Conjugate of a tubulysin analog with branched linkers

A composition comprising at least one side chain-linkaged conjugate compound of the Formula (I):

• wherein
• “-” represents a single bond;
• n is 1 to 30;
• T is a cell-binding molecule, which is an antibody;
• L1 and L2 are, the same or different, independently selected from O, NH, N, S, P, NNH, NHNH, N(R3), N(R3)N(R3′), CH, CO, C(O)NH, C(O)O, NHC(O)NH, NHC(O)O, polyethyleneoxy unit of formula (OCH2CH2)pOR3, or (OCH2CH—(CH3))pOR3, or NH(CH2CH2O)pR3, or NH(CH2CH(CH3)O)pR3, or N[(CH2CH2O)pR3]—[(CH2CH2O)p′R3′], or (OCH2CH2)pCOOR3, or CH2CH2(OCH2CH2)pCOOR3, wherein p and p′ are independently an integer selected from 0 to about 1000, or combination thereof;
• C1-C8 alkyl;
• C2-C8 heteroalkyl, alkylcycloalkyl, or heterocycloalkyl;
• C3-C8 aryl, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl;
• or (Aa)r, r=1-12 (one to 12 amino acid units), which is composed from natural or unnatural amino acids, or the same or different sequences of dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit;
• R3 and R3′ are independently H, C1-C8 alkyl;
• C2-C8 heteroalkyl, or heterocyclic;
• C3-C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl;
• W is a stretcher unit having C1-C18, normally a self-immolative spacer, a peptidic unit, a hydrazone, a disulfide, a thioether, an ester, or an amide bond;
• w is 1 or 2 or 3;
• V1 and V2 are independently a spacer unit and selected from O, NH, S, C1-C8 alkyl, C2-C8 heteroalkyl, alkenyl, or alkynyl, C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, or alkylcarbonyl, or (Aa)r, r=1-12 (one to 12 amino acid units), which is composed from a natural or unnatural amino acid, or the same or different sequences of dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit;
• or (CH2CH2O)p, p is 0-1000;
• and v1 and v2 are independently 0, 1 or 2, but v1 and v2 are not 0 at the same time;
• when v1 or v2 is 0, it means one of the side chain Q1 or Q2 fragment is absent;
• Q1 and Q2 are independently a C2-C100 polycarboxylacid;
• a C2-C100 polyalkylamine;
• a C6-C100 oligosaccharide or polysaccharide, a C6-C100 zwitterionic betaines or zwitterionic poly(sulfobetaine)) (PSB)s that consist of a quaternary ammonium cation and a sulfonate anion;
• a C6-C100 biodegradable polymer composed of poly (lactic/glycolic acid) (PLGA), poly(acrylates), chitosan, copolymer of N-(2-hydroxypropyl)methacrylamide, poly[2-(methacryloyloxy)ethyl phosphorylcholine] (PMPC), poly-L-glutamic acid, poly(lactide-co-glycolide) (PLG), poly(lactide-co-glycolide), poly(ethylene glycol)(PEG), poly(propylene glycol)(PPG), poly(lactide-co-glycolide), poly(ethylene glycol)-modified peptides, poly(ethylene glycol)-containing an amino acid or peptides, poly(ethylene glycol)-modified lipids, poly(ethylene glycol)-modified alkylcarboxic acid, poly(ethylene glycol)-modified alkylamine, poly(lactide-co-glycolide, hyaluronic acid (HA) (glycosaminoglycan), heparin/heparan sulfate (HSGAGs), chondroitin sulfate/dermatan sulfate (CSGAGs), poly(ethylene glycol)-modified alkylsulfate, poly(ethylene glycol)-modified alkylphosphate, or poly(ethylene glycol)-modified alkyl quaternary ammonium;
• alternatively, any one or more of W, Q1, Q2, L1, L2, V1, or V2 can be independently absent but Q1, and Q2 are not absent at the same time;
• D is tubulysin analog having the following formula (II):
• or a pharmaceutically acceptable salt, hydrates, or hydrated salt;
• or a polymorphic crystalline structure;
• or an optical isomer, racemate, diastereomer or enantiomer thereof,
• wherein - - - - - is a linkage site that links to W independently;
• wherein R1, R2, R3, and R4 are independently H, C1˜C8 alkyl;
• C2˜C8 heteroalkyl, or heterocyclic;
• C3˜C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl;
• or R1R2, R1R3, R2R3, R3R4, R5R6, R11R12 or R13R14 form a 3˜7 membered carbocyclic, cycloalkyl, heterocyclic, heterocycloalkyl, aromatic or heteroaromatic ring system;
• R1 and R2 can be independently absent when they link to W independently or simultaneously, Y1 is N or CH;
• wherein R5, R6, R8, R10 and R11 are independently H, or C1˜C4 alkyl or heteroalkyl;
• wherein R7 is independently H, R14, —R14C(═O)X1R15;
• or —R14X1R15;
• X1 is O, S, S—S, NH, CH2 or NR14;
• wherein R9 is selected from H, OH, —O—, ═O, —OR14, —OC(═O)R14, —OC(═O)NHR14—, —OC(═O)R14SSR15—, OP(═O)(OR14)—, —OC(═O)NR14R15, OP(═O)(OR14), or OR14OP(═O)(OR15);
• wherein R11 is independently H, R14, —R14C(═O)R16, —R14X2R16, —R14C(═O)X2;
• wherein X2 is —O—, —S—, —NH—, —N(R14)—, —O—R14—, —S—R14—, —S(═O)—R14—, or —NHR14;
• wherein R12 is R15, —OH, —SH,—NH2, NH, NHNH2, —NH(R15), —OR15, —R15COR16, —R15COOR16, —R15C(O)NH2, —R15C(O)NHR17, —SR16, R15S(═O)R16, —R15P(═O)(OR17)2, —R15OP(═O)(OR17)2, —CH2OP(═O)(OR17)2, —R15SO2R17, —R15X2R16, or —R15C(═O)X2, where X2 is —O—, OH, SH, —S—, NH2, —NH—, —N(R15)—, —O—R15—, —S—R15—, —S(═O)—R15—, CH2 or —NHR15—;
• R13 and R14 are independently H, O, S, NH, N(R15), NHNH, —OH, —SH, —NH2, NH, NHNH2, —NH(R15), —OR15, CO, —COX2, —COX2R16, R17, F, Cl, Br, I, SR16, NR16R17, N═NR16, N═R16, NO2, SOR16R17, SO2R16, SO3R16, OSO3R16, PR16R17, POR16R17;
• PO2R16R17, OP(O)(OR17)2, OCH2OP(O)(OR17)2, OC(O)R17, OC(O)OP(O)(OR17)2, PO(OR16)(OR17), OP(O)(OR17)OP(O)(OR17)2, OC(O)NHR17, —O—(C4-C12 glycoside), —N—(C4-C12 glycoside);
• C1˜C8 alkyl, heteroalkyl;
• C2-C8 alkenyl, alkynyl, heteroalkyl, or heterocycloalkyl;
• C3-C8 aryl, Ar-alkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, or heteroaryl, or C2-C8 ester, ether, or amide;
• or peptide containing 1-8 amino acids (NH(Aa)1˜8 or CO(Aa)1˜8 (N-terminal or C-terminal 1-8 the same or different amino acids), or polyethyleneoxy unit of formula (OCH2CH2)p or (OCH2CH(CH3))p, wherein p is an integer from 0 to about 1000, or any combination of above groups thereof;
• X2 is O, S, S—S, NH, CH2, OH, SH, NH2, CHR14 or NR14;
• R15, R16 and R17 is independently H, C1˜C8 alkyl, heteroalkyl;
• C2-C8 alkenyl, alkynyl, heteroalkyl, or heterocycloalkyl;
• C3-C8 aryl, Ar-alkyl, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, alkylcarbonyl, or Na+, K+, Cs+, Li+, Ca2+, Mg+, Zn2+, N+(R1)(R2)(R3)(R4), or HN+(C2H5OH)3 salt;
• Y1 and Y2 are independently N or CH;
• q is 0 or 1;
• when q=0, Y3 does not exist, Y4, Y5, Y6 and Y7 are independently CH, N, NH, O, S, or N(R1), thus Y2, Y4, Y5, Y6 and Y7 form a heteroaromatic ring of furan, pyrrole, thiophene, thiazole, oxazole, imidazole, pyrazole, triazole, tetrazole, thiadiazole;
• when q=1, Y3, Y4, Y5, Y6 and Y7 are independently CH or N, thus Y2, Y3, Y4, Y5, Y6 and Y7 form aromatic ring of benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, tetrazine, or pentazine;
• and
• wherein D/T ratio is 3.4 to 4.1.