DRUG ANTIBODY CONJUGATES

Abstract

Drug conjugates having formula [D-(X).sub.b-(AA).sub.w-(T).sub.g-(L)-].sub.n-Ab wherein: D is a drug moiety having the following formula (I) or a pharmaceutically acceptable salt, 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.

##STR00001##

Claims

1-119. (canceled)

120. 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)-(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, ##STR00128## wherein: the wavy line indicates the point of covalent attachment to (X).sub.b if any, or (AA), if any, or to (T).sub.g if any, or to (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 hydrogen, CH.sub.2OH, CH.sub.2OC(?O)R.sub.c, CH.sub.2NH.sub.2, and CH.sub.2NHProt.sup.NH; 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; R.sub.c is selected from substituted or unsubstituted C.sub.1-C.sub.12 alkyl, substituted or unsubstituted C.sub.2-C.sub.12alkenyl, and substituted or unsubstituted C.sub.2-C.sub.12 alkynyl; and Prot.sup.NH is a protecting group for amino; 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.

121. The drug conjugate according to claim 120, wherein D is a compound of formula: ##STR00129## or a pharmaceutically acceptable salt or ester thereof.

122. The drug conjugate according to claim 120, wherein D is a compound of formula: ##STR00130## or a pharmaceutically acceptable salt or ester thereof.

123. The drug conjugate according to claim 120, 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.

124. The drug conjugate according to claim 120, wherein L is a linker group selected from the group consisting of: ##STR00131## 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), 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-Cis 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-Cis arylene-C.sub.1-C.sub.12 alkylene- wherein the arylene group is in one or more rings which may optionally be substituted with one or more substituents R.sub.x, C.sub.1-C.sub.12 alkylene-(C.sub.3-C.sub.8 carbocyclo)-, (C.sub.3-C.sub.8 carbocyclo)-C.sub.1-C.sub.12 alkylene-, C.sub.5-C.sub.14 heterocyclo- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, C.sub.1-C.sub.12 alkylene-(C.sub.5-C.sub.14 heterocyclo)- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, (C.sub.5-C.sub.14 heterocyclo)-C.sub.1-C.sub.12 alkylene- wherein said heterocyclo group may be a saturated or unsaturated group having one or more rings and comprising at least one oxygen, nitrogen or sulphur atom in said ring(s), said group optionally being substituted with one or more substituents R.sub.x, (OCH.sub.2CH.sub.2).sub.r and CH.sub.2(OCH.sub.2CH.sub.2).sub.r, wherein each of the above alkylene substituents whether alone or attached to another moiety the carbon chain may optionally be substituted by one or more substituents R.sub.x; R.sub.30 is a C.sub.1-C.sub.6 alkylene- group; M is selected from the group consisting of C.sub.1-C.sub.6 alkylene-, C.sub.1-C.sub.6 alkylene-(C.sub.3-C.sub.8 carbocyclo)-, (CH.sub.2CH.sub.2O).sub.s, C.sub.1-C.sub.6 alkylene-(C.sub.3-C.sub.8 carbocyclo)-CON(H or C.sub.1-C.sub.6 alkyl)-C.sub.1-C.sub.6 alkylene-, phenylene which may optionally be substituted with one or more substituents R.sub.x, phenylene-C.sub.1-C.sub.6 alkylene- wherein the phenylene moiety may optionally be substituted with one or more substituents R.sub.x and C.sub.1-C.sub.6 alkylene-CON(H or C.sub.1-C.sub.6 alkyl)C.sub.1-C.sub.6 alkylene-; Q is selected from the group consisting of N(H or C.sub.1-C.sub.6 alkyl)phenylene- and N(H or C.sub.1-C.sub.6 alkyl)-(CH.sub.2).sub.s; r is an integer ranging from 1 to 10; and s is an integer ranging from 1 to 10; or wherein L is a linker group selected from the group consisting of: ##STR00132## 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), 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.8 carbocyclo)- and phenylene which may optionally be substituted with one or more substituents R.sub.x; and r is an integer ranging from 1-6.

125. The drug conjugate according to claim 120, selected from the formulas (IV), (V) and (VI): ##STR00133## 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)-(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)-; or selected from the formulas (IV), (V) and (VI): ##STR00134## 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; 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.7 carbocyclo)-.

126. The drug conjugate according to claim 120, wherein (AA), is of formula (II): ##STR00135## 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, ##STR00136## and w is an integer ranging from 0 to 12; or wherein (AA), 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; or wherein w is 0 or 2, and where w is 2, then (AA), is of formula (III): ##STR00137## 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.

127. The drug conjugate according to claim 120, wherein X is an extending group selected from: where D is covalently attached via an amine group: COO(C.sub.1-C.sub.6 alkylene)NH; COOCH.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)NHCOOCH.sub.2-(phenylene which may optionally be substituted with one or more substituents R.sub.x)NH; COCH.sub.2NHCOCH.sub.2NH; 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: CONH(C.sub.1-C.sub.6 alkylene)NH; COOCH.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)NHCOOCH.sub.2-(phenylene which may optionally be substituted with one or more substituents R.sub.x)NH; COCH.sub.2NHCOCH.sub.2NH; COCH.sub.2NH; CONH(C.sub.1-C.sub.6 alkylene)S; CONH(C.sub.1-C.sub.6 alkylene)NHCO(C.sub.1-C.sub.6 alkylene)S; and b is 0 or 1; or wherein X is an extending group selected from the group consisting of: where D is covalently attached via an amine group: COO(C.sub.2-C.sub.4 alkylene)NH; COOCH.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)NHCOOCH.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.2NHCOCH.sub.2NH; 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: CONH(C.sub.2-C.sub.4 alkylene)NH; COOCH.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)NHCOOCH.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.2NHCOCH.sub.2NH; CONH(C.sub.2-C.sub.4 alkylene)S; CONH(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S; and b is 0 or 1; or wherein X is an extending group selected from the group consisting of: where D is covalently attached via an amine group: COOCH.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.3S; COO(CH.sub.2).sub.3NHCO(CH.sub.2).sub.2S; or where D is covalently attached via an hydroxy group: COOCH.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.3S; CONH(CH.sub.2).sub.3NHCO(CH.sub.2).sub.2S; and b is 0 or 1.

128. The drug conjugate according to claim 120, wherein T is an extending group selected from the group consisting of CO(C.sub.1-C.sub.6 alkylene)-NH, CO(C.sub.1-C.sub.6 alkylene)-[O(C.sub.2-C.sub.6 alkylene)].sub.jNH, COO(C.sub.1-C.sub.6 alkylene)-[O(C.sub.2-C.sub.6 alkylene)].sub.jNH; where j is an integer from 1 to 25, and g is 0 or 1; or wherein T is an extending group selected from the group consisting of CO(C.sub.1-C.sub.4 alkylene)NH, CO(C.sub.1-C.sub.4 alkylene)-[O(C.sub.2-C.sub.4 alkylene)].sub.jNH, COO(C.sub.1-C.sub.4 alkylene)-[O(C.sub.2-C.sub.4 alkylene)].sub.jNH, where j is an integer from 1 to 10; and g is 0 or 1; or wherein T is an extending group selected from the group consisting of CO(C.sub.1-C.sub.4 alkylene)NH, CO(C.sub.1-C.sub.4 alkylene)-[O(C.sub.2-C.sub.4 alkylene)].sub.jNH, COO(C.sub.1-C.sub.4 alkylene)-[O(C.sub.2-C.sub.4 alkylene)].sub.jNH; where j is an integer from 1 to 5; and g is 0 or 1.

129. The drug conjugate according to claim 120, wherein D is a drug moiety of formula (IH) 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, R.sub.4 is selected from hydrogen, CH.sub.2OH, and CH.sub.2NH.sub.2; and Y is NH or O; or wherein D is a drug moiety of formula (IH) 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; R.sub.4 is hydrogen or CH.sub.2OH; and Y is NH or O; or wherein D is a drug moiety of formula (IH), 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 methoxy; R.sub.4 is hydrogen; and Y is NH or O.

130. The drug conjugate according to claim 120, wherein D is selected from: ##STR00138## 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), if any, or to (T).sub.g if any, or to (L).

131. The drug conjugate according to claim 120, wherein the moiety Ab comprising at least one antigen binding site is an antigen-binding peptide; including wherein the moiety Ab comprising at least one antigen binding site is an antibody, a single domain antibody or an antigen-binding fragment thereof; or wherein the moiety Ab comprising at least one antigen binding site is a monoclonal antibody, polyclonal antibody or bispecific antibody and/or wherein the antibody or an antigen-binding fragment thereof is derived from a human, mouse or rabbit; or wherein the moiety Ab comprising at least one antigen binding site 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; or wherein the moiety Ab comprising at least one antigen binding site 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; or wherein the moiety Ab comprising at least one antigen binding site is a monoclonal antibody which immunospecifically binds to cancer cell antigens, viral antigens, antigens of cells that produce autoimmune antibodies associated with autoimmune disease, or microbial antigens; or wherein the moiety Ab comprising at least one antigen binding site is an antibody selected from the group consisting of Alemtuzumab, Anetumab, Atezolizumab, Avelumab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab, Coltuximab, 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, Panitumumab, Pembrolizumab, Pertuzumab, Pinatuzumab, Polatuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab, Siltuximab, Sirtratumab, Sofituzumab, Vadastuximab, Vorsetuzumab, Trastuzumab or other an anti-HER2 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; or wherein the moiety Ab comprising at least one antigen binding site is an antibody selected from the group consisting of Alemtuzumab, Anetumab, Atezolizumab, Avelumab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab, 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, Panitumumab, Pembrolizumab, Pertuzumab, Polatuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab, Siltuximab, Sirtratumab, Vadastuximab, Vorsetuzumab, Trastuzumab or other an anti-HER2 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; or wherein the moiety Ab comprising at least one antigen binding site is an antibody selected from the group consisting of Alemtuzumab, Atezolizumab, Avelumab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab, Daratumumab, Denosumab, Dinutuximab, Durvalumab, Elotuzumab, Gemtuzumab, Ibritumomab, Inotuzumab, Ipilimumab, Labetuzumab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Panitumumab, Pembrolizumab, Pertuzumab, Ramucirumab, Rovalpituzumab, Siltuximab, Trastuzumab or another anti-HER2 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD30 antibody, or an antigen-binding fragment or an immunologically active portion thereof; or wherein the moiety Ab comprising at least one antigen binding site is an aptamer, including a nucleic acid or a peptide aptamer.

132. The drug conjugate according to claim 120, that is an antibody drug conjugate selected from the group consisting of: ##STR00139## ##STR00140## wherein n is from 2 to 6 and each and is independently selected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab or another an anti-HER2 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD30 antibody, or an antigen-binding fragment or an immunologically active portion thereof.

133. The drug conjugate according to claim 120, that is an antibody drug conjugate in isolated or purified form.

134. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 or of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-H, wherein: L.sub.1 is a linker selected from the group of formulas consisting of: ##STR00141## wherein each of the the wavy lines indicates the point of covalent attachment to (T).sub.g if any, or (AA), 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 OC(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-Cis 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; wherein for compounds of formula D-(X-).sub.b(AA).sub.w-(T).sub.g-H, b+w+g?0; each of D, R.sub.x, X, T, and AA is as defined in claim 120; including wherein the compound of formula D-X-(AA).sub.w-(T).sub.g-L.sub.1 is selected from: ##STR00142##

135. A compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-L.sub.1 or of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-H, wherein each of D, X, AA, T, L.sub.1, b, g and w are as defined in claim 120; but further wherein if the compound is a compound of formula D-(X).sub.b-(AA).sub.w-(T).sub.g-H then b+w+g?0.

136. The drug conjugate according to claim 120, wherein b+g+w is not 0; or wherein b+w is not 0; or wherein when w is not 0, then b is 1; or wherein when w is 0, then b is 1.

137. The compound according to claim 134, wherein b+g+w is not 0; or wherein b+w is not 0; or wherein when w is not 0, then b is 1; or wherein when w is 0, then b is 1.

138. The compound according to claim 135, wherein b+g+w is not 0; or wherein b+w is not 0; or wherein when w is not 0, then b is 1; or wherein when w is 0, then b is 1.

139. The drug conjugate according to claim 120, wherein, unless otherwise defined, if substituted, substituted groups are substituted with one or more substituents R.sub.x that are independently 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.12alkyl 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).

140. The compound according to claim 134, wherein, unless otherwise defined, if substituted, substituted groups are substituted with one or more substituents R.sub.x that are independently 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).

141. The compound according to claim 135, wherein, unless otherwise defined, if substituted, substituted groups are substituted with one or more substituents R.sub.x that are independently 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).

142. A method of manufacturing an antibody drug conjugate comprising utilizing a drug moiety D as defined in claim 120 or a compound according to claim 134 or 135.

143. A drug conjugate according to claim 120, for use as a medicament.

144. A method of treating cancer comprising administering a therapeutically effective amount of a drug conjugate according to claim 120 to a patient in need thereof; wherein the cancer includes lung cancer including NSCLC, gastric cancer, colorectal cancer, breast cancer, pancreas carcinoma, endometrial cancer, bladder cancer, cervical cancer, esophageal cancer, gallbladder cancer, uterine cancer, salivary duct cancer, ovarian cancer, kidney cancer, leukaemia, multiple myeloma, and lymphoma; optionally wherein the cancer is a HER2 positive cancer or a HER2 positive lung cancer including HER2 positive NSCLC, HER2 positive gastric cancer, HER2 positive colorectal cancer, HER2 positive breast cancer, HER2 positive pancreas carcinoma, HER2 positive endometrial cancer, HER2 positive bladder cancer, HER2 positive cervical cancer, HER2 positive esophageal cancer, HER2 positive gallbladder cancer, HER2 positive uterine cancer, HER2 positive salivary duct cancer or HER2 positive ovarian cancer.

145. A pharmaceutical composition comprising the drug conjugate according to claim 120 and a pharmaceutically acceptable carrier.

146. The drug conjugate according to claim 120, wherein n is in the range of 1-12, 1-8, 3-8, 3-6, or 3-5 or n is 1, 2, 3, 4, 5 or 6.

147. A process for the preparation of a drug antibody conjugate according to claim 120, comprising conjugating a moiety Ab comprising at least one antigen binding site and a drug D, Ab and D being as defined in claim 120; including wherein the preparation of a drug antibody conjugate of formula (G) or (G): ##STR00143## said process comprising the following steps: (i) reacting a drug D-H of formula (IH)-H: ##STR00144## wherein the substituents in the definitions of (IH)-H are as defined in claim 120 with a compound of formula (D) or (E): ##STR00145## to give a compound of formula (F) or (F), respectively: ##STR00146## (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: ##STR00147## 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: ##STR00148##

148. A compound of formula (IA) ##STR00149## 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 hydrogen, CH.sub.2OH, CH.sub.2OC(?O)Re, CH.sub.2NH.sub.2 and CH.sub.2NHProt.sup.NH; 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.12alkenyl, and substituted or unsubstituted C.sub.2-C.sub.12 alkynyl; R.sub.c 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; and Prot.sup.NH is a protecting group for amino; with the proviso that when R.sub.4 is hydrogen, then Y is O.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[1055] The invention is diagrammatically illustrated, by way of example, in the accompanying drawings in which:

[1056] FIG. 1 is a schematic illustration of one process according to the present invention wherein conjugation to the antibody is via free thiol groups;

[1057] FIG. 2 is a schematic illustration of one process according to the present invention wherein conjugation to the antibody is via free amino groups.

EXAMPLES

[1058] The present invention is further illustrated by way of the following, non-limiting examples. In the examples, the following abbreviations are used: [1059] CDI, 1,1-Carbonyldiimidazole [1060] DIPEA, N,N-Diisopropylethylamine [1061] Hex, Hexane [1062] EtOAc, Ethyl acetate [1063] DCM, Dichloromethane [1064] NMP, N-Methyl-2-pyrrolidone [1065] DMF, Dimethylformamide [1066] EDC, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride [1067] EDTA, Ethylenediaminetetraacetic acid [1068] MeOH, Methanol [1069] DTT, Dithiothreitol [1070] Py, Pyridine [1071] THF, Tetrahydrofuran [1072] TCEP, Tris[2-carboxyethyl]phosphine hydrochloride [1073] MC, 6-Maleimidocaproyl [1074] Fmoc, 9-Fluorenylmethoxycarbonyl [1075] Cit, Citrulline [1076] Val, Valine [1077] DMSO, Dimethylsulfoxide [1078] Trt, Triphenylmethyl [1079] HOBt, 1-Hydroxybenzotriazole [1080] DIPCDI, N,N-Diisopropylcarbodiimide [1081] TFA, Trifluoroacetic acid [1082] PABOH, 4-Aminobenzyl alcohol [1083] bis-PNP, bis(4-Nitrophenyl) carbonate [1084] NAC, N-Acetylcysteine [1085] SEC, Size-Exclusion Chromatography [1086] HPLC, High Performance Liquid Chromatography [1087] ADC, Antibody Drug Conjugate [1088] ATCC, American Type Culture Collection [1089] DMEM, Dulbecco's Modified Eagle's Medium [1090] RPMI, Rosmell Park Memorial Institute Medium [1091] ITS, Insulin-transferrin-sodium selenite media supplement [1092] FCS, Fetal Calf Serum [1093] SRB, Sulforhodamine B [1094] PBS, Phosphate Buffered Saline [1095] DR, Dose-Response [1096] UV, Ultraviolet [1097] SMCC, Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate [1098] LAR, Linker to Antibody Ratio

Example 1. Synthesis of Drugs

[1099] Compounds 1 and 2 were obtained following the procedures described in WO2003066638 (Examples 69 and 65, respectively, at pages 112-116).

[1100] Compound 4 was obtained following the procedure described in WO2003066638 (Example 12, at pages 61-62).

[1101] Compounds 8-S and 8-R were obtained following the procedure described in WO2018197663 (Example 8, at pages 97-98).

[1102] Compound 16-S was obtained following the procedure described in WO2018197663 (Example 19, at page 117).

Example 1-1

[1103] ##STR00090##

[1104] To a solution of 4 (35 mg, 0.054 mmol) in acetic acid (0.7 mL, 0.08 M) was added L-Tryptophanol (36 mg, 0.189 mmol, Sigma-Aldrich). The reaction mixture was stirred a 50? C. for 3 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 Na.sub.2SO.sub.4. Flash chromatography (Hexane:EtOAc, 1:1) gives pure compound 5-S (28 mg, 63%).

[1105] R.sub.f=0.25 (Hexane:EtOAc, 1:1).

[1106] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.72 (s, 1H), 7.35 (d, J=7.9 Hz, 1H), 7.26 (d, J=7.9 Hz, 1H), 7.12 (t, J=7.9 Hz, 1H), 7.02 (t, J=8.0 Hz, 1H), 6.62 (s, 1H), 6.25 (s, 1H), 6.03 (s, 1H), 5.91-5.80 (m, 1H), 5.75 (s, 1H), 5.17-5.04 (m, 3H), 4.60 (s, 1H), 4.41 (s, 1H), 4.36 (d, J=11.5 Hz, 1H), 4.29 (dd, J=11.7, 2.1 Hz, 1H), 4.22 (d, J=2.7 Hz, 1H), 3.81 (s, 3H), 3.59-3.44 (m, 3H), 3.35 (dd, J=11.1, 9.0 Hz, 1H), 2.97-2.64 (m, 5H), 2.61 (dd, J=15.3, 4.6 Hz, 1H), 2.43-2.29 (m, 1H), 2.37 (s, 3H), 2.28 (s, 3H), 2.05 (s, 3H).

[1107] ESI-MS m/z: Calcd. for C.sub.44H.sub.45N.sub.5O.sub.9S: 819.3. Found: 820.3 (M+1).sup.+.

##STR00091##

[1108] To a solution of 5-S (26 mg, 0.032 mmol) in CH.sub.2Cl.sub.2 was added PdCl.sub.2(PPh.sub.3).sub.2 (13 mg, 0.02 mmol), acetic acid (0.069 mL, 1.2 mmol) and HSnBu.sub.3 (0.17 mL, 0.64 mmol). The reaction mixture was stirred a 23? C. for 2 h. The crude was concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 1:9 to 9:1) gives pure compound 6-S (17 mg, 68%).

[1109] R.sub.f=0.15 (Hexane:EtOAc, 1:1).

[1110] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.74 (s, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.26 (d, J=7.9 Hz, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.02 (t, J=7.9 Hz, 1H), 6.63 (s, 1H), 6.26 (s 1H), 6.03 (s, 1H), 5.82 (s, 1H), 5.15 (d, J=11.6 Hz, 1H), 4.59 (s, 1H), 4.50 (d, J=5.1 Hz, 1H), 4.41 (s, 1H), 4.29 (dd, J=11.7, 2.1 Hz, 1H), 4.22 (d, J=2.7 Hz, 1H), 3.90-3.81 (m, 1H), 3.80 (s, 3H), 3.67-3.49 (m, 1H), 3.49 (d, J=5.2 Hz, 1H), 3.36 (dd, J=11.0, 9.1 Hz, 1H), 3.11-2.85 (m, 3H), 2.60 (dd, J=15.3, 4.5 Hz, 1H), 2.42 (d, J=15.3 Hz, 1H), 2.38-2.22 (m, 2H), 2.35 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H).

[1111] ESI-MS m/z: Calcd. for C.sub.41H.sub.41N.sub.5O.sub.9S: 779.3. Found: 780.2 (M+1).sup.+.

##STR00092##

[1112] To a solution of 6-S (14 mg, 0.018 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 1.3 mL, 0.015 M) was added AgNO.sub.3 (61 mg, 0.36 mmol). After 17 h at 23? C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of brine 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 pure 7-S (3 mg, 22%).

[1113] R.sub.f=0.15 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

[1114] .sup.1H NMR (500 MHz, CD.sub.3OD): ? 7.70 (s, 1H), 7.35 (d, J=7.9 Hz, 1H), 7.26 (d, J=7.9 Hz, 1H), 7.11 (t, J=8.2 Hz, 1H), 7.02 (t, J=8.2 Hz, 1H), 6.63 (s, 1H), 6.23 (s, 1H), 6.01 (s, 1H), 5.76 (s, 1H), 5.26 (d, J=11.5 Hz, 1H), 4.92 (s, 1H), 4.54 (s, 1H), 4.48 (s, 2H), 4.37 (d, J=5.3 Hz, 1H), 4.21 (d, J=10.2 Hz, 1H), 3.80 (s, 3H), 3.67-3.50 (m, 4H), 3.36 (t, J=10.2 Hz, 1H), 3.04-2.82 (m, 3H), 2.61 (dd, J=15.2, 5.8 Hz, 1H), 2.42-2.28 (m, 2H), 2.36 (s, 3H), 2.27 (s, 3H), 2.02 (s, 3H).

[1115] ESI-MS m/z: Calcd. for C.sub.40H.sub.42N.sub.4O.sub.10S: 770.3. Found: 753.2 (M?H.sub.2O+1).sup.+.

Example 1-2

[1116] ##STR00093##

[1117] To a solution of 4 (400 mg, 0.62 mmol) in acetic acid (8 mL, 0.08 M) was added 8-S (468 mg, 2.13 mmol). The reaction mixture was stirred a 52? C. for 17 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 Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) gives pure compound 9-S (325 mg, 62%).

[1118] R.sub.f=0.30 (Hexane:EtOAc, 1:1).

[1119] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.65 (s, 1H), 7.16 (d, J=8.6 Hz, 1H), 6.78 (s, 1H), 6.77 (m, 1H), 6.62 (s, 2H), 6.23 (d, J=1.3 Hz, 2H), 6.02 (d, J=1.3 Hz, 2H), 5.85 (dddd, J=17.1, 10.2, 6.8, 5.8 Hz, 1H), 5.75 (s, 1H), 5.15-5.00 (m, 3H), 4.59 (s, 1H), 4.43-4.22 (m, 4H), 3.80 (s, 3H), 3.78 (s, 3H), 3.53 (d, J=12.9 Hz, 2H), 3.46 (d, J=5.0 Hz, 1H), 3.38 (s, 1H), 2.93 (s, 1H), 2.86 (d, J=4.4 Hz, 1H), 2.85-2.70 (m, 2H), 2.58 (dd, J=15.2, 4.6 Hz, 1H), 2.42-2.30 (m, 2H), 2.37 (s, 3H), 2.26 (s, 3H), 2.04 (s, 3H).

[1120] ESI-MS m/z: Calcd. for C.sub.45H.sub.47N.sub.5O.sub.1OS: 849.9. Found: 850.3 (M+1).sup.+.

##STR00094##

[1121] To a solution of 9-S (325 mg, 0.38 mmol) in CH.sub.2Cl.sub.2 was added PdCl.sub.2(PPh.sub.3).sub.2 (160 mg, 0.23 mmol), acetic acid (0.82 mL, 14.2 mmol) and HSnBu.sub.3 (1.7 mL, 6.27 mmol). The reaction mixture was stirred a 23? C. for 1.5 h. The crude was concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 1:9 to 9:1) gives pure compound 10-S (180 mg, 59%).

[1122] R.sub.f=0.15 (Hexane:EtOAc, 1:1).

[1123] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.19 (s, 1H), 6.79 (m, 2H), 6.65 (s, 1H), 6.26 (s, 1H), 6.03 (d, J=1.4 Hz, 2H), 5.77 (d, J=11.5 Hz, 1H), 5.10 (s, 1H), 4.59 (s, 1H), 4.48 (d, J=4.9 Hz, 1H), 4.39-4.29 (m, 3H), 3.79 (s, 3H), 3.79 (s, 3H), 3.64-3.33 (m, 4H), 3.03-2.90 (m, 4H), 2.59 (d, J=14.6 Hz, 2H), 2.44-2.32 (m, 2H), 2.37 (s, 3H), 2.26 (s, 3H), 2.04 (s, 3H).

[1124] ESI-MS m/z: Calcd. for C.sub.42H.sub.43N.sub.5O.sub.1OS: 809.3. Found: 810.3 (M+1).sup.+.

##STR00095##

[1125] To a solution of 10-S (180 mg, 0.22 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 16 mL, 0.015 M) was added AgNO.sub.3 (756 mg, 4.40 mmol). After 18 h at 23? C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of brine and NaHCO.sub.3, stirred for 15 min, diluted with CH.sub.2C.sub.2, stirred for 5 min, and extracted with CH.sub.2C.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 pure 11-S (100 mg, 56%).

[1126] R.sub.f=0.35 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

[1127] .sup.1H NMR (500 MHz, CD.sub.3OD): ? 7.15 (dd, J=8.8, 0.6 Hz, 1H), 6.82 (dd, J=2.5, 0.6 Hz, 1H), 6.68 (dd, J=8.9, 2.5 Hz, 1H), 6.56 (s, 1H), 6.27 (d, J=1.3 Hz, 1H), 6.08 (d, J=1.3 Hz, 1H), 5.31 (d, J=11.5 Hz, 1H), 4.62-4.55 (m, 1H), 4.44 (ddtd, J=4.9, 1.5, 1.0, 0.5 Hz, 2H) 4.38-4.27 (m, 1H), 4.25-4.18 (m, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.64 (d, J=4.8 Hz, 1H), 3.61-3.42 (m, 3H), 3.13-2.95 (m, 3H), 2.80 (dd, J=10.4, 5.4 Hz, 2H), 2.68 (dd, J=15.1, 4.2 Hz, 2H), 2.55 (d, J=15.4 Hz, 1H), 2.51-2.36 (m, 3H), 2.34 (s, 3H), 2.29 (s, 3H), 2.00 (s, 3H).

[1128] .sup.13C NMR (126 MHz, CD.sub.3OD): ? 172.6, 169.2, 155.1, 148.0, 147.2, 144.7, 142.4, 142.1, 133.1, 132.6, 132.2, 131.1, 128.2, 125.5, 122.2, 122.0, 116.3, 112.9, 112.8, 111.4, 109.0, 103.5, 100.9, 91.0, 66.6, 65.0, 61.8, 60.3, 59.2, 57.1, 56.1, 51.7, 47.2, 45.5, 43.8, 39.0, 28.2, 25.4, 20.6, 16.3, 9.5.

[1129] ESI-MS m/z: Calcd. for C.sub.41H.sub.44N.sub.4O.sub.11S: 800.3. Found: 783.4 (M?H.sub.2O+1).sup.+.

[1130] (+)-HR-ESI-TOF-MS m z 800.2796 [M+H].sup.+ (calcd. for C.sub.41H.sub.44N.sub.4O.sub.11S: 800.2727).

Example 1-3

[1131] ##STR00096##

[1132] To a solution of 4 (400 mg, 0.62 mmol) in acetic acid (8 mL, 0.08 M) was added 8-R (468 mg, 2.13 mmol). The reaction mixture was stirred at 52? C. for 17 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.2C.sub.2. The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1) gives pure compound 9-R (390 mg, 77%).

[1133] R.sub.f=0.30 (Hexane:EtOAc, 1:1).

[1134] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.64 (s, 1H), 7.14 (d, J=8.8 Hz, 1H), 6.81 (d, J=2.6 Hz, 1H), 6.74 (dd, J=8.8, 2.4 Hz, 1H), 6.59 (s, 1H), 6.18 (d, J=1.4 Hz, 1H), 5.97 (d, J=1.4 Hz, 1H), 5.91-5.80 (m, 1H), 5.79 (s, 1H), 5.15-4.92 (m, 3H), 4.62 (s, 1H), 4.42-4.23 (m, 2H), 4.23-4.03 (m, 3H), 3.79 (s, 3H), 3.78 (s, 3H), 3.68-3.48 (m, 2H), 3.43 (d, J=5.1 Hz, 2H), 3.01-2.68 (m, 3H), 2.57-2.41 (m, 3H), 2.39 (s, 3H), 2.25 (s, 3H), 2.22-2.20 (m, 1H), 2.07 (s, 3H).

[1135] ESI-MS m/z: Calcd. for C.sub.45H.sub.47N.sub.5O.sub.1OS: 849.9. Found: 850.4 (M+1).sup.+.

##STR00097##

[1136] To a solution of 9-R (390 mg, 0.46 mmol) in CH.sub.2Cl.sub.2 was added PdCl.sub.2(PPh.sub.3).sub.2 (193 mg, 0.28 mmol), acetic acid (1.0 mL, 17.2 mmol) and HSnBu.sub.3 (2.04 mL, 7.60 mmol). The reaction mixture was stirred a 23? C. for 1.5 h. The crude was concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 1:9 to 9:1) gave pure compound 10-R (210 mg, 57%).

[1137] R.sub.f=0.15 (Hexane:EtOAc, 1:1).

[1138] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.16 (d, J=8.8 Hz, 1H), 6.82 (d, J=2.4 Hz, 1H), 6.76 (dd, J=8.8, 2.5 Hz, 1H), 6.62 (s, 1H), 6.22 (d, J=1.5 Hz, 1H), 6.00 (d, J=1.5 Hz, 1H), 5.03 (d, J=11.5 Hz, 1H), 4.62 (s, 1H), 4.51 (d, J=5.0 Hz, 1H), 4.36 (s, 1H), 4.23-4.11 (m, 3H), 3.85 (dd, J=8.1, 4.1 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.72-3.57 (m, 1H), 3.45 (d, J=5.2 Hz, 2H), 3.12 (d, J=17.6 Hz, 1H), 2.99 (dd, J=17.9, 9.6 Hz, 1H), 2.54 (s, 2H), 2.46 (d, J=14.7 Hz, 2H), 2.38 (s, 3H), 2.33-2.19 (m, 1H), 2.26 (s, 3H), 2.08 (s, 3H).

[1139] ESI-MS m/z: Calcd. for C.sub.42H.sub.43N.sub.5O.sub.1OS: 809.3. Found: 810.5 (M+1).sup.+.

##STR00098##

[1140] To a solution of 10-R (210 mg, 0.26 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 18 mL, 0.015 M) was added AgNO.sub.3 (883 mg, 5.20 mmol). After 18 h at 23? C., the reaction was quenched with a mixture 1:1 of saturated aqueous solutions of brine 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 pure 11-R (140 mg, 66%).

[1141] R.sub.f=0.30 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

[1142] .sup.1H NMR (500 MHz, CD.sub.3OD): ? 7.13 (dd, J=8.8, 0.6 Hz, 1H), 6.82 (d, J=8.8 Hz, 1H), 6.67 (dd, J=8.8, 2.5 Hz, 1H), 6.61 (s, 1H), 6.25 (d, J=1.3 Hz, 1H), 6.07 (d, J=1.4 Hz, 1H), 5.21 (d, J=11.3 Hz, 1H), 4.80-4.72 (m, 2H), 4.58 (s, 1H), 4.45 (d, J=5.4 Hz, 1H), 4.21 (d, J=2.7 Hz, 1H), 4.16-4.06 (m, 1H), 3.75 (s, 3H), 3.72 (s, 3H), 3.64 (d, J=7.7 Hz, 2H), 3.55-3.48 (m, 3H), 3.16 (d, J=17.6 Hz, 1H), 3.03 (dd, J=17.7, 9.8 Hz, 1H), 2.76-2.63 (m, 2H), 2.32 (s, 3H), 2.29 (s, 3H), 2.25-2.11 (m, 2H), 2.04 (s, 3H).

[1143] .sup.13C NMR (126 MHz, CD.sub.3OD): ? 171.6, 153.6, 146.6, 145.9, 143.4, 141.3, 140.9, 132.1, 131.1, 130.8, 129.7, 126.4, 121.2, 120.7, 114.8, 112.1, 111.5, 110.0, 108.8, 107.6, 107.6, 102.1, 99.5, 89.6, 65.4, 63.1, 60.1, 59.0, 57.8, 55.9, 54.7, 52.7, 45.9, 26.6, 25.1, 24.2, 19.4, 19.1, 14.8, 13.0, 8.2.

[1144] ESI-MS m/z: Calcd. for C.sub.41H.sub.44N.sub.4O.sub.11S: 800.3. Found: 783.3 (M?H.sub.2O+1).sup.+.

[1145] (+)-HR-ESI-TOF-MS m z 800.2781 [M+H].sup.+ (calcd. for C.sub.41H.sub.44N.sub.4O.sub.11S: 800.2727).

Example 1-4

[1146] ##STR00099##

[1147] To a solution of 4 (350 mg, 0.54 mmol) in acetic acid (7 mL, 0.08 M) was added 2-benzofuran-3-yl-ethylamine hydrochloride (12) (1.52 g, 7.70 mmol, Sigma Aldrich). The reaction mixture was stirred at 52? C. for 72 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) yields pure 13 (180 mg, 42%).

[1148] R.sub.f=0.5 (Hexane:EtOAc, 1:1).

[1149] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.39-7.29 (m, 2H), 7.23-7.07 (m, 2H), 6.64 (s, 1H), 6.19 (d, J=1.3 Hz, 1H), 6.04 (d, J=1.3 Hz, 1H), 5.97-5.80 (m, 1H), 5.78 (s, 1H), 5.19-4.97 (m, 3H), 4.54 (s, 1H), 4.36 (dd, J=4.8, 1.6 Hz, 1H), 4.31 (s, 1H), 4.20 (dd, J=11.4, 1.9 Hz, 2H), 3.80 (s, 3H), 3.59-3.49 (m, 1H), 3.47 (dd, J=7.0, 2.9 Hz, 1H), 3.25 (ddd, J=11.4, 8.1, 5.0 Hz, 1H), 3.04 (d, J=18.0 Hz, 1H), 2.98-2.72 (m, 5H), 2.59-2.49 (m, 2H), 2.37 (s, 3H), 2.27 (s, 3H), 2.23-2.12 (m, 1H), 2.07 (s, 3H).

[1150] ESI-MS m/z: Calcd. for C.sub.43H.sub.42N.sub.4O.sub.9S: 790.9. Found: 791.5 (M+1).sup.+.

##STR00100##

[1151] To a solution of 13 (320 mg, 0.40 mmol) in CH.sub.2Cl.sub.2 was added PdCl.sub.2(PPh.sub.3).sub.2 (170 mg, 0.24 mmol), acetic acid (0.86 mL, 15 mmol) and SnBu.sub.3H (1.78 mL, 6.60 mmol). The reaction mixture was stirred at 23? C. for 1.5 h. The crude was concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 1:9 to 9:1) affords pure 14 (130 mg, 43%).

[1152] R.sub.f=0.15 (Hexane:EtOAc, 1:1).

[1153] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.40-7.31 (m, 2H), 7.19-7.10 (m, 2H), 6.65 (s, 1H), 6.19 (d, J=1.5 Hz, 1H), 6.04 (d, J=1.5 Hz, 1H), 5.05 (d, J=11.5 Hz, 1H), 4.43 (s, 1H), 4.51-4.47 (m, 1H), 4.30 (s, 1H), 4.21 (d, J=2.3 Hz, 2H), 3.86-3.76 (m, 2H), 3.79 (d, J=1.9 Hz, 2H), 3.46 (d, J=4.7 Hz, 1H), 3.29-3.22 (m, 1H), 3.19 (d, J=17.9 Hz, 1H), 2.99 (dd, J=17.9, 9.4 Hz, 1H), 2.83 (s, 1H), 2.53 (dt, J=7.9, 4.8 Hz, 2H), 2.35 (s, 3H), 2.33-2.23 (m, 1H), 2.27 (s, 3H), 2.20-2.14 (m, 1H), 2.07 (m, 3H).

[1154] ESI-MS m/z: Calcd. for C.sub.40H.sub.38N.sub.4O.sub.9S: 750.8. Found: 751.9 (M+1).sup.+.

##STR00101##

[1155] To a solution of 14 (130 mg, 0.17 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 12 mL, 0.015 M) was added AgNO.sub.3 (578 mg, 3.40 mmol). After 3 h at 23? C., a mixture 1:1 of saturated aqueous solutions of brine 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 pure 15 (80 mg, 64%).

[1156] R.sub.f=0.25 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

[1157] .sup.1H NMR (500 MHz, CD.sub.3OD): ? 7.45-7.34 (m, 2H), 7.26-7.09 (m, 2H), 6.60 (s, 1H), 6.06 (d, J=1.1 Hz, 1H), 6.24 (d, J=1.1 Hz, 1H), 5.24 (d, J=11.5 Hz, 1H), 4.74 (s, 1H), 4.52 (s, 1H), 4.47 (d, J=4.9 Hz, 1H), 4.19-4.09 (m, 2H), 3.74 (s, 3H), 3.64 (d, J=9.2 Hz, 1H), 3.57 (d, J=4.9 Hz, 1H), 3.43-3.37 (m, 1H), 3.20-3.09 (m, 1H), 3.04 (dd, J=17.8, 9.5 Hz, 1H), 2.96-2.90 (m, 1H), 2.83 (d, J=15.4 Hz, 1H), 2.59-2.56 (m, 2H), 2.34 (s, 3H), 2.30 (s, 3H), 2.10-2.02 (m, 1H), 2.05 (s, 3H).

[1158] .sup.13C NMR (126 MHz, CD.sub.3OD): ? 171.9, 170.7, 156.0, 150.5, 148.7, 147.0, 144.8, 142.4, 142.1, 132.6, 131.2, 128.6, 125.5, 124.7, 123.8, 122.3, 121.2, 120.2, 116.8, 114.9, 114.0, 112.3, 103.5, 91.4, 90.7, 63.7, 62.3, 60.4, 58.7, 57.1, 47.2, 43.5, 40.8, 39.3, 28.2, 21.5, 20.6, 16.2, 9.6.

[1159] ESI-MS m/z: Calcd. for C.sub.39H.sub.39N.sub.3O.sub.10S: 741.8. Found: 724.9 (M?H.sub.2O+1).sup.+.

[1160] (+)-HR-ESI-TOF-MS m/z: 741.2416 [M+H].sup.+ (calcd. for C.sub.39H.sub.39N.sub.3O.sub.10S: 741.2356).

Example 1-5

[1161] ##STR00102##

[1162] To a solution of 4 (150 mg, 0.24 mmol) in CH.sub.3CN (15 mL, 0.016 M) was added 16-S (230 mg, 1.20 mmol) and Cyanuric Chloride (TCT) (45 mg, 30%). The reaction mixture was stirred for 24 h at 85? C. and then 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, from 9:1 to 1:9) gives pure 17-S (145 mg, 73% yield).

[1163] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.35 (dt, J=8.2, 0.9 Hz, 1H), 7.31 (ddd, J=7.6, 1.5, 0.7 Hz, 1H), 7.20 (ddd, J=8.4, 7.2, 1.5 Hz, 1H), 7.13 (td, J=7.4, 1.1 Hz, 1H), 6.62 (s, 1H), 6.20 (d, J=1.5 Hz, 1H), 6.05 (d, J=1.4 Hz, 1H), 5.85 (m, 1H), 5.74 (s, 1H), 5.16-5.08 (m, 3H), 4.58 (s, 1H), 4.40-4.32 (m, 2H), 4.28 (dd, J=11.5, 2.2 Hz, 1H), 4.19 (d, J=2.9 Hz, 1H), 3.80 (s, 3H), 3.58-3.53 (m, 1H), 3.50 (dd, J=11.3, 4.1 Hz, 2H), 3.42-3.30 (m, 1H), 2.96 (s, 1H), 2.90-2.73 (m, 4H), 2.58 (dd, J=15.7, 4.9 Hz, 1H), 2.52 (d, J=15.0 Hz, 1H), 2.37 (s, 3H), 2.36-2.26 (m, 2H), 2.28 (s, 3H), 2.04 (s, 3H).

[1164] ESI-MS m/z: 821.3 (M+H).sup.+.

##STR00103##

[1165] To a solution of 17-S (140 mg, 0.17 mmol) in CH.sub.2Cl.sub.2 was added PdCl.sub.2(PPh.sub.3).sub.2 (19 mg, 0.027 mmol), acetic acid (0.097 mL, 1.70 mmol) and SnBu.sub.3H (1.65 mL, 6.12 mmol). The reaction mixture was stirred for 3 h at 23? C. The crude was concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 1:9 to 9:1) affords pure 18-S (94 mg, 71% yield).

[1166] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.35 (dt, J=8.2, 0.9 Hz, 1H), 7.31 (dt, J=7.6, 1.0 Hz, 1H), 7.20 (ddd, J=8.3, 7.2, 1.5 Hz, 1H), 7.13 (td, J=7.4, 1.1 Hz, 1H), 6.62 (s, 1H), 6.20 (d, J=1.4 Hz, 1H), 6.05 (d, J=1.4 Hz, 1H), 5.09 (dd, J=11.5, 1.1 Hz, 1H), 4.58 (s, 1H), 4.52 (d, J=5.0 Hz, 1H), 4.39-4.35 (m, 1H), 4.27 (dd, J=11.5, 2.1 Hz, 1H), 4.20 (d, J=2.6 Hz, 1H), 3.85 (d, J=18.3 Hz, 1H), 3.79 (s, 3H), 3.54-3.44 (m, 2H), 3.34 (dd, J=11.2, 9.2 Hz, 1H), 3.04-2.97 (m, 2H), 2.92 (tt, J=8.6, 4.3 Hz, 1H), 2.60-2.47 (m, 2H), 2.35 (s, 3H), 2.34-2.28 (m, 2H), 2.28 (s, 3H), 2.05 (s, 3H).

[1167] ESI-MS m/z: 781.3 (M+H).sup.+.

##STR00104##

[1168] To a solution of 18-S (90 mg, 0.11 mmol) in CH.sub.3CN:H.sub.2O (1.39:1, 8 mL, 0.015 M) was added AgNO.sub.3 (580 mg, 3.45 mmol). After 18 h at 23? C., a mixture 1:1 of saturated aqueous solutions of brine 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 pure 19-S (60 mg, 68% yield).

[1169] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.35 (d, J=8.1 Hz, 1H), 7.32-7.28 (m, 1H), 7.19 (td, J=8.3, 7.8, 1.4 Hz, 1H), 7.16-7.09 (m, 1H), 6.58 (s, 1H), 6.18 (d, J=1.5 Hz, 1H), 6.04 (d, J=4.6 Hz, 1H), 5.18 (d, J=11.3 Hz, 1H), 4.91 (s, 1H), 4.63 (s, 1H), 4.60-4.46 (m, 2H), 4.18 (d, J=10.8 Hz, 2H), 3.83-3.71 (m, 2H), 3.78 (s, 1H), 3.69 (s, 1H), 3.56-3.44 (m, 2H), 3.32 (t, J=10.3 Hz, 1H), 3.08-2.86 (m, 2H), 2.54 (dd, J=15.6, 5.0 Hz, 2H), 2.37-2.23 (m, 2H), 2.32 (s, 3H), 2.27 (s, 3H), 2.04 (s, 3H).

[1170] ESI-MS m/z: 754.3 (M?H.sub.2O+H).sup.+.

Example 2. Synthesis of Linkers

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

[1171] ##STR00105##

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

[1172] ##STR00106##

[1173] 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.2Cit-O-TrtCl-resin. The loading was calculated: 1.15 mmol/g.

[1174] The NH.sub.2Cit-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.2Cit-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.2Val-Cit-O-TrtCl-resin.

[1175] 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.2Val-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).

[1176] 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.

[1177] .sup.1H NMR (500 MHz, DMSO-d.sub.6): ? 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).

[1178] 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

[1179] ##STR00107##

[1180] 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%).

[1181] .sup.1H NMR (500 MHz, DMSO-d.sub.6): ? 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).

[1182] 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

[1183] ##STR00108##

[1184] 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%).

[1185] R.sub.f=0.40 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

[1186] .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): ? 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).

[1187] .sup.13C NMR (125 MHz, CDCl.sub.3/CD.sub.3OD) ? 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.

[1188] ESI-MS m/z: Calcd. for C.sub.35H.sub.43N.sub.7O.sub.11: 737.3. Found: 738.3 (M+H).sup.+.

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

[1189] ##STR00109##

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

[1190] ##STR00110##

[1191] 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.

[1192] The NH.sub.2Cit-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.2Cit-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).

[1193] 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.2Val-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).

[1194] 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.2PEG4-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?15 mL?0.5 min).

[1195] 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.

[1196] .sup.1H NMR (300 MHz, CDCl.sub.3): ? 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).

[1197] ESI-MS m/z: 673.3 (M+H).sup.+.

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

[1198] ##STR00111##

[1199] 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.

[1200] .sup.1H NMR (500 MHz, DMSO-d.sub.6): ? 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).

[1201] ESI-MS m/z: 778.4 (M+H).sup.+.

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

[1202] ##STR00112##

[1203] 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).

[1204] .sup.1H NMR (500 MHz, CD.sub.3OD): ? 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).

[1205] .sup.13C NMR (75 MHz, CDCl.sub.3/CD.sub.3OD): ? 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.

[1206] ESI-MS m/z: 943.4 (M+H).sup.+.

[1207] 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

[1208] ##STR00113##

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

[1209] ##STR00114##

[1210] 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?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.

[1211] 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).

[1212] 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.2Val-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).

[1213] 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.2PEG4-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?15 mL?0.5 min).

[1214] 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.

[1215] .sup.1H NMR (500 MHz, CDCl.sub.3): ? 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

[1216] ##STR00115##

[1217] 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.

[1218] .sup.1H NMR (500 MHz, DMSO-d.sub.6): ? 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

[1219] ##STR00116##

[1220] 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).

[1221] .sup.1H NMR (500 MHz, CDCl.sub.3): ? 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).

[1222] .sup.13C NMR (75 MHz, CD.sub.3OD): ? 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.

[1223] ESI-MS m/z: 857.3 (M+H).sup.+.

[1224] R.sub.f=0.45 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

Example 3: Synthesis of a Compounds of Formula D-X-(AA).SUB.w.-(T).SUB.g.-L.SUB.1

Preparation of Compound DL-1

[1225] ##STR00117##

[1226] To a solution of 1 (15 mg, 0.019 mmol) and L1 (14 mg, 0.019 mmol) in 1-methyl-2-pyrrolidone (NMP) (1 mL, 0.019 M) was added DIPEA (3 ?L, 0.019 mmol) at 23? C. After 72 h, EtOAc was added and the reaction mixture was washed with water and the organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by HPLC preparative to yield pure DL1 (7.5 mg, 29% yield).

[1227] .sup.1H NMR (500 MHz, CD.sub.3OD): ? 7.58 (d, J=8.6 Hz, 1H), 7.50 (d, J=8.6 Hz, 1H), 7.32 (d, J=8.6 Hz, 1H), 7.21 (d, J=8.6 Hz, 1H), 7.11 (dd, J=8.7, 1.8 Hz, 1H), 6.82 (t, J=2.0 Hz, 1H), 6.77 (s, 2H), 6.67 (ddd, J=8.9, 2.5, 1.3 Hz, 1H), 6.58 (s, 1H), 6.23 (dd, J=3.0, 1.3 Hz, 1H), 6.07 (t, J=1.4 Hz, 1H), 5.64 (ddd, J=12.4, 4.9, 1.8 Hz, 1H), 5.21 (dd, J=22.0, 11.1 Hz, 1H), 5.19-5.11 (m, 1H), 5.14-5.04 (m, 1H), 5.04-4.96 (m, 1H), 4.75 (s, 1H), 4.70 (s, 1H), 4.58 (s, 1H), 4.50 (ddd, J=8.7, 5.1, 3.3 Hz, 1H), 4.30 (d, J=3.1 Hz, 1H), 4.22-4.11 (m, 3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.58-3.53 (m, 1H), 3.50-3.44 (m, 2H), 3.35 (s, 3H), 3.24-3.17 (m, 2H), 3.11 (ddd, J=13.7, 10.6, 6.6 Hz, 1H), 3.02 (dd, J=17.5, 9.8 Hz, 1H), 2.90-2.84 (m, 2H), 2.76 (dd, J=15.3, 2.4 Hz, 1H), 2.59 (dd, J=7.0, 4.9 Hz, 2H), 2.36-2.24 (m, 6H), 2.14-2.07 (m, 1H), 2.10-1.97 (m, 4H), 2.04 (s, 3H), 1.93-1.86 (m, 1H), 1.79-1.71 (m, 1H), 1.66-1.60 (m, 2H), 1.59-1.53 (m, 4H), 1.35-1.25 (m, 4H), 0.97 (m, 6H).

[1228] ESI-MS m/z: 1352.2 (M?H.sub.2O+H).sup.+.

Preparation of Compound DL-2

[1229] ##STR00118##

[1230] To a solution of 2 (21 mg, 0.027 mmol) in Dimethylformamide (DMF) (2 mL, 0.013 M) was added L1 (22 mg, 0.029 mmol), 1-Hydroxybenzotriazole (HOBt, 3.9 mg, 0.029 mmol) and DIPEA (26 ?L, 0.15 mmol) at 23? C. After 72 h, EtOAc was added and the reaction mixture was washed with water and the organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified by HPLC preparative to yield pure DL2 (3.5 mg, 9% yield).

[1231] .sup.1H NMR (400 MHz, CDCl.sub.3): ? 7.74 (d, J=7.8 Hz, 1H), 7.47 (dd, J=21.6, 8.1 Hz, 2H), 7.23 (d, J=7.8 Hz, 1H), 7.12 (d, J=8.2 Hz, 1H), 7.07 (d, J=8.2 Hz, 1H), 6.77 (s, 2H), 6.64 (s, 2H), 6.54 (s, 1H), 6.16 (s, 1H), 5.97 (s, 1H), 5.63 (d, J=17.2 Hz, 1H), 5.11 (d, J=12.5 Hz, 1H), 5.01 (s, 1H), 4.90 (d, J=12.2 Hz, 1H), 4.66 (s, 1H), 4.50 (s, 1H), 4.29-4.19 (m, 2H), 4.13-4.08 (m, 1H), 3.74 (s, 3H), 3.70 (s, 3H), 3.68 (s, 3H), 3.43 (t, J=7.1 Hz, 2H), 3.34 (t, J=1.9 Hz, 1H), 3.33 (s, 2H), 3.08 (s, 2H), 2.98-2.72 (m, 5H), 2.50 (d, J=16.0 Hz, 1H), 2.33 (s, 3H), 2.26 (s, 3H), 2.22-2.14 (m, 3H), 1.99 (s, 3H), 1.81 (s, 1H), 1.63-1.50 (t, J=7.4 Hz, 4H), 1.48-1.39 (m, 4H), 1.28-1.19 (m, 3H), 0.90-0.86 (m, 6H).

[1232] ESI-MS m/z: 1379.5 (M+H).sup.+.

Example 4: Preparation of Antibody-Drug Conjugates (ADCs)

[1233] 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 4a Preparation of Anti-CD13 Monoclonal Antibody

[1234] 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, MA). 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.

[1235] 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 ?g/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 ?m 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 DL1

(a) Preparation of Trastuzumab

[1236] 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 (17.0 mg/mL) was determined by measuring the absorbance at 280 nm.

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

[1237] Trastuzumab solution (0.5 mL, 8.5 mg, 56.6 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 (34 ?L, 170 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 5.0.

(c) Preparation of ADC1

[1238] To the solution of partially reduced Trastuzumab (0.2 mL, 2 mg, 13.3 nmol), DMA was added (39.4 ?L) followed by addition of a freshly prepared solution of DL1 (10 mM in DMA, 10.6 ?L, 106 nmol, 8 eq.). The conjugation reaction was stirred for 30 min at 20? C. and the excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 10.6 ?L, 106 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 ADC1 was concentrated to a final concentration of 3.9 mg/mL as determined by UV and 370 ?L (1.44 mg, 9.6 nmol, 72%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (94%).

Preparation of Antibody-Drug Conjugate ADC2 with Trastuzumab and Compound DL2

(a) Preparation of Trastuzumab

[1239] 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.

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

[1240] 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 (34.2 ?L, 171 ?mol, 3 eq.) The reduction reaction was left to stir for 90 min at 20? C. Immediately after the reduction, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 6.7.

(c) Preparation of ADC2

[1241] To the solution of partially reduced Trastuzumab (171 ?L, 1.71 mg, 11.4 nmol), DMA was added (33.6 ?L) followed by addition of a freshly prepared solution of DL2 (10 mM in DMA, 9.1 ?L, 91 nmol, 8 eq.). The conjugation reaction was stirred for 30 min at 20? C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 9.1 ?L, 91 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 ADC2 was concentrated to a final concentration of 5.14 mg/mL as determined by UV and 300 ?L (1.5 mg, 10 nmol, 87%) ADC solution was obtained. HIC HPLC runs were performed to determine the percentage of conjugation reaction (75%).

Preparation of Antibody-Drug Conjugate ADC3 with Trastuzumab and Compound DL1

(a) Preparation of Trastuzumab

[1242] 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 (16.5 mg/mL) was determined by measuring the absorbance at 280 nm.

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

[1243] Trastuzumab solution (0.5 mL, 8.25 mg, 55 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8). 14 mM solution of Traut's reagent was added (47.1 ?L, 660 nmol, 12 eq.), and the reaction stirred for 2 h at 20? C. The mixture was buffer exchanged using two Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 0.85 mL (9.7 mg/mL). Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 5.5.

(c) Preparation of ADC3

[1244] To the solution of thiol-activated Trastuzumab (200 ?L, 1.94 mg, 12.9 nmol), DMA was added (38 ?L) followed by addition of a freshly prepared solution of DL1 (10 mM in DMA, 12 ?L, 120 nmol, 9.3 eq.). The conjugation reaction was stirred for 2 h at 25? C. and purified by desalting using a Sephadex G25 NAP-5 column into PBS buffer. The final target product ADC3 was concentrated to a final concentration of 1.48 mg/mL as determined by UV and 340 ?L (0.5 mg, 3.3 nmol, 25%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC4 with Trastuzumab and DL2

(a) Preparation of Trastuzumab

[1245] 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.

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

[1246] Trastuzumab solution (0.85 mL, 14.5 mg, 96.6 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8). 14 mM solution of Traut's reagent was added (69 ?L, 966 nmol, 10 eq.), and the reaction stirred for 2 h at 20? C. The mixture was buffer exchanged using two Sephadex G25 NAP-5 columns into PBS buffer, and concentrated to a volume of 1.45 mL (10 mg/mL). Immediately after, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 3.7.

(c) Preparation of ADC4

[1247] To the solution of thiol-activated Trastuzumab (290 ?L, 2.9 mg, 19.3 nmol), DMA was added (57.1 ?L) followed by addition of a freshly prepared solution of DL2 (10 mM in DMA, 15.4 ?L, 154 nmol, 8 eq.). The conjugation reaction was stirred for 2 h at 25? C. and purified by desalting using a Sephadex G25 NAP-5 column into PBS buffer. The final target product ADC4 was concentrated to a final concentration of 3.82 mg/mL as determined by UV and 315 ?L (1.2 mg, 8.0 nmol, 41%) ADC solution was obtained.

Example 5. In Vitro Bioassays for the Detection of Antitumor Activity of the Drugs of the Invention

[1248] 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

[1249]

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-231 HTB-26 human breast breast adenocarcinoma PSN1 CRM-CRL- human pancreas pancreas 3211 adenocarcinoma

Evaluation of Cytotoxic Activity Using the SBR Colorimetric Assay

[1250] 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).

[1251] This form of assay employs SBS-standard 96-well cell culture microplates (Faircloth et al. Methods in Cell Science, 1988, 11(4), 201-205; Mosmann et al. Journal of Immunological Methods, 1983, 65 (1-2), 55-63. 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.

[1252] Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin at 37? C., 5% C.sub.02 and 98% humidity. For the experiments, cells were harvested from subconfluent cultures using trypsinization and resuspended in fresh medium before counting and plating.

[1253] Cells were seeded in 96 well microtiter plates, at 5?103 cells per well in aliquots of 150 ?L, 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 ?L aliquots of 4? stock solutions in complete culture medium plus 4% DMSO) using ten serial dilutions (concentrations ranging from 10 to 0.00262 ?g/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. Effects on cell growth and survival were estimated by applying the NCI algorithm (Boyd MR and Paull KD. Drug Dev. Res. 1995, 34, 91-104).

[1254] Using the mean?SD of triplicate cultures, a dose-response curve was automatically generated using nonlinear regression analysis. Three reference parameters were calculated (NCI algorithm) by automatic interpolation: 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).

[1255] The in vitro cytostatic (ability to delay or arrest tumor cell growth) or cytotoxic (ability to kill tumor cells) of compounds 1, 2, 3 and ET722 and other payloads of this invention, have been disclosed in WO2003066638 (compounds 64, 60, 59 and 63, respectively, at pages 149-151).

[1256] Tables 3-6 illustrate data on the biological activity of the drugs of the present invention together with biological activity of the closest prior art compounds.

TABLE-US-00004 TABLE 3 Biological activity (Molar) Drug Reference compound [00119] 14 R.sub.0 = H, R.sub.1 = CN 15 R.sub.0 = H, R.sub.1 = OH [00120] ET-722 MDA-MB- MDA- A549 HT29 231 PSN1 A549 HT29 MB-231 PSN1 GI.sub.50 14 8.79E?09 8.52E?09 6.66E?09 9.72E?09 TGI 1.73E?08 9.19E?09 1.13E?08 1.33E?08 LC.sub.50 >1.33E?07 >1.33E?07 2.93E?08 2.26E?08 GI.sub.50 15 1.21E?08 8.76E?09 7.14E?09 9.98E?09 ET- 1.35E?09 1.35E?09 8.91E?10 1.48E?09 TGI 2.56E?08 9.57E?09 1.16E?08 1.48E?08 722 1.35E?09 1.48E?09 1.48E?09 2.16E?09 LC.sub.50 >1.35E?07 >1.35E?07 2.43E?08 4.58E?08 >1.35E?07 >1.35E?07 2.56E?09 3.10E?09

TABLE-US-00005 TABLE 4 Biological activity (Molar) Drug Reference compound [00121] 6-S R.sub.0 = H, R.sub.1 = CN 7-S R.sub.0 = H, R.sub.1 = OH [00122] ET-722 MDA-MB- MDA- A549 HT29 231 PSN1 A549 HT29 MB-231 PSN1 GI.sub.50 6-S 8.08E?09 3.33E?09 2.95E?09 3.72E?09 TGI 1.12E?08 3.59E?09 6.03E?09 5.77E?09 LC.sub.50 >1.28E?07 >1.28E?07 2.44E?08 1.09E?08 GI.sub.50 7-S 8.17E?09 3.37E?09 2.85E?09 3.11E?09 ET- 1.35E?09 1.35E?09 8.91E?10 1.48E?09 TGI 1.28E?08 3.63E?09 4.28E?09 4.15E?09 722 1.35E?09 1.48E?09 1.48E?09 2.16E?09 LC.sub.50 >1.30E?07 >1.30E?07 6.88E?09 6.62E?09 >1.35E?07 >1.35E?07 2.56E?09 3.10E?09

TABLE-US-00006 TABLE 5 Biological activity (Molar) Drug Reference compound [00123] 10-S R.sub.0 = H, R.sub.1 = CN 11-S R.sub.0 = H, R.sub.1 = OH [00124] [00125] 10-R R.sub.0 = H, R.sub.1 = CN 2 R.sub.0 = H, R.sub.1 = CN 11-R R.sub.0 = H, R.sub.1 = OH 1 R.sub.0 = H, R.sub.1 = OH MDA- MDA- A549 HT29 MB-231 PSN1 A549 HT29 MB-231 PSN1 GI.sub.50 10-S 4.32E?08 1.23E?08 1.20E?08 8.64E?09 2 1.28E?08 5.13E?09 5.00E?09 2.05E?09 TGI 1.05E?07 1.23E?08 1.23E?08 1.48E?08 1.28E?08 5.64E?09 5.26E?09 3.08E?09 LC.sub.50 >1.23E?06 >1.23E?06 1.36E?08 >1.23E?06 >1.28E?06 1.28E?06 5.77E?09 5.00E?09 GI.sub.50 10-R 4.32E?09 8.64E?10 6.79E?10 7.53E?10 TGI 1.20E?08 1.61E?09 1.21E?09 1.48E?09 LC.sub.50 >1.23E?07 >1.23E?07 3.09E?09 >1.23E?07 GI.sub.50 11-S 6.62E?08 1.37E?08 1.05E?08 1.62E?08 1 1.82E?09 1.28E?09 7.52E?10 1.22E?09 TGI >1.25E?07 2.50E?08 1.87E?08 2.37E?08 3.37E?09 1.43E?09 1.30E?09 1.69E?09 LC.sub.50 >1.25E?07 >1.25E?07 4.50E?08 >1.25E?07 7.78E?09 >1.30E?07 2.46E?09 2.34E?09 GI.sub.50 11-R 1.50E?08 2.00E?09 1.62E?09 2.12E?09 TGI 4.50E?08 3.62E?09 2.87E?09 3.62E?09 LC.sub.50 >1.25E?07 >1.25E?07 7.24E?09 1.50E?08

TABLE-US-00007 TABLE 6 Biological activity (Molar) Drug Reference compound [00126] 18-S R.sub.0 = H, R.sub.1 = CN 19-S R.sub.0 = H, R.sub.1 = OH [00127] ET-722 MDA-MB- MDA- A549 HT29 231 PSN1 A549 HT29 MB-231 PSN1 GI.sub.50 18-S 8.08E?09 3.33E?09 2.95E?09 3.72E?09 TGI 1.12E?08 3.59E?09 6.03E?09 5.77E?09 LC.sub.50 >1.28E?07 >1.28E?07 2.44E?08 1.09E?08 GI.sub.50 19S 8.17E?09 3.37E?09 2.85E?09 3.11E?09 ET- 1.35E?09 1.35E?09 8.91E?10 1.48E?09 TGI 1.28E?08 3.63E?09 4.28E?09 4.15E?09 722 1.35E?09 1.48E?09 1.48E?09 2.16E?09 LC.sub.50 >1.30E?07 >1.30E?07 6.88E?09 6.62E?09 >1.35E?07 >1.35E?07 2.56E?09 3.10E?09

Example 6: Demonstrating the Cytotoxicity of the Antibody-Drug Conjugates of the Present Invention

Bioassays for the Detection of Antitumor Activity

[1257] 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

[1258] 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?), 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), or RPMI-1640 (HCC-1954), all media supplemented with 10% Fetal Calf Serum (FCS), 2 mM L-glutamine and 100 units/mL penicillin and streptomycin.

Cytotoxicity Assay

[1259] 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.

[1260] 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 IC.sub.50 value (the concentration of compound causing 50% cell death as compared to the control cell survival) was calculated.

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

[1261] The in vitro cytotoxicity of the ACD 1 along with the parent cytotoxic compounds 1 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

[1262] The in vitro cytotoxicity 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 g/mL (3.33E-07-8.74E-11). 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 7 where results corresponding to the geometric mean of the IC.sub.50 values obtained in three independent experiments are presented.

TABLE-US-00008 TABLE 7 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, ?g/mL >50 >50 >50 >50 IC.sub.50, M >3.4E?07 >3.4E?07 >3.4E?07 >3.4E?07

Cytotoxicity of 1

[1263] The cytotoxicity of payload 1 was evaluated against the different tumor cell lines by performing triplicated 10-points, 2.5-fold dilution DR curves ranging from 100 to 0.03 ng/mL (1.26E-07-3.3E-11 M.

[1264] As shown in Table 8, 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 8.82E-10 to 1.95E-09 M). The geometric mean IC.sub.50 value across the whole cell panel was 1.32E-09 M.

TABLE-US-00009 TABLE 8 Summary of the in vitro cytotoxicity of 1 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, ?g/mL 8.60E?04 1.50E?03 6.80E?04 1.20E?03 IC.sub.50, M 1.12E?09 1.95E?09 8.82E?10 1.56E?09

Cytotoxicity of ADC1

[1265] The cytotoxicity of ADC1 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 ?g/mL to 26 ng/mL (6.67E-07-1.75 E-10 M). The evaluation was performed in three independent experiments, Table 9 summarizes the results corresponding to the geometric mean of the IC.sub.50 values obtained in three independent experiments. As observed in Table 9, ADC1 showed a cytotoxicity which is similar to that shown by the parent drug 1 only in HER-2 positive cells. However, in HER2 negative cells such toxicity is significantly lower: nearly 8-fold lower according to the selectivity ratio obtained by dividing the mean IC.sub.50 values in HER2 negative cells between that in HER2 positive cells. This selectivity leads us to conclude that the conjugate ADC1 is acting through the interaction of the antibody with the membrane associates HER2 receptor on the tumor cells, followed by intracellular delivery of the cytotoxic drug.

TABLE-US-00010 TABLE 9 Summary of in vitro activity of ADC1 IC.sub.50 in IC.sub.50 in HER2 positive HER2 negative HER2+ HER2? SK- HCC- MDA- (geom. (geom. Selectivity BR-3 1954 MB-231 MCF-7 Mean) Mean) ratio IC.sub.50 9.00E?01 1.00E+00 5.70E+00 1.00E+01 9.49E?01 7.55E+00 8.0 (?g/mL) IC.sub.50 6.00E?09 6.67E?09 3.80E?08 6.67E?08 6.33E?09 5.03E?08 (M))

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

[1266] The in vitro cytotoxicity of the ADC2 along with the parent cytotoxic compound 2 were evaluated against four different human breast cancer cell lines over-expressing or not the HER2 receptor, including XK-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 monoclonal antibody Trastuzumab described above.

Cytotoxicity of 2

[1267] The cytotoxicity of the intermediate compound 2 was evaluated against the different tumor cell lines by performing triplicated 10-points, 2.5-fold dilution DR curves ranging from 100 to 0.03 ng/mL (1.26E-07-3.3E-11 M. 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, 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 8.85E-10 to 2-31E-09 M). The geometric mean with IC.sub.50 value across the whole cell panel was 1.53E-09 M.

TABLE-US-00011 TABLE 10 Summary of the in vitro cytotoxicity of 2 HER2 positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC.sub.50, ?g/mL 9.60E?04 1.80E?03 6.90E?04 1.70E?03 IC.sub.50, M 1.23E?09 2.31E?09 8.85E?10 2.18E?09

Cytotoxicity of ADC2

[1268] The cytotoxicity of ADC2 was evaluated against the different tumor cell lines by performing triplicate 10-points 2.5-fold dilution DR curves ranging from 100 ?g/mL to 26 ng/mL (6.67E-07-1.75E-10 M). The evaluation was performed in three independent experiments, Table 11 summarized the results corresponding to the geometric mean of the IC.sub.50 values obtained in the three independent experiments. As observed in Table 11, ADC2 showed a cytotoxicity which is similar to that shown by the parent drug 2 only in HER2-positive cells. However, in HER2-negative cells such toxicity is significantly lower according to the selectivity ratio obtained by dividing the mean IC.sub.50 in HER2-negative cells between that in HER2-positive cells. This selectivity leads us to conclude that ADC2 is acting through the interaction of the antibody with the membrane associates HER2 receptor on the tumor cells, followed by intracellular delivery of the cytotoxic drug.

TABLE-US-00012 TABLE 11 Summary of the in vitro cytotoxicity of ADC2 IC.sub.50 in IC.sub.50 in HER2 positive HER2 negative HER2+ HER2? SK- HCC- MDA- (geom. (geom. Selectivity BR-3 1954 MB-231 MCF-7 Mean) Mean) ratio IC.sub.50 8.50E+00 1.80E+01 >1.0E+02 >1.0E+02 1.24E+01 >1.0E+02 >8.09 (?g/mL) IC.sub.50 5.67E?08 1.20E?07 >6.67E?07 >6.67E?07 8.25E?08 >6.67E?07 (M))

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

[1269] The in vitro cytotoxicity of ADC3 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.

Cytotoxicity of ADC3

[1270] The cytotoxicity of ADC3 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 ?g/mL to 26 ng/mL (6.67E-07-1.75E-10 M). The evaluation was performed in three independent experiments, Table 12 summarizes the results corresponding to the geometric mean of the IC.sub.50 values obtained in three independent experiments. As observed in Table 12, ADC3 showed a cytotoxicity which is similar to that shown by the parent drug 1 only in HER2 positive cells. However, in HER2 negative cells such toxicity is significantly lower, nearly 56-fold lower according to the selectivity ratio obtained by dividing the mean IC50 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-00013 TABLE 12 In vitro activity of ADC3 IC.sub.50 in IC.sub.50 in HER2 positive HER2 negative HER2+ HER2? SK- HCC- MDA- (geom. (geom. Selectivity BR-3 1954 MB-231 MCF-7 Mean) Mean) ratio IC.sub.50 2.50E?01 2.70E?01 1.40E+01 1.70E+01 2.60E?01 1.94E+00 55.7 (?g/mL) IC.sub.50 1.67E?09 1.80E?09 9.33E?08 1.00E?07 1.73E?09 9.66E?08 (M))

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

[1271] The in vitro cytotoxicity of the ADC4 along was evaluated against four different human breast cancer cell lines over-expressing or not the HER2 receptor, including SK-BR-3, HCC-1954 (HER-2 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 ADC4

[1272] The cytotoxicity of ADC4 was evaluated against the different tumor cell lines by performing triplicate 10-points, 2.5-fold dilution DR curves ranging from 100 ?g/mL to 26 ng/mL (6.67E-07-1.75E-10 M). The evaluation was performed in three different experiments, Table 13 summarizes the results corresponding to the geometric mean of the IC.sub.50 values obtained in three different experiments. As observed in Table 13, ADC4 showed a cytotoxicity which is similar to that shown by the parent drug 2 only in HER2 positive cells. However, in HER2 negative cells such toxicity in significantly lower: nearly 14-fold lower according to the selectivity ration 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 ADC4 is acting through the interaction of the antibody with the membrane associated HER2 receptor on the tumor cells, followed by intercellular delivery of the cytotoxic drug.

TABLE-US-00014 TABLE 13 In vitro activity of ADC4 IC.sub.50 in IC.sub.50 in HER2 positive HER2 negative HER2+ HER2? SK- HCC- MDA- (geom. (geom. Selectivity BR-3 1954 MB-231 MCF-7 Mean) Mean) ratio IC.sub.50 3.10E?01 6.30E?01 7.00E+00 5.40E+00 4.42E?01 6.15E+00 13.91 (?g/mL) IC.sub.50 2.07E?09 4.20E?09 4.67E?08 3.60E?08 2.95E?09 4.10E?08 (M))