Thieno-indole moieties and methods of treating using the same

Abstract

The present invention relates to a novel class of alkylating agents comprising a thieno-indole moiety linked to a DNA-binding moiety, which have cytotoxic activity and are useful in treating diseases such as cancer, cellular proliferation disorders and viral infections. The present invention also provides methods for preparing these compounds, pharmaceutical compositions comprising them and methods of treating diseases utilizing such compounds or the pharmaceutical composition containing them. The invention also relates to the use of this novel class of alkylating agents in the preparation of conjugates. The present invention also relates to methods of treating ovarian cancer by administration of compounds of formula (II): ##STR00001##

Claims

1. A method for treating ovarian cancer, which comprises administering to a mammal in need thereof an effective amount of a compound of formula (II) ##STR00166## wherein R1 and R2 taken together form a group (D) or (G): ##STR00167## wherein R5 is hydrogen, linear or branched C.sub.1-C.sub.4 alkyl, linear or branched C.sub.1-C.sub.4 hydroxyalkyl or linear or branched C.sub.1-C.sub.4 aminoalkyl; R3 and R4 are, each independently, hydrogen or a group selected from an optionally substituted linear or branched C.sub.1-C.sub.4 alkyl and linear or branched C.sub.1-C.sub.4 hydroxyalkyl; R6 is a leaving group; T is null or N; BM of formula (V): ##STR00168## wherein: X is null, linear or branched C.sub.1-C.sub.4 alkyl, linear or branched C.sub.2-C.sub.4 alkenyl or linear or branched C.sub.2-C.sub.4 alkynyl; Y is an optionally substituted aryl or heteroaryl selected from the group consisting of ##STR00169## ##STR00170## Y is an optionally substituted aryl or heteroaryl selected from ##STR00171## ##STR00172## wherein R15, R16 and R20 are independently hydrogen, halogen, hydroxy, NO.sub.2, an optionally substituted linear or branched C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy, cyano,COOH, CONHR3, NC(O)OR3, C(NH)NH.sub.2 or NR3R4, and R3 and R4 are as defined above; U is a moiety of formula (VI) or (VII): ##STR00173## wherein R3 is as defined above; q is an integer from 0 to 4; L1 is hydrogen or L, wherein L is null or a moiety selected from NHCOR9 (Xa); NHCONHR9 (Xb); NHCOOR9 (Xc); NHR9 (Xd); ##STR00174## wherein R9 and R10 are, each independently, hydrogen, hydroxy or an optionally substituted group selected from linear or branched C.sub.1-C.sub.4 alkyl, linear or branched C.sub.1-C.sub.4 hydroxyalkyl, linear or branched C.sub.1-C.sub.4 sulfhydrylalkyl and linear or branched C.sub.1-C.sub.4 aminoalkyl, when W1 and Z1 are null, or R9 and R10 are null when at least one of W1 and Z1 is not null, n is an integer from 0 to 2 and n1 is an integer from 0 to 4; W1 is null or a system comprising one or more groups independently selected from ##STR00175## ##STR00176## wherein one of R9 and R10 is null and the other is as defined above, R11 and R12 are, each independently, hydrogen, halogen, methyl, ethyl or linear or branched C.sub.1-C.sub.4 hydroxymethyl, m is an integer from 0 to 3, and A.sub.1 is CH.sub.2, CH.sub.2NR12 or NR12, wherein R12 is as defined above; Z1 is null or a peptidic linker (Z.sub.a), a non peptidic linker (Z.sub.b) or hybrid linker(Z.sub.c), wherein Z.sub.a is selected from a single amino acid, a dipeptide, a tripeptide, a tetrapeptide and an oligopeptide moiety comprising natural L-amino acids, unnatural D-amino acids, synthetic amino acids or any combination thereof; Z.sub.b is selected from ##STR00177## wherein one of R9 and R10 is null and the other is as defined above and p is an integer from 1 to 20; and Z.sub.c is a group of formula
Z.sub.aZ.sub.b or Z.sub.bZ.sub.a wherein Z.sub.a and Z.sub.b are as defined above; provided that a compound of formula (II) wherein L1 is hydrogen is excluded when 1) both T and X are null, q is 0 and Y is an heterocyclyl moiety of formula (VIII), (VIII) or (VIII): ##STR00178## or 2) both T and X are null, q is 1, U is a group of formula (VII), Y is an heterocyclyl moiety of formula (IX) ##STR00179## and Y is an heterocyclyl moiety of formula (VIII) ##STR00180## wherein Q is NH or O, and R21 is hydrogen or a group selected from N(C.sub.2H.sub.5).sub.2 and C(NH)NH.sub.2; with the proviso that in a compound of formula (II) when L1 is L, at least one among L, W1 and Z1 is not null; or the pharmaceutically acceptable salts thereof, or a compound of formula (III) or (IV) ##STR00181## wherein R1 and R2 taken together form a group (D) or (G): ##STR00182## wherein R5 is hydrogen, linear or branched C.sub.1-C.sub.4 alkyl, linear or branched C.sub.1-C.sub.4 hydroxyalkyl or linear or branched C.sub.1-C.sub.4 aminoalkyl; R3 and R4 are, each independently, hydrogen or a group selected from an optionally substituted linear or branched C.sub.1-C.sub.4 alkyl and linear or branched C.sub.1-C.sub.4 hydroxyalkyl; R6 is a leaving group; T is null or N; BM is of formula (V): ##STR00183## wherein: X is null, linear or branched C.sub.1-C.sub.4 alkyl, linear or branched C.sub.2-C.sub.4 alkenyl or linear or branched C.sub.2-C.sub.4 alkynyl, Y is an optionally substituted aryl or heteroaryl selected from the group consisting of ##STR00184## ##STR00185## Y is an optionally substituted aryl or heteroaryl selected from ##STR00186## ##STR00187## wherein R15, R16 and R20 are independently hydrogen, halogen, hydroxy, NO.sub.2, an optionally substituted linear or branched C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy, cyano,COOH, CONHR3, NC(O)OR3, C(NH)NH.sub.2 or NR3R4, and R3 and R4 are as defined above, U is a moiety of formula (VI) or (VII) ##STR00188## wherein R3 is as defined above, and q is an integer from 0 to 4; A is an atom selected from O, NH, CO; A is null or A, wherein A is as defined above; L is null or a moiety selected from NHCOR9 (Xa); NHCONHR9 (Xb); NHCOOR9 (Xc); NHR9 (Xd); ##STR00189## wherein R9 and R10 are, each independently, hydrogen, hydroxy or an optionally substituted group selected from linear or branched C.sub.1-C.sub.4 alkyl, linear or branched C.sub.1-C.sub.4 hydroxyalkyl, linear or branched C.sub.1-C.sub.4 sulfhydrylalkyl and linear or branched C.sub.1-C.sub.4 aminoalkyl, when W, Z and RM are null, or R9 and R10 are null when at least one of W, Z and RM is not null, n is an integer from 0 to 2 and n1 is an integer from 0 to 4; L1 is hydrogen or L, wherein L is as defined above; W and W1 are independently null or a system comprising one or more groups independently selected from ##STR00190## ##STR00191## wherein one of R9 and R10 is null and the other is as defined above, R11 and R12 are, each independently, hydrogen, halogen, methyl, ethyl or linear or branched C.sub.1-C.sub.4 hydroxymethyl, m is an integer from 0 to 3, and A.sub.1 is CH.sub.2, CH.sub.2NR12 or NR12, wherein R12 is as defined above; Z and Z1 are independently null or a peptidic linker (Z.sub.a), a non peptidic linker (Z.sub.b) or a hybrid linker (Z.sub.c), wherein Z.sub.a is selected from a single amino acid, a dipeptide, a tripeptide, a tetrapeptide and an oligopeptide moiety comprising natural L-amino acids, unnatural D-amino acids, synthetic amino acids or any combination thereof; Z.sub.b is selected from ##STR00192## wherein one of R9 and R10 is null and the other is as defined above and p is an integer from 1 to 20; and Z.sub.c is a group of formula
Z.sub.aZ.sub.b or Z.sub.bZ.sub.a wherein Z.sub.a and Z.sub.b are as defined above; RM and RM1 are independently null or a moiety selected from ##STR00193## ##STR00194## wherein R13 is C.sub.1-C.sub.3 alkyl or an electron-withdrawing group selected from the group consisting of NO.sub.2 and CN; r is an integer from 0 to 7; and R11 and R12 are as defined above, said RM being attached to one or more of A, L, W or Z groups and said RM1 being attached to one or more of L1, W1 or Z1 groups; provided that 1) a compound of formula (IV) is excluded when A is null and RM1 is null; 2) a compound of formula (III) or (IV) is excluded when a) both T and X are null, q is 0 and Y is an heterocyclyl moiety of formula (VIII).sup.IV ##STR00195## wherein Q is O, S, NR14, wherein R14 is hydrogen, C.sub.1-C.sub.4 alkyl or C.sub.1-C.sub.4 hydroxyalkyl; Q1 is CH or N; R7 and R8 are independently hydrogen, halogen, hydroxy, C.sub.1-C.sub.4 alkoxy, cyano, NCOOR3, C(NH)NH.sub.2 or NR3R4, wherein R3 and R4 are as defined above; or b) both T and X are null, q is 1 or 2, U is a group of formula (VII), Y is a heterocyclyl moiety of formula (IX): ##STR00196## and Y is a heterocyclyl moiety of formula (VIII).sup.IV ##STR00197## wherein Q, Q1, R7 and R8 are as defined above; with the proviso that when L1 is L, at least one among L, W1, Z1 and RM1 is not null; and with the proviso that when A is A, at least one among L, W, Z and RM is not null; or the pharmaceutically acceptable salts thereof.

2. The method according to claim 1 wherein the mammal in need thereof is a human.

3. The method according to claim 1, wherein the compound of formula (II), (III) or (IV), as defined in claim 1, is selected from the group consisting of: N-(6-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide, N-(5-{[(8R)-8-(chloromethyl)-4-hydroxy-I-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1-methyl-1H-pyrrol-3-yl)-1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide, (2E)-1-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one, (2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one, (8R)N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide, (8S)N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide, N-(3-{(1E)-3-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide, N-(3-{(1E)-3-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide, N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide, tert-butyl {2-[(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate, (8S)-6-({5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8S)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino)}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate hydrochloride, (8S)-8-chloromethyl)-6-({5-[(1H-indol-2-ylcarbonyl)amino]-1H-indol-2-yl}carbonyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl piperazine-1-carboxylate, (8R)-6-({5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8R)-6-[(5-1{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate hydrochloride, (8S)-8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8R)-8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8S)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino})-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, (8R)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4 ({[{3-[({[(8R)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, [(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanone, [(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanone, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N.sup.5-carbamoyl-N-[4-({[({3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N.sup.6-carbamoyl-N-[4-({[{3-[[({(8R)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-omithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N.sup.5-carbamoyl-N-[4-(({[(3-{[({(8S)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N.sup.5-carbamoyl-N-[4-({[(3-{([({(8R)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, (2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-(1H-indol-3-yl)prop-2-en-1-one, N-(2-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1-methyl-1H-indol-5-yl)-1-methyl-1H-indole-2-carboxamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({-[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methy)carbamoyl]oxy}methy)phenyl]glycinamide, L-valyl-N.sup.5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L -ornithinamide, L-valyl-N.sup.5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl)}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L -valyl-N.sup.5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl)}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-,7, 10,13-tetraoxa-16-azanonadecan-1-oyl]-L -valyl-N.sup.5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-1-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-omithinamide and N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N.sup.5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide.

Description

(1) FIG. 1 shows the mass spectrum of the conjugate A1 and reports the molecular weight (m/z) on the x axis while intensity expressed as counts per second (cps) is reported on the y axis.

(2) FIG. 2 shows the HPLC profile of the conjugate A1 and reports the time (min) on the x axis while UV absorbance (mAU) is reported on the y axis.

(3) FIG. 3 shows the HPLC profile after 2 h treatment of conjugate A1 with cathepsin and reports the time (min) on the x axis while UV absorbance (mAU) is reported on the y axis.

(4) FIG. 4a shows the mass spectrum of the released Compd. 37 and reports the molecular weight (m/z) on the x axis while intensity expressed as counts per second (cps) is reported on the y axis.

(5) FIG. 4b shows the mass spectrum of the released Compound A3, a precursor of 37, and reports the molecular weight (m/z) on the x axis while intensity expressed as counts per second (cps) is reported on the y axis.

RELEASE OF A DRUG MOIETY FROM A CONJUGATE

(6) As an example, that is not intended to limit the scope of the invention, the release of a compound of formula (II) from the conjugate was performed in presence of cathepsin as reported below.

(7) The conjugate A1 was incubated with 0.2 unit of cathepsin B in sodium acetate buffer pH 5.5 and 1 mM Cys for 2 hours at 40 C.

(8) Disappearance of the conjugate A1 and release of the corresponding compound of formula (II), i.e. compd. 37, as well as of its precursor A3, confirms the breaking of the Z peptidic linker of the conjugate.

(9) Complete release of the compound of formula (II) from the conjugate has been observed by HPLC ESI-MS analysis.

(10) The compounds of the present invention can be administered either as single agents or, alternatively, in combination with known anticancer treatments such as radiation therapy or chemotherapy regimen, in combination with cytostatic or cytotoxic agents, antibiotic-type agents, alkylating agents, antimetabolite agents, hormonal agents, immunological agents, interferon-type agents, cyclooxygenase inhibitors (e.g. COX-2 inhibitors), matrix metalloprotease inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor agents, anti-HER agents, anti-EGFR agents, anti-angiogenesis agents (e.g. angiogenesis inhibitors), famesyl transferase inhibitors, ras-raf signal transduction pathway inhibitors, cell cycle inhibitors, other cdks inhibitors, tubulin binding agents, topoisomerase I inhibitors, topoisomerase II inhibitors, and the like.

(11) If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described below and the other pharmaceutically active agent within the approved dosage range.

(12) Compounds of formula (I), (II), (III) or (IV) may be used sequentially with known anticancer agents when a combination formulation is inappropriate.

(13) The compounds of formula (I), (II), (III) or (IV) of the present invention, suitable for administration to a mammal, e.g., to humans, can be administered by the usual routes and the dosage level depends upon the age, the weight, the conditions of the patient and the administration route.

(14) For example, a suitable dosage adopted for oral administration of a compound of formula (I) may range from about 1 to about 300 mg per dose, from 1 to 5 times daily. The compounds of the invention can be administered in a variety of dosage forms, e.g., orally, in the form of tablets, capsules, sugar or film coated tablets, liquid solutions or suspensions; rectally in the form of suppositories; parenterally, e.g., subcutaneous, intramuscularly, or through intravenous and/or intrathecal and/or intraspinal injection or infusion.

(15) The present invention also includes pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof in association with a pharmaceutically acceptable excipient, which may be a carrier or a diluent

(16) The pharmaceutical compositions containing the compounds of the invention are usually prepared following conventional methods and are administered in a suitable pharmaceutical form. For example, the solid oral forms may contain, together with the active compound, diluents, e.g., lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g., silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents, e.g., starches, arabic gum, gelatine methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disintegrating agents, e.g., starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. These pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

(17) The liquid dispersions for oral administration may be, e.g., syrups, emulsions and suspensions. As an example, the syrups may contain, as carrier, saccharose or saccharose with glycerine and/or mannitol and sorbitol.

(18) The suspensions and the emulsions may contain, as examples of carriers, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. The suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, if desired, a suitable amount of lidocaine hydrochloride. The solutions for intravenous injections or infusions may contain, as a carrier, sterile water or preferably they may be in the form of sterile, aqueous, isotonic, saline solutions or they may contain propylene glycol as a carrier. The suppositories may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. cocoa butter, polyethylene glycol, a polyoxyethylene sorbitan fatty acid ester surfactant or lecithin.

(19) With the aim at better illustrating the present invention, without posing any limitation to it, the following examples are now given.

EXAMPLES

(20) The synthetic preparation of some compounds of formula (I) of the invention is described in the following examples. The compounds of the present invention, as prepared according to the following examples, were also characterized by .sup.1H-NMR and/or by Exact mass data ESI(+).

(21) .sup.1H-NMR spectra were recorded at a constant temperature of 25 C. on a Varian INOVA 500 spectrometer (operating at 499.8 MHz for .sup.1H, 125.8 for .sup.13C and 50.6 MHz for .sup.15N) and equipped with 5 mm .sup.1H{.sup.13C, .sup.15N} z axis PFG Indirect Detection Cold-Probe or alternatively with 5 mm .sup.1H{.sup.13C-.sup.15N} z axis PFG Triple Resonanace Probe.

(22) Chemical shifts were referenced with respect to the residual solvents signals. Data are reported as follows: chemical shift (), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, br. s=broad singlet, td=triplet of doublet, dd=doublet of doublets, ddd=doublet of doublets of doublets, m=multiplet), coupling constants (Hz), and number of protons.

(23) Exact mass data ESI(+) were obtained on a Waters Q-Tof Ultima mass spectrometer directly connected with a Agilent 1100 micro-HPLC system as previously described (M. Colombo, F. Riccardi-Sirtori, V. Rizzo, Rapid Commun. Mass Spectrom. 2004, 18, 511-517).

(24) In the examples below as well as throughout the application, the following abbreviations have the following meanings. If not defined, the terms have their generally accepted meanings.

(25) TABLE-US-00001 ABBREVIATIONS DCC N,N-dicyclohexylcarbodiimide DBU diazabicycloundecene DCM dichloromethane DIPEA N,N-diisopropyethylamine DMAP N,N-dimethylaminopyridine DMF N,N-dimethylformamide DMSO dimethyl sulfoxide EDCl N-ethyl-N,N-diisopropyl carbodiimide hydrochloride EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Et.sub.2O diethyl ether EtOAc ethyl acetate EtOH ethanol HCl hydrochloric acid HOBt 1H-benzotriazol-1-ol LiHMDS lithium bia(trimethylsilyl)amide MeOH methanol Na.sub.2SO.sub.4 sodium sulfate NaHCO.sub.3 sodium hydrogen carbonate NaOH sodium hydroxide TEA triethylamine TFA trifluoro acetic acid THF tetrahydrofurane

Example 1

(26) Step b, Step a

N-(6-{[(8S)-8-{chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6]-yl}carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide [(II)] (compd. 1)

(27) ##STR00087##
Step b

(28) A solution of (8S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ol (XVII) prepared as reported in the GB2344818 (14.2 mg, 0.0563 mmol) was dissolved in dry DMF (1.5 mL), and treated with EDCI (43 mg, 4 eq.) and 3-[(1H-indol-6-ylcarbonyl)amino]-1H-indole-6-carboxylic acid (XVI) (27 mg, 1.5 eq.) The mixture was stirred for 16 h at room temperature and then was quenched by adding saturated aqueous NaCl. Isolation of the product was performed by extraction with EtOAc (4) and subsequent washing of the combined organic layers with aqueous 2M HCl (3), saturated aqueous Na.sub.2CO.sub.3 (3) and saturated aqueous NaCl (3). Organic layer was dried (Na.sub.2SO.sub.4), concentrated under vacuum. The crude residue was purified by flash chromatography (hexane-acetone 6:4) to afford the title compound (18.7 mg, 60%).

(29) ESI MS: m/z 555 (MH+)

(30) ##STR00088##

(31) Analogously, by using the corresponding carboxylic acids, the following compounds have been prepared: N-(5-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1-methyl-1 H-pyrrol-3-yl)-1-methyl-4-{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}1H-pyrrole-2-carboxamide [(II)](compd. 2)

(32) ##STR00089##

(33) ESI MS: m/z 650 (MH+)

(2E)-1-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[,2-e]indol-6-yl]-3-1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one [(II)] (compd. 3)

(34) ##STR00090##

(35) ESI MS: m/z 424 (MH+)

(2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8 dihydro-6H-thieno[3,2-e]indol-6-yl]-3-1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one [(II)] (compd. 4)

(36) ##STR00091##

(37) ESI MS: m/z 424 (MH+)

N-(3-{(1E)-3-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2]indol-6-yl]-3 oxoprop-1-en-1-yl}1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide [(II)] (compd. 13)

(38) ##STR00092##

(39) ESI MS: m/z 582 (MH+)

N-(3-{(1E) 3-[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]-3-oxoprop-1-en-1-yl}1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide [(II)] (compd. 14)

(40) ##STR00093##

(41) ESI MS: m/z 582 (MH+)

(2E)-1-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6 yl]3-1H-indol-3-yl)prop-2-en-1-one [(II)] (compd. 43)

(42) ##STR00094##

(43) ESI MS: m/z 423 (MH+)

(44) .sup.1H NMR (500 MHz, acetone-d6) ppm 2.60 (d, J=1.0 Hz, 3 H) 3.58 (m, 1 H) 3.88 (m, J=11.2, 1.9 Hz, 1 H) 4.2 (m, 1 H) 4.46 (t, J=9.5 Hz, 1 H) 4.63 (d, J=10.6 Hz, 1 H) 7.07 (d, J=15.4 Hz, 1 H) 7.24 (m, 2 H) 7.32 (m, 1 H) 7.53 (m, 1 H) 7.91 (m, 1 H) 8.02 (m, 2 H) 8.13 (br. s., 1 H) 9.26 (br. s., 1 H) 10.81 (br. s., 1 H)

N-(2-{[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro 6H-thieno[3,2-e]indol-6 yl]carbonyl}-1-methyl-1H-indo-5-yl)-1-methyl-1H-indole-2-carboxamide [(II)] (compd. 44)

(45) ##STR00095##

(46) ESI MS: m/z 583 (MH+)

(47) Step a

N-(6-{[(3bR,4aS)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-3-yl)-1H-indole-6-carboxamide [(I)] (compd. 5)

(48) Compound 1 (25 mg, 0.045 mmol) was dissolved in DMF (2 mL), and treated with a solution of NaHCO.sub.3 in water (1 mL, 15 mg NaHCO.sub.3/mL). The reaction mixture was stirred for 2 h, EtOAc was added and the resulting organic layer was washed with brine (4), dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The crude residue was purified by flash chromatography (hexane-acetone 6:4) to afford compound 5 (19 mg, 82%).

(49) ##STR00096##

(50) ESI MS: m/z 519 (MH+)

(51) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 1.75 (t, J=4.8 Hz, 1 H) 2.27 (s, 3 H) 2.32 (dd, J=7.6, 4.6 Hz, 1 H) 3.40 (dt, J=7.5, 4.9 Hz, 1 H) 4.13 (d, J=11.3 Hz, 1 H) 4.30 (dd, J=11.1, 4.7 Hz, 1 H) 5.78 (s, 1 H) 6.57 (br. s., 1 H) 7.32 (dd, J=8.4, 0.7 Hz, 1 H) 7.44 (s, 1 H) 7.52 (t, J=2.7 Hz, 1 H) 7.67 (d, J=8.2 Hz, 1 H) 7.79 (m, 2 H) 8.00 (d, J=8.2 Hz, 1 H) 8.22 (d, J=2.7 Hz, 2 H) 9.51 (s, 1 H) 10.45 (br. s, 1 H) 10.61 (br. s, 1 H)

(52) Analogously, by using the corresponding carboxylic acids, the following compounds have been prepared:

1-methyl-N-(1-methyl-5-{[(3bS,4aR)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-pyrrol-3-yl)-4-4{[(1-methyl-4-nitro-1H-pyrrol-2-yl)carbonyl]amino}-1H-pyrrole-2-carboxamide [(I)] (compd. 6)

(53) ##STR00097##

(54) ESI MS: m/z 614 (MH+)

(55) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 1.52 (t, J=4.9 Hz, 1 H) 2.21 (s, 3 H) 2.28 (dd, J=7.6, 4.3 Hz, 1 H) 3.43 (dt, J=7.8, 5.2 Hz, 1 H) 3.76 (s, 3 H) 3.86 (s, 3 H) 3.96 (s, 3 H) 4.16 (d, J=10.7 Hz, 1 H) 4.29 (dd, J=10.6, 4.8 Hz, 1 H) 6.30 (s, 1 H) 6.77 (d, J=1.7 Hz, 1 H) 7.08 (d, J=1.8 Hz, 1 H) 7.25 (d, J=1.7 Hz, 1 H) 7.48 (d, J=1.7 Hz, 1 H) 7.58 (t, J=1.9 Hz, 2 H) 8.19 (d, J=1.8 Hz, 1 H) 10.00 (s, 1 H) 10.30 (s, 1 H)

(3bS,4aR)-3-methyl-6-[(2E)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-enoyl]-4,4a,5,6-tetrahydro-8H-cyclopropa[c]thieno[3,2-e]indo-8-one [(I)] (compd. 7)

(56) ##STR00098##

(57) ESI MS: m/z 388 (MH+)

(58) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 1.45 (t, J=4.8 Hz, 1 H) 2.19 (dd, J=7.6, 4.5 Hz, 1 H) 2.26 (d, J=0.7 Hz, 3 H) 3.44 (dt, J=7.7, 4.8 Hz, 1 H) 4.40 (dd, J=10.4, 4.8 Hz, 1 H) 4.51 (d, J=10.3 Hz, 1 H) 6.98 (d, J=15.4 Hz, 1 H) 7.08 (br. s., 1 H) 7.24 (dd, J=7.9, 4.7 Hz, 1 H) 7.44 (s, 1 H) 7.98 (d, J=15.4 Hz, 1 H) 8.09 (s, 1 H) 8.34 (dd, J=4.8, 1.5 Hz, 1 H) 8.41 (dd, J=7.8, 1.3 Hz, 1 H) 11.30 (s, 1 H)

(3bR,4aS)-3-methyl-6-[(2E)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)prop-2-enoyl]-4,4a,5,6-tetrahydro-8H-cyclopropa[c]thieno[3,2-e]indol-8-one [(I)] (compd. 8)

(59) ##STR00099##

(60) ESI MS: m/z 388 (MH+)

(61) .sup.1H NMR (500 MHz, acetone-d6) ppm 1.45 (t, J=4.9 Hz, 1 H) 2.16-2.21 (m, 1 H) 2.26 (s, 3 H) 3.44 (dt, J=7.7, 4.9 Hz, 1 H) 4.40 (dd, J=10.4, 5.1 Hz, 1 H) 4.51 (d, J=10.4 Hz, 1 H) 6.98 (d, J=15.4 Hz, 1 H) 7.08 (br. s., 1 H) 7.24 (dd, J=7.8, 4.5 Hz, 1 H) 7.44 (s, 1 H) 7.98 (d, J=15.4 Hz, 1 H) 8.09 (s, 1 H) 8.34 (dd, J=4.7, 1.1 Hz, 1 H) 8.41 (d, J=7.8 Hz, 1 H) 11.29 (br. s., 1 H)

N-(3-{(1E)-3-[(3bR,4aS)-3-methyl-8-oxo 4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide [(I)] (compd. 15)

(62) ##STR00100##

(63) ESI MS: m/z 546 (MH+)

N-(3-{(1E)-3-[(3bR,4aS)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]-3-oxoprop-1-en-1-yl}-1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-indole-2-carboxamide [(I)] (compd. 16)

(64) ##STR00101##

(65) ESI MS: m/z 546 (MH+)

(66) .sup.1H NMR (500 MHz, acetone-d6) ppm 1.58 (m, 1H) 2.24 (m, 1H) 2.27 (s, 3 H) 3.47 (dt, J=7.6, 4.9 Hz, 1 H) 4.39 (dd, J=10.2, 4.9 Hz, 1 H) 4.48 (m, 1 H) 6.96 (d, J=15.1 Hz, 1 H) 7.01 (s, 1 H) 7.12 (m, 2 H) 7.28 (m, 1 H) 7.40 (d, J=1.8 Hz, 1 H) 7.45 (s, 1 H) 7.61 (d, J=8.3 Hz, 1 H) 7.70 (d, J=8.1 Hz, 1 H) 7.98 (m, 1 H) 8.09 (d, J=2.8 Hz, 1 H) 8.76 (d, J=2.0 Hz, 1 H) 8.87 (d, J=2.0 Hz, 1 H) 9.76 (br. s., 1 H) 10.97 (br. s., 1 H) 11.32 (br. s., 1 H)

1-methyl-N-(1-methyl-2-{[(3bS,4aR)-3-methy-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide [(I)] (compd. 45)

(67) ##STR00102##

(68) ESI MS: m/z 547 (MH+)

(69) .sup.1H NMR (500 MHz, acetone-d6) ppm 1.65 (t, J=4.9 Hz, 1 H) 2.27 (d, J=12.4 Hz, 1 H) 2.27 (d, J=0.7 Hz, 3 H) 3.43 (dt, J=7.6, 4.89 Hz, 1 H) 3.93 (s, 3 H) 4.12 (s, 3 H) 4.38 (d, J=11.1 Hz, 1 H) 4.51 (dd, J=10.8, 4.8 Hz, 1 H) 6.52 (s, 1 H) 7.08 (s, 1 H) 7.13 (m, 1 H) 7.26 (s, 1 H) 7.32 (td, J=7.7, 1.0 Hz, 1 H) 7.47 (d, J=1.0 Hz, 1 H) 7.54 (m, 2 H) 7.66 (d, J=7.8 Hz, 1 H) 7.71 (dd, J=9.1, 2.0 Hz, 1 H) 8.29 (d, J=2.0 Hz, 1 H) 9.54 (s, 1 H)

Example 2

tert-butyl {2-[(2-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1 methyl-7,8-dihydro-6H-thieno[32-e]indol-6-yl]carbonyl}-1 H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate [(II)] (compd. 18)

(70) Step b, Deprotection, Step a

(71) ##STR00103##
Step b

(72) A solution of (8S)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-ol ((XVII), 11.4 mg, 0.045 mmol), prepared as reported in GB2344818, was dissolved in dry DMF (1 mL), and treated with EDCI (35 mg, 4 eq.) and 5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indole-2-carboxylic acid (XVI) (29 mg, 1.5 eq.) The mixture was stirred for 16 h at room temperature and then was quenched by adding saturated aqueous NaCl. Isolation of the product was performed by extraction with EtOAc (4) and subsequent washing of the combined organic layers with aqueous 2M HCl (3), saturated aqueous Na.sub.2CO.sub.3 (3) and saturated aqueous NaCl (3). Organic layer was dried (Na.sub.2SO.sub.4), concentrated under vacuum to give the title compd. 18, that is then purified by flash chromatography hexane-acetone 1:1).

(73) ##STR00104##

(74) ESI MS: m/z 670 (MH.sup.+)

(75) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 1.50 (s, 9 H) 1.79 (dt, J=6.4, 3.3 Hz, 1 H) 2.61 (s, 2 H) 3.53-3.66 (m, 1 H) 3.89 (dd, J=11.4, 2.8 Hz, 1 H) 4.25 (m, 1 H) 4.73 (m, 1 H) 4.84 (d, J=10.6 Hz, 1 H) 7.21 (s, 1 H) 7.26 (s, 1 H) 7.34 (m, 2 H) 7.49 (d, J=8.8 Hz, 1 H) 7.56 (m, 1 H) 7.61 (m, 1 H) 7.96 (br. s., 2 H) 8.21 (br. s., 1 H) 8.34 (s, 1 H) 9.28 (s, 1 H) 9.52 (s, 1 H) 10.73 (br. s., 1 H) 10.80 (br. s., 1 H).

(76) By deprotection, the following compound has been obtained:

5-amino-N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-5a,7,8,8a-tetrahydro-6H-thieno[3,2-e]indo-6-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide [(II)] (compd. 19)

(77) ##STR00105##

(78) A solution of compd. 18 (18 mg, 0.0268 mmol) in 3.5 M HCl-EtOAc (5 mL) was stirred for 30 minutes before removing the solvent under a steady stream of nitrogen and affording hydrochloride derivative of compd. 2 (15 mg, 89%).

(79) ESI MS: m/z 570 (MH.sup.+)

(80) Step a

tert-butyl {2-[(2-{[(3bR,4aS)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate [(I)] (compd. 20)

(81) ##STR00106##

(82) Compd. 18 (30 mg, 0.045 mmol) was dissolved in DMF (1 mL), and treated with aqueous NaHCO.sub.3 (0.5 mL, 15 mg NaHCO.sub.3/mL). The reaction mixture was stirred for 2 h, EtOAc was added and the resulting organic layer was washed with brine (4), dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The crude residue was purified by flash chromatography (hexane-acetone 6:4) to afford the final compd. 20 (9 mg, 31%).

(83) ESI MS: m/z 634 (MH.sup.+)

(84) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 1.41 (s, 9 H) 1.56 (t, J=4.8 Hz, 1 H) 2.22 (m, 1 H) 2.29 (d, J=0.9 Hz, 3 H) 3.52 (m, 1 H) 4.69 (m, 2 H) 7.06 (s, 1 H) 7.26 (d, J=1.5 Hz, 1 H) 7.28 (d, J=1.8 Hz, 1 H) 7.36 (dd, J=8.8, 1.8 Hz, 1 H) 7.48 (m, 1 H) 7.49 (d, J=8.8 Hz, 1 H) 7.58 (m, 1 H) 7.63 (m, 1 H) 7.96 (br. s., 1 H) 8.26 (br. s., 1 H) 8.35 (s, 1 H) 9.55 (s, 1 H) 10.77 (br. s., 1 H) 10.95 (br. s., 1 H)

(85) By deprotection the following compound has been prepared:

5-amino-N-(2-{[(3bR,4aS)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide [(I)] (compd. 21)

(86) ##STR00107##

(87) ESI MS: m/z 534 (MH.sup.+)

(88) By analogue procedure and using the suitable starting material the following compounds have been prepared:

tert-butyl {2-[(2-{[(3bS,4aR)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl}carbamate [(I)] (compd. 25)

(89) ##STR00108##

(90) ESI MS: m/z 634 (MH.sup.+)

5-amino-N-(2-{[(3bS,4aR)-3-methy oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide [(1)] (compd. 26)

(91) ##STR00109##

(92) ESI MS: m/z 534 (MH.sup.+)

N-(2-{[(3bR,4aS)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide [(I)] (compd. 29)

(93) ##STR00110##

(94) ESI MS: m/z 564 (MH.sup.+)

N-(2-{[(3bS,4aR)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indol-6(8H)-yl]carbonyl}-1H-indol-5-yl)-5-nitro-1H-indole-2-carboxamide [(I)] (compd. 30)

(95) ##STR00111##

(96) ESI MS: m/z 564 (MH.sup.+)

Example 3

(97) Step b, Deprotection, Step a

(98) ##STR00112##
Step b

The Intermediate

(8R)N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-carboxamide

(99) A solution of 4-amino-N-[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]-1-methyl-1H-pyrrole-2-carboxamide, prepared as reported in J.Med. Chem. 2004, 47, 2611-2623, (155 mg, 0.341 mmol), triethylamine (0.048 mL, 0.341 mmol) and CDI (300 mg, 1.71 mmol) in dry DMF (5 mL) was stirred at room temperature for 1 h. After evaporation of the solvent, the residue was treated with THF and stirred overnight. The precipitate (comp XVI) was filtered and dried under vacuum at 50 C. and treated with (8R)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole (comp (XVII) (15.5 mg, 0.045 mmol, prepared as reported in the GB2344818) in dry DMF (2 mL) in the presence of NaHCO.sub.3 (4 mg, 0.047 mmol) and were stirred under nitrogen atmosphere at room temperature for 16 h. After evaporation of the solvent the residue was purified by flash chromatography (DCM-MeOH 95:5) to afford the intermediate (30 mg, 80%).

(100) ##STR00113##

(101) ESI MS: m/z 824 (MH+)

(102) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 2.31 (t, J=7.2 Hz, 2 H) 2.53 (s, 3 H) 3.53 (t, J=10.4 Hz, 1 H) 3.80 (s, 3 H) 3.85 (s, 3 H) 3.87 (s, 3 H) 4.12 (m, 1 H) 4.18 (m, 1 H) 4.32 (d, J=10.4 Hz, 1 H) 5.27 (s, 2 H) 6.83 (d, J=1.7 Hz, 1 H) 6.84 (m, 1 H) 7.05 (m, 2 H) 7.13 (d, J=1.5 Hz, 1 H) 7.20 (d, J=1.5 Hz, 1 H) 7.25 (d, J=1.5 Hz, 1 H) 7.36 (m, 2 H) 7.41 (s, 1 H) 7.43 (m, 2 H) 7.50 (d, J=7.3 Hz, 2 H) 7.99 (m, 2 H) 8.77 (s, 1 H) 9.91 (s, 1 H) 9.93 (s, 1 H)

(103) Analogously, by using the corresponding acyl derivative, the following compounds have been prepared:

(8S)N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl)carbamoyl}-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-yl)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide

(104) ##STR00114##

(105) ESI MS: m/z 824 (MH+)

N-(2-{[(8S)-4-(benzyloxy)-8-(chloromethyl)-1-methy-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide

(106) ##STR00115##

(107) ESI MS: m/z 758 (MH+)

(108) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 1.74 (m, 4 H) 2.59 (m, 4 H) 2.63 (d, J=1.0 Hz, 3 H) 2.86 (m, 2H) 3.65 (dd, J=11.4, 9.6 Hz, 1 H) 3.93 (dd, J=11.4, 2.4 Hz, 1 H) 4.12 (t, J=6.1 Hz, 2 H) 4.29 (m, 1 H) 4.77 (m, 1 H) 4.87 (m, 1 H) 5.36 (m, 2 H) 6.94 (dd, J=8.8, 2.3 Hz, 1 H) 7.15 (d, J=2.3 Hz, 1 H) 7.25 (m, 2 H) 7.35 (m, 1 H) 7.38 (d, J=1.0 Hz, 1 H) 7.43 (m, 2 H) 7.49 (d, J=9.1 Hz, 1 H) 7.58 (m, 3 H) 7.63 (m, 1 H) 8.18 (br. s., 1 H) 8.37 (d, J=1.3 Hz, 1 H) 9.52 (s, 1 H) 10.77 (br. s., 1 H) 10.87 (br. s., 1 H)

[(8S)-4-(benzyloxy)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanone hydrochloride

(109) ##STR00116##

(110) ESI MS: m/z 600 (MH+)

(111) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 2.06 (m, 4H) 2.62 (d, J=1.0 Hz, 3 H) 3.02-3.32 (m, 2 H) 3.56 (br. s., 2 H) 3.63 (dd, J=11.4, 9.6 Hz, 1 H) 3.91 (dd, J=11.4, 2.4 Hz, 1 H) 4.27 (m, 1 H) 4.57 (br. s., 2 H) 4.73 (dd, J=10.2, 8.2 Hz, 1 H) 4.84 (m, 1 H) 5.35 (m, 2 H) 7.05 (dd, J=8.8, 2.3 Hz, 1 H) 7.16 (d, J=1.7 Hz, 1 H) 7.31 (d, J=1.8 Hz, 1 H) 7.35 (m, 1 H) 7.38 (d, J=1.0 Hz, 1 H) 7.43 (m, 2 H) 7.52 (d, J=9.1 Hz, 1 H) 7.57 (d, J=7.6 Hz, 2 H) 8.16 (br. s., 1 H) 10.80 (br. s., 1 H) 13.38 (br. s., 1 H)

(112) Deprotection

(8R)N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide [(II)] (compd. 10)

(113) A solution of the intermediate (15 mg, 0.0182 mmol), 25% aq. HCO.sub.2NH.sub.4 (0.15 mL) and 10% PdC (15 mg) in THF (3 mL) was stirred for 3 h under nitrogen atmosphere. The reaction mixture was filtered through Celite and concentrated to yield after chromatographic purification (DCM-MeOH 10:1) compound 10 (6.7 mg, 50%).

(114) ##STR00117##

(115) ESI MS: m/z 734 (MH+)

(116) Analogously the following compounds have been prepared:

(8S)N-(5-{[5-{5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indole-6-carboxamide [(II)] (compd. 11)

(117) ##STR00118##

(118) ESI MS: m/z 734 (MH+)

N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide [(II)] (compd. 17)

(119) ##STR00119##

(120) ESI MS: m/z 668 (MH+)

(121) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 2.19-2.25 (m, 4 H) 2.60 (s, 3 H) 3.39 (br. s., 2 H) 3.81 (m, 2 H) 3.88 (dd, J=11.1, 2.5 Hz, 1 H) 3.91-3.96 (br. s, 2 H) 4.24 (m, 1 H) 4.51 (m, 1 H) 4.72 (dd, J=10.8, 7.8 Hz, 1 H) 4.80-4.82 (d, J=10.8, 1 H) 7.01 (dd, J=8.9, 2.4 Hz, 1 H) 7.22 (s, 1 H) 7.26 (d, J=2.3 Hz, 1 H) 7.28 (s, 1 H) 7.33 (s, 1 H) 7.49 (d, J=8.9 Hz, 1 H) 7.55 (m, 1 H) 7.60 (m, 1 H) 7.90 (br. s., 1 H) 8.29 (d, J=1.8 Hz, 1 H)

[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}methanone hydrochloride [(II)] (compd. 37)

(122) ##STR00120##

(123) ESI MS: m/z 510 (MH+)

(124) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 2.10-2.29 (m, 4 H) 2.58 (d, J=1.3 Hz, 3 H) 3.33 (m, 2 H) 3.54 (dd, J=11.4, 10.1 Hz, 1 H) 3.80 (m, 2 H) 3.89 (m, 3 H) 4.20 (m, 1 H) 4.51 (m, 2 H) 4.68 (dd, J=10.2, 8.2 Hz, 1 H) 4.77 (m, 1 H) 7.04 (dd, J=8.9, 2.4 Hz, 1 H) 7.12 (s, 1 H) 7.29 (d, J=2.3 Hz, 1 H) 7.31 (d, J=0.5 Hz, 1 H) 7.49 (d, J=8.9 Hz, 1 H) 7.87 (br. s., 1 H)

[(8R)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]{5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl})methanone hydrochloride [(II)] (compd. 38)

(125) ##STR00121##

(126) ESI MS: m/z 510 (MH+)

(127) Step a

(128) A solution of compound 10 (5 mg, 0.0068 mmol), in DMF (5 mL), was treated with a solution of NaHCO.sub.3 in water (3 mL, 15 mg NaHCO.sub.3/mL). The reaction mixture was stirred for 4 h, EtOAc was added and the resulting organic layer was washed with brine (4), dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The crude residue was purified by flash chromatography (DCM-MeOH 10:1) to afford the compound 9 (3.9 mg, 82%).

(3bS,4aR)-N-(5-{[5-({5-[(3-amino-3-oxopropyl)carbamoyl]-1-methyl-1H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indole-6(8H)-carboxamide [(I)] (compd. 9)

(129) ##STR00122##

(130) ESI MS: m/z 698 (MH+)

(131) .sup.1H NMR (500 MHz, dichloromethane-d.sub.2) ppm 1.37 (m, 1H) 2.12 (dd, J=7.6, 4.6 Hz, 1 H) 2.18 (d, J=0.9 Hz, 3 H) 2.47 (t, J=6.6 Hz, 2 H) 3.26 (dt, J=7.6, 5.0 Hz, 1 H) 3.53 (1 , J=6.6 Hz, 2 H) 3.85 (s, 3 H) 3.88 (s, 6 H) 4.05 (d, J=10.4 Hz, 1 H) 4.14 (m, 1H) 6.64 (d, J=2.1 Hz, 1 H) 6.73 (s, 1 H) 6.82 (m, 2 H) 6.99 (d, J=1.8 Hz, 1 H) 7.17 (d, J=1.8 Hz, 1 H) 7.20 (d, J=1.8 Hz, 1 H) 7.29 (d, J=0.9 Hz, 1 H)

(132) Analogously the following compounds have been prepared:

(3bR,4aS)N-(5-{5-({5-[(3-amino-3-oxopropyl)carbamoyl-1-methyl-H-pyrrol-3-yl}carbamoyl)-1-methyl-1H-pyrrol-3-yl]carbamoyl}-1-methyl-1H-pyrrol-3-yl)-3-methyl-8-oxo-4a,5-dihydro-4H-cyclopropa[c]thieno[3,2-e]indole-6(8H)carboxamide [(I)] (compd. 12)

(133) ##STR00123##

(134) ESI MS: m/z 698 (MH+)

Example 4

(135) Step c, Deprotection

(136) ##STR00124##
Step c

(8S)-6-({5-[({5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate [(II)](compd. 22)

(137) ##STR00125##

(138) To a solution of tert-butyl {2[(2-[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)carbamoyl]-1H-indol-5-yl)carbamate, (compd. 18) (42 mg, 0.064 mmol) in dry DCM (6 mL) 4-methylpiperazine-1-carbonyl chloride hydrochloride (XIII) (39 mg, 0.193 mmol) and N,N-dimethylaminopyridine (27 mg, 0.212 mmol) were added. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 16 h. The solvent was evaporated and the residue was dissolved in EtOAc, the resulting organic layer was washed with brine (3), dried (Na.sub.2SO.sub.4) and concentrated in vacuo. The crude residue was purified by flash chromatography (DCM-MeOH 95:5) to afford the compd. 22 (30 mg, 59%).

(139) ESI MS: m/z 796 (MH.sup.+)

(140) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 2.22 (s, 3 H) 2.65 (d, J=0.9 Hz, 3 H) 2.39-2.91 (m, 4H) 3.72 (dd, J=11.6, 9.5 Hz, 1 H) 3.96 (dd, J=11.6, 3.1 Hz, 1 H) 3.49-4.14 (m, 4H) 4.37 (m, 1 H) 4.79-4.85 (m, 1 H) 4.87-4.93 (m, 1 H) 7.27 (d, J=1.5 Hz, 1 H) 7.28 (s, 1 H) 7.34-7.38 (m, 1 H) 7.41 (s, 1 H) 7.50 (d, J=8.8 Hz, 1 H) 7.56 (d, J=8.5 Hz, 1 H) 7.62-7.65 (m, 1 H) 7.96 (br. s., 1 H) 8.38 (s, 1 H) 9.60 (s, 1 H) 10.86 (br. s., 1 H) 10.90 (br. s., 1 H)

(141) By analogous procedure the following products have been prepared:

(8R)-6-[5-{[(5-[(tert-butoxycarbonyl)amino]-1H-indol-2-yl)carbonyl]amino}-1H-indo-2-yl)carbonyl]-8-(chloromethyl)-1-methy-7,8-dihydro-6H-thieno[3,2-e]indo-4-yl 4-methylpiperazine-1-carboxylate [(II)](compd. 27)

(142) ##STR00126##

(143) ESI MS: m/z 796 (MH.sup.+)

(8S)-8-(chloromethyl)-1-methy-6-[(5-{[(5-nitro-1H-idol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl-4-methylpiperazine-1-piperazine-1-carboxylate [(II)] (compd. 31)

(144) ##STR00127##

(145) ESI MS: m/z 726 (MH.sup.+)

(8R) 8-(chloromethyl)-1-methyl-6-[(5-{[(5-nitro-1H-indol-2-yl)carbonyl]amino}-1H-indo-2-yl)carbonyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate [(II)] (compd. 32)

(146) ##STR00128##

(147) ESI MS: m/z 726 (MH.sup.+)

(148) Deprotection

(8S)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indo-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate hydrochloride [(II)] (compd. 23)

(149) ##STR00129##

(150) A solution of compd. 22 (22 mg, 0.0276 mmol) in 3.5 M HCl-EtOAc (5 mL) was stirred for 30 minutes before removing the solvent under a steady stream of nitrogen and affording the desired product as hydrochloride salt (18 mg, 89%).

(151) ESI MS: m/z 696 (MH.sup.+)

(152) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 2.59 (s, 3 H) 2.76 (br. s, 3H) 3.05-3.42 (m, 4H) 3.75 (dd, J=10.8, 7.9 Hz, 1 H) 3.95-4.01 (m, 1 H) 3.58-4.08 (m, 4H) 4.32-4.40 (m, 1 H) 4.67 (d, J=11.1 Hz, 1 H) 4.75-4.83 (m, 1 H) 6.92 (br. s., 1 H) 7.21 (br. s., 1 H) 7.23 (s, 1 H) 7.30 (br. s., 1 H) 7.37 (d, J=8.4 Hz, 1 H) 7.49-7.52 (m, 1 H) 7.55 (s, 1 H) 7.57-7.60 (m, 1 H) 8.16 (s, 1 H) 8.24 (s, 1 H) 10.15 (br. s., 1 H) 11.67 (br. s., 1H) 11.70 (s, 1 H)

(153) By analogous procedure the following products have been prepared:

(8R)-6-[(5-{[(5-amino-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-8-(chloromethyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate hydrochloride [(II)] (compd. 28)

(154) ##STR00130##

(155) ESI MS: m/z 697 (MH.sup.+)

(8S)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-ethyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate [II)] (compd. 33)

(156) ##STR00131##

(157) ESI MS: m/z 697 (MH.sup.+)

(8R)-8-(chloromethyl)-6-[(5-{[(5-hydroxy-1H-indol-2-yl)carbonyl]amino}-1H-indol-2-yl)carbonyl]-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl 4-methylpiperazine-1-carboxylate [(II)] (compd. 34)

(158) ##STR00132##

(159) ESI MS: m/z 697 (MH.sup.+)

Example 5

(160) Step c, Deprotection

(161) ##STR00133##
Step c

Preparation of tert-butyl (8S)-8-(chloromethyl)-6-({5-[(1H-indol-2-ylcarbonyl)amino]-1H-indol-2-yl}carbonyl) -1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl piperazine-1,4-dicarboxylate [(II)]

(162) ##STR00134##

N-(2-{[(8S)-8-chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-1H-indole-2-carboxamide (111 mg, 0.2 mmol), prepared as reported in GB2344818, was dissolved in dry DCM (15 mL) and to this solution tert-butyl 4-(chlorocarbonyl)piperazine-1-carboxylate (100 mg, 0.4 mmol) and N,N-dimethylaminopyridine (55 mg, 0.45 mmol) were added. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 16 h. The solvent was evaporated and the residue was dissolved in EtOAc, the resulting organic layer was washed with brine (4), dried (Na2SO4) and concentrated in vacuo. The crude residue was purified by flash chromatography (hexane-acetone 7:3) to afford the title compound (30 mg, 19%).

(163) ESI MS: m/z 767 (MH.sup.+)

(164) .sup.1H NMR (500 MHz, acetone-d.sub.6) ppm 1.48 (s, 9 H) 2.65 (d, J=1.01 Hz, 3 H) 3.55 (br. s., 4 H) 3.60 (br. s., 2 H) 3.73 (dd. J=11.36, 9.59 Hz, 1 H) 3.79 (br. s., 2 H) 3.97 (dd, J=11.36, 2.78 Hz, 1 H) 4.33-4.42 (m, 1 H) 4.80-4.86 (m, 1 H) 4.89-4.94 (m, 1 H) 7.10 (t, J=7.19 Hz, 1 H) 7.23-7.27 (m, 1 H) 7.28 (s, 1 H) 7.34 (d, J=1.51 Hz, 1 H) 7.42 (s, 1 H) 7.53-7.57 (m, 1 H) 7.60 (d, J=8.08 Hz, 1 H) 7.61-7.65 (m, 1 H) 7.67 (d, J=8.08 Hz, 1 H) 8.29 (s, 1 H) 8.38 (d, J=1.26 Hz, 1 H) 9.56 (s, 1 H) 10.89 (br. s., 2 H).

(165) Deprotection

(8S)-8-(chloromethyl)-6-({5-[(1H-indo-2-yl}carbonyl)amino]-1H-indo-2-ylcarbonyl)-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl piperazine-1-carboxylate [(II)] (compd. 24)

(166) ##STR00135##

(167) A solution of the intermediate (25 mg, 0.0326 mmol) in 3.5 M HCl-EtOAc (5 mL) was stirred for 2 h. After evaporation of the solvent under a steady stream of nitrogen, the residue was dried in vacuo to afford the compd. 9 (11 mg, 48%).

(168) ESI MS: m/z 667 (MH.sup.+)

Example 6

(169) Step e

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)] (compd. 35)

(170) ##STR00136##
Step e

(171) To a solution of N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide (Compd 17) (5 mg, 0.0071 mmol) in DCM (1 ml) and THF (0.5 mL) were added 4-nitrophenyl chloroformate (3.1 mg, 0.0156 mmol) and triethylamine (2.2 mL, 0.0156 mmol). The resulting mixture was stirred at room temperature for 6 hours. N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N.sup.5-carbamoyl-N-{4-[({[2,2-dimethyl-3-(methylamino)propyl] (methyl)carbamoyl}oxy)methyl]phenyl}-L-omithinamide (XX) (16 mg, 0.018 mmol) and triethylamine (3 mL, 0.021 mmol) were added. The mixture thus obtained was stirred at room temperature overnight, diluted with DCM and washed with a saturated solution of aqueous NaHCO.sub.3. The crude residue was purified by flash chromatography (DCM-MeOH 100:15) to afford the title compound (2 mg, 20%).

(172) ESI MS: m/z 1422 (MH+)

(173) .sup.1H NMR (500 MHz, methanol-d.sub.3) ppm 0.82-1.45 (m 29H) 2.68 (s, 3H) 2.90-3.61 (m, 20 H) 3.84-4.34 (m, 8H) 4.49 (m, 1H) 5.04-5.14 (m, 2H) 6.70-6.77 (m, 2H) 7.01 (dd, J=9.0, 2.4, 1H) 7.18-7.71 (m, 11H) 8.11 (br. s., 2H)

(174) Analogously the following compounds have been prepared:

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1 yl)hexanoyl]-L-valyl-N5-carbamoyl-N[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-omithinamide [(IV)] (Compd 36).

(175) ##STR00137##

(176) ESI MS: m/z 1424 (MH.sup.+)

N-[6-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)] (Compd. 39)

(177) ##STR00138##

(178) ESI MS: m/z 1264 (MH.sup.+)

N-[6-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-({[{3-[({[(8R)-8-(chloromethyl)-1-methyl-6-({5-[2-pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)] (Compd 40).

(179) ##STR00139##

(180) ESI MS: m/z 1264 (MH+)

N-[6-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-({[(3-{[({(8S)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{5-[2-pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)] (Compd 41).

(181) ##STR00140##

(182) ESI MS: m/z 1290 (MH+)

N-[6-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-({[(3-{[({(8R)-8-(chloromethyl)-1-methyl-6-[(2E)-3-{-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}prop-2-enoyl]-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl}oxy)carbonyl](methy)amino}-2,2-dimethylpropyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)] (Compd. 42)

(183) ##STR00141##

(184) ESI MS: m/z 1290 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide [(IV)] (Compd 46)

(185) ##STR00142##

(186) ESI MS: m/z 1483 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{3-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]-2,2-dimethylpropyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide [(IV)] (Compd 47)

(187) ##STR00143##

(188) ESI MS: m/1325 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide [(IV)] (Compd 48)

(189) ##STR00144##

(190) ESI MS: m/z 1441 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1 yl)hexanoyl]-L-phenylalanyl-L-leucyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methy-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indo-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]glycinamide [(IV)](Compd 49)

(191) ##STR00145##

(192) ESI MS: m/z 1283 (MH+)

Example 7

(193) Step c, Deprotection, Step g

(194) ##STR00146## ##STR00147##
Step c

N-[(9H fluoren-9-ylmethoxy)carbonyl]-L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2 yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(II)]

(195) ##STR00148##

(196) To a solution of N-(2-{[(8S)-8-(chloromethyl)-4-hydroxy-1-methyl-7,8-dihydro-6H-thieno[3,2-e]indol-6-yl]carbonyl}-1H-indol-5-yl)-5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indole-2-carboxamide hydrochloride (13 mg, 0.02 mmol) in THF/DCM 1:1 (2 mL) at 0 C. under nitrogen atmosphere, were added 4-nitrophenyl chloroformate (20 mg, 0.1 mmol) and triethylamine (20 mL, 0.14 mmol). The mixture was stirred at room temperature for 6 hours, solvent was evaporated and the residue was isolated by filtration after treatment with diethyether. The resulting solid compound was diluted with 1 mL of DCM (10% DMF) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N.sup.5-carbamoyl-N-{4-[({methyl[2- (methylamino)ethyl]carbamoyl}oxy)methyl]phenyl}-L-omithinamide hydrochloride (38 mg, 0.05 mmol) and trietylamine (7 mL 0.05 mmol) were added. The mixture was stirred overnight, solvents were evaporated and title compound (12 mg, 42% yield) was isolated by column chromatography purification (DCM/MeOH 8:2).

(197) ESI MS: m/z 1409 (MH+)

(198) .sup.1H NMR (500 MHz, methanol-d.sub.4) ppm 2.07 (br. s., 4 H) 2.58 (s, 3 H) 2.93-3.09 (m, 6 H) 3.40-3.69 (m, 8 H) 4.11-4.40 (m, 3 H) 4.43-4.81 (m, 3 H) 4.99-5.17 (m, 2 H) 6.92-7.44 (m, 14 H) 7.46-7.64 (m, 4 H) 7.75 (br. s., 3 H) 8.01-8.22 (m, 2 H) 8.01-8.06 (m, 1 H)

(199) Analogously the following compound has been prepared:

N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-chloromethyl)-1-methyl-6-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide

(200) ##STR00149##

(201) ESI MS: m/z 1251 (MH+)

(202) .sup.1H NMR (500 MHz, methanol-d.sub.4) ppm 0.94-1.01 (m, 6 H) 1.51-1.95 (m, 4 H) 2.12 (br. s., 5 H) 2.48-2.62 (m, 3 H) 2.89-3.05 (m, 4 H) 3.40-3.54 (m, 4 H) 3.46-3.65 (m, 4 H) 3.58-3.74 (m, 2 H) 3.75-4.05 (m, 1 H) 4.08-4.42 (m, 3 H) 4.50 (m, 1 H) 4.55-4.81 (m, 4 H) 4.96-5.20 (m, 2 H) 6.92-7.81 (m, 17 H) 8.04 (br. s., 1 H)

(203) Deprotection

L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(II)] (Compd 50)

(204) ##STR00150##

(205) To a solution of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N.sup.5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methy)amino]ethyl}methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide (11 mg, 0.0078 mmol) in DMF (1 mL) was added piperidine (4 mL, 0.039 mmol). The mixture was stirred 1 hour at room temperature, solvent was evaporated and the resulting title compound was used without further purification.

(206) Analogously the following compound has been prepared:

L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-chloromethyl)-1-methyl-6-({5-[2-pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(II)] (Compd 51)

(207) ##STR00151##
Step g

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8 dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)] (Compd 52)

(208) ##STR00152##

(209) To a solution of L-valyl-N.sup.5-carbamoyl-N-[4-({[{2-[({(8S)-8-chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino)ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide (4.6 mg, 0.0039 mmol) in 1 mL of DCM (10% DMF), were added 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (3.6 mg, 0.0117 mmol) and triethylamine (2.2 mL, 0.0156 mmol). The mixture was stirred at room temperature for 3 hours, solvents were evaporated and the title compound (2.2 mg, 41% yield) was isolated by column chromatography purification (DCM/MeOH 8:2).

(210) ESI MS: m/z 1380 (MH+)

(211) .sup.1H NMR (500 MHz, methanol-d.sub.4) ppm 1.98 (br. s., 4 H) 2.60-2.63 (m, 3 H) 2.89-3.11 (m, 6 H) 3.39-3.72 (m, 10H) 4.07-4.75 (m, 7H) 4.98-5.17 (m, 2H) 6.74 (br. s., 2 H) 7.00 (m, 2 H) 7.07-7.45 (m, 8 H) 7.52 (br. s., 3 H) 8.07-8.11 (m, 1 H)

(212) Analogously the following compounds have been prepared:

N-[19-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-oyl]-L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)] (Compd 53)

(213) ##STR00153##

(214) ESI MS: m/z 1585 (MH+)

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-({[{2-[({[(8S)-8-(chloromethyl)-1-methyl-({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)](Compd 54)

(215) ##STR00154##

(216) ESI MS: m/z 1222 (MH+)

(217) .sup.1H NMR (400 MHz, dmf-d7) ppm 0.95 (t, J=7.8 Hz, 6 H) 2.16 (m, 1 H) 2.66 (br. s., 3 H) 2.99-3.09 (m, 3 H) 3.09-3.29 (m, 3 H) 3.59 (br. s., 2 H) 3.69 (br. s., 2 H) 3.81 (br. s., 2 H) 4.04 (d, J=10.7 Hz, 1 H) 4.31-4.47 (m, 2 H) 4.61 (br. s., 1 H) 4.83 (br. s., 2 H) 5.11 (d, J=15.7 Hz, 2 H) 5.60 (s, 2 H) 6.29 (br. s., 1 H) 7.00 (m, 3 H) 7.30 (m, 2 H) 7.53 (d, J=8.5 Hz, 2 H) 7.88 (d, J=8.2 Hz, 1 H) 8.13 (d, J=7.8 Hz, 1 H) 8.27 (s, 1 H) 10.1 (m, 1 H) 11.61 (br. s., 1 H)

Preparation of the Intermediate

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-{4-[({[2,2-dimethyl-3-(methylamino)propyl] (methyl)carbamoyl}oxy)methyl]phenyl}-L-ornithinamide (XX)

(218) ##STR00155##
Step 1

N,N-(2,2-dimethylpropane-1,3-diyl)diformamide

(219) ##STR00156##

(220) In a round bottomed flask, commercially available 2,2-dimethylpropane-1,3-diamine (851 mg, 8.32 mmol) was reacted with ethylformiate (4.2 ml). The reaction mixture was stirred at 60 C. for 3 hours, until no starting material was detectable (TLC analysis, MeOH:CH.sub.2Cl.sub.2=2:8). The reaction mixture was then evaporated under vacuum, affording the crude product (1.4 g, oil).

(221) ESI MS: m/z 159 (MH+)

(222) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.79 (s, 6 H) 2.92 (d, J=6.41 Hz, 4 H) 7.94 (br. s., 2 H) 8.05-8.09 (m, 2 H)

(223) Step 2

N,N,2,2-tetramethylpropane-1,3-diamine

(224) ##STR00157##

(225) In a dried round bottomed flask, containing N,N-(2,2-dimethylpropane-1,3-diyl)diformamide (1.4 g, 8.85 mmol) cooled at 0 C. under argon atmosphere, a solution (24 ml) of Lithium Aluminium Hydride in tetrahydrofuran (1M) was added. The reaction mixture was stirred at room temperature for 28 hours. After cooling at 0 C., a solution of water in tetrahydrofuran was added, and the resulting mixture was filtered on a celite pad. The filtrate was dried over anhydrous sodium sulfate, filtered and the product thus obtained as a tetrahydrofuran solution was used without further purification in the next step.

(226) Step 3

2-(biphenyl-4-yl)propan-2-yl [2,2-dimethyl-3-(methylamino)propyl]methylcarbamate

(227) ##STR00158##

(228) Commercial methyl 4-[({[2-(biphenyl-4-yl)propan-2-yl]oxy}carbonyl)oxy]benzoate (5.54 mmol, 2.16 g) was added to the tetrahydrofuran solution of N,N,2,2-tetramethylpropane-1,3-diamine. The reaction mixture was stirred at room temperature for 24 hours. The solvent was then evaporated under vacuum and the residue was purified by flash chromatography (MeOH:CH.sub.2Cl.sub.2=1:9) affording the desired product (848 mg).

(229) MS (ESI): 369 (MH+)

(230) .sup.1H NMR (600 MHz, DMSO-d.sub.6) ppm 0.73-0.83 (m, 5 H) 0.91 (br. s., 3 H) 1.65-1.77 (m, 6 H) 2.78 (br. s., 1 H) 7.32-7.38 (m, 1 H) 7.41 (d, J=8.43 Hz, 2 H) 7.46 (t, J=7.69 Hz, 2 H) 7.58-7.69 (m, 4 H)

(231) Analogously the following compound has been prepared:

2-(biphenyl-4-yl)propan-2-yl methyl[2-(methylamino)ethyl]carbamate

(232) ##STR00159##

(233) MS (ESI): 327 (MH+)

(234) .sup.1H NMR (401 MHz, DMSO-d.sub.6) ppm 1.70-1.75 (m, 6 H) 2.23-2.39 (m, 3 H) 7.30-7.39 (m, 1 H) 7.40-7.49 (m, 4 H) 7.58-7.63 (m, 2 H) 7.63-7.68 (m, 2 H)

(235) Step 4

N-[6-(2,5-dioxo-25-dihydro-1H-pyrrol-1-yl)hexanoyl]L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa-4,8-diazadodec-1-yl]phenyl}-N5-carbamoyl-L-ornithinamide

(236) ##STR00160##

(237) To a solution of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N.sup.5-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-omithinamide (0.492 mmol, 363 mg) in anhydrous dimethylsulfoxide (1.0 ml), a solution of 2-(biphenyl-4-yl)propan-2-yl [2,2-dimethyl-3-(methylamino)propyl]methylcarbamate (0.393 mmol, 145 mg) in anhydrous dimethylsulfoxide (0.9 ml) and triethylamine (1.5 mmol, 150 mg) were added. The reaction mixture was stirred at room temperature until no starting material was detectable, then treated with n-hexane (36 ml), and the crude residue was used without further purification in the next step.

(238) MS (ESI): 967 (MH+).

(239) Analogously the following compound has been prepared

N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl]phenyl}N5-carbamoyl-L-ornithinamide

(240) ##STR00161##

(241) MS (ESI): 954 (MH+)

(242) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 0.86 (dd, J=13.34, 6.79 Hz, 6 H) 1.69 (br. s., 6 H) 1.86-2.05 (m, 1 H) 2.76 (m, J=9.30 Hz, 2 H) 2.92 (d, J=14.95 Hz, 3 H) 3.01 (m, J=6.10 Hz, 1 H) 3.15-3.15 (m, 0 H) 3.38-3.53 (m, 2 H) 3.93 (t, J=7.63 Hz, 1 H) 4.18-4.26 (m, 2 H) 4.26-4.34 (m, 1 H) 4.42 (br. s., 1 H) 4.92-5.06 (m, 2 H) 5.39 (s, 2 H) 5.96 (t, J=6.02 Hz, 1 H) 7.18-7.48 (m, 12 H) 7.52-7.66 (m, 6 H) 7.70-7.77 (m, 2 H) 7.88 (d, J=7.47 Hz, 2 H) 8.11 (br. s., 1 H) 10.06 (br. s., 1 H)

(243) Step 5

The Title Intermediate

(244) The crude product N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2, 10-dioxa-4,8-diazadodec-1-yl]phenyl}-N.sup.5-carbamoyl-L-ornithinamide was treated with dichloroacetic acid (7.8 mmol). The reaction mixture was stirred at room temperature, until no starting material was detectable, then purified by flash column chromatography (EtOH:CH.sub.2Cl.sub.2=1.5:8.5) on silica gel, affording the desired product (44.0 mg, white solid).

(245) MS (ESI): 729 (MH+)

(246) .sup.1H NMR (500 MHz, METHANOL-d.sub.4) ppm 0.97 (d, J=4.42 Hz, 3 H) 0.98 (d, J=4.27 Hz, 3 H) 1.04 (s, 6 H) 1.23-1.35 (m, 4 H) 1.50-1.68 (m, 7 H) 1.76 (dtd, J=13.90, 9.26, 9.26, 5.11 Hz, 1 H) 1.86-1.95 (m, 1 H) 2.07 (dq, J=13.90, 6.86 Hz, 1 H) 2.26-2.31 (m, 2 H) 2.57 (s, 3 H) 2.67 (s, 2 H) 3.04 (s, 3 H) 3.11 (dt, J=13.50, 6.67 Hz, 1 H) 3.16-3.22 (m, 1 H) 3.23 (br. s., 2 H) 3.48 (t, J=7.09 Hz, 2 H) 4.14 (d, J=7.47 Hz, 1 H) 4.49 (dd, J=9.07, 5.11 Hz, 1 H) 5.13 (s, 2 H) 5.94 (s, 3 H) 6.75-6.82 (m, 1 H) 7.36 (d, J=8.54 Hz, 2 H) 7.60 (d, J=8.69 Hz, 2 H).

Preparation of Intermediate

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-{4-[({methyl[2-(methylamino)ethyl]carbamoyloxy}methyl]phenyl}-L-ornithinamide

(247) ##STR00162##
Step 6

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa-4,8-diazadodec-1-yl]phenyl}-N5-carbamoyl-L-omithinamide

(248) ##STR00163##

(249) To a solution of the product N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-val-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1yl]phenyl}-N.sup.5-carbamoyl-L-ornithinamide [prepared as reported above under step 4] (0.063 mmol, 60 mg) in anhydrous dimethylformamide (0.8 ml), piperidine (0.32 mmol, 27.2 mg) was added. The reaction mixture was stirred at room temperature until no starting material was detectable. The solvent was evaporated under vacuum and the crude thus obtained was used without further purification in the next step.

(250) MS (ESI): 732 (MH+)

(251) Step 7

N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl]phenyl}-N5-carbamoyl-L-omithinamide

(252) ##STR00164##

(253) To a solution of the crude product N-[6-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa-4,8-diazadodec-1-yl]phenyl}-N.sup.5-carbamoyl-L-omithinamide (0.0629 mmol) in anhydrous dimethylformamide (1.5 ml), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (0.189 mmol, 58.3 mg) and triethylamine (0.252 mmol, 25.5 mg) were added. The reaction mixture was stirred at room temperature until no starting material was detectable. The solvent was evaporated under vacuum and the crude was purified by flash column chromatography (eluant:EtOH:CH.sub.2Cl.sub.2=1:9) on silica gel (230-400 mesh), affording the desidered product (34 mg, white wax)

(254) MS (ESI): 925 (MH+)

(255) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 0.83 (dd, J=15.79, 6.79 Hz, 6 H) 1.31-1.40 (m, 1 H) 1.60 (br. s., 1 H) 1.69 (s, 6 H) 1.90-2.02 (m, 1 H) 2.05-2.23 (m, 2 H) 2.89-2.96 (m, 3 H) 3.01 (br. s., 1 H) 3.34-3.38 (m, 2 H) 3.46 (m, J=8.24 Hz, 2 H) 4.19 (t, J=7.40 Hz, 1 H) 4.38 (br. s., 1 H) 4.95-5.05 (m, 2 H) 5.40 (s, 2 H) 5.97 (d, J=12.20 Hz, 1 H) 6.98-7.02 (m, 2 H) 7.30 (d, J=8.85 Hz, 2 H) 7.37 (dd, J=15.40, 7.93 Hz, 3 H) 7.45 (m, J=7.17 Hz, 2 H) 7.52-7.67 (m, 6 H) 7.80 (d, J=8.24 Hz, 1 H) 8.08 (d, J=6.71 Hz, 1 H) 9.76-10.18 (m, 1 H)

(256) Step 8

The Title Intermediate

(257) Reacting the intermediate prepared under step 6 under reaction conditions reported above in step 5, the title compound as a white powder was obtained

(258) ##STR00165##