U.S. patent application number 16/618110 was filed with the patent office on 2020-05-07 for process for preparing intermediate of antibody drug conjugate.
The applicant listed for this patent is REMEGEN, LTD.. Invention is credited to Jianmin FANG, Changjiang HUANG, Haohua JIE, Xuejing YAO, Hui YE, Shizhong ZHAI.
Application Number | 20200138968 16/618110 |
Document ID | / |
Family ID | 68616215 |
Filed Date | 2020-05-07 |
![](/patent/app/20200138968/US20200138968A1-20200507-C00001.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00002.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00003.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00004.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00005.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00006.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00007.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00008.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00009.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00010.png)
![](/patent/app/20200138968/US20200138968A1-20200507-C00011.png)
View All Diagrams
United States Patent
Application |
20200138968 |
Kind Code |
A1 |
HUANG; Changjiang ; et
al. |
May 7, 2020 |
PROCESS FOR PREPARING INTERMEDIATE OF ANTIBODY DRUG CONJUGATE
Abstract
The present disclosure relates to a process for preparing an
intermediate of antibody-drug conjugate. Compared with the
conventional art, the process for preparing an intermediate of the
antibody-drug conjugate provided by the present disclosure
significantly reduces the feed loss for drug moiety, such as
MMAD/MMAE or MMAF, etc., as drug moiety is involved in the last
step of the reaction, thereby effectively reducing production
costs, as well as increasing production efficiency. In addition,
the process provided by the present disclosure is simple,
environmentally friendly and suitable for large-scale
industrialization.
Inventors: |
HUANG; Changjiang; (Yantai,
CN) ; YE; Hui; (Yantai, CN) ; YAO;
Xuejing; (Yantai, CN) ; JIE; Haohua; (Yantai,
CN) ; ZHAI; Shizhong; (Yantai, CN) ; FANG;
Jianmin; (Yantai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REMEGEN, LTD. |
Yantai |
|
CN |
|
|
Family ID: |
68616215 |
Appl. No.: |
16/618110 |
Filed: |
May 20, 2019 |
PCT Filed: |
May 20, 2019 |
PCT NO: |
PCT/CN2019/087641 |
371 Date: |
November 28, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/65 20170801;
A61K 38/07 20130101; A61P 35/00 20180101; A61K 47/6889 20170801;
C07K 16/30 20130101; C07K 16/28 20130101; A61K 47/6803 20170801;
A61K 47/6807 20170801 |
International
Class: |
A61K 47/65 20060101
A61K047/65; A61K 38/07 20060101 A61K038/07 |
Foreign Application Data
Date |
Code |
Application Number |
May 21, 2018 |
CN |
201810487856.4 |
Claims
1. A process for preparing an intermediate of antibody-drug
conjugate comprising a linker and a drug moiety, in which the
intermediate of antibody-drug conjugate is Py-MAA-Val-Cit-PAB-D,
the Py-MAA-Val-Cit-PAB in the intermediate is the linker and the D
in the intermediate represents the attached drug moiety, and the
attached drug moiety contains free amino group, wherein it
comprises the following reactions: ##STR00042## ##STR00043##
2. The process according to claim 1, wherein Reaction A: dissolving
Py-MAA-Val-Cit-PAB-OH in a solvent, adding bis(4-nitrophenyl)
carbonate, stirring until dissolved or the solution is slightly
turbid, and then adding an organic base; Reaction B: after
completion of Reaction A, adding a triazole catalyst, the drug
moiety D and an organic base, and after completion of the reaction,
carrying out purification to obtain Py-MAA-Val-Cit-PAB-D.
3. The process according to claim 1, wherein the drug moiety D is
an auristatin class cytotoxic agent, an anthramycin class cytotoxic
agent, an anthracycline class cytotoxic agent or a puromycin class
cytotoxic agent.
4. The process according to claim 3, wherein the auristatin class
cytotoxic agent is selected from the group consisting of MMAE,
MMAF, MMAD and a derivative thereof; the anthramycin class
cytotoxic agent is anthramycin or a derivative thereof; the
anthracycline class cytotoxic agent is selected from the group
consisting of daunorubicin, doxorubicin, epirubicin, idarubicin,
valrubicin, mitoxantrone and a derivative thereof; and the
puromycin class cytotoxic agent is puromycin or a derivative
thereof.
5. The process according to claim 4, wherein the structure of the
Py-MAA-Val-Cit-PAB-D is represented by Formulas (1-11):
##STR00044## ##STR00045## ##STR00046## ##STR00047##
6. The process according to claim 2, wherein the solvent is a polar
solvent and/or a non-polar solvent, the polar solvent is one or
more selected from the group consisting of DMF, DMA and NMP; the
non-polar solvent is one or more selected from the group consisting
of dichloromethane and carbon tetrachloride.
7. The process according to claim 1, wherein the organic base is
one or more selected from the group consisting of
N,N-diisopropylethylamine, triethylamine and pyridine, preferably
one or two selected from the group consisting of
N,N-diisopropylethylamine and pyridine.
8. The process according to claim 1, wherein the triazole catalyst
is one or more selected from the group consisting of
1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole and ethyl
1-hydroxy-1H-1,2,3-triazole-4-carboxylate, preferably
1-hydroxybenzotriazole.
9. The process according to claim 1, comprising: in the Reaction A,
controlling the temperature of the reaction system to be in the
range from 5 to 15.degree. C. prior to the addition of
bis(4-nitrophenyl) carbonate, preferably 10.degree. C.; and further
preferably, in the Reaction A, controlling the temperature of the
reaction system to be in the range from 20 to 35.degree. C. after
addition of the organic base, preferably 30.degree. C.
10. The process according to claim 1, comprising: in the Reaction
B, controlling the temperature of the reaction system to be in the
range from 5 to 15.degree. C. prior to the addition of the triazole
catalyst, preferably 10.degree. C.; further preferably, in the
Reaction B, controlling the temperature of the reaction system to
be in the range from 15 to 30.degree. C. after the addition of the
organic base, preferably 20.degree. C.
11. The preparation process according to claim 1, wherein, in the
Reaction A, the molar ratio of Py-MAA-Val-Cit-PAB-OH.
bis(4-nitrophenyl) carbonate and the organic base is 1:1-5:1-3.
12. The process according to claim 1, wherein, in the Reaction B,
the molar ratio of the triazole catalyst, MMAE, the organic base
and Py-MAA-Val-Cit-PAB-OH is 1-3:1-3:1.5-30:1.
13. The process according to claim 1, comprising: after completion
of the Reaction B, carrying out pre-HPLC purification to obtain
Py-MAA-Val-Cit-PAB-D.
14. The process according to claim 1, wherein purification is
carried out in Reaction A by the following method to obtain
Py-MAA-Val-Cit-PAB-PNP: (1) centrifuging the reaction solution of
Reaction A to obtain a supernatant; (2) adding solvent 1 to the
supernatant, and stirring well; (3) adding solvent 2 dropwise at 0
to 10.degree. C., and stirring well; (4) filtering under suction to
obtain a filter cake; (5) re-dissolving the filter cake in a mixed
solution of solvent 3 and solvent 4; (6) adding the resultant
dropwise to solvent 5 at 0 to 10.degree. C., and stirring; (7)
filtering under suction to obtain an off-white powder, which is
purified Py-MAA-Val-Cit-PAB-PNP; wherein the solvent 1 is a
moderately polar solvent, preferably selected from the group
consisting of ethyl acetate, dichloromethane, methanol, methyl
tert-butyl ether and the like, most preferably ethyl acetate; the
solvent 2 is a weakly polar solvent, preferably selected from the
group consisting of petroleum ether, n-hexane, n-pentane,
cyclohexane and the like, most preferably petroleum ether; the
solvent 3 and solvent 4 are solvents in which
Py-MAA-Val-Cit-PAB-PNP is easy to dissolve, preferably selected
from the group consisting of acetic acid, trifluoroacetic acid,
formic acid, methanol, ethanol and the like, most preferably acetic
acid and methanol; the solvent 5 is preferably selected from the
group consisting of acetonitrile, ethyl acetate, dichloromethane,
water and the like, most preferably water.
15. The process according to claim 1, wherein the reactions are
carried out under the protection of nitrogen.
Description
FIELD
[0001] The present invention relates to the field of pharmaceutical
preparation, specifically to a process for preparing an
intermediate of antibody-drug conjugate.
BACKGROUND
[0002] Antibody-drug conjugate (abbreviated as ADC) is a class of
anti-tumor drugs consisting of three components: antibody, linker
and drug. The number of drugs attached to the antibody
(Drug-antibody Ratio, DAR) determines the homogeneity of the
antibody-drug conjugate. Excessive number of attached drugs will
lead to unstable pharmacodynamics, increased drug metabolism,
decreased half-life, and increased systemic toxicity.
[0003] The DAR value is primarily determined by the linker.
Classical ADCs have two coupling methods: one is amino coupling and
the other is thiol coupling, which is currently the most commonly
used coupling method. Amino coupling is the coupling of a drug to a
lysine (Lys) residue of an antibody via a linker. Since there are
80 lysines on one antibody, 30 of which can be linked, the ADC
formed by lysine coupling has a DAR value of 0 to 30, and the
product homogeneity is extremely poor. Thiol coupling is to open
the interchain disulfide bonds in the antibody first to give free
cysteine (Cys) residues, before being coupled to a linker-drug
complex capable of pairing with a cysteine residue. Since a single
antibody only has 4 pairs of interchain disulfide bonds, 8 free
cysteine residues would be generated after the interchain disulfide
bonds are all opened. Therefore, the ADCs formed by cysteine
residue coupling have DAR values of 0 to 8. Although thiol coupling
can better control the number of linkages per antibody, the
cleavage of the interchain disulfide bond greatly reduces the
stability of the antibody.
[0004] Currently, in addition to the classical amino coupling and
thiol coupling, there is a new coupling method: bridging coupling.
The bridging coupling method is a new coupling method developed
based on thiol coupling, in which the linker is simultaneously
coupled to two free thiol groups on the antibody, and the DAR value
of the antibody-drug conjugate produced in this manner is 0 to 4. A
variety of linkers capable of being covalently linked to an
antibody by way of bridging coupling are disclosed in a Chinese
patent publication CN107921030A, which discloses an intermediate of
antibody-drug conjugate (Py-MAA-Val-Cit-PAB-MMAE) (wherein Py is
triacryloylhexahydro triazine, its CAS is 959-52-4, and is
available from J&K Scientific and Nanjing Chemlin Chemical
Industry Co., Ltd.) as shown below on page 42 of the
specification.
##STR00001##
[0005] In addition, a process for preparing an intermediate of
antibody-drug conjugate (Py-MAA-Val-Cit-PAB-MMAD) in which the Drug
is MMAD (Demethyldolastatin) is also disclosed on page 32 of the
patent application:
##STR00002##
[0006] The process involves first coupling Val-Cit-PAB with Drug
(MMAD) to form a Val-Cit-PAB-MMAD conjugate, and then reacting with
Py-MAA to form an antibody-drug conjugate intermediate
Py-MAA-Val-Cit-PAB-MMAD. Due to high price for the drug (such as
MMAD/MMAE or MMAF, etc.) used in the antibody-drug conjugate and
great feed loss for the production of drug using the
above-mentioned process, the production cost is high, making it not
suitable for industrial mass production.
SUMMARY
[0007] The present disclosure provides a process for preparing an
intermediate of antibody-drug conjugate, specifically provides a
process for preparing an intermediate of antibody-drug conjugate
comprising a linker and a drug moiety. The intermediate of
antibody-drug conjugate is Py-MAA-Val-Cit-PAB-D, in which
Py-MAA-Val-Cit-PAB is the linker, and D represents the attached
drug moiety. The process is characterized in that it comprises the
following reactions:
##STR00003## ##STR00004##
[0008] Further, the preparation process comprises the following
reaction procedure:
[0009] Reaction A: dissolving Py-MAA-Val-Cit-PAB-OH in a solvent,
adding bis(4-nitrophenyl) carbonate, stirring until dissolved or
the solution is slightly turbid, and then adding an organic
base;
[0010] Reaction B: after completion of Reaction A, adding a
triazole catalyst, the drug moiety D, and an organic base, and
after completion of the reaction, carrying out purification to
obtain Py-MAA-Val-Cit-PAB-D. In one embodiment, pre-HPLC is used
for the purification.
[0011] Further, the drug moiety D is an auristatin class cytotoxic
agent, an anthramycin class cytotoxic agent, an anthracycline class
cytotoxic agent or a puromycin class cytotoxic agent.
[0012] In one embodiment, the auristatin class cytotoxic agent is
selected from the group consisting of MMAE, MMAF, MMAD and a
derivative thereof; the anthramycin class cytotoxic agent is
anthramycin; the anthracycline class cytotoxic agent is selected
from the group consisting of daunorubicin, doxorubicin, epirubicin,
idarubicin, valrubicin and mitoxantrone; and the puromycin class
cytotoxic agent is puromycin.
[0013] In one embodiment, the Py-MAA-Val-Cit-PAB-D has the
structures as represented by Formulas (1-11):
##STR00005## ##STR00006## ##STR00007## ##STR00008##
[0014] Further, the solvent is a polar solvent and/or a non-polar
solvent. The polar solvent is one or more selected from the group
consisting of DMF, DMA and NMP; the non-polar solvent is one or
more selected from the group consisting of dichloromethane and
carbon tetrachloride.
[0015] Further, the organic base is one or more selected from the
group consisting of N,N-diisopropylethylamine, triethylamine and
pyridine, In one embodiment, one or two selected from the group
consisting of N,N-diisopropylethylamine and pyridine.
[0016] Further, the triazole catalyst is one or more selected from
the group consisting of 1-hydroxybenzotriazole,
1-hydroxy-7-azabenzotriazole, ethyl
1-hydroxy-1H-1,2,3-triazole-4-carboxylate, In one embodiment
1-hydroxybenzotriazole.
[0017] Further, the reaction temperature of the Reaction A is 5 to
35.degree. C., and the reaction duration is 2 to 10 hours.
[0018] In one embodiment, in the Reaction A, controlling the
temperature of the reaction system to be in a range from 5 to
15.degree. C. prior to the addition of bis(4-nitrophenyl)
carbonate, and further In one embodiment 10.degree. C.
[0019] In one embodiment, in the Reaction A, controlling the
temperature of the reaction system to be in a range from 20 to
35.degree. C. after the addition of the organic base, In one
embodiment 30.degree. C.
[0020] Further, the reaction temperature of the Reaction B ranges
from 5 to 30.degree. C., and the reaction duration ranges from 15
to 48 hours.
[0021] In one embodiment, in the Reaction B, controlling the
temperature of the reaction system to be in a range from 5 to
15.degree. C. prior to the addition of the triazole catalyst, In
one embodiment 10.degree. C.
[0022] In one embodiment, in the Reaction B, controlling the
temperature of the reaction system to be in a range from 15 to
30.degree. C. after the addition of the organic base, In one
embodiment 20.degree. C. Further, the molar ratio of
Py-MAA-Val-Cit-PAB-OH, bis(4-nitrophenyl) carbonate and the organic
base in Reaction A is 1:1-5:1-3.
[0023] Further, the molar ratio of the triazole catalyst, MMAE, the
organic base and Py-MAA-Val-Cit-PAB-OH in the Reaction B is
1-3:1-3:1.5-30:1.
[0024] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of an antibody-drug conjugate, which is
Py-MAA-Val-Cit-PAB-MMAF, and its structure is as represented by
Formula (2):
##STR00009##
[0025] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which is
Py-MAA-Val-Cit-PAB-MMAD, and its structure is as represented by
Formula (3):
##STR00010##
[0026] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which has a structure as
represented by Formula (4):
##STR00011##
[0027] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which has a structure as
represented by Formula (5):
##STR00012##
[0028] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which has a structure as
represented by Formula (6):
##STR00013##
[0029] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which has a structure as
represented by Formula (7):
##STR00014##
[0030] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which has a structure as
represented by Formula (8):
##STR00015##
[0031] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which has a structure as
represented by Formula (9):
##STR00016##
[0032] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which has a structure as
represented by Formula (10):
##STR00017##
[0033] Further, the present disclosure also provides using any one
of the above-mentioned preparation processes to prepare an
intermediate of antibody-drug conjugate, which has a structure as
represented by Formula (11):
##STR00018##
[0034] In some particular embodiments, the process comprises the
following steps:
[0035] Reaction A: dissolving Py-MAA-Val-Cit-PAB in DMF, adding
bis(p-nitrophenyl)carbonate, stirring until they are dissolved or
slightly turbid before adding N,N-diisopropylethylamine dropwise,
separating and purifying to obtain Py-MAA-Val-Cit-PAB-PNP;
[0036] Reaction B: dissolving Py-MAA-Val-Cit-PAB-PNP in DMF, adding
1-hydroxybenzotriazole, MMAE, N,N-diisopropylethylamine and
pyridine, carrying out pre-HPLC purification after completion of
the reaction to obtain compound Py-MAA-Val-Cit-PAB-MMAE as
represented by Formula (1).
[0037] wherein the structure of the Py-MAA-Val-Cit-PAB-PNP is shown
as follow:
##STR00019##
[0038] Furthermore, the separation and purification in the
above-mentioned reaction process may be as follows: [0039] (1)
centrifuging the reaction solution of Reaction A to obtain a
supernatant; [0040] (2) adding solvent 1 to the supernatant, and
stirring well; [0041] (3) adding solvent 2 dropwise at 0 to
10.degree. C., and stirring well; [0042] (4) filtering under
suction to obtain a filter cake; [0043] (5) re-dissolving the
filter cake in a mixed solution of solvent 3 and solvent 4; [0044]
(6) adding the resultant dropwise to solvent 5 at 0 to 10.degree.
C., and stirring; [0045] (7) filtering under suction to obtain an
off-white powder, which is purified Py-MAA-Val-Cit-PAB-PNP.
[0046] Thereinto, the solvent 1 is a moderately polar solvent, In
one embodiment selected from the group consisting of ethyl acetate,
dichloromethane, methanol, methyl tert-butyl ether and the like,
most In one embodiment ethyl acetate;
[0047] the solvent 2 is a weakly polar solvent, In one embodiment
selected from the group consisting of petroleum ether, n-hexane,
n-pentane, cyclohexane and the like, most In one embodiment
petroleum ether;
[0048] the solvents 3 and 4 are solvents in which
Py-MAA-Val-Cit-PAB-PNP is easy to dissolve, In one embodiment
selected from the group consisting of acetic acid, trifluoroacetic
acid, formic acid, methanol, ethanol and the like, most In one
embodiment acetic acid and methanol;
[0049] the solvent 5 is a solvent in which Py-MAA-Val-Cit-PAB-PNP
is poorly soluble, In one embodiment selected from the group
consisting of acetonitrile, ethyl acetate, dichloromethane, water
and the like, most In one embodiment water.
[0050] The volume/weight ratio of the solvent 1, the solvent 2, the
solvent 3 and the solvent 4 and Py-MAA-Val-Cit-PAB is 40-100:
80-200:5-15: 1-3:15-30:1.
[0051] In some particular embodiments, the above-mentioned
separation and purification may be as follows: [0052] (1)
centrifuging the intermediate product A to obtain a supernatant;
[0053] (2) adding ethyl acetate to the supernatant, and stirring
well; [0054] (3) adding petroleum ether dropwise at 0 to 10.degree.
C., and stirring well; [0055] (4) filtering under suction, and
taking the filter cake; [0056] (5) re-dissolving the filter cake in
a mixed solution of acetic acid and methanol; [0057] (6) adding
purified water dropwise at 0 to 10.degree. C., and stirring; [0058]
(7) filtering under suction to obtain a off-white powder, which is
purified Py-MAA-Val-Cit-PAB-PNP.
[0059] Compared with the process for preparing an intermediate of
the antibody-drug conjugate (Py-MAA-Val-Cit-PAB-MMAD) disclosed by
the Chinese patent publication CN107921030A on page 43 of the
specification, the process for preparing an intermediate of the
antibody-drug conjugate provided by the present disclosure
significantly reduces the feed loss for drug moiety (such as
MMAD/MMAE or MMAF, etc.) as drug moiety is involved in the last
step of the reaction, thereby effectively reducing production
costs, as well as increasing production efficiency. In addition,
the process provided by the present disclosure is simple,
environmentally friendly and suitable for large-scale
industrialization.
DETAILED DESCRIPTION
Abbreviations
TABLE-US-00001 [0060] Abbreviations Structure/English name Py
##STR00020## Triacryloylhexahydro triazine Py-MAA ##STR00021##
Triacryloylhexahydro triazine-mercaptoacetic acid Py-MAA-Val-
Cit-PAB-OH ##STR00022## Triacryloylhexahydro
triazine-mercaptoacetic acid-valine-citrulline-p-aminobenzyl
alcohol Py-MAA-Val- Cit-PAB-PNP ##STR00023## Triacryloylhexahydro
triazine-mercaptoacetic
acid-valine-citrulline-p-aminobenzyloxycarbonyl-p-nitrophenyl ester
Py-MAA-Val- Cit-PAB-MMAE ##STR00024## Triacryloylhexahydro
triazine-mercaptoacetic
acid-valine-citrulline-p-aminobenzyloxycarbonyl Monomethyl
auristatin E MMAE ##STR00025## MMAD ##STR00026## MMAF ##STR00027##
NPC ##STR00028## Bis(4-nitrophenyl) carbonate Anthramycin
##STR00029## Daunorubicin ##STR00030## Doxorubicin ##STR00031##
Epirubicin ##STR00032## Idarubicin ##STR00033## Valrubicin
##STR00034## Mitoxantrone ##STR00035## puromycin ##STR00036##
Definitions
[0061] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as interpreted by a person having
ordinary skill in the art.
[0062] Although numerical ranges and parameter approximations are
shown in a broad scope in the present disclosure, the numerical
values shown in specific embodiments are described as being as
accurate as possible. However, any numerical value inherently
contains certain errors due to the standard deviations present in
their respective measurements. In addition, all ranges disclosed
herein are to be interpreted as encompassing any and all
sub-ranges. For example, the stated range of "1 to 10" should be
considered to contain any and all subranges between the minimum 1
and the maximum 10 (including the endpoints); that is all subranges
starting with a minimum of 1 or greater, such as 1 to 6.1, and
subranges ending with a maximum of 10 or less, such as 5.5 to 10.
In addition, any reference that is referred to as "incorporated
herein" is interpreted to be incorporated in its entirety.
[0063] In addition, it should be noted that, as used in the
specification, the singular forms include the plural forms of the
subject matter, unless clearly and explicitly limited to one
subject matter. The term "or" can be used interchangeably with the
term "and/or" unless the context clearly dictates otherwise.
[0064] The term "antibody-drug conjugate" as used in the present
disclosure represents a compound in which an antibody/antibody
functional fragment, a linker, and a drug moiety are chemically
linked together, and the structure thereof usually consists of
three parts: an antibody or antibody ligand, a drug moiety, and a
linker that couples the antibody or antibody ligand and the drug.
Currently the preparation of antibody-drug conjugates is usually
divided into two steps: the first step is to chemically react a
linker with a drug moiety to form a "linker-drug" conjugate, and
the second step is to covalently couple the linker part of the
"linker-drug" conjugate to an antibody/antibody functional fragment
via a thiol or amino group. The term "intermediate of antibody-drug
conjugate" as used in the present disclosure refers to the
"linker-drug" conjugate as described above, and further, the
"intermediate of antibody-drug conjugate" referred to in the
present disclosure generally refers to those "linker-drug"
conjugate in which a linker and a drug are coupled together by a
"--CO--NH--" bond via amine transesterification.
[0065] The terms "linker" and "linker part" as used in the present
disclosure refers to the part of an antibody-drug conjugate that
links an antibody to a drug, which may be cleavable or
non-cleavable. A cleavable linker (i.e., a linker which is
cleavable or a biodegradable linker) can be cleaved inside or on
the target cell so as to release the drug. In some embodiments, the
linker of the present disclosure is selected from cleavable
linkers, such as a disulfide-based linker (which is selectively
cleaved in tumor cells which have higher concentration of
sulfhydryl groups), a peptide linker (which is cleaved by an enzyme
in tumor cells), or a hydrazone linker. In another embodiments, the
linker of the present disclosure is selected from non-cleavable
linkers (i.e. a linker which is not cleavable), such as a thioether
linker. In another embodiments, the linker of the present
disclosure is a combination of a cleavable linker and a
non-cleavable linker.
[0066] The terms "drug" and "drug moiety" as used in the present
disclosure generally refers to any compound having desired
biological activity and reactive functional group for the
preparation of the conjugates of the present disclosure. The
desired biological activity includes diagnosing, curing,
alleviating, treating, preventing diseases in humans or other
animals. As novel drugs are continuously discovered and developed,
these novel drugs should also be included in the drugs of the
present disclosure. Specifically, the drug includes, but is not
limited to, cytotoxic drugs, cell differentiation factors, stem
cell trophic factors, steroid drugs, drugs for treating autoimmune
diseases, anti-inflammatory drugs, or drugs for infectious
diseases. More specifically, the drug includes, but is not limited
to, tubulin inhibitors or DNA, RNA damaging agents.
EXAMPLES
[0067] The followings are examples of the methods and compositions
of the present disclosure. It should be understood that various
other embodiments may be implemented in light of the above
definitions and the general description.
Example 1 Preparation of Py-MAA
##STR00037##
[0069] Compound Py (1.87 g, 7.51 mmol) (CAS: 959-52-4) and
Et.sub.3N (104 .mu.L, 0.75 mmol) were dissolved in anhydrous
CH.sub.2Cl.sub.2 (40 mL), a solution of thioglycolic acid (103.9
.mu.L, 1.50 mmol) in CH.sub.2Cl.sub.2 (40 mL) was added dropwise.
After completion of the addition, the temperature of the system was
raised to room temperature and it was stirred overnight. After
completion of the reaction, the solvent was removed in vacuo, and
the crude product was purified by column chromatography to obtain
white solid Py-MAA (1.87 g, 40.1%).
Example 2 Comparative Example
[0070] Preparation of Py-MAA-Val-Cit-PAB-MMAE
(which is the process disclosed on page 32 of the specification of
the Chinese patent publication CN107921030A).
[0071] The procedure and method are shown as below:
##STR00038##
[0072] (1) Preparation of Fmoc-Val-Cit-PAB-MMAE
[0073] Fmoc-Val-Cit-PAB-PNP (388.4 mg, 0.507 mmol) was taken and
dissolved in DMF (40 m) under the protection of nitrogen at
0.degree. C., and HOBt (68.4 mg, 0.507 mmol) and compound MMAE
(400.0 mg, 0.558 mmol) were added. After 15 minutes, pyridine (8
ml) and DIPEA (106.2 .mu.L, 0.608 mmol) were added and reacted at
0.degree. C. for 30 minutes. The temperature was increased to room
temperature and it was stirred for 3 hours. The reaction mixture
was supplemented with DIPEA (106.2 .mu.L, 0.608 mmol) and the
reaction was continued at room temperature for 24 hours. After
completion of the reaction, the mixture was concentrated in vacuo.
The crude product was purified by column chromatography to obtain a
white solid compound Fmoc-Val-Cit-PAB-MMAE (325 mg, yield
47.7%).
[0074] (2) Preparation of Val-Cit-PAB-MMAE
[0075] Compound Fmoc-Val-Cit-PAB-MMAE (325 mg, 0.289 mmol) was
dissolved in DMF (6 mL), before diethylamine (3 mL) was added, and
the reaction was continued with stirring for 2.5 hour. After
completion of the reaction, the solvent was removed under reduced
pressure to obtain a crude product Val-Cit-PAB-MMAE (220 mg, yield:
67.8%), which can be used for the next reaction directly without
purification.
3) Synthesis of Py-MAA-Val-Cit-PAB-MMAE
[0076] Compound Py-MAA (80.2 mg, 0.235 mmol), HOBt (31.8 mg, 0.235
mmol) and DIC (29.6 mg, 0.235 mmol) were taken and dissolved in DMF
(10 mL) and cooled to 0.degree. C. in an ice bath. Compound
Val-Cit-PAB-MMAE (220.0 mg, 0.196 mmol) and DIPEA (25.3 mg, 0.196
mmol) were added to the reaction solution, the temperature was
raised to room temperature and the reaction was performed for 24
hours. After completion of the reaction, the crude product was
purified by column chromatography
(methanol:dichloromethane=60:1-30:1) to obtain a white solid
compound Py-MAA-Val-Cit-PAB-MMAE (147.4 mg, yield 52%). LCMS m/z
(ES+), 1445.78 (M+H)+.
[0077] Py-MAA-Val-Cit-PAB-MMAE was prepared by the process for
preparing Py-MAA-Val-Cit-PAB-MMAD as disclosed on page 32 of the
specification of Chinese patent publication CN107921030A. The feed
amount of MMAE was 400 mg, and the obtained Py-MAA-Val-Cit-PAB-MMAE
was 147.4 mg.
Example 3 Improved Preparation Method of
Py-MAA-Val-Cit-PAB-MMAE
[0078] (1) Preparation of Py-MAA-Val-Cit-PAB
##STR00039##
[0079] Compound Py-MAA (10.00 g, 29.33 mmol) was put in
tetrahydrofuran (200 mL), before N,N'-Carbonyldiimidazole (7.13 g,
44.00 mmol) and Val-Cit-PAB-OH (13.34 g, 35.20 mmol) were added,
and stirred at room temperature for 24 hours. Petroleum ether (200
mL) was added, stirred for 0.5 hours, and filtered to obtain a
white solid. The white solid was purified by preparative high
performance liquid chromatography, the resultant was subjected to
rotary evaporation to obtain Py-MAA-Val-Cit-PAB-OH (6.67 g, 32.4%,
a white solid powder).
[0080] (2) Preparation of Py-MAA-Val-Cit-PAB-PNP
##STR00040##
[0081] Py-MAA-Val-Cit-PAB-OH (10.00 g, 1.0 eq) and DMF
(V.sub.DMF/W.sub.Py-MAA-Val-Cit-PAB=15-25 mL/g) were added into a
three neck flask under the protection of nitrogen, stirred under
the protection of nitrogen until the solid was dissolved or the
solution was slightly turbid. The temperature was reduced to
10.degree. C. for the three neck flask, NPC (1.5-2.5 eq, 6.50-10.83
g) solid was added in one portion and stirred until dissolved or
until the solution was slightly turbid. DIPEA (1.0-1.5 eq,
1.84-2.76 g) was added dropwise under the protection of nitrogen.
The temperature was raised to 30.degree. C., and the reaction was
monitored by UPLC until the purity of Py-MAA-Val-Cit-PAB was less
than 5.0%, then the reaction was terminated.
[0082] The reaction solution was centrifuged, and the supernatant
was poured into a beaker of suitable size, into which EA
(V.sub.EA/W.sub.Py-MAA-Val-Cit-PAB=60.+-.2) was added and stirred
with an electric mixer; PE
(V.sub.PE/W.sub.Py-MAA-Val-Cit-PAB=120.+-.2) was added dropwise at
0 to 6.degree. C., and stirred for 20 to 30 minutes after dropwise
addition. After suction filtration, the filter cake was taken, and
dissolved with acetic acid (V.sub.AcOH/W.sub.Py-MAA-Val-Cit-PAB=7)
and methanol (V.sub.MeOH/W.sub.Py-MAA-Val-Cit-PAB) to obtain a
solution.
[0083] Purified water (V.sub.water/W.sub.Py-MAA-Val-Cit-PAB=21) was
poured into a beaker of suitable size, mechanically stirred, and
cooled to about 3.degree. C. The above-mentioned solution was added
dropwise to purified water and stirred for 15 minutes after the
dropwise addition. After suction filtration, the filter cake was
obtained and lyophilized to give Py-MAA-Val-Cit-PAB-PNP (a white to
off-white powder, 10.69 g, yield 86.54%).
[0084] (3) Preparation of Py-MAA-Val-Cit-PAB-MMAE
##STR00041##
[0085] Py-MAA-Val-Cit-PAB-PNP (9.00 g, 1.0 eq) and DMF
(V.sub.DMF/W.sub.Py-MAA-Val-Cit-PAB-PNP=15.0-25.0 mL/g) were added
into a reaction vessel, stirred under the protection of nitrogen
until clear or slightly turbid. The internal temperature was
maintained at 10.degree. C., while HOBt (2.10 g, 1.5 eq) was added
and stirred for 20 minutes. MMAE (8.93 g, 1.2 eq) was added, and
the stirring was continued for 20 minutes. A mixed solution of
DIPEA (1.34 g, 1.0 eq) and pyridine (V.sub.pyridineV.sub.DIPEA=5)
was added dropwise, and the temperature was raised to maintain the
internal temperature at 20.degree. C. After 36 hours of reaction,
UPLC monitoring was performed once every other hour, and when the
purity of MMAE in the reaction solution was <2.00%, the reaction
was terminated.
[0086] A suitable sized beaker was taken, into which a saturated
aqueous sodium chloride solution (referred to as brine,
V.sub.brine/V.sub.reaction solution=10) was added, and the
temperature was reduced until the internal temperature was
5.degree. C. The reaction solution was added dropwise to saturated
sodium chloride solution. A crude product was obtained by
centrifugation, and purified using preparative high performance
liquid chromatography. After lyophilization,
Py-MAA-Val-Cit-PAB-MMAE (a white powder, 9.79 g, yield 65.27%) was
obtained.
[0087] In the present example, the feed amount of MMAE was 8.93 g,
with the obtained amount of Py-MAA-Val-Cit-PAB-MMAE being 9.79
g.
[0088] Comparing the processes provided in Example 2 and Example 3
in terms of the feed amount of MMAE and the mass of the final
product (the comparison results are shown in Table 1), it can be
seen from Table 1 that the final obtained amount of
Py-MAA-Val-Cit-PAB-MMAE prepared by the preparation process of
Example 2 was 147.4 mg while the feed amount of MMAE was 400 mg;
the final obtained amount of Py-MAA-Val-Cit-PAB-MMAE prepared by
the preparation process of Example 3 was 9.79 g while the feed
amount of MMAE was 8.93 g.
TABLE-US-00002 TABLE 1 Comparison of Feed Amount of MMAE and
Obtained Amount of Final Product Input-output Percentage Ratio of
Reduction MMAE in Feed Final (Feed Amount Amount Obtained of
MMA/Final compared Amount of Obtained with Feed Py-MAA- Amount of
Comparative Amount Val-Cit- Py-MAA-Val- Example Examples of MMAE
PAB-MMAE Cit-PAB-MMAE (Example 2) Example 2 400 mg 147.4 mg 2.71 --
Example 3 8.93 g 9.79 g 0.91 66.4%
[0089] The input-output rate of MMAE represents how many units of
MMAE are required to be fed in order to obtain each unit mass of
Py-MAA-Val-Cit-PAB-MMAE. As can be seen from Table 1, by using the
method of Example 2, 2.71 unit mass of MMAE is required to obtain 1
unit mass of Py-MAA-Val-Cit-PAB-MMAE; while by adopting the method
of Example 3, only 0.91 unit mass of MMAE is required to be fed.
The feed amount reduced by 66.4%, which significantly reduces
production costs.
[0090] The above described is merely one embodiment, which is only
illustrative and not limiting the combinations of features
necessary to practice the invention. The provided title is not to
limit the various embodiments of the invention. Terms such as
"comprising" and "including" are not intended to limit. In
addition, unless otherwise indicated, plural forms is included when
there is no numeral modification, and "or" means "and/or". Unless
otherwise defined herein, the meaning of all technical and
scientific terms used herein is the same as what is commonly
interpreted by a person having ordinary skill in the art.
[0091] All publications and patents mentioned in this application
are hereby incorporated by reference. Numerous modifications and
variations of the described methods and compositions of the present
invention are obvious to a person having ordinary skill in the art.
Although the invention has been described by way of embodiments, it
should be understood that the invention as claimed should not be
unduly limited to these specific embodiments. In fact, those
numerous modifications and variations used to practice the modes of
the present invention, which are obvious to a person having
ordinary skill in the art, are intended to be included within the
scope of the appended claims.
* * * * *