Bioactive conjugate, preparation method therefor and use thereof

Abstract

The disclosure relates to a bioactive molecule conjugate, preparation methods and use thereof, particularly relates to a novel bioactive molecule conjugate obtained by improving coupling of the drug and the targeting moiety in an ADC or SMDC, as well as its preparation method and use in the manufacture of a medicament for the treatment of a disease associated with an abnormal cell activity.

1 - 91 . (canceled) 
     
     
         92 . A compound of formula (I) or a pharmaceutically acceptable salt thereof,
                       wherein, T is a fragment of a bioactive molecule,   L 1  is selected from
                     
 an amino acid, a peptide composed of 2-10 amino acids, an oligosaccharide, -(CH 
 2 ) t1 -, -(CH 2 CH 2 O) t1 -(CH 2 ) t2 -,
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
   wherein each of R, R′, R 1  and R 2  is independently hydrogen, deuterium, halogen, a carboxylic acid group, a sulfonic acid group, cyano, C 1-6  alkyl, halogenated C 1-6  alkyl, C 1-6  alkyl substituted with cyano, C 1-6  alkoxy, C 2-10  alkenyl, C 2-10  alkynyl, C 3-6  cycloalkyl, 6-10 membered aryl or 5-12 membered heteroaryl, each Z 1  is independently an amino acid or a peptide composed of 2-10 amino acids, each of t 1  and t 2  is independently 0, 1, 2, 3, 4, 5 or 6, each of x 1  and x 2  is independently 0, 1, 2, 3, 4, 5 or 6, each x 3  is independently 0, 1, 2, 3 or 4, and L 1  is bonded to T at the position 1 of L 1 ;   L 2  is selected from
                     
                     
                     
 an amino acid, a peptide composed of 2-10 amino acids, an oligosaccharide, -(CH 
 2 ) t1 -, -(CH 2 CH 2 O) t1 -(CH 2 ) t2 -,
                     
                     
                     
                     
                     
                     
 
 wherein each of R 3 , R 4 , R 5  and R 6  is independently selected from hydrogen, deuterium, halogen, a carboxylic acid group, a sulfonic acid group, CN, C 1-6  alkyl, halogenated C 1-6  alkyl, C 1-6  alkyl substituted with cyano, C 1-6  alkoxy, C 2-10  alkenyl, C 2-10  alkynyl or C 3 - 6  cycloalkyl or R 3  and R 4 , R 5  and R 6  or R 3  and R 5  together with the carbon atoms attached thereto form a 3-8 membered ring, each of t 1  and t 2  is independently 0, 1, 2, 3, 4, 5 or 6, each of y 1  and y 2  is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L 2  is bonded to L 1  at the position 1 of L 2 ;
 L 3  is selected from the following groups optionally substituted with one or more R 7 : 5-12 membered heteroarylene, amino, 3-8 membered cycloalkylene, 3-8 aliphatic heterocyclylene, 6-12 membered bridged heterocyclylene, 6-12 membered spiroheterocyclylene, 6-12 membered fused heterocyclylene, 6-10 membered arylene or 3-8 membered cycloalkylene-W-; wherein W is oxygen or NR 8 , R 7  is independently selected from hydrogen, deuterium, halogen, =O, CN, carboxyl, sulfonic acid group, C 1-6  alkyl, halogenated C 1-6  alkyl, C 1-6  alkyl substituted with cyano, C 1-6  alkoxy, C 2-10  alkenyl or C 2-10  alkynyl, R 8  is independently selected from hydrogen, deuterium, C 1-6  alkyl, C 3-6  cycloalkyl, C 1-6  alkoxy or cyano C 1-2  alkyl; 
 L 4  is selected from
                     
 wherein Z 
 5  is selected from C 2 - 6  alkynyl, C 2 - 6  alkenyl, amido group, sulfuryl, sulfinyl, 6-10 membered arylene or 5-6 membered heteroarylene; Z 2  is selected from C 1-6  alkylene, C 2-10  alkenylene, C 2-10  alkynylene, C 3-8  cycloalkylene, 6-10 membered arylene or 5-14 membered heteroarylene; R 9  is selected from hydrogen or C 1-6  alkyl; Z 3  is absent or selected from C 1-6  alkylene, halogenated C 1-6  alkylene or C 1-6  alkylene substituted with alkoxy; or R 9  and Z 3  together with nitrogen atom attached thereto form a 4-8 membered heterocyclyl; α is independently 0, 1, 2, 3,4, 5 or 6; and L 4  is bonded to E at the position 2 of L 4 ; 
 E is selected from the following groups optionally substituted with one or more R 12 : pyrimidylene, quinolylene or pyrrolo[2,3-d]pyrimidylene; wherein R 12  is independently selected from hydrogen, deuterium, halogen, CN, nitro, C 1-6  alkyl or halogenated C 1-6  alkyl; 
 G is a leaving group for nucleophilic substitutions; 
 each of m 1 , m 2 , and m 3  is independently 0, 1, 2, 3, 4,5, 6, 7, 8, 9 or 10. 
 
     
     
         93 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein,
 L 1  is selected from
                     
 Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, a peptide composed of 2-5 amino acids, 
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
                     
 wherein each of R, R′, R 
 1  and R 2  is independently hydrogen, deuterium, C 1-6  alkyl, C 2-10  alkenyl, C 2-10  alkynyl or C 3-6  cycloalkyl, Z 1  is Lys, Val, Cit, Phe, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg or Ala-Ala-Asn, x 1  is 0, 1, 2 or 3, and x 3  is 0, 1, 2, 3 or 4.   
     
     
         94 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein,
 L 2  is selected from
                     
                     
                     
 Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, a peptide composed of 2-5 amino acids, 
                     
                     
                     
                     
                     
                     
 wherein each of R 
 3 , R 4 , R 5  and R 6  is independently selected from hydrogen, deuterium, halogen, a carboxylic acid group, a sulfonic acid group, CF 3 , CN, CH 2 CN, C 1-4  alkyl, C 1-4  alkoxy, C 2-6  alkenyl, C 2-6  alkynyl or C 3-6  cycloalkyl, each of y 1  and y 2  is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L 2  is bonded to L 1  at the position 1 of L 2 ;and   m 1  is 0, 1, 2 or 3.   
     
     
         95 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein,
 L 3  is selected from the following groups optionally substituted with one or more R 7 : 5-12 membered heteroarylene, amino, 3-8 membered cycloalkylene, 3-8 aliphatic heterocyclylene, 6-12 membered bridged heterocyclylene, 6-12 membered spiroheterocyclylene, 6-12 membered fused heterocyclylene, 6-10 membered arylene or 3-8 membered cycloalkylene-W-; wherein W is oxygen or NR 8 , R 7  is independently selected from hydrogen, deuterium, halogen, =O, CF 3 , CN, CH 2 CN, carboxyl, sulfonic acid group, C 1-   4  alkyl, C 1-4 alkoxy, C 2-6  alkenyl or C 2-6  alkynyl; and   m 2  is 0, 1, 2 or 3.   
     
     
         96 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein L 4  is selected from
                     
                     
                     
                     
                     
                     
                     
 wherein Z 
 4  is 6-10 membered arylene or 5-6 membered heteroarylene; R 10  is selected from hydrogen or C 1-6  alkyl; Z 2  is selected from C 1-6  alkylene, C 2-10  alkenylene, C 2-10  alkynylene or C 3-8  cycloalkylene; R 9  is selected from hydrogen or C 1-6  alkyl; Z 3  is absent or selected from C 1-6  alkylene; or R 9  and Z 3  together with the nitrogen atom attached thereto form a 4-8 membered heterocyclylene; α is independently 0, 1, 2, 3, 4, 5 or 6, and L 4  is bonded to E at the position 2 of L 4 ; and 
 m 3  is 0, 1, 2 or 3. 
 
     
     
         97 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein, E is selected from pyrimidylene optionally substituted with one or more R 12 ; wherein R 12  is independently selected from hydrogen or deuterium. 
     
     
         98 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein, G is selected from alkyl sulfonyl, halogen, OMs, OTs, OTf, nitro or the following groups optionally substituted with one or more R 13 : alkylthio, arylthio, heteroarylthio, alkyl sulfinyl, aryl sulfinyl, heteroaryl sulfinyl, aryl sulfonyl or heteroaryl sulfonyl; wherein R 13  is independently selected from hydrogen, deuterium, halogen, CN, nitro, C 1-6  alkyl, halogenated C 1-6  alkyl, C 1-6  alkoxy, 6-10 membered aryl or 5-12 membered heteroaryl. 
     
     
         99 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein, in
                       G is methylsulfonyl, E is pyrimidylene, and m   3  is 1. 
     
     
         100 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein -[L 1 -(L 2 ) m1 -(L 3 ) m2 -(L 4 ) m3 -E]-G is selected from the following fragments:                                                                                                                                                                                                     or . 
     
     
         101 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein, T is a fragment of a bioactive molecule, and the bioactive molecule is selected from a metal complex; a glycopeptide antibiotic; a DNA topoisomerase inhibitor; a drug interfering with DNA synthesis; a drug acting on a structural protein; a tumor cell signaling pathway inhibitor; a proteasome inhibitor; a histone deaceylase inhibitor; a tumor angiogenesis inhibitor; a cyclin inhibitor; a maytansine derivative; a calicheamicin derivative; a auristatin derivative; a pyrrolobenzodiazepine dimers (PBD) derivative; melphalan; mitomycin C; or chlorambucil; or other active substances which inhibit the growth of tumor cells, promote the apoptosis or necrosis of tumor cells. 
     
     
         102 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein T is selected from
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       .   
     
     
         103 . The compound or the pharmaceutically acceptable salt of  claim 92 ,
 wherein, T is a fragment of a bioactive molecule;   L 1  is selected from an amino acid, a peptide composed of 2-10 amino acids, -(CH 2 ) t1 -,
                     
                     
 t 
 1  is 0, 1, 2, 3, 4, 5 or 6;   L 2  is selected from
                     
                     
 an amino acid, a peptide composed of 2-10 amino acids, or 
                     
 y 
 1  and y 2  is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;   L 3  is selected from 5-12 membered heteroarylene or amino;   L 4  is selected from
                     
 wherein Z 
 2  is C 1-6  alkylene; Z 3  is absent or C 1-6  alkylene; R 9  is hydrogen; α is independently 0, 1, 2, 3,4, 5 or 6;   E is pyrimidylene;   G is a leaving group for nucleophilic substitutions; and   each of m 1 , m 2 , and m 3  is independently 0, 1, 2, 3, 4,5, 6, 7, 8, 9 or 10.   
     
     
         104 . The compound or the pharmaceutically acceptable salt of  claim 92 ,
 wherein, T is a fragment of a bioactive molecule;   L 1  is selected from an amino acid, a peptide composed of 2-10 amino acids, -(CH 2 ) t1 -,
                     
                     
 t 
 1  is 0, 1, 2, 3, 4, 5 or 6;   L 2  is selected from
                     
                     
 an amino acid, a peptide composed of 2-10 amino acids, or 
                     
 y 
 1  and y 2  is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;   L 3  is selected from 5-12 membered heteroarylene or amino;
                     
                     
                     
 wherein m 
 4  is an integer from 0 to 6, methylsulfonyl is a substituent on a carbon atom adjacent to a nitrogen atom in the pyrimidine ring; and   each of m 1 , m 2 , and m 3  is independently 0, 1, 2, 3, 4,5, 6, 7, 8, 9 or 10.   
     
     
         105 . The compound or the pharmaceutically acceptable salt of  claim 92 ,
 wherein, T is a fragment of a bioactive molecule;   L 1  is
                     
 or -(CH 
 2 ) t1 -,   wherein each of R, R′ and R 1  is independently hydrogen, deuterium, halogen, a carboxylic acid group, a sulfonic acid group, cyano, C 1-6  alkyl, halogenated C 1-6  alkyl, C 1-6  alkyl substituted with cyano, C 1-6  alkoxy, C 2-10  alkenyl, C 2-10  alkynyl, C 3-6  cycloalkyl, 6-10 membered aryl or 5-12 membered heteroaryl, Z 1  is independently an amino acid or a peptide composed of 2-10 amino acids, t 1  is 0, 1, 2, 3, 4, 5 or 6, each of x 1 , and x 2  is independently 0, 1, 2, 3, 4, 5 or 6, each x 3  is independently 0, 1, 2, 3 or 4, and L 1  is bonded to T at the position 1 of L 1 ;   L 2  is selected from
                     
 an amino acid, a peptide composed of 2-10 amino acids; y 
 1  is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L 2  is bonded to L 1  at the position 1 of L 2 ;   L 3  is 5-12 membered heteroarylene;   L 4  is
                     
 wherein Z 
 5  is selected from C 2-6  alkynyl, C 2-6  alkenyl, amido group, sulfuryl, sulfinyl, 6-10 membered arylene or 5-6 membered heteroarylene; Z 2  is selected from C 1-6  alkylene, C 2-10  alkenylene, C 2-10  alkynylene, C 3-8  cycloalkylene, 6-10 membered arylene or 5-14 membered heteroarylene; R 9  is selected from hydrogen or C 1-6  alkyl; Z 3  is absent or selected from C 1-6  alkylene, halogenated C 1-6  alkylene or C 1-6  alkylene substituted with alkoxy; or R 9  and Z 3  together with nitrogen atom attached thereto form a 4-8 membered heterocyclyl; α is independently 0, 1, 2, 3,4, 5 or 6; and L 4  is bonded to E at the position 2 of L 4 ;   E is pyrimidylene optionally substituted with one or more R 12 ; wherein R 12  is independently selected from hydrogen, deuterium, halogen, CN, nitro, C 1-6  alkyl or halogenated C 1-6  alkyl;   G is a leaving group for nucleophilic substitutions; and   each of m 1 , m 2 , and m 3  is independently 0, 1, 2, 3, 4,5, 6, 7, 8, 9 or 10.   
     
     
         106 . The compound or the pharmaceutically acceptable salt of  claim 92 ,
 wherein T is
                     
   L 1  is
                     
 and L 
 1  is bonded to T at the position 1 of L 1 ;   L 2  is
                     
 y 
 1  is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L 2  is bonded to L 1  at the position 1 of L 2 ; m 1  is 1;   L 3  is triazolylene; m 2  is 0 or 1;   L 4  is
                     
                     
 m 
 3  is 1; and L 4  is bonded to E at the position 2 of L 4 ;   E is pyrimidylene; and   G is methylsulfonyl.   
     
     
         107 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein -[L 1 -(L 2 ) m1 -(L 3 ) m2 -(L 4 ) m3 -E]-G is the following fragments: . 
     
     
         108 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein the compound is selected from                                                                                             . 
     
     
         109 . The compound or the pharmaceutically acceptable salt of  claim 92 , wherein the compound is . 
     
     
         110 . A pharmaceutical composition comprising the compound of  claim 92 , and one or more pharmaceutical excipients. 
     
     
         111 . A method of treating a disease associated with an abnormal cell activity, comprising administering an effective amount of the compound of  claim 92  to an individual in need thereof. 
     
     
         112 . A compound or the pharmaceutically acceptable salt thereof, wherein the compound is
                                                                                                               wherein R 14  is selected from acyl or sulfonyl, which is substituted with R 15 , and R 15  is selected from C 1-6  alkyl, halogenated C 1-6  alkyl, 6-10 membered aryl or 5-12 membered heteroaryl; R 16  is selected from hydrogen, deuterium, C 1-6  alkyl or C 1-6  alkyl substituted with R 17 , and R 17  is selected from aryl or heteroaryl, including but not limited to phenyl and pyridyl, and m 11  is 0, 1 or 2.   
     
     
         113 . A pharmaceutical composition comprising the compound of  claim 112 , and one or more pharmaceutical excipients. 
     
     
         114 . A method of treating a disease associated with an abnormal cell activity, comprising administering an effective amount of the compound of  claim 112  to an individual in need thereof. 
     
     
         115 . A compound, which is:
                                                                                                                                                                                                                                                                                                                                                                                                               6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid, 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid, or methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate.   
     
     
         116 . A process of preparing compound T030:
                       comprising reacting belotecan hydrochloride with methanesulfonyl chloride.   
     
     
         117 . A process for preparing a compound, comprising deprotecting a protective form of the compound, wherein the compound is:         and the protective form of the compound is         
. 
     
     
         118 . A process for preparing the protective form of the compound according to  claim 117 , comprising reacting an azide compound with an alkynyl compound, wherein the azide compound is   and the alkynyl compound is:
                     
                     
                     
                     
 
. 
     
     
         119 . A process for preparing the azide compound according to  claim 118 , comprising reacting compound T030:
                       with (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-6(((4-methoxyphenyl)benzhydryl)amino)acetamide.   
     
     
         120 . A process for preparing the alkynyl compound according to  claim 118 , comprising reacting an carboxylic acid with propargylamine, wherein the carboxylic acid is:
                                                                                         .   
     
     
         121 . A process for preparing the carboxylic acid according to  claim 120 , comprising reacting a 2-methylthiopyrimidine compound with m-chloroperoxybenzoic acid, wherein the 2-methylthiopyrimidine compound is
                                                                                         .   
     
     
         122 . A process for preparing the 2-methylthiopyrimidine compound according to  claim 121 , wherein the 2-methylthiopyrimidine compound is 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid, comprising hydrolyzing methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate. 
     
     
         123 . A process for preparing methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate according to  claim 122 , comprising reacting methyl 5-hexynoate with 5-bromo-2-methylthiopyrimidine. 
     
     
         124 . A process for preparing an amide compound, comprising reacting an carboxylic acid with an amine, wherein the carboxylic acid is:
                                                                                         .   
     
     
         125 . A conjugate comprising a fragment of a bioactive molecule, a linker, and a targeting moiety, wherein the targeting moiety is linked to the linker via an active group to form a conjugate; wherein the conjugate has a structure shown in formula (II):
                       wherein, A is a targeting moiety; γ is an integer or a decimal from 1 to 10; and   the rest groups are as defined in  claim 92 .   
     
     
         126 . A pharmaceutical composition comprising the conjugate of  claim 125 , and one or more pharmaceutical excipients. 
     
     
         127 . A method of treating a disease associated with an abnormal cell activity, comprising administering an effective amount of the conjugate of  claim 125  to an individual in need thereof. 
     
     
         128 . A method for preparing the conjugate of  claim 125  comprising a step of coupling the linker of the compound of formula (I) with an active group of the targeting moiety. 
     
     
         129 . A conjugate comprising a fragment of a bioactive molecule, a linker, and a targeting moiety, wherein the targeting moiety is linked to the linker via an active group to form a conjugate; wherein the targeting moiety is an anti-Trop-2 monoclonal antibody of antibody M1, M2 or M3;
 wherein the antibody comprises:
 the heavy chain variable region of the antibody M1 which has the amino acid sequence of SEQ ID No.: 11; and the light chain variable region of the antibody M1 which has the amino acid sequence of SEQ ID No.: 12; 
 the heavy chain variable region of the antibody M2 which has the amino acid sequence of SEQ ID No.: 13; and the light chain variable region of the antibody M2 wchih has the amino acid sequence of SEQ ID No. : 14; or 
 the heavy chain variable region of the antibody M3 which has the amino acid sequence of SEQ ID No. : 15; and the light chain variable region of the antibody M3 which has the amino acid sequence of SEQ ID No.: 16. 
   
     
     
         130 . A pharmaceutical composition comprising the conjugate of  claim 129 , and one or more pharmaceutical excipients. 
     
     
         131 . A method of treating a disease associated with an abnormal cell activity, comprising administering an effective amount of the conjugate of  claim 129  to an individual in need thereof.