Acetylenedicarboxyl linkers and their uses in specific conjugation of a cell-binding molecule

1. A bridge linker compound of Formula (I)

wherein:

the acetylenedicarboxyl group in Formula (I) is capable of reacting with a pair of sulfur atoms of a cell-binding agent;

Z 1 and Z 2 are the same or different and represent a functional group that enables the bridge linker compound to react with a cytotoxic drug, to form a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, secondary, tertiary, or quaternary amine, imine, cycloheteroalkyl, heteroaromatic, alkyloxime or amide bond;

R 1 and R 2 are the same or different, and are absent, linear alkyl having from 1-6 carbon atoms; branched or cyclic alkyl having from 3 to 6 carbon atoms; linear, branched or cyclic alkenyl or alkynyl having from 3 to 6 carbon atoms; ester, ether, or amide having from 2 to 6 carbon atoms; or polyethyleneoxy unit of formula (OCH 2 CH 2 ) p , wherein p is an integer from 1 to about 1000, or combination thereof;

or R 1 and R 2 are respectively a chain of atoms selected from C, N, O, S, Si, and P, which covalently connects to X 1 ′ or X 2 ′ and Z 1 or Z 2 ;

X 1 ′ and X 2 ′ are independently NH, N(R 3 ), O, S or CH 2 ; R 3 is H, linear alkyl having from 1-6 carbon atoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear, branched or cyclic alkenyl or alkynyl, ester, ether, or amide having from 2 to 6 carbon atoms; or polyethyleneoxy unit of formula (OCH 2 CH 2 ) p , wherein p is an integer from 1 to about 1000, or combination thereof,

wherein Z 1 and Z 2 each are represented by one of following structures:

wherein X 1 is F, Cl, Br, I or Lv; X 2 is O, NH, N(R 1 ), or CH 2 ;

R 5 and R 3 are H, R 1 , aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by —R 1 , -halogen, —OR 1 , —SR 1 , —NR 1 R 2 , —NO 2 , —S(O)R 1 , —S(O) 2 R 1 , or —COOR 1 ;

R 3 ′ is a linear alkyl having from 1-6 carbon atoms; branched or cyclic alkyl having from 3 to 6 carbon atoms; linear, branched or cyclic alkenyl or alkynyl having from 3 to 6 carbon atoms, aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by —R 1 , -halogen, —OR 1 , —SR 1 , —NR 1 R 2 , —NO 2 , —S(O)R 1 , —S(O) 2 R 1 , or —COOR 1 ;

Lv is a leaving group selected from the group consisting of nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate, an anhydride and an intermediate molecule generated from any of the above with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions.

2. The bridge linker compound according to claim 1 , wherein R 1 or R 2 is 6-maleimidocaproyl (MC), maleimido propanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), lysine-phenylalanine (lys-phe), p-aminobenzyloxycarbonyl (PAB), 4-thio-pentanoate (SPP), 4-(N-maleimidomethyl)cyclo-hexane-1-carboxylate (MCC), 4-thio-butyrate (SPDB), maleimidoethyl (ME), 4-thio-2-hydroxysulfonyl-butyrate (2-Sulfo-SPDB), pyridinyl-dithiol (PySS), alkoxy amino (AOA), ethyleneoxy (EO), 4-methyl-4-dithio-pentanoic (MPDP), azido (N 3 ), alkynyl, dithio, peptides, or (4-acetyl)aminobenzoate (SIAB).

3. The bridge linker compound according to claim 1 , wherein Z 1 and Z 2 are represented by following structures:

wherein X 1 is F, Cl, Br, I or Lv; X is O, NH, N(R 1 ), or CH 2 ; R 5 and R 3 are H, R 1 , aromatic, heteroaromatic, or aromatic group wherein one or several H atoms are replaced independently by —R 1 , -halogen, —OR 1 , —SR 1 —, —NR 1 R 2 , —NO 2 , —S(O)R 1 , —S(O) 2 R 1 , or —COOR 1 ; Lv is a leaving group selected from nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluorophenol; monofluorophenol; pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; -hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate, an anhydrides or an intermediate molecule generated from any of the above with a condensation reagent for peptide coupling reactions, or for Mitsunobu reactions.

4. The bridge linker compound according to claim 1 , wherein R 1 or R 2 comprises a peptide of 1˜20 units of natural or unnatural amino acids, a p-aminobenzyl unit, a 6-maleimidocaproyl unit, a disulfide unit, a thioether unit, a hydrozone unit, a triazole unit, or an alkoxime unit.

5. The bridge linker compound according to claim 1 , wherein R 1 or R 2 is cleavable by a protease.

6. The bridge linker compound of claim 3 , wherein the condensation reagent is EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide), DCC (dicyclohexyl-carbodiimide), N,N′-diisopropylcarbodiimide (DIC), N-cyclohexyl-N′-(2-morpholinoethyl)carbodimide metho-p-toluenesulfonate (CMC, or CME-CDI), 1,1′-carbonyldiimidazole (CDI), TBTU (O-(benzotria-zol-1-yl)N,N,N′,N′-tetramethyluronium tetrafluoroborate), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), (benzotriazol-1-yloxy)tris(dimethyl-amino)phosphonium hexafluorophosphate (BOP), (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate, 1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino) methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium 3-oxide hexafluorophosphate (HDMA), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate, (PyCloP), fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N,N′,N′-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate, O-(2-Oxo-1(2H)pyridyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TPTU), S-(1-oxido-2-pyridyl)-N,N,N′,N′-tetramethylthiuronium tetrafluoroborate, O-[(ethoxycarbonyl) cyano-methylenamino]-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylidenamino-oxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), O-(benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene) uronium hexafluorophosphate (HBPyU), N-benzyl-N′-cyclohexylcarbodiimide, dipyrrolidino(N-succinimidyloxy)-carbenium hexafluoro-phosphate (HSPyU), chlorodipyrrolidinocarbenium hexafluorophos-phate (PyClU), 2-chloro-1,3-dimethylimidazolidinium tetrafluoroborate (CIB), (benzotriazol-1-yloxy) dipiperidinocarbenium hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TCTU), bromotris(dimethylamino)-phosphonium hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA®), 2-morpholinoethyl isocyanide (MEI), N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyanomethylamino]-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N,N,N′,N′-tetramethyl-O—(N-succinimyl)uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-borate (TDBTU), 1,1′-(azodicarbonyl)dipiperidine (ADD), di-(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), or diethyl azodicarboxylate (DEAD).

7. A method for preparing the bridge linker compound of Formula (I) of claim 1 , comprising condensing an acetylenedicarboxylic acid, or derivative of the acetylenedicarboxylic acid, with a compound containing a terminal 1° or 2° amine, alcohol, or thiol, as shown in the following scheme (Ia);

wherein X is X 1 H or X 2 H; X 1 and X 2 are independently NH, N(R 3 ), O, or S; R is R 1 or R 2 ; R 1 , R 2 and R 3 are the same described in claim 1 ;

Lv 1 and Lv 2 are the same or different and are independently OH; F; Cl; Br; I; nitrophenol; N-hydroxysuccinimide (NHS); phenol; dinitrophenol; pentafluorophenol; tetrafluorophenol; difluoro-phenol; monofluorophenol: pentachlorophenol; triflate; imidazole; dichlorophenol; tetrachlorophenol; 1-hydroxybenzotriazole; tosylate; mesylate; 2-ethyl-5-phenylisoxazolium-3′-sulfonate, an anhydride; or an intermediate molecule generated from any of the above with a condensation reagent for peptide coupling reactions, or for Mitsunohu reactions.

8. A method for preparing the bridge linker compound of Formula (I) of claim 1 , comprising condensing bis(trimethylsilyl)acetylene, or acetylene bis-magnesiums, or a di-metal acetylide with an acid halide or acid anhydride, depicted as following reaction equations (Ib), (Ic), (Id), (Ie), (If), (Ig) and (Ih):

wherein M is Na, K, Li, Cu, CuLi, Sn, Ti, Ca, Mg or Zn.

9. The method of claim 7 , wherein the condensation reagent is EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide), DCC (dicyclohexyl-carbodiimide), N,N′-diisopropylcarbodiimide (DIC), N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC, or CME-CDI), 1,1′-carbonyldiimidazole (CDI), TBTU (O-(benzotria-zol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), (benzotriazol-1-yloxy)tris(dimethyl-amino)phosphonium hexafluorophosphate (BOP), (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), diethyl cyanophosphonate (DEPC), chloro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate, 1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), 1-[(dimethylamino)(morpholino) methylene]-1H-[1,2,3]triazolo[4,5-b]pyridine-1-ium 3-oxide hexafluorophosphate 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH), N,N,N′,N′-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate, O-(2-Oxo-1(2H)pyridyl)-N,N,N′N′-tetramethyluronium tetrafluoroborate (TPTU), S-(1-oxido-2-pyridyl)-N,N,N′,N′-tetramethylthiuronium tetrafluoroborate, O-[(ethoxycarbonyl) cyano-methylenamino]-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HOTU), (1-cyano-2-ethoxy-2-oxoethylidenamino-oxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), O-(benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene) uronium hexafluorophosphate (HBPyU), N-benzyl-N′-cyclohexylcarbodiimide, dipyrrolidino(N-succinimidyloxy)-carbenium hexafluoro-phosphate (HSPyU), chlorodipyrrolidinocarbenium hexafluorophosphate (PyClU), 2-chloro-1,3-dimethylimidazolidinium tetrafluoroborate (CIB), (benzotriazol-1-yloxy) dipiperidinocarbenium hexafluorophosphate (HBPipU), O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TCTU), bromotris(dimethylamino)-phosphonium hexafluorophosphate (BroP), propylphosphonic anhydride (PPACA®), 2-morpholinoethyl isocyanide (MEI), N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium hexafluorophosphate (HSTU), 2-bromo-1-ethyl-pyridinium tetrafluoroborate (BEP), O-[(ethoxycarbonyl)cyanomethylamino]-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TOTU), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (MMTM, DMTMM), N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU), O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TDBTU), 1,1′-(azodicarbonyl)dipiperidine (ADD), di-(4-chlorobenzyl) azodicarboxylate (DCAD), di-tert-butyl azodicarboxylate (DBAD), diisopropyl azodicarboxylate (DIAD), or diethyl azodicarboxylate (DEAD).