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.

Claims

1-91. (canceled)

92. A compound of formula (I) or a pharmaceutically acceptable salt thereof, ##STR00369## wherein, T is a fragment of a bioactive molecule, L.sub.1 is selected from ##STR00370## an amino acid, a peptide composed of 2-10 amino acids, an oligosaccharide, -(CH .sub.2).sub.t1-, -(CH.sub.2CH.sub.2O).sub.t1-(CH.sub.2).sub.t2-, ##STR00371## ##STR00372## ##STR00373## ##STR00374## ##STR00375## ##STR00376## ##STR00377## ##STR00378## ##STR00379## ##STR00380## ##STR00381## ##STR00382## ##STR00383## ##STR00384## ##STR00385## ##STR00386## ##STR00387## ##STR00388## ##STR00389## ##STR00390## wherein each of R, R′, R.sub.1 and R.sub.2 is independently hydrogen, deuterium, halogen, a carboxylic acid group, a sulfonic acid group, cyano, C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl, C.sub.1-6 alkyl substituted with cyano, C.sub.1-6 alkoxy, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-6 cycloalkyl, 6-10 membered aryl or 5-12 membered heteroaryl, each Z.sub.1 is independently an amino acid or a peptide composed of 2-10 amino acids, each of t.sub.1 and t.sub.2 is independently 0, 1, 2, 3, 4, 5 or 6, each of x.sub.1 and x.sub.2 is independently 0, 1, 2, 3, 4, 5 or 6, each x.sub.3 is independently 0, 1, 2, 3 or 4, and L.sub.1 is bonded to T at the position 1 of L.sub.1; L.sub.2 is selected from ##STR00391## ##STR00392## ##STR00393## an amino acid, a peptide composed of 2-10 amino acids, an oligosaccharide, -(CH .sub.2).sub.t1-, -(CH.sub.2CH.sub.2O).sub.t1-(CH.sub.2).sub.t2-, ##STR00394## ##STR00395## ##STR00396## ##STR00397## ##STR00398## ##STR00399## wherein each of R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is independently selected from hydrogen, deuterium, halogen, a carboxylic acid group, a sulfonic acid group, CN, C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl, C.sub.1-6 alkyl substituted with cyano, C.sub.1-6 alkoxy, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl or C.sub.3-.sub.6 cycloalkyl or R.sub.3 and R.sub.4, R.sub.5 and R.sub.6 or R.sub.3 and R.sub.5 together with the carbon atoms attached thereto form a 3-8 membered ring, each of t.sub.1 and t.sub.2 is independently 0, 1, 2, 3, 4, 5 or 6, each of y.sub.1 and y.sub.2 is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L.sub.2 is bonded to L.sub.1 at the position 1 of L.sub.2; L.sub.3 is selected from the following groups optionally substituted with one or more R.sub.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.sub.8, R.sub.7 is independently selected from hydrogen, deuterium, halogen, =O, CN, carboxyl, sulfonic acid group, C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl, C.sub.1-6 alkyl substituted with cyano, C.sub.1-6 alkoxy, C.sub.2-10 alkenyl or C.sub.2-10 alkynyl, R.sub.8 is independently selected from hydrogen, deuterium, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.1-6 alkoxy or cyano C.sub.1-2 alkyl; L.sub.4 is selected from ##STR00400## wherein Z .sub.5 is selected from C.sub.2-.sub.6 alkynyl, C.sub.2-.sub.6 alkenyl, amido group, sulfuryl, sulfinyl, 6-10 membered arylene or 5-6 membered heteroarylene; Z.sub.2 is selected from C.sub.1-6 alkylene, C.sub.2-10 alkenylene, C.sub.2-10 alkynylene, C.sub.3-8 cycloalkylene, 6-10 membered arylene or 5-14 membered heteroarylene; R.sub.9 is selected from hydrogen or C.sub.1-6 alkyl; Z.sub.3 is absent or selected from C.sub.1-6 alkylene, halogenated C.sub.1-6 alkylene or C.sub.1-6 alkylene substituted with alkoxy; or R.sub.9 and Z.sub.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.sub.4 is bonded to E at the position 2 of L.sub.4; E is selected from the following groups optionally substituted with one or more R.sub.12: pyrimidylene, quinolylene or pyrrolo[2,3-d]pyrimidylene; wherein R.sub.12 is independently selected from hydrogen, deuterium, halogen, CN, nitro, C.sub.1-6 alkyl or halogenated C.sub.1-6 alkyl; G is a leaving group for nucleophilic substitutions; each of m.sub.1, m.sub.2, and m.sub.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.sub.1 is selected from ##STR00401## Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, a peptide composed of 2-5 amino acids, ##STR00402## ##STR00403## ##STR00404## ##STR00405## ##STR00406## ##STR00407## ##STR00408## ##STR00409## ##STR00410## ##STR00411## ##STR00412## ##STR00413## wherein each of R, R′, R .sub.1 and R.sub.2 is independently hydrogen, deuterium, C.sub.1-6 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl or C.sub.3-6 cycloalkyl, Z.sub.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.sub.1 is 0, 1, 2 or 3, and x.sub.3 is 0, 1, 2, 3 or 4.

94. The compound or the pharmaceutically acceptable salt of claim 92, wherein, L.sub.2 is selected from ##STR00414## ##STR00415## ##STR00416## Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, a peptide composed of 2-5 amino acids, ##STR00417## ##STR00418## ##STR00419## ##STR00420## ##STR00421## ##STR00422## wherein each of R .sub.3, R.sub.4, R.sub.5 and R.sub.6 is independently selected from hydrogen, deuterium, halogen, a carboxylic acid group, a sulfonic acid group, CF.sub.3, CN, CH.sub.2CN, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl or C.sub.3-6 cycloalkyl, each of y.sub.1 and y.sub.2 is independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L.sub.2 is bonded to L.sub.1 at the position 1 of L.sub.2;and m.sub.1 is 0, 1, 2 or 3.

95. The compound or the pharmaceutically acceptable salt of claim 92, wherein, L.sub.3 is selected from the following groups optionally substituted with one or more R.sub.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.sub.8, R.sub.7 is independently selected from hydrogen, deuterium, halogen, =O, CF.sub.3, CN, CH.sub.2CN, carboxyl, sulfonic acid group, C.sub.1-.sub.4 alkyl, C.sub.1-4alkoxy, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl; and m.sub.2 is 0, 1, 2 or 3.

96. The compound or the pharmaceutically acceptable salt of claim 92, wherein L.sub.4 is selected from ##STR00423## ##STR00424## ##STR00425## ##STR00426## ##STR00427## ##STR00428## ##STR00429## wherein Z .sub.4 is 6-10 membered arylene or 5-6 membered heteroarylene; R.sub.10 is selected from hydrogen or C.sub.1-6 alkyl; Z.sub.2 is selected from C.sub.1-6 alkylene, C.sub.2-10 alkenylene, C.sub.2-10 alkynylene or C.sub.3-8 cycloalkylene; R.sub.9 is selected from hydrogen or C.sub.1-6 alkyl; Z.sub.3 is absent or selected from C.sub.1-6 alkylene; or R.sub.9 and Z.sub.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.sub.4 is bonded to E at the position 2 of L.sub.4; and m.sub.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.sub.12; wherein R.sub.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.sub.13: alkylthio, arylthio, heteroarylthio, alkyl sulfinyl, aryl sulfinyl, heteroaryl sulfinyl, aryl sulfonyl or heteroaryl sulfonyl; wherein R.sub.13 is independently selected from hydrogen, deuterium, halogen, CN, nitro, C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl, C.sub.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 ##STR00430## G is methylsulfonyl, E is pyrimidylene, and m .sub.3 is 1.

100. The compound or the pharmaceutically acceptable salt of claim 92, wherein -[L.sub.1-(L.sub.2).sub.m1-(L.sub.3).sub.m2-(L.sub.4).sub.m3-E]-G is selected from the following fragments: ##STR00431## ##STR00432## ##STR00433## ##STR00434## ##STR00435## ##STR00436## ##STR00437## 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 ##STR00438## ##STR00439## ##STR00440## ##STR00441## ##STR00442## ##STR00443## ##STR00444## ##STR00445## ##STR00446## ##STR00447## ##STR00448## ##STR00449## ##STR00450## ##STR00451## ##STR00452## ##STR00453## ##STR00454## ##STR00455## ##STR00456## ##STR00457## ##STR00458## ##STR00459## ##STR00460## ##STR00461## .

103. The compound or the pharmaceutically acceptable salt of claim 92, wherein, T is a fragment of a bioactive molecule; L.sub.1 is selected from an amino acid, a peptide composed of 2-10 amino acids, -(CH.sub.2).sub.t1-, ##STR00462## ##STR00463## t .sub.1 is 0, 1, 2, 3, 4, 5 or 6; L.sub.2 is selected from ##STR00464## ##STR00465## an amino acid, a peptide composed of 2-10 amino acids, or ##STR00466## y .sub.1 and y.sub.2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; L.sub.3 is selected from 5-12 membered heteroarylene or amino; L.sub.4 is selected from ##STR00467## wherein Z .sub.2 is C.sub.1-6 alkylene; Z.sub.3 is absent or C.sub.1-6 alkylene; R.sub.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.sub.1, m.sub.2, and m.sub.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.sub.1 is selected from an amino acid, a peptide composed of 2-10 amino acids, -(CH.sub.2).sub.t1-, ##STR00468## ##STR00469## t .sub.1 is 0, 1, 2, 3, 4, 5 or 6; L.sub.2 is selected from ##STR00470## ##STR00471## an amino acid, a peptide composed of 2-10 amino acids, or ##STR00472## y .sub.1 and y.sub.2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; L.sub.3 is selected from 5-12 membered heteroarylene or amino; ##STR00473## ##STR00474## ##STR00475## wherein m .sub.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.sub.1, m.sub.2, and m.sub.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.sub.1 is ##STR00476## or -(CH .sub.2).sub.t1-, wherein each of R, R′ and R.sub.1 is independently hydrogen, deuterium, halogen, a carboxylic acid group, a sulfonic acid group, cyano, C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl, C.sub.1-6 alkyl substituted with cyano, C.sub.1-6 alkoxy, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.3-6 cycloalkyl, 6-10 membered aryl or 5-12 membered heteroaryl, Z.sub.1 is independently an amino acid or a peptide composed of 2-10 amino acids, t.sub.1 is 0, 1, 2, 3, 4, 5 or 6, each of x.sub.1, and x.sub.2 is independently 0, 1, 2, 3, 4, 5 or 6, each x.sub.3 is independently 0, 1, 2, 3 or 4, and L.sub.1 is bonded to T at the position 1 of L.sub.1; L.sub.2 is selected from ##STR00477## an amino acid, a peptide composed of 2-10 amino acids; y .sub.1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L.sub.2 is bonded to L.sub.1 at the position 1 of L.sub.2; L.sub.3 is 5-12 membered heteroarylene; L.sub.4 is ##STR00478## wherein Z .sub.5 is selected from C.sub.2-6 alkynyl, C.sub.2-6 alkenyl, amido group, sulfuryl, sulfinyl, 6-10 membered arylene or 5-6 membered heteroarylene; Z.sub.2 is selected from C.sub.1-6 alkylene, C.sub.2-10 alkenylene, C.sub.2-10 alkynylene, C.sub.3-8 cycloalkylene, 6-10 membered arylene or 5-14 membered heteroarylene; R.sub.9 is selected from hydrogen or C.sub.1-6 alkyl; Z.sub.3 is absent or selected from C.sub.1-6 alkylene, halogenated C.sub.1-6 alkylene or C.sub.1-6 alkylene substituted with alkoxy; or R.sub.9 and Z.sub.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.sub.4 is bonded to E at the position 2 of L.sub.4; E is pyrimidylene optionally substituted with one or more R.sub.12; wherein R.sub.12 is independently selected from hydrogen, deuterium, halogen, CN, nitro, C.sub.1-6 alkyl or halogenated C.sub.1-6 alkyl; G is a leaving group for nucleophilic substitutions; and each of m.sub.1, m.sub.2, and m.sub.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 ##STR00479## L.sub.1 is ##STR00480## and L .sub.1 is bonded to T at the position 1 of L.sub.1; L.sub.2 is ##STR00481## y .sub.1 is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and L.sub.2 is bonded to L.sub.1 at the position 1 of L.sub.2; m.sub.1 is 1; L.sub.3 is triazolylene; m.sub.2 is 0 or 1; L.sub.4 is ##STR00482## ##STR00483## m .sub.3 is 1; and L.sub.4 is bonded to E at the position 2 of L.sub.4; E is pyrimidylene; and G is methylsulfonyl.

107. The compound or the pharmaceutically acceptable salt of claim 92, wherein -[L.sub.1-(L.sub.2).sub.m1-(L.sub.3).sub.m2-(L.sub.4).sub.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 ##STR00484## ##STR00485## ##STR00486## ##STR00487## ##STR00488## wherein R.sub.14 is selected from acyl or sulfonyl, which is substituted with R.sub.15, and R.sub.15 is selected from C.sub.1-6 alkyl, halogenated C.sub.1-6 alkyl, 6-10 membered aryl or 5-12 membered heteroaryl; R.sub.16 is selected from hydrogen, deuterium, C.sub.1-6 alkyl or C.sub.1-6 alkyl substituted with R.sub.17, and R.sub.17 is selected from aryl or heteroaryl, including but not limited to phenyl and pyridyl, and m.sub.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: ##STR00489## ##STR00490## ##STR00491## ##STR00492## ##STR00493## ##STR00494## ##STR00495## ##STR00496## ##STR00497## ##STR00498## ##STR00499## ##STR00500## ##STR00501## ##STR00502## ##STR00503## ##STR00504## ##STR00505## 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: ##STR00506## 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: ##STR00507## ##STR00508## ##STR00509## ##STR00510## .

119. A process for preparing the azide compound according to claim 118, comprising reacting compound T030: ##STR00511## 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: ##STR00512## ##STR00513## ##STR00514## ##STR00515## .

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 ##STR00516## ##STR00517## ##STR00518## ##STR00519## .

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: ##STR00520## ##STR00521## ##STR00522## ##STR00523## .

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): ##STR00524## 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.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0209] FIG. 1 shows a TIC (total ion chromatogram) of BT001002.

[0210] FIG. 2 shows a deconvolution diagram of a coupled light chain of BT001002.

[0211] FIG. 3 shows a deconvolution diagram of a coupled heavy chain of BT001002.

[0212] FIG. 4 shows a TIC (total ion chromatogram) of BT001004.

[0213] FIG. 5 shows a deconvolution diagram of a coupled light chain of BT001004.

[0214] FIG. 6 shows a deconvolution diagram of a coupled heavy chain of BT001004.

[0215] FIG. 7 shows a SEC chromatogram of BT001002.

[0216] FIG. 8 shows a SEC chromatogram of molecular weight Marker of BT001002.

[0217] FIG. 9 shows a SEC chromatogram of BT001004.

[0218] FIG. 10 shows a deconvolution diagram of a coupled light chain of BT001012.

[0219] FIG. 11 shows a deconvolution diagram of a coupled heavy chain of BT001012.

[0220] FIG. 12 shows a deconvolution diagram of a coupled light chain of BT001013.

[0221] FIG. 13 shows a deconvolution diagram of a coupled heavy chain of BT001013.

[0222] FIG. 14 shows a deconvolution diagram of a coupled light chain of BT001018.

[0223] FIG. 15 shows a deconvolution diagram of a coupled heavy chain of BT001018.

[0224] FIG. 16 shows a deconvolution diagram of a coupled light chain of BT001021.

[0225] FIG. 17 shows a deconvolution diagram of a coupled heavy chain of BT001021.

[0226] FIG. 18 shows a deconvolution diagram of a coupled light chain of BT001023.

[0227] FIG. 19 shows a deconvolution diagram of a coupled heavy chain of BT001023.

[0228] FIG. 20 shows a deconvolution diagram of a coupled light chain of BT001040.

[0229] FIG. 21 shows a deconvolution diagram of a coupled heavy chain of BT001040.

[0230] FIG. 22 shows a deconvolution diagram of a coupled light chain of BT001041.

[0231] FIG. 23 shows a deconvolution diagram of a coupled heavy chain of BT001041.

[0232] FIG. 24 shows a deconvolution diagram of a coupled light chain of BT001042.

[0233] FIG. 25 shows a deconvolution diagram of a coupled heavy chain of BT001042.

[0234] FIG. 26 shows a deconvolution diagram of a coupled light chain of BT001043.

[0235] FIG. 27 shows a deconvolution diagram of a coupled heavy chain of BT001043.

[0236] FIG. 28 shows a deconvolution diagram of a coupled light chain of BT001044.

[0237] FIG. 29 shows a deconvolution diagram of a coupled heavy chain of BT001044.

[0238] FIG. 30 shows a deconvolution diagram of a coupled light chain of BT001046.

[0239] FIG. 31 shows a deconvolution diagram of a coupled heavy chain of BT001046.

[0240] FIG. 32 shows a deconvolution diagram of a coupled light chain of BT001047.

[0241] FIG. 33 shows a deconvolution diagram of a coupled heavy chain of BT001047.

[0242] FIG. 34 shows a SEC chromatogram of BT001012.

[0243] FIG. 35 shows a SEC chromatogram of BT001013.

[0244] FIG. 36 shows a SEC chromatogram of BT001018.

[0245] FIG. 37 shows a SEC chromatogram of BT001021.

[0246] FIG. 38 shows a SEC chromatogram of BT001023.

[0247] FIG. 39 shows a SEC chromatogram of BT001042.

[0248] FIG. 40 shows a SEC chromatogram of BT001043.

[0249] FIG. 41 shows a SEC chromatogram of BT001044.

[0250] FIG. 42 shows a SEC chromatogram of BT001046.

[0251] FIG. 43 shows a SEC chromatogram of BT001047.

[0252] FIG. 44 shows changes in growth of tumor volume of each group of mice in a NCI-N87 human gastric cancer model.

[0253] FIG. 45 shows changes in body weight of each group of mice in a NCI-N87 human gastric cancer model.

[0254] FIG. 46 shows changes in growth of tumor volume of each group of mice in an HCC1806 human breast cancer model.

[0255] FIG. 47A shows changes in growth of tumor volume of each group of mice in a xenograft model of HCC827 human non-small cell lung cancer.

[0256] FIG. 47B shows changes in body weight of each group of mice in a xenograft model of HCC827 human non-small cell lung cancer.

[0257] FIG. 48A shows changes in growth of tumor volume of each group of mice in a xenograft model of NCI-N87 human gastric cancer.

[0258] FIG. 48B shows changes in body weight of each group of mice in a xenograft model of NCI-N87 human gastric cancer.

[0259] FIG. 49A shows changes in growth of tumor volume of each group of mice in a tumor-bearing mice model of MDA-MB-231 human breast cancer.

[0260] FIG. 49B shows changes in body weight of each group of mice in a tumor-bearing mice model of MDA-MB-231 human breast cancer.

SPECIFIC MODE FOR CARRYING OUT THE INVENTION

[0261] The disclosure will be further illustrated in combination with specific embodiments, but the disclosure is not limited thereto. It should be understood by a person skilled in the art that various modifications or improvements can be made according to the teachings of the disclosure without departing from the basic idea and scope of the disclosure.

[0262] Abbreviations in the invention have the following meanings:

TABLE-US-00014 OMs methylsulfonyloxy FA Formic acid OTs Trifluoromethylsulfonyloxy ACN Acetonitrile OTf p-toluenesulfonyloxy CCK8 reagent 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt TBS Tert-butyldimethylsilyl FBS Fetal bovine serum MMT p-methoxytriphenylmethyl DMSO Dimethyl sulfoxide PB/PBS Phosphate buffered saline

Preparation Solutions

[0263] The structures of compounds described in the following examples were determined by nuclear magnetic resonance (.sup.1H NMR) or mass spectrometry (MS).

[0264] Nuclear magnetic resonance (.sup.1H NMR) was determined by using a Bruker 400 MHz NMR spectrometer. Deuterated methanol (CD.sub.3OD), deuterated chloroform (CDCl.sub.3) or deuterated dimethyl sulfoxide (DMSO-D.sub.6) was the solvent for determination, and tetramethylsilane (TMS) was an internal standard substance.

[0265] Abbreviations in nuclear magnetic resonance (NMR) spectra used in the examples were shown below.

[0266] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d.sub.6: deuterated dimethyl sulfoxide. δ value was expressed in ppm.

[0267] Mass spectra (MS) were determined using Agilent (ESI) mass spectrometer (model: Agilent 6120B).

[0268] Preparative liquid chromatography: Method A: [0269] Chromatographic column: Daisogel C18 10 .Math.m 100×250 mm [0270] Mobile phase A: water; Mobile phase B: acetonitrile

TABLE-US-00015 Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 70.0 30.0 300.0 8.00 70.0 30.0 50.00 20.0 80.0

Method B: [0271] Chromatographic column: Daisogel C18 10 .Math.m 50×250 mm [0272] Mobile phase A: water; Mobile phase B: acetonitrile

TABLE-US-00016 Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 70.0 30.0 80.0 8.00 70.0 30.0 50.00 20.0 80.0

Method C: [0273] Chromatographic column: Daisogel C18 10 .Math.m 50×250 mm [0274] Mobile phase A: water containing 0.1% trifluoroacetic acid; Mobile phase B: acetonitrile

TABLE-US-00017 Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL/min] 0.00 90.0 10.0 80.0 10.00 90.0 10.0 50.00 60.0 40.0

Method D: chromatographic column: Waters SunFire C18 5 .Math.m19×250 mm [0275] Mobile phase A: acetonitrile; Mobile phase B: water containing 0.05% formic acid [0276] Time: 0 min-Y; Mobile phase A: 10%-90%; Flow rate: 28 mL/min

I. Synthesis Of Bioactive Molecules

Example 1: Synthesis of (2S)-N-((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-((1-((4-aminobenzyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-propionyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-heptanoyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyramide (T001)

[0277]

[0278] Step 1: synthesis of tert-butyl (4-((2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5 methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl) carbamate

[0279] At room temperature, 1-hydroxybenzotriazole (2.0 mg, 14.74 .Math.mol) was dissolved in N,N-dimethylformamide (4 mL), cooled to 0° C., and then tert-butyl 4-methylaminobenzyl carbamate (4.0 mg, 16.1 .Math.mol), N,N-diisopropylethylamine (8.5 mg, 66.8 .Math.mol), ((2R, 3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine (10.0 mg, 13.5 .Math.mol, commercially available) were successively added. After being stirred for 5 min, 1H-benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (10.0 mg, 20.1 .Math.mol) was added thereto, and stirred at 0° C. for 1 h. The reaction of raw materials was monitored by high performance liquid chromatography-mass spectrometry. After the raw materials were consumed up, the reaction solution was purified by preparative liquid chromatography (method D) to obtain the title compound (9.0 mg of white solid). ESI-MS (m/z): 950.5 [M + H] .sup.+.

[0280] Step 2: synthesis of (2S)-N-((3R,4S,5S)-1-((2S)-2-((1R,2R)-3-((1-((4-aminobenzyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-propionyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-heptanoyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutanamide

[0281] At room temperature, tert-butyl (4-((2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-3-phenylpropanamide)methyl)phenyl) carbamate (9.0 mg, 0.02 mmol) was dissolved in 1,4-dioxane (0.5 mL), cooled to 0° C., and then the hydrogen chloride solution in dioxane (1 mL, 4.0 M) was added and reacted at room temperature for 3 hours under stirring. The reaction of raw materials was monitored by high performance liquid chromatography-mass spectrometry. After the raw materials were consumed up, the solvent was evaporated under reduced pressure, and the crude product was purified by preparative liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (5.0 mg of white solid). ESI-MS (m/z): 850.5 [M + H] .sup.+.

Example 2: Synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-((4-aminobenzyl)amino) 1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyramide (T011)

[0282]

[0283] Step 1: synthesis of tert-buty (S)-(4-((2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionamido) methyl)phenyl) l carbamate

[0284] At 0° C., 4-aminobenzylamine (222 mg, 1.0 mmol) and N-methylmorpholine (306 mg, 1.5 mmol) were added to a solution of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionicacid (387 mg, 1.0 mmol) in N,N-dimethylformamide (5 mL), then 1-hydroxybenzotriazole (203 mg, 1.5 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (288 mg, 1.5 mmol) were successively added. The resulting mixture was reacted overnight at 0° C. The reaction solution was poured into water (50 mL), and a white solid was precipitated. The solid was filtered, the filter cake was washed with water (20 mL×3). The solid was purified by silica gel column chromatography to obtain the title compound (a 380 mg white solid). ESI-MS (m/z): 592.3 [M + H].sup.+.

[0285] Step 2: synthesis of tert-butyl (S)-(4-((2-amino-3-phenylpropionamido) methyl)phenyl) carbamate

[0286] Lithium hydroxide monohydrate (21 mg, 0.51 mmol) was dissolved in water (1 mL) and added to a tetrahydrofuran (2 mL) solution of tert-butyl (S)-(4-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionamido)methyl)phenyl) carbamate (102 mg, 0.17 mmol). The resulting mixture was reacted at room temperature for 2 hours. The reaction solution was added with water (20 mL) and extracted with ethyl acetate (30 mL×4). The organic phases were combined, washed with saturated saline (30 mL × 2) and dried over anhydrous sodium sulfate. Then the desiccant was removed by filtration, the solvent was evaporated under reduced pressure, and the residues were purified by preparative liquid chromatography (method D) to obtain the title compound (65 mg of white solid). ESI-MS (m/z): 370.2 [M + H].sup.+.

[0287] Step 3: synthesis of (4-((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-((methylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy -5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)carbamate

[0288] At 0° C., tert-butyl (S)-(4-((2-amino-3-phenylpropionamido)methyl)phenyl) carbamate (15 mg, 0.04 mmol) and N-methylmorpholine (12 mg, 0.12 mmol) were added to a solution of (2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl-3-methoxy-2-methylpropionic acid (24 mg, 0.04 mmol) in N,N-dimethylformamide (2 mL), then 1-hydroxybenzotriazole (8 mg, 0.06 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (12 mg, 0.06 mmol) were successively added. The resulting mixture was reacted overnight at 0° C. The reaction solution was purified by preparative liquid chromatography (method D) to obtain the title compound (24 mg of white solid). ESI-MS (m/z): 950.6 [M + H].sup.+.

[0289] Step 4: synthesis of (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-((4-aminobenzyl)amino)-1-oxo-3-phenylprop-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyramide

[0290] Trifluoroacetic acid (0.5 mL) was added to a solution of (4-((S)-2-((2R,3R)-3-((S)-1-(3R,4S,5S)-4-((S)-3-(methylamino)-3-methylbutyrylamino)-N, 3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropionamido)methyl)phenyl) carbamate (14.0 mg, 0.015 mmol) in dichloromethane (1.5 mL). The resulting mixture was reacted at room temperature for 1 h. Then the solvent was evaporated under reduced pressure, and the residue was purified by preparative liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (4.2 mg of white solid). ESI-MS (m/z): 850.6 [M + H].sup.+.

[0291] The following molecules can be synthesized by a similar synthetic method:

TABLE-US-00018 Name Structure ESI-MS T012 850.6 T013 864.6 T015 760.6 T021 851.6

Example 3: Synthesis of (S)-N-(2-(4-ethyl-4-hydroxyl-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylacetamide

[0292]

[0293] Belotecan hydrochloride (1.0 g, 2.13 mmol) and triethylamine (0.65 g, 0.9 mL) were dissolved in dichloromethane (50 mL) at room temperature, and acetic anhydride (0.22 g, 2.13 mmol) was slowly added dropwise. The resulting mixture was reacted at room temperature for 1h. The organic phase was washed with water (10 mL×2) and dried over anhydrous sodium sulfate. Insoluble substances were removed by filtration, the solvent was evaporated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=50/1) to obtain the title compound (1 g). ESI-MS (m/z): 476.2[M+H].sup.+.

Example 4: Synthesis of (S)-N-(2-(4-ethyl-4-hydroxyl-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide

[0294]

[0295] Methylsulfonyl chloride (462 mg, 12.77 mmol, purity:about 70%) was added dropwise to a solution of belotecan hydrochloride (3 g, 6.38 mmol) and triethylamine (2.58 g, 25.54 mmol) in dichloromethane (40 mL). The resulting mixture was reacted at room temperature for 2 h. Suction filtration was performed, and the filter cake was washed three times with dichloromethane (3 mL) to obtain the title compound (2.2 g).

[0296] Structural characterization data are as follows:

[0297] .sup.1H NMR (400 MHz, DMSO-d6) δ 8.32 (d, J = 8.4 Hz, 1H), 8.20 (dd, J = 8.4, 1.2 Hz, 1H), 7.93-7.84 (m, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.35 (s, 1H), 6.56 (s, 1H), 5.44 (d, J = 9.2 Hz, 4H), 3.98 (p, J = 6.7 Hz, 1H), 3.50 (t, J = 8.0 Hz, 2H), 3.42-3.35 (m, 2H), 3.00 (s, 3H), 1.93-1.82 (m, 2H), 1.15 (d, J = 6.7 Hz, 6H), 0.88 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 512.2 [M+H].sup.+ . [α].sub.D.sup.20 is +28.19° (c =0.101 g/100 mL, CH.sub.3CN).

[0298] The rest bioactive molecules without illustration of synthetic method are commercially available or can be prepared by the method disclosed in the prior art.

II. Synthesis Of Compounds Containing Bioactive Molecules And Linkers

Example 5: Synthesis of (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyrylamido)-3-methylbutyrylamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidovaleramide

[0299]

[0300] Step 1: synthesis of tert-butyl 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl) butyrate (compound 1-2)

[0301] At room temperature, compound 1-1 (500 mg, 3.27 mmol) was dissolved in N,N-dimethylformamide (10 mL), sodium hydride (130 mg, 3.27 mmol) was slowly added in batches thereto. The resulting mixture was stirred at room temperature for 10 min, followed by the dropwise addition of t-butyl 4-bromobutyrate (725 mg, 3.27 mmol), and then reacted at room temperature for 2 hours. The reaction was quenched with saturated ammonium chloride aqueous solution, and extracted with ethyl acetate (50 mL×3). Then organic phases were combined, washed with saturated saline solution (50 mL×3) and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the title compound (500 mg). ESI-MS (m/z): 296.1 [M + H].sup.+.

[0302] Step 2: synthesis of 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (compound 1-3)

[0303] At room temperature, compound 1-2 (500 mg, 1.69 mmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (3 mL) was added and reacted at room temperature for 4 hours. Then the solvent was evaporated under reduced pressure to obtain the title compound (400 mg). ESI-MS (m/z): 240.1 [M + H].sup.+.

[0304] Step 3: sysnthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((tert-butoxycarbonyl)amino)methyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (compound 1-5)

[0305] At room temperature, 4-(N-Boc-aminomethyl)-aniline (6.0 g, 27 mmol), compound 1-4 (3.35 g, 6.75 mmol), and 2-ethyoxyl-1-ethoxycarboxyl-1,2-dihydroquinoline (3.34 g, 13.5 mmol) were dissolved in the mixed solvent of dichloromethane (140 mL) and methanol (70 mL), then warmed to 45° C. and reacted at the temperature for 8.0 hours. After being cooled to room temperature, a large amount of solid was precipitated, which was subject to suction filtration to obtain the title compound (3.65 g). ESI-MS (m/z): 701.4 [M + H].sup.+.

[0306] Step 4: synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(aminomethyl)phenyl))amino)-1-oxo-5-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (compound 1-6)

[0307] At room temperature, trifluoroacetic acid (15 mL) was added to compound 1-5 (3.0 g, 4.29 mmol) and stirred at room temperature for 1.0 h. Then the solvent was evaporated under reduced pressure to obtain a yellow oil. Anhydrous diethyl ether (20 mL) was added, and a large amount of solid was precipitated. After vigorous stirring for 0.5 h, suction filtration was carried out to obtain the trifluoroacetate of the title compound (3.06 g). ESI-MS (m/z): 601.3 [M + H].sup.+.

[0308] Step 5: synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((R)-2-((t-butyloxycarboryl)amino)-3-phenylpropionamido)methyl)phenyl)amino-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (compound 1-7)

[0309] At room temperature, Boc-D-phenylalanine (1.1 g, 4.2 mmol) and the trifluoroacetate of compound 1-6 (3.0 g, 4.2 mmol) were dissolved in N,N-dimethylformamide (40 mL), cooled to 0° C., and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 g, 6.3 mmol), 1-hydroxybenzotriazole (0.9 g, 6.3 mmol) and N-methylmorpholine (1.7 g, 16.8 mmol) were successively added. The reaction system was stirred for 1.0 h at the temperature. The reaction solution was then added dropwise to ice water (400 mL) and stirred vigorously for 0.5 h, a large amount of solid was precipitated, and suction filtration was carried out to obtain the title compound (3.3 g). ESI-MS (m/z): 848.4 [M + H].sup.+.

[0310] Step 6: synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((R)-2-amino-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxobutyl-2-yl)-carbamate (compound 1-8)

[0311] At room temperature, compound 1-7 (3.0 g, 3.3 mmol) was dissolved in trifluoroacetic acid (30 mL) and stirred at room temperature for 1.0 h. The solvent was evaporated under reduced pressure to obtain a yellow oil. Anhydrous diethyl ether (100 mL) was added and stirred vigorously for 0.5 h, and a large amount of solid was precipitated. Suction filtration was carried out to obtain the trifluoroacetate of the title compound (2.1 g). ESI-MS (m/z): 748.4 [M + H].sup.+.

[0312] Step 7: synthesis of (9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyrylamido)-N,3-dimethylamino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (compound 1-9)

[0313] At room temperature, (2R3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-(dimethylamino)-3-butyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl)-3-methoxy-2-methylpropionic acid (1.3 g, 2.17 mmol) and trifluoroacetate of compound 1-8 (1.8 g, 2.17 mmol) were dissolved in N,N-dimethylformamide (20 mL), cooled to 0° C., then 1-hydroxybenzotriazole (440 mg, 3.26 mmol) and N-methylmorpholine (658 mg, 6.51 mmol) were successively added, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (624 mg, 1.38 mmol) was added at last, the reaction solution was stirred at 0° C. for 5 hours, and purified by preparative liquid chromatography (method D) to obtain the title compound (1.8 g). ESI-MS (m/z): 1329.2 [M + H].sup.+.

[0314] Step 8: synthesis of (S)-2-((S)-2-amino-3-butyrylamino)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidovaleramide (compound 1-10)

[0315] At room temperature, compound 1-9 (500 mg, 0.38 mmol) was dissolved in N,N-dimethylformamide (5 mL), added with piperidine (324 mg, 3.8 mmol) and stirred at room temperature for 3 h. Then the purification was performed on preparative liquid chromatography (method D) to obtain the title compound (350 mg). ESI-MS (m/z): 1107.2 [M + H].sup.+.

[0316] Step 9: synthesis of (S)-2-((S)-2-(4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyrylamido)-3-methylbutyrylamido)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidovaleramide (compound TL001)

[0317] At room temperature, compound 1-10 (60 mg, 0.054 mmol) and 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (26 mg, 0.066 mmol) were dissolved in N,N-dimethylformamide (3 mL), cooled to 0° C., and N,N-diisopropylethylamine (105 mg, 0.81 mmol) and 1H-benzotriazole-1-oxytripyrrolidinophosphonium hexafluorophosphate (281 mg, 0.54 mmol) were successively added. The reaction system was stirred at room temperature for 3 hours. Then purificaiton was performed on preparative liquid chromatography (method D) to obtain the title compound (30 mg). ESI-MS (m/z): 664.5 [M/2 + H].sup.+.

Example 6: (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butyrylamido)-butyrylamido)-5-ureidovaleramide

[0318]

[0319] Step 1: synthesis of 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric acid (compound 2-2)

[0320] At room temperature, 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl) butyric acid (300 mg, 1.25 mmol) was dissolved in methanol (8 mL), sodium methanethiol (351 mg, 5.02 mmol) was added in one batch, and then warmed to 50° C. and reacted overnight. Purification was performed on preparative liquid chromatography (method D) to obtain the title compound (120 mg). ESI-MS (m/z): 252.1 [M + H].sup.+.

[0321] Step 2: synthesis of (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butyrylamido)-butyrylamido)-5-ureidovaleramide (compound 2-3)

[0322] Operations similar to those described in step 9 of example 5, except that 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl) butyric acid was replaced with 4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl) butyric acid, were carried out, and purification was performed by using preparative liquid chromatography (method D) to obtain the title compound (20 mg). ESI-MS (m/z): 670.5 [M/2 + H].sup.+.

[0323] Step 3: synthesis of (S)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-butyrylamido)-butyrylamido)-5-ureidovaleramide (compound TL002)

[0324] At room temperature, compound 2-3 (20 mg, 0.015 mmol) was dissolved in dichloromethane (2 mL), and m-chloroperoxybenzoic acid (4.0 mg, 0.022 mmol) was added. The resulting mixture was reacted at room temperature for 2 hours. Purification was performed on preparative liquid chromatography (method D) to obtain the title compound (5.0 mg). ESI-MS (m/z): 686.5 [M/2 + H].sup.+.

Example 7: N-((S)-1-(((S)-1-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0325]

[0326] Step 1: synthesis of methyl 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate (compound 3-2)

[0327] At room temperature, methyl 5-hexynoate (500 mg, 3.97 mmol) and 5-bromo-2-methylthiopyrimidine were dissolved in N,N-dimethylformamide (3 ml), then triethylamine (3 ml), cuprous iodide (75 mg, 0.4 mmol) and Bis (triphenylphosphine) palladium (II) dichloride (279 mg, 0.4 mmol) were successively added. The resulting mixture was heated to 95° C. under nitrogen protection and reacted for 6 h under stirring, quenched with water, and extracted with ethyl acetate (20 mL×3). Organic phases were combined, washed with saturated saline (20 mL×2) and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure. Purification was performed on preparative liquid chromatography (method D) to obtain the title compound (300 mg). ESI-MS (m/z): 251.3 [M + H].sup.+.

[0328] Step 2: synthesis of 6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoic acid (compound 3-3)

[0329] At room temperature, compound 3-2 (200 mg, 0.8 mmol) was dissolved in a mixed solution of tetrahydrofuran and water (4 mL/4 mL), and lithium hydroxide monohydrate (235 mg, 5.6 mmol) was added, and reacted at room temperature under stirring for 4 h, then diluted with water and extracted with ethyl acetate (20 ml x 2). The aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, and extracted with ethyl acetate (20 mL×3), then organic phases were combined, washed with saturated saline (20 mL×2) and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure to obtain the title compound (120 mg).

[0330] Step 3: synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid (compound 3-4)

[0331] At room temperature, compound 3-3 (20 mg, 0.085 mmol) was dissolved in dichloromethane (4 mL), and m-chloroperoxybenzoic acid (22 mg, 0.127 mmol) was added for reaction at room temperature overnight under stirring. Purification was performed on preparative liquid chromatography (method D) to obtain the title compound (20 mg). ESI-MS (m/z): 269.1 [M + H].sup.+.

[0332] Step 4: N-((S)-1-(((S)-1-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxybutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide (compound TL003)

[0333] Operations similar to those described in step 9 of example 5, except that 4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl) butyric acid was replaced with 6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic acid, were carried out, and purification was performed by using preparative liquid chromatography (method D) to obtain the title compound (14 mg). ESI-MS (m/z): 679.0 [M/2 + H].sup.+.

Example 8: (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)-ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′\,4\′\,6,7]-indolizino[1,2-b]-quinolin-4-yl(4-((S)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9,36-azatetracosan-41-amido)benzyl)carbonate

[0334]

[0335] Step 1: synthesis of methyl (S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)-(9H-fluorenyl) carbamate (compound 19-2)

[0336] At room temperature, Fmoc-L-citrulline (5.0 g, 12.58 mmol), p-aminobenzyl alcohol (6.20 g, 50.32 mmol) and 2-ethoxy-1-ethoxycarboxyl-1, 2-dihydroquinoline (6.22 g, 25.16 mmol) were dissolved in dichloromethane (100 mL), and heated to 45° C. and reacted for 6 h. The reaction solution was concentrated under reduced pressure, and beaten with anhydrous diethyl ether (100 mL) to obtain the title compound (6.0 g). ESI-MS (m/z): 503.3[M+H].sup.+.

[0337] Step 2: synthesis of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide (compound 19-3)

[0338] At room temperature, compound 19-2 (1.0 g, 1.99 mmol) was dissolved in N,N-dimethylformamide (8 mL), and piperidine (339 mg, 3.98 mmol) was added dropwise for reaction at room temperature for 30 min, then dichloromethane (10 mL) was added, followed by stirring for 10 min. The reaction solution was concentrated under reduced pressure, and purified by flash column chromatography to obtain the title compound (400 mg). ESI-MS (m/z): 281.2[M+H].sup.+.

[0339] Step 3: synthesis of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxa-6-azatriacetamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide (compound 19-4)

[0340] Compound 19-3 (150 mg, 0.54 mmol) and 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatricycloundecanoic acid (296 mg, 0.54 mmol) were dissolved in dichloromethane (10 mL) and cooled to 0° C., then 2-ethoxy-1-ethoxycarboxyl-1,2-dihydroquinoline (145 mg, 0.58 mmol) was added. The resulting mixture was moved to room temperature and reacted overnight. The reaction solution was concentrated under reduced pressure, and purified by flash column chromatography to obtain the title compound (200 mg). ESI-MS (m/z): 817.5[M+H].sup.+.

[0341] Step 4: synthesis of 4-((S)-35-azido-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9-azatetracosane)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound 19-5)

[0342] At room temperature, (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide(200 mg, 0.39 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0° C., a solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 ml) was added, and then a solution of triphosgene (116 mg, 0.39 mmol) in dichloromethane (1.0 ml) was slowly added dropwise. The resulting mixture was reacted at 0° C. for 1 h under stirring. A solution of the compound 19-4 (159 mg, 0.18 mmol) in dichloromethane (2.0 mL) was added to the reaction solution and reacted at room temperature for 1 h. Purification was perfomed on preparative high performance liquid chromatography (method D) to obtain the title compound (160 mg). ESI-MS (m/z): 678.0[M/2+H].sup.+.

[0343] Step 5: synthesis of 4-((S)-35-amino-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9-azatetracosane)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound 19-6)

[0344] At room temperature, compound 19-5 (80 mg, 0.059 mmol) was dissolved in tetrahydrofuran (1.0 ml) and cooled to 0° C., then a solution of 4-dimethylaminopyridine (573 mg, 4.69 mmol) in dichloromethane (1.0 ml) was added, and platinum dioxide (15 mg, 0.059 mmol) was added in one batch under nitrogen protection, then air was substituted with hydrogen for three times and reacted at room temperature for 6 hours. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product which was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (40 mg). ESI-MS (m/z): 665.0[M/2+H].sup.+.

[0345] Step 6: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9,36-azatetracosan-41-amido)benzyl)carbonate (compound TL019)

[0346] Compound 19-6 (30 mg, 0.016 mmol) and 6-(2-methylsulfonylpyrimidin-5-yl)-5-hexynoic acid (6.4 mg, 0.024 mmol) were dissolved in N,N-dimethylformamide (1 mL) and cooled to 0° C., then benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate (16.5 mg, 0.032 mmol), N,N-diisopropylethylamine (6.2 mg, 0.047 mmol) were successively added. The resulting mixture was reacted at room temperature for 2 hours. Purification was performed on preparative high performance liquid chromatography (method D) to obtain the title compound (10 mg). ESI-MS (m/z): 790.0[M/2+H].sup.+.

Example 9: (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-4-yl-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl))-5-hexynamido)butyramido-5-ureidovalerylamido)benzyl)carbonate

[0347]

[0348] Step 1: methyl ((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidovalerylamido-2-yl)amino))-3-methyl-butyramido-2-yl)-(9H-fluorenyl) carbamate

[0349] Operations similar to those described in step 1 of example 8 were carried out to obtain the title compound (310 mg), except that compound 19-1 was replaced with compound 28-1. ESI-MS (m/z):602.3[M+H].sup.+.

[0350] Step 2: synthesis of (S)-2-((S)-2-amino-3-methylbutyramido)-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide (compound 28-2)

[0351] Operations similar to those described in step 2 of example 8 were carried out to obtain the title compound (150 mg), except that compound 19-2 was replaced with compound 28-2. ESI-MS (m/z): 380.3[M+H].sup.+.

[0352] Step 3: synthesis of N-((S)-1-(((S)-1-((4-hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide(compound 28-4)

[0353] At room temperature, benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate (313 mg, 0.6 mmol) and N,N-diisopropylethylamine (194 mg, 1.50 mmol) were added to a solution of 6-(2-methylsulfonylpyrimidin-5-yl)-5-hexynoic acid (135 mg, 0.5 mmol) and (2S)-2-(((2S)-2-amino-3-methyl-butyryl)amino)-N-(4-(hydroxymethyl)phenyl)-5-ureido-valeramide (190 mg, 0.5 mmol) in N,N-dimethylformamide (10 mL) and reacted at room temperature for 3 hours under stirring. The reaction solution was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (78 mg). ESI-MS (m/z): 630.3 [M + H].sup.+.

[0354] Step 4: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-4-yl-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl))-5-hexynamido)butyramido-5-ureidovalerylamido)benzyl)carbonate (compound TL028)

[0355] Operations similar to those described in step 4 of example 8 were carried out to obtain the title compound (1.76 mg), except that compound 19-4 was replaced with compound 28-4. ESI-MS (m/z): 1167.4 [M + H].sup.+.

Example 10: (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-4-yl-(4-((2S,SS)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazotritriacontylamido)benzyl)carbonate

[0356]

[0357] Step 1: synthesis of (S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-azatetracosyl)-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide (compound 29-1)

[0358] Operations similar to those described in step 3 of example 8 were carried out to obtain the title compound (180 mg), except that compound 19-3 was replaced with compound 28-3. ESI-MS (m/z): 916.5[M+H].sup.+.

[0359] Step 2: synthesis of 4-((2S,SS)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatritriacontylamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound 29-2)

[0360] Operations similar to those described in step 4 of example 8 were carried out to obtain the title compound (30 mg), except that compound 19-4 was replaced with compound 29-1. ESI-MS (m/z): 727.5 [M/2+H].sup.+.

[0361] Step 3: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinolin-4-yl-(4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazotritriacontylamido)benzyl)carbonate (compound TL029)

[0362] At room temperature, compound 29-2 (20 mg, 0.014 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(2-propyn-1-yl)-5-hexynamide (4.3 mg, 0.014 mmol) were dissolved in a mixed solvent (1 mL/0.25 mL) of dimethyl sulfoxide and water, then cuprous bromide (3.95 mg, 0.027 mmol) was added and reacted for 1 h under stirring. Purification was performed on preparative high performance liquid chromatography (method D) to obtain the title compound (15 mg). ESI-MS (m/z): 880.0[M/2+H].sup.+.

Example 11: (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3\′\,4\′:6,7]indolizino[1,2-b]quinolin-4-yl(4-((2S\,5S)-5-isopropyl-45-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,40-tetraoxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6, 12,39-tetraazapentatetracontane-44-carbamoyl)benzyl)carbonate

[0363]

[0364] Step 1: synthesis of (S)-2-((S)-35-amino-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide

[0365] At 20° C., compound 29-1 (400 mg, 0.44 mmol) was dissolved in methanol and tetrahydrofuran (2.0 mL: 4.0 mL). After complete dissolution, platinum dioxide (40 mg) was added in one batch under nitrogen protection, then the mixed solution was subject to hydrogen substitution for three times. Hydrogenation was conducted at 20° C. for 2 hours. The reaction solution was filtered. The filter cake was washed with methanol. The filtrate was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (200 mg). ESI-MS (m/z): 890.4[M+H].sup.+.

[0366] Step 2: synthesis of N-((6S,9S)-1-amino-6-((4-(hydroxymethyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonoxy-2,7,10,16-tetraazadotetracont-42-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide

[0367] At 20° C., compound 22-1 (250 mg, 0.28 mmol) was dissolved in N,N-dimethylformamide (1.0 mL), then HATU (160 mg, 0.42 mmol) and N,N-diisopropylethylamine (109 mg, 0.84 mmol) were successively added, followed by stirring overnight at room temperature. Purification was performed on preparative high performance liquid chromatography (method D) to obtain the title compound (250 mg).

[0368] Step 3: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-((2S,5S)-5-isopropyl-45-(2-(methylsulfonyl)pyrimidin-5-yl)-4,7,11,40-tetraoxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-3,6, 12,39-tetraazapentatetracontane-44-carbamoyl)benzyl)carbonate (compound TL022)

[0369] At 20° C., (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (70 mg, 0.14 mmol) was dissolved in dichloromethane (4.0 mL) and cooled to 0° C., then a solution of p-dimethylaminopyridine (200 mg, 1.64 mmol) in dichloromethane (1.0 ml) was added, and a solution of triphosgene (40.6 mg, 0.14 mmol) in dichloromethane (1.0 ml) was slowly added dropwise. The resulting mixture was reacted at 0° C. for 1 h under stirring. The unreacted triphosgene was blown off with nitrogen, and a solution of compound 22-2 (139 mg, 012 mmol) in dichloromethane (2.0 mL) was added to the reaction solution and reacted at 0° C. for 1 h under stirring. Purification was performed on preparative high performance liquid chromatography (method D) to obtain the title compound (1.5 mg). ESI-MS (m/z): 839.5[M/2+H].sup.+.

Example 12: 4-((S)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkynamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0370]

[0371] Step 1: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadotetracontyl-41-alkynamido)benzylcarbonate

[0372] At room temperature, 6-(-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (12 mg, 0.045 mmol) was dissolved in dichloromethane (2 mL), then 2-(7-azobenzotriazol)-N,N,N′,N′-tetramethylureahexafluorophosphate (21.2 mg, 0.056 mmol) and N,N-diisopropylethylamine (8.6 mg, 0.067 mmol) were added and stirred for 10 min, and compound 24-1 (35 mg, 0.022 mmol) was added and reacted for 1h under stirring. Purification was performed on preparative high performance liquid chromatography (method B) to obtain the title compound (20 mg). ESI-MS (m/z): 1821.8[M+H].sup.+.

[0373] Step 2: synthesis of 4-((S)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkynamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound TL024)

[0374] At room temperature, compound 24-2 (20 mg, 0.011 mmol) was dissolved in acetonitrile (1 mL), and a solution of trifluoroacetic acid (0.5 ml) in acetonitrile (0.5 ml) was added dropwise and stirred for 20 min. Purification was performed on preparative high performance liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (12 mg). ESI-MS (m/z): 1549.6[M+H].sup.+.

Example 13: Synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl-4-((2S,5S)-5-isopropyl-2-methyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatriacontazanamido)benzyl)carbonate

[0375]

[0376] Step 1: preparation of (S)-(9H-fluoren-9-yl)-methyl(1-((4-(hydroxymethyl)phenyl)amino)-1 -oxopropyl-2-yl)carbamate

[0377] At room temperature, 2-ethoxy-1-ethoxycarboxyl-1,2-dihydroquinoline (1.31 g, 5.30 mmol) and p-aminobenzyl alcohol (593 mg, 4.82 mmol) were added to a solution of the compound 30-1 (1.5 g, 4.82 mmol) in dichloromethane (35 mL) and reacted for 3 hours under stirring. Purification was performed on silica gel column chromatography to obtain the title compound (1.8 g). ESI-MS (m/z): 417.2 [M+H].sup.+

[0378] Step 2: preparation of (S)-2-amino-N-(4-(hydroxymethyl)phenyl)propionamide

[0379] At room temperature, ethylenediamine (5 mL) was added to a solution of compound 30-2 (1.8 g, 4.32 mmol) in dichloromethane (20 mL) and reacted for 2 hours. Purification was performed on silica gel column chromatography to obtain the title compound (820 mg). ESI-MS (m/z): 195.1 [M+H]+

[0380] Step 3: preparation of (9H-fluoren-9-yl)-methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-carbamate

[0381] At room temperature, (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butyricacid (875 mg, 2.58 mmol), O-benzotriazolyl-tetramethyluronium hexafluorophosphate (1.45 g, 3.83 mmol), N,N-diisopropylethylamine (1.00 g, 7.74 mmol) and 1-hydroxybenzotriazole (525 mg, 3.89 mmol) were successively added to a solution of the compound 30-3 (503 mg, 2.58 mmol) in dichloromethane (2 mL) and reacted for 4 hours under stirring. Purification was performed on silica gel column chromatography to obtain the title compound (1.1 g). ESI-MS (m/z): 516.2 [M+H]+

[0382] Step 4: preparation of (S)-2-amino-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)-3-methylbutanamide

[0383] At room temperature, ethylenediamine (2 mL) was added to a solution of the compound 30-4 (1.1 g, 2.13 mmol) in dichloromethane (8 mL) and reacted for 1 h under stirring. Purification was performed on silica gel column chromatography to obtain the title compound (610 mg). ESI-MS (m/z): 294.2 [M+H].sup.+

[0384] Step 5: preparation of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-diazapentatriacontamido)-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutyramide

[0385] At room temperature, O-benzotriazolyl-tetramethyluroniumhexafluorophosphate (160 mg, 0.42 mmol), 1-hydroxybenzotriazole (57 mg, 0.42 mmol), N,N-diisopropylethylamine (109 mg, 0.84 mmol) and 32-azido-5-oxo-3.9,12,15,18,21,24,27,30-nonoxy-6-azatricyclodecane-1-acid (156 mg, 0.28 mmol) were added to a solution of compound 30-5 (84 mg, 0.28 mmol) in dichloromethane (3 mL) and reacted for 4 hours under stirring. Purification was performed on silica gel column chromatography to obtain the title compound (163 mg). ESI-MS (m/z): 830.4 [M+H].sup.+

[0386] Step 6: preparation of 4-((2S,5S)-38 azido-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatriacontamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0387] Under nitrogen protection and at 0° C., a solution of triphosgene (16 mg, 0.05 mmol) in dichloromethane (0.3 mL) was added dropwise to a mixed solution of 4-dimethylaminopyridine (65 mg, 0.53 mmol) and (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[30′,4′:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (45 mg, 0.09 mmol) in dichloromethane (0.7 mL) and reacted at 0° C. for 1 h. Then a solution of the compound 30-6 (73 mg, 0.09 mmol) in dichloromethane (1 mL) was added dropwise to the reaction solution and reacted at 0° C. for 1 h. Purification was performed on silica gel column chromatography to obtain the title compound (33 mg). ESI-MS (m/z): 1367.6 [M+H].sup.+

[0388] Step 7: preparation of (S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7indolizino[1,2-b]quinolin-4-yl-4-((2S,5S)-5-isopropyl-2-methyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,11-trioxo-9,15,18,21,24,27,30,33,36-nonoxy-3,6,12-triazatriacontazanamido)benzylcarbonate (compound TL030)

[0389] \\At room temperature, cuprous bromide (5 mg, 0.04 mmol) and compound 30-7 (20 mg, 15 umol) were added dropwise to a solution of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynylamide (9 mg, 0.007 mmol) in water and N,N-dimethylformamide (0.2 ml: 0.8 ml) and reacted for 4 hours under stirring. Purification was performed on preparative high performance liquid chromatography (method D) to obtain the title compound (4.15 mg). ESI-MS (m/z): 1672.7 [M+H].sup.+

Example 14: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11 -(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0390]

[0391] Step 1: synthesis of 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide

[0392] At 25° C., prop-2-ynyl-1-amine (189 mg, 3.4 mmol) and compound 3-4 (800 mg, 2.83 mmol) were dissolved in dichloromethane (10 mL), then N,N-diisopropylethylamine (738 mg, 5.67 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (1.63 g, 4.25 mmol) were successively added and reacted for 2 hours under stirring. The reaction solution was concentrated under reduced pressure, and the residue was purified by flash silica gel column chromatography (ethyl acetate/petroleum ether =3/1) to obtain the title compound (700 mg). ESI-MS (m/z): 306.1[M+H].sup.+.

[0393] Step 2: synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazo-3,9-diazapentatriacontamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamide)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-2H-pyrano[2,3-b]-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0394] At 25° C. and under nitrogen protection, T-030 (250 mg, 0.49 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0° C., then a solution of 4-dimethylaminopyridine (478 mg, 3.91 mmol) in dichloromethane (3 mL) was added, followed by the slow and dropwise addition of a solution of triphosgene (72 mg, 0.24 mmol) in dichloromethane (10 mL) and reacted at 0° C. for 20 min under stirring. The reaction solution was bubbled with nitrogen for 20 min, then a solution of (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 (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added and reacted at 0° C. for 1 h under stirring. The reaction solution was concentrated under reduced pressure, the residue was purified by preparative high performance liquid chromatography (method A) to obtain the title compound (500 mg). ESI-MS (m/z): 1597.5[M+H].sup.+.

[0395] Step 3: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl)carbonate

[0396] At room temperature, compound 33-1 (14 mg, 0.05 mmol) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), followed by an addition of cuprous bromide (11 mg, 0.08 mmol) and reacted for 1 h under stirring. Purification was performed on preparative high performance liquid chromatography (method B) to obtain the title compound (30 mg). ESI-MS (m/z): 815.9[(M-273)/2+H].sup.+.

[0397] Step 4: synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound TL033)

[0398] Compound 33-2 (30 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (0.2 mL) was added to the reaction solution and reacted at room temperature for 30 min. Purification was performed on preparative high performance liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (20.0 mg). Identification of the title compound is as follows:

[0399] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.18 (s, 1H), 9.10 (s, 2H), 8.38 (t, J= 5.56 Hz, 1H), 8.32 (d, J = 8.40 Hz, 1H), 8.22 - 8.20 (m, 2H), 8.09 (t, J = 5.68 Hz, 1H), 7.91 - 7.87 (m, 2H), 7.82 -7.78 (m, 1H), 7.69 (brs, 3H), 7.61 (d, J= 8.56 Hz, 2H), 7.32 (d, J= 8.56 Hz, 2H), 7.06 (s, 1H), 5.56 (d, J = 16.96 Hz, 1H), 5.51 (d, J = 16.96 Hz, 1H), 5.47 (d, J = 19.28 Hz, 1H), 5.42 (d, J = 19.28 Hz, 1H), 5.14 (d, J= 12.20 Hz, 1H), 5.07 (d, J= 12.16 Hz, 1H), 4.48 (t, J= 5.24 Hz, 2H), 4.46 - 4.43 (m, 1H), 4.29 (d, J = 5.60 Hz, 2H), 4.08 - 3.95 (m, 5H), 3.79 (t, J = 5.28 Hz, 2H), 3.51 - 3.43 (m, 32H), 3.40 (s, 3H), 3.39 - 3.35 (m, 2H), 3.30 - 3.26 (m, 2H), 3.00 (s, 3H), 2.82 - 2.74 (m, 2H), 2.56 (t, J= 7.08 Hz, 2H), 2.29 (t, J= 7.36 Hz, 2H), 2.23 - 2.13 (m, 2H), 1.82 (p, J= 7.24 Hz, 2H), 1.78 -1.63 (m, 2H), 1.61-1.49 (m, 2H), 1.42 -1.27 (m, 2H), 1.15 (d, J= 6.80 Hz, 3H), 1.13 (d, J = 6.76 Hz, 3H), 0.90 (t, J = 7.32 Hz, 3H). ESI-MS (m/z): 816.0[M/2+H].sup.+. [α].sub.D.sup.20 is -19.55° (c =1.000 g/100 mL, CH.sub.3CN).

Example 15: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl((S)-11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-carbonate

[0400]

[0401] Step 1: synthesis of 4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo 6,12,15,18,21,24,27-nonoxy-((S)-9-((tert-butyldimethylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1,2,3,4-tetrahydroquinolin-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0402] At room temperature, compound 34-1 (100 mg, 0.2 mmol) was dissolved in anhydrous dichloromethane (2 ml) under nitrogen protection, then cooled to 0° C., followed by an addition of a solution of 4-dimethylaminopyridine (144 mg, 1.18 mmol) in anhydrous dichloromethane (0.5 ml), then a solution of triphosgene (41 mg, 0.14 mmol) in dry dichloromethane (0.5 ml) was slowly added dropwise. The resulting mixture was reacted at 0° C. for 1h under stirring. Then a solution of (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(160 mg, 0.15 .Math.mol) in dry dichloromethane (0.5 mL) was added to the reaction solution and reacted at room temperature for 1h. Purification was performed on preparative high performance liquid chromatography (method B) to obtain the title compound (60 mg). ESI-MS (m/z): 1592.7[M+H].sup.+.

[0403] Step 2: synthesis of (S)-9-(tert-butyldimethylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl-4-((S)-2-(4-(((6-2-(methylsulfonyl)pyrimidin-5-yl)-35-(4-((6-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-dioxo 6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)carbonate

[0404] At room temperature, compound 34-2 (40 mg, 0.03 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (11.50 mg, 0.04 mmol) were dissolved in dimethyl sulfoxide and water (0.5 ml: 0.1 ml), and cuprous bromide (9.01 mg, 0.06 mmol) was added. The resulting mixture was reacted for 1h under stirring. Purification was performed on preparative high performance liquid chromatography (method B) to obtain the title compound (20 mg). ESI-MS (m/z): 1897.5[M+H].

[0405] Step 3: synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl((S)-11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-carbonate(compound TL034)

[0406] At room temperature, compound 34-3 (30 mg, 0.018 mmol) was dissolved in acetonitrile and water (0.4 mL: 0.1 mL), then a mixed solution of trifluoroacetic acid and acetonitrile (0.5 mL: 0.5 mL) was added dropwise, and stirred at room temperature for 2 hours. Purification was performed on preparative high performance liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (12 mg). ESI-MS (m/z): 1511.5[M+H].sup.+.

Example 16: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0407]

[0408] Step 1: synthesis of ((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1,2,3,6-triazacycloheptane-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0409] Operations similar to those described in step 1 of example 15 were carried out to obtain the title compound (60 mg), except that compound 34-1 was replaced with compound 35-1. ESI-MS (m/z): 1561.5[M+H].sup.+.

[0410] Step 2: synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)-4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido]carbonate

[0411] A synthetic method similar to that as described in step 2 of example 15 was adopted to obtain the title compound (20 mg), except that compound 34-2 was replaced with compound 35-2. ESI-MS (m/z): 1866.5[M+H].

[0412] Step 3: synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound TL035)

[0413] A synthetic method similar to that as described in step 3 of example 15 was adopted to obtain the trifluoroacetate of the title compound (4.9 mg), except that compound 34-3 was replaced with compound 35-3. ESI-MS (m/z): 1594.5[M+H].sup.+.

Example 17: Synthesis of 4-((S,Z)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadotetracontyl-41-alkenamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0414]

[0415] Step 1: synthesis of (Z)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enoic acid

[0416] At 20° C., compound 3-4 (200 mg, 0.67 mmol) was dissolved in methanol (8.0 mL), and a Lindlar catalyst (20 mg) was added under nitrogen protection, then the solution was subject to hydrogen substitution for three times. Hydrogenation was conducted at 20° C. for 3 hours. After filtration, the filtrate was subject to spin drying to obtain the title compound (150 mg). ESI-MS (m/z):271.1 [M+H].sup.+.

[0417] Step 2: synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-((S,Z)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadotetracontyl-41-alkenamido)benzylcarbonate

[0418] At room temperature, compound 45-2 (8 mg, 0.030 mmol) was dissolved in dichloromethane (2 mL), then 2-(7-azobenzotriazol)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (14.9 mg, 0.039 mmol) and N,N-diisopropylethylamine (8.8 mg, 0.068 mmol) were added. The reaction solution was stirred at room temperature for 10 min, then compound 48-1 (30 mg, 0.020 mmol) was added and reacted at room temperature for 1 h under stirring. Purificaiton was performed on preparative high performance liquid chromatography (method B) to obtain the title compound (30 mg). ESI-MS (m/z): 1787.8[M+H].sup.+.

[0419] Step 3: synthesis of 4-((S,Z)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkenamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound TL045)

[0420] At room temperature, compound 45-3 (30 mg, 0.017 mmol) was dissolved in acetonitrile (1 ml), and a solution of trifluoroacetic acid (0.5 ml) in acetonitrile (0.5 ml) was added dropwise. The reaction solution was stirred at room temperature for 20 min. Purificaiton was performed on preparative high performance liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (9 mg). ESI-MS (m/z): 1515.6[M+H].sup.+.

Example 18: 4-((S)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkynamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0421]

[0422] Step 1: synthesis of (S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadotetracontyl-41-alkynamido)benzylcarbonate

[0423] A synthetic method similar to that described in step 1 of example 12 was adopted to obtain the title compound (15 mg), except that compound 24-1 was replaced with compound 48-1. ESI-MS (m/z): 1785.8[M+H].sup.+.

[0424] Step 2: synthesis of 4-((S)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkynamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (compound TL048)

[0425] A synthetic method similar to that described in step 2 of example 12 was adopted to obtain the trifluoroacetate of the title compound (11.35 mg), except that compound 24-2 was replaced with compound 48-2. ESI-MS (m/z): 1513.7[M+H].sup.+.

Example 19: 4-((S)-2-(4-aminobutyl)-35-(4-((2-(2-((methylsulfonyl)pyrimidin-5-yl)thiazol-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0426]

[0427] Step 1: synthesis of 2-(2-(methylthio)pyrimidin-5-yl)thiazol-4-carboxylic acid

[0428] Compound 49-1 (100 mg, 0.40 mmol), 2-bromo-4-thiazolecarboxylic acid (99.01 mg, 0.48 mmol), potassium carbonate (137.03 mg, 0.99 mmol) and [1,1′-bis(diphenylphosphino)ferrocenyl]palladium dichloride (29.02 mg, 0.04 mmol) were dissolved in N,N-dimethylformamide (4 mL) and water (1 ml), under nitrogen protection, the reaction system was heated to 100° C. and stirred for 4 h. Then the reaction solution was cooled to room temperature and dropped into water. After filtration, the filtrate was collected and extracted with ethyl acetate (10 mL× 3). The aqueous phase was collected and adjusted with dilute hydrochloric acid to pH=3 to precipitate a solid, and filtered. The filter cake was collected to obtain the title compound (70 mg). ESI-MS (m/z):254.0[M+H].sup.+.

[0429] Step 2: synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazol-4-carboxylic acid

[0430] Compound 49-2 (73 mg, 0.29 mmol) was dissolved in dichloromethane (15 mL), and m-chloroperoxybenzoic acid (175.53 mg, 0.87 mmol, 85%) was added. The reaction system was stirred overnight at room temperature. The solvent was concentrated under reduced pressure. Purificaiton was performed on preparative high performance liquid chromatography (method D) to obtain the title compound (20 mg). ESI-MS (m/z):286.0[M+H].sup.+.

[0431] Step 3: synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)thiazo-4-carboxamide

[0432] Compound 49-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (2 mL), and O-(7-benzotriazol)-N,N,N,N-tetramethyluronium hexafluorophosphate (39.98 mg, 0.11 mmol) was added. The obtained reaction system was cooled to 0° C., then N, N-diisopropylethylamine (22.65 mg, 0.18 mmol) and propargylamine (4.63 mg, 0.09 mmol) were added thereto. Thereaction solution was stirred at room temperature for 3 hours. Purificaiton was performed on preparative high performance liquid chromatography (method D) to obtain the title compound (10 mg). ESI-MS (m/z):323.0[M+H].sup.+.

[0433] Step 4: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl-(4-((S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyl)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazol-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonazeza-3,9-diazapentatriacontamino)benzyl)carbonate

[0434] At room temperature, compound 33-1 (30 mg, 0.02 mmol) and compound 49-4 (9.08 mg, 0.03 mmol) were dissolved in dimethyl sulfoxide and water (2 mL/0.5 mL), and cuprous bromide (5.39 mg, 0.04 mmol) was added and reacted for 2 hours under stirring. After filtration, the filtrate was purified by preparative high performance liquid chromatography (method B) to obtain the title compound (20 mg). ESI-MS (m/z): 1647.3[M+H-273].sup.+.

[0435] Step 5: synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((2-(2-((methylsulfonyl)pyrimidin-5-yl)thiazol-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrone[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0436] At room temperature, compound 49-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.2 mL) was added dropwise. The obtained reaction solution was stirred at room temperature for 20 min. The reaction solution was then concentrated. The residue was purified by preparative high performance liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (8 mg). ESI-MS (m/z): 1647.9[M+H].sup.+.

Example 20: 4-((S)-2-(4-aminobutyric acid)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)-oxazol-4-formylamino)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0437]

[0438] Step 1: synthesis of ethyl 2-(2-(methylthio)pyrimidin-5-yl)oxazol-4-carboxylate

[0439] At 25° C., ethyl 2-bromooxazol-4-carboxylate (100 mg, 0.45 mmol) and compound 49-1 (126 mg, 0.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane and water (4 mL/2 mL), then potassium carbonate (125 mg, 0.9 mmol) and [1,1′-bis(diphenylphosphino)ferrocen]palladium dichloride (33 mg, 0.05 mmol) were successively added, under N.sub.2 protection, the mixture was heated to 90° C. and reacted for 3 hours. The reaction solution was filtered through diatomite. The filtrate was diluted with water (50 mL) and extracted with ethyl acetate (30 mL×3). The organic phases were combined and dried. The desiccant was removed by filtration, the filtrate was concentrated under reduced pressure to obtain a crude product which was purified by preparative thin layer chromatography (petroleum ether/ethyl acetate =2/1) to obtain the title compound (40 mg). ESI-MS (m/z):266.1[M+H].sup.+.

[0440] Step 2: synthesis of 2-(2-(methylthio)pyrimidin-5-yl)oxazol-4-carboxylic acid

[0441] At 25° C., compound 50-1 (50 mg, 0.19 mmol) was dissolved in a mixed solvent of tetrahydrofuran and water (4 mL/2 mL), after complete dissolution, lithium hydroxide monohydrate (40 mg, 0.94 mmol) was added thereto and reacted at 25° C. for 1 h. The reaction solution was diluted with water (15 mL) and extracted with ethyl acetate (20 mL× 2). The aqueous phase was adjusted with 1N dilute hydrochloric acid to pH=2-3, then extracted with a mixed solvent of dichloromethane/methanol (v:v=10:1) (20 mL×3). The organic phases were combined, washed with saturated saline (30 mL×1) and dried over anhydrous sodium sulfate. The desiccant was removed by filtration and the filtrate was concentrated to obtain the title compound (40 mg), which was directly used in further reaction without purification. ESI-MS (m/z):238.1 [M+H].sup.+.

[0442] Step 3: synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazol-4-carboxylic acid

[0443] At 25° C., compound 50-2 (40 mg, 0.17 mmol) was dissolved in dichloromethane (6 mL), after complete dissolution, m-chloroperoxybenzoic acid (29 mg, 0.17 mmol) was added thereto and reacted at 25° C. for 14 hours under stirring. The reaction solution was concentrated, and the residue was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (20 mg). ESI-MS (m/z):269.9[M+H].sup.+.

[0444] Step 4: synthesis of 2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazol-4-formamide

[0445] At 25° C., compound 50-3 (20 mg, 0.07 mmol) was dissolved in dichloromethane (4 mL), then O-(7-azabenzotriazol-1-yl)-N,N,N′,N′- tetramethyluronium hexafluorophosphate (42 mg, 0.11 mmol) and N,N-diisopropylethylamine (19 mg, 0.15 mmol) were successively added and stirred for 5 min, followed by an addition of propargylamine (5.0 mg, 0.09 mmol) and then the resulting mixture was stirred at room temperature for 30 min. The reaction solution was concentrated, the residue was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (5.0 mg). ESI-MS (m/z): 306.9 [M+H].sup.+.

[0446] Step 5: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazol-4-formylamino)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl)carbonate

[0447] At 25° C., compound 50-4 (6.0 mg, 0.02 mmol) and compound 33-1 (30 mg, 0.02 mmol) were dissolved in a mixed solvent (2 mL/0.5 mL) of dimethyl sulfoxide and water, and cuprous bromide (5.0 mg, 0.04 mmol) was added in one batch. The resulting mixture was reacted at room temperature for 2 hours. The reaction solution was filtered and purified by preparative high performance liquid chromatography (method B) to obtain the title compound (25 mg). ESI-MS (m/z): 1631.3[(M-273+H].sup.+.

[0448] Step 6: synthesis of 4-((S)-2-(4-aminobutyric acid)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazol-4-formylamino)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0449] At 25° C., compound 50-5 (20 mg, 0.01 mmol) was dissolved in dichloromethane (2.0 mL). After complete dissolution, the reaction mixture was added with trifluoroacetic acid (0.2 mL) and reacted at 25° C. for 10 min. The reaction solution was concentrated, and the residue was purified by preparative high performance liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (3.0 mg). ESI-MS (m/z):816.5[M/2 + H].sup.+.

Example 21: N-((1-((6S,9S)-1-amino-6-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,SS)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxy-13,19,22,25,28,31,34,37,40-nonaoxa-2,7,10,16-tetraazaanthracen-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0450]

[0451] Step 1: synthesis of 9-fluorenylmethyl ((S)-1-(((S)-1-((4-(((S)2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyrylamido)-N,3-dimethylbutyrylamido)-3-methoxy-5-methylheptanoyl)pyrro-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate

[0452] At room temperature, compound 51-1 (100 mg, 0.17 mmol) and 9-fluorenylmethyl ((S)-1-(((S)-1-((4-(((S)-2-amino-3-phenylpropanamido)methyl)phenyl)amino)-1-oxo-5-pentylureido-2-yl)amino)-3-methyl-1-oxobutan-2-yl) carbamate trifluoroacetate (144 mg, 0.17 mmol) were dissolved in N,N-dimethylformamide (2 mL) and cooled to 0° C., then 1-hydroxybenzotriazole (34 mg, 0.25 mmol), N-methylmorpholine (51 mg, 0.51 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(48 mg, 0.25 mmol) were successively added. After addition, the reaction solution was stirred at 0° C. for 5 hours. The reaction solution was poured into water (20 mL) to precipitate a white solid, followed by suction filtration. The filter cake was washed and dried to obtain the title compound (200 mg). ESI-MS (m/z): 1329.2 [M + H].sup.+.

[0453] Step 2: synthesis of (S)-2-((S)-2-amino-3-butyrylamino)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-butyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phenyl)-5-ureidovaleramide

[0454] At room temperature, compound 51-2 (200 mg, 0.12 mmol) was dissolved in N, N-dimethylformamide (5 mL), and piperidine (0.5 mL) was added. The reaction solution was stirred at room temperature for 2 hours, and then purified by preparative high performance liquid chromatography (method D) to obtain the title compound (65 mg). ESI-MS (m/z): 1107.2 [M + H].sup.+.

[0455] Step 3: synthesis of (S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)-N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)-5-ureidovaleramide

[0456] 32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatricarboxylic acid (33.1 mg, 0.06 mmol) was dissolved in N,N-dimethylformamide (5 mL), then O-(7-azabenzotriazol)-N,N,N,N-tetramethyluronium hexafluorophosphate (38 mg, 0.10 mmol) and N,N-diisopropylethylamine (26 mg, 0.20 mmol) were added. The reaction solution was stirred at room temperature for 10 min, then cooled to 0° C., and added with compound 51-3 (55 mg, 0.05 mmol). The reaction solution was stirred at room temperature for 2 hours, and purified by preparative high performance liquid chromatography (method D) to obtain the title compound (56 mg). ESI-MS (m/z): 821.8 [M/2 + H].sup.+.

[0457] Step 4: synthesis of N-((1-((6S,9S)-1-amino-6-((4-(((S)-2-((2R,3R)-3-((S)1-((3R,4S,SS)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanamido)methyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxy-13,19,22,25,28,31,34,37,40-nonaoxa-2,7,10,16-tetraazaanthracen-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamide

[0458] At room temperature, compound 51-4 (56 mg, 0.04 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-5-hexynamide (16 mg, 0.05 mmol) were dissolved in a mixed solution of dimethyl sulfoxide and water (2 mL/0.5 mL), and cuprous bromide (10 mg, 68.17 umol) was added. The obtained mixture was stirred for 2 hours, and then filtered. The filtrate was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (50 mg). ESI-MS (m/z): 974.3[M/2+H].sup.+.

Example 22: 4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7,1 1-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12-triazadotetracontyl)benzyl-((S)-1-(((S)-1-(((3R,4S,SS)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0459]

[0460] Step 1: 4-((2S,SS)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6, 12-triazadotetracontyl)benzyl-((S)-1-(((S)1-(((3R,4S,SS)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0461] At room temperature, compound 53-1 (100 mg, 0.09 mmol) was dissolved in N,N-dimethylformamide (3 mL), then 1-hydroxybenzotriazole (13 mg, 0.09 mmol), N,N-diisopropylethylamine (36 mg, 0.28 mmol) and compound 52-1 (67 mg, 0.09 mol) were added. The reaction solution was stirred at room temperature for 16 hours, and then purified by preparative high performance liquid chromatography (method D) to obtain the title compound (120 mg). ESI-MS (m/z): 830.1[M/2+H].sup.+.

[0462] Step 2: synthesis of 4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,7, 11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonaoxa-3,6, 12-triazadotetracontyl)benzyl-((S)-1-(((S)-1-(((3R,4S,SS)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0463] At room temperature, compound 52-2 (22 mg, 0.07 mmol) was dissolved in a mixed solution of dimethyl sulfoxide and water (3 mL/0.3 mL), then cuprous bromide (18 mg, 0.13 mmol) was added and stirred for 1 h. The reaction solution was filtered. The filtrate was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (92 mg). ESI-MS (m/z): 982.8[M/2+H].sup.+.

Example 23: Synthesis of (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,SS)-4-((S)-N,3-dimethyl-2-((S)-3-methyl2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-carbamoyl)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatriacontamino)butyrylamino)-5-ureidovalerylamino)benzyl)oxy)carbonyl)amino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine

[0464]

[0465] Step 1: synthesis of 4-((2S,5S)-38-azido-5-isopropyl-4,7,11-trioxo-2-(3-ureidopropyl)-9,15,18,21,24,27,30,33,36-nonoxy-\3,6,12-triazaoctatriacontamino)benzyl-(4-nitrophenyl)-carbonate

[0466] \\At 25° C., compound 29-1 (500 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (10 mL), and added with N,N-diisopropylethylamine (141 mg, 1.09 mmol), then followed by a dropwise addition of a solution of di(p-nitrobenzol)carbonate (332 mg, 1.09 mmol) in dichloromethane (1 mL). After the addition, the mixture was reacted at 25° C. for 3 hours under stirring. The reaction solution was purified by reverse column (C18) chromatography (acetonitrile/water=1:2) to obtain the title compound (400 mg). ESI-MS (m/z):1081.9[M+H].sup.+.

[0467] \\\Step 2: synthesis of (S)-2-((2R\,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((((4-((S)-2-((S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatriacontamido)-3-methylbutyrylamino)-5-ureidopentanoylamino)benzyl)oxy)carbonyl)(methyl)amino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine

[0468] At 25° C., compound 53-1 (60 mg, 0.06 mmol) and ((2R)-3-((2S)-1-((3R, 5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butyrylamino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methypropionyl)-L-phenylalanine (41 mg, 0.06 mmol) were dissolved in N,N-dimethylformamide (2 mL). After complete dissolution, 1-hydroxybenzotriazole (8 mg, 0.06 mmol) was added. After the addition, the mixture was stirred at 25° C. for 16 hours. The reaction solution was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (38 mg). ESI-MS (m/z): 837.2[M/2+H].sup.+.

[0469] Step 3: synthesis of (S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-carbamoyl)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azatriacontamino)butyrylamino)-5-ureidovalerylamino)benzyl)oxy)carbonyl)amino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine

[0470] At 25° C., 2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazol-4-formamide (9 mg, 0.03 mmol) and compound 53-2 (50 mg, 0.03 mmol) were dissolved in a mixed solvent of dimethyl sulfoxide and water (1 mL/0.25 mL). After complete dissolution, cuprous bromide (11 mg, 0.08 mmol) was added. After the addition, the mixture was stirred for 1 h under N.sub.2 protection. Filtration was then conducted, and the filtrate was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (25 mg). ESI-MS (m/z): 989.9[M/2+H].sup.+.

Example 24: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0471]

[0472] Step 1: synthesis of (S)-4-(35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyryl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl-(4-nitrophenyl)-carbonate

[0473] At room temperature, compound 54-1 1 g, 0.95 mmol) was dissolved in dichloromethane (20 ml), then added with N,N-diisopropylethylamine (488 mg, 3.77 mmol), followed by a dropwise addition of a solution of di-(p-nitrophenyl)-carbonate (860 mg, 2.83 mmol) in dichloromethane (10 mL). The resulting reaction solution was stirred at room temperature for 6 hours and purified by silica gel column chromatography (dichloromethane/methanol=40/1) to obtain the title compound (900 mg). ESI-MS (m/z): 953.0[M+H-273].sup.+.

[0474] Step 2: synthesis of 4-((S)-35-azide-2-(4-(((4-methoxyphenyl) benzhydryl)amino)butyryl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl((S)-1-(((S)-1-(((3R,4S,SS)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0475] At room temperature, to compound 54-2 (2 ml) were added 1-hydroxybenzotriazole (33 mg, 0.25 mmol), N, N-diisopropylethylamine (48 mg, 0.37 mmol), and then compound 52-1 (88 mg, 0.12 mmol). The obtained reaction solution was stirred at room temperature for 16 hours and then purified by preparative high performance liquid chromatography (method B) to obtain the title compound (150 mg). ESI-MS (m/z): 1803.6[M+H].sup.+.

[0476] Step 3: synthesis of 4-((S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyryl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl((S)-1-(((S)-1-(((3R,4S,SS)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0477] At room temperature, compound 54-3 (100 mg, 0.06 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-5-hexynamide (26 mg, 0.08 mmol) were dissolved in dimethyl sulfoxide (2 mL) and water (0.5 mL), then cuprous bromide (16 mg, 0.11 mmol) was added and stirred for 2 hours. Filtration was then performed, and the filtrate was purified by preparative high performance liquid chromatography (method B) to obtain the title compound (70 mg). ESI-MS (m/z): 1936.6[M+H-273].sup.+.

[0478] Step 4: synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

[0479] At room temperature, compound 54-4 (70 mg, 0.04 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.2 mL) was added dropwise. The obtained reaction solution was stirred at room temperature for 20 min, then concentrated, and the residue was purified by preparative high performance liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (55 mg). ESI-MS (m/z): 918.8[M/2+H].sup.+.

Example 25: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3\′\,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0480]

[0481] Step 1: synthesis of methyl 4-(2-(methylthio)pyrimidin-5-yl)benzoate

[0482] At 25° C., compound 49-1 (252 mg, 1.0 mmol), water (3 mL), Pd(dppf)Cl.sub.2 (40 mg, 0.05 mmol) and potassium carbonate (277 mg, 2.0 mmol) were added successively to a solution of methyl p-bromobenzoate (215 mg, 1.0 mmol) in 1,4-dioxane (5 mL) and stirred at 80° C. for 4 hours. The reaction solution was extracted with ethyl acetate (30 mL×3). The organic phases were combined and dried, then the insoluble substances were removed by filtration, and the residue was purified by silica gel column chromatography to obtain the title compound (220 mg). ESI-MS (m/z):261.0[M+H].sup.+.

[0483] Step 2: synthesis of 4-(2-(methylthio)pyrimidin-5-yl)benzoic acid

[0484] At 25° C., lithium hydroxide monohydrate (322 mg, 7.68 mmol) and water (3 ml) were respectively added to a solution of compound 55-1 (500 mg, 1.92 mmol) in tetrahydrofuran (3 ml) and stirred for 4 hours. The reaction solution was adjusted with 1N hydrochloric acid to pH=3-4, and extracted with ethyl acetate (20 mL×3). The organic phases were combined and dried. The insoluble substances were removed by filtration, and the residue was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (430 mg). ESI-MS (m/z):246.9[M+H].sup.+.

[0485] Step 3: synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)benzoic acid

[0486] At 25° C., m-chloroperoxybenzoic acid (420 mg, 2.44 mmol) was added to a solution of compound 55-2 (200 mg, 0.81 mmol) in dichloromethane (5 ml), and stirred for 5 hours, and then purified by silica gel column chromatography to obtain the title compound (180 mg). ESI-MS (m/z):279.0[M+H].sup.+.

[0487] Step 4: synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)benzamide

[0488] At 25° C., benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (100 mg, 0.26 mmol) was added to a solution of compound 55-3 (50 mg, 0.18 mmol) in dichloromethane (10 mL), and stirred for 30 min, then propynylamine (10 mg, 0.2 mmol) and N,N-diisopropylethylamine (70 mg, 0.5 mmol) were added to the reaction solution and reacted for 2.5 h under stirring. The reaction solution was purified by silica gel column chromatography to obtain the title compound (20 mg). ESI-MS (m/z):316.0[M+H].sup.+.

[0489] Step 5: synthesis of (S)-4-ethyl-11-(2-(N-isopropylmethanesulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl)carbonate

[0490] At 25° C. and under N.sub.2 protection, cuprous iodide (10 mg, 0.05 mmol) and water (2 mL) were successively added to dimethyl sulfoxide solution (2 mL) of compound 55-4 (16 mg, 0.05 mmol) and compound 33-1 (80 mg, 0.05 mmol) and reacted for 1 h under stirring. Purificaiton (method B) was performed to obtain the title compound (79 mg). ESI-MS (m/z): 1641.5[M-273+H].sup.+.

[0491] Step 6: synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benzamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonamido)ethyl)-\3,14-dioxo-\3,4,12,14-tetrahydro-1H-pyrano[3\′\,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbonate

[0492] At 25° C., compound 55-5 (55 mg, 0.029 mmol) was added to trifluoroacetic acid (0.5 mL) in a mixed solvent of water/acetonitrile (0.1 mL/0.5 mL), and reacted for 15 min under stirring. The reaction solution was purified by preparative high performance liquid chromatography (method C) to obtain the trifluoroacetate of the title compound (42 mg). ESI-MS (m/z):821.0[M/2+H].sup.+.

Example 26: N-((1-((6S,9S)-1-amino-6-((4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,SS)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonoxy-2,7,10,16-tetraazadotetracont-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide

[0493]

[0494] Step 1: synthesis of 32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azadotriacontanoic acid

[0495] At 20° C., compound 56-1 (750 mg, 1.28 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynylamide (496 mg, 1.54 mmol) were dissolved in dimethyl sulfoxide (10 mL), and cuprous bromide (465 mg, 3.21 mmol) was added in one batch. After the addition, the mixture was reacted for 12 hours under stirring. The reaction solution was filtered, and the filtrate was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (500 mg). ESI-MS (m/z): 860.4[M+H].sup.+.

[0496] Step 2: synthesis of (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)amino)-1-oxo-5-ureidopentanoylamino-2-yl)amino)-3-methyl-1 -oxobutan-2-yl)carbamate

[0497] At 25° C., (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(4-aminophenyl)-3-azidopropyl-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptyl-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamine(185 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL), then HATU (137 mg, 0.36 mmol) was added and stirred for 5 min, followed by an addition of (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)-5-ureidopentanoic acid (131 mg, 0.26 mmol). The mixture was stirred at room temperature for 30 min. The reaction solution was directly used in further reaction. ESI-MS (m/z): 626.0[M/2+H].sup.+.

[0498] Step 3: synthesis of (S)-2-((S)-2-amino-3-methylbutyrylamino)-N-(4-((S)-3-azido-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)-5-ureidovaleramide

[0499] At 25° C., diethylamine (0.5 mL) was added to the reaction solution obtained in step 2, and stirred for reaction for 30 min after the addition. The reaction solution was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (70 mg). ESI-MS (m/z): 515.0[M/2+H].sup.+.

[0500] Step 4: synthesis of N-((1-((6S,9S)-1-amino-6-((4-((S)-3-azido-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)carbamoyl)-9-isopropyl-1,8,11,15-tetraoxo-13,19,22,25,28,31,34,37,40-nonoxy-2,7,10,16-tetraazadotetracont-42-yl)-1H-1,2,3-triazol-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide

[0501] At 25° C., (S)-2-((S)-2-amino-3-methylbutyrylamino)-N-(4-((S)-3-azido-2-((2R,3R)-3-((S)- 1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)propyl)phenyl)-5-ureidovaleramide (95 mg, 0.092 mmol) and 32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azadotriacontanic acid (79 mg, 0.092 mmol) were dissolved in N,N-dimethylformamide (4 mL), and HATU (70 mg, 0.184 mmol) was added in one batch. The mixture was stirred at room temperature for 1 h. The reaction solution was purified by preparative high performance liquid chromatography (method D) to obtain the title compound (30 mg). ESI-MS (m/z): 935.8[M/2+H].sup.+.

III. Coupling of the Compound Containing the Bioactive Molecule and the Linker With an Antibody

Example 27: Preparation of BT001002

[0502] 0.3 mL of antibody Sacituzumab (anti-Trop-2, 33.5 mg/mL) was diluted with 0.25 ml of a solution (pH 7.6) containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate, to which 0.45 ml of a solution (pH 7.6) containing 20 mM PB and 150 mM NaCl was added and evenly mixed. The mixture was adjusted with 1 M K.sub.2HPO.sub.4 solution to pH=7.4, and then 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added and evenly mixed, which was allowed to stand at room temperature for 30 min. To the solution system, TL003 dissolved in dimethyl sulfoxide was added in an amount of 15 equiv. and evenly mixed, which was allowed to stand at room temperature for 2 hours. After the addition, 6.1 .Math.l of 100 mM cysteine was added to terminate the reaction. At last, the buffer was replaced with a 20 mM PB buffer solution of pH 6.44 by G-25 gel column to obtain the coupling product of TL003 with Sacituzumab, which was named as BT001002.

Example 28: Preparation of BT001004

[0503] 0.285 mL of Sacituzumab (anti-Trop-2, 17.6 mg/mL) was diluted with 0.095 mL of a diluent (a solution containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate, pH 7.6). Then the diluted solution was adjusted with 1 M Na.sub.2HPO.sub.4 solution to pH 7.4, and 10 mM TCEP solution was added and evenly mixed, which was allowed to stand at room temperature for 30 min. To the solution system, TL019 dissolved in dimethyl sulfoxide was added in an amount of 9 equiv. and evenly mixed, which was allowed to stand at room temperature for 2 h. At last, the buffer was replaced with a PBS buffer solution of pH 6.5 by G-25 gel column to obtain the coupling product of TL019 with Sacituzumab, which was named as BT001004.

Example 29: Preparation of BT001012

[0504] A method similar to that described in example 27 was adopted to obtain the coupling product of TL024 with Sacituzumab, which was named as BT001012, except that TL003 was replaced by trifluoroacetate of TL024.

Example 30: Preparation of BT001013

[0505] A method similar to that described in example 27 was adopted to obtain the coupling product of TL048 with Sacituzumab, which was named as BT001013, except that TL003 was replaced by trifluoroacetate of TL048.

Example 31: Preparation of BT001018

[0506] A method similar to that described in example 27 was adopted to obtain the coupling product of TL030 with Sacituzumab, which was named as BT001018, except that TL003 was replaced by TL030.

Example 32: Preparation of BT001021

[0507] 0.3 mL of Sacituzumab (anti-Trop-2, 33.5 mg/mL) was diluted with 0.25 ml of a solution (pH 7.6) containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate, then 0.45 mL of a solution (pH 7.6) containing 20 mM PB and 150 mM NaCl was added and evenly mixed. The mixture was adjusted with 1 M Na.sub.2HPO.sub.4 solution to pH=7.4, then 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added and evenly mixed, which was allowed to stand at room temperature for 30 min. To the solution system, trifluoroacetate of TL033 dissolved in dimethyl sulfoxide was added in an amount of 10 equiv. and evenly mixed, which was allowed to stand at room temperature for 2 hours. Then 6.1.Math.l of 100 mM cysteine was added to terminate the reaction. At last, the buffer was replaced by a PBS buffer solution of pH 6.5 by G-25 gel column to obtain the coupling product of TL033 with Sacituzumab, which was named as BT001021.

Example 33: Preparation of BT001022

[0508] A method similar to that described in example 27 was adopted to obtain the coupling product of TL034 with Sacituzumab, which was named as BT001022, except that TL003 was replaced by trifluoroacetate of TL034.

Example 34: Preparation of BT001023

[0509] A method similar to that described in example 27 was adopted to obtain the coupling product of TL035 with Sacituzumab, which was named as BT001023, except that TL003 was replaced by trifluoroacetate of TL035.

Example 35: Preparation of BT001032

[0510] A method similar to that described in example 27 was adopted to obtain the coupling product of TL045 with Sacituzumab, which was named as BT001032, except that TL003 was replaced by trifluoroacetate of TL045.

Example 36: Preparation of BT001033

[0511] A method similar to that described in example 27 was adopted to obtain the coupling product of TL033 with antibody M1, which was named as BT001033, except that TL003 was replaced by trifluoroacetate of TL033 and Sacituzumab was replaced by antibody M1.

Example 37: Preparation of BT001034

[0512] A method similar to that described in example 27 was adopted to obtain the coupling product of TL033 with antibody M2, which was named as BT001034, except that TL003 was replaced by trifluoroacetate of TL033 andSacituzumab was replaced by antibody M2.

Example 38: Preparation of BT001035

[0513] 0.3 mL of antibody M3 (anti-Trop-2, 33.5 mg/mL) was diluted with 0.25 ml of a solution (pH 7.6) containing 20 mM PB, 150 mM NaCl and 20 mM sodium edetate, then 0.45 mL of a solution (pH 7.6) containing 20 mM PB and 150 mM NaCl was added and evenly mixed. The mixture was adjusted with 1 M Na.sub.2HPO.sub.4 solution to pH=7.4, then 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added and evenly mixed, which was allowed to stand at room temperature for 30 min. To the solution system, trifluoroacetate of TL033 dissolved in dimethyl sulfoxide was added in an amount of 10 equiv. and evenly mixed. The resulting mixture was allowed to stand at room temperature for 2 hours. Then 6.1 .Math.l of 100 mM cysteine was added to terminate the reaction. At last, the buffer was replaced with a PBS buffer solution of pH 6.5 by G-25 gel column to obtain a coupling product of TL033 with antibody M3, which was named as BT001035.

Example 39: Preparation of BT001036

[0514] A method similar to that described in example 27 was adopted to obtain the coupling product of TL033 with Trastuzumab, which was named as BT001036, except that TL003 was replaced by trifluoroacetate of TL033 and Sacituzumab was replaced by Trastuzumab.

Example 40: Preparation of BT001040

[0515] A method similar to that described in example 27 was adopted to obtain the coupling product of TL049 with Sacituzumab, which was named as BT001040, except that TL003 was replaced by trifluoroacetate of TL049.

Example 41: Preparation of BT001041

[0516] A method similar to that described in example 27 was adopted to obtain the coupling product of TL050 with Sacituzumab, which was named as BT001041, except that TL003 was replaced by trifluoroacetate of TL050.

Example 42: Preparation of BT001042

[0517] A method similar to that described in example 27 was adopted to obtain the coupling product of TL051 with Sacituzumab, which was named as BT001042, except that TL003 was replaced by TL051.

Example 43: Preparation of BT001043

[0518] A method similar to that described in example 27 was adopted to obtain the coupling product of TL052 with Sacituzumab, which was named as BT001043, except that TL003 was replaced by TL052.

Example 44: Preparation of BT001044

[0519] A method similar to that described in example 27 was adopted to obtain the coupling product of TL053 with Sacituzumab, which was named as BT001044, except that TL003 was replaced by TL053.

Example 45: Preparation of BT001045

[0520] A method similar to that described in example 27 was adopted to obtain the coupling product of TL054 with Sacituzumab, which was named as BT001045, except that TL003 was replaced by trifluoroacetate of TL054.

Example 46: Preparation of BT001046

[0521] A method similar to that described in example 27 was adopted to obtain the coupling product of TL055 with Sacituzumab, which was named as BT001046, except that TL003 was replaced by trifluoroacetate of TL055.

Example 47: Preparation of BT001047

[0522] A method similar to that described in example 27 was adopted to obtain the coupling product of TL056 with Sacituzumab, which was named as BT001047, except that TL003 was replaced by TL056.

Example 48: Determination of Molecular Weight of BT001002 by LC-MS

[0523] The molecular weight of BT001002 obtained by coupling was analyzed by LC-MS. LC conditions: [0524] Liquid chromatographic column: ACQUITU UPLC® Protein BEH C4 1.7 .Math.m, 2.1mm x 100 mm; [0525] Mobile phase A: 0.1%FA/98%H.sub.2O/2%ACN; Mobile phase B: 0.1%FA/2%H.sub.2O/98%ACN; [0526] Flow rate: 0.25 mL/min; Sample room temperature: 8° C.; Column temperature: 60° C.; Sample size: 1 .Math.g;

TABLE-US-00019 Time (min.) 1 7 8 9 13 Mobile phase A (%volume) 90 20 20 90 90 Mobile phase B (%volume) 10 80 80 10 10

[0527] MS conditions: [0528] Mass spectrometer model: Triple TOF 5600+; [0529] GS160; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10 m/z600-5000;

[0530] Results were shown in FIGS. 1-3.

TABLE-US-00020 Theoretical molecular weight and measured molecular weight of BT001002 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.0 24610.7 25887.3 27163.9 28440.5 Measured value Not detected 24611.1 Not detected Not detected Not detected HC Theoretical value 50734.0 52010.6 53287.2 54563.8 55840.4 Measured value Not detected Not detected Not detected 54563.1 Not detected

[0531] In the table, mAb stands for a monoclonal antibody; LC stands for the light chain of an antibody; HC stands for the heavy chain of an antibody; DAR1 stands for a conjugate containing a light chain/heavy chain of an antibody and a bioactive molecule; DAR2 stands for a conjugate containing a light chain/heavy chain of an antibody and two bioactive molecules; DAR3 stands for a conjugate containing a light chain/heavy chain of an antibody and three bioactive molecules; DAR4 stands for a conjugate containing a light chain/heavy chain of an antibody and four bioactive molecules; glycoform stands for the structure of glycan of the two heavy chains: G0F stands for fucosylation and free of galactosylation. The mAb, LC, HC, DAR1, DAR2, DAR3, DAR4, and G0F hereinafter are as described above.

[0532] As can be seen from FIGS. 1-3, both the molecular weights of the light chain and the heavy chain of the antibody are changed after being coupled with TL003, wherein the light chain was coupled with 1 bioactive molecule and the heavy chain was coupled with 3 bioactive molecules. Therefore, it could be inferred that the DAR of the antibody to the bioactive molecule was 8.

Example 49: Determination of Molecular Weight of BT001004 by LC-MS

[0533] The molecular weight of BT001004 obtained from coupling was analyzed by LC-MS.

[0534] LC conditions: [0535] Liquid chromatographic column: ACQUITU UPLC® Protein BEH C18 1.7 .Math.m, 2.1 mm × 100 mm; [0536] Mobile phase A: 0.1%FA/98%H.sub.2O/2% ACN; Mobile phase B: 0.1%FA/2%H.sub.2O/98% ACN; [0537] Flow rate: 0.25 mL/min; Sample room temperature: 8° C.; Column temperature: 60° C.; Sample size: 1 .Math.g;

TABLE-US-00021 Time (min.) 2 20 22 25 26 30 Mobile phase A (%volume) 80 60 10 10 80 80 Mobile phase B (%volume) 20 40 90 90 20 20

[0538] MS conditions: [0539] Mass spectrometer model: Triple TOF 5600+; [0540] GS160; GS2 60; CUR30; TEM 350; ISVF5500; DP300; CE10; m/z 600-5000;

[0541] Results were shown in FIGS. 4-6.

TABLE-US-00022 Theoretical molecular weight and measured molecular weight of BT001004 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24832.7 26331.4 27830.0 29328.7 Measured value 23334.9 24833.7 Not detected Not detected Not detected HC Theoretical value 50734.0 52232.6 53731.3 55230.0 56728.6 Measured value Not detected 52232.1 53730.8 55229.3 Not detected

[0542] LC stands for the light chain of an antibody; and HC stands for the heavy chain of an antibody.

[0543] As can be seen from FIGS. 4-6, in BT001004, the light chain of the antibody was coupled with 0-1 bioactive molecule (LC and DAR1 accounted for 14% and 86%, respectively), and the heavy chain was coupled with 1-3 bioactive molecules (DAR1, DAR2 and DAR3 accounted for 13%, 19% and 68%, respectively). Therefore, it could be calculated that the DAR of the antibody to the bioactive molecule was 7.0.

Example 50: Determination of Molecular Weight of BT001012 by LC-MS

[0544] A method similar to that as described in example 48 was adopted, and results were shown in FIGS. 10 and 11.

[0545] The theoretical molecular weight and measured molecular weight of the light chain and heavy chain of BT001012 obtained by coupling TL024 and the antibody (calculated from main glycoform G0F) were shown in the table below:

TABLE-US-00023 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24803.7 26273.4 27743.0 29212.7 Measured value 23334.8 24804.6 Not detected Not detected Not detected HC Theoretical value 50734.0 52203.6 53673.3 55143.0 56612.6 Measured value Not detected 52203.0 53672.2 55141.8 Not detected

[0546] As can be seen from FIGS. 10 and 11, in BT001012, the light chain of the antibody was coupled with 0-1 toxin (LC and DAR1 accounted for 12.9% and 87.1%, respectively), and the heavy chain was coupled with 1-3 toxins (DAR1, DAR2 and DAR3 accounted for 13.4%, 10.8% and 75.8%, respectively). Therefore, it could be calculated that the DAR of the antibody to toxins was 7.0.

Example 51: Determination of Molecular Weight of BT001013 by LC-MS

[0547] A method similar to that as described in example 48 was adopted, and results were shown in FIGS. 12 and 13.

[0548] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001013 obtained from coupling TL048 and the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00024 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24767.7 26201.3 27634.9 29068.5 Measured value 23334.6 24768.6 Not detected Not detected Not detected HC Theoretical value 50734.0 52167.6 53601.2 55034.8 56468.4 Measured value Not detected 52166.9 53600.8 55034.3 56468.2

[0549] As can be seen from FIGS. 12 and 13, in BT001013, the light chain of the antibody was coupled with 0-1 toxin (LC and DAR1 accounted for 6.8% and 93.2%, respectively), and the heavy chain was coupled with 1-4 toxins (DAR1, DAR2, DAR3 and DAR4 accounted for 12.8%, 12.8%, 64.9% and 9.5%, respectively). Therefore, it could be calculated that the DAR of the antibody to toxins was 7.3.

Example 52: Determination of Molecular Weight of BT001018 by LC-MS

[0550] A method similar to that as described in example 48 was adopted, and results were shown in FIGS. 14 and 15.

[0551] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001018 obtained from coupling TL030 and the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00025 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24926.8 26519.6 28112.3 29705.1 Measured value 23335.6 24928.2 Not detected Not detected Not detected HC Theoretical value 50734.0 52326.7 53919.5 55512.3 57105.0 Measured value 50736.3 52328.0 53920.7 55513.3 Not detected

[0552] As can be seen from FIGS. 14 and 15, in BT001018, the light chain of the antibody was coupled with 0-1 toxin (LC and DAR1 accounted for 55.3% and 44.7%, respectively), and the heavy chain was coupled with 1-3 toxins (DAR1, DAR2 and DAR3 accounted for 19.6%, 23.3% and 49.6%, respectively). Therefore, it could be calculated that the DAR of the antibody to toxins was 5.2.

Example 53: Determination of Molecular Weight of BT001021 by LC-MS

[0553] The molecular weight of the coupled BT001021 was analyzed by LC-MS.

[0554] LC conditions: [0555] Liquid chromatographic column: ACQUITU UPLC® Protein BEH C4 1.7 .Math.m, 2.1 mm × 100 mm; [0556] Mobile phase A: 0.1%FA/98%H2O/2%ACN; Mobile phase B: 0.1%FA/2%H2O/98%ACN; [0557] Flow rate: 0.25 mL/min; Sample room temperature: 8° C.; Column temperature: 60° C.; Sample size: 1 .Math.g;

TABLE-US-00026 Time (min.) 1 7 8 9 13 Mobile phase A (%volume) 90 20 20 90 90 Mobile phase B (%volume) 10 80 80 10 10

[0558] MS conditions: [0559] Mass spectrometer model: Triple TOF 5600+; [0560] GS160; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10 m/z600-5000;

[0561] Results were shown in FIGS. 16 and 17.

[0562] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001021 obtained from coupling TL033 and the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00027 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24884.8 26435.5 27986.3 29537.0 Measured value 23334.6 24885.9 Not detected Not detected Not detected HC Theoretical value 50734.0 52284.7 53835.5 55386.2 56937.0 Measured Not 52284.3 53834.5 55385.4 Not value detected detected

[0563] As can be seen from FIGS. 16 and 17, in BT001021, the light chain of the antibody was coupled with 0-1 toxin (LC and DAR1 accounted for 4.5% and 95.5%, respectively), and the heavy chain wascoupled with 1-3 toxins (DAR1, DAR2 and DAR3 accounted for 15.3%, 17.6% and 67.1%, respectively). Therefore, it could be calculated that the DAR of the antibody to toxins was 6.9.

Example 54: Determination of Molecular Weight of BT001023 by LC-MS

[0564] A method similar to that as described in example 48 was adopted, and results were shown in FIGS. 18 and 19.

[0565] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001023 obtained by coupling TL035 and the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00028 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24848.7 26363.4 27878.1 29392.7 Measured value 23335.4 24850.2 Not detected Not detected Not detected HC Theoretical value 50733.98 52248.7 53763.3 55278.0 56792.7 Measured value 50735.2 52249.1 53764.1 55278.6 Not detected

[0566] As can be seen from FIGS. 18 and 19, in BT001023, the light chain of the antibody was coupled with 0-1 toxin (LC and DAR1 accounted for 15% and 85%, respectively), and the heavy chain was coupled with 0-3 toxins (HC, DAR1, DAR2 and DAR3 accounted for 6.7%, 16.7%, 12.7% and 63.9%, respectively). Therefore, it could be calculated that the DAR of the antibody to toxins was 6.4.

Example 55: Determination of Molecular Weight of BT001040 by LC-MS

[0567] The molecular weight of BT001040 obtained by coupling was analyzed by LC-MS. [0568] Liquid chromatographic column: Thermo MabPacTM RP 4 .Math.m, 3.0 mm* 100 mm [0569] Mobile phase A: 0.1%FA/98%H2O/2% ACN; Mobile phase B: 0.1%FA/2%H2O/98% ACN [0570] Flow rate: 0.25 mL/min; Sample room temperature: 8° C.; Column temperature: 60° C.; Sample size: 1 .Math.g

TABLE-US-00029 Time (min.) 2 20 22 25 26 30 Mobile phase A (%volume) 80 60 10 10 80 80 Mobile phase B (%volume) 20 40 90 90 20 20

[0571] MS conditions: [0572] Mass spectrometer model: Triple TOF 5600+ [0573] GS135; GS2 35; CUR30; TEM 350; ISVF5000; DP250; m/z 600-5000

[0574] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001040 obtained by coupling TL049 with the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00030 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24901.8 26469.5 28037.3 29605.0 Measured value 23334.2 24902.8 Not detected Not detected Not detected HC Theoretical value 50733.98 52301.7 53869.5 55437.2 57005.0 Measured value Not detected 52301.6 53869.2 55437.4 57005.3

[0575] As can be seen from FIGS. 20 and 21, in BT001040, the light chain of the antibody was coupled with 0-1 bioactive molecule (LC and DAR1 accounted for 4.9% and 95.1%, respectively), and the heavy chain was coupled with 1-4 bioactive molecules (DAR1, DAR2, DAR3 and DAR4 accounted for 16.5%, 14.3%, 52.6% and 16.6%, respectively). Therefore, it could be calculated that the DAR of the antibody to bioactive molecules was 7.3.

Example 56: Determination of Molecular Weight of BT001041 by LC-MS

[0576] A method similar to that as described in example 55 was adopted, and results were shown in FIGS. 22 and 23.

[0577] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001041 obtained by coupling TL050 with the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00031 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24885.7 26437.4 27989.1 29540.8 Measured value 23334.5 24886.9 Not detected Not detected Not detected HC Theoretical value 50733.98 52285.7 53837.3 55389.0 56940.7 Measured value Not detected 52285.2 53836.9 55389.1 56940.9

[0578] As can be seen from FIGS. 22 and 23, the light chain of the antibody in BT001041 was coupled with 0-1 bioactive molecule (LC and DAR1 accounted for 10.5% and 89.5%, respectively), and the heavy chain was coupled with 1-4 bioactive molecules (DAR1, DAR2, DAR3 and DAR4 accounted for 21.3%, 14.8%, 57.9% and 6.0%, respectively). Therefore, it could be calculated that the DAR of the antibody to bioactive molecules was 6.8.

Example 57: Determination of Molecular Weight of BT001042 by LC-MS

[0579] A method similar to that as described in example 55 was adopted, and results were shown in FIGS. 24 and 25.

[0580] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001042 obtained by coupling TL051 with the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00032 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 25202.3 27070.6 28938.9 30807.2 Measured value 23335.2 25203.8 Not detected Not detected Not detected HC Theoretical value 50733.98 52602.3 54470.6 56338.9 58207.2 Measured value Not detected 52602.8 54471.0 56339.8 Not detected

[0581] As can be seen from FIGS. 24 and 25, the light chain of the antibody in BT001042 was coupled with 0-1 bioactive molecule (LC and DAR1 accounted for 14.9% and 85.1%, respectively), and the heavy chain was coupled with 1-3 bioactive molecules (DAR1, DAR2 and DAR3 accounted for 19.7%, 9.4% and 70.9%, respectively). Therefore, it could be calculated that the DAR of the antibody to bioactive molecules was 6.7.

Example 58: Determination of Molecular Weight of BT001043 by LC-MS

[0582] A method similar to that as described in example 55 was adopted, and results were shown in FIGS. 26 and 27.

[0583] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001043 obtained by couplingTL052 with the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00033 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 25202.3 27100.6 28983.9 30867.2 Measured value 23335.5 25221.1 Not detected Not detected Not detected HC Theoretical value 50733.98 52617.3 54500.6 56383.9 58267.2 Measured value Not detected 52620.4 54505.8 56391.5 Not detected

[0584] As can be seen from FIGS. 26 and 27, the light chain of the antibody in BT001043 was coupled with 0-1 bioactive molecule (LC and DAR1 accounted for 9.1% and 90.9%, respectively), and the heavy chain was coupled with 1-3 bioactive molecules (DAR1, DAR2 and DAR3 accounted for 20.1%, 11.4% and 68.4%, respectively). Therefore, it could be calculated that the DAR of the antibody to bioactive molecules was 6.8.

Example 59: Determination of Molecular Weight of BT001044 by LC-MS

[0585] A method similar to that as described in example 55 was adopted, and results were shown in FIGS. 28 and 29.

[0586] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001044 obtained by coupling TL053 with the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00034 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 25233.3 27132.5 29031.8 30931.1 Measured value 23335.5 25234.7 Not detected Not detected Not detected HC Theoretical value 50733.98 52633.2 54532.5 56431.7 58331.0 Measured value Not detected 52634.1 54532.9 56432.1 Not detected

[0587] As can be seen from FIGS. 28 and 29, in BT001044, the light chain of the antibody was coupled with 0-1 bioactive molecule (LC and DAR1 accounted for 23.0% and 77.0%, respectively), and the heavy chain was coupled with 1-3 bioactive molecules (DAR1, DAR2 and DAR3 accounted for 19.4%, 11.4% and 69.3%, respectively). Therefore, it could be calculated that the DAR of the antibody to bioactive molecules was 6.5.

Example 60: Determination of Molecular Weight of BT001046 by LC-MS

[0588] A method similar to that as described in example 55 was adopted, and results were shown in FIGS. 30 and 31.

[0589] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001046 obtained by coupling TL055 with the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00035 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 24894.8 26455.5 28016.2 29577.0 Measured value 23335.5 24896.7 Not detected Not detected Not detected HC Theoretical value 50733.98 52294.7 53855.4 55416.2 56976.9 Measured value 50735.4 52296.1 53856.9 55418.0 Not detected

[0590] As can be seen from FIGS. 30 and 31, in BT001046, the light chain of the antibody was coupled with 0-1 bioactive molecule (LC and DAR1 accounted for 33.8% and 66.2%, respectively), and the heavy chain was coupled with 0-3 bioactive molecules (DAR0, DAR1, DAR2 and DAR3 accounted for 21.9%, 6.1%, 9.6% and 62.3%, respectively). Therefore, it could be calculated that the DAR of the antibody to bioactive molecules was 5.6.

Example 61: Determination of Molecular Weight of BT001047 by LC-MS

[0591] A method similar to that as described in example 55 was adopted, and results were shown in FIGS. 32 and 33.

[0592] The theoretical molecular weight and measured molecular weight of the light chain and the heavy chain of BT001047 obtained by coupling TL056 with the antibody (calculated based on main glycoform G0F) were shown in the table below:

TABLE-US-00036 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical value 23334.04 25124.2 26914.4 28704.6 30494.8 Measured value 23335.5 25126.3 Not detected Not detected Not detected HC Theoretical value 50733.98 52524.2 54314.3 56104.5 57894.7 Measured value 50735.4 52524.6 54314.1 56106.5 Not detected

[0593] As can be seen from FIGS. 32 and 33, in BT001047, the light chain of the antibody was coupled with 0-1 bioactive molecule (LC and DAR1 accounted for 13.7% and 86.3%, respectively), and the heavy chain was coupled with 1-3 bioactive molecules (DAR1, DAR2 and DAR3 accounted for 22.2%, 13.5% and 64.3%, respectively). Therefore, it could be calculated that the DAR of the antibody to bioactive molecules was 6.6.

Example 62: Size Exclusion Chromatography Analysis

[0594] The coupling reaction was monitored by SEC-HPLC, and the conjugates were tested by SEC.

[0595] Chromatographic conditions: [0596] Liquid chromatographic column: TOSOH TSKgel SuperSW mAb, 4 .Math.m, 7.8 mm × 300 mm; [0597] Mobile phase: 100 mmol/L Na.sub.2HPO.sub.4, 100 mmol/L NaCl, 5% isopropanol, pH7.0; [0598] Flow rate: 0.5 ml/min; Detection wavelength: 280 nm; Column temperature: room temperature; Sample room temperature: 8° C.; [0599] Sample size: 30 .Math.g; Isocratic operation: 30 min.

[0600] The SEC chromatogram and molecular weight Marker SEC chromatogram of BT001002 obtained by coupling TL003 with the antibody were shown in FIGS. 7 and 8 respectively. According to the molecular weight Marker, it is confirmed that the molecular weight of the main peak of the coupling product is about 150 kD, i.e., for BT001002 obtained by coupling TL003 with the antibody, the light chain and the heavy chain are not dissociated, and the antibody still maintains its integral structure.

[0601] The SEC chromatogram of BT001004 obtained by coupling TL019 with the antibody is shown in FIG. 9. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001004 obtained by coupling TL019 with the antibody still maintains the integral structure of the antibody.

[0602] The SEC chromatogram of BT001012 obtained by coupling TL024 with the antibody is shown in FIG. 34. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001012 obtained by coupling TL024 with the antibody still maintains the integral structure of the antibody.

[0603] The SEC chromatogram of BT001013 obtained by coupling TL048 with the antibody is shown in FIG. 35. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001013 obtained by coupling TL048 with the antibody still maintains an integral structure of the antibody.

[0604] The SEC chromatogram of BT001018 obtained by coupling TL030 with the antibody is shown in FIG. 36. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001018 obtained by coupling TL030 with the antibody still maintains an integral structure of the antibody.

[0605] The SEC chromatogram of BT001021 obtained by coupling TL033 with the antibody is shown in FIG. 37. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001021 obtained by coupling TL033 with the antibody still maintains an integral structure of the antibody.

[0606] The SEC chromatogram of BT001023 obtained by coupling TL035 with the antibody is shown in FIG. 38. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001023 obtained by coupling TL035 with the antibody still maintains an integral structure of the antibody.

[0607] The SEC chromatogram of BT001042 obtained by coupling TL051 with an antibody is shown in FIG. 39. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001042 obtained by coupling TL051 with the antibody still maintains the integral structure of the antibody.

[0608] The SEC chromatogram of BT001043 obtained by coupling TL052 with the antibody is shown in FIG. 40. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150kD, i.e., BT001043 obtained by coupling TL052 with the antibody still maintains the integral structure of the antibody.

[0609] The SEC chromatogram of BT001044 obtained by coupling TL053 with the antibody is shown in FIG. 41. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001044 obtained by coupling TL053 with the antibody still maintains the integral structure of the antibody.

[0610] The SEC chromatogram of BT001046 obtained by coupling TL055 with the antibody is shown in FIG. 42. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001046 obtained by coupling TL055 with the antibody still maintains the integral structure of the antibody.

[0611] The SEC chromatogram of BT001047 obtained by coupling TL056 with the antibody is shown in FIG. 43. According to the retention time and peak area ratio in the SEC, it is confirmed that the molecular weight of the main coupling product is about 150 kD, i.e., BT001047 obtained by coupling TL056 with the antibody still maintains the integral structure of the antibody.

Example 63: Test of Inhibitory Effects of Bioactive Molecules and Antibody Drug Conjugates on Activity of Cells in vitro

[0612] First, tumor cells MDA-MB-468 (Trop-2 positive cell lines) and HCC1806 (Trop-2 positive cell lines) were cultured. The bioactive molecules and ADC molecules disclosed in the disclosure were co-cultured with the tumor cells, then a CCK8 reagent (Dojindo Molecular Technologies, Inc., Cat: CK04, Lot:JJ744) was added . The activity of dehydrogenase in mitochondria was tested through readings (detection wave length was 450 nm) from a microplate reader (manufacturer: Molecular Devices, model: SpectraMax M2) so as to evaluate the inhibitory effect of ADC on cell proliferation. Sources of the tumor cells were shown in table 1.

TABLE-US-00037 Cell name Tumor type Source MDA-MB-468 Breast cancer Concortis HCC1806 Breast cancer Cobioer Biosciences Co., Ltd.

[0613] In-vitro cell activity testing: bioactive molecules or ADCs were diluted (12 concentration gradients) with corresponding test media (containing 2% FBS). The tumor cells were trypsinized with trypsin by a conventional method, collected and counted, and then resuspended with corresponding test media (containing 2% FBS). Diluted bioactive molecules or ADCs were added to a 96-well plate, and then cells were added. 20 .Math.L of the CCK8 reagent was added to each well and reacted for 4 h, and readings (detection wavelength was 450 nm) were taken from a microplate reader. Experimental conditions and test results were shown in Table 2 and Table 3.

TABLE-US-00038 Killing effects of bioactive molecules on cells Name Cell name EC.sub.50(nM) T001 MDA-MB-468 (7500 cells/well, 4 days) 1.126 T011 0.211 T012 HCC1806 (7500 cells/well, 3 days) 1.621 T013 0.414 T015 7.428 T-028 6.016 T-030 6.734

[0614] The test results indicated that all of the bioactive molecules had killing effects on the tumor cells.

TABLE-US-00039 Killing effects of conjugates (ADCs) on cell lines Name Cell name EC50(nM) BT001002 MDA-MB-468 (10000 cells/well, 3 days) 0.072 BT001004 HCC1806 (7500 cells/well, 3 days) 6.139 BT001012 14.41 BT001013 48.01 BT001018 13.42 BT001021 13.15 BT001022 23.43 BT001023 21.65 BT001040 11.08 BT001041 10.34 BT001042 0.0186 BT001043 0.062 BT001044 0.0051 BT001046 0.81 BT001047 0.23

[0615] The test results indicated that ADC molecules obtained by a novel coupling way had killing effects on tumor cells, indicating that the ADCs formed by the novel coupling method had killing effects on the tumor cells, and the novel coupling method was workable in the synthesis of ADC molecules.

Example 64: Pharmacodynamic Test of Antibody Drug Conjugates and Bioactive Molecules In vivo

Drugs Under Test

[0616] Drug name, source and preparation method: [0617] BT001021, liquid aliquots were stored at -20° C. at a concentration of 5.44 mg/ml, and diluted with physiological saline by dosage before use to obtain a test solution; [0618] Immu-132 (prepared according to example 2 of WO2015/012904A2, DAR=5.4, also described as IMMU-132), liquid aliquots were stored at -20° C. at a concentration of 13.158 mg/ml, and diluted with physiological saline by dosage before use to obtain a test solution; [0619] Solid powder of T-030 was prepared with 100% DMSO (Sigma) into a solution at a concentration of 5.2 mg/mL, and liquid aliquots were stored at -20° C., and diluted with physiological saline to a desired dose before use to obtain a test solution; [0620] Solid powder of SN-38 (also described as SN38) was prepared with 100% DMSO (Sigma) into a solution at a concentration of 3.23 mg/ml, liquid aliquots were stored at -20° C., and diluted with physiological saline by dosage before use to obtain a test solution.

[0621] Note: Toxin was prepared and administered in an equimolar ratio of ADC samples.

[0622] Structures of T-030, SN-38 and Immu-132 were as follows:

##STR00367##

##STR00368##

Experimental Animals and Cell Lines

[0623] Balb/c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., production license No.: SCXK (Beijing) 2016-0011); Gastric cancer cell line NCI-N87 (ATCC), breast cancer cell line HCC1806 (COBIOER Nanjing).

Experimental Grouping and Evaluation Method

[0624] Tumor-bearing mice (6 mice/group) with tumor volume of 100 - 200 mm.sup.3 were randomly grouped (the number of groups was determined according to sample number). The administration volume was 10 ml/kg, and the administration route was tail intravenous injection. The mice were administered twice a week, and tumor diameter was measured with a vernier caliper, and tumor volume was calculated based on the following calculation formula: V = 0.5 a × b.sup.2, wherein a and b stand for the long diameter and short diameter of a tumor respectively. Animal deaths were observed and recorded every day.

[0625] The tumor growth inhibition rate TGI (%) was calculated from the following formula to evaluate tumor inhibitory effect of antibody drug conjugates:

[00001]$\text{TGI}\left(\%\right)=\left[1\text{-}\left({\text{V}}_{\text{Tend}}{\text{-V}}_{\text{Tstart}}\right)/\left({\text{V}}_{\text{Cend}}{\text{-V}}_{\text{Cstart}}\right)\right]*100\%$

[0626] wherein, V.sub.Tend: average tumor volume at the end of the experiment in the treatment group [0627] V.sub.Tstart: average tumor volume at the begining of administration in the treatment group [0628] V.sub.Cend: average tumor volume at the end of the experiment in the control group [0629] V.sub.Cstart: average tumor volume at the begining of administration in the control group

[0630] In the following experimental examples 1 and 2, the inhibition of the antibody conjugate BT001021 on tumor proliferation of tumor-bearing mice constructed by subcutaneous xenograft of human tumor cells was evaluated. Specifically, in the experimental examples 1 and 2, tumor-bearing mice models were constructed by subcutaneous xenograft of a human gastric cancer cell line NCI-N87 or a human triple negative breast cancer cell line HCC1806. After the tumor volume was about 100 mm.sup.3, the mice were randomly grouped, and intravenously administered with BT001021 twice a week for a total of 6 times. Changes in tumor volume and animal body weight were measured twice a week to evaluate the efficacy (tumor inhibitory effect) of the antibody drug conjugate on tumor-bearing mice.

Experimental Example 1. Inhibition of NCI-N87 by Antibody Drug Conjugates and Bioactive Molecules

Experimental Methods

[0631] NCI-N87 cells were cultured in a 1640 culture medium containing 10% fetal bovine serum at 37° C. and 5% CO.sub.2. NCI-N87 cells in the exponential growth stage were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into female Balb/c-nu mice to construct gastric cancer models. When the mean tumor volume was about 90 mm.sup.3, the mice were randomly grouped into a physiological saline group, a BT001021 (3 mg/kg, IV, BIW×3W) group, a positive drug Immu-132 (3 mg/kg, IV, BIW×3W) group, a T030 group and a SN38 group according to the tumor size, followed by tail intravenous injection of corresponding drugs twice a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. Specific results were shown in Table 4, FIGS. 44 and 45.

Conclusion

[0632] In the experimental example, a human gastric cancer cell line NCI-N87 was used to construct subcutaneous xenograft models of human gastric cancer, and the efficacy of BT001021 in the NCI-N87 human gastric cancer tumor-bearing mice models was evaluated.

[0633] Experimental results showed that BT001021 (3 mg/kg, IV, BIW×3W) could significantly inhibit the tumor growth of xenograft model mice of NCI-N87 gastric cancer, and tumor regression occurred at the end of administration, with anti-tumor activity superior to that of positive control Immu-132. Neither animal death nor significant animal weight loss occurred in all treatment groups during the observation period, indicating that BT001021 had no significant toxicity.

TABLE-US-00040 NCI-N87 model of gastric cancer Group No. Regimen D21 after administration Tumor volume (mm.sup.3) (x ±S) Tumor growth inhibition rate (%) P value (vs group 1) Group 1 Physiological saline 405.67± 91.81 Group 2 BT001021 42.78± 21.87 114.61 0.0000 Group 3 Immu-132 259.04± 42.41 46.46 0.0053 Group 4 T-030 339.02± 152.80 21.48 0.3813 Group 5 SN-38 416.11±195.15 -2.75 0.9079

Experimental Example 2. Inhibition of HCC1806 by Antibody Drug Conjugates

Experimental Methods

[0634] HCC1806 cells were cultured in a 1640 culture medium containing 10% fetal bovine serum at 37° C. and 5% CO.sub.2. HCC1806 cells in the exponential growth stage were collected, resuspended in PBS in a suitable concentration, and inoculated subcutaneously into female Balb/c-nu to construct breast cancer models. When the mean tumor volume was about 130 mm.sup.3, the mice were randomly grouped into a physiological saline group, a BT001021 (10 mg/kg, IV, BIW×3W) group and a positive drug Immu-132 (10 mg/kg, IV, BIW×3W) group according to the tumor size, followed by tail intravenous injection of corresponding drugs twice a week for a total of 5 times. After administration, the tumor volume of the mice was observed and measured regularly. Specific results were shown in Table 5 and FIG. 46.

Conclusion

[0635] In the experimental example, a human breast cancer cell line HCC1806 was used to construct subcutaneous xenograft models of human breast cancer, and the efficacy of BT001021 in the HCC1806 human breast cancer tumor-bearing mice models was evaluated.

[0636] Experimental results showed that BT001021 (10 mg/kg, IV, BIW×3W) could significantly inhibit the tumor growth of xenograft model mice of HCC1806 breast cancer, with anti-tumor activity superior to that of positive Immu-132.

TABLE-US-00041 HCC1806 model of breast cancer D17 after administration Group No. Regimen Tumor Volume (mm.sup.3) (x̅ ±S) Tumor growth inhibition rate (%) P value (vs group 1) Group 1 Physiological saline 2638.22±553.81 Group 2 BT001021 1260.87± 415.60 54.93 0.0006 Group 3 Immu-132 2347.05± 317.79 11.62 0.2901

[0637] According to Table 4, Table 5 and FIGS. 44-46, the antibody drug BT001021 of the invention could significantly inhibit tumor growth in NCI-N87 mice models. It was significantly superior to Immu-132 at the same dosage, and had neither significant weight loss nor significant drug toxicity. In the HCC1806 mice models, when the dosage increased to 10 mg/kg due to high malignancy of the tumor, Immu-132 did not show significant inhibitory activity, whereas BT001021 could significantly inhibit tumor growth. The results indicated that BT001021 of the invention had good efficacy and excellent safety.

[0638] In the subcutaneous xenograft models of the experimental examples 1 and 2, the anti-tumor activity of BT001021 was significantly superior to that of Immu-132 at the same dosage, suggesting that BT001021 had the potential to treat solid tumors, and was expected to benefit more patients clinically than Immu-132.

Experimental Example 3. Inhibition of HCC827 by Antibody Drug Conjugates

[0639] The experimental example 3 was used to evaluate the inhibitory effect of BT001021 and BT001035 on proliferation of tumor-bearing mice models constructed by subcutaneous xenograft human tumor cells of HCC827 non-small cell lung cancer. Specifically, in the experiment, tumor-bearing mice models were constructed by subcutaneous xenograft of a human non-small cell lung cancer cell line HCC827. After the tumor volume was about 100 mm.sup.3, the mice were randomly grouped, and intravenously administrated with BT001021 and BT001035 twice a week for a total of 6 times. Then changes in tumor volume and animal body weight were measured twice a week to calculate the efficacy (tumor inhibitory effect) of BT001021 and BT001035 on the tumor-bearing mice.

Experimental Methods

[0640] HCC827 cells were cultured in a 1640 culture medium containing 10% fetal bovine serum at 37° C. and 5% CO.sub.2. HCC827 cells in the exponential growth stage were collected, resuspended in PBS in a suitable concentration, and inoculated subcutaneously into female Balb/c-nu mice to construct xenograft models of lung cancer. When the mean tumor volume was about 80 mm.sup.3, the mice were randomly grouped into a physiological saline group, a positive drug Immu-132 (10 mg/kg, IV, BIW×3W) group, a BT001021 (10 mg/kg, IV, BIW×3W) group and a BT001035 (10 mg/kg, IV, BIW×3W) group according to the tumor size, followed by tail intravenous injection of corresponding drugs twice a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. Results were shown in Table 6, FIG. 47A and FIG. 47B.

Conclusion

[0641] Experimental results showed that BT001021 and BT001035 could significantly inhibit the tumor growth of xenograft model mice of HCC827 non-small cell lung cancer, and tumor regression occurred at the end of administration, with anti-tumor activity superior to that of the positive control Immu-132 group. During the observation period, no animal death and significant animal weight loss occurred in all treatment groups, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all drugs to be evaluated.

TABLE-US-00042 HCC827 model of lung cancer D21 after administration Group No. Regimen Tumor Volume (mm.sup.3) (x̅ ±S) TGI(%) P value(vs group 1) 1 Physiological saline 515.25±165.09 2 Immu-132 145.94±19.72 85.19 0.0003 3 BT001021 40.26±8.36 108.70 0.0001 4 BT001035 48.61±9.99 106.95 0.0000

[0642] According to Table 6, FIG. 47A and FIG. 47B, both BT001021 and BT001035 had significant inhibitory activity on tumor growth during the evaluation period, and they are significantly superior to that of Immu-132 at the same dosage. During treatment, no significant weight loss and drug toxicity were observed in all groups. The results indicated that both BT001021 and BT001035 had excellent anti-tumor activity.

[0643] In the subcutaneous xenograft models, the anti-tumor activity of both BT001021 and BT001035 was significantly superior to that of Immu-132 at the same dosage, suggesting that both BT001021 and BT001035 had the potential to treat solid tumors, and was expected to benefit more patients clinically than Immu-132.

Experimental Example 4. Inhibition of NCI-N87 by Antibody Drug Conjugates

[0644] The experimental example 4 was used to evaluate the inhibition of the antibody drug conjugate BT001036 on tumor proliferation of tumor-bearing mice constructed by subcutaneous xenograft of human tumor cells. Specifically, in the experiment, tumor-bearing mice models were constructed by subcutaneous xenograft of a human gastric cancer cell line NCI-N87. After the tumor volume was about 140 mm.sup.3, the mice were randomly grouped, and intravenously administrated with BT001036 twice a week for a total of 6 times. Changes in tumor volume and animal body weight were measured twice a week to evaluate the efficacy (tumor inhibitory effect) of the antibody drug conjugate on tumor-bearing mice.

Experimental Methods

[0645] NCI-N87 cells were cultured in a 1640 culture medium containing 10% fetal bovine serum at 37° C. and 5% CO.sub.2. NCI-N87 cells in the exponential growth stage were collected, resuspended in PBS in a suitable concentration, and inoculated subcutaneously into female Balb/c-nu mice to construct xenograft models of gastric cancer. When the mean tumor volume was about 140 mm.sup.3, the mice were randomly grouped into a physiological saline group, a BT001036 (1.5 mg/kg, IV, BIW×3W) group and a BT001036 (3 mg/kg, IV, BIW×3W) group according to the tumor size, followed by tail intravenous injection of corresponding drugs twice a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. Specific results were shown in Table 7, FIG. 48A and FIG. 48B.

TABLE-US-00043 NCI-N87 model of gastric cancer D21 after administration Group No. Regimen Tumor Volume (mm.sup.3) x̅ (±S) TGI(%) P value (vs group 1) Group 1 Physiological saline 601.29±198.92 Group 2 BT001036 (1.5 mg/kg) 150.87±112.84 97.78 0.0017 Group 3 BT001036 (3 mg/kg) 2.74± 0.64 129.95 0.0000

Conclusion:

[0646] In the experimental example, subcutaneous xenograft models of human gastric cancer were constructed by subcutaneous xenograft of a human gastric cancer cell line NCI-N87, and the efficacy of BT001036 in the NCI-N87 human gastric cancer tumor-bearing mice models was evaluated.

[0647] Experimental results showed that both the high and low dosags of BT001036 (1.5 mg/kg, 3 mg/kg) could significantly inhibit the tumor growth of xenograft model mice of NCI-N87 gastric cancer, and tumor regression occurred at the end of administration, with excellent anti-tumor activity. During the observation period, no animal death and significant animal weight loss occurred in all treatment groups, and no significant drug toxicity was observed. During the treatment period, the mice showed good tolerance to all drugs to be evaluated.

Experimental Example 5. Inhibition of MDA-MB-231 by Antibody Drug Conjugates

[0648] The experimental example 5 was used to evaluate the inhibitory effect of BT001021 on the proliferation of tumor-bearing mice models constructed by subcutaneous xenograft human tumor cells of MDA-MB-231 breast cancer. Specifically, in the experiment, tumor-bearing mice models were constructed by subcutaneous xenograft of a human breast cancer cell line MDA-MB-231. After the tumor volume was about 130 mm.sup.3, the mice were randomly grouped, and intravenously administreatd with BT001021 twice a week for a total of 6 times. Then changes in tumor volume and animal body weight were measured to calculate the efficacy (tumor inhibitory effect) of BT001021 on the tumor-bearing mice.

Experimental Methods

[0649] NCI-MDA-MB-231 cells were cultured in a RPMI1640 culture medium containing 10% fetal bovine serum at 37° C. and 5% CO.sub.2. MDA-MB-231 cells in the exponential growth stage were collected, resuspended in PBS in a suitable concentration, and inoculated subcutaneously into female Balb/c-nu mice to construct xenograft models of lung cancer. When the mean tumor volume was about 130 mm.sup.3, the mice were randomly grouped into a physiological saline group and a BT001021 (3 mg/kg) group according to the tumor size, followed by tail intravenous injection of corresponding drugs twice a week for a total of 6 times. After administration, the tumor volume and body weight of the mice were observed and measured regularly. Results were shown in Table 8, FIG. 49A and FIG. 49B.

Conclusion

[0650] Results showed that BT001021 could significantly inhibit the tumor growth of xenograft model mice of MDA-MB-231 breast cancer, and tumor regression occurred at the end of administration. During the observation period, neither animal death nor significant animal weight loss occurred in all treatment groups, no significant drug toxicity was observed, either. During the treatment period, the mice showed good tolerance to all drugs to be evaluated.

TABLE-US-00044 MDA-MB-231 model of breast cancer D33 after administration Group No. Regimen Tumor Volume (mm.sup.3) x̅ (±S) TGI (%) P value (vs group 1) 1 Physiological saline 744.53±306.66 2 BT001021 43.96±7.72 114.18 0.0119

[0651] In the subcutaneous xenograft models, BT001021 had significant anti-tumor activity. During the observation period, neither animal death nor significant animal weight loss occurred in all treatment groups, no significant drug toxicity was observed, either. During the treatment period, the mice showed good tolerance to all drugs to be evaluated.

Example 65: Test of Pharmacokinetics of Antibody Drug Conjugates and Bioactive Molecules in vivo

[0652] Experimental example 6 was used to evaluate pharmacokinetics of antibody drug conjugates and bioactive molecules in vivo. Specifically, in the experiment, a tumor-bearing mice model was constructed by subcutaneous xenograft of a human gastric cancer cell line NCI-N87 to Balb/c-nu mice. After the tumor volume was 100-200 mm.sup.3, the mice were randomly grouped, and intravenously administrated with a single dose of BT001021 and T-030. The concentration of the T-030 in tumor tissues and serum was determined to evaluate the pharmacokinetics in vivo of the antibody conjugate BT001021 and the bioactive molecule T-030 in the tumor-bearing mice.

Drugs Under Test

[0653] Drug names and preparation methods: [0654] BT001021, liquid aliquots were stored at -20° C. at a concentration of 20 mg/ml, and diluted with physiological saline to the desired doses before use to obtain a test solution; [0655] T-030, which was prepared into 1 mg/ml with dimethyl sulfoxide and diluted with physiological saline to a desired dose to obtain a test solution.

Experimental Animals and Cell Lines

[0656] Balb/c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., production license No.: SCXK (Beijing) 2016-0011); Gastric cancer cell line NCI-N87 (ATCC).

Experimental Groups and Evaluation Method

[0657] Tumor-bearing mice (4 mice/group) with tumor volume of 100 - 200 mm.sup.3 were randomly grouped (the number of groups was determined according to sample number), and the administration route was single tail intravenous injection.

Experimental Example 6. Test of Pharmacokinetics of BT001021 and T-030 in Tumor-Bearing Mice in vivo

Experimental Method

[0658] NCI-N87 cells were cultured in a 1640 culture medium containing 10% heat-inactivated fetal bovine serum at 37° C. and 5% CO.sub.2. NCI- N87 cells in the exponential growth stage were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into Balb/c-nu mice to construct xenograft models of lung cancer. When the mean tumor volume was about 100-200 mm.sup.3, the mice were randomly grouped into a physiological saline group, a T-030 (0.23 mg/kg, IV, single dose) group and a BT001021 (10 mg/kg, IV, single dose) group according to the tumor size, followed by tail intravenous injection of corresponding drugs. For the T-030 group, serum and tumor tissues were collected 1 h, 2 h, 4 h, 8 h, 24 h and 72 h after administration (T-030 was not detected in the serum and tumor tissues 72 h after administration, therefore, serum and tumor tissues were not collected 168 h after administration). For BT001021 group, serum and tumor tissues were collected 1 h, 2 h, 4 h, 8 h, 24 h, 72 h and 168 h after administration to test the concentration of the T-030 in the serum and the tumors by LC-MS/MS. Specific results were shown in Table 9. The administration dose of the T-030 (0.23 mg/kg) was converted to isomolar dose (10 mg/kg) of BT001021.

TABLE-US-00045 Pharmacokinetic parameters of T-030 in tumors and serum of tumor-bearing mice after intravenous administration of T-030 and BT001021 Regimen T-030 (0.23 mg/kg) administration group BT001021 administration (10 mg/kg) group Tumor Serum Tumor Serum AUC.sub.last(h*ng/ml) 3.85 5.58 850.1 174.97 C.sub.max(ng/ml) 1.20 1.81 7.82 11.7 MRT.sub.INF(h) 1.52 1.11 64.24 42.9 T.sub.max(h) 1.00 1.00 8.00 1.00 T.sub.½(h) 2.55 1.76 93.14 44.35

Conclusion:

[0659] AUC.sub.last of the drug in the tumors and serum of BT001021 (10 mg/kg) administration group was 850.1 h*ng/ml and 174.97 h*ng/ml, respectively, whereas the AUC.sub.last of the drug in the tumors and serum of the T-030 administration group was 3.85 h*ng/ml and 5.58 h*ng/ml, respectively. The comparison indicated that the exposure doses of T-030 of BT001021 administration group was significantly increased compared with those of the T-030 administration group. In addition, the exposure doses of the bioactive molecule T-030 in the tumors of BT001021 administration group was significantly higher than those in the serum, whereas, the exposure doses of the active biomolecule in the serum and tumors of the T-030 administration group was basically the same, indicating that the antibody drug conjugate (BT001021) had high tumor tissue targetability.

[0660] C.sub.max of the bioactive molecule T-030 in the tumors and serum of BT001021 (10 mg/kg) administration group was 7.82 ng/ml and 11.7 ng/ml, respectively, whereas the C.sub.max of the bioactive molecule T-030 in the tumors and serum of the T-030 administration group was 1.20 ng/ml and 1.81 ng/ml, respectively, indicating that the antibody drug conjugate (BT001021) had higher concentration of the bioactive molecule (T-030) in tumor tissues and serum.

[0661] T.sub.½ of the bioactive molecule T-030 in the tumors of BT001021 (10 mg/kg) administration group was 93.14 h, whereas the T.sub.½ of the bioactive molecule T-030 in the tumors of the T-030 administration group was 2.55 h, indicating that the antibody drug conjugate (BT001021) had a longer half life in the tumor tissues.

[0662] In conclusion, BT001021 had significant tumor tissue targetability and good pharmacokinetic properties compared with the corresponding bioactive molecule (T-030).

Experimental Example 7. Test of Pharmacokinetics of Antibody Drug Conjugates BT001021 and Immu-132 in vivo

[0663] In the experiment, tumor-bearing mice models were constructed by subcutaneous xenograft of a human gastric cancer cell line NCI-N87 to Balb/c-nu mice. After the tumor volume was 100-200 mm.sup.3, the mice were randomly grouped, and intravenously given a single dose of BT001021 and Immu-132. The concentration of the bioactive molecule T-030 and the SN-38 corresponding to BT001021 and Immu-132 in tumor tissues and serum was respectively measured to evaluate the pharmacokinetics of the antibody conjugates BT001021 and Immu-132 in tumor-bearing mice in vivo.

Drugs Under Test

[0664] Drug names and preparation methods: [0665] BT001021, liquid aliquots were stored at -20° C. at a concentration of 20 mg/ml, and diluted with physiological saline to the desired doses before use to obtain the test solution; [0666] Immu-132 was diluted with physiological saline to the desired dose to obtain the test solution.

Experimental Animals and Cell Lines

[0667] Balb/c-nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., production license No.: SCXK (Beijing) 2016-0011); Gastric cancer cell line NCI-N87 (ATCC).

Experimental Groups and Evaluation Method

[0668] Tumor-bearing mice (4 mice/group) with tumor volume of 100 - 200 mm.sup.3 were randomly grouped (the number of groups was determined according to sample number), and the administration route was single tail intravenous injection.

Experimental Methods

[0669] NCI-N87 cells were cultured in a 1640 culture medium containing 10% heat-inactivated fetal bovine serum at 37° C. and 5% CO.sub.2. NCI- N87 cells in the exponential growth stage were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously into Balb/c-nu mice to construct an xenograft model of lung cancer. When the mean tumor volume was about 100-200 mm.sup.3, the mice were randomly grouped into a BT001021 (5 mg/kg, IV, single dose) group and an Immu-132 (5 mg/kg, IV, single dose) group according to the tumor size, followed by tail intravenous injection of corresponding drugs. Serum and tumor tissues were collected at 2 h, 24 h, 48 h and 72 h after administration respectively to test the concentration of the T-030 or SN-38 in the serum and tumors by LC-MS/MS.

TABLE-US-00046 Pharmacokinetic parameters of T-030 and SN-38 in tumors and serum of tumor-bearing mice after intravenous administration of BT001021 and Immu-132 Regimen Immu-132 (5 mg/kg) administration group BT001021 (5 mg/kg) administration group Tumor Serum Tumor Serum AUC.sub.last (h*ng/ml) 116.8 422.7 427.2 115.3 C.sub.max (ng/ml) 2.8 19.58 6.8 5.08 MRT.sub.last (h) 21.5 15.66 31.9 17.6

Conclusion

[0670] AUC.sub.last of small toxin molecules in the tumors and serum of BT001021 administration group was 427.2 h*ng/ml and 115.3 h*ng/ml respectively, whereas the AUC.sub.last of small toxin molecules in the tumors and serum of Immu-132 administration group was 116.8 h*ng/ml and 422.7 h*ng/ml respectively. C.sub.max of small toxin molecules in the tumors of BT001021 administration group was 6.8 ng/ml, whereas the C.sub.max of small toxin molecules in the tumors of Immu-132 administration group was 2.8 ng/ml. The results showed that BT001021 had better tumor tissue targetability, better pharmacokinetic properties and better therapeutic window, compared with Immu-132.

[0671] Even though specific modes for carrying out the invention have been described in detail, it should be understood by a person skilled in the art that various modifications and alternatives can be made to the details according to all published teachings, and such changes are within the protection scope of the invention. The full scope of the invention is given by the attached claims and any equivalent thereof.