U.S. patent application number 16/758980 was filed with the patent office on 2021-04-08 for bioactive conjugate, preparation method therefor and use thereof.
This patent application is currently assigned to SICHUAN KELUN-BIOTECH BIOPHARMACEUTICAL CO., LTD.. The applicant listed for this patent is SICHUAN KELUN-BIOTECH BIOPHARMACEUTICAL CO., LTD.. Invention is credited to Jiaqiang CAI, Xu CAO, Hanwen DENG, Dengnian LIU, Liping Liu, Shuai SONG, Qiang TIAN, Jing WANG, Jingyi WANG, Lichun WANG, Liang XIAO, Tongtong XUE, Zhouning YANG, Haimin YU, Guoqing ZHONG.
Application Number | 20210101906 16/758980 |
Document ID | / |
Family ID | 1000005476182 |
Filed Date | 2021-04-08 |
![](/patent/app/20210101906/US20210101906A2-20210408-C00001.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00002.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00003.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00004.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00005.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00006.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00007.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00008.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00009.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00010.png)
![](/patent/app/20210101906/US20210101906A2-20210408-C00011.png)
View All Diagrams
United States Patent
Application |
20210101906 |
Kind Code |
A2 |
CAI; Jiaqiang ; et
al. |
April 8, 2021 |
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.
Inventors: |
CAI; Jiaqiang; (Chengdu,
CN) ; SONG; Shuai; (Chengdu, CN) ; XUE;
Tongtong; (Chengdu, CN) ; XIAO; Liang;
(Chengdu, CN) ; DENG; Hanwen; (Chengdu, CN)
; TIAN; Qiang; (Chengdu, CN) ; WANG; Jing;
(Chengdu, CN) ; LIU; Dengnian; (Chengdu, CN)
; Liu; Liping; (Chengdu, CN) ; YU; Haimin;
(Chengdu, CN) ; YANG; Zhouning; (Chengdu, CN)
; CAO; Xu; (Chengdu, CN) ; ZHONG; Guoqing;
(Chengdu, CN) ; WANG; Lichun; (Chengdu, CN)
; WANG; Jingyi; (Chengdu, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SICHUAN KELUN-BIOTECH BIOPHARMACEUTICAL CO., LTD. |
Chengdu |
|
CN |
|
|
Assignee: |
SICHUAN KELUN-BIOTECH
BIOPHARMACEUTICAL CO., LTD.
Chengdu
CN
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20200347075 A1 |
November 5, 2020 |
|
|
Family ID: |
1000005476182 |
Appl. No.: |
16/758980 |
Filed: |
December 10, 2018 |
PCT Filed: |
December 10, 2018 |
PCT NO: |
PCT/CN2018/120125 PCKC 00 |
371 Date: |
April 24, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 47/6803 20170801; A61P 35/00 20180101; C07D 401/14 20130101;
C07K 16/30 20130101; A61K 47/6851 20170801; C07K 16/32 20130101;
C07D 403/12 20130101; C07D 403/14 20130101; C07D 491/22
20130101 |
International
Class: |
C07D 491/22 20060101
C07D491/22; C07D 403/12 20060101 C07D403/12; C07D 403/14 20060101
C07D403/14; C07D 401/14 20060101 C07D401/14; A61P 35/00 20060101
A61P035/00; A61K 47/68 20060101 A61K047/68; C07K 16/32 20060101
C07K016/32; C07K 16/30 20060101 C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 15, 2017 |
CN |
201711347535.6 |
Mar 20, 2018 |
CN |
201810230346.9 |
Sep 14, 2018 |
CN |
201811071947.6 |
Claims
1.-91. (canceled)
92. A compound as shown in formula (I) or a pharmaceutically
acceptable salt thereof,
T-[L.sub.1-(L.sub.2).sub.m1-(L.sub.3).sub.m2-(L.sub.4).sub.m3-E]-G
formula (I) wherein, T is a fragment of a bioactive molecule,
preferably a fragment of a molecule with antitumor bioactivity;
L.sub.1 is selected from ##STR00226## 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--, ##STR00227##
##STR00228## ##STR00229## 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
(e.g., --CH.sub.2CN), 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 x3 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 ##STR00230## 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--, ##STR00231##
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.3-6 cycloalkyl or R.sub.3/R.sub.4, R.sub.5/R.sub.6 or
R.sub.3/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, .dbd.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.9 is
independently selected from hydrogen, deuterium, C.sub.1-6 alkyl,
C.sub.3-6 cycloalkyl, C.sub.1-6 alkoxy or cyano 01-2 alkyl, and
L.sub.3 is bonded to L.sub.2 at the position 1 of L.sub.3; L.sub.4
is selected from ##STR00232## wherein Z.sub.5 is preferably
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.3-9
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; .alpha. 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 ##STR00233## Val, Cit, Phe,
Lys, D-Val, Leu, Gly, Ala, Asn, a peptide composed of 2-5 amino
acids, ##STR00234## ##STR00235## 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; preferably, L.sub.1
is selected from ##STR00236## Val, Cit, Phe, Lys, D-Val, Leu, Gly,
Ala, Asn, Cit-Val, Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys,
Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, ##STR00237##
wherein each of R, R' and R.sub.1 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, and each of x.sub.1 and x.sub.3 is independently 0, 1,
2 or 3; more preferably, L.sub.1 is selected from ##STR00238## Lys,
Cit, Cit-Val, Val-Ala, Lys-Val or ##STR00239## wherein each of R,
R' and R.sub.1 is independently hydrogen, deuterium or C.sub.1-4
alkyl, Z.sub.1 is Lys, Cit, Cit-Val, Cit-Ala, Val-Ala or Lys-Val,
and each of x.sub.1 and x.sub.3 is independently 0, 1 or 2; more
preferably, L.sub.1 is selected from ##STR00240## Lys, Cit,
Cit-Val, Val-Ala, Lys-Val, ##STR00241## more preferably, L.sub.1 is
selected from ##STR00242##
94. The compound or the pharmaceutically acceptable salt of claim
92, wherein, L.sub.2 is selected from ##STR00243## Val, Cit, Phe,
Lys, D-Val, Leu, Gly, Ala, Asn, a peptide composed of 2-5 amino
acids, ##STR00244## 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; m.sub.1
is 0, 1, 2 or 3; preferably, L.sub.2 is selected from ##STR00245##
Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val,
Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys,
Gly-Gly-Arg, Ala-Ala-Asn, ##STR00246## wherein each of R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 is independently selected from
hydrogen, deuterium, halogen, carboxylic acid group, 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;
m.sub.1 is 0, 1 or 2; more preferably, L.sub.2 is selected from
##STR00247## wherein each of R.sub.3, R.sub.4, R.sub.5 and R.sub.6
is independently selected from hydrogen, deuterium or C.sub.1-4
alkyl, 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; m.sub.1 is 1; more preferably, L.sub.2 is selected from
##STR00248## more preferably, L.sub.2 is selected from
##STR00249##
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, .dbd.O, CF.sub.3, CN, CH.sub.2CN,
carboxyl, sulfonic acid group, C.sub.1-4 alkyl, C.sub.1-4alkoxy,
C.sub.2-6 alkenyl or C.sub.2-6 alkynyl; preferably, the 3-8
aliphatic heterocyclylene, 6-12 membered bridged heterocyclylene,
6-12 membered spiroheterocyclylene or 6-12 membered fused
heterocyclylene has one or more nitrogen atoms; preferably, the 3-8
membered aliphatic heterocyclylene, 6-12 membered bridged
heterocyclylene, 6-12 membered spiroheterocyclylene or 6-12
membered fused heterocyclylene has one or more quaternized nitrogen
atoms; preferably, the 3-8 membered aliphatic heterocyclylene, 6-12
membered bridged heterocyclylene, 6-12 membered
spiroheterocyclylene or 6-12 membered fused heterocyclylene has one
or more nitrogen atoms, and at least one nitrogen atom is
substituted with .dbd.O; R.sub.8 is independently selected from
hydrogen, deuterium, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.3-6
alkynyl, C.sub.3-6 cycloalkyl, C.sub.1-6 alkoxy or cyano 01-2
alkyl; m.sub.2 is 0, 1, 2 or 3; preferably, L.sub.3 is selected
from the following groups optionally substituted with one or more
R.sub.7: 5-10 membered heteroarylene, amino or 3-6 membered
aliphatic heterocyclylene; wherein R.sub.7 is independently
selected from hydrogen, deuterium, halogen, .dbd.O, CF.sub.3, CN,
CH.sub.2CN, carboxyl, sulfonic acid group, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl;
preferably, the 3-6 membered aliphatic heterocyclylene has one or
more nitrogen atoms; preferably, the 3-6 membered aliphatic
heterocyclylene has one or more quaternized nitrogen atoms;
preferably, the 3-6 membered aliphatic heterocyclylene has one or
more nitrogen atoms, and at least one nitrogen atom is substituted
with .dbd.O; m.sub.2 is 0, 1 or 2; more preferably, L.sub.3 is
selected from 5-6 membered heteroarylene optionally substituted
with one or more R.sub.7; wherein R.sub.7 is independently selected
from hydrogen, deuterium, halogen, .dbd.O, CF.sub.3, CN,
CH.sub.2CN, carboxyl, sulfonic acid group, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl; m.sub.2
is 1; more preferably, L.sub.3 is selected from the following
groups optionally substituted with one or more R.sub.7:
triazolylene, pyrazolylene, amino or N-methylpiperidylene; wherein
R.sub.7 is independently selected from hydrogen, deuterium,
halogen, .dbd.O, CF.sub.3, CN, CH.sub.2CN, carboxyl, sulfonic acid
group, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.2-6 alkenyl or
C.sub.2-6 alkynyl; m.sub.2 is 0 or 1; more preferably, L.sub.3 is
selected from triazolylene. m.sub.2 is 0 or 1.
96. The compound or the pharmaceutically acceptable salt of claim
92, wherein L.sub.4 is selected from ##STR00250## 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
03-8 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; .alpha. 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; m.sub.3 is 0, 1, 2 or 3; preferably,
L.sub.4 is selected from ##STR00251## Z.sub.4 is a benzene ring,
and 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.3-9 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; a 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; m.sub.3 is 0,
1, 2 or 3; more preferably, L.sub.4 is selected from ##STR00252##
and Z.sub.4 is 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
03-9 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; .alpha. 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; m.sub.3 is 0, 1, 2 or 3; more
preferably, L.sub.4 is selected from ##STR00253## ##STR00254##
m.sub.3 is 1; more preferably, L.sub.4 is selected from
##STR00255## m.sub.3 is 1; more preferably, L.sub.4 is selected
from ##STR00256## m.sub.3 is 1.
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; preferably, G is selected from
methylsulfonyl, F, Cl, Br, I, OMs, OTs, OTf, ethylsulfonyl,
p-toluenesulfonyl or naphthalenesulfonyl; more preferably, G is
selected from methylsulfonyl, F, Cl, Br, OMs, OTs or
p-toluenesulfonyl; more preferably, G is selected from Cl or
methylsulfonyl.
99. The compound or the pharmaceutically acceptable salt of claim
92, wherein, in ##STR00257## G is preferably methylsulfonyl, E is
preferably pyrimidylene, m.sub.3 is 1; preferably ##STR00258## is:
(1) ##STR00259## wherein m.sub.4 is preferably an integer from 0 to
6, methylsulfonyl is a substituent on a carbon atom adjacent to a
nitrogen atom in the pyrimidine ring; (2) ##STR00260## wherein
m.sub.5 is preferably an integer from 0 to 6, methylsulfonyl is a
substituent on a carbon atom adjacent to a nitrogen atom in the
pyrimidine ring; (3) ##STR00261## wherein m.sub.6 is preferably an
integer from 0 to 6, methylsulfonyl is a substituent on a carbon
atom adjacent to a nitrogen atom in the pyrimidine ring; (4)
##STR00262## wherein m.sub.7 is an integer from 1 to 5,
methylsulfonyl is a substituent on a carbon atom adjacent to a
nitrogen atom in the pyrimidine ring; (5) ##STR00263## wherein
m.sub.8 is an integer from 1 to 5, methylsulfonyl is a substituent
on a carbon atom adjacent to a nitrogen atom in the pyrimidine
ring; (6) ##STR00264## wherein m.sub.9 is an integer from 1 to 5,
R.sub.13 is selected from hydrogen or C.sub.1-6 alkyl,
methylsulfonyl is a substituent on a carbon atom adjacent to a
nitrogen atom in the pyrimidine ring; (7) ##STR00265## wherein
m.sub.10 is an integer from 0 to 6, and Z.sub.4 is selected from
5-6 membered heteroarylene; methylsulfonyl is a substituent on a
carbon atom adjacent to a nitrogen atom in the pyrimidine ring; (8)
##STR00266## Z.sub.4 is selected from pyridylene, pyrimidylene,
pyrazolylene, thiazolylene, oxazolylene or triazolylene;
methylsulfonyl is a substituent on a carbon atom adjacent to a
nitrogen atom in the pyrimidine ring; or, (9) ##STR00267## Z.sub.4
is selected from oxazolylene or thiazolylene, methylsulfonyl is a
substituent on a carbon atom adjacent to a nitrogen atom in the
pyrimidine ring; preferably, ##STR00268##
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: ##STR00269## ##STR00270##
##STR00271## ##STR00272## ##STR00273## ##STR00274##
##STR00275##
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 such as a
platinum metal complex (e.g., oxaliplatin) or a gold metal complex;
a glycopeptide antibiotic such as bleomycin or pingyangmycin; a DNA
topoisomerase inhibitor such as a topoisomerase I inhibitor (e.g.,
camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38,
irinotecan, topotecan, bellotencian or rubitecan) or a
topoisomerase II inhibitor (e.g., actinomycin D, doxorubicin,
duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or
etoposide); a drug interfering with DNA synthesis, such as
methotrexate, 5-fluorouracil, cytarabine, gemcitabine,
mercaptopurine, pentostatin, fludarabine, cladribine or narabine; a
drug acting on a structural protein, such as a tubulin inhibitor, a
vinblastine alkaloid, a vincristine, vinblastine, paclitaxel,
docetaxel or cabazitaxel; a tumor cell signaling pathway inhibitor
such as a serine/threonine kinase inhibitor, a tyrosine kinase
inhibitor, a aspartokinase inhibitor or a histidine kinase
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; preferably, the bioactive molecule is selected from
##STR00276## 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, 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
selected from 0, 1, or 2; preferably, the bioactive molecule is
selected from ##STR00277## wherein R.sub.14 is selected from acyl
or sulfonyl, which are 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, C.sub.1-6 alkyl
substituted with R.sub.17, and R.sub.17 is selected from aryl or
heteroaryl, and m.sub.11 is selected from 0, 1 or 2; preferably,
the bioactive molecule is selected from ##STR00278## ##STR00279##
##STR00280## more preferably, the bioactive molecule is selected
from ##STR00281## more preferably, the bioactive molecule is
selected from ##STR00282## more preferably, the bioactive molecule
is selected from ##STR00283## more preferably, the bioactive
molecule is selected from ##STR00284##
102. The compound or the pharmaceutically acceptable salt of claim
92, wherein T is selected from ##STR00285## ##STR00286##
##STR00287## ##STR00288## ##STR00289## ##STR00290## preferably, T
is selected from ##STR00291## ##STR00292## ##STR00293##
##STR00294## more preferably, T is selected from ##STR00295## more
preferably, T is selected from ##STR00296## more preferably, T is
##STR00297##
103. The compound or the pharmaceutically acceptable salt of claim
92, wherein the compound is selected from ##STR00298## ##STR00299##
##STR00300## ##STR00301## ##STR00302## ##STR00303## ##STR00304##
##STR00305## ##STR00306## ##STR00307## ##STR00308## ##STR00309##
##STR00310## ##STR00311## ##STR00312## ##STR00313## ##STR00314##
##STR00315## ##STR00316## ##STR00317## ##STR00318## ##STR00319##
##STR00320##
104. 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 (e.g., a thiol group) to
form a conjugate; wherein the conjugate has a structure shown in
formula (II):
{T-[L.sub.1-(L.sub.2).sub.m1-(L.sub.3).sub.m2-(L.sub.4).sub.m3-E]}.sub..g-
amma.-A formula (II) wherein, A is a targeting moiety (e.g., a
small molecule ligand, a protein, a polypeptide or a non-protein
reagent (e.g., saccharide, RNA or DNA)); .gamma. is an integer or a
decimal from 1 to 10; preferably, .gamma. is an integer or a
decimal from 5 to 8 (e.g., 5, 6, 7 or 8); the rest groups are as
defined in claim 92.
105. The conjugate of claim 104, wherein, a target of A is selected
from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII,
Mesothelin, Folate receptor1, Mucin 1, CD138, CD20, CD19, CD30,
SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor,
STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium
phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein,
AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16,
0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4,
CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20R.alpha., Brevican,
EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5,
CD72, LY64, FcRH1, IRTA2, TENB2, integrin .alpha.5.beta.6,
.alpha.4.beta.7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin,
EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3,
Claudin18.2, BMPR1B, Tyrol, c-Met, ApoE, CD1 Ic, CD40, CD45
(PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7,
PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX,
LRRC15, MCPT8, MMP10, NOG, SERPINEI, STAT1, TGFBR1, CTSS, PGF,
VEGFA, C1QA, C.sub.1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5,
CXCL10, CXCL11, IF16, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1,
PDK1, AKR1C.sub.1, AKR1C.sub.2, CADM1, CDH11, COL6A3, CTGF, HMOX1,
KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1,
TGFB2, CD1 Ib, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2,
IGF-1R, RNF43, NaPi2b or BCMA; or, A is a small molecule ligand
such as a folic acid derivative, a glutamate urea derivative, a
somatostatin derivative, an arylsulfonamide derivative (e.g., a
carbonic anhydrase IX inhibitor), a polyene connecting two
aliphatic indoles, a cyanine dye or IR-783 or a derivative thereof;
preferably, A is selected from ##STR00321##
106. The conjugate of claim 104, wherein, A is an antibody such as
a monoclonal antibody or an antigen binding fragment thereof,
wherein the monoclonal antibody or the antigen binding fragment
thereof comprises Fab, Fab', F(ab').sub.2, Fd, Fv, dAb, a
complementary determinant fragment, a single chain antibody (e.g.,
scFv), a non-human antibody, a humanized antibody, a chimeric
antibody, a completely humanized antibody, a probody, a bispecific
antibody or a multispecific antibody; preferably, A is an anti-Her
2 monoclonal antibody or an anti-Trop-2 monoclonal antibody,
preferably, the anti-Trop-2 monoclonal antibody is selected from
antibodies of Sacituzumab, M1, M2 or M3; preferably, the anti-Her 2
monoclonal antibody is selected from Trastuzumab or Pertuzumab;
wherein, a heavy chain of the Sacituzumab has an amino acid
sequence setting forth in SEQ ID No.: 19; and a light chain has an
amino acid sequence setting forth in SEQ ID No.: 20; a heavy chain
variable region of antibody M1 has an amino acid sequence setting
forth in SEQ ID No.: 11; and a light chain variable region has an
amino acid sequence setting forth in SEQ ID No.: 12; a heavy chain
variable region of the antibody M2 has an amino acid sequence
setting forth in SEQ ID No.: 13; and a light chain variable region
has an amino acid sequence setting forth in SEQ ID No.: 14; a heavy
chain variable region of the antibody M3 has an amino acid sequence
setting forth in SEQ ID No.: 15; and a light chain variable region
has an amino acid sequence setting forth in SEQ ID No.: 16; heavy
chain constant regions of the antibodies M1, M2 and M3 have amino
acid sequences setting forth in SEQ ID No.: 10; and light chain
constant regions have amino acid sequences setting forth in SEQ ID
No.: 9; more preferably, A is an anti-Her 2 monoclonal antibody or
an anti-Trop-2 monoclonal antibody, preferably, the anti-Trop-2
monoclonal antibody is selected from Sacituzumab, and the anti-Her
2 monoclonal antibody is selected from Trastuzumab or
Pertuzumab.
107. The conjugate of claim 104, wherein, A is selected from a RGD
peptide that recognizes cell surface integrin receptor; a growth
factor that recognizes cell surface growth factor receptor, such as
EGF, PDGF or VEGF; or a peptide capable of recognizing functional
cell surface plasminogen activator, bombesin, bradykinin,
somatostatin or prostate-specific membrane antigen receptor;
preferably, A is selected from a CD40 ligand, a CD30 ligand, an
OX40 ligand, a PD-1 ligand, an ErbB ligand, a Her2 ligand, a
TACSTD2 ligand or a DR5 ligand.
108. The conjugate of claim 104, wherein, the conjugate is selected
from: ##STR00322## ##STR00323## ##STR00324## ##STR00325##
##STR00326## ##STR00327## ##STR00328## ##STR00329## ##STR00330##
##STR00331## wherein, .gamma. is an integer or a decimal from 1 to
10, and mAb is an anti-Trop-2 monoclonal antibody or an anti-Her 2
monoclonal antibody; preferably, the anti-Trop-2 monoclonal
antibody is selected from antibodies of Sacituzumab, M1, M2 or M3,
and the anti-Her 2 monoclonal antibody is selected from Trastuzumab
or Pertuzumab; preferably, .gamma. is an integer or a decimal from
5 to 8 (e.g., 5, 6, 7 or 8); preferably, the conjugate is selected
from: ##STR00332## ##STR00333## ##STR00334## ##STR00335##
##STR00336## ##STR00337## ##STR00338## ##STR00339## wherein,
.gamma. is an integer or a decimal from 1 to 10, and mAb is an
anti-Trop-2 monoclonal antibody or an anti-Her 2 monoclonal
antibody; preferably, the anti-Trop-2 monoclonal antibody is
selected from Sacituzumab, and the anti-Her 2 monoclonal antibody
is selected from Trastuzumab or Pertuzumab; preferably, .gamma. is
an integer or a decimal from 5 to 8 (e.g., 5, 6, 7 or 8).
109. The conjugate of claim 104, wherein the conjugate is selected
from: ##STR00340## ##STR00341## ##STR00342## ##STR00343## wherein,
A1 is Sacituzumab, and .gamma. is an integer or a decimal from 1 to
10; preferably, .gamma. is an integer or a decimal from 5 to 8;
preferably, the conjugate is selected from: ##STR00344##
##STR00345## wherein, A1 is Sacituzumab, and .gamma. is an integer
or a decimal from 1 to 10; and preferably, .gamma. is an integer or
a decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8; more preferably,
the conjugate is selected from: ##STR00346## wherein, A1 is
Sacituzumab, and .gamma. is an integer or a decimal from 1 to 10;
and preferably, .gamma. is an integer or a decimal from 5 to 8,
such as an integer or a decimal from 6-7, 6-7.5, 6-8, 6.5-7,
6.5-7.5, 6.5-8, 7-8 or 7.5-8.
110. The conjugate of claim 104, wherein the conjugate is selected
from: ##STR00347## ##STR00348## ##STR00349## ##STR00350## wherein,
A2 is Trastuzumab, and .gamma. is an integer or a decimal from 1 to
10; and preferably, .gamma. is an integer or a decimal from 5 to 8,
such as an integer or a decimal from 6-7, 6-7.5, 6-8, 6.5-7,
6.5-7.5, 6.5-8, 7-8 or 7.5-8; preferably, the conjugate is selected
from: ##STR00351## ##STR00352## wherein, A2 is Trastuzumab, and
.gamma. is an integer or a decimal from 1 to 10; preferably,
.gamma. is an integer or a decimal from 5 to 8, such as an integer
or a decimal from 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or
7.5-8; more preferably, the conjugate is: ##STR00353## wherein A2
is Trastuzumab, and .gamma. is an integer or a decimal from 1 to
10; preferably, .gamma. is an integer or a decimal from 5 to 8,
such as an integer or a decimal from 6-7, 6-7.5, 6-8, 6.5-7,
6.5-7.5, 6.5-8, 7-8 or 7.5-8.
111. The conjugate of claim 104, wherein the conjugate is selected
from: ##STR00354## ##STR00355## ##STR00356## ##STR00357## wherein
A3 is Pertuzumab, and .gamma. is an integer or a decimal from 1 to
10; preferably, .gamma. is an integer or a decimal from 5 to 8,
such as an integer or a decimal from 6-7, 6-7.5, 6-8, 6.5-7,
6.5-7.5, 6.5-8, 7-8 or 7.5-8; preferably, the conjugate is selected
from: ##STR00358## ##STR00359## wherein A3 is Pertuzumab, and
.gamma. is an integer or a decimal from 1 to 10; and preferably,
.gamma. is an integer or a decimal from 5 to 8, such as an integer
or a decimal from 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or
7.5-8.
112. The conjugate of claim 104, wherein the conjugate is selected
from: ##STR00360## ##STR00361## ##STR00362## ##STR00363## wherein,
A4 is antibody M1, and .gamma. is an integer or a decimal from 1 to
10; and preferably, .gamma. is an integer or a decimal from 5 to 8,
such as an integer or a decimal from 6-7, 6-7.5, 6-8, 6.5-7,
6.5-7.5, 6.5-8, 7-8 or 7.5-8; preferably, the conjugate is:
##STR00364## wherein A4 is antibody M1, and .gamma. is an integer
or a decimal from 1 to 10; and preferably, .gamma. is an integer or
a decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
113. The conjugate of claim 104, wherein the conjugate is selected
from: ##STR00365## ##STR00366## ##STR00367## ##STR00368## wherein,
A5 is antibody M2, and .gamma. is an integer or a decimal from 1 to
10; and preferably, .gamma. is an integer or a decimal from 5 to 8,
such as an integer or a decimal from 6-7, 6-7.5, 6-8, 6.5-7,
6.5-7.5, 6.5-8, 7-8 or 7.5-8; preferably, the conjugate is:
##STR00369## wherein, A5 is antibody M2, and .gamma. is an integer
or a decimal from 1 to 10; and preferably, .gamma. is an integer or
a decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
114. The conjugate of claim 104, wherein the conjugate is selected
from: ##STR00370## ##STR00371## ##STR00372## ##STR00373## wherein,
A6 is antibody M3, and .gamma. is an integer or a decimal from 1 to
10; and preferably, .gamma. is an integer or a decimal from 5 to 8,
such as an integer or a decimal from 6-7, 6-7.5, 6-8, 6.5-7,
6.5-7.5, 6.5-8, 7-8 or 7.5-8; preferably, the conjugate is:
##STR00374## wherein, A6 is antibody M3, and .gamma. is an integer
or a decimal from 1 to 10; and preferably, .gamma. is an integer or
a decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
115. A method for preparing the conjugate of claim 104 comprising a
step of coupling the linker of the compound of formula (I) with an
active group of the targeting moiety; preferably, the method
comprising a step of coupling the linker of the compound of formula
(I) with an active group of the targeting moiety to form a C--S
bond; preferably, the targeting moiety of the conjugate is an
anti-Her 2 monoclonal antibody or an anti-Trop-2 monoclonal
antibody or an active fragment or mutant thereof; preferably, the
anti-Trop-2 monoclonal antibody is selected from antibodies of
Sacituzumab, M1, M2 or M3, and the anti-Her 2 monoclonal antibody
is selected from Trastuzumab or Pertuzumab; preferably, the
targeting moiety of the conjugate is an anti-Her 2 monoclonal
antibody or an anti-Trop-2 monoclonal antibody or an active
fragment or mutant thereof; preferably, the anti-Trop-2 monoclonal
antibody is selected from Sacituzumab, and the anti-Her 2
monoclonal antibody is selected from Trastuzumab or Pertuzumab;
preferably, the molar ratio of the targeting moiety of the
conjugate to the compound of formula (I) is 1:(1-20); preferably,
the coupling is carried out in water and/or an organic solvent;
preferably, the organic solvent is selected from
N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone,
nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol)
or any combination thereof; preferably, the method further
comprising a step of purifying a coupling product; preferably, the
coupling product is purified by chromatography; preferably, the
chromatography comprises one or more of ion exchange
chromatography, hydrophobic chromatography, reversed phase
chromatography or affinity chromatography.
116. A pharmaceutical composition comprising the conjugate of claim
104, and one or more pharmaceutical excipients.
117. A method of treating a disease associated with an abnormal
cell activity (e.g., cancer), comprising a step of administering an
effective amount of the conjugate of claim 104 to an individual in
need thereof; preferably, the cancer is selected from a solid tumor
or a non-solid tumor, such as esophageal cancer (e.g., esophageal
adenocarcinoma, esophageal squamous cell carcinoma), a brain tumor,
lung cancer (e.g., small cell lung cancer, non-small cell lung
cancer), squamous cell carcinoma, bladder cancer, stomach cancer,
ovarian cancer, peritoneal cancer, pancreatic cancer, breast
cancer, head and neck cancer, cervical cancer, endometrial cancer,
colorectal cancer, liver cancer, kidney cancer, non Hodgkin's
lymphoma, central nervous system tumors (e.g., neuroglioma,
glioblastoma multiforme, glioma or sarcoma), prostate cancer or
thyroid cancer.
118. The conjugate of claim 104 wherein, A is a targeting moiety
(e.g., a small molecule ligand, a protein, a polypeptide or a
non-protein reagent (e.g., saccharide, RNA or DNA)); .gamma. is an
integer or a decimal from 1 to 10; preferably, .gamma. is an
integer or a decimal from 5 to 8 (e.g., 5, 6, 7 or 8); the rest
groups are as defined in claim 103.
119. The pharmaceutical composition, comprising the conjugate of
claim 108, and one or more pharmaceutical excipients.
120. A method of treating a disease associated with an abnormal
cell activity (e.g., cancer), comprising a step of administering an
effective amount of the conjugate of claim 108 to an individual in
need thereof; preferably, the cancer is selected from a solid tumor
or a non-solid tumor, such as esophageal cancer (e.g., esophageal
adenocarcinoma, esophageal squamous cell carcinoma), a brain tumor,
lung cancer (e.g., small cell lung cancer, non-small cell lung
cancer), squamous cell carcinoma, bladder cancer, stomach cancer,
ovarian cancer, peritoneal cancer, pancreatic cancer, breast
cancer, head and neck cancer, cervical cancer, endometrial cancer,
colorectal cancer, liver cancer, kidney cancer, non Hodgkin's
lymphoma, central nervous system tumors (e.g., neuroglioma,
glioblastoma multiforme, glioma or sarcoma), prostate cancer or
thyroid cancer.
121. A method of treating a disease associated with an abnormal
cell activity (e.g., cancer), comprising a step of administering an
effective amount of the pharmaceutical composition of claim 116 to
an individual in need thereof; preferably, the cancer is selected
from a solid tumor or a non-solid tumor, such as esophageal cancer
(e.g., esophageal adenocarcinoma, esophageal squamous cell
carcinoma), a brain tumor, lung cancer (e.g., small cell lung
cancer, non-small cell lung cancer), squamous cell carcinoma,
bladder cancer, stomach cancer, ovarian cancer, peritoneal cancer,
pancreatic cancer, breast cancer, head and neck cancer, cervical
cancer, endometrial cancer, colorectal cancer, liver cancer, kidney
cancer, non Hodgkin's lymphoma, central nervous system tumors
(e.g., neuroglioma, glioblastoma multiforme, glioma or sarcoma),
prostate cancer or thyroid cancer.
Description
FIELD OF THE INVENTION
[0001] The disclosure belongs to the technical field of medical
technology, and relates to a bioactive molecule conjugate,
preparation method thereof, and use in the prevention and/or
treatment of a disease associated with an abnormal cell activity,
including but not limited to the use in the prevention and/or
treatment of a neoplastic disease.
BACKGROUND ART
[0002] Chemotherapy was once a standard therapy for cancer, but
bioactive molecules having high killing effect can mistakenly kill
normal cells, resulting in serious side effects. Targeted therapy
has become a hot research topic in the field of oncology due to the
targetability and anti-tumor activity. Since the 20th century,
breakthroughs have been made in the development of anti-tumor drugs
and tumor targeted therapies using bio-macromolecular drugs (e.g.,
therapeutic antibodies or antibody fragments) and targeted small
molecule ligands. However, despite of their high targetability,
bio-macromolecular drugs have limited curative effects on solid
tumors; in addition, bioactive molecules often lack targetability
and accidentally injure normal cells and cause serious toxic and
side effects, despite of their high killing effect on cancer
cells.
[0003] Recent studies have found that therapeutic antibodies can be
linked to bioactive molecules to form antibody-drug conjugates
(ADCs). The ADC combine the targeting effect of antibodies and the
activity of bioactive molecules making it a "biological missile".
The ADC is guided by antibodies to bind to target cells, and then
is internalized by cells to release drugs thereby treating relevant
diseases. Due to the specificity and targetability to tumor cell
related targets, the application values of antibodies not only are
reflected in the treatment, but also become an ideal carrier for
drug targeted delivery, and reduce side effects of drugs. Small
molecule drug conjugates (SMDCs) are designed on the basis of same
principle as antibody-drug conjugates (ADCs); that is, coupling
bioactive molecules with some small molecule ligands which can
selectively bind to receptors on the surfaces of tumor cells
through chemical processes, thereby improving the targetability of
effector molecules (bioactive molecules) to tumor cells. The
difference between the SMDCs and the ADCs is that the SMDCs use
small molecule ligands instead of antibodies, and there is not yet
SMDC available on the market.
[0004] Currently, there are four commercially available ADCs:
Mylotarg (Gemtuzumab Ozogamicin), Adcetris (Brentuximab Vedotin,
CD30 monoclonal antibody-MMAE), Kadcyla (Trastuzumab Emtansine) and
Besponsa (Inotuzumab ozogamicin, CD22 monoclonal
antibody-calicheamicin). An ADC generally consists of an antibody,
a bioactive molecule and a linker. The bioactive molecule is
covalently coupled to the antibody via the linker; the antibody
(e.g., monoclonal antibodies) can specifically recognize a specific
target on the surface of a tumor cell, thus guiding the ADC to
reach the surface of cancer cell and enabling the ADC to enter the
cancer cell through endocytosis effect; then the bioactive molecule
is released in the cancer cell to achieve the effect of
specifically killing the cancer cell without damaging normal tissue
cells.
[0005] Lysine is the most common linking site in antibodies, and
.epsilon.-amino groups thereof can react with activated carboxyl
groups of linkers to form amide bonds. Techniques for site-specific
coupling are currently available, that is, carboxyl groups of
linkers are activated, and then form amide bonds with specific
lysine .epsilon.-amino groups in antibodies to complete the
coupling. However, such amide bonds are prone to hydrolysis under
the action of enzymes in vivo, as a result, bioactive molecules and
antibodies dissociate before reaching target cells resulted in
increasing toxicity while losing targetability of ADCs.
[0006] Thio groups of antibody cysteine usually exist in the form
of disulfide bonds. The disulfide bonds in the antibody can be
opened to provide multiple free sulfhydryl groups as coupling
sites. One method of coupling with the sulfhydryl groups of the
antibody is Michael addition reaction between the free sulfhydryl
groups of the antibody and maleimide, or two Michael addition
reactions between a specific substrate and free sulfhydryl groups
of the antibody to form a sulfur bridge bond in a unique structure.
However, many literatures have reported that ADCs obtained by
thiol-Michael addition methods will undergo reverse Michael
additions in systemic circulation, resulting in toxic reactions.
The patent WO2016142049 discloses amatoxins as bioactive molecules,
and structure comprising bioactive molecules having the structure
of methylsulfonyl-substituted benzobisoxadiazole and linkers, but
details of coupling with antibodies are not specifically
described.
CONTENTS OF THE INVENTION
[0007] The invention discloses a novel bioactive molecule
conjugate, which is obtained by improving the coupling way of the
drug and the targeting moiety in an ADC or SMDC. The conjugate has
high stability, extremely high coupling efficiency (90%) and high
DAR (5-8). The disclosure is based on the above findings. Through
intensive research, it was surprisingly found that, the ADC of the
invention, e.g. BT001021 (example 32), after intravenous
administration, the exposure of the bioactive small molecular toxin
in tumor is significantly higher than that in plasma, whereas
Immu-132 has significantly higher plasma exposure than tumor
exposure under the same administration route. Therefore, the ADC of
the invention has a better therapeutic window than Immu-132. We
were also surprised that the ADC of the invention has a better
efficacy than Immu-132 in animal models of gastric cancer, breast
cancer and non-small cell lung cancer.
[0008] A first aspect of the disclosure provides a compound as
shown in formula (I) or a pharmaceutically acceptable salt
thereof,
T-[L.sub.1-(L.sub.2).sub.m1-(L.sub.3).sub.m2-(L.sub.4).sub.m3-E]-G
formula (I)
[0009] wherein, T is a fragment of a bioactive molecule, preferably
a fragment of a molecule with antitumor bioactivity;
[0010] L.sub.1 is selected from an amino acid, a peptide composed
of 2-10 amino acids, an oligosaccharide, --(CH.sub.2)t.sub.1-,
--(CH.sub.2CH.sub.2O).sub.t1--(CH.sub.2).sub.t2--,
##STR00001## ##STR00002## ##STR00003##
[0011] wherein each of R, R', R.sub.1 and R.sub.2 is independently
H (hydrogen), D (deuterium), halogen, a carboxylic acid group, a
sulfonic acid group, cyano, C.sub.1-6 alkyl, halogenated C.sub.1-6
alkyl (e.g., --CF.sub.3), C.sub.1-6 alkyl substituted with cyano
(e.g., --CH.sub.2CN), 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;
[0012] L.sub.2 is selected from 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--,
##STR00004##
wherein each of R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is
independently selected from H (hydrogen), D (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.3-6 cycloalkyl, or R.sub.3/R.sub.4, R.sub.5/R.sub.6 or
R.sub.3/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;
[0013] L.sub.3 is selected from the following groups optionally
substituted with one or more R.sub.7: 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 (e.g., phenylene or
naphthylene), 5-12 membered heteroarylene or 3-8 membered
cycloalkylene-W--; wherein W is oxygen or NR.sub.8, R.sub.7 is
independently selected from H (hydrogen), D (deuterium), halogen,
.dbd.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 H (hydrogen), D (deuterium),
C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.1-6 alkoxy or cyano
C.sub.1-2 alkyl, and L.sub.3 is bonded to L.sub.2 at the position 1
of L.sub.3;
[0014] L.sub.4 is selected from
##STR00005##
wherein Z.sub.5 is preferably selected from C.sub.2-6 alkenylidene,
C.sub.2-6 alkynylidene, 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 H (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;
.alpha. 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;
[0015] E is selected from the following groups optionally
substituted with one or more R.sub.12: 6-10 membered arylene or
5-14 membered heteroarylene; wherein R.sub.12 is independently
selected from H (hydrogen), D (deuterium), halogen, CN, nitro,
C.sub.1-6 alkyl or halogenated C.sub.1-6 alkyl;
[0016] G is a leaving group for nucleophilic substitutions; such
as, halogen, sulfonyl, sulfonic acid ester group, nitro, etc.;
[0017] 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.
[0018] In some preferred embodiments, L.sub.1 is selected from Val,
Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, a peptide composed of 2-5
amino acids,
##STR00006## ##STR00007##
wherein each of R, R', R.sub.1 and R.sub.2 is independently H
(hydrogen), D (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 Val, Cit,
Phe, Lys, 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 x3 is 0,
1, 2, 3 or 4.
[0019] In some preferred embodiments, L.sub.1 is selected from Val,
Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Cit-Val, Val-Ala,
Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys,
Gly-Gly-Arg, Ala-Ala-Asn,
##STR00008##
wherein each of R, R' and R.sub.1 is independently H (hydrogen), D
(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 Val, Cit, Phe, Lys,
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, and each of x.sub.1 and x.sub.3 is
independently 0, 1, 2 or 3.
[0020] In some preferred embodiments, L.sub.1 is selected from Lys,
Cit, Cit-Val, Val-Ala, Lys-Val,
##STR00009##
wherein each of R, R' and R.sub.1 is independently H (hydrogen), D
(deuterium) or C.sub.1-4 alkyl, Z.sub.1 is Cit, Lys, Cit-Val,
Cit-Ala, Val-Ala or Lys-Val, and each of x.sub.1 and x.sub.3 is
independently 0, 1 or 2.
[0021] In some preferred embodiments, L.sub.1 is selected from Lys,
Cit, Cit-Val, Val-Ala, Lys-Val,
##STR00010##
[0022] In some preferred embodiments, L.sub.1 is selected from
##STR00011##
In some preferred embodiments, L.sub.2 is selected from Val, Cit,
Phe, Lys, D-Val, Leu, Gly, Ala, Asn, a peptide composed of 2-5
amino acids,
##STR00012##
wherein each of R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is
independently selected from H (hydrogen), D (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;
[0023] m.sub.1 is 0, 1, 2 or 3.
[0024] In some preferred embodiments, L.sub.2 is selected from Val,
Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Cit-Val,
Val-Ala, Lys-Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys,
Gly-Gly-Arg, Ala-Ala-Asn,
##STR00013##
wherein each of R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is
independently selected from H (hydrogen), D (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;
[0025] m.sub.1 is 0, 1 or 2.
[0026] In some preferred embodiments, L.sub.2 is selected from
##STR00014##
wherein each of R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is
independently selected from H (hydrogen), D (deuterium) or
C.sub.1-4 alkyl, 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;
[0027] m.sub.1 is 1.
[0028] In some preferred embodiments, L.sub.2 is selected from
##STR00015##
[0029] In some preferred embodiments, L.sub.2 is selected from
##STR00016##
[0030] In some preferred embodiments, L.sub.3 is selected from the
following groups optionally substituted with one or more R.sub.7:
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, 5-12 membered heteroarylene or 3-8 membered
cycloalkylene-W--; wherein W is oxygen or NR.sub.8, R.sub.7 is
independently selected from H (hydrogen), D (deuterium), halogen,
.dbd.O, CF.sub.3, CN, CH.sub.2CN, carboxyl, sulfonic acid group,
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.2-6 alkenyl or C.sub.2-6
alkynyl; preferably, the 3-8 aliphatic heterocyclylene, 6-12
membered bridged heterocyclylene, 6-12 membered
spiroheterocyclylene or 6-12 membered fused heterocyclylene has one
or more nitrogen atoms; preferably, the 3-8 membered aliphatic
heterocyclylene, 6-12 membered bridged heterocyclylene, 6-12
membered spiroheterocyclylene or 6-12 membered fused
heterocyclylene has one or more quaternized nitrogen atoms;
preferably, the 3-8 membered aliphatic heterocyclylene, 6-12
membered bridged heterocyclylene, 6-12 membered
spiroheterocyclylene or 6-12 membered fused heterocyclylene has one
or more nitrogen atoms, and at least one nitrogen atom is
substituted with .dbd.O; R.sub.8 is independently selected from H
(hydrogen), D (deuterium), C.sub.1-6 alkyl, C.sub.2-6 alkenyl,
C.sub.3-6 alkynyl, C.sub.3-6 cycloalkyl, C.sub.1-6 alkoxy or cyano
C.sub.1-2 alkyl;
[0031] m.sub.2 is 0, 1, 2 or 3.
[0032] In some preferred embodiments, L.sub.3 is selected from the
following groups optionally substituted with one or more R.sub.7:
amino, 3-6 membered aliphatic heterocyclylene or 5-10 membered
heteroarylene; wherein R.sub.7 is independently selected from H
(hydrogen), D (deuterium), halogen, .dbd.O, CF.sub.3, CN,
CH.sub.2CN, carboxyl, sulfonic acid group, C.sub.1-4 alkyl,
C.sub.1-4 alkoxy, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl;
preferably, the 3-6 membered aliphatic heterocyclylene has one or
more nitrogen atoms; preferably, the 3-6 membered aliphatic
heterocyclylene has one or more quaternized nitrogen atoms;
preferably, the 3-6 membered aliphatic heterocyclylene has one or
more nitrogen atoms, and at least one nitrogen atom is substituted
with .dbd.O;
[0033] m.sub.2 is 0, 1 or 2.
[0034] In some preferred embodiments, L.sub.3 is selected from the
following groups optionally substituted with one or more R.sub.7:
amino or 5-6 membered heteroarylene; wherein R.sub.7 is
independently selected from H (hydrogen), D (deuterium), halogen,
.dbd.O, CF.sub.3, CN, CH.sub.2CN, carboxyl, sulfonic acid group,
C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.2-6 alkenyl or C.sub.2-6
alkynyl; m.sub.2 is 0 or 1.
[0035] In some preferred embodiments, L.sub.3 is selected from the
following groups optionally substituted with one or more R.sub.7:
amino, N-methylpiperidylene, pyrazolylene or triazolylene; wherein
R.sub.7 is independently selected from H (hydrogen), D (deuterium),
halogen, .dbd.O, CF.sub.3, CN, CH.sub.2CN, carboxyl, sulfonic acid
group, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, C.sub.2-6 alkenyl or
C.sub.2-6 alkynyl; m.sub.2 is 0 or 1.
[0036] In some preferred embodiments, L.sub.3 is selected from
triazolylene; m.sub.2 is 0 or 1.
[0037] In some preferred embodiments, L.sub.3 is selected from
##STR00017##
m.sub.2 is 0 or 1. preferably, L.sub.3 is bonded to L.sub.2 at the
position 1 of L.sub.3.
[0038] In some preferred embodiments, L.sub.3 is selected from the
following groups optionally substituted with one or more R.sub.7:
amino,
##STR00018##
[0039] R.sub.7 is independently selected from H (hydrogen), D
(deuterium), .dbd.O, CN, CH.sub.2CN, methyl or CF.sub.3;
[0040] W is NR.sub.B, and R.sub.8 is selected from H (hydrogen), D
(deuterium), C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.3-6 alkynyl
or C.sub.3-6 cycloalkyl.
[0041] In some preferred embodiments, L.sub.3 is selected from
##STR00019##
[0042] wherein each R.sub.q is independently selected from
C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.3-6 alkynyl or C.sub.3-8
cycloalkyl; .beta..sub.1 is 0, 1 or 2; and .beta..sub.2 is 1, 2 or
3.
[0043] In some preferred embodiments, L.sub.3 is selected from
##STR00020##
[0044] In some preferred embodiments, L.sub.4 is selected from
##STR00021##
wherein Z.sub.4 is 6-10 membered aryl or 5-6 membered heteroaryl;
R.sub.10 is selected from H (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 H (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; a 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;
[0045] m.sub.3 is 0, 1, 2 or 3.
[0046] In some preferred embodiments, L.sub.4 is selected from
wherein Z.sub.4 is a benzene ring, and R.sub.10 is selected from H
(hydrogen) and 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 H (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; .alpha. 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;
[0047] m.sub.3 is 0, 1, 2 or 3.
[0048] In some preferred embodiments, L.sub.4 is selected from
##STR00022##
Z.sub.4 is 5-6 membered heteroarylene; R.sub.10 is selected from H
(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 H (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; .alpha. 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;
[0049] m3 is 0, 1, 2 or 3.
[0050] In some preferred embodiments, L.sub.4 is selected from
##STR00023## ##STR00024##
m.sub.3 is 1.
[0051] In some preferred embodiments, L.sub.4 is selected from
##STR00025##
m.sub.3 is 1.
[0052] In some preferred embodiments, L.sub.4 is selected from
##STR00026##
[0053] m.sub.3 is 1.
[0054] In some preferred embodiments, E is selected from 5-10
membered heteroarylene optionally substituted with one or more
R.sub.12; wherein R.sub.12 is independently selected from H
(hydrogen), D (deuterium), halogen, CN, nitro, C.sub.1-4 alkyl or
halogenated C.sub.1-4 alkyl.
[0055] In some preferred embodiments, 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 H (hydrogen), D
(deuterium), halogen, CN, nitro, C.sub.1-2 alkyl or halogenated
C.sub.1-2 alkyl.
[0056] In some preferred embodiments, E is selected from
pyrimidinyl optionally substituted with one or more R.sub.12;
wherein R.sub.12 is independently selected from H (hydrogen) or D
(deuterium).
[0057] In some preferred embodiments, G is selected from halogen,
OMs, OTs, OTf, nitro, or anyone of the following groups which is
optionally substituted with one or more R.sub.13: alkylthio,
arylthio, heteroarylthio, alkyl sulfinyl, aryl sulfinyl, heteroaryl
sulfinyl, alkyl sulfonyl, aryl sulfonyl or heteroaryl sulfonyl;
wherein Ria is independently selected from H (hydrogen), D
(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.
[0058] In some preferred embodiments, G is selected from F, Cl, Br,
I, OMs, OTs, OTf, methylsulfonyl, ethylsulfonyl, p-toluenesulfonyl
or naphthalenesulfonyl.
[0059] In some preferred embodiments, G is selected from F, Cl, Br,
OMs, OTs, methylsulfonyl or p-toluenesulfonyl.
[0060] In some preferred embodiments, G is selected from Cl or
methylsulfonyl.
[0061] In some preferred embodiments, in
##STR00027##
G is preferably methylsulfonyl, E is preferably pyrimidylene,
m.sub.3 is 1.
[0062] In some preferred embodiments,
##STR00028##
wherein m.sub.4 is preferably an integer from 0 to 6,
methylsulfonyl is a substituent on a carbon atom adjacent to a
nitrogen atom in the pyrimidine ring.
[0063] In some preferred embodiments,
##STR00029##
wherein m.sub.5 is preferably an integer from 0 to 6,
methylsulfonyl is a substituent on a carbon atom adjacent to a
nitrogen atom in the pyrimidine ring.
[0064] In some preferred embodiments,
##STR00030##
wherein m.sub.6 is preferably an integer from 0 to 6,
methylsulfonyl is a substituent on a carbon atom adjacent to a
nitrogen atom in the pyrimidine ring.
[0065] In some preferred embodiments,
##STR00031##
wherein m.sub.7 is an integer from 1 to 5, methylsulfonyl is a
substituent on a carbon atom adjacent to a nitrogen atom in the
pyrimidine ring.
[0066] In some preferred embodiments,
##STR00032##
wherein m.sub.8 is an integer from 1 to 5, methylsulfonyl is a
substituent on a carbon atom adjacent to a nitrogen atom in the
pyrimidine ring.
[0067] In some preferred embodiments,
##STR00033##
wherein m.sub.9 is an integer from 1 to 5, Ria is selected from
hydrogen or C.sub.1-6 alkyl, methylsulfonyl is a substituent on a
carbon atom adjacent to a nitrogen atom in the pyrimidine ring.
[0068] In some preferred embodiments,
##STR00034##
wherein m.sub.10 is an integer from 0 to 6, and Z.sub.4 is selected
from 5-6 membered heteroarylene; methylsulfonyl is a substituent on
a carbon atom adjacent to a nitrogen atom in the pyrimidine
ring.
[0069] In some preferred embodiments,
##STR00035##
Z.sub.4 is selected from pyridylene, pyrimidylene, pyrazolylene,
thiazolylene, oxazolylene or triazolylene; methylsulfonyl is a
substituent on a carbon atom adjacent to a nitrogen atom in the
pyrimidine ring; more preferably, m.sub.10 is an integer from
0-6.
[0070] In some preferred embodiments,
##STR00036##
Z.sub.4 is selected from pyridylene, pyrimidylene, pyrazolylene or
triazolylene. More preferably, m.sub.10 is an integer from 0-6.
[0071] In some preferred embodiments,
##STR00037##
Z.sub.4 is selected from oxazolylene or thiazolylene, and
methylsulfonyl is a substituent of a carbon atom adjacent to a
nitrogen atom in the pyrimidine ring. More preferably, m.sub.10 is
an integer from 0-6.
[0072] In some preferred embodiments,
##STR00038##
wherein m.sub.10 is an integer from 0-6, and Z.sub.4 is selected
from 6-10 membered arylene; and methylsulfonyl is a substituent of
a carbon atom adjacent to a nitrogen atom. More preferably,
m.sub.10 is an integer from 0-6.
[0073] In some preferred embodiments,
##STR00039##
wherein m.sub.10 is an integer from 0-6, and Z.sub.4 is a benzene
ring.
[0074] In some preferred embodiments,
##STR00040##
[0075] In some preferred embodiments,
##STR00041##
in formula (I) is selected from the following fragments:
##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046##
##STR00047## ##STR00048##
[0076] In some preferred embodiments, T is a fragment of a
bioactive molecule. In some preferred embodiments, the bioactive
molecule is selected from a metal complex, such as a platinum metal
complex (e.g., oxaliplatin) or a gold metal complex; a glycopeptide
antibiotic such as bleomycin or pingyangmycin; a DNA topoisomerase
inhibitor, such as a topoisomerase I inhibitor (e.g., camptothecin,
hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan,
topotecan, bellotencian or rubitecan) or a topoisomerase II
inhibitor (e.g., actinomycin D, adriamycin, doxorubicin,
duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin or
etoposide); a drug interfering with DNA synthesis, such as
methotrexate, 5-fluorouracil, cytarabine, gemcitabine,
mercaptopurine, pentostatin, fludarabine, cladribine or narabine; a
drugs acting on a structural protein, such as a tubulin inhibitor,
a vinblastine alkaloid, a vincristine, vinblastine, paclitaxel,
docetaxel or cabazitaxel; a tumor cell signaling pathway inhibitor,
such as a serine/threonine kinase inhibitor, a tyrosine kinase
inhibitor, a aspartokinase inhibitor or a histidine kinase
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; chlorambucil; and other active substances which inhibit the
growth of tumor cells, promote the apoptosis or necrosis of tumor
cells.
[0077] In some preferred embodiments, the bioactive molecule is
selected from
##STR00049##
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 H (hydrogen), D
(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 mu is 0, 1 or
2.
[0078] In some preferred embodiments, the bioactive molecule is
selected from
##STR00050##
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.10 is selected from H (hydrogen), D
(deuterium), C.sub.1-6 alkyl, 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 or pyridyl, and mu is 0, 1, or
2.
[0079] In some preferred embodiments, the bioactive molecule is
selected from
##STR00051## ##STR00052##
[0080] In some preferred embodiments, the bioactive molecule is
selected from
##STR00053## ##STR00054##
[0081] In some preferred embodiments, the bioactive molecule is
selected from
##STR00055##
[0082] In some preferred embodiments, the bioactive molecule is
selected from
##STR00056##
[0083] In some preferred embodiments, the bioactive molecule is
selected from
##STR00057##
[0084] In some preferred embodiments, T is selected from
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063##
[0085] In some preferred embodiments, T is selected from
##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068##
[0086] In some preferred embodiments, T is selected from
##STR00069##
[0087] In some preferred embodiments, T is selected from
##STR00070##
[0088] In some preferred embodiments, T is selected from
##STR00071##
[0089] In some preferred embodiments, the compound shown in formula
(I) is selected from
##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076##
##STR00077## ##STR00078## ##STR00079## ##STR00080## ##STR00081##
##STR00082## ##STR00083##
[0090] In some preferred embodiments, the compound is selected
from
##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088##
##STR00089## ##STR00090##
[0091] In a second aspect, the disclosure provides a conjugate,
comprising a bioactive molecule, a linker and a targeting moiety.
The targeting moiety is linked to the linker via an active group
(e.g., a thiol group) to form a conjugate.
[0092] In some preferred embodiments, the structure of the
conjugate is shown in formula (II):
{T-[L.sub.1-(L.sub.2).sub.m1-(L.sub.3).sub.m2-(L.sub.4).sub.m3-E]}.sub..-
gamma.-A formula (II)
[0093] where, wherein, A is a targeting moiety (e.g., a small
molecule ligand, a protein, a polypeptide or a non-protein reagent
(e.g., saccharide, RNA or DNA)); .gamma. is an integer or a decimal
from 1 to 10; preferably, .gamma. is an integer or a decimal from 5
to 8 (e.g., 5, 6, 7 or 8);
[0094] the rest groups are as described in the first aspect of the
disclosure.
[0095] In some preferred embodiments, a target of A is selected
from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII,
Mesothelin, Folate receptor1, Mucin 1, CD138, CD20, CD19, CD30,
SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor,
STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium
phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein,
AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L.sub.1, TACSTD2, DR5,
E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783,
STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20R.alpha.,
Brevican, EphB2R, A5LG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5,
HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, integrin
.alpha.5.beta.6, .alpha.4.beta.7, FGF2, FGFR2, Her3, CD70, CA6,
DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4,
ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2, BMPR1B, E16,
STEAP1, Tyrol, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783,
STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20R.alpha.,
Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB,
P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1 lc, CD40, CD45
(PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7,
PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX,
LRRC15, MCPT8, MMP10, NOG, SERPINET, STAT1, TGFBR1, CTSS, PGF,
VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNIP3, AIF1,
CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3,
PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1,
KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1,
TGFB2, CD1 lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2,
IGF-1R, RNF43, NaPi2b, BCMA or TENB2.
[0096] In some preferred embodiments, A is a small molecule ligand,
such as a folic acid derivative, a glutamate urea derivative, a
somatostatin derivative, an arylsulfonamide derivative (e.g., a
carbonic anhydrase IX inhibitor), a polyene connecting two
aliphatic indoles, a cyanine dye or IR-783 or a derivative
thereof.
[0097] In some preferred embodiments, A is selected from
##STR00091##
[0098] In some preferred embodiments, A is an antibody such as a
monoclonal antibody or an antigen binding fragment thereof, wherein
the monoclonal antibody or the antigen binding fragment thereof
comprises Fab, Fab', F(ab').sub.2, Fd, Fv, dAb, a complementary
determinant fragment, a single chain antibody (e.g., scFv), a
non-human antibody, a humanized antibody, a chimeric antibody, a
completely humanized antibody, a probody, a bispecific antibody or
a multispecific antibody.
[0099] In some preferred embodiments, A is an anti-Her 2 monoclonal
antibody, such as Trastuzumab, Pertuzumab; or an anti-Trop-2
monoclonal antibody, such as Sacituzumab.
[0100] In some preferred embodiments, A is an anti-Trop-2
monoclonal antibody, such as antibody M1, M2 or M3.
TABLE-US-00001 Anti- body M1 M2 M3 Heavy GYTFTNY GYTFTNY GYTFTNY
chain (SEQ ID No.: 1) (SEQ ID No.: 1) (SEQ ID No.: 1) CDR1 Heavy
NTDSGE NTDSGE NTDSGE chain (SEQ ID No.: 2) (SEQ ID No.: 2) (SEQ ID
No.: 2) CDR2 Heavy GGFGSSYWYFDV GGFGSSYWYFDV GGFGSSYWYFDV chain
(SEQ ID No.: 3) (SEQ ID No.: 3) (SEQ ID No.: 3) CDR3 Light
KASQDVSSAVA KASQDVSSAVA KASQDVSIAVA chain (SEQ ID No.: 4) (SEQ ID
No.: 4) (SEQ ID No.: 8) CDR1 Light SASYRYT SASYRYT SASYRYT chain
(SEQ ID No.: 5) (SEQ ID No.: 5) (SEQ ID No.: 5) CDR2 Light
QQHYSTPLT QQHYITPLT QQHYSTPLT chain (SEQ ID No.: 6) (SEQ ID No.: 7)
(SEQ ID No.: 6) CDR3
[0101] The assignment of amino acids in each region or domain can
follow Chothia & Lesk (1987) J. Mol. Biol. 196:901-917;
definition of Chothia et al. (1989) in Nature 342: 878-883.
[0102] 1. Heavy Chain and Light Chain Sequences of the
Hydrophobically Modified Antibody M1
[0103] Amino acid sequence of heavy chain variable region of M1:
(121 aa)
TABLE-US-00002 (SEQ ID No.: 11)
QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGW
INTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGG
FGSSYWYFDVWGQGSLVTVSS
[0104] Amino acid sequence of light chain variable region of M1:
(107 aa)
TABLE-US-00003 (SEQ ID No.: 12)
DIQLTQSPSSLSASVGDRVSITCKASQDVSSAVAWYQQKPGKAPKLLIYS
ASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYSTPLTFGA GTKVEIK
[0105] 2. Heavy Chain and Light Chain Sequences of the
Hydrophobically Modified Antibody M2
[0106] Amino acid sequence of heavy chain variable region of M2:
(121 aa)
TABLE-US-00004 (SEQ ID No.: 13)
QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGW
INTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGG
FGSSYWYFDVWGQGSLVTVSS
[0107] Amino acid sequence of light chain variable region of M2:
(107 aa)
TABLE-US-00005 (SEQ ID No.: 14)
DIQLTQSPSSLSASVGDRVSITCKASQDVSSAVAWYQQKPGKAPKLLIYS
ASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGA GTKVEIK
[0108] 3. Heavy Chain and Light Chain Sequences of the
Hydrophobically Modified Antibody M3
[0109] Amino acid sequence of heavy chain variable region of M3:
(121 aa)
TABLE-US-00006 (SEQ ID No.: 15)
QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGW
INTDSGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGG
FGSSYWYFDVWGQGSLVTVSS
[0110] Amino acid sequence of light chain variable region of M3:
(107 aa)
TABLE-US-00007 (SEQ ID No.: 16)
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYS
ASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYSTPLTFGA GTKVEIK
[0111] Sequence of light chain constant regions of M1, M2, M3: (107
aa)
TABLE-US-00008 (SEQ ID No.: 9)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC
[0112] Sequence of heavy chain constant regions of M1, M2, M3: (330
aa)
TABLE-US-00009 (SEQ ID No.: 10)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0113] Terminal Lys of heavy chains is easily deleted, but such
deletion does not affect bioactivity. See Dick, L. W. et al.,
Biotechnol. Bioeng., 100: 1132-1143. The above monoclonal
antibodies M1, M2, M3 and sequences or fragments thereof with
deleted Lys at terminal of heavy chains all belong to the M1, M2,
M3 monoclonal antibodies of this invention.
[0114] In some preferred embodiments, A is selected from a RGD
peptide that recognizes cell surface integrin receptor; a growth
factor that recognizes cell surface growth factor receptor, such as
EGF, PDGF or VEGF; or a peptide capable of recognizing functional
cell surface plasminogen activator, bombesin, bradykinin,
somatostatin or prostate-specific membrane antigen receptor.
[0115] In some preferred embodiments, A is selected from CD40
ligand, CD30 ligand, OX40 ligand, PD-1 ligand, ErbB ligand, Her2
ligand, TACSTD2 ligand, or DR5 ligand.
[0116] In some preferred embodiments, the conjugate is selected
from:
##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096##
##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101##
##STR00102##
[0117] wherein, .gamma. is an integer or a decimal from 1 to 10,
and mAb is an anti-Trop-2 monoclonal antibody or an anti-Her 2
monoclonal antibody; preferably, the anti-Trop-2 monoclonal
antibody is selected from antibodies of Sacituzumab, M1, M2 or M3,
and the anti-Her 2 monoclonal antibody is Trastuzumab or
Pertuzumab; preferably, .gamma. is an integer or a decimal from 5
to 8 (e.g., 5, 6, 7 or 8).
[0118] In some preferred embodiments, the conjugate is selected
from:
##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107##
##STR00108##
[0119] wherein, .gamma. is an integer or a decimal from 1 to 10,
and mAb is an anti-Trop-2 monoclonal antibody or an anti-Her 2
monoclonal antibody; preferably, the anti-Trop-2 monoclonal
antibody is selected from Sacituzumab, and the anti-Her 2
monoclonal antibody is selected from Trastuzumab or Pertuzumab;
preferably, .gamma. is an integer or a decimal from 5 to 8 (e.g.,
5, 6, 7 or 8).
[0120] In some preferred embodiments, the conjugate is:
##STR00109## ##STR00110## ##STR00111## ##STR00112## ##STR00113##
##STR00114##
[0121] wherein, A1 is Sacituzumab, and .gamma. is an integer or a
decimal from 1 to 10; preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0122] In some preferred embodiments, the conjugate is:
##STR00115## ##STR00116## ##STR00117## ##STR00118##
[0123] wherein, A1 is Sacituzumab, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0124] In some preferred embodiments, the conjugate is:
##STR00119## ##STR00120##
[0125] wherein, A1 is Sacituzumab, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0126] In some preferred embodiments, the conjugate is:
##STR00121##
[0127] wherein, A1 is a fragment of Sacituzumab, and .gamma. is an
integer or a decimal from 1 to 10; and preferably, .gamma. is an
integer or a decimal from 5 to 8, such as an integer or a decimal
from 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0128] In some preferred embodiments, the conjugate is:
##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126##
##STR00127##
[0129] wherein, A2 is Trastuzumab, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0130] In some preferred embodiments, the conjugate is:
##STR00128## ##STR00129## ##STR00130## ##STR00131##
[0131] wherein, A2 is Trastuzumab, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0132] In some preferred embodiments, the conjugate is:
##STR00132##
[0133] wherein, A2 is Trastuzumab, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0134] In some preferred embodiments, the conjugate is:
##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137##
##STR00138##
[0135] wherein, A3 is Pertuzumab, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0136] In some preferred embodiments, the conjugate is:
##STR00139## ##STR00140## ##STR00141## ##STR00142##
[0137] wherein, A3 is Pertuzumab, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0138] In some preferred embodiments, the conjugate is:
##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147##
##STR00148##
[0139] wherein, A4 is antibody M1, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0140] In some preferred embodiments, the conjugate is:
##STR00149##
[0141] wherein, A4 is antibody M1, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0142] In some preferred embodiments, the conjugate is:
##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154##
##STR00155##
[0143] wherein, A5 is antibody M2, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0144] In some preferred embodiments, the conjugate is:
##STR00156##
[0145] wherein, A5 is antibody M2, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0146] In some preferred embodiments, the conjugate is:
##STR00157## ##STR00158## ##STR00159## ##STR00160##
##STR00161##
[0147] wherein A6 is antibody M3, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0148] In some preferred embodiments, the conjugate is:
##STR00162##
[0149] wherein A6 is antibody M3, and .gamma. is an integer or a
decimal from 1 to 10; and preferably, .gamma. is an integer or a
decimal from 5 to 8, such as an integer or a decimal from 6-7,
6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-8 or 7.5-8.
[0150] In another aspect, the disclosure provides a method for
preparing the conjugate of the second aspect, comprising a step of
coupling the linker of the compound of formula (I) with an active
group of the targeting moiety.
[0151] In some preferred embodiments, the method comprises a step
of opening a disulfide bond of the targeting moiety by a reductant
(e.g., TCEP) to obtain a sulfhydryl group.
[0152] In some preferred embodiments, the method comprises a step
of forming a C--S bond between the linker of the compound of
formula (I) and the sulfhydryl group of the targeting moiety.
[0153] In some preferred embodiments, the targeting moiety is an
anti-Her 2 monoclonal antibody (e.g., Trastuzumab, Pertuzumab) or
an anti-Trop-2 monoclonal antibody (e.g., Sacituzumab, M1, M2 or
M3), or an active fragment or mutant thereof.
[0154] In some preferred embodiments, the molar ratio of the
targeting moiety to the compound of formula (I) is 1:(1-20);
preferably, the coupling is carried out in water and/or an organic
solvent; preferably, the organic solvent is selected from
N,N-dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone,
nitriles (e.g., acetonitrile), alcohols (e.g., methanol, ethanol)
or any combination thereof.
[0155] In some preferred embodiments, the method further comprises
a step of purifying the coupling product; preferably, the coupling
product is purified by chromatography (e.g., one or more of ion
exchange chromatography, hydrophobic chromatography, reverse phase
chromatography or affinity chromatography).
[0156] In another aspect, the disclosure provides a pharmaceutical
composition comprising the compound of the first aspect of the
disclosure or a pharmaceutically acceptable salt thereof, or the
conjugate of the second aspect, and one or more pharmaceutical
excipients.
[0157] In another aspect, the disclosure provides use of the
compound of the first aspect or a pharmaceutically acceptable salt
thereof or the conjugate of the second aspect in the manufacturer
of a medicament for the treatment of a disease associated with an
abnormal cell activity (e.g., cancer).
[0158] In some preferred embodiments, the cancer is a solid tumor
or a non-solid tumor, such as esophageal cancer (e.g., esophageal
adenocarcinoma, esophageal squamous cell carcinoma), a brain tumor,
lung cancer (e.g., small cell lung cancer, non-small cell lung
cancer), squamous cell carcinoma, bladder cancer, stomach cancer,
ovarian cancer, peritoneal cancer, pancreatic cancer, breast
cancer, head and neck cancer, cervical cancer, endometrial cancer,
colorectal cancer, liver cancer, kidney cancer, non Hodgkin's
lymphoma, central nervous system tumors (e.g., neuroglioma,
glioblastoma multiforme, glioma or sarcoma), prostate cancer and
thyroid cancer.
[0159] In another aspect, the disclosure provides use of the
compound of the first aspect or a pharmaceutically acceptable salt
thereof or the conjugate or pharmaceutical composition of the
second aspect in treating a disease associated with an abnormal
cell activity (e.g., cancer).
[0160] In another aspect, the disclosure provides a method of
treating a disease associated with an abnormal cell activity (e.g.,
cancer), comprising a step of administering an effective amount of
the compound of the first aspect or a pharmaceutically acceptable
salt thereof or the conjugate or pharmaceutical composition of the
second aspect to an individual in need thereof.
[0161] Unless otherwise specified, all scientific and technical
terms used in the disclosure have the meanings commonly understood
by those skilled in the art. Moreover, cell culture, molecular
genetics, nucleic acid chemistry and immunology laboratory
procedures used herein are all routine steps widely used in the
corresponding art. In addition, definitions and explanations of
relevant terms are given below for a better understanding of the
disclosure.
[0162] In the disclosure, the pharmaceutical excipients refer to
excipients and additives used in drug manufacturing and
formulating, and are substances that have been reasonably evaluated
in terms of safety and are contained in pharmaceutical preparations
in addition to active ingredients. In addition to being used as
excipients, carriers and stability enhancers, pharmaceutical
excipients also have important functions such as solubilization,
sustained release, and are important ingredients that may affect
the quality, safety and efficacy of drugs. Pharmaceutical
excipients can be divided by sources into natural substances,
semi-synthetic substances and full-synthetic substances; divided by
effects and uses into solvents, propellants, solubilizers,
cosolvents, emulsifiers, colorants, binders, disintegrants, filling
agents, lubricants, wetting agents, osmotic pressure regulators,
stabilizers, glidants, flavoring agents, preservatives, suspending
agents, coating materials, aromatics, anti-adhesion agents,
antioxidants, chelating agents, penetration enhancers, pH
regulators, buffers, plasticizers, surfactants, foaming agents,
defoamers, thickeners, inclusion agents, humectants, absorbents,
diluents, flocculants and deflocculants, filter aids and release
retardants; and divided by administration routes into oral
administration, injection, mucosal, transdermal or local
administration, nasal or oral inhalation and ophthalmic
administration. The same pharmaceutical excipient can be used for
pharmaceutical preparations with different administration routes,
and has different effects and uses.
[0163] The pharmaceutical composition can be formulated into
various suitable dosage forms depending on administration routes,
such as tablets, capsules, granules, oral solutions, oral
suspensions, oral emulsions, powders, tinctures, syrups,
injections, suppositories, ointments, creams, pastes, ophthalmic
preparations, pills, subdermals, aerosols, powders and sprays. The
pharmaceutical composition or suitable dosage forms may contain
0.01 mg to 1000 mg of the compound of the disclosure or a
pharmaceutically acceptable salt or conjugate thereof, suitably 0.1
mg to 800 mg, preferably 0.5 to 500 mg, preferably 0.5 to 350 mg,
and particularly preferably 1 to 1-250 mg.
[0164] The pharmaceutical composition can be administered in the
form of injections, including liquids for injection, sterilized
powders for injection, and concentrated solutions for injection.
Acceptable carriers and solvents include water, Ringer's solution
and isotonic sodium chloride solution. In addition, sterilized
non-volatile oil can also be used as a solvent or suspending
medium, such as monoglyceride or diglyceride.
[0165] In the disclosure, the term "individual" include a human
individual or a non-human animal. Exemplary human individual
includes a human individual with a disease (e.g., a disease
described herein) (referred to as a patient) or a normal
individual. The term "non-human animal" in the disclosure includes
all vertebrates, such as a non-mammal (e.g., a bird, an amphibian
and a reptile) and a mammal, such as a non-human primate, a
domestic animal, and/or a domesticated animal (e.g., a sheep, a
dog, a cat, a cow and a pig).
[0166] In the disclosure, the term "effective amount" refers to the
amount of the compound that, after being administered, relieves one
or more symptoms of the treated disease to some extent.
[0167] In the disclosure, the term "conjugate" refers to a
substance obtained by linking a bioactive molecule with a targeting
moiety. In some embodiments of the disclosure, the bioactive
molecule is linked to the targeting moiety via a linker. The linker
can be cleaved in a specific environment (e.g., an intracellular
low pH environment) or under a specific action (e.g., the action of
lysosomal protease), thereby dissociating the bioactive molecule
from the target moiety. In some embodiments of the disclosure, the
linker comprises cleavable or non-cleavable units, such as a
peptide or disulfide bond. In some embodiments of the disclosure,
the bioactive molecule is linked directly to the targeting moiety
via a covalent bond that can be cleaved under a specific
environment or action, thereby dissociating the bioactive molecule
from the targeting moiety.
[0168] In the disclosure, the terms "bioactive substance" and
"bioactive molecule" refer to a substance that inhibits or prevents
cell functions and/or cause cell death or destruction. In some
embodiments of the disclosure, the bioactive substance or bioactive
molecule in the conjugate is a molecule with anti-tumor
bioactivity. For example: a radioisotope such as At.sup.211,
I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 or a radioisotope of Lu; a metal
complex such as a platinum metal complex, a gold metal complex or
oxaliplatin; a glycopeptide antibiotic such as bleomycin or
pingyangmycin; a DNA topoisomerase inhibitor such as a
topoisomerase I inhibitor, e.g., camptothecin, hydroxycamptothecin,
9-aminocamptothecin, SN-38, irinotecan, topotecan, bellotencian or
rubitecan, or a topoisomerase II inhibitor, e.g., actinomycin D,
adriamycin, doxorubicin, duocarmycin, daunorubicin, mitoxantrone,
podophyllotoxin, etoposide and so on; a drug interfering with DNA
synthesis, such as methotrexate, 5-fluorouracil, cytarabine,
gemcitabine, mercaptopurine, pentostatin, fludarabine, cladribine,
narabine and so on; a drug acting on a structural protein, such as
a tubulin inhibitor, a vinblastine alkaloid, a vincristine,
vinblastine, paclitaxel, docetaxel, cabazitaxel and so on; a tumor
cell signaling pathway inhibitor such as a serine/threonine kinase
inhibitor, a tyrosine kinase inhibitor, a aspartokinase inhibitor
or a histidine kinase inhibitor and so on; also includes a
proteasome inhibitor; a histone deaceylase inhibitor; a tumor
angiogenesis inhibitor; a cyclin inhibitor; a maytansine
derivative; a calicheamicin derivative; a auristatin derivative; a
Pyrrolobenzodiazepines (PBD) derivative; melphalan; mitomycin C;
chlorambucil; and other active substances which inhibit the growth
of tumor cells, promote the apoptosis or necrosis of tumor cells;
an enzymes and fragment thereof, such as karyolytic enzyme; an
antibiotic; a toxin such as a small molecule toxin or an
enzymatically active toxin originated from bacterium, fungus,
plants or animals, including fragment and/or variant thereof; a
growth inhibitor; and a drug module. The term "toxin" refers to a
substance that has deleterious effects on the growth or
proliferation of cells.
[0169] In the disclosure, the term "small molecule" refers to a
small molecule drug with bioactivity.
[0170] In the disclosure, the term "linker" refers to a fragment
linking a bioactive molecule with a targeting moiety.
[0171] In the disclosure, the term "targeting moiety" refers to a
moiety of the conjugate that can specifically bind to a target (or
a portion of the target) on the cell surface. The conjugate can be
delivered to a specific cell population by interaction between the
targeting moiety and the target.
[0172] In the disclosure, the conjugate can be referred to as a
"drug-antibody conjugate" when the targeting moiety of the
conjugate is an antibody. In the disclosure, the "drug-antibody
conjugate" and "immune conjugate" are interchangeable.
[0173] In the disclosure, the term "antibody" is interpreted in its
broadest sense, including a complete monoclonal antibody,
polyclonal antibody, and a multispecific antibody (e.g., a
bispecific antibody) formed from at least two complete antibodies,
provided that the antibody has required bioactivity. In the
disclosure, the terms "antibody" and "immunoglobulin" are
interchangeable.
[0174] In the disclosure, the term "monoclonal antibody" refers to
an antibody from a group of substantially uniform antibodies, i.e.,
antibodies that make up the group are identical except for a small
number of possible natural mutations. A monoclonal antibody has
high specificity for one determinant (epitope) of an antigen, while
a comparative polyclonal antibody contains different antibodies for
different determinants (epitopes). In addition to specificity, the
monoclonal antibody has the advantage of being free from
contamination by other antibodies during synthesis. The modifier
"monoclonal" here indicates that the antibody is characterized by
coming from a substantially uniform antibody group and should not
be construed as being prepared by a special method.
[0175] In some embodiments of the disclosure, the monoclonal
antibody also specifically includes a chimeric antibody, i.e., a
portion of a heavy chain and/or a light chain is the same as or
homologous to a type, a class, or a subclass of antibodies, while
the rest is the same as or homologous to another type, another
class, or another subclass of antibodies, provided the antibody has
required bioactivity (see, e.g., U.S. Pat. No. 4,816,567; and
Morrison et al., 1984, PNAS, 81: 6851-6855). The chimeric antibody
available in the disclosure includes a primatized antibody
containing a variable region antigen binding sequence from a
non-human primate (e.g., an ancient monkey or an orangutan) and a
human constant region sequence.
[0176] The term "antibody fragment" refers to a portion of the
antibody, preferably an antigen binding region or a variable
region. Examples of antibody fragment includes Fab, Fab', F(ab')2,
Fd, Fv, dAb and complementary determinant fragment, diabody, linear
antibody and single chain antibody molecule.
[0177] The term "bispecific antibody", also known as "bifunctional
antibody conjugate", refers to a conjugate formed by a first
antibody (fragment) and a second antibody (fragment) through a
coupling arm. The conjugate retains the activity of each antibody
and thus has bifunctional and bispecific properties.
[0178] The term "multispecific antibody" includes, for example, a
trispecific antibody which is an antibody having three different
antigen binding specificities, and a tetraspecific antibody which
is an antibody having four different antigen binding
specificities.
[0179] The term "complete antibody" refers to an antibody
containing an antigen binding variable region, a light chain
constant region (CL) and heavy chain constant regions (CH1, CH2 and
CH3). The constant regions can be natural sequences (e.g., human
natural constant region sequences) or amino acid sequence variants
thereof. The complete antibody is preferably a complete antibody
having one or more effector functions.
[0180] The term "probody" is a modified antibody comprising an
antibody or antibody fragment that can specifically bind to a
target thereof and can be coupled with a masked group, and the
masked group here refers that the cleavage constant for the binding
capacity of the antibody or antibody fragment to the target is at
least 100 times or 1000 times or 10000 times greater than that for
the binding capacity of an antibody or antibody fragment not
coupled with a masked group to a target thereof.
[0181] In the disclosure, a "humanized" form of a non-human (e.g.,
mouse) antibody refers to a chimeric antibody that contains minimal
non-human immunoglobulin sequences. Most of the humanized
antibodies are those in which residues in hypervariable regions of
human recipient immunoglobulins are substituted with residues in
non-human (e.g., mice, rats, rabbits or non-human primates)
hypervariable regions (donor antibodies) with required specificity,
affinity and functions. In some embodiments, residues in frame
regions (FRs) of human immunoglobulins are also substituted with
non-human residues. Furthermore, the humanized antibody can also
contain residues not present in recipient antibodies or donor
antibodies. Such modifications are made to further optimize
antibody performance. A humanized antibody generally contains at
least one variable region, typically two variable regions, in which
all or almost all hypervanable loops correspond to non-human
immunoglobulins, while all or almost all FRs are those of human
immunoglobulin sequences. A humanized antibody can also contain at
least a portion of an immunoglobulin constant region (Fc, usually
human immunoglobulin Fc). For details, see, for example, Jones et
al., 1986, Nature, 321: 522-525; Riechmann et al., 1988, Nature,
332: 323-329; and Presta, 1992, Curr Op Struct Bwl 2: 593-596.
[0182] Complete antibody can be classified into different "classes"
according to amino acid sequences of heavy chain constant regions.
The main five classes are IgA, IgD, IgE, IgG and IgM, and several
of which can also be further classified into different "subclasses"
(isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
Different classes of heavy chain constant regions of antibodies are
called .alpha., .beta., .epsilon., .gamma. and .mu. respectively.
Different classes of subunit structures and 3D configurations of
immunoglobulins are well known in the art.
[0183] In the disclosure, although amino acid substitutions in
antibodies are substituted with L-amino acids in most cases, the
embodiments are not limited thereto. In some embodiments, an
antibody peptide chain can contain one or more D-amino acids.
Peptides containing D-amino acids may be more stable and less
degradable in oral cavity, intestinal tract or plasma than peptides
containing only L-amino acids.
[0184] Monoclonal antibody used in the disclosure can be produced
by multiple methods. For example, the monoclonal antibody used in
the disclosure can be obtained by hybridoma methods using multiple
species (including cells of mice, hamsters, rats and humans) (see,
for example, Kohler et al., 1975, Nature, 256: 495), or by
recombinant DNA techniques (see, for example, U.S. Pat. No.
4,816,567), or isolated from phage antibody libraries (see, for
example, Clackson et al., 1991, Nature, 352: 624-628; and Marks et
al., 1991, Journal of Molecular Biology, 222: 581-597). Monoclonal
antibody that can be used in the disclosure includes, but is not
limited to anti-Her 2 monoclonal antibody such as Trastuzumab and
Pertuzumab, or anti-Trop-2 monoclonal antibody such as Sacituzumab
(i.e., Isactuzumab or hRS7 antibody), M1, M2 or M3.
[0185] In some preferred embodiments, a target of A is selected
from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII,
Mesothelin, Folate receptor1, Mucin 1, CD138, CD20, CD19, CD30,
SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelin receptor,
STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium
phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein,
AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L.sub.1, TACSTD2, DR5,
E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783,
STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20R.alpha.,
Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD22, CD79a, CXCR5,
HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, integrin
.alpha.5.beta.6, .alpha.4.beta.7, FGF2, FGFR2, Her3, CD70, CA6,
DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4,
ROR1, SLITRK6, 5T4, ENPP3, SLC39A6, Claudin18.2, BMPR1B, E16,
STEAP1, Tyrol, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783,
STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20R.alpha.,
Brevican, EphB2R, ASLG659, PSCA, GEDA, CD22, CD79a, CXCR5, HLA-DOB,
P2X5, CD72, LY64, FcRH1, IRTA2, c-Met, ApoE, CD1 lc, CD40, CD45
(PTPRC), CD49D (ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7,
PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, IL-6, ACTA2, COL7A1, LOX,
LRRC15, MCPT8, MMP10, NOG, SERPINE1, STAT1, TGFBR1, CTSS, PGF,
VEGFA, C1QA, C1QB, ANGPTL4, EGLN, ANGPTL4, EGLN3, BNIP3, AIF1,
CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3,
PGK1, PDK1, AKR1C1, AKR1C2, CADM1, CDH11, COL6A3, CTGF, HMOX1,
KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1,
TGFB2, CD1 lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2,
IGF-1R, RNF43, NaPi2b, BCMA and TENB2.
[0186] In some embodiments of the disclosure, the target of the
targeting moiety A is selected from a RGD peptide that recognizes
cell surface integrin receptor; a growth factor that recognizes
cell surface growth factor receptor, such as EGF, PDGF or VEGF; and
a peptide capable of recognizing functional cell surface
plasminogen activator, bombesin, bradykinin, somatostatin or
prostate-specific membrane antigen receptor.
[0187] In some embodiments of the disclosure, the target of the
targeting moiety A is selected from a CD40 ligand, a CD30 ligand,
an OX40 ligand, a PD-1 ligand, an ErbB ligand, a Her2 ligand, a
TACSTD2 ligand and a DR5 ligand.
[0188] In some embodiments of the disclosure, the targeting moiety
A is an anti-Her 2 monoclonal antibody, such as Trastuzumab or
Pertuzumab; or an anti-Trop-2 monoclonal antibody, such as
Sacituzumab, M1, M2 or M3.
[0189] In some embodiments of the disclosure, the targeting moiety
is Trastuzumab or Pertuzumab. Tratuzumab is an anti-Her 2
monoclonal antibody, an amino acid sequence thereof is known to a
person skilled in the art, for a schematic sequence thereof, refer
to, for example, CN103319599.
[0190] In some embodiments of the disclosure, terminal Lys of heavy
chains of the targeting moiety is easily deleted, but such deletion
does not affect bioactivity. See Dick, L. W. et al., Biotechnol.
Bioeng., 100: 1132-1143. For example, the targeting moiety is an
anti-Trop-2 monoclonal antibody, such as Sacituzumab, M1, M2 or M3
deleted terminal Lys of heavy chains, for example, the targeting
moiety is an anti-Her 2 monoclonal antibody, such as Trastuzumab or
Pertuzumab deleted terminal Lys of heavy chains.
[0191] Exemplary heavy and light chain sequences of Trastuzumab,
refer to SEQ ID No.: 17 and SEQ ID No.:18. In the disclosure, heavy
and light chain sequences of Trastuzumab referred to or involved
are described using the sequences shown in SEQ ID No.: 17 and SEQ
ID No.: 18, respectively. Exemplary heavy and light chain sequences
of Pertuzumab, refer to SEQ ID No.: 16 and SEQ ID No.: 15 of U.S.
Pat. No. 7,560,111.
TABLE-US-00010 (heavy chain sequence) SEQ ID No.: 17
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (K) (light chain
sequence) SEQ ID No.: 18
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0192] In some embodiments of the disclosure, the anti-Trop-2
antibody of the targeting moiety is RS7 (i.e., Sacituzumab of the
disclosure) described in U.S. Pat. No. 7,517,964; and hRS7 (i.e.,
Sacituzumab of the disclosure) described in US2012/0237518. The
anti-Trop-2 antibody available in the disclosure can also be
obtained by screening through carrier design, construction and
construction of an antibody library displaying antibodies as
disclosed in CN103476941A, or can be obtained by screening a
G-MAB.RTM. library of Sorrento Therapeutics, Inc.
[0193] For the heavy chain sequence and light chain amino acid
sequence of the monoclonal antibody Sacituzumab, refer to, for
example, SEQ ID No.: 19 and SEQ ID No.:20, respectively.
TABLE-US-00011 (heavy chain sequence) SEQ ID No.: 19
QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGW
INTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGG
FGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (K)
[0194] Terminal K (or lys) of heavy chains is easily deleted, but
such deletion does not affect bioactivity. See Dick, L. W. et al.,
Biotechnol. Bioeng., 100: 1132-1143.
TABLE-US-00012 (light chain sequence) SEQ ID No.: 20
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYS
ASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGA
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0195] In the disclosure, ErbB2 and Her2/neu are interchangeable,
both of which represent human Her2 proteins of natural sequences
(Genebank CAS No.: X03363, see, for example, Semba et al., 1985,
PNAS, 82: 6497-6501; and Yamamoto et al., 1986, Nature, 319:
230-234) and functional derivatives thereof, such as amino acid
sequence variants. ErbB2 represents a gene encoding human Her2 and
neu represents a gene encoding rat p185neu. In some embodiments,
the compound or conjugate of the disclosure can inhibit or kill
cells that express ErbB2 receptors, such as breast cancer cells,
ovarian cancer cells, gastric cancer cells, endometrial cancer
cells, salivary gland cancer cells, lung cancer cells, kidney
cancer cells, colon cancer cells, thyroid cancer cells, pancreatic
cancer cells, bladder cancer cells or liver cancer cells.
[0196] In the disclosure, Trop-2 or TROP2 refers to human
trophoblast cell-surface antigen 2, also known as TACSTD2, M1S1,
GA733-1, EGP-1, which is a cell surface receptor expressed in many
human tumors (e.g., breast cancer, colorectal cancer, lung cancer,
pancreatic cancer, ovarian cancer, prostate cancer and cervical
cancer). In some embodiments, the compound or conjugates of the
disclosure can inhibit or kill cells that express TROP2 receptors,
such as breast cancer cells, colorectal cancer cells, lung cancer
cells, pancreatic cancer cells, ovarian cancer cells, prostate
cancer cells or cervical cancer cells.
[0197] As used herein,
##STR00163##
contained in the conjugate of the invention indicates a specific
linking mode of a sulfhydryl group and a linker in the antibody
when the targeting moiety is an antibody.
[0198] As used herein, the term "C.sub.1-6 alkyl" refers to linear
or branched alkyl containing 1-6 carbon atoms, including, for
example, "C.sub.1-4 alkyl" and "C.sub.1-3 alkyl". Specific examples
include, but are not limited to methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl,
2-methyl butyl, neopentyl, 1-ethyl propyl, n-hexyl, isohexyl,
3-methyl pentyl, 2-methyl pentyl, 1-methyl pentyl, 3,3-dimethyl
butyl, 2,2-dimethyl butyl, 1,1-dimethyl butyl, 1,2-dimethyl butyl,
1,3-dimethyl butyl, 2,3-dimethyl butyl, 2-ethyl butyl and
1,2-dimethyl propyl.
[0199] As used herein, the term "C.sub.2-6 alkenyl" refers to
linear, branched or cyclic alkenyl containing at least a double
bond and 2-6 carbon atoms, including, for example, "C.sub.2-4
alkenyl". Examples thereof include, but are not limited to vinyl,
1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl,
1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl,
1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl,
cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl and
1,4-cyclohexadienyl.
[0200] As used herein, the term "C.sub.2-6 alkynyl" refers to
linear or branched alkynyl containing at least a triple bond and
2-6 carbon atoms, including, for example, "C.sub.2-4 alkynyl".
Examples thereof include, but are not limited to ethynyl, propynyl,
2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl,
3-hexynyl and 5-methyl-2-hexynyl.
[0201] As used herein, the term "halogen" includes fluorine,
chlorine, bromine and iodine.
[0202] As used herein, the terms "3-8 membered cycloalkyl" or
"C.sub.3-8 cycloalkyl" refers to saturated cyclic alkyl containing
3-8 carbon atoms, including, for example, "3-6 membered
cycloalkyl", "4-6 membered cycloalkyl", "5-7 membered cycloalkyl"
or "5-6 membered cycloalkyl". Specific examples include, but are
not limited to cyclopropanyl, cyclobutylalkyl, cyclopentanyl,
cyclohexyl, cycloheptyl and cyclooctadecyl.
[0203] As used herein, the term "C.sub.1-6 alkoxy" refers to a
group having a structure of C.sub.1-6 alkyl-O--, wherein C.sub.1-6
alkyl is as defined previously. Specific examples include, but are
not limited to methoxy, ethoxy, propoxy, isopropoxy, n-propoxy,
isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy and
hexyloxy.
[0204] As used herein, the term "3-8 membered aliphatic
heterocyclyl" refers to a cyclic group containing 3-8 ring-forming
atoms (at least one of which is a heteroatom, such as a nitrogen
atom, an oxygen atom or a sulfur atom). Optionally, the
ring-forming atoms (e.g., carbon atoms, nitrogen atoms or sulfur
atoms) in the cyclic structure can be substituted with oxygen. The
"3-8 membered aliphatic heterocyclyl" includes, for example, "3-8
membered nitrogen-containing aliphatic heterocyclyl," "3-8 membered
oxygen-containing aliphatic heterocyclyl," "3-6 membered aliphatic
heterocyclyl," "3-6 membered oxygen-containing aliphatic
heterocyclyl," "4-7 membered aliphatic heterocyclyl," "4-6 membered
aliphatic heterocyclyl," "5-7 membered aliphatic heterocyclyl,"
"5-6 membered aliphatic heterocyclyl," "5-6 membered
nitrogen-containing aliphatic heterocyclyl," including but not
limited to oxiranyl, oxocyclobutyl, pyrrolidinyl,
tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl and
homopiperazinyl.
[0205] As used herein, the term "6-12 membered spirocyclyl" refers
to a cyclic structure containing 6-12 ring-forming carbon atoms and
formed by two or more cyclic structures sharing one carbon atom.
Optionally, the carbon atoms in the cyclic structure can be
substituted with oxygen. The "6-12 membered spirocyclyl" includes,
for example, "6-11 membered spirocyclyl", "6-10 membered
spirocyclyl", "7-10 membered spirocyclyl", "7-9 membered
spirocyclyl", "7-8 membered spirocyclyl", "9-10 membered
spirocyclyl" and "3-10 membered spirocyclyl". Specific examples
include, but are not limited to
##STR00164##
[0206] As used herein, the term "6-12 membered bridged cyclyl"
refers to a cyclic structure containing 6-12 ring-forming carbon
atoms and formed by two or more cyclic structures sharing two
nonadjacent carbon atoms, Optionally, the carbon atoms in the
cyclic structure can be substituted with oxygen. The "6-12 membered
bridged cyclyl" includes, for example, "6-11 membered bridged
cyclyl", "5-10 membered bridged cyclyl", "7-10 membered bridged
cyclyl", "7-9 membered bridged cyclyl", "7-8 membered bridged
cyclyl", "9-10 membered bridged cyclyl" and "3-10 membered bridged
cyclyl". Specific examples include, but are not limited to
##STR00165##
[0207] As used herein, the term "6-12 membered fused cyclyl" refers
to a cyclic structure containing 6-12 ring-forming carbon atoms and
formed by two or more cyclic structures sharing two adjacent atoms,
including "6-11 membered fused cyclyl", "6-10 membered fused
cyclyl", "6-8 membered fused cyclyl", "10-12 membered fused
cyclyl", "7-10 membered fused cyclyl". Examples thereof include,
but are not limited to
##STR00166##
[0208] As used herein, the term "6-12 membered spiroheterocyclyl"
refers to a cyclic structure containing 6-12 ring-forming atoms (at
least one of which is a heteroatom, such as a nitrogen atom, an
oxygen atom or a sulfur atom) and formed by two or more cyclic
structures sharing one ring-forming atom. Optionally, the
ring-forming atoms (e.g., carbon atoms, nitrogen atoms or sulfur
atoms) in the cyclic structure can be substituted with oxygen. The
"6-12 membered spiroheterocyclyl" includes, for example, "6-11
membered spiroheterocyclyl", "5-10 membered spiroheterocyclyl",
"7-11 membered spiroheterocyclyl", "7-10 membered
spiroheterocyclyl", "7-9 membered spiroheterocyclyl", "7-8 membered
spiroheterocyclyl", "9-10 membered spiroheterocyclyl" and "3-10
membered spiroheterocyclyl". Specific examples include, but are not
limited to
##STR00167##
[0209] As used herein, the term "6-12 membered bridged
heterocyclyl" refers to a cyclic structure containing 6-12
ring-forming atoms (at least one of which is a heteroatom, such as
a nitrogen atom, an oxygen atom or a sulfur atom) and formed by two
or more cyclic structures sharing two nonadjacent ring-forming
atoms. Optionally, the ring-forming atoms (e.g., carbon atoms,
nitrogen atoms or sulfur atoms) in the cyclic structure can be
substituted with oxygen. The "6-12 membered bridged heterocyclyl"
includes, for example, "6-11 membered bridged heterocyclyl", "6-9
membered bridged heterocyclyl", "6-10 membered bridged
heterocyclyl", "7-10 membered bridged heterocyclyl", "7-9 membered
bridged heterocyclyl", "7-8 membered bridged heterocyclyl", "8
membered bridged heterocyclyl", "9-10 membered bridged
heterocyclyl" and "3-10 membered bridged heterocyclyl". Specific
examples include, but are not limited to
##STR00168##
[0210] As used herein, the term "6-12 membered fused heterocyclyl"
refers to a cyclic structure containing 6-12 ring-forming atoms (at
least one of which is a heteroatom, such as a nitrogen atom, an
oxygen atom or a sulfur atom) and formed by two or more cyclic
structures sharing two adjacent atoms. Optionally, the ring-forming
atoms (e.g., carbon atoms, nitrogen atoms or sulfur atoms) in the
cyclic structure can be substituted with oxygen. The "6-12 membered
fused heterocyclyl" includes, for example, "6-11 membered fused
heterocyclyl," "5-10 membered fused heterocyclyl", "7-10 membered
fused heterocyclyl", "3-10 membered fused heterocyclyl", "3-10
membered nitrogen-containing fused heterocyclyl", "9-10 membered
fused heterocyclyl", "9-10 membered nitrogen-containing fused
heterocyclyl" and "6-12 membered oxygen-containing fused
heterocyclyl". Specific examples include, but are not limited to
tetrahydroimidazo [4,5-c]pyridyl, 3,4-dihydroquinazolinyl,
1,2-dihydroquinoxalinyl, benzo[d][1,3]dioxolyl,
1,3-dihydroisobenzofuranyl, 4H-1,3-benzoxazinyl,
4,6-dihydro-1H-furo[3,4-d]imidazolyl,
3a,4,6,6a-tetrahydro-1H-furo[3,4-d]imidazolyl,
4,6-dihydro-1H-thieno[3,4-d]imidazolyl, 4,6-dihydro-1H-pyrrolo
[3,4-d]imidazolyl, benzoimidazolidinyl,
octahydro-benzo[d]imidazolyl, decahydroquinolyl,
hexahydrothienoimidazolyl, hexahydrofuroimidazolyl,
4,5,6,7-tetrahydro-1H-benzo[d]imidazolyl,
octahydrocyclopenteno[c]pyrrolyl, dihydroindolyl,
dihydroisoindolyl, benzooxazolidinyl, benzothiazolidinyl,
1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl and
4H-1,3-benzoxazinyl.
[0211] As used herein, the term "aryl" refers to a monocyclic or
polycyclic hydrocarbonyl having aromaticity, such as 6-20 membered
aryl, 6-10 membered aryl and 5-8 membered aryl. Specific examples
include but are not limited to phenyl, naphthyl, anthracenyl and
phenanthryl. The "6-20 membered aryl" refers to aryl containing
6-20 ring-forming atoms.
[0212] As used herein, the term "heteroaryl" refers to a cyclic
group having aromaticity, wherein at least one ring-forming atom is
a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur
atom. Optionally, the ring-forming atoms (e.g., carbon atoms,
nitrogen atoms or sulfur atoms) in the cyclic structure can be
substituted with oxygen. Specific examples include, but are not
limited to 5-10 membered heteroaryl, 5-10 membered
nitrogen-containing heteroaryl, 6-10 membered oxygen-containing
heteroaryl, 6-8 membered nitrogen-containing heteroaryl and 5-8
membered oxygen-containing heteroaryl, such as furyl, thienyl,
pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl,
isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl,
1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridone,
4-pyridone, pyrimidinyl, 1,4-dioxacyclohexadienyl, 2H-1,2-oxazinyl,
4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl,
4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl,
1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl,
1,3-diazcycloheptatrienyl and azacyclooctatetraenyl.
Beneficial Effects of the Invention
[0213] The disclosure obtains a kind of novel bioactive molecule
conjugate by improving the coupling way of drugs and targeting
moieties in ADCs or SMDCs. In some embodiments of the disclosure,
the bioactive molecule conjugate is obtained through nucleophilic
substitutions of the heteroaryl ring on the ADC linker by the free
sulfhydryl in the antibody. The conjugate obtained by the coupling
can achieve at least one of the following technical effects:
[0214] (1) high stability;
[0215] (2) high DAR, the DAR values of the conjugate can reach 5-8
in some embodiments;
[0216] (3) extremely high coupling efficiency, the coupling
efficiency can reach 90% in some embodiments;
[0217] (4) the conjugate obtained by the coupling can effectively
improve the stability of the drug in the circulation and reduce
unexpected dissociation of the drug in non-target cells;
[0218] (5) the conjugate can also increase effective release of the
bioactive molecule in cells to attain the purposes of decreasing
toxicity and increasing efficacy;
[0219] (6) the conjugate has good tumor tissue targetability;
and
[0220] (7) the conjugate has good efficacy on animal models of
tumors.
[0221] In addition, the coupling method described in the disclosure
has broad application scope and can be widely used in coupling
bioactive molecules with antibodies or targeted small molecule
ligands.
BRIEF DESCRIPTION OF THE DRAWINGS
[0222] FIG. 1 shows a TIC (total ion chromatogram) of BT001002.
[0223] FIG. 2 shows a deconvolution diagram of a coupled light
chain of BT001002.
[0224] FIG. 3 shows a deconvolution diagram of a coupled heavy
chain of BT001002.
[0225] FIG. 4 shows a TIC (total ion chromatogram) of BT001004.
[0226] FIG. 5 shows a deconvolution diagram of a coupled light
chain of BT001004.
[0227] FIG. 6 shows a deconvolution diagram of a coupled heavy
chain of BT001004.
[0228] FIG. 7 shows a SEC chromatogram of BT001002.
[0229] FIG. 8 shows a SEC chromatogram of molecular weight Marker
of BT001002.
[0230] FIG. 9 shows a SEC chromatogram of BT001004.
[0231] FIG. 10 shows a deconvolution diagram of a coupled light
chain of BT001012.
[0232] FIG. 11 shows a deconvolution diagram of a coupled heavy
chain of BT001012.
[0233] FIG. 12 shows a deconvolution diagram of a coupled light
chain of BT001013.
[0234] FIG. 13 shows a deconvolution diagram of a coupled heavy
chain of BT001013.
[0235] FIG. 14 shows a deconvolution diagram of a coupled light
chain of BT001018.
[0236] FIG. 15 shows a deconvolution diagram of a coupled heavy
chain of BT001018.
[0237] FIG. 16 shows a deconvolution diagram of a coupled light
chain of BT001021.
[0238] FIG. 17 shows a deconvolution diagram of a coupled heavy
chain of BT001021.
[0239] FIG. 18 shows a deconvolution diagram of a coupled light
chain of BT001023.
[0240] FIG. 19 shows a deconvolution diagram of a coupled heavy
chain of BT001023.
[0241] FIG. 20 shows a deconvolution diagram of a coupled light
chain of BT001040.
[0242] FIG. 21 shows a deconvolution diagram of a coupled heavy
chain of BT001040.
[0243] FIG. 22 shows a deconvolution diagram of a coupled light
chain of BT001041.
[0244] FIG. 23 shows a deconvolution diagram of a coupled heavy
chain of BT001041.
[0245] FIG. 24 shows a deconvolution diagram of a coupled light
chain of BT001042.
[0246] FIG. 25 shows a deconvolution diagram of a coupled heavy
chain of BT001042.
[0247] FIG. 26 shows a deconvolution diagram of a coupled light
chain of BT001043.
[0248] FIG. 27 shows a deconvolution diagram of a coupled heavy
chain of BT001043.
[0249] FIG. 28 shows a deconvolution diagram of a coupled light
chain of BT001044.
[0250] FIG. 29 shows a deconvolution diagram of a coupled heavy
chain of BT001044.
[0251] FIG. 30 shows a deconvolution diagram of a coupled light
chain of BT001046.
[0252] FIG. 31 shows a deconvolution diagram of a coupled heavy
chain of BT001046.
[0253] FIG. 32 shows a deconvolution diagram of a coupled light
chain of BT001047.
[0254] FIG. 33 shows a deconvolution diagram of a coupled heavy
chain of BT001047.
[0255] FIG. 34 shows a SEC chromatogram of BT001012.
[0256] FIG. 35 shows a SEC chromatogram of BT001013.
[0257] FIG. 36 shows a SEC chromatogram of BT001018.
[0258] FIG. 37 shows a SEC chromatogram of BT001021.
[0259] FIG. 38 shows a SEC chromatogram of BT001023.
[0260] FIG. 39 shows a SEC chromatogram of BT001042.
[0261] FIG. 40 shows a SEC chromatogram of BT001043.
[0262] FIG. 41 shows a SEC chromatogram of BT001044.
[0263] FIG. 42 shows a SEC chromatogram of BT001046.
[0264] FIG. 43 shows a SEC chromatogram of BT001047.
[0265] FIG. 44 shows changes in growth of tumor volume of each
group of mice in a NCI-N87 human gastric cancer model.
[0266] FIG. 45 shows changes in body weight of each group of mice
in a NCI-N87 human gastric cancer model.
[0267] FIG. 46 shows changes in growth of tumor volume of each
group of mice in an HCC1806 human breast cancer model.
[0268] 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.
[0269] FIG. 47B shows changes in body weight of each group of mice
in a xenograft model of HCC827 human non-small cell lung
cancer.
[0270] FIG. 48A shows changes in growth of tumor volume of each
group of mice in a xenograft model of NCI-N87 human gastric
cancer.
[0271] FIG. 48B shows changes in body weight of each group of mice
in a xenograft model of NCI-N87 human gastric cancer.
[0272] 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.
[0273] 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
[0274] 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.
[0275] Abbreviations in the invention have the following
meanings:
TABLE-US-00013 OMs methylsulfonyloxy FA Formic acid OTs
Trifluoromethyl- ACN Acetonitrile sulfonyloxy CCK8
2-(2-methoxy-4-nitrophenyl)- OTf p-toluenesulfonyloxy reagent
3-(4-nitropheny1)-5- TBS Tert-butyldimethylsilyl
(2,4-disulfophenyl)-2H- MMT p-methoxytriphenyl- tetrazole
monosodium salt methyl FBS Fetal bovine serum PB/PBS Phosphate
buffered DMSO Dimethyl sulfoxide saline
[0276] Preparation Solutions
[0277] The structures of compounds described in the following
examples were determined by nuclear magnetic resonance (.sup.1H
NMR) or mass spectrometry (MS).
[0278] 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.
[0279] Abbreviations in nuclear magnetic resonance (NMR) spectra
used in the examples were shown below.
[0280] 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. .delta. value was
expressed in ppm.
[0281] Mass spectra (MS) were determined using Agilent (ESI) mass
spectrometer (model: Agilent 6120B).
[0282] Preparative Liquid Chromatography:
[0283] Method A:
[0284] Chromatographic column: Daisogel C18 10 .mu.m 100.times.250
mm
[0285] Mobile phase A: water; Mobile phase B: acetonitrile
TABLE-US-00014 Time Mobile phase A Mobile phase B Flow rate [min]
[%] [%] [mL/min] 0.00 70.0 30.0 300.0 8.00 70.0 30.0 50.00 20.0
80.0
[0286] Method B:
[0287] Chromatographic column: Daisogel C18 10 .mu.m 50.times.250
mm
[0288] Mobile phase A: water; Mobile phase B: acetonitrile
TABLE-US-00015 Time Mobile phase A Mobile phase B Flow rate [min]
[%] [%] [mL/min] 0.00 70.0 30.0 80.0 8.00 70.0 30.0 50.00 20.0
80.0
[0289] Method C:
[0290] Chromatographic column: Daisogel C18 10 .mu.m 50.times.250
mm
[0291] Mobile phase A: water containing 0.1% trifluoroacetic acid;
Mobile phase B: acetonitrile
TABLE-US-00016 Time Mobile phase A Mobile phase B Flow rate [min]
[%] [%] [mL/min] 0.00 90.0 10.0 80.0 10.00 90.0 10.0 50.00 60.0
40.0
[0292] Method D: chromatographic column: Waters SunFire C18 5 .mu.m
19.times.250 mm
[0293] Mobile phase A: acetonitrile; Mobile phase B: water
containing 0.05% formic acid
[0294] Time: 0 min-16 min; Mobile phase A: 10%-90%; Flow rate: 28
mL/min
[0295] 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-methylbu-
tyrylamino)-N,3-dimethylbutyramide (T001)
##STR00169##
[0296] Step 1: Synthesis of Tert-Butyl
(4-((2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-me-
thylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenyl-
propionamido)methyl)phenyl) carbamate
[0297] At room temperature, 1-hydroxybenzotriazole (2.0 mg, 14.74
.mu.mol) was dissolved in N,N-dimethylformamide (4 mL), cooled to
0.degree. C., and then tert-butyl 4-methylaminobenzyl carbamate
(4.0 mg, 16.1 .mu.mol), N,N-diisopropylethylamine (8.5 mg, 66.8
.mu.mol), ((2R,
3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyryla-
mino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-y-
l)-3-methoxy-2-methylpropionyl)-L-phenylalanine (10.0 mg, 13.5
.mu.mol, commercially available) were successively added. After
being stirred for 5 min,
1H-benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (10.0 mg, 20.1 .mu.mol) was added thereto, and
stirred at 0.degree. 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.+.
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-methylbu-
tyrylamino)-N,3-dimethylbutanamide
[0298] At room temperature, tert-butyl
(4-((2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-me-
thylbutyrylamino)-N,3-dimethylbutyryl)-3-methoxy-5-methylheptanoyl)pyrroli-
din-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.degree. 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-(dimethyla-
mino)-3-methylbutyrylamino)-N,3-dimethylbutyramide (T011)
##STR00170##
[0299] Step 1: Synthesis of Tert-Buty
(S)-(4-((2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropionam-
ido) methyl)phenyl) 1 Carbamate
[0300] At 0.degree. 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.degree. 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.times.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.+.
Step 2: Synthesis of Tert-Butyl
(S)-(4-((2-amino-3-phenylpropionamido) methyl)phenyl) carbamate
[0301] 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-phenylpropionami-
do)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.times.4). The organic phases were combined, washed
with saturated saline (30 mL.times.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.+.
Step 3: Synthesis of
(4-((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-3-((methylamino)-3-methylb-
utyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrroli-
din-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionamido)methyl)phen-
yl)carbamate
[0302] At 0.degree. 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-methylbuty-
rylamino)-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.degree. 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.+.
Step 4: Synthesis of
(S)--N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((S)-1-((4-aminobenzyl)amino)-1-ox-
o-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-methylbut-
yrylamino)-N,3-dimethylbutyramide
[0303] 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-methylbut-
yrylamino)-N,
3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-me-
thoxy-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.+.
[0304] The following molecules can be synthesized by a similar
synthetic method:
TABLE-US-00017 Name Structure ESI-MS T012 ##STR00171## 850.6 T013
##STR00172## 864.6 T015 ##STR00173## 760.6 T021 ##STR00174##
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
##STR00175##
[0306] 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 1 h. The organic phase was washed with water (10
mL.times.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 (1g).
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-isopropylmethanesulfonam-
ide
##STR00176##
[0308] 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).
[0309] Structural characterization data are as follows:
[0310] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 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.+.
[.alpha.].sub.D.sup.20 is +28.19.degree. (c=0.101 g/100 mL,
CH.sub.3CN).
[0311] 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-me-
thoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-
-phenylpropionamido)methyl)phenyl)-5-ureidovaleramide
##STR00177## ##STR00178## ##STR00179##
[0312] Step 1: Synthesis of Tert-Butyl
4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl) butyrate (Compound
1-2)
[0313] 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.times.3). Then organic phases
were combined, washed with saturated saline solution (50
mL.times.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.+.
Step 2: Synthesis of
4-(4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric Acid (Compound
1-3)
[0314] 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.+.
[0315] Step 3: Synthesis 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)
[0316] 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.degree. 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.+.
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)
[0317] 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.+.
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)
[0318] 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.degree. 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.+.
Step 6: Synthesis of
(9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((R)-2-amino-3-phenylpropion-
amido)methyl)phenyl)amino)-1-oxo-5-ureidopentyl-2-yl)amino)-3-methyl-1-oxo-
butyl-2-yl)-carbamate (Compound 1-8)
[0319] 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.+.
Step 7: Synthesis of
(9H-fluoren-9-yl)-methyl-((S)-1-(((S)-1-((4-(((S)-2-((2R,3R)-3-((S)-1-((3-
R,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)
[0320] At room temperature,
(2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-(dimethylamino)-3-butyrylamino-
)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl)-3-meth-
oxy-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.degree. 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.degree. 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.+.
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-dimethylbutyrylam-
ino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl)-3-methoxy-2-methylpropionamid-
o)-3-phenylpropionamido)methyl)phenyl)-5-ureidovaleramide (Compound
1-10)
[0321] 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.+.
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-me-
thoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-
-phenylpropionamido)methyl)phenyl)-5-ureidovaleramide (Compound
TL001)
[0322] 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.degree. 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 purification 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-phen-
ylpropionamido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H--
pyrrolo
[2,3-d]pyrimidin-7-yl)-butyrylamido)-butyrylamido)-5-ureidovaleram-
ide
##STR00180##
[0323] Step 1: Synthesis of
4-(4-(methylthio)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)butyric Acid
(Compound 2-2)
[0324] 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.degree. 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.+.
Step 2: Synthesis of
(S)--N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethyl-
amino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylh-
eptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropiona-
mido)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)
[0325] 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.+.
Step 3: Synthesis of
(S)--N-(4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(dimethyl-
amino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylh-
eptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropiona-
mido)methyl)phenyl)-2-((S)-3-methyl-2-(4-(4-(methylsulfonyl)-7H-pyrrolo
[2,3-d]pyrimidin-7-yl)-butyrylamido)-butyrylamido)-5-ureidovaleramide
(Compound TL002)
[0326] 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-((3R,4S,5S)-4-(-
(S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyrylamin-
o)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropiona-
mido-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-hexynamid-
e
##STR00181##
[0327] Step 1: Synthesis of methyl
6-(2-(methylthio)pyrimidin-5-yl)-5-hexynoate (Compound 3-2)
[0328] 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.degree. C. under nitrogen
protection and reacted for 6 h under stirring, quenched with water,
and extracted with ethyl acetate (20 mL.times.3). Organic phases
were combined, washed with saturated saline (20 mL.times.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.+.
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.times.2). The aqueous phase was adjusted to pH=3 with 1N
hydrochloric acid, and extracted with ethyl acetate (20
mL.times.3), then organic phases were combined, washed with
saturated saline (20 mL.times.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).
Step 3: Synthesis of
6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynoic Acid (Compound
3-4)
[0330] 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.+.
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-methylpropionamid-
o)-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)
[0331] 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-di-
oxo-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-ureido-
propyl)-6,12,15,18,21,24,27,30,33-nonoxy-3,9,36-azatetracosan-41-amido)ben-
zyl)carbonate
##STR00182## ##STR00183##
[0332] Step 1: Synthesis of methyl
(S)-(1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)-(9H-fl-
uorenyl) carbamate (Compound 19-2)
[0333] 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.degree. 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.+.
Step 2: Synthesis of
(S)-2-amino-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide
(Compound 19-3)
[0334] 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.+.
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)
[0335] 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.degree. 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.+.
Step 4: Synthesis of
4-((S)-35-azido-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-no-
noxy-3,9-azatetracosane)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfony-
lamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizi-
no[1,2-b]quinolin-4-yl)carbonate (Compound 19-5)
[0336] 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-isopropylmethanesulfonami-
de (200 mg, 0.39 mmol) was dissolved in dichloromethane (10 mL) and
cooled to 0.degree. 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.degree. 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 performed 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.+.
Step 5: Synthesis of
4-((S)-35-amino-4,8-dioxo-2-(3-ureidopropyl)-6,12,15,18,21,24,27,30,33-no-
noxy-3,9-azatetracosane)benzyl((S)-4-ethyl-11-(2-(N-isopropylmethylsulfony-
lamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizi-
no[1,2-b]quinolin-4-yl)carbonate (Compound 19-6)
[0337] At room temperature, compound 19-5 (80 mg, 0.059 mmol) was
dissolved in tetrahydrofuran (1.0 ml) and cooled to 0.degree. 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.+.
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)
[0338] 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.degree. 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-di-
oxo-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-4-y-
l-(4-((S)-2-((S)-3-methyl-2-(6-(2-(methylsulfonyl)pyrimidin-5-yl))-5-hexyn-
amido)butyramido-5-ureidovalerylamido)benzyl)carbonate
##STR00184##
[0339] Step 1: methyl
((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidovalerylamid-
o-2-yl)amino))-3-methyl-butyramido-2-yl)-(9H-fluorenyl)
carbamate
[0340] 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.+.
Step 2: Synthesis of
(S)-2-((S)-2-amino-3-methylbutyramido)-N-(4-(hydroxymethyl)phenyl)-5-urei-
dovaleramide (Compound 28-2)
[0341] 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.+.
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-h-
exynamide (Compound 28-4)
[0342] 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.+.
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)buty-
ramido-5-ureidovalerylamido)benzyl)carbonate (Compound TL028)
[0343] 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,5-
S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)m-
ethyl)-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
##STR00185##
[0344] Step 1: Synthesis of
(S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonox-
y-3,9-azatetracosyl)-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleramide
(Compound 29-1)
[0345] 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.+.
Step 2: 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-triazatritriacontylamido)benzyl((S)-4-ethy-
l-11-(2-(N-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahyd-
ro-1H-pyrano[3',4',6,7]indolizino[1,2-b]quinolin-4-yl)carbonate
(Compound 29-2)
[0346] 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.+.
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,5-
S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hexynamido)m-
ethyl)-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)
[0347] 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,5-
S)-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-tetraazapentat-
etracontane-44-carbamoyl)benzyl)carbonate
##STR00186##
[0348] Step 1: Synthesis of
(S)-2-((S)-35-amino-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonox-
y-3,9-diazapentatriacontamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidovaleram-
ide
[0349] At 20.degree. 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.degree. 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.+.
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-tetraazadotet-
racont-42-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide
[0350] At 20.degree. 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).
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,5-
S)-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-tetraazapentat-
etracontane-44-carbamoyl)benzyl)carbonate (compound TL022)
[0351] At 20.degree. 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-isopropylmethanesulfonami-
de (70 mg, 0.14 mmol) was dissolved in dichloromethane (4.0 mL) and
cooled to 0.degree. 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.degree. 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.degree. 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-trio-
xo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkyn-
amido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)ethyl)-3,14-dio-
xo-3,4,12,14-tetrahydro-1H-pyrone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl-
)carbonate
##STR00187##
[0352] 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)pyrim-
idin-5-yl)-4,8,37-trioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazado-
tetracontyl-41-alkynamido)benzylcarbonate
[0353] 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 1 h 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.+.
Step 2: Synthesis of
4-((S)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trio-
xo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkyn-
amido)benzyl-((S)-4-ethyl-11-(2-(N-isopropylmethylsulfonyl)ethyl)-3,14-dio-
xo-3,4,12,14-tetrahydro-1H-pyrone[3',4':6,7]indolizino[1,2-b]quinolin-4-yl-
)carbonate (Compound TL024)
[0354] 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
##STR00188##
[0355] Step 1: Preparation of
(S)-(9H-fluoren-9-yl)-methyl(1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-
yl-2-yl)carbamate
[0356] 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.+
Step 2: Preparation of
(S)-2-amino-N-(4-(hydroxymethyl)phenyl)propionamide
[0357] 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]+
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
[0358] 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]+
Step 4: Preparation of
(S)-2-amino-N--((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxoprop-2-yl)-3--
methylbutanamide
[0359] 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.+
Step 5: preparation of
(S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-diazapentatriacon-
tamido)-N--((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-met-
hylbutyramide
[0360] 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.+
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-isopropylmethyl
sulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]-
indolizino[1,2-b]quinolin-4-yl)carbonate
[0361] Under nitrogen protection and at 0.degree. 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[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethan-
esulfonamide (45 mg, 0.09 mmol) in dichloromethane (0.7 mL) and
reacted at 0.degree. 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.degree. 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.+
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)
[0362] 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-6,12,15,18,21,24,27,30,3-
3-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropy-
lmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':
6,7]indolizino[1,2-b]quinolin-4-yl)carbonate
##STR00189## ##STR00190##
[0363] Step 1: Synthesis of
6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide
[0364] At 25.degree. 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.+.
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-te-
trahydro-2H-pyrano[2,3-b]-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4--
yl)carbonate
[0365] At 25.degree. C. and under nitrogen protection, T-030 (250
mg, 0.49 mmol) was dissolved in dichloromethane (10 mL) and cooled
to 0.degree. 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.degree. 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)acetamid-
e (518 mg, 0.49 mmol) in dichloromethane (7 mL) was added and
reacted at 0.degree. 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.+.
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-(methylsul-
fonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-diox-
o-6,12,15,18,21,24,27,30,33-nonoxy-3,9-diazapentatriacontamido)benzyl)carb-
onate
[0366] 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.+.
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-6,12,15,18,21,24,27,30,3-
3-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropy-
lmethylsulfonamido)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)
[0367] 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:
[0368] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 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.+. [.alpha.].sub.D.sup.20 is -19.55.degree.
(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-6,12,15,18,21,24,27,30,3-
3-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
##STR00191##
[0369] Step 1: Synthesis of
4-((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-d-
ioxo
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-pyr-
ano[3',4': 6,7]indolizino[1,2-b]quinolin-4-yl)carbonate
[0370] 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.degree. 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.degree. C. for 1 h
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)acetamid-
e (160 mg, 0.15 .mu.mol) in dry dichloromethane (0.5 mL) was added
to the reaction solution and reacted at room temperature for 1 h.
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.+.
Step 2: Synthesis of
(S)-9-(tert-butyldimethylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14-tet-
rahydro-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
[0371] 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 1 h 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].
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-6,12,15,18,21,24,27,30,3-
3-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)
[0372] 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-6,12,15,18,21,24,27,30,3-
3-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropy-
lmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':-
6,7]indolizino[1,2-b]quinolin-4-yl)carbonate
##STR00192##
[0373] Step 1: Synthesis of
((S)-35-azido-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dio-
xo-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-tetrahydr-
o-1,2,3,6-triazacycloheptane-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-
-4-yl)carbonate
[0374] 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.+.
Step 2: Synthesis of
(S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo3,4,12,14-tetrah-
ydro-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)pyri-
midin-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
[0375] 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].
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-6,12,15,18,21,24,27,30,3-
3-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropy-
lacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indo-
lizino[1,2-b]quinolin-4-yl)carbonate (Compound TL035)
[0376] 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-tr-
ioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadotetracontyl-41-alken-
amido)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)carb-
onate
##STR00193##
[0377] Step 1: synthesis of
(Z)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-enoic Acid
[0378] At 20.degree. 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.degree. 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.+.
Step 2: Synthesis of
(S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetra-
hydro-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-triazadotetraco-
ntyl-41-alkenamido)benzylcarbonate
[0379] 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. Purification 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.+.
Step 3: Synthesis of
4-((S,Z)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-tr-
ioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alk-
enamido)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)ca-
rbonate (Compound TL045)
[0380] 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.
Purification 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-trio-
xo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkyn-
amido)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)carb-
onate
##STR00194##
[0381] Step 1: Synthesis of
(S)-4-ethyl-11-(2-(N-isopropylacetamido)ethyl)-3,14-dioxo-3,4,12,14-tetra-
hydro-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-triazadotetracont-
yl-41-alkynamido)benzylcarbonate
[0382] 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.+.
Step 2: Synthesis of
4-((S)-2-(4-aminobutyl)-42-(2-(methylsulfonyl)pyrimidin-5-yl)-4,8,37-trio-
xo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9,36-triazadedotetracontyl-41-alkyn-
amido)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)carb-
onate (Compound TL048)
[0383] 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)thia-
zol-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,2-
4,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl((S)-4-ethyl-11-(2-(N-
-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyro-
ne[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate
##STR00195##
[0384] Step 1: Synthesis of
2-(2-(methylthio)pyrimidin-5-yl)thiazol-4-carboxylic Acid
[0385] 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.degree. 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.times.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.+.
Step 2: synthesis of
2-(2-(methylsulfonyl)pyrimidin-5-yl)thiazol-4-carboxylic Acid
[0386] 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. Purification 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.+.
Step 3: Synthesis of
2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)thiazo-4-carboxami-
de
[0387] 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.degree. C., then N, N-diisopropylethylamine (22.65 mg,
0.18 mmol) and propargylamine (4.63 mg, 0.09 mmol) were added
thereto. The reaction solution was stirred at room temperature for
3 hours. Purification 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.+.
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-(methylsulfon-
yl)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)benz-
yl)carbonate
[0388] 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.+.
Step 5: synthesis of
4-((S)-2-(4-aminobutyl)-35-(4-((2-(2-((methylsulfonyl)pyrimidin-5-yl)thia-
zol-4-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,2-
4,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl((S)-4-ethyl-11-(2-(N-
-isopropylmethylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyro-
ne[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate
[0389] 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)m-
ethyl)-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-isopropylmethylsul-
fonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indoli-
zino[1,2-b]quinolin-4-yl)carbonate
##STR00196##
[0390] Step 1: synthesis of ethyl
2-(2-(methylthio)pyrimidin-5-yl)oxazol-4-carboxylate
[0391] At 25.degree. 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.degree. 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.times.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.+.
Step 2: Synthesis of
2-(2-(methylthio)pyrimidin-5-yl)oxazol-4-carboxylic Acid
[0392] At 25.degree. 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.degree. C. for 1
h. The reaction solution was diluted with water (15 mL) and
extracted with ethyl acetate (20 mL.times.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.times.3). The organic phases were combined, washed with
saturated saline (30 mL.times.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.+.
Step 3: Synthesis of
2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazol-4-carboxylic Acid
[0393] At 25.degree. 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.degree. 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.+.
Step 4: Synthesis of
2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazol-4-formamid-
e
[0394] At 25.degree. 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.+.
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-(methylsulf-
onyl)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)benzy-
l)carbonate
[0395] At 25.degree. 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.+.
Step 6: Synthesis of 4-((S)-2-(4-aminobutyric
acid)-35-(4-((2-(2-(methylsulfonyl)pyrimidin-5-yl)oxazol-4-formylamino)me-
thyl)-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-isopropylmethylsulf-
onamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indoliz-
ino[1,2-b]quinolin-4-yl)carbonate
[0396] At 25.degree. 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.degree. 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,5S)-4-((S)-
-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-me-
thoxy-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-hexynamid-
e
##STR00197##
[0397] 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-meth-
ylheptanoyl)pyrro-2-yl)-3-methoxy-2-methylpropionamido)-3-phenylpropionami-
do)methyl)phenyl)amino)-1-oxo-5-ureidopent-2-yl)amino)-3-methyl-1-oxobutan-
-2-yl) carbamate
[0398] 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.degree. 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.degree. 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.+.
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-dimethylbutyrylam-
ino)-3-methoxy-5-methylheptanoyl)pyrro-2-yl)-3-methoxy-2-methylpropionamid-
o)-3-phenylpropionamido)methyl)phenyl)-5-ureidovaleramide
[0399] 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.+.
Step 3: Synthesis of
(S)-2-((S)-35-azido-2-isopropyl-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonao-
xa-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-dimethylbutanamid-
o)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanam-
ido)-3-phenylpropanamido)methyl)phenyl)-5-ureidovaleramide
[0400]
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.degree. 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.+.
Step 4: synthesis of
N-((1-((6S,9S)-1-amino-6-((4-(((S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-
-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-me-
thoxy-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-hexynamid-
e
[0401] 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-hex-
ynamido)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,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-m-
ethyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)car-
bamate
##STR00198##
[0402] Step 1:
4-((2S,5S)-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,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)(methyl)carbamate
[0403] 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.+.
Step 2: synthesis of
4-((2S,5S)-5-isopropyl-38-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-hex-
ynamido)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,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-m-
ethyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)car-
bamate
[0404] 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,5S)-4-((S)--N,3-dimethyl-2-((S)-3-methyl-
2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methylsulfonyl)pyrimid-
in-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)b-
enzyl)oxy)carbonyl)amino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-
-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine
##STR00199##
[0405] 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-nitrophe-
nyl)-carbonate
[0406] At 25.degree. 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.degree. 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.+.
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-meth-
ylbutyrylamino)-5-ureidopentanoylamino)benzyl)oxy)carbonyl)(methyl)amino)--
3-methylbutyrylamino)-N,3-dimethylbutyrylamino)-3-methoxy-5-methylheptyl)p-
yrrolidin-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine
[0407] At 25.degree. 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)buty-
rylamino)-3-methoxy-5-methylheptyl)pyrrolidine-2-yl)-3-methoxy-2-methyprop-
ionyl)-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.degree. 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.+.
Step 3: Synthesis of
(S)-2-((2R,3R)-3-((2S)-1-((3R,4S,5S)-4-((S)--N,3-dimethyl-2-((S)-3-methyl-
2-(methyl(((4-((S)-2-((S)-3-methyl-2-(32-(4-((6-(2-(methylsulfonyl)pyrimid-
in-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)b-
enzyl)oxy)carbonyl)amino)butyrylamino)-3-methoxy-5-methylheptyl)pyrrolidin-
-2-yl)-3-methoxy-2-methylpropionyl)-L-phenylalanine
[0408] At 25.degree. C.,
2-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-oxazol-4-formamid-
e (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-he-
xynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,3-
3-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-methox-
y-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-y-
l)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-
(methyl)carbamate
##STR00200##
[0409] Step 1: Synthesis of
(S)-4-(35-azido-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyryl)-4,8-dio-
xo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamino)benzyl-(4-
-nitrophenyl)-carbonate
[0410] 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.+.
Step 2: Synthesis of
4-((S)-35-azide-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyryl)-4,8-dio-
xo-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-phenylp-
ropan-2-yl)amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methox-
y-5-methyl-1-oxyheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-
-3-methyl-1-oxobutan-2-yl)(methyl)carbamate
[0411] 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.+.
Step 3: Synthesis of
4-((S)-2-(4-(((4-methoxyphenyl)benzhydryl)amino)butyryl)-35-(4-((6-(2-(me-
thylsulfonyl)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)ben-
zyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-p-
henylpropan-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
[0412] 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.+.
Step 4: Synthesis of
4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)-5-he-
xynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,3-
3-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-methox-
y-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxyheptan-4-y-
l)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-
(methyl)carbamate
[0413] 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].+-..
Example 25: Synthesis of
4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benza-
mido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-no-
noxy-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmeth-
ylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]i-
ndolizino[1,2-b]quinolin-4-yl)carbonate
##STR00201##
[0414] Step 1: Synthesis of Methyl
4-(2-(methylthio)pyrimidin-5-yl)benzoate
[0415] At 25.degree. 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.degree. C. for 4 hours. The reaction solution was
extracted with ethyl acetate (30 mL.times.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.+.
Step 2: Synthesis of 4-(2-(methylthio)pyrimidin-5-yl)benzoic
Acid
[0416] At 25.degree. 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.times.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.+.
Step 3: Synthesis of 4-(2-(methylsulfonyl)pyrimidin-5-yl)benzoic
Acid
[0417] At 25.degree. 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.+.
Step 4: Synthesis of
4-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)benzamide
[0418] At 25.degree. 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.+.
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-(methylsul-
fonyl)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)carbonat-
e
[0419] At 25.degree. 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. Purification (method B) was performed to obtain the
title compound (79 mg). ESI-MS (m/z): 1641.5 [M-273+H].sup.+.
Step 6: Synthesis of
4-((S)-2-(4-aminobutyl)-35-(4-((4-(2-(methylsulfonyl)pyrimidin-5-yl)benza-
mido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-no-
noxy-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropylmeth-
ylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]i-
ndolizino[1,2-b]quinolin-4-yl)carbonate
[0420] At 25.degree. 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,5S)-
-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutyrylamino)-N,3-dimethylbutyryl-
amino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropion-
amido)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-triazo-
l-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide
##STR00202## ##STR00203##
[0421] 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
[0422] At 20.degree. 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.+.
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-di-
methylbutyrylamino)-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
[0423] At 25.degree. C.,
(S)--N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((S)-1-(4-aminophenyl)-3-azidopro-
pyl-2-ye
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-methylbutyryla-
mino)-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.+.
Step 3: Synthesis of
(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-methox-
y-2-methylpropionamido)propyl)phenyl)-5-ureidovaleramide
[0424] At 25.degree. 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.+.
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-dimethylbutyryl-
amino)-3-methoxy-5-methylheptyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropion-
amido)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-triazo-
l-4-yl)methyl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynylamide
[0425] At 25.degree. 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-methox-
y-2-methylpropionamido)propyl)phenyl)-5-ureidovaleramide (95 mg,
0.092 mmol) and
32-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methy-
l)-1H-1,2,3-triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonoxy-6-azadotri-
acontanic 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.+.
[0426] III. Coupling of the Compound Containing the Bioactive
Molecule and the Linker with an Antibody
Example 27: Preparation of BT001002
[0427] 0.3 mL of antibody Sacituzumab (anti-Trop-2, 33.5 mg/mL) was
diluted with 0.25 m.sub.1 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 1M 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
.mu.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.
##STR00204##
Example 28: Preparation of BT001004
[0428] 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 1M 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 2h. 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.
##STR00205##
Example 29: Preparation of BT001012
[0429] 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.
##STR00206##
Example 30: Preparation of BT001013
[0430] 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.
##STR00207##
Example 31: Preparation of BT001018
[0431] 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.
##STR00208##
Example 32: Preparation of BT001021
[0432] 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 1M 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 .mu.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.
##STR00209##
Example 33: Preparation of BT001022
[0433] 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.
##STR00210##
Example 34: Preparation of BT001023
[0434] 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.
##STR00211##
Example 35: Preparation of BT001032
[0435] 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.
##STR00212##
Example 36: Preparation of BT001033
[0436] 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.
##STR00213##
Example 37: Preparation of BT001034
[0437] 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 and Sacituzumab was replaced by antibody
M2.
##STR00214##
Example 38: Preparation of BT001035
[0438] 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 1M 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 .mu.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.
##STR00215##
Example 39: Preparation of BT001036
[0439] 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.
##STR00216##
Example 40: Preparation of BT001040
[0440] 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.
##STR00217##
Example 41: Preparation of BT001041
[0441] 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.
##STR00218##
Example 42: Preparation of BT001042
[0442] 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.
##STR00219##
Example 43: Preparation of BT001043
[0443] 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.
##STR00220##
Example 44: Preparation of BT001044
[0444] 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.
##STR00221##
Example 45: Preparation of BT001045
[0445] 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.
##STR00222##
Example 46: Preparation of BT001046
[0446] 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.
##STR00223##
Example 47: Preparation of BT001047
[0447] 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.
##STR00224##
Example 48: Determination of Molecular Weight of BT001002 by
LC-MS
[0448] The molecular weight of BT001002 obtained by coupling was
analyzed by LC-MS.
[0449] LC conditions:
[0450] Liquid chromatographic column: ACQUITU UPLC.RTM. Protein BEH
C4 1.7 .mu.m, 2.1 mm.times.100 mm;
[0451] Mobile phase A: 0.1% FA/98% H.sub.2O/2% ACN; Mobile phase B:
0.1% FA/2% H.sub.2O/98% ACN;
[0452] Flow rate: 0.25 mL/min; Sample room temperature: 8.degree.
C.; Column temperature: 60.degree. C.; Sample size: 1 .mu.g;
TABLE-US-00018 Time (min.) 1 7 8 9 13 Mobile phase A 90 20 20 90 90
(% volume) Mobile phase B 10 80 80 10 10 (% volume)
[0453] MN conditions:
[0454] Mass spectrometer model: Triple TOF 5600+;
[0455] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10
m/z600-5000; Results were shown in FIG. 1-3.
Theoretical Molecular Weight and Measured Molecular Weight of
BT001002
TABLE-US-00019 [0456] Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC
Theoretical 23334.0 24610.7 25887.3 27163.9 28440.5 value Measured
Not 24611.1 Not Not Not value detected detected detected detected
HC Theoretical 50734.0 52010.6 53287.2 54563.8 55840.4 value
Measured Not Not Not 54563.1 Not value detected detected detected
detected
[0457] 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.
[0458] 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
[0459] The molecular weight of BT001004 obtained from coupling was
analyzed by LC-MS.
[0460] LC conditions:
[0461] Liquid chromatographic column: ACQUITU UPLC.RTM. Protein BEH
C18 1.7 .mu.m, 2.1 mm.times.100 mm;
[0462] Mobile phase A: 0.1% FA/98% H.sub.2O/2% ACN; Mobile phase B:
0.1% FA/2% H.sub.2O/98% ACN;
[0463] Flow rate: 0.25 mL/min; Sample room temperature: 8.degree.
C.; Column temperature: 60.degree. C.; Sample size: 1 .mu.g;
TABLE-US-00020 Time (min.) 2 20 22 25 26 30 Mobile phase A 80 60 10
10 80 80 (% volume) Mobile phase B 20 40 90 90 20 20 (% volume)
[0464] MS conditions:
[0465] Mass spectrometer model: Triple TOF 5600+;
[0466] GS1 60; GS2 60; CUR30; TEM 350; ISVF5500; DP300; CE10; m/z
600-5000;
[0467] Results were shown in FIG. 4-6.
Theoretical Molecular Weight and Measured Molecular Weight of
BT001004
TABLE-US-00021 [0468] Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC
Theoretical 23334.04 24832.7 26331.4 27830.0 29328.7 value Measured
23334.9 24833.7 Not Not Not value detected detected detected HC
Theoretical 50734.0 52232.6 53731.3 55230.0 56728.6 value Measured
Not 52232.1 53730.8 55229.3 Not value detected detected
[0469] LC stands for the light chain of an antibody; and HC stands
for the heavy chain of an antibody.
[0470] 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
[0471] A method similar to that as described in example 48 was
adopted, and results were shown in FIGS. 10 and 11.
[0472] 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-00022 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 24803.7 26273.4 27743.0 29212.7 value Measured 23334.8
24804.6 Not Not Not value detected detected detected HC Theoretical
50734.0 52203.6 53673.3 55143.0 56612.6 value Measured Not 52203.0
53672.2 55141.8 Not value detected detected
[0473] 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
[0474] A method similar to that as described in example 48 was
adopted, and results were shown in FIGS. 12 and 13.
[0475] 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-00023 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 24767.7 26201.3 27634.9 29068.5 value Measured 23334.6
24768.6 Not Not Not value detected detected detected HC Theoretical
50734.0 52167.6 53601.2 55034.8 56468.4 value Measured Not 52166.9
53600.8 55034.3 56468.2 value detected
[0476] 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
[0477] A method similar to that as described in example 48 was
adopted, and results were shown in FIGS. 14 and 15.
[0478] 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-00024 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 24926.8 26519.6 28112.3 29705.1 value Measured 23335.6
24928.2 Not Not Not value detected detected detected HC Theoretical
50734.0 52326.7 53919.5 55512.3 57105.0 value Measured 50736.3
52328.0 53920.7 55513.3 Not value detected
[0479] 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
[0480] The molecular weight of the coupled BT001021 was analyzed by
LC-MS.
[0481] LC conditions:
[0482] Liquid chromatographic column: ACQUITU UPLC.RTM. Protein BEH
C4 1.7 .mu.m, 2.1 mm.times.100 mm;
[0483] Mobile phase A: 0.1% FA/98% H.sub.2O/2% ACN; Mobile phase B:
0.1% FA/2% H.sub.2O/98% ACN;
[0484] Flow rate: 0.25 mL/min; Sample room temperature: 8.degree.
C.; Column temperature: 60.degree. C.; Sample size: 1 .mu.g;
TABLE-US-00025 Time (min.) 1 7 8 9 13 Mobile phase A 90 20 20 90 90
(% volume) Mobile phase B 10 80 80 10 10 (% volume)
[0485] MS conditions:
[0486] Mass spectrometer model: Triple TOF 5600+;
[0487] GS1 60; GS2 60; CUR30; TEM600; ISVF5000; DP300; CE10
m/z600-5000;
[0488] Results were shown in FIGS. 16 and 17.
[0489] 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-00026 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 24884.8 26435.5 27986.3 29537.0 value Measured 23334.6
24885.9 Not Not Not value detected detected detected HC Theoretical
50734.0 52284.7 53835.5 55386.2 56937.0 value Measured Not 52284.3
53834.5 55385.4 Not value detected detected
[0490] 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
was coupled 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
[0491] A method similar to that as described in example 48 was
adopted, and results were shown in FIGS. 18 and 19.
[0492] 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-00027 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 24848.7 26363.4 27878.1 29392.7 value Measured 23335.4
24850.2 Not Not Not value detected detected detected HC Theoretical
50733.98 52248.7 53763.3 55278.0 56792.7 value Measured 50735.2
52249.1 53764.1 55278.6 Not value detected
[0493] 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
[0494] The molecular weight of BT001040 obtained by coupling was
analyzed by LC-MS.
[0495] Liquid chromatographic column: Thermo MabPac.TM. RP 4 .mu.m,
3.0 mm*100 mm
[0496] Mobile phase A: 0.1% FA/98% H.sub.2O/2% ACN; Mobile phase B:
0.1% FA/2% H.sub.2O/98% ACN
[0497] Flow rate: 0.25 mL/min; Sample room temperature: 8.degree.
C.; Column temperature: 60.degree. C.; Sample size: 1 .mu.g
TABLE-US-00028 Time (min.) 2 20 22 25 26 30 Mobile phase A 80 60 10
10 80 80 (% volume) Mobile phase B 20 40 90 90 20 20 (% volume)
[0498] MS conditions:
[0499] Mass spectrometer model: Triple TOF 5600+
[0500] GS1 35; GS2 35; CUR30; TEM 350; ISVF5000; DP250; m/z
600-5000
[0501] 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-00029 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 24901.8 26469.5 28037.3 29605.0 value Measured 23334.2
24902.8 Not Not Not value detected detected detected HC Theoretical
50733.98 52301.7 53869.5 55437.2 57005.0 value Measured Not 52301.6
53869.2 55437.4 57005.3 value detected
[0502] 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
[0503] A method similar to that as described in example 55 was
adopted, and results were shown in FIGS. 22 and 23.
[0504] 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-00030 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 24885.7 26437.4 27989.1 29540.8 value Measured 23334.5
24886.9 Not Not Not value detected detected detected HC Theoretical
50733.98 52285.7 53837.3 55389.0 56940.7 value Measured Not 52285.2
53836.9 55389.1 56940.9 value detected
[0505] 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
[0506] A method similar to that as described in example 55 was
adopted, and results were shown in FIGS. 24 and 25.
[0507] 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-00031 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 25202.3 27070.6 28938.9 30807.2 value Measured 23335.2
25203.8 Not Not Not value detected detected detected HC Theoretical
50733.98 52602.3 54470.6 56338.9 58207.2 value Measured Not 52602.8
54471.0 56339.8 Not value detected detected
[0508] 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
[0509] A method similar to that as described in example 55 was
adopted, and results were shown in FIGS. 26 and 27.
[0510] The theoretical molecular weight and measured molecular
weight of the light chain and the heavy chain of BT001043 obtained
by coupling TL052 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
23334.04 25202.3 27100.6 28983.9 30867.2 value Measured 23335.5
25221.1 Not Not Not value detected detected detected HC Theoretical
50733.98 52617.3 54500.6 56383.9 58267.2 value Measured Not 52620.4
54505.8 56391.5 Not value detected detected
[0511] 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
[0512] A method similar to that as described in example 55 was
adopted, and results were shown in FIGS. 28 and 29.
[0513] 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-00033 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 25233.3 27132.5 29031.8 30931.1 value Measured 23335.5
25234.7 Not Not Not value detected detected detected HC Theoretical
50733.98 52633.2 54532.5 56431.7 58331.0 value Measured Not 52634.1
54532.9 56432.1 Not value detected detected
[0514] 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
[0515] A method similar to that as described in example 55 was
adopted, and results were shown in FIGS. 30 and 31.
[0516] 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-00034 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 24894.8 26455.5 28016.2 29577.0 value Measured 23335.5
24896.7 Not Not Not value detected detected detected HC Theoretical
50733.98 52294.7 53855.4 55416.2 56976.9 value Measured 50735.4
52296.1 53856.9 55418.0 Not value detected
[0517] 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
[0518] A method similar to that as described in example 55 was
adopted, and results were shown in FIGS. 32 and 33.
[0519] 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-00035 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Theoretical
23334.04 25124.2 26914.4 28704.6 30494.8 value Measured 23335.5
25126.3 Not Not Not value detected detected detected HC Theoretical
50733.98 52524.2 54314.3 56104.5 57894.7 value Measured 50735.4
52524.6 54314.1 56106.5 Not value detected
[0520] 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
[0521] The coupling reaction was monitored by SEC-HPLC, and the
conjugates were tested by SEC.
[0522] Chromatographic Conditions:
[0523] Liquid chromatographic column: TOSOH TSKgel SuperSW mAb, 4
.mu.m, 7.8 mm.times.300 mm;
[0524] Mobile phase: 100 mmol/L Na.sub.2HPO.sub.4, 100 mmol/L NaCl,
5% isopropanol, pH7.0;
[0525] Flow rate: 0.5 ml/min; Detection wavelength: 280 nm; Column
temperature: room temperature; Sample room temperature: 8.degree.
C.;
[0526] Sample size: 30 .mu.g; Isocratic operation: 30 min.
[0527] 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 150kD, 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.
[0528] 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 150kD, i.e.,
BT001004 obtained by coupling TL019 with the antibody still
maintains the integral structure of the antibody.
[0529] 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 150kD, i.e.,
BT001012 obtained by coupling TL024 with the antibody still
maintains the integral structure of the antibody.
[0530] 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 150kD, i.e.,
BT001013 obtained by coupling TL048 with the antibody still
maintains an integral structure of the antibody.
[0531] 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 150kD, i.e.,
BT001018 obtained by coupling TL030 with the antibody still
maintains an integral structure of the antibody.
[0532] 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 150kD, i.e.,
BT001021 obtained by coupling TL033 with the antibody still
maintains an integral structure of the antibody.
[0533] 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 150kD, i.e.,
BT001023 obtained by coupling TL035 with the antibody still
maintains an integral structure of the antibody.
[0534] 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 150kD, i.e.,
BT001042 obtained by coupling TL051 with the antibody still
maintains the integral structure of the antibody.
[0535] 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.
[0536] 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 150kD, i.e.,
BT001044 obtained by coupling TL053 with the antibody still
maintains the integral structure of the antibody.
[0537] 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 150kD, i.e.,
BT001046 obtained by coupling TL055 with the antibody still
maintains the integral structure of the antibody.
[0538] 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 150kD, 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
[0539] 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-00036 TABLE 1 Cell name Tumor type Source MDA-MB-468
Breast cancer Concortis HCC1806 Breast cancer Cobioer Biosciences
Co., Ltd.
[0540] 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 .mu.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-00037 TABLE 2 Killing effects of bioactive molecules on
cells Name Cell name EC.sub.50(nM) T001 MDA-MB-468 1.126 T011 (7500
cells/well, 4 days) 0.211 T012 1.621 T013 0.414 T015 7.428 T-028
HCC1806 6.016 T-030 (7500 cells/well, 3 days) 6.734
[0541] The test results indicated that all of the bioactive
molecules had killing effects on the tumor cells.
TABLE-US-00038 TABLE 3 Killing effects of conjugates (ADCs) on cell
lines Name Cell name EC50(nM) BT001002 MDA-MB-468 0.072 (10000
cells/well, 3 days) BT001004 HCC1806 6.139 BT001012 (7500
cells/well, 3 days) 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
[0542] 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
[0543] Drugs Under Test
[0544] Drug name, source and preparation method:
[0545] BT001021, liquid aliquots were stored at -20.degree. C. at a
concentration of 5.44 mg/ml, and diluted with physiological saline
by dosage before use to obtain a test solution;
[0546] Immu-132 (prepared according to example 2 of
WO2015/012904A2, DAR=5.4, also described as IMMU-132), liquid
aliquots were stored at -20.degree. C. at a concentration of 13.158
mg/ml, and diluted with physiological saline by dosage before use
to obtain a test solution;
[0547] 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.degree. C., and diluted with
physiological saline to a desired dose before use to obtain a test
solution;
[0548] 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.degree. C., and diluted
with physiological saline by dosage before use to obtain a test
solution.
[0549] Note: Toxin was prepared and administered in an equimolar
ratio of ADC samples.
[0550] Structures of T-030, SN-38 and Immu-132 were as follows:
##STR00225##
[0551] Experimental Animals and Cell Lines
[0552] 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).
[0553] Experimental Grouping and Evaluation Method
[0554] 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.times.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.
[0555] The tumor growth inhibition rate TGI (%) was calculated from
the following formula to evaluate tumor inhibitory effect of
antibody drug conjugates:
TGI
(%)=[1-(V.sub.Tend-V.sub.Tstart)/(V.sub.Cend-V.sub.Cstart)]*100%
[0556] wherein, V.sub.Tend: average tumor volume at the end of the
experiment in the treatment group
[0557] V.sub.Tstart: average tumor volume at the beginning of
administration in the treatment group
[0558] V.sub.Cend: average tumor volume at the end of the
experiment in the control group
[0559] V.sub.Cstart: average tumor volume at the beginning of
administration in the control group
[0560] 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
[0561] Experimental Methods:
[0562] NCI-N87 cells were cultured in a 1640 culture medium
containing 10% fetal bovine serum at 37.degree. 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.times.3W) group, a positive drug
Immu-132 (3 mg/kg, IV, BIW.times.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.
[0563] Conclusion:
[0564] 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.
[0565] Experimental results showed that BT001021 (3 mg/kg, IV,
BIW.times.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
TABLE-US-00039 TABLE 4 NCI-N87 model of gastric cancer D 21 after
administration Tumor growth P Group Tumor volume inhibition value
(vs No. Regimen (mm.sup.3) (x .+-. S) rate (%) group 1) Group 1
Physiological 405.67 .+-. 91.81 saline 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
[0566] Experimental Methods:
[0567] HCC1806 cells were cultured in a 1640 culture medium
containing 10% fetal bovine serum at 37.degree. 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.times.3W) group and a positive drug Immu-132 (10
mg/kg, IV, BIW.times.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.
[0568] Conclusion:
[0569] 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.
[0570] Experimental results showed that BT001021 (10 mg/kg, IV,
BIW.times.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-00040 TABLE 5 HCC1806 model of breast cancer D 17 after
administration Tumor growth P Group Tumor Volume inhibition value
(vs No. Regimen (mm.sup.3) (x .+-. S) rate (%) group 1) Group 1
Physiological 2638.22 .+-. 553.81 saline Group 2 BT001021 1260.87
.+-. 415.60 54.93 0.0006 Group 3 Immu-132 2347.05 .+-. 317.79 11.62
0.2901
[0571] 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.
[0572] 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
[0573] 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.
[0574] Experimental Methods:
[0575] HCC827 cells were cultured in a 1640 culture medium
containing 10% fetal bovine serum at 37.degree. 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.times.3W)
group, a BT001021 (10 mg/kg, IV, BIW.times.3W) group and a BT001035
(10 mg/kg, IV, BIW.times.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.
[0576] Conclusion:
[0577] 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-00041 TABLE 6 HCC827 model of lung cancer D 21 after
administration Group Tumor Volume TGI P value No. Regimen
(mm.sup.3) (x .+-. S) (%) (vs group 1) 1 Physiological 515.25 .+-.
165.09 saline 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
[0578] 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.
[0579] 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
[0580] 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.
[0581] Experimental methods: NCI-N87 cells were cultured in a 1640
culture medium containing 10% fetal bovine serum at 37.degree. 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.times.3W) group and a BT001036 (3 mg/kg, IV, BIW.times.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-00042 TABLE 7 NCI-N87 model of gastric cancer D 21 after
administration Group Tumor Volume TGI P value No. Regimen
(mm.sup.3) x (.+-.S) (%) (vs group 1) Group 1 Physiological 601.29
.+-. 198.92 saline Group 2 BT001036 150.87 .+-. 112.84 97.78 0.0017
(1.5 mg/kg) Group 3 BT001036 2.74 .+-. 0.64 129.95 0.0000 (3
mg/kg)
[0582] Conclusion:
[0583] 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.
[0584] Experimental results showed that both the high and low
dosages 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
[0585] 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 administered 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.
[0586] Experimental Methods:
[0587] NCI-MDA-MB-231 cells were cultured in a RPMI1640 culture
medium containing 10% fetal bovine serum at 37.degree. 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.
[0588] Conclusion:
[0589] 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-00043 TABLE 8 MDA-MB-231 model of breast cancer D 33 after
administration Group Tumor Volume TGI P value No. Regimen
(mm.sup.3) x (.+-.S) (%) (vs group 1) 1 Physiological 744.53 .+-.
306.66 saline 2 BT001021 43.96 .+-. 7.72 114.18 0.0119
[0590] 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
[0591] 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.
[0592] Drugs Under Test
[0593] Drug Names and Preparation Methods:
[0594] BT001021, liquid aliquots were stored at -20.degree. C. at a
concentration of 20 mg/ml, and diluted with physiological saline to
the desired doses before use to obtain a test solution;
[0595] 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.
[0596] Experimental Animals and Cell Lines:
[0597] 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).
[0598] Experimental Groups and Evaluation Method:
[0599] 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
[0600] Experimental Method:
[0601] NCI-N87 cells were cultured in a 1640 culture medium
containing 10% heat-inactivated fetal bovine serum at 37.degree. 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 1h, 2h, 4h, 8h, 24h and 72h after administration
(T-030 was not detected in the serum and tumor tissues 72h after
administration, therefore, serum and tumor tissues were not
collected 168h after administration). For BT001021 group, serum and
tumor tissues were collected 1h, 2h, 4h, 8h, 24h, 72h and 168h
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-00044 TABLE 9 Pharmacokinetic parameters of T-030 in
tumors and serum of tumor-bearing mice after intravenous
administration of T-030 and BT001021 T-030 (0.23 mg/kg) BT001021
(10 mg/kg) administration group administration group Regimen 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.1/2(h) 2.55 1.76 93.14
44.35
[0602] conclusion:
[0603] 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.
[0604] 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.
[0605] T.sub.1/2 of the bioactive molecule T-030 in the tumors of
BT001021 (10 mg/kg) administration group was 93.14 h, whereas the
Tin 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.
[0606] 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
[0607] 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.
[0608] Drugs Under Test
[0609] Drug Names and Preparation Methods:
[0610] BT001021, liquid aliquots were stored at -20.degree. C. at a
concentration of 20 mg/ml, and diluted with physiological saline to
the desired doses before use to obtain the test solution;
[0611] Immu-132 was diluted with physiological saline to the
desired dose to obtain the test solution.
[0612] Experimental Animals and Cell Lines:
[0613] 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).
[0614] Experimental Groups and Evaluation Method:
[0615] 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.
[0616] Experimental Methods:
[0617] NCI-N87 cells were cultured in a 1640 culture medium
containing 10% heat-inactivated fetal bovine serum at 37.degree. 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 2h, 24h, 48h and 72h 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-00045 TABLE 10 Pharmacokinetic parameters of T-030 and
SN-38 in tumors and serum of tumor-bearing mice after intravenous
administration of BT001021 and Immu-132 Immu-132 (5 mg/kg) BT001021
(5 mg/kg) administration group administration group Regimen 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
[0618] Conclusion:
[0619] 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.
[0620] 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 changed are within the protection scope of the invention. The
full scope of the invention is given by the attached claims and any
equivalent thereof.
Sequence CWU 1
1
2017PRTartificialHeavy chain CDR1 of antibodies of M1, M2 and M3
1Gly Tyr Thr Phe Thr Asn Tyr1 526PRTartificialHeavy chain CDR2 of
antibodies of M1, M2 and M3 2Asn Thr Asp Ser Gly Glu1
5312PRTartificialHeavy chain CDR3 of antibodies of M1, M2 and M3
3Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val1 5
10411PRTartificialLight chain CDR1 of antibodies of M1 and M2 4Lys
Ala Ser Gln Asp Val Ser Ser Ala Val Ala1 5 1057PRTartificialLight
chain CDR2 of antibodies of M1, M2 and M3 5Ser Ala Ser Tyr Arg Tyr
Thr1 569PRTartificialLight chain CDR3 of antibodies of M1 and M3
6Gln Gln His Tyr Ser Thr Pro Leu Thr1 579PRTartificialLight chain
CDR3 of antibody M2 7Gln Gln His Tyr Ile Thr Pro Leu Thr1
5811PRTartificialLight chain CDR1 of antibody M3 8Lys Ala Ser Gln
Asp Val Ser Ile Ala Val Ala1 5 109107PRTartificialLight chain
constant region of antibodies of M1, M2 and M3 9Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
10510330PRTartificialHeavy chain constant region of antibodies of
M1, M2 and M3 10Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 325 33011121PRTartificialHeavy chain variable region of
antibody M1 11Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln
Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Asp Ser Gly Glu Pro
Thr Tyr Thr Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Asp
Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Ser Ser Leu Lys
Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Gly Gly Phe Gly
Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln Gly Ser Leu
Val Thr Val Ser Ser 115 12012107PRTartificialLight chain variable
region of antibody M1 12Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser
Gln Asp Val Ser Ser Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Ala
Gly Thr Lys Val Glu Ile Lys 100 10513121PRTartificialHeavy chain
variable region of antibody M2 13Gln Val Gln Leu Gln Gln Ser Gly
Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys
Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr
Asp Ser Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55 60Lys Gly Arg Phe
Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln
Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala
Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105
110Gln Gly Ser Leu Val Thr Val Ser Ser 115
12014107PRTartificialLight chain variable region of antibody M2
14Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ser
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
His Tyr Ile Thr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Val Glu
Ile Lys 100 10515121PRTartificialHeavy chain variable region of
antibody M3 15Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln
Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Asp Ser Gly Glu Pro
Thr Tyr Thr Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Asp
Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Ser Ser Leu Lys
Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Gly Gly Phe Gly
Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln Gly Ser Leu
Val Thr Val Ser Ser 115 12016107PRTartificialLight chain variable
region of antibody M3 16Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser
Gln Asp Val Ser Ile Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu 85 90 95Thr Phe Gly Ala
Gly Thr Lys Val Glu Ile Lys 100 10517450PRTartificialHeavy chain
amino acid sequences of Trastuzumab 17Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330
335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly
Lys 45018214PRTartificialLight chain amino acid sequences of
Trastuzumab 18Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Val Asn Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21019451PRTartificialHeavy chain amino acid sequences of
Sacituzumab 19Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln
Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro
Thr Tyr Thr Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Asp
Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Ser Ser Leu Lys
Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Gly Gly Phe Gly
Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110Gln Gly Ser Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375
380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45020214PRTartificialLight chain amino acid sequences of
Sacituzumab 20Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp
Val Ser Ile Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val
Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
210
* * * * *