 2.15-2.07 (m, 1H), 1.95-1.88 (m, 1H), 1.79-1.70 (m, 1H), 1.67-1.50 (m, 2H), 0.99 (d, J=7.0 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H).

    [1167] ESI-MS m/z: 778.4 (M+H).sup.+.

    (c) Preparation of LIN 2: MC2-PEG4-Val-Cit-PABC-PNP

    [1168] ##STR00240##

    [1169] To a solution of LIN 2-2 (1.73 g, 2.22 mmol) and bis(4-nitrophenyl) carbonate (bis-PNP) (3.38 g, 11.12 mmol) in DCM:DMF (8:2, 75 mL) was added DIPEA (1.16 mL, 6.07 mmol) at 23° C. The reaction mixture was stirred for 19 h at 23° C. and poured onto silica gel column (CH.sub.2Cl.sub.2:CH.sub.3OH, from 50:1 to 10:1) to afford pure LIN 2 (945 mg, 45% yield).

    [1170] .sup.1H NMR (500 MHz, CD3OD): δ 8.22 (d, J=9.2 Hz, 2H), 7.61 (d, J=8.6 Hz, 2H), 7.34 (d, J=9.2 Hz, 2H), 7.33 (d, J=8.6 Hz, 2H), 6.67 (s, 2H), 4.57-4.47 (m, 1H), 4.23-4.12 (m, 1H), 3.78-3.76 (m, 12H), 3.63-3.50 (m, 16H), 3.49-3.41 (m, 2H), 3.34-3.25 (m, 2H), 3.18-3.03 (m, 2H), 2.51 (t, J=5.9 Hz, 2H), 2.45 (t, J=7.2 Hz, 2H), 2.13-1.99 (m, 1H), 1.92-1.84 (m, 1H), 1.73-1.62 (m, 1H), 1.55-1.45 (m, 2H), 0.92 (d, J=6.8 Hz, 3H), 0.90 (d, J=6.8 Hz, 3H).

    [1171] .sup.13C NMR (75 MHz, CDCl.sub.3/CD3OD): δ 174.4, 172.9, 172.4, 172.4, 171.6, 170.9, 170.8, 170.7, 163.7, 155.8, 155.7, 152.5, 145.4, 138.8, 134.1, 131.3, 130.4, 129.2, 128.7, 125.7, 124.9, 121.8, 119.8 (×2), 115.1, 70.2 (×2), 70.1 (×2), 70.0, 69.9, 69.8, 69.0, 66.9, 59.2, 53.5, 39.0, 36.0, 34.4, 34.1, 30.4, 29.0, 18.5, 17.5.

    [1172] ESI-MS m/z: 943.4 (M+H).sup.+.

    [1173] R.sub.f=0.20 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    Preparation of LIN 3: MC2-PEG4-Val-Ala-PABC-PNP

    [1174] ##STR00241##

    (a) Preparation of LIN 3-1: MC2-PEG4-Val-Ala-OH

    [1175] ##STR00242##

    [1176] Cl-TrtCl-resin (5 g, 1.49 mmol/g) was placed in a filter plate. To the resin was added CH.sub.2Cl.sub.2 (25 mL) and the mixture was stirred for 1 h at 23° C. The solvent was eliminated by filtration over vacuum. A solution of Fmoc-Ala-OH (2.31 g, 7.41 mmol) and DIPEA (4.28 mL, 24.61 mmol) in CH.sub.2Cl.sub.2 (20 mL) was added and the mixture was stirred for 10 min at 23° C. DIPEA (8.60 mL, 49.37 mmol) was additionally added and the reaction mixture was stirred for 1 h at 23° C. The reaction was stopped by addition of MeOH (10 mL) and stirred 15 min at 23° C. The Fmoc-Ala-O-TrtCl-resin was subjected to the following washing/treatments: CH.sub.2Cl.sub.2 (5 x 15 mL×0.5 min), DMF (5×15 mL×0.5 min), piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min), DMF (5×15 mL×0.5 min), CH.sub.2Cl.sub.2 (5×15 mL×0.5 min). The loading was calculated: 1.34 mmol/g.

    [1177] The NH.sub.2-Ala-O-TrtCl-resin was washed with DMF (5×15 mL×0.5 min) and a solution of Fmoc-Val-OH (9.09 g, 26.79 mmol) and HOBt (3.62 g, 26.79 mmol) in DMF (25 mL) was added to the NH.sub.2-Ala-O-TrtCl-resin followed by addition DIPCDI (4.14 mL, 26.79 mmol) at 23° C. The mixture was stirred for 1.5 h at 23° C. The reaction was stopped by washing with DMF (5×15 mL×0.5 min). The Fmoc-Val-Ala-O-TrtCl-resin was treated with piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min) and washed with DMF (5×15 mL×0.5 min).

    [1178] A solution of 15-(9-Fluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoic acid (Fmoc-NH-PEG4-OH) (4.90 g, 8.75 mmol) and HOBt (1.35 g, 9.98 mmol) in DMF (30 mL) was added to the NH.sub.2-Val-Ala-O-TrtCl-resin followed by addition DIPCDI (1.55 mL, 10.0 mmol) at 23° C. The reaction mixture was stirred for 22 h at 23° C. The reaction was stopped by washing with DMF (5×15 mL×0.5 min). The Fmoc-NH-PEG4-Val-Ala-O-TrtCl-resin was treated with piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min) and washed with DMF (5×15 mL×0.5 min).

    [1179] A solution of 3-(Maleimido)propionic acid (MC2-OH) (4.53 g, 26.78 mmol) and HOBt (3.62 g, 26.77 mmol) in DMF (30 mL) was added to the NH.sub.2-PEG4-Val-Ala-O-TrtCl-resin followed by addition of DIPCDI (4.15 mL, 26.80 mmol) at 23° C. The reaction mixture was stirred for 2 h at 23° C. The reaction was stopped by washing with DMF (5×15 mL×0.5 min) and CH.sub.2Cl.sub.2 (5 x 15 mL×0.5 min).

    [1180] The peptide was cleaved from the resin by treatments with TFA:CH.sub.2Cl.sub.2 (1:99, 5×50 mL). The resin was washed with CH.sub.2Cl.sub.2 (7×50 mL×0.5 min). The combined filtrates were evaporated to dryness under reduced pressure, the solid obtained was triturated with Et.sub.2O and filtrated to obtain L 3-1 (4.73 g, 87% yield) as a white solid.

    [1181] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 7.67 (bs, 1H), 7.31 (d, J=8.9 Hz, 1H), 7.17 (d, J=7.0 Hz, 1H), 6.85 (t, J=5.6 Hz, 1H), 6.72 (s, 2H), 4.51 (q, J=7.1 Hz, 1H), 4.38 (dd, J=8.9, 6.9 Hz, 1H), 3.84 (t, J=7.1 Hz, 2H), 3.75 (t, J=5.9 Hz, 2H), 3.69-3.59 (m, 12H), 3.55 (t, J=5.1 Hz, 2H), 3.41 (qd, J=5.0, 1.7 Hz, 2H), 2.62-2.49 (m, 4H), 2.19-2.01 (m, 1H), 1.44 (d, J=7.2 Hz, 3H), 0.95 (d, J=11.9 Hz, 1H), 0.94 (d, J=11.9 Hz, 1H).

    (b) Preparation of LIN 3-2: MC2-PEG4-Val-Ala-PABOH

    [1182] ##STR00243##

    [1183] To a solution of LIN 3-1 (1.84 g, 3.13 mmol) and 4-aminobenzyl alcohol (PABOH) (0.77 g, 6.27 mmol) in CH.sub.2Cl.sub.2 (70 mL) was added a solution of HOBt (0.84 g, 6.27 mmol) in DMF (5 mL) followed by addition of DIPCDI (0.97 mL, 6.27 mmol) at 23° C. The reaction mixture was stirred for 5 h at 23° C., Et.sub.2O (150 mL) was added, and the solid obtained was filtrated under vacuum to obtain crude LIN 3-2 (1.74 g, 81% yield) which was used in the next step without further purification.

    [1184] .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 7.58 (d, J=8.5 Hz, 2H), 7.30 (d, J=8.5 Hz, 2H), 6.81 (s, 2H), 4.56 (s, 2H), 4.52-4.41 (m, 1H), 4.21 (d, J=6.7 Hz, 1H), 3.91 (p, J=6.5 Hz, 1H), 3.81-3.67 (m, 4H), 3.65-3.54 (m, 12H), 3.49 (t, J=5.5 Hz, 2H), 2.56 (dd, J=6.6, 5.5 Hz, 2H), 2.46 (t, J=6.9 Hz, 2H), 2.12 (h, J=6.8 Hz, 1H), 1.45 (d, J=7.2 Hz, 3H), 1.00 (d, J=12.1 Hz, 3H), 0.98 (d, J=12.1 Hz, 3H).

    (c) Preparation of LIN 3: MC2-PEG4-Val-Ala-PABC-PNP

    [1185] ##STR00244##

    [1186] To a solution of LIN 3-2 (1.74 g, 2.51 mmol) and bis(4-nitrophenyl) carbonate (bis-PNP) (3.82 g, 12.57 mmol) in CH.sub.2Cl.sub.2:DMF (8:1, 70 mL) was added DIPEA (1.31 mL, 7.54 mmol) at 23° C. The reaction mixture was stirred for 20 h at 23° C. and poured onto silica gel column (CH.sub.2Cl.sub.2:CH.sub.3OH, from 50:1 to 10:1) to afford pure LIN 3 (1.26 g, 59% yield).

    [1187] .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.82 (s, 1H), 8.27 (d, J=9.2 Hz, 2H), 7.73 (d, J=8.6 Hz, 2H), 7.38 (d, J=9.1 Hz, 4H), 7.15 (dd, J=21.8, 7.2 Hz, 2H), 6.69 (s, 2H), 6.62 (t, J=5.7 Hz, 1H), 5.24 (s, 2H), 4.67 (p, J=7.2 Hz, 1H), 4.24 (dd, J=6.8, 5.7 Hz, 1H), 3.91-3.76 (m, 2H), 3.71 (ddd, J=10.1, 6.1, 4.3 Hz, 1H), 3.66-3.54 (m, 14H), 3.53 (t, J=5.1 Hz, 1H), 3.46-3.33 (m, 2H), 2.76-2.57 (m, 1H), 2.57-2.42 (m, 2H), 2.33-2.19 (m, 1H), 1.46 (d, J=7.1 Hz, 3H), 1.01 (d, J=12.1 Hz, 3H), 1.00 (d, J=12.1 Hz, 3H).

    [1188] .sup.13C NMR (75 MHz, CD3OD): δ 173.0, 172.1, 171.6 (×2), 170.7, 163.8, 155.7, 152.5, 145.4, 140.3, 138.9, 134.1, 130.4, 129.1, 125.6, 124.8, 121.9, 119.7, 115.1, 70.2, 70.1 (×3), 70.0, 69.9, 69.8, 69.0, 66.9, 59.1, 53.4, 49.7, 39.0, 36.0, 34.3, 34.1, 30.4, 18.3, 17.3, 16.6.

    [1189] ESI-MS m/z: 857.3 (M+H).sup.+.

    [1190] R.sub.f=0.45 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

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

    Preparation of Compound DL-1

    [1191] ##STR00245##

    [1192] To a solution of 11-R (100 mg, 0.12 mmol) and LIN 1 (465 mg, 0.63 mmol) in N-Methyl-2-pyrrolidone (NMP) (15 mL) was added N,N-diisopropylethylamine (DIPEA) (111 μL, 0.63 mmol) at 23° C. The reaction mixture was stirred for 3 days at 23° C., diluted with EtOAc (50 mL) and washed with H.sub.2O (4×30 mL) and a saturated aqueous solution of NaCl (30 mL). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 90:10) to obtain DL 1 which was purified by HPLC preparative to yield pure DL 1 (69 mg, 40% yield).

    [1193] .sup.1H NMR (400 MHz, CD3OD/CDCl.sub.3): δ 7.85 (d, J=7.9 Hz, 1H), 7.59 (d, J=8.1 Hz, 2H), 7.36 (d, J=8.2 Hz, 2H), 7.30 (d, J=7.9 Hz, 1H), 7.22 (d, J=8.2 Hz, 1H), 7.01 (t, J=7.6 Hz, 1H), 6.91 (t, J=7.5 Hz, 1H), 6.72 (s, 2H), 6.58 (s, 1H), 6.26 (s, 1H), 6.07 (s, 1H), 5.25-5.14 (m, 2H), 5.14-5.01 (m, 2H), 4.67 (bs, 1H), 4.51 (d, J=6.7 Hz, 2H), 4.28 (dd, J=16.2, 7.1 Hz, 4H), 4.21-4.05 (m, 3H), 3.71 (s, 3H), 3.51-3.40 (m, 2H), 3.36-3.32 (m, 2H), 3.23-2.99 (m, 2H), 2.99-2.72 (m, 2H), 2.65 (d, J=14.9 Hz, 2H), 2.28 (s, .3H), 2.25 (s, .3H), 2.10 (s, .3H), 2.04 (s, .3H), 1.96-1.83 (m, 1H), 1.80-1.68 (m, 2H), 1.65-1.50 (m, 10H), 1.35-1.23 (m, 2H), 0.95 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H).

    [1194] .sup.13C NMR (75 MHz, CD3OH/CDCl.sub.3): δ 174.9, 172.5, 171.5, 171.1, 170.7, 169.5, 160.8, 157.4, 148.6, 146.1, 143.6, 141.1, 140.9, 138.0, 136.8, 133.9, 132.6, 130.5, 129.8, 129.6, 128.6, 126.0, 121.6, 120.4, 119.8, 119.0, 118.6, 118.0, 117.8, 116.7, 113.5, 112.8, 110.9, 109.1, 102.3, 66.0, 63.1, 62.9, 61.6, 60.2, 59.9, 59.2, 59.1, 58.9, 54.6, 54.6, 53.5, 50.7, 45.3, 42.1, 40.5, 37.1, 35.3, 30.3, 29.1, 27.9, 26.3, 26.0, 25.0, 24.5, 23.6, 19.4, 18.6, 17.7, 15.1, 8.6.

    [1195] ESI-MS m/z: 1391.4 (M+H).sup.+.

    [1196] R.sub.f=0.40 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    Preparation of Compound DL 2

    [1197] ##STR00246##

    [1198] To a solution of 11-R (50 mg, 0.063 mmol) and LIN 2 (118 mg, 0.12 mmol) in CH.sub.2Cl.sub.2 (2 mL) was added N,N-diisopropylethylamine (DIPEA) (22 μL, 0.12 mmol) at 23° C. The reaction mixture was stirred for 18 h at 23° C. and poured onto silica gel column (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 90:10) to yield DL 2 which was purified by HPLC preparative to afford pure DL 2 (30 mg, 30% yield).

    [1199] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.63 (d, J=7.9 Hz, 1H), 7.59 (d, J=8.1 Hz, 2H), 7.39 (d, J=8.2 Hz, 2H), 7.30 (d, J=7.9 Hz, 1H), 7.23 (d, J=8.2 Hz, 1H), 7.01 (t, J=7.6 Hz, 1H), 6.92 (t, J=7.5 Hz, 1H), 6.76 (s, 2H), 6.58 (s, 1H), 6.28 (s, 1H), 6.09 (s, 1H), 5.25-5.14 (m, 2H), 5.14-5.01 (m, 2H), 4.67 (bs, 1H), 4.51 (d, J=6.7 Hz, 2H), 4.36-4.24 (m, 4H), 4.23-4.15 (m, 3H), 3.73 (s, 3H), 3.75-3.69 (m 3H), 3.58-3.50 (m, 14H), 3.51-3.40 (m, 2H), 3.36-3.32 (m, 2H), 3.23-3.05 (m, 2H), 2.99-2.88 (m, 2H), 2.68 (d, J=14.9 Hz, 2H), 2.56-2.41 (m, 2H), 2.29 (s, .3H), 2.27 (s, .3H), 2.10 (s, .3H), 2.05 (s, .3H), 1.96-1.83 (m, 1H), 1.80-1.68 (m, 2H), 1.65-1.50 (m, 10H), 1.35-1.23 (m, 2H), 0.96 (d, J=6.8 Hz, 3H), 0.95 (d, J=6.8 Hz, 3H).

    [1200] .sup.13C NMR (75 MHz, CD3OD): δ 173.0, 172.34, 171.6, 171.5 (×2), 170.8 (×2), 170.7, 169.4, 160.8, 157.4, 155.8, 148.7, 148.5, 146.2, 146.1, 143.7, 141.2, 141.1, 140.9, 138.4, 136.9, 134.0, 131.3, 129.8, 128.7, 128.5, 126.0, 121.5, 120.5, 119.9, 119.7, 118.5, 117.7, 112.7, 102.4, 70.1 (×5), 70.0 (×2), 69.9, 69.8, 69.7, 69.0, 68.9, 66.8, 61.6, 59.9, 59.2, 54.6, 54.0, 53.5, 40.4, 40.0, 39.0, 36.0, 35.1, 34.4, 34.1, 30.4, 29.0, 26.4, 24.6, 23.6, 19.3, 18.5, 17.4, 15.1, 8.4.

    [1201] ESI-MS m/z: 1596.6 (M+H).sup.+.

    [1202] R.sub.f=0.48 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    Preparation of Compound DL 3

    [1203] ##STR00247##

    [1204] To a solution of 11-R (50 mg, 0.063 mmol) and LIN 3 (108 mg, 0.12 mmol) in CH.sub.2Cl.sub.2 (2 mL) was added N,N-diisopropylethylamine (DIPEA) (22 μL, 0.12 mmol) at 23° C. The reaction mixture was stirred for 18 h at 23° C. and poured onto silica gel column (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 90:10) to yield DL 3 which was purified by HPLC preparative to afford pure DL 3 (25 mg, 26% yield).

    [1205] .sup.1H NMR (400 MHz, CD3OD): δ 7.64 (d, J=8.2 Hz, 2H), 7.39 (d, J=8.4 Hz, 2H), 7.30 (dt, J=7.8, 1.0 Hz, 1H), 7.23 (dt, J=8.4, 0.9 Hz, 1H), 7.01 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 6.91 (ddd, J=7.9, 7.0, 1.0 Hz, 1H), 6.76 (s, 2H), 6.61 (s, 1H), 6.28 (s, 1H), 6.10 (s, 1H), 5.26-5.15 (m, 1H), 5.07 (dd, J=12.1, 4.7 Hz, 2H), 4.66 (s, 1H), 4.57 (s, 1H), 4.47 (t, J=7.1 Hz, 1H), 4.36 (s, 1H), 4.32-4.22 (m, 2H), 4.18 (d, J=6.5 Hz, 2H), 3.75-3.68 (m, 6H), 3.72 (s, 3H), 3.59-3.46 (m, 8H), 3.44 (t, J=5.3 Hz, 2H), 3.33-3.25 (m, 10H), 3.15 (dd, J=9.7, 5.0 Hz, 1H), 2.95 (d, J=17.9 Hz, 1H), 2.81 (dd, J=18.0, 9.9 Hz, 2H), 2.73-2.59 (m, 2H), 2.52 (t, J=6.1 Hz, 2H), 2.43 (t, J=6.9 Hz, 2H), 2.29 (s, 3H), 2.27 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 1.45 (t, J=8.7 Hz, 2H), 0.99 (d, J=9.9 Hz, 3H), 0.97 (d, J=9.9 Hz, 3H).

    [1206] .sup.13C NMR (75 MHz, CD3OD): δ 173.1, 172.1, 172.1, 171.6, 171.5, 170.7, 169.4, 148.7, 146.1, 143.6, 140.9, 138.1, 136.9, 135.7, 134.0, 134.0, 132.7, 127.1, 126.0, 121.5, 120.5, 119.8, 119.7, 119.1, 118.5, 117.6, 113.4, 110.8, 105.8, 102.4, 99.9, 86.9, 70.1, 70.1, 70.1, 70.0, 70.0, 69.9, 69.8, 69.0, 67.7, 66.8, 65.9, 63.0, 62.0, 61.6, 60.1, 59.9, 59.2, 59.1, 54.7, 54.6, 50.8, 49.6, 42.1, 40.3, 40.0, 39.0, 36.0, 34.3, 34.1, 30.4, 28.8, 23.6, 19.2, 18.4, 17.3, 16.6, 14.9, 8.4.

    [1207] ESI-MS m/z: 1511.2 (M+H).sup.+.

    [1208] R.sub.f=0.50 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

    Preparation of Compound DL 4

    [1209] ##STR00248##

    [1210] To a solution of 46-R (26 mg, 0.032 mmol) and LIN 1 (48 mg, 0.64 mmol) in DMF (2 mL) was added N,N-diisopropylethylamine (DIPEA) (12 μL, 0.64 mmol) at 23° C. The reaction mixture was stirred for 18 hours at 23° C., diluted with EtOAc (50 mL) and washed with H.sub.2O (4×30 mL) and a saturated aqueous solution of NaCl (30 mL). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue was purified by HPLC preparative to yield pure DL 4 (14 mg, 31% yield).

    [1211] .sup.1H NMR (400 MHz, CD3OD): δ 7.88 (d, J=7.9 Hz, 1H), 7.60 (d, J=8.2 Hz, 2H), 7.39-7.36 (m, 3H), 7.20-7.11 (m, 3H), 6.74 (s, 2H), 6.57 (s, 1H), 6.24 (s, 1H), 6.08 (s, 1H), 5.30-5.05 (m, 2H), 4.64 (s, 1H), 4.50 (d, J=6.1 Hz, 2H), 4.34-4.27 (m, 4H), 4.21-4.13 (m, 3H), 3.70 (s, 3H), 3.45 (t, J=7.2 Hz, 2H), 3.19-3.06 (m, 4H), 2.93-2.76 (m, 2H), 2.70-2.56 (m, 2H), 2.29-2.19 (m, 4H), 2.25 (s, 3H), 2.29-2.11 (m, 2H), 2.11 (s, 3H), 2.06 (s, 3H), 1.93-1.72 (m, 2H), 1.64-1.52 (m, 9H), 1.32-1.26 (m, 3H), 0.96-0.93 (m, 8H).

    [1212] .sup.13C NMR (100 MHz, CD3OD): δ 173.4, 171.1, 169.7, 169.3, 169.1, 167.7, 159.3, 155.9, 153.5, 147.2, 147.1, 144.3, 142.1, 139.6, 139.2, 136.6, 132.5, 131.1, 129.1, 128.3, 127.2, 125.6, 122.7, 121.0, 119.2 (×2), 118.3, 117.5, 117.4, 112.2, 112.1, 111.7, 109.7, 100.8, 97.0, 64.6, 61.7, 60.0, 58.9, 58.4, 57.7 (×2), 57.4, 53.2, 53.1, 52.0, 48.9, 43.6, 40.5, 39.0, 35.6, 33.8, 28.8, 28.1, 27.6, 26.5, 24.6, 23.6, 22.6, 22.2, 21.5, 17.9, 17.1, 16.2, 13.6, 7.1.

    [1213] ESI-MS m/z: 1392.4 (M+H).sup.+.

    Preparation of Compound DL 5

    [1214] ##STR00249##

    [1215] To a solution of DL 3 (30 mg, 0.026 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 6 mL, 0.015 M) was added AgNO.sub.3 (132 mg, 0.79 mmol). After 18 h at 23° C., the reaction mixture was quenched with an aqueous solution of NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2 (×3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified in an automatic system for flash chromatography (SiO.sub.2, CH.sub.2Cl.sub.2:CH.sub.3OH, from 95:5 to 50:50) to obtain pure DL 5 (34 mg, 87%).

    [1216] .sup.1H NMR (400 MHz, CD3OD): 7.64 (d, J=8.4 Hz, 2H), 7.38 (d, J=8.3 Hz, 2H), 7.29 (d, J=7.8 Hz, 1H), 7.22 (d, J=8.1 Hz, 1H), 7.00 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 6.91 (ddd, J=8.0, 7.0, 1.1 Hz, 1H), 6.75 (s, 2H), 6.64 (s, 1H), 6.25 (d, J=1.4 Hz, 1H), 6.06 (d, J=1.4 Hz, 1H), 5.23-5.06 (m, 3H), 4.60 (s, 1H), 4.47 (q, J=7.1 Hz, 1H), 4.31 (dd, J=16.3, 4.1 Hz, 2H), 4.18 (d, J=6.6 Hz, 1H), 4.12 (d, J=11.6 Hz, 1H), 3.76-3.66 (m, 2H), 3.61 (d, J=5.3 Hz, 1H), 3.57-3.48 (m, 16H), 3.44 (t, J=7.2 Hz, 4H), 3.27 (t, J=5.3 Hz, 3H), 3.13 (dd, J=13.4, 6.7 Hz, 1H), 2.96 (d, J=18.0 Hz, 1H), 2.85 (dd, J=18.1, 9.3 Hz, 1H), 2.75-2.61 (m, 2H), 2.56-2.48 (m, 2H), 2.43 (t, J=6.9 Hz, 2H), 2.28 (s, 6H), 2.24 (s, 1H), 2.14 (s, 3H), 2.12-2.05 (m, 2H), 2.04 (s, 3H), 1.43 (d, J=7.2 Hz, 3H), 0.99 (d, J=6.8 Hz, 3H), 0.96 (d, J=6.8 Hz, 3H).

    [1217] .sup.13C NMR (100 MHz, CD3OD): δ 173.1, 172.1, 171.7, 171.6, 171.5, 170.7, 169.3, 162.5, 157.5, 148.9, 146.1, 144.1, 141.4, 140.9, 138.2, 136.9, 134.0, 132.7, 131.2, 129.7, 128.4, 126.0, 121.4, 120.1, 119.8, 119.7, 118.5, 117.6, 114.3, 112.5, 110.8, 102.3, 90.5, 70.1 (×2), 70.0 (×2), 69.9, 69.8, 69.0, 66.8, 65.9, 63.1, 60.2, 59.2, 59.1, 57.9, 55.6, 55.1, 53.6, 50.8, 49.6, 45.4, 42.1, 40.3, 39.5, 39.0, 35.9, 34.3, 34.0 (×2), 29.3, 24.7, 23.2, 19.1, 18.3, 17.3, 16.5, 15.0, 8.2.

    [1218] ESI-MS m/z: 1483.4 (M−H.sub.2O+H).sup.+.

    Preparation of Compound DL 6

    [1219] ##STR00250##

    [1220] To a solution of DL 1 (50 mg, 0.035 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 2.39 mL, 0.015 M) was added AgNO.sub.3 (181 mg, 1.07 mmol). After 18 h at 23° C., the reaction mixture was quenched with an aqueous solution of NaHCO.sub.3:NaCl (1:1) and extracted with CH.sub.2Cl.sub.2 (×3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by HPLC preparative to obtain pure DL 6 (23 mg, 47% yield).

    [1221] .sup.1H NMR (400 MHz, CD3OD): δ 7.62 (d, J=8.1 Hz, 2H), 7.41 (d, J=8.1 Hz, 2H), 7.39 (d, J=8.2 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 7.12 (ddd, J=8.0, 7.0, 0.9 Hz, 1H), 7.01 (ddd, J=8.0, 7.0, 0.9 Hz, 1H), 6.82 (s, 1H), 6.75 (s, 2H), 6.32 (d, J=1.3 Hz, 1H), 6.12 (d, J=1.3 Hz, 1H), 5.32 (d, J=11.8 Hz, 1H), 5.24-5.12 (m, 3H), 4.81 (m, 2H), 4.65 (s, 1H), 4.47 (s, 1H), 4.31 (d, J=11.6 Hz, 1H), 4.18 (d, J=9.0 Hz, 1H), 4.12 (d, J=7.4 Hz, 1H), 3.93-3.83 (m, 2H), 3.76 (s, 3H), 3.65 (d, J=12.3 Hz, 1H), 3.44 (t, J=7.1 Hz, 2H), 3.23-2.99 (m, 2H), 2.92 (d, J=15.9 Hz, 1H), 2.62 (s, 3H), 2.45 (d, J=15.5 Hz, 1H), 2.35 (s, 3H), 2.30 (s, 3H), 2.28 (d, J=13.3 Hz, 6H), 2.07 (s, 3H), 2.13-2.00 (m, 2H), 1.90 (m, 1H), 1.80-1.70 (m, 2H), 1.65-1.50 (m, 4H), 1.34-1.22 (m, 2H), 0.96 (d, J=6.8 Hz, 3H), 0.95 (d, J=6.8 Hz, 3H).

    [1222] .sup.13C NMR (100 MHz, CD3OD): δ 175.0, 172.6, 171.1, 170.9, 169.2, 169.1, 160.9, 157.7, 149.5, 147.0, 146.0, 144.8, 141.7, 141.2, 137.3, 136.8, 133.9, 132.5, 130.5, 129.8, 129.6, 128.5, 127.9, 122.6, 120.3, 119.8, 119.2, 118.4, 117.9, 116.7, 113.1, 112.8, 111.1, 108.4, 102.7, 101.5, 89.1, 66.2, 63.1, 62.9, 61.6, 59.3, 56.9, 56.1, 55.5, 54.7, 53.6, 50.7, 45.3, 42.2, 39.0, 38.9, 37.0, 35.1, 30.1, 29.3, 27.9, 26.5, 26.3, 26.0, 25.0, 23.1, 19.1, 18.4, 17.6, 15.2, 8.3.

    [1223] ESI-MS m/z: 1364.4 (M−H.sub.2O+H).sup.+.

    Preparation of Compound DL 7

    [1224] ##STR00251##

    [1225] To a solution of 12-R (100 mg, 0.12 mmol) and LIN 2 (180 mg, 0.19 mmol) in Dimethylformamide (DMF) (2 mL, 0.06 M) was added N,N-Diisopropylethylamine (DIPEA) (90 μL, 0.51 mmol) at 23° C. After 18 hours the reaction mixture was purified by HPLC preparative to yield pure DL 7 (125 mg, 62% yield).

    [1226] .sup.1H NMR (400 MHz, CD3OD): δ 7.65 (d, J=8.1 Hz, 2H), 7.41 (d, J=8.1 Hz, 2H), 7.39 (d, J=8.2 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 7.13 (ddd, J=8.2, 7.0, 1.1 Hz, 1H), 7.01 (ddd, J=8.2, 7.0, 1.1 Hz, 1H), 6.82 (s, 1H), 6.76 (s, 2H), 6.32 (d, J=1.3 Hz, 1H), 6.12 (d, J=1.3 Hz, 1H), 5.32 (d, J=11.8 Hz, 1H), 5.18 (d, J=5.7 Hz, 2H), 4.82 (m, 2H), 4.64 (d, J=2.8 Hz, 1H), 4.47 (s, 1H), 4.33 (dd, J=12.0, 2.1 Hz, 1H), 4.22-4.12 (m, 2H), 3.96 (bs, 1H), 3.88 (d, J=5.0 Hz, 1H), 3.78-3.67 (m, 8H), 3.60-3.52 (m, 14H), 3.56-3.41 (m, 3H), 3.27 (t, J=5.4 Hz, 2H), 3.22-3.04 (m, 4H), 2.93 (d, J=16.0 Hz, 1H), 2.70-2.58 (m, 3H), 2.55 (t, J=6.9 Hz, 2H), 2.43 (t, J=6.9 Hz, 2H), 2.36 (s, 3H), 2.30 (s, 3H), 2.15-2.07 (m, 1H), 2.06 (s, 3H), 2.02 (s, 2H), 1.95-1.87 (m, 1H), 1.80-1.70 (m, 1H), 1.65-1.50 (m, 2H), 0.97 (d, J=6.8 Hz, 3H), 0.96 (d, J=6.8 Hz, 3H).

    [1227] .sup.13C NMR (125 MHz, CD3OD): δ 173.1, 172.5, 171.6, 171.0, 170.7 (×2), 169.3, 168.5, 160.9, 160.0, 149.6, 147.1, 144.9, 141.8, 141.3, 138.2, 137.4, 134.1, 132.4, 128.6, 127.9, 125.2, 122.9, 120.2, 119.9, 119.4, 118.1 (×2), 117.1, 114.8, 113.2, 112.7, 111.3, 108.3, 102.7, 89.0, 70.2, 70.1 (×2), 70.0, 69.9, 69.8, 69.0, 66.9, 66.3, 65.5, 65.4, 61.9, 59.4, 56.9, 56.1, 55.4 (×2), 54.6, 53.7, 43.7, 42.4, 39.1, 39.0, 36.0, 34.4, 34.1, 31.4, 30.3, 28.9, 26.5, 23.1, 22.3, 19.2, 18.5, 17.5, 15.3, 13.2, 8.5.

    [1228] ESI-MS m/z: 1570.4 (M−H.sub.2O+H).sup.+.

    Preparation of Compound DL 8

    [1229] ##STR00252##

    [1230] To a solution of 11-S (30 mg, 0.037 mmol) and LIN 1 (56 mg, 0.075 mmol) in Dimethylformamide (DMF) (2 mL, 0.018 M) was added N,N-Diisopropylethylamine (DIPEA) (26 μL, 0.15 mmol) and 1-Hydroxybenzotriazole (HOBt, 10 mg, 0.075 mmol) at 23° C. After 18 hours the reaction mixture was purified by HPLC preparative to yield pure DL 8 (30 mg, 58% yield).

    [1231] .sup.1H NMR (400 MHz, CD3OD): δ 7.59 (d, J=8.1 Hz, 2H), 7.36 (d, J=8.2 Hz, 2H), 7.28 (d, J=7.9 Hz, 1H), 7.23 (d, J=8.2 Hz, 1H), 7.00 (t, J=7.6 Hz, 1H), 6.91 (t, J=7.5 Hz, 1H), 6.74 (s, 2H), 6.50 (s, 1H), 6.27 (s, 1H), 6.09 (s, 1H), 5.20-5.03 (m, 2H), 4.65 (bs, 1H), 4.54-4.46 (m, 1H), 4.43-4.37 (m, 1H), 4.34-4.30 (m, 1H), 4.17-4.12 (m, 1H), 3.75 (s, 3H), 3.45 (t, J=7.0 Hz, 4H), 3.32-3.23 (m, 2H), 3.38 (d, J=7.6 Hz, 2H), 3.23-2.99 (m, 2H), 3.21-2.97 (m, 3H), 2.94-2.83 (m, 3H), 2.61-2.53 (m, 2H), 2.48-2.34 (m, 2H), 2.28 (s, .3H), 2.27-2.22 (m, 1H), 2.21 (s, .3H), 2.11 (s, .3H), 2.08-2.02 (m, 1H), 1.99 (s, .3H), 1.91-1.82 (m, 1H), 1.77-1.68 (m, 1H), 1.65-1.50 (m, 6H), 1.31-1.24 (m, 2H), 0.94 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8 Hz, 3H).

    [1232] .sup.13C NMR (100 MHz, CD3OD): δ 174.9, 174.8, 172.5, 172.0, 171.2, 170.8, 169.4, 160.8, 157.5, 148.6, 146.0, 143.6, 140.9, 140.8, 138.0, 136.6, 136.5, 134.0, 132.7, 130.2, 129.9, 129.7, 128.7, 128.4, 126.3, 121.6, 120.5, 120.0, 119.8, 119.3, 118.7, 118.0, 117.7, 113.5, 112.9, 111.0, 107.8, 102.4, 65.9, 63.5, 61.4, 60.6, 59.7, 59.3, 59.2, 59.1, 58.8, 54.6, 54.6, 53.4, 44.8, 42.3, 40.6, 38.3, 37.1, 35.3 (×2), 30.3, 29.1, 28.0, 26.3, 26.0, 25.1, 24.0, 23.7, 19.5, 18.6, 17.7, 15.2, 8.6.

    [1233] ESI-MS m/z: 1391.4 (M+H).sup.+.

    Preparation of Compound DL 9

    [1234] ##STR00253##

    [1235] To a solution of 11-S (110 mg, 0.13 mmol) and LIN-3 (119 mg, 0.13 mmol) in Dimethylformamide (DMF) (4 mL, 0.032 M) was added N,N-Diisopropylethylamine (DIPEA) (97 μL, 0.55 mmol) and 1-Hydroxybenzotriazole (HOBt, 38 mg, 0.27 mmol) at 23° C. After 18 hours the reaction mixture was purified by HPLC preparative to yield pure DL 9 (120 mg, 57% yield).

    [1236] .sup.1H NMR (400 MHz, CD3OD): δ 7.62 (d, J=8.6 Hz, 2H), 7.36 (d, J=8.5 Hz, 2H), 7.28 (d, J=8.2 Hz, 1H), 7.23 (d, J=8.2 Hz, 1H), 7.00 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 6.91 (td, J=7.5, 7.0, 1.1 Hz, 1H), 6.75 (s, 2H), 6.50 (s, 1H), 6.26 (d, J=1.3 Hz, 1H), 6.08 (d, J=1.4 Hz, 1H), 5.19 (d, J=11.4 Hz, 1H), 5.09 (d, J=10.4 Hz, 2H), 4.65 (s, 1H), 4.48 (p, J=6.9 Hz, 1H), 4.42-4.38 (m, 2H), 4.34-4.30 (m, 2H), 4.22-4.14 (m, 1H), 3.78-3.64 (m, 5H), 3.61-3.50 (m, 8H), 3.45 (t, J=5.4 Hz, 2H), 3.38 (d, J=5.1 Hz, 1H), 3.33-3.23 (m, 3H), 3.02 (dd, J=13.5, 5.5 Hz, 1H), 2.89 (d, J=9.4 Hz, 2H), 2.62-2.33 (m, 7H), 2.27 (s, 3H), 2.22 (s, 3H), 2.12 (s, 3H), 2.15-2.05 (m, 1H), 1.99 (m, 3H), 1.42 (d, J=7.1 Hz, 3H), 0.97 (dd, J=6.8 Hz, 3H), 0.95 (dd, J=6.8 Hz, 3H).

    [1237] .sup.13C NMR (100 MHz, CD3OD): δ 173.1 (×2), 172.1, 171.9, 171.6, 171.5, 171.5, 170.7, 169.3, 148.6, 146.0, 143.6, 140.9, 140.8, 138.1, 136.5, 134.1, 132.8, 130.3, 129.8, 128.4, 126.3, 121.6, 120.5, 120.0, 119.7, 119.3, 118.7, 118.0, 117.7, 113.5, 112.9, 111.0, 107.8, 102.4, 70.2, 70.1 (×2), 70.0 (×2), 69.8, 69.1, 66.9, 65.9, 63.5, 61.4, 60.6, 59.7, 59.3, 59.2, 59.1, 58.8, 54.7, 54.6, 49.6, 42.3, 40.5, 39.1, 39.0, 36.0, 34.4 (×2), 34.1, 30.4, 24.0, 23.7, 19.4, 18.5, 17.4, 16.8, 15.1, 8.5.

    [1238] ESI-MS m/z: 1511.4 (M+H).sup.+.

    Preparation of Compound DL 10

    [1239] ##STR00254##

    [1240] To a solution of 12-S (30 mg, 0.058 mmol) and LIN 1 (98 mg, 0.13 mmol) in N-Methyl-2-pyrrolidone (NMP) (4 mL, 0.014 M) was added N,N-Diisopropylethylamine (DIPEA) (83 μL, 0.13 mmol) at 23° C. After 18 hours the reaction mixture was diluted with EtOAc (25 mL) and washed with H.sub.2O (4×25 mL) and an aqueous saturated solution of NaCl. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (CH.sub.2Cl.sub.2:CH.sub.3OH, from 99:1 to 90:10) and the compound obtained purified by HPLC preparative to yield pure DL 10 (11 mg, 21% yield).

    [1241] .sup.1H NMR (400 MHz, CD3OD): δ 7.61 (d, J=8.1 Hz, 2H), 7.39 (d, J=8.1 Hz, 2H), 7.35 (d, J=8.2 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 7.08 (ddd, J=8.0, 7.0, 0.9 Hz, 1H), 6.98 (ddd, J=8.0, 7.0, 0.9 Hz, 1H), 6.76 (s, 2H), 6.66 (s, 1H), 6.29 (d, J=1.3 Hz, 1H), 6.14 (d, J=1.3 Hz, 1H), 5.13 (q, J=12.3 Hz, 2H), 4.93-.4.81 (m, 3H), 4.69 (s, 1H), 4.48 (s, 1H), 4.23 (d, J=10.0 Hz, 1H), 4.13 (dd, J=7.5, 4.0 Hz, 1H), 3.90 (d, J=5.0, 1H), 3.76 (s, 3H), 3.49-3.41 (m, 2H), 3.40-3.27 (m, 1H), 3.28-3.24 (m, 4H), 3.22-3.05 (m, 4H), 2.80-2.65 (m, 3H), 2.63 (s, 3H), 2.29 (s, 3H), 2.28 (s, 3H), 2.13-2.00 (m, 2H), 2.03 (s, 3H), 1.93-1.83 (m, 1H), 1.79-1.69 (m, 2H), 1.65-1.51 (m, 6H), 1.34-1.22 (m, 2H), 0.96 (d, J=6.8 Hz, 3H), 0.95 (d, J=6.8 Hz, 3H).

    [1242] .sup.13C NMR (100 MHz, CD3OD): δ 174.9, 172.7, 172.6, 171.1, 170.8 (×2), 169.1, 160.9, 149.4, 146.6, 144.7, 142.3, 141.5, 141.0, 138.1, 136.8, 133.9, 133.5, 132.6, 128.5, 127.5, 125.9, 122.2, 120.9, 119.8, 117.8, 113.2, 113.0, 111.2, 107.3, 102.6, 89.2, 66.2, 61.7, 61.3, 60.2, 59.4, 59.3, 57.4, 56.9, 55.8, 55.7, 55.2, 54.8, 53.5, 42.0, 39.1, 37.1, 35.2, 31.7, 30.2, 29.4, 29.1, 29.0 (×2), 27.9, 26.4, 26.0, 25.0, 23.3, 22.4, 19.2, 18.5, 17.7, 15.2, 13.2, 8.5.

    [1243] ESI-MS m/z: 1364.4 (M−H.sub.2O+H).sup.+.

    Preparation of Compound DL 11

    [1244] ##STR00255##

    [1245] To a solution of DL 9 (90 mg, 0.059 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 4 mL, 0.015 M) was added AgNO.sub.3 (298 mg, 1.78 mmol). After 21 h at 23° C., the reaction mixture was quenched with an aqueous solution of NaHCO.sub.3:NaCl (1:1) and extracted with CH.sub.2Cl.sub.2 (×3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified in an automatic system for flash chromatography (SiO.sub.2, CH.sub.2Cl.sub.2:CH.sub.3OH, from 95:5 to 50:50) to obtain pure DL 11 (65 mg, 73% yield).

    [1246] .sup.1H NMR (400 MHz, CD3OD): δ 7.62 (d, J=8.6 Hz, 1H), 7.37 (d, J=8.5 Hz, 2H), 7.28 (d, J=7.8 Hz, 1H), 7.23 (d, J=8.2 Hz, 1H), 6.99 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 6.90 (ddd, J=8.0, 7.0, 1.1 Hz, 1H), 6.76 (s, 2H), 6.52 (s, 1H), 6.25 (d, J=1.3 Hz, 1H), 6.06 (d, J=1.3 Hz, 1H), 5.28 (d, J=11.4 Hz, 1H), 5.09 (d, J=10.4 Hz, 1H), 4.59 (s, 1H), 4.47 (q, J=7.1 Hz, 1H), 4.33 (dd, J=11.5, 1.9 Hz, 1H), 4.33-4.25 (m, 2H), 4.19 (d, J=6.6 Hz, 1H), 3.75 (s, 3H), 3.75-3.67 m, 3H), 3.61 (d, J=5.3 Hz, 1H), 3.58-3.51 (m, 12H), 3.45 (dd, J=10.6, 5.2 Hz, 2H), 3.34 (s, 3H), 3.27 (t, J=5.5 Hz, 3H), 3.04 (dd, J=13.4, 6.7 Hz, 1H), 2.88-2.82 (m, 3H), 2.64-2.35 (m, 7H), 2.27 (s, 3H), 2.23 (s, 3H), 2.17-2.04 (m, 1H), 2.09 (s, 3H), 2.01 (d, J=7.2 Hz, 1H), 1.98 (s, 3H), 1.42 (d, J=7.2 Hz, 3H), 0.98 (dd, J=6.8 Hz, 3H), 0.95 (dd, J=6.8 Hz, 3H).

    [1247] .sup.13C NMR (100 MHz, CD3OD): δ 173.1, 172.1, 172.0, 171.6, 171.5, 170.7, 169.4, 157.6, 148.6, 145.8, 143.7, 141.1, 140.8, 138.1, 136.5, 134.1, 132.8, 130.4, 130.0, 129.8, 128.4, 126.4, 126.2, 121.5, 120.9, 120.1, 119.8, 118.6, 117.7, 114.9, 112.4, 111.0, 107.7, 102.1, 91.0, 70.2, 70.1 (×2), 70.0 (×2), 69.8, 69.1, 66.9, 65.9, 63.5, 60.5, 59.3, 59.1, 58.2, 55.2, 54.7, 52.9, 49.6, 44.9, 42.6, 40.0, 39.0, 38.3, 36.0, 34.4, 34.1, 30.4, 29.4, 24.0, 23.3, 19.4, 18.5, 17.4, 16.8, 15.1, 8.4.

    [1248] ESI-MS m/z: 1483.4 (M−H.sub.2O+H).sup.+.

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

    [1249] 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.

    Example 2a Preparation of Anti-CD13 Monoclonal Antibody

    [1250] Anti-CD13 monoclonal antibodies were obtained following well known procedures commonly used in the art. Briefly BALB/c mice were immunized with human endothelial cells isolated from umbilical cord. To that end, 1.5E7 of the cells were injected to the mice intraperitoneally on days −45 and −30 and intravenously on day −3. On day 0 spleen from these animals were removed and spleen cells were fused with SP2 mouse myeloma cells at a ratio of 4:1 according to standard techniques to produce the hybridoma and distributed on 96-well tissue culture plates (Costar Corp., Cambridge, Mass.). After 2 weeks hybridoma culture supernatants were harvested and their reactivity against the cell line used in the immunization step was tested by flow cytometry. Positive supernatants were assayed by immunofluorescence staining the corresponding cells used as antigens. Hybridomas showing a specific staining, immunoprecipitation pattern and cell distribution were selected and cloned and subcloned by limiting dilution.

    [1251] Once the clones were selected, cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37° C. during 3-4 days until the medium turned pale yellow. At that point, two thirds of the medium volume were removed, centrifuged at 1,000×g for 10 min to pellet the cells and the supernatant was either centrifuged again for further cleaning at 3,000×g for 10 min or filtered through 22 μm pore size membranes. The clarified supernatant was subjected to precipitation with 55% saturation ammonium sulphate and the resulting pellet was resuspended in 100 mM Tris-HCl pH 7.8 (1 mL per 100 mL of the original clarified supernatant) and dialyzed at 4° C. for 16-24 h against 5 L of 100 mM Tris-HCl pH 7.8 with 150 mM NaCl, changing the dialyzing solution at least three times. The dialyzed material was finally loaded onto a Protein A-Sepharose column and the corresponding monoclonal antibody was eluted with 100 mM sodium citrate pH 3.0 or alternatively with 1M glycine pH 3.0. Those fractions containing the antibody were neutralized with 2M Tris-HCl pH 9.0 and finally dialyzed against PBS and stored at −80° C. until its use.

    Preparation of Antibody-Drug Conjugate ADC1 with Trastuzumab and DL 1

    (a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab

    [1252] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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 (13.9 mg/mL) was determined by measuring the absorbance at 280 nm.

    [1253] Trastuzumab solution (0.33 mL, 4.6 mg, 30.6 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 (17.5 μL, 87.6 nmol, 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 4.0.

    (b) Preparation of ADC 1

    [1254] To the solution of partially reduced Trastuzumab (140 μL, 1.4 mg, 9 nmol), N,N-Dimethylacetamide (DMA) was added (28.2 μL) followed by addition of a freshly prepared solution of DL 1 (10 mM in DMA, 6.8 μL, 67.5 nmol, 7.5 eq.) Upon addition of DL 1, the solution turned turbid. The conjugation reaction was stirred for 30 min at 20° C. and the turbidity vanished during the conjugation reaction. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5 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 1 was concentrated to a final concentration of 6.05 mg/mL as determined by UV and 232 μL (1.4 mg, 9.3 nmol, 103%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (89%).

    Preparation of Antibody-Drug Conjugate ADC 2 with Trastuzumab and DL 2

    (a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab

    [1255] Trastuzumab solution (0.33 mL, 4.6 mg, 30.6 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 (17.5 μL, 87.6 nmol, 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 3.9.

    (b) Preparation of ADC 2

    [1256] To the solution of partially reduced Trastuzumab (140 μL, 1.4 mg, 9 nmol), N,N-Dimethylacetamide (DMA) was added (28.2 μL) followed by addition of a freshly prepared solution of DL 2 (10 mM in DMA, 6.8 μL, 67.5 nmol, 7.5 eq.) Upon addition of DL 2 the solution was stirred for 30 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5 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 2 was concentrated to a final concentration of 5.19 mg/mL as determined by UV and 270 μL (1.4 mg, 9.3 nmol, 103%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (65%).

    Preparation of Antibody-Drug Conjugate ADC 3 with Trastuzumab and Compound DL 3

    (a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab

    [1257] Trastuzumab solution (0.33 mL, 4.6 mg, 30.6 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 (17.5 μL, 87.6 nmol, 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 3.9.

    (b) Preparation of ADC 3

    [1258] To the solution of partially reduced Trastuzumab (140 μL, 1.4 mg, 9 nmol), N,N-Dimethylacetamide (DMA) was added (28.2 μL) followed by addition of a freshly prepared solution of DL 3 (10 mM in DMA, 6.8 μL, 67.5 nmol, 7.5 eq.) Upon addition of DL 3 the solution was stirred for 30 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5 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 5.15 mg/mL as determined by UV and 280 μL (1.44 mg, 9.6 nmol, 107%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (93%).

    Preparation of Antibody-Drug Conjugate ADC 4 with Traut's-Modified Trastuzumab and Compound DL 1

    (a) Reaction of Trastuzumab with 2-iminothiolane hydrochloride (Traut's reagent) to give thiol-activated Trastuzumab

    [1259] Trastuzumab solution (0.65 mL, 9 mg, 60 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 (64.4 μL, 900 nmol, 15 eq.) and the reaction stirred for 2 h at 25° C. The mixture was buffer exchanged using two Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 1.2 mL (7.5 mg/mL). Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 7.9.

    (b) Preparation of ADC 4

    [1260] To the solution of thiol-activated Trastuzumab (300 μL, 2.25 mg, 15 nmol), DMA was added (59.8 μL) followed by addition of a freshly prepared solution of DL 1 (10 mM in DMA, 22.5 μL, 225 nmol, 15 eq.). Upon addition of DL 1, the solution turned turbid. 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 4 was concentrated to a final concentration of 3.49 mg/mL as determined by UV and 252 μL (0.88 mg, 5.86 nmol, 39%) ADC solution was obtained.

    Preparation of Antibody-Drug Conjugate ADC 5 with Trastuzumab and Compound DL 4

    (a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab

    [1261] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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.

    [1262] Trastuzumab solution (0.5 mL, 8.55 mg, 57 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 (24.5 μL, 122.4 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.

    (b) Preparation of ADC 5

    [1263] To the solution of partially reduced Trastuzumab (200 μL, 1.9 mg, 13.2 nmol), N,N-Dimethylacetamide (DMA) was added (42.1 μL) followed by addition of a freshly prepared solution of DL 4 (10 mM in DMA, 7.9 μL, 79.2 nmol, 6 eq.) Upon addition of DL 4, the solution turned turbid. The conjugation reaction was stirred for 30 min at 20° C. and the turbidity vanished during the conjugation reaction. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 7.9 μL, 79.2 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 5.30 mg/mL as determined by UV and 290 μL (1.54 mg, 1.0 nmol, 81%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (91%).

    Preparation of Antibody-Drug Conjugate ADC 6 with Trastuzumab and Compound DL 5

    (a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab

    [1264] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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.

    [1265] Trastuzumab solution (0.55 mL, 9.7 mg, 64.6 nmol) was diluted to a concentration of 12.8 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, 146 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.

    (b) Preparation of ADC 6

    [1266] To the solution of partially reduced Trastuzumab (140 μL, 1.8 mg, 12 nmol), N,N-Dimethylacetamide (DMA) was added (28.2 μL) followed by addition of a freshly prepared solution of DL 5 (10 mM in DMA, 6.8 μL, 67.5 nmol, 5.6 eq.) Upon addition of DL 5 the solution was stirred for 30 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5 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 6 was concentrated to a final concentration of 4.29 mg/mL as determined by UV and 320 μL (1.37 mg, 9.1 nmol, 76%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (83%).

    Preparation of Antibody-Drug Conjugate ADC 7 with Anti-CD13 and Compound DL 1

    (a) Partial Reduction of Anti-CD13 to Give Partially Reduced Anti-CD13

    [1267] Anti-CD13 solution (0.5 mL, 8.2 mg, 54.6 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 (31.9 μL, 159 nmol, 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 4.7.

    (b) Preparation of ADC 7

    [1268] To the solution of partially reduced Anti-CD13 (200 μL, 2.0 mg, 13.3 nmol), N,N-Dimethylacetamide (DMA) was added (40 μL) followed by addition of a freshly prepared solution of DL 1 (10 mM in DMA, 10 μL, 100 nmol, 7.5 eq.) Upon addition of DL 1 the solution was stirred for 30 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 10 μL, 100 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 7 was concentrated to a final concentration of 5.58 mg/mL as determined by UV and 350 μL (1.95 mg, 13 nmol, 98%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (90%).

    Preparation of Antibody-Drug Conjugate ADC 8 with Anti-CD13 and Compound DL 3

    (a) Partial Reduction of Anti-CD13 to Give Partially Reduced Anti-CD13

    [1269] Anti-CD13 solution (0.5 mL, 8.2 mg, 54.6 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 (31.9 μL, 159 nmol, 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 4.7.

    (b) Preparation of ADC 8

    [1270] To the solution of partially reduced Anti-CD13 (200 μL, 2.0 mg, 13.3 nmol), N,N-Dimethylacetamide (DMA) was added (40 μL) followed by addition of a freshly prepared solution of DL 3 (10 mM in DMA, 10 μL, 100 nmol, 7.5 eq.) Upon addition of DL 3 the solution was stirred for 30 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 10 μL, 100 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 8 was concentrated to a final concentration of 5.83 mg/mL as determined by UV and 380 μL (2.21 mg, 14.7 nmol, 111%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (94%).

    Preparation of Antibody-Drug Conjugate ADC 9 with Anti-CD13 and Compound DL 5

    (a) Partial Reduction of Anti-CD13 to Give Partially Reduced Anti-CD13

    [1271] Anti-CD13 solution (0.5 mL, 8.2 mg, 54.6 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 (31.9 μL, 159 nmol, 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 4.7.

    (b) Preparation of ADC 9

    [1272] To the solution of partially reduced Anti-CD13 (200 μL, 2.0 mg, 13.3 nmol), N,N-Dimethylacetamide (DMA) was added (40 μL) followed by addition of a freshly prepared solution of DL 5 (10 mM in DMA, 10 μL, 100 nmol, 7.5 eq.) Upon addition of DL 5 the solution was stirred for 30 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 10 μL, 100 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 9 was concentrated to a final concentration of 5.82 mg/mL as determined by UV and 380 μL (2.21 mg, 14.7 nmol, 111%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (89%).

    Preparation of Antibody-Drug Conjugate ADC 10 with Anti-CD13 and Compound DL 2

    (a) Partial Reduction of Anti-CD13 to Give Partially Reduced Anti-CD13

    [1273] Anti-CD13 solution (0.5 mL, 8.2 mg, 54.6 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 (31.9 μL, 159.6 nmol, 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 4.7.

    (b) Preparation of ADC 10

    [1274] To the solution of partially reduced Anti-CD13 (200 μL, 2 mg, 13.3 nmol), DMA was added (40 μL) followed by addition of a freshly prepared solution of DL 2 (10 mM in DMA, 10 μL, 100 nmol, 7.5 eq.). Upon addition of DL 2, the solution turned turbid. 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, 10 μL, 100 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 10 was concentrated to a final concentration of 6.61 mg/mL as determined by UV and 250 μL (1.65 mg, 11 nmol, 85%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (23%).

    Preparation of Antibody-Drug Conjugate ADC 11 with Trastuzumab and Compound DL 6

    (a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab

    [1275] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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 (13.9 mg/mL) was determined by measuring the absorbance at 280 nm.

    [1276] Trastuzumab solution (0.33 mL, 4.6 mg, 30.6 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 (17.5 μL, 87.6 nmol, 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 4.0.

    (b) Preparation of ADC 11

    [1277] To the solution of partially reduced Trastuzumab (140 μL, 1.4 mg, 9 nmol), DMA was added (28.2 μL) followed by addition of a freshly prepared solution of DL 6 (10 mM in DMA, 6.8 μL, 67.5 nmol, 7.5 eq.). Upon addition of DL 6, the solution turned turbid. 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, 6.8 μL, 67.5 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 11 was concentrated to a final concentration of 6.14 mg/mL as determined by UV and 218 μL (1.33 mg, 8.9 nmol, 99%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (38%).

    Preparation of Antibody-Drug Conjugate ADC 12 with Trastuzumab and Compound DL 7

    (a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab

    [1278] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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.

    [1279] Trastuzumab solution (0.5 mL, 8.5 mg, 57 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 (24.5 μL, 122.4 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.

    (b) Preparation of ADC 12

    [1280] To the solution of partially reduced Trastuzumab (200 μL, 2 mg, 13.2 nmol), DMA was added (42.1 μL) followed by addition of a freshly prepared solution of DL 7 (10 mM in DMA, 7.9 μL, 79 nmol, 6 eq.). Upon addition of DL 7, the solution turned turbid. 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, 7.9 μL, 79 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 12 was concentrated to a final concentration of 5.38 mg/mL as determined by UV and 270 μL (1.45 mg, 9.6 nmol, 72%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (76%).

    Preparation of Antibody-Drug Conjugate ADC 13 with Traut's Modified Trastuzumab and Compound DL 2

    (a) Reaction of Trastuzumab with 2-iminothiolane (Traut's Reagent) to give thiol-activated Trastuzumab

    [1281] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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.

    [1282] Trastuzumab solution (0.5 mL, 8.0 mg, 53.7 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 (14 mM, 46.0 μL, 644 nmol, 12 eq.), and the reaction stirred for 2 h at 20° C. The mixture was buffer exchanged using Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 0.8 mL. Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.4.

    (b) Preparation of ADC 13

    [1283] To the solution of thiol-activated Trastuzumab (200 μL, 2.0 mg, 13 nmol), DMA was added (37 μL) followed by addition of a freshly prepared solution of DL 2 (10 mM in DMA, 13 μL, 130 nmol, 10 eq.). Upon addition of DL 2, the solution turned turbid. 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 13 was concentrated to a final concentration of 2.83 mg/mL as determined by UV and 340 μL (0.96 mg, 6.4 nmol, 49%) ADC solution was obtained.

    Preparation of Antibody-Drug Conjugate ADC 14 with Traut's modified Trastuzumab and Compound DL 3

    (a) Reaction of Trastuzumab with 2-iminothiolane (Trauct's Reagent) to give thiol-activated Trastuzumab

    [1284] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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.

    [1285] Trastuzumab solution (0.5 mL, 8.0 mg, 53.7 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 (14 mM, 46.0 μL, 644 nmol, 12 eq.), and the reaction stirred for 2 h at 20° C. The mixture was buffer exchanged using Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 0.8 mL. Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.4.

    (b) Preparation of ADC 14

    [1286] To the solution of thiol-activated Trastuzumab (200 μL, 2.0 mg, 13 nmol), DMA was added (37 μL) followed by addition of a freshly prepared solution of DL 3 (10 mM in DMA, 13 μL, 130 nmol, 10 eq.). Upon addition of DL 3, the solution turned turbid. 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 14 was concentrated to a final concentration of 0.75 mg/mL as determined by UV and 380 μL (0.28 mg, 1.9 nmol, 15%) ADC solution was obtained.

    Preparation of Antibody-Drug Conjugate ADC 15 with Traut's modified Trastuzumab and Compound DL 5

    (a) Reaction of Trastuzumab with 2-Iminothiolane (Traut's Reagent) to Give Thiol-Activated Trastuzumab

    [1287] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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.

    [1288] Trastuzumab solution (0.5 mL, 8.0 mg, 53.7 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 (14 mM, 46.0 μL, 644 nmol, 12 eq.), and the reaction stirred for 2 h at 20° C. The mixture was buffer exchanged using Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 0.8 mL. Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.4.

    (b) Preparation of ADC 15

    [1289] To the solution of thiol-activated Trastuzumab (200 μL, 2.0 mg, 13 nmol), DMA was added (37 μL) followed by addition of a freshly prepared solution of DL 5 (10 mM in DMA, 13 μL, 130 nmol, 10 eq.). Upon addition of DL 5, the solution turned turbid. 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 15 was concentrated to a final concentration of 1.79 mg/mL as determined by UV and 440 μL (0.79 mg, 5.2 nmol, 40%) ADC solution was obtained.

    Preparation of Antibody-Drug Conjugate ADC 16 with Traut's modified Trastuzumab and Compound DL 6

    (a) Reaction of Trastuzumab with 2-iminothiolane hydrochloride (Traut's reagent) to give thiol-activated Trastuzumab

    [1290] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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.

    [1291] Trastuzumab solution (0.25 mL, 4.3 mg, 28.5 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 (14 mM, 24.4 μL, 342 nmol, 12 eq.), and the reaction stirred for 2 h at 20° C. The mixture was buffer exchanged using Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 0.43 mL. Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.6.

    (b) Preparation of ADC 16

    [1292] To the solution of thiol-activated Trastuzumab (200 μL, 2.0 mg, 13 nmol), DMA was added (37 μL) followed by addition of a freshly prepared solution of DL 6 (10 mM in DMA, 13 μL, 130 nmol, 10 eq.). Upon addition of DL 6, the solution turned turbid. 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 16 was concentrated to a final concentration of 5.63 mg/mL as determined by UV and 230 μL (1.29 mg, 8.6 nmol, 66%) ADC solution was obtained.

    Preparation of Antibody-Drug Conjugate ADC 17 with Traut's modified Trastuzumab and Compound DL 8

    (a) Reaction of Trastuzumab with 2-iminothiolane (Traut's reagent) to give thiol-activated Trastuzumab

    [1293] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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 (17.7 mg/mL) was determined by measuring the absorbance at 280 nm.

    [1294] Trastuzumab solution (1.5 mL, 26.5 mg, 177 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8) and fractionated in two vials (1.3 mL each). Traut's reagent was added (14 mM, 61.8 μL, 866 nmol, 10 eq.) in each vial, and the reactions stirred for 2 h at 20° C. The reactions were mixed and buffer exchanged using Sephadex G25 NAP-10 columns into PBS buffer, and concentrated to a volume of 2.6 mL. Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 5.6.

    (b) Preparation of ADC 17

    [1295] To the solution of thiol-activated Trastuzumab (500 μL, 5.0 mg, 33 nmol), DMA was added (98.6 μL) followed by addition of a freshly prepared solution of DL 8 (10 mM in DMA, 26.4 μL, 264 nmol, 8 eq.). Upon addition of DL 8, the solution turned turbid. 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 17 was concentrated to a final concentration of 3.21 mg/mL as determined by UV and 390 μL (1.25 mg, 8.3 nmol, 25%) ADC solution was obtained.

    Preparation of Antibody-Drug Conjugate ADC 18 with Traut's modified Trastuzumab and Compound DL 9

    (a) Reaction of Trastuzumab with 2-iminothiolane (Traut's reagent) to give thiol-activated Trastuzumab

    [1296] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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 (17.7 mg/mL) was determined by measuring the absorbance at 280 nm.

    [1297] Trastuzumab solution (1.5 mL, 26.5 mg, 177 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8) and fractionated in two vials (1.3 mL each). Traut's reagent was added (14 mM, 61.8 μL, 866 nmol, 10 eq.) in each vial, and the reactions stirred for 2 h at 20° C. The reactions were mixed and buffer exchanged using Sephadex G25 NAP-10 columns into PBS buffer, and concentrated to a volume of 2.6 mL. Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 5.6.

    (b) Preparation of ADC 18

    [1298] To the solution of thiol-activated Trastuzumab (500 μL, 5.0 mg, 33 nmol), DMA was added (98.6 μL) followed by addition of a freshly prepared solution of DL 9 (10 mM in DMA, 26.4 μL, 264 nmol, 8 eq.). Upon addition of DL 9, the solution turned turbid. 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 18 was concentrated to a final concentration of 3.16 mg/mL as determined by UV and 390 μL (1.23 mg, 8.2 nmol, 25%) ADC solution was obtained.

    Preparation of Antibody-Drug Conjugate ADC 19 with Traut's modified Trastuzumab and Compound DL 10

    (a) Reaction of Trastuzumab with 2-iminothiolane (Traut's reagent) to give thiol-activated Trastuzumab

    [1299] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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 (17.7 mg/mL) was determined by measuring the absorbance at 280 nm.

    [1300] Trastuzumab solution (1.5 mL, 26.5 mg, 177 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8) and fractionated in two vials (1.3 mL each). Traut's reagent was added (14 mM, 61.8 μL, 866 nmol, 10 eq.) in each vial, and the reactions stirred for 2 h at 20° C. The reactions were mixed and buffer exchanged using Sephadex G25 NAP-10 columns into PBS buffer, and concentrated to a volume of 2.6 mL. Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 5.6.

    (b) Preparation of ADC 19

    [1301] To the solution of thiol-activated Trastuzumab (500 μL, 5.0 mg, 33 nmol), DMA was added (98.6 μL) followed by addition of a freshly prepared solution of DL 10 (10 mM in DMA, 26.4 μL, 264 nmol, 8 eq.). Upon addition of DL 10, the solution turned turbid. 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 19 was concentrated to a final concentration of 11.3 mg/mL as determined by UV and 290 μL (3.2 mg, 21.3 nmol, 64%) ADC solution was obtained.

    Preparation of Antibody-Drug Conjugate ADC 20 with Traut's modified Trastuzumab and Compound DL 11

    (a) Reaction of Trastuzumab with 2-iminothiolane (Traut's reagent) to give thiol-activated Trastuzumab

    [1302] Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised 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 (17.7 mg/mL) was determined by measuring the absorbance at 280 nm.

    [1303] Trastuzumab solution (1.5 mL, 26.5 mg, 177 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8) and fractionated in two vials (1.3 mL each). Traut's reagent was added (14 mM, 61.8 μL, 866 nmol, 10 eq.) in each vial, and the reactions stirred for 2 h at 20° C. The reactions were mixed and buffer exchanged using Sephadex G25 NAP-10 columns into PBS buffer, and concentrated to a volume of 2.6 mL. Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 5.6.

    (b) Preparation of ADC 20

    [1304] To the solution of thiol-activated Trastuzumab (500 μL, 5.0 mg, 33 nmol), DMA was added (98.6 μL) followed by addition of a freshly prepared solution of DL 11 (10 mM in DMA, 26.4 μL, 264 nmol, 8 eq.). Upon addition of DL 11, the solution turned turbid. 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 20 was concentrated to a final concentration of 3.73 mg/mL as determined by UV and 440 μL (1.6 mg, 10.6 nmol, 32%) ADC solution was obtained. Example 3: Demonstrating the Cytotoxicity of the Antibody-Drug Conjugates of the Present Invention

    Bioassays for the Detection of Antitumor Activity

    [1305] 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.

    Cell Lines and Cell Culture

    [1306] The following human cell lines were obtained from the American Type Culture Collection (ATCC): SK-BR-3 (ATCC HB-30), HCC-1954 (ATCC CRL-2338) (Breast cancer, HER2+); MDA-MB-231 (ATCC HTB-26) and MCF-7 (ATCC HTB-22) (Breast cancer, HER2-), HT-1080 (ATCC CCL-121, fibrosarcoma, CD13+), Raji (ATCC CCL-86, Burkitt's lymphoma, CD13-) and RPMI 8226 (ATCC CRM-CCL-155, myeloma, CD13-). The human acute promyelocytic leukemia cell line NB 4 (ACC 207, CD13+) was obtained from the Leibniz-Institut DSMZ (Braunschweig, Germany). Cells were maintained at 37° C., 5% CO.sub.2 and 95% humidity in Dulbecco's Modified Eagle's Medium (DMEM) (for SK-BR-3, MDA-MB-231 and MCF-7 cells), Eagle's Minimum Essential Medium (EMEM) (for HT-1080 cells) or RPMI-1640 (for the rest of the cell lines), all media supplemented with 10% Fetal Calf Serum (FCS), 2 mM L-glutamine and 100 units/mL penicillin and streptomycin.

    Cytotoxicity Assay

    [1307] 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 in V. Vichai and K. Kirtikara. 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.

    [1308] For HT-1080, NB 4, Raji and RPMI-8226 cells, an alternative colorimetric assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used for quantitative measurement of cell viability as described by T. Mosmann in “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays”, J Immunol Methods, 1983, 65: 55-63. Briefly, cells were seeded in 96-well trays and treated as above and after 72 hours of exposure to the tested substances cellular viability was estimated from conversion of MTT (Sigma, St Louis, Mo., USA) to its coloured reaction product, MTT-formazan, which was dissolved in DMSO to measure its absorbance at 540 nm in a microplate spectrophotometer.

    [1309] Cell survival was expressed 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 curve from which the IC50 value (the concentration of compound causing 50% cell death as compared to the control cell survival) was calculated.

    Bioactivity Example 1—Cytotoxicity of the Conjugate ADC 1 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

    [1310] The in vitro cytotoxicity of the ADC 1 along with the parent cytotoxic compounds DL 1 and 11-R 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.

    Cytotoxicity of Trastuzumab

    [1311] 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 10 where results corresponding to the geometric mean of the IC.sub.50 values obtained in three independent experiments are presented.

    TABLE-US-00011 TABLE 10 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

    Cytotoxicity of 11-R

    [1312] The cytotoxicity of the intermediate compound 11-R 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 (1.26E-07-3.3E-11 M).

    [1313] As shown in Table 11, 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 low nanomolar range, from 0.4 to 1.4 ng/mL (5.04E-10 to 1.70E-09 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.79 ng/mL (9.94E-10 M), with the standard geometric deviation being 1.8 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00012 TABLE 11 Summary of the in vitro cytotoxicity of 11-R HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 5.76E−04 1.35E−03 3.99E−04 1.24E−03 IC.sub.50, M 7.27E−10 1.70E−09 5.04E−10 1.57E−09

    Cytotoxicity of DL 1

    [1314] The cytotoxicity of the intermediate compound DL 1 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 (7.58E-06-1.99E-09 M).

    [1315] As shown in Table 12, 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 high nanomolar range, from 0.07 to 0.43 μg/mL (5.23E-08 to 3.11 E-07 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.16 μg/mL (1.15E-07 M), with the standard geometric deviation being 2.1 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00013 TABLE 12 Summary of the in vitro cytotoxicity of DL 1 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 1.16E−01 1.80E−01 7.28E−02 4.33E−01 IC.sub.50, M 8.31E−08 1.29E−07 5.23E−08 3.11E−07

    Cytotoxicity of ADC 1

    [1316] 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). The evaluation was performed in three independent experiments, FIG. 3 shows a representative DR curve corresponding to one of these experiments and Table 13 summarizes the results corresponding to the geometric mean of the IC.sub.50 values obtained in the three independent experiments.

    [1317] As observed in Table 13, ADC 1 showed a cytotoxicity which is similar to that shown by the parent drug 11-R only in HER2-positive cells. However, in HER2-negative cells such toxicity is significantly lower: nearly 40-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-00014 TABLE 13 Summary of the in vitro cytotoxicity of ADC 1 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 3.20E−01 1.38E+00 1.36E+01 4.90E+01 6.64E−01 2.58E01  38.8 IC.sub.50 (M) 2.14E−09 9.20E−09 9.04E−08 3.27E−07 4.44E−09 1.72E−07

    [1318] To graphically compare the cytotoxicity of the monoclonal antibody Trastuzumab alone with that of the conjugate ADC 1, histograms showing the percentages of cell survival after treatment of the different cell lines with the monoclonal antibody alone (20 μg/mL) or ADC 1 at 16 or 2.5 μg/mL, are shown in FIG. 4. As observed, at similar concentrations (20 versus 16 μg/mL) Trastuzumab showed negligible cytotoxicity regardless the HER2 expression levels of the cell lines (cell survival between 68% and 100%), whilst the ADC 1 showed a potent antiproliferative effect against the HER2-expressing cells HCC-1954 and SK-BR-3 (cell survival 23% and 8% respectively) and negligible against the HER2-negative cell line MCF-7 (100% cell survival). The effect of the ADC on MDA-MB-231 is noticeable at this concentration (22% cell survival) but it is very modest at lower concentrations (83% cell survival at 2.5 μg/mL) whereas it is still remarkable in the HER2-expressing cells (30% cell survival for both SK-BR-3 and HCC-1954).

    [1319] These results clearly demonstrated the remarkable cytotoxicity and specificity of the ADC 1 against HER2 expressing human tumor cells in vitro.

    Bioactivity Example 2—Cytotoxicity of the Conjugate ADC 2 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

    [1320] The in vitro cytotoxicity of the ADC 2 along with the parent cytotoxic compound DL 2 was 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. The results are also compared with the parent cytotoxic compound 11-R and the monoclonal antibody Trastuzumab described above.

    Cytotoxicity of DL 2

    [1321] 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 (6.26E-06-1.64E-09 M).

    [1322] As shown in Table 14, the cytotoxicity of this compound was similar in all the tumor cell lines regardless of their HER2 expression, with IC50 values in the sub-micromolar range, from 0.2 to 0.47 μg/mL (1.25E-07 to 2.94E-07 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.28 μg/mL (1.73E-07 M), with the standard geometric deviation being 1.5 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00015 TABLE 14 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 2.00E−01 3.10E−01 2.00E−01 4.69E−01 IC.sub.50, M 1.25E−07 1.94E−07 1.25E−07 2.94E−07

    Cytotoxicity of ADC 2

    [1323] 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). FIG. 5 shows the DR curve corresponding to this experiment and Table 15 summarizes the IC.sub.50 values obtained.

    [1324] As observed in Table 15, ADC 2 showed higher cytotoxicity in HER2-positive cells than in HER2-negative cells: the ADC is nearly 10-fold more potent in the former according to the selectivity ratio obtained by dividing the mean IC.sub.50 value in HER2-negative cells between that in HER2-positive cells. Although this selectivity is rather modest, it still 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-00016 TABLE 15 Summary of the in vitro cytotoxicity of ADC 2 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 2.00E+00 5.60E+00 1.70E+01 4.01E+01 3.34E+00 2.61E+01 7.8 IC.sub.50 (M) 1.33E−08 3.73E−08 1.13E−07 2.67E−07 2.23E−08 1.74E−07

    [1325] To graphically compare the cytotoxicity of the monoclonal antibody Trastuzumab alone with that of the conjugate ADC 2, histograms showing the percentages of cell survival after treatment of the different cell lines with the monoclonal antibody alone (20 μg/mL) or ADC 2 at 16 or 2.5 μg/mL, are shown in FIG. 6. As observed, at similar concentrations (20 versus 16 μg/mL) Trastuzumab showed negligible cytotoxicity disregarding the HER2 expression levels of the cell lines (cell survival between 68% and 100%), whilst the ADC 2 showed a significant antiproliferative effect against the HER2-expressing cells HCC-1954 and SK-BR-3 (cell survival 31% and 40% respectively), such effect being negligible against the HER2-negative cell line MCF-7 (94% cell survival). The effect of the ADC on the HER2-negative cell line MDA-MB-231 is also significant at this concentration, (58% cell survival) but it is null at lower concentrations (100% cell survival at 2.5 μg/mL) whereas it is still remarkable in the HER2-expressing cells (63% cell survival for HCC-1954 and 51% for SK-BR-3).

    Bioactivity Example 3—Cytotoxicity of the Conjugate ADC 3 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

    [1326] The in vitro cytotoxicity of the ADC 3 along with the parent cytotoxic compound DL 3 was 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. The results are also compared with the parent cytotoxic compound 11-R and the monoclonal antibody Trastuzumab described above.

    Cytotoxicity of DL 3

    [1327] The cytotoxicity of the intermediate compound DL 3 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 (6.62E-06-1.74E-09 M).

    [1328] As shown in Table 16, the cytotoxicity of this compound was similar in all the tumor cell lines regardless of their HER2 expression, with IC50 values in the sub-micromolar range, from 0.15 to 0.28 μg/mL (9.93E-08 to 1.85E-07 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.2 μg/mL (1.33E-07 M), with the standard geometric deviation being 1.4 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00017 TABLE 16 Summary of the in vitro cytotoxicity of DL 3 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 1.60E−01 2.40E−01 1.50E−01 2.79E−01 IC.sub.50, M 1.06E−07 1.59E−07 9.93E−08 1.85E−07

    Cytotoxicity of ADC 3

    [1329] The cytotoxicity of the ADC 3 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 50 μg/mL to 13 ng/mL (3.33E-07-8.74E-11 M). FIG. 7 shows the DR curve corresponding to this experiment and Table 17 summarizes the IC.sub.50 values obtained.

    [1330] As observed in Table 17, ADC 3 showed a cytotoxicity which only in HER2-positive cells is similar to that shown by the parent drug 11-R. However, in HER2-negative cells such toxicity is significantly lower: 23-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 as previously stated for ADC 1 and ADC 2.

    TABLE-US-00018 TABLE 17 Summary of the in vitro cytotoxicity of ADC 3 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 6.71E−01 8.10E−01 1.30E+01 2.21E+01 7.37E−01 1.70E+01 23.0 IC.sub.50 (M) 4.47E−09 5.40E−09 8.67E−08 1.47E−07 4.91E−09 1.13E−07

    [1331] To graphically compare the cytotoxicity of the monoclonal antibody Trastuzumab alone with that of the conjugate ADC 3, histograms showing the percentages of cell survival after treatment of the different cell lines with the monoclonal antibody alone (20 μg/mL) or ADC 3 at 20 or 3 μg/mL, are shown in FIG. 8. As observed, at 20 μg/mL Trastuzumab showed negligible cytotoxicity regardless the HER2 expression levels of the cell lines (cell survival between 68% and 100%), whilst the ADC 3 showed a potent antiproliferative effect against the HER2-expressing cells HCC-1954 and SK-BR-3 (cell survival 23% and 32% respectively) and only marginal against the HER2-negative cell line MCF-7 (88% cell survival). The effect of the ADC on MDA-MB-231 is noticeable at this concentration (32% cell survival) but it is negligible at lower concentrations (91% cell survival at 3 μg/mL) whereas it is still remarkable in the HER2-expressing cells (34% cell survival for HCC-1954 and 45% for SK-BR-3).

    Bioactivity Example 4—Cytotoxicity of the Conjugate ADC 4 Against HER2 Positive and Negative Breast Cancer Cells

    [1332] The in vitro cytotoxicity of the ADC ADC 4 was 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 ADC were performed. The results are also compared with the parent cytotoxic compounds 11-R and DL 1 as well as the monoclonal antibody Trastuzumab described above.

    Cytotoxicity of ADC 4

    [1333] 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 50 μg/mL to 13 ng/mL (3.33E-07-8.74E-11 M). FIG. 9 shows the DR curve corresponding to this experiment and Table 18 summarizes the IC50 values obtained.

    [1334] As observed in Table 18, ADC 4 showed a cytotoxicity which only in HER2-positive cells is similar to that shown by the parent drug 11-R. However, in HER2-negative cells such cytotoxicity is nearly 100-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. As already stated for ADC 1, such 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. Moreover, these results obtained with ADC 4 demonstrate that the antitumoral potential endowed by the payload is similar regardless of the antibody residue involved in the covalent bond, either Cys or Lys.

    TABLE-US-00019 TABLE 18 Summary of the in vitro cytotoxicity of ADC 4 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 3.20E−02 1.50E−01 3.50E+00 8.10E+00 6.92E−02 5.32E+00 76.86 IC.sub.50 (M) 2.13E−10 1.00E−09 2.33E−08 5.40E−08 4.62E−10 3.55E−08

    [1335] To graphically compare the cytotoxicity of the monoclonal antibody Trastuzumab alone with that of the conjugate ADC 4, histograms showing the percentages of cell survival after treatment of the different cell lines with the monoclonal antibody alone at 3 μg/mL or with the ADC at 3 or 0.2 μg/mL, are shown in FIG. 10. As observed, at 3 μg/mL Trastuzumab showed scarce cytotoxicity irrespective of the HER2 expression levels of the cell lines (cell survival between 60% and 90%), whilst the ADC 4 showed a potent antiproliferative effect against the HER2-expressing cells HCC-1954 and SK-BR-3 (cell survival 11% and 23% respectively) and nearly no effect on the HER2-negative cell line MCF-7. The effect of the ADC on the cell line MDA-MB-231 is noticeable at this concentration (55% cell survival) but it is negligible at lower concentrations (97-100% cell survival at 0.2 μg/mL) whereas it is clearly remarkable in the HER2-expressing cells (38% cell survival for HCC-1954 and 26% for SK-BR-3).

    Bioactivity Example 5—Cytotoxicity of the Conjugate ADC 6 and Related Reagents Against Her2 Positive and Negative Breast Cancer Cells

    [1336] The in vitro cytotoxicity of the ADC 6 along with the parent cytotoxic compounds DL 5 and 12 R and 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.

    Cytotoxicity of 12-R

    [1337] The cytotoxicity of the intermediate compound 12-R 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 (1.28E-07-3.83E-11 M).

    [1338] As shown in Table 19, where results corresponding to the geometric mean of the IC50 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 low nanomolar range, from 0.4 to 1.3 ng/mL (5.62E-10 to 1.62E-09 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.71 ng/mL (9.06E-10 M), with the standard geometric deviation being 1.7 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00020 TABLE 19 Summary of the in vitro cytotoxicity of 12-R HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 4.41E−04 1.27E−03 4.93E−04 9.24E−04 IC.sub.50, M 5.62E−10 1.62E−09 6.29E−10 1.18E−09

    Cytotoxicity of DL 5

    [1339] The cytotoxicity of the intermediate compound DL 5 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 (6.66E-06-1.73E-09 M).

    [1340] As shown in Table 20, 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, from 0.10 to 0.19 μg/mL (6.53E-08 to 1.27E-07 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.13 μg/mL (8.84E-08 M), with the standard geometric deviation being 1.4 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00021 TABLE 20 Summary of the in vitro cytotoxicity of DL 5 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 9.81E−02 1.91E−01 9.81E−02 1.70E−01 IC.sub.50, M 6.53E−08 1.27E−07 6.53E−08 1.13E−07

    Cytotoxicity of ADC 6

    [1341] The cytotoxicity of the ADC 6 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). FIG. 11 shows the DR curve obtained and Table 21 summarizes the deduced IC.sub.50 values.

    [1342] As observed in Table 21, the cytotoxicity of ADC 6 is comparable to that shown by the parent drug 12-R only in HER2-positive cells. However, in HER2-negative cells such toxicity is lower: nearly 10-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. These data 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-00022 TABLE 21 Summary of the in vitro cytotoxicity of ADC 6 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 6.50E−01 4.10E+00 1.20E+01 1.70E+01 1.63E00  1.43E01  8.8 IC.sub.50 (M) 4.33E−09 2.73E−08 8.00E−08 1.13E−07 1.08E−08 9.51E−08

    Bioactivity Example 6—Cytotoxicity of the Conjugate ADC 11 and Related Reagents Against Her2 Positive and Negative Breast Cancer Cells

    [1343] The in vitro cytotoxicity of the ADC 11 along with the parent cytotoxic compounds DL 6 and 12 R and 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.

    Cytotoxicity of DL 6

    [1344] The cytotoxicity of the intermediate compound DL 6 was evaluated against the different tumor cell lines by performing triplicated 10-points, 2.5-fold dilution DR curves ranging from 1 μg/mL to 0.26 ng/mL (7.23E-07-1.88E-10 M).

    [1345] As shown in Table 22, the cytotoxicity of this compound was similar in all the tumor cell lines regardless of their HER2 expression, with IC50 values in the high nanomolar range, from 0.04 to 0.3 μg/mL (3.1 E-08 to 1.97E-07 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.11 μg/mL (7.89E-08 M), with the standard geometric deviation being 2.31 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00023 TABLE 22 Summary of the in vitro cytotoxicity of DL 6 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, μg/mL 7.09E−02 1.71E−01 4.29E−02 2.73E−01 IC.sub.50, M 5.13E−08 1.24E−07 3.10E−08 1.97E−07

    Cytotoxicity of ADC 11

    [1346] The cytotoxicity of the ADC 11 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 50 μg/mL to 13 ng/mL (3.33E-07-8.74E-11 M). Table 23 summarizes the deduced IC.sub.50 values.

    TABLE-US-00024 TABLE 23 Summary of the in vitro cytotoxicity of ADC 11 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 1.10E+01 3.60E+01 >5.00E+01 >5.00E+01 1.99E+01 >5.00E+01 >2.5 IC.sub.50 (M) 7.33E−08 2.40E−07 >3.33E−07 >3.33E−07 1.33E−07 >3.33E−07

    Bioactivity Example 7—Cytotoxicity of the Conjugate ADC 12 Against Her2 Positive and Negative Breast Cancer Cells

    [1347] The in vitro cytotoxicity of the ADC 12 was 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.

    [1348] The cytotoxicity of the ADC 12 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 24 summarizes the deduced IC50 values. As observed, ADC 12 is significantly more active in in HER2-positive cells, yielding a noticeable selectivity ratio higher than 20.

    TABLE-US-00025 TABLE 24 Summary of the in vitro cytotoxicity of ADC 12 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 1.80E−01 2.90E−01 1.91E+00 1.30E+01 2.28E−01 4.98E+00 21.8 IC.sub.50 (M) 1.20E−09 1.93E−09 1.27E−08 8.67E−08 1.52E−09 3.32E−08

    Bioactivity Example 8—Cytotoxicity of the Conjugate ADC 13 Against Her2 Positive and Negative Breast Cancer Cells

    [1349] The cytotoxicity of the ADC 13 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 25 summarizes the deduced IC.sub.50 values. As observed, ADC 13 is significantly more active in HER2-positive cells, yielding an outstanding selectivity ratio close to 100.

    TABLE-US-00026 TABLE 25 Summary of the in vitro cytotoxicity of ADC 13 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 1.50E−01 2.60E−01 1.10E+01 2.90E+01 1.97E−01 1.78E+01 90.43 IC.sub.50 (M) 1.00E−09 1.73E−09 7.33E−08 1.93E−07 1.32E−09 1.19E−07

    Bioactivity Example 9—Cytotoxicity of the Conjugate ADC 14 Against Her2 Positive and Negative Breast Cancer Cells

    [1350] The cytotoxicity of the ADC 14 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 26 summarizes the deduced IC50 values. Remarkably, ADC 14 has shown a cytotoxic activity in the low nM range comparable to that of the parental drug 11R but only in HER2-expressing cells, whilst it has failed to show any activity in HER2-negative cells within the range of concentrations tested, thus confirming an outstanding selectivity.

    TABLE-US-00027 TABLE 26 Summary of the in vitro cytotoxicity of ADC 14 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 1.10E−01 1.80E−01 >2.51E+01 >2.51E+01 1.41E−01 >2.51E+01 >178 IC.sub.50 (M) 7.33E−10 1.20E−09 >1.67E−07 >1.67E−07 9.38E−10 >1.67E−07

    Bioactivity Example 10—Cytotoxicity of the Conjugate ADC 15 Against Her2 Positive and Negative Breast Cancer Cells

    [1351] The cytotoxicity of the ADC 15 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 50 μg/mL to 13 ng/mL (3.33E-07-8.74E-11 M). Table 27 summarizes the deduced IC.sub.50 values. As observed, ADC 15 is significantly more active in in HER2-positive cells, yielding an outstanding selectivity ratio close to 200.

    TABLE-US-00028 TABLE 27 Summary of the in vitro cytotoxicity of ADC 15 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 8.00E−02 1.80E−01 2.10E+01 2.21E+01 1.20E−01 2.15E+01 179.4 IC.sub.50 (M) 5.33E−10 1.20E−09 1.40E−07 1.47E−07 8.00E−10 1.43E−07

    Bioactivity Example 11—Cytotoxicity of the Conjugate ADC 16 Against Her2 Positive and Negative Breast Cancer Cells

    [1352] The cytotoxicity of the ADC 16 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 28 summarizes the deduced IC.sub.50 values. As observed, ADC 16 is significantly more active in in HER2-positive cells, yielding an outstanding selectivity ratio above 100.

    TABLE-US-00029 TABLE 28 Summary of the in vitro cytotoxicity of ADC 16 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 4.01E−02 8.00E−01 1.30E+01 2.70E+01 1.79E−01 1.87E+01 104.7 IC.sub.50 (M) 2.67E−10 5.33E−09 8.67E−08 1.80E−07 1.19E−09 1.25E−07

    Bioactivity Example 12—Cytotoxicity of the Conjugate ADC 17 Against Her2 Positive and Negative Breast Cancer Cells

    [1353] The cytotoxicity of the ADC 17 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 29 summarizes the deduced IC.sub.50 values.

    TABLE-US-00030 TABLE 29 Summary of the in vitro cytotoxicity of ADC 17 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 7.40E+00 1.40E+01 4.40E+01 4.20E+01 1.02E+01 4.30E+01 4.22 IC.sub.50 (M) 4.93E−08 9.33E−08 2.93E−07 2.80E−07 6.78E−08 2.86E−07

    Bioactivity Example 13—Cytotoxicity of the Conjugate ADC 18 Against Her2 Positive and Negative Breast Cancer Cells

    [1354] The cytotoxicity of the ADC 18 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 30 summarizes the deduced IC.sub.50 values.

    TABLE-US-00031 TABLE 30 Summary of the in vitro cytotoxicity of ADC 18 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 4.40E+00 2.21E+01 4.31E+01 9.71E+01 9.84E+00 6.46E+01 6.57 IC.sub.50 (M) 2.93E−08 1.47E−07 2.87E−07 6.47E−07 6.56E−08 4.31E−07

    Bioactivity Example 14—Cytotoxicity of the Conjugate ADC 19 Against Her2 Positive and Negative Breast Cancer Cells

    [1355] The cytotoxicity of the ADC 19 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 31 summarizes the deduced IC.sub.50 values.

    TABLE-US-00032 TABLE 31 Summary of the in vitro cytotoxicity of ADC 19 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 8.30E+00 3.41E+01 >1.00E+02 >1.00E+02 1.68E+01 >1.00E+02 >5.95 IC.sub.50 (M) 5.53E−08 2.27E−07 >6.67E−07 >6.67E−07 1.12E−07 >6.67E−07

    Bioactivity Example 15—Cytotoxicity of the Conjugate ADC 20 Against Her2 Positive and Negative Breast Cancer Cells

    [1356] The cytotoxicity of the ADC 20 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 32 summarizes the deduced IC.sub.50 values.

    TABLE-US-00033 TABLE 32 Summary of the in vitro cytotoxicity of ADC 20 IC50 in IC50 in HER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC.sub.50 (μg/mL) 2.21E+00 1.61E+01 5.40E+01 4.01E+01 5.95E+00 4.65E+01 7.82 IC.sub.50 (M) 1.47E−08 1.07E−07 3.60E−07 2.67E−07 3.97E−08 3.10E−07

    Bioactivity Example 16—Cytotoxicity of the Conjugate ADC 7 and Related Reagents Against CD13 Positive and Negative Cancer Cells

    Cytotoxicity of 11-R

    [1357] The cytotoxicity of the intermediate compound 11-R 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 0.03 ng/mL (1.26E-07-3.3E-11 M).

    [1358] As shown in Table 33, where results corresponding to the geometric mean of the IC50 values obtained in three different experiments are presented, the cytotoxicity of this compound was similar in all the tumor cell lines regardless of their CD13 expression, with IC.sub.50 values in the low nanomolar range, from 0.5 to 1.2 ng/mL (5.8E-10 to 1.51 E-09 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.84 ng/mL (1.06E-09 M) with the standard geometric deviation being 1.5 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00034 TABLE 33 Summary of the in vitro cytotoxicity of 11-R CD13 positive CD13 negative HT-1080 NB-4 Raji RPMI8226 IC.sub.50, μg/mL 1.20E−03 4.60E−04 9.67E−04 9.28E−04 IC.sub.50, M 1.51E−09 5.80E−10 1.22E−09 1.17E−09

    Cytotoxicity of ADC 7

    [1359] The cytotoxicity of the ADC 7 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 34 summarizes the deduced IC.sub.50 values. Remarkably, ADC 7 has shown a cytotoxic activity in the low nM range comparable to that of the parental drug 11-R but only in CD13-expressing cells, whereas its activity in CD13-negative cells is rather modest. Consequently, an outstanding selectivity fair above 100 can be observed as a function of CD13 expression.

    TABLE-US-00035 TABLE 34 Summary of the in vitro cytotoxicity of ADC 7 IC50 in IC50 in CD13+ CD13− CD13 positive CD13 negative (geom. (geom. Selec. HT-1080 NB-4 Raji RPMI8226 mean) mean ratio IC.sub.50 (μg/mL) 1.30E−01 2.70E−01 2.21E+01 4.10E+01 1.87E−01 3.00E+01 160.4 IC.sub.50 (M) 8.67E−10 1.80E−09 1.47E−07 2.73E−07 1.25E−09 2.00E−07

    Bioactivity Example 17—Cytotoxicity of the Conjugate ADC 8 Against CD13 Positive and Negative Cancer Cells

    [1360] The cytotoxicity of the ADC 8 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 35 summarizes the deduced IC50 values. ADC 8 has shown a cytotoxic activity in the low nM range comparable to that of the parental drug 11-R but only in CD13-expressing cells, whereas its activity in CD13-negative cells is rather modest. Consequently, an outstanding selectivity close to 200 was obtained as a function of CD13 expression.

    TABLE-US-00036 TABLE 35 Summary of the in vitro cytotoxicity of ADC 8 IC50 in IC50 in CD13+ CD13− CD13 positive CD13 negative (geom. (geom. Selec. HT-1080 NB-4 Raji RPMI8226 mean) Mean) ratio IC.sub.50 (μg/mL) 1.61E−01 2.51E−01 4.31E+01 3.30E+01 2.01E−01 3.77E+01 188 IC.sub.50 (M) 1.07E−09 1.67E−09 2.87E−07 2.20E−07 1.34E−09 2.51E−07

    Bioactivity Example 18—Cytotoxicity of the Conjugate ADC 9 and Related Reagents Against Cd13 Positive and Negative Cancer Cells

    Cytotoxicity of 12-R

    [1361] The cytotoxicity of the intermediate compound 12-R 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 (1.28E-07-3.83E-11 M).

    [1362] As shown in Table 36, 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 CD13 expression, with IC.sub.50 values in the low nanomolar range, from 0.3 to 1.1 ng/mL (4.21E-10 to 1.40E-09 M). The geometric mean IC.sub.50 value across the whole cell panel was 0.6 ng/mL (7.8E-10 M), with the standard geometric deviation being 1.7 in agreement with the homogeneity of results across the four cell lines.

    TABLE-US-00037 TABLE 36 Summary of the in vitro cytotoxicity of 12-R. CD13 positive CD13 negative HT-1080 NB-4 Raji RPMI8226 IC.sub.50, μg/mL 1.10E−03 3.30E−04 4.70E−04 8.30E−04 IC.sub.50, M 1.40E−09 4.21E−10 6.00E−10 1.06E−09

    Cytotoxicity of ADC-9

    [1363] The cytotoxicity of the ADC 9 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 37 summarizes the deduced IC.sub.50 values. ADC 9 has shown a cytotoxic activity in the low nM range comparable to that of the parental drug 12-R but only in CD13-expressing cells, whereas its activity in CD13-negative cells is rather modest. Consequently, an outstanding selectivity ratio above 200 was yielded as a function of CD13 expression.

    TABLE-US-00038 TABLE 37 Summary of the in vitro cytotoxicity of ADC 9 IC50 in IC50 in CD13+ CD13− CD13 positive CD13 negative (geom. (geom. Selec. HT-1080 NB-4 Raji RPMI8226 mean) Mean) ratio IC.sub.50 (μg/mL) 1.20E−01 5.21E−02 1.70E+01 1.70E+01 7.90E−02 1.70E+01 214 IC.sub.50 (M) 8.00E−10 3.47E−10 1.13E−07 1.13E−07 5.27E−10 1.13E−07

    Bioactivity Example 19—Cytotoxicity of the Conjugate ADC 10 Against CD13 Positive and Negative Cancer Cells

    [1364] The cytotoxicity of the ADC 10 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 38 summarizes the deduced IC.sub.50 values. ADC 10 has shown a cytotoxic activity in the low nM range comparable to that of the parental drug 11-R but only in CD13-expressing cells, whereas its activity in CD13-negative cells is rather modest. Consequently, a remarkable selectivity close to 100 was obtained as a function of CD13 expression.

    TABLE-US-00039 TABLE 38 Summary of the in vitro cytotoxicity of ADC 10 IC50 in IC50 in CD13+ CD13− CD13 positive CD13 negative (geom. (geom. Selec. HT1080 NB-4 Raji RPMI 8226 mean) mean ratio IC.sub.50 (μg/mL) 9.71E−02 9.71E−01 3.30E+01 2.81E+01 3.07E−01 3.04E+01 99.14 IC.sub.50 (M) 6.47E−10 6.47E−09 2.20E−07 1.87E−07 2.05E−09 2.03E−07

    Example 4: Demonstrating the In Vivo Efficacy of the Antibody-Drug Conjugates of the Present Invention

    [1365] Trastuzumab-based antibody drug conjugate ADC 1 was tested in several in vivo models. ADC-1 batches used in these studies were prepared using 2.2 eq of TCEP (ADC 1 2.2 TCEP) or 3 eq of TCEP (ADC-1), These batches were manufactured using the procedures described above with the exception that the final purification by size exclusion chromatography (SEC) was carried out using a Hi Load 26/600 Superdex™ 200 μg column and PBS (pH 7.4) as eluent.

    [1366] ADC-1 and ADC 1 2.2 TCEP were evaluated in a breast HER2 positive model, BT-474 together with their payload, compound 11-R. Of note, in spite of the low dose (suboptimal) administered to mice bearing tumors in this experiment, encouraging positive result was obtained (See FIG. 12). Therefore, a set of new experiments, aimed at evaluating the antitumor activity in breast and non-breast Her2 positive models were performed. ADC-1 was evaluated at a higher dose level in the breast tumor model, JIMT-1 (See FIG. 13), in one ovarian tumor model, SK-OV-3 (FIG. 14) and in a gastric model, Gastric-008, a patient derived xenograft (PDX), (FIG. 16). Additionally ADC-1 2.2 TCEP) was evaluated in another gastric model, N87 (FIG. 15).

    [1367] Briefly, 4 to 6 week-old athymic nu/nu (N87, Gastric-008 or SK-OV-3) or SCID (BT-474 or JIMT-1) mice were subcutaneously implanted with either tumor cell suspension (JIMT-1 or N87) or tumor fragments (BT-474, Gastric-008 or SK-OV3) previously generated in donor mice.

    [1368] Tumor dimension and body weight was 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. 2,000 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.

    [1369] The animals were implanted as above described and when tumors reached ca. 150-200 mm.sup.3, tumor bearing animals (N=8-10/group) were randomly allocated into treatment groups according to the following experimental design:

    TABLE-US-00040 Tumor Group Dose (mg/kg) JIMT-1 Control 0.0 11-R 5.0 ADC-1 30.0 ADC-1 10.0 ADC-1 5.0 N87 Control 0.0 11-R 5.0 ADC-1 (2.2 TCEP) 30.0 ADC-1 (2.2 TCEP) 10.0 ADC-1 (2.2 TCEP) 5.0 SK-OV-3 Control 0.0 11-R 5.0 ADC-1 30.0 ADC-1 10.0 ADC-1 5.0 Gastric-008 Control 0.0 11-R 5.0 ADC-1 30.0 ADC-1 10.0 ADC-1 5.0 BT-474 Control 0.0 11-R 5.0 ADC-1 (2.2 TCEP) 6.5 ADC-1 2.24 ADC-1 (2.2 TCEP) 1.6