U.S. patent application number 16/630080 was filed with the patent office on 2021-07-29 for oligopeptide linker intermediate and preparation method thereof.
This patent application is currently assigned to MABPLEX INTERNATIONAL, LTD.. The applicant listed for this patent is MABPLEX INTERNATIONAL, LTD.. Invention is credited to Changjiang HUANG, Lele LI.
Application Number | 20210230218 16/630080 |
Document ID | / |
Family ID | 1000005551675 |
Filed Date | 2021-07-29 |
United States Patent
Application |
20210230218 |
Kind Code |
A1 |
LI; Lele ; et al. |
July 29, 2021 |
OLIGOPEPTIDE LINKER INTERMEDIATE AND PREPARATION METHOD THEREOF
Abstract
The invention provides a new oligopeptide linker intermediate
and a preparation method thereof. The preparation method of the
oligopeptide intermediate is easily carried out under mild reaction
conditions, and since almost no side reactions occur in the
reaction, the method produces a high-purity product with fewer
impurities and easy to be purified, achieving unexpected technical
effects.
Inventors: |
LI; Lele; (Yantai, Shandong,
CN) ; HUANG; Changjiang; (Yantai, Shandong,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MABPLEX INTERNATIONAL, LTD. |
China (Shandong) |
|
CN |
|
|
Assignee: |
MABPLEX INTERNATIONAL, LTD.
China (Shandong)
CN
|
Family ID: |
1000005551675 |
Appl. No.: |
16/630080 |
Filed: |
October 23, 2019 |
PCT Filed: |
October 23, 2019 |
PCT NO: |
PCT/CN2019/112671 |
371 Date: |
January 10, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 5/1027 20130101;
A61K 47/6803 20170801; A61K 47/65 20170801; C07K 5/0827 20130101;
C07K 5/06191 20130101; C07K 1/063 20130101 |
International
Class: |
C07K 5/06 20060101
C07K005/06; C07K 5/08 20060101 C07K005/08; C07K 5/10 20060101
C07K005/10; A61K 47/68 20060101 A61K047/68; A61K 47/65 20060101
A61K047/65; C07K 1/06 20060101 C07K001/06 |
Claims
1. An oligopeptide linker intermediate having the structure
represented by formulas (1)-(4): ##STR00048## wherein, the
AA.sub.1, AA.sub.2, AA.sub.3, AA.sub.4 are any amino acid.
2. The oligopeptide linker intermediate according to claim 1,
wherein the AA.sub.1, AA.sub.2, AA.sub.3, and AA.sub.4 are each
independently selected from the group consisting of -valine-
(-Val-), -citrulline -(-Cit-), -alanine- (-Ala-), -lysine- (-Lys-),
-lysine(trityl)- (-Lys(Trt)-),
-lysine(monomethoxytrityl)-(-Lys(Mmt)-),
-lysine(fluorenylmethyloxycarbonyl)- (-Lys(Fmoc)-), -arginine-
(-Arg-), -phenylalanine- (-Phe-), -glycine- (-Gly-), -leucine-
(-Leu-) and -isoleucine- (-Ile-).
3. The oligopeptide linker intermediate according to claim 2,
wherein the -AA.sub.1-AA.sub.2- is selected from the group
consisting of -valine-citrulline- (-Val-Cit-),
-valine-alanine-(-Val-Ala-), -valine-lysine- (-Val-Lys-),
-valine-lysine(trityl)- (-Val-Lys(Trt)-),
-valine-lysine(monomethoxytrityl)-(-Val-Lys(Mmt)-),
-valine-lysine(fluorenylmethyloxycarbonyl)-(-Val-Lys(Fmoc)-),
-valine-arginine- (-Val-Arg-),
-phenylalanine-citrulline-(-Phe-Cit-), -phenylpropyl-lysine-
(-Phe-Lys-), -phenylalanine-lysine(trityl)-(-Phe-Lys(Trt)-),
-phenylalanine-lysine(monomethoxytrityl)- (-Phe-Lys (Mmt)-),
-phenylalanine-lysine (fluorenylmethyloxycarbonyl)-
(-Phe-Lys(Fmoc)-), leucine-citrulline- (-Leu-Cit-),
isoleucine-citrulline- (-Ile-Cit-) and -phenylalanine-arginine-
(-Phe-Arg-); the -AA.sub.1-AA.sub.2-AA.sub.3- is
-phenylalanine-arginine-arginine-(-Ala-Arg-Arg-); and the
-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is selected from the group
consisting of
-glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-),
-glycine-phenylalanine-leucine-glycine- (-Gly-Phe-Leu-Gly-) and
-alanine-leucine-alanine-leucine (-Ala-Leu-Ala-Leu-).
4. The oligopeptide linker intermediate according to claim 3,
wherein the oligopeptide linker intermediate includes the following
structure: ##STR00049## ##STR00050## ##STR00051## ##STR00052##
5. An antibody-drug conjugate having a linker, wherein the
precursor of the linker used in the antibody-drug conjugate is the
oligopeptide linker intermediate according to claim 1.
6. A method for preparing the oligopeptide linker intermediate
according to claim 1, wherein the method for preparing the
oligopeptide linker intermediate of formulas (1) to (3) comprising:
1) performing a condensation reaction between a carbonylation
reagent, 2-(trimethylsilyl)ethanol and amino acid AA.sub.1, and
then amino acid AA.sub.2, or amino acid AA.sub.1, amino acid
AA.sub.2 and amino acid AA.sub.3 in sequence, or amino acid
AA.sub.1, amino acid AA.sub.2, amino acid AA.sub.3 and amino acid
AA.sub.4 in sequence, to obtain a
2-(trimethylsilyl)ethoxycarbonyl-oligopeptide condensate; 2)
reacting the resulting
2-(trimethylsilyl)ethoxycarbonyl-oligopeptide condensate and
p-aminobenzyl alcohol to obtain a
2-(trimethylsilyl)ethoxycarbonyl-oligopeptide-p-aminobenzyl alcohol
condensate, wherein the carbonylation reagent is any compound
containing a carbonyl group; or a method for preparing the
oligopeptide linker intermediate of formula (4) comprising: 1')
performing a condensation reaction between a carbonylation reagent,
2-(trimethylsilyl)ethanol and amino acid AA.sub.1, amino acid
AA.sub.2, amino acid AA.sub.3 and amino acid AA.sub.4 in sequence,
to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide
condensate, wherein the carbonylation reagent is any compound
containing a carbonyl group.
7. The method according to claim 6, wherein the oligopeptide linker
intermediate of formula (1) is obtained via the following reaction
path: ##STR00053## wherein the method comprises the following
steps: 1) performing a condensation reaction between a
carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid
AA.sub.1 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-amino acid
condensate; 2) performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino
acid AA.sub.2 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; and 3)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and
p-aminobenzyl alcohol to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide-p-aminobenzyl alcohol
condensate.
8. The method according to claim 6, wherein the oligopeptide linker
intermediate of formula (2) is obtained via the following reaction
path: ##STR00054## wherein the method comprises the following
steps: 1) performing a condensation reaction between a
carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid
AA.sub.1 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-amino acid
condensate; 2) performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino
acid AA.sub.2 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; 3)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino
acid AA.sub.3 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; and 4)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and
p-aminobenzyl alcohol to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tripeptide-p-aminobenzyl alcohol
condensate.
9. The method according to claim 6, wherein the oligopeptide linker
intermediate of formula (3) is obtained via the following reaction
path: ##STR00055## wherein the method comprises the following
steps: 1) performing a condensation reaction between a
carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid
AA.sub.1 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-amino acid
condensate; 2) performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino
acid AA.sub.2 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; 3)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino
acid AA.sub.3 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; 4)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and amino
acid AA.sub.4 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate; and 5)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate and
p-aminobenzyl alcohol to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide-p-aminobenzyl alcohol
condensate.
10. (canceled)
11. The method according to claim 6, wherein the oligopeptide
linker intermediate of formula (4) is obtained via the following
reaction path: ##STR00056## 1) performing a condensation reaction
between a carbonylation reagent, 2-(trimethylsilyl)ethanol, and
amino acid AA.sub.1 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate; 2)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino
acid AA.sub.2 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; 3)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino
acid AA.sub.3 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; and 4)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and amino
acid AA.sub.4 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate.
12. The method according to claim 6, wherein the carbonylation
reagent has a structure of formula (5): ##STR00057## wherein: the
R.sub.1 and R.sub.2 are each independently selected from the group
consisting of: ##STR00058##
13. The method according to claim 12, wherein the carbonylation
reagent is selected from the group consisting of: ##STR00059##
14. The method according to claim 6, wherein the AA.sub.1,
AA.sub.2, AA.sub.3, and AA.sub.4 are each independently selected
from the group consisting of: -valine-(-Val-), -citrulline
-(-Cit-), -alanine- (-Ala-), -lysine- (-Lys-), -lysine(trityl)-
(-Lys(Trt)-), -lysine(monomethoxytrityl)-(-Lys(Mmt)-),
-lysine(fluorenylmethyloxycarbonyl)- (-Lys(Fmoc)-), -arginine-
(-Arg-), -phenylalanine- (-Phe-), -glycine- (-Gly-), -leucine-
(-Leu-) and -isoleucine- (-Ile-).
15. The method according to claim 14, wherein the
-AA.sub.1-AA.sub.2- is selected from the group consisting of
-valine-citrulline- (-Val-Cit-), -valine-alanine-(-Val-Ala-),
-valine-lysine- (-Val-Lys-), -valine-lysine(trityl)-
(-Val-Lys(Trt)-),
-valine-lysine(monomethoxytrityl)-(-Val-Lys(Mmt)-),
-valine-lysine(fluorenylmethyloxycarbonyl)-(-Val-Lys(Fmoc)-),
-valine-arginine- (-Val-Arg-),
-phenylalanine-citrulline-(-Phe-Cit-), -phenylpropyl-lysine-
(-Phe-Lys-), -phenylalanine-lysine(trityl)-(-Phe-Lys(Trt)-),
-phenylalanine-lysine(monomethoxytrityl)- (-Phe-Lys (Mmt)-),
-phenylalanine-lysine (fluorenylmethyloxycarbonyl)-
(-Phe-Lys(Fmoc)-), leucine-citrulline- (-Leu-Cit-),
isoleucine-citrulline- (-Ile-Cit-) and -phenylalanine-arginine-
(-Phe-Arg-); the -AA.sub.1-AA.sub.2-AA.sub.3- is
-phenylalanine-arginine-arginine-(-Ala-Arg-Arg-); and the
-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is selected from the group
consisting of
-glycine-glycine-phenylalanine-glycine-(-Gly-Gly-Phe-Gly-),
-glycine-phenylalanine-leucine-glycine- (-Gly-Phe-Leu-Gly-), and
-alanine-leucine-alanine-leucine (-Ala-Leu-Ala-Leu-).
16. The method of claim 15, wherein the -AA.sub.1-AA.sub.2- is
selected from the following structures: ##STR00060## ##STR00061##
##STR00062##
17. The method according to claim 15, wherein the
-AA.sub.1-AA.sub.2-AA.sub.3- is ##STR00063##
18. The method according to claim 15, wherein the
-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is selected from the
following structures: ##STR00064##
19. The method according to claim 6, wherein solvent used in the
condensation reaction is a polar solvent or non-polar solvent;
preferably, the solvent is one or more selected from the group
consisting of tetrahydrofuran, dioxane, acetonitrile, DMF, DMSO,
DMAc, DMPU, HMPA, ethylene glycol dimethyl ether, diethyl ether,
tert-butyl methyl ether, tert-butanol, water, ethyl acetate,
methanol, ethanol, isopropanol, dichloromethane, chloroform, and
carbon tetrachloride; more preferably, the solvent is one or more
selected from the group consisting of tetrahydrofuran, dioxane,
acetonitrile, DMF, DMSO, water, methanol, dichloromethane,
chloroform, carbon tetrachloride and ethanol.
20-21. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of antibody-drug
conjugates, and in particular, to a method for preparing an
oligopeptide linker and an intermediate thereof.
BACKGROUND OF THE INVENTION
[0002] Antibody-Drug Conjugate (ADC), as a new type of biological
missile, achieves an advantageous combination of the targeting
effect of monoclonal antibodies and the cytotoxicity effect of
small molecule drugs, and has now become one of the fastest
developing fields in tumor targeting therapy. The three components
(an antibody, a cytotoxin and a linker) of an ADC constitute a
targeted drug delivery system, in which the construction and
optimization of linkers are crucial for the development of such
system. Linkers are the basis for the effective delivery of
cytotoxic drugs through ADCs, and the key factor in determining the
toxicity of ADC products, because the premature release of drugs
into the blood circulation may lead to systemic toxicity and a
lower therapeutic index. By optimizing the existing coupling
technology, developing stable linkers, and exploring new toxin
release mechanisms, it is possible to solve the problems such as
off-target action of toxins and drug resistance commonly observed
in ADC, thereby further improving the efficacy and safety.
[0003] At the present stage, oligopeptide-based cleavable ADCs
which are degraded under the action of cathepsin B in the lysosomes
of target cells to release toxins to ultimately kill tumors are the
main focus (Reference 1: Wang Y, Fan S, Zhong W, et al. Development
and properties of valine-alanine based antibody-drug conjugates
with monomethyl auristatine as the potent payload[J]. International
journal of molecular sciences, 2017, 18(9): 1860.). Among them,
oligopeptide linkers (such as valine-citrulline dipeptide (Val-Cit,
VC)) have been widely used in various ADC drugs. In the five
antibody-conjugated drugs currently approved (as of July 2019)
(Table 1), the linkers of Brentuximab vedotin of Seattle &
Takeda and Polatuzumab vedotin-piiq of Genentech are oligopeptide
linkers (valine-citrulline dipeptide linker).
TABLE-US-00001 TABLE 1 Commercially available antibody drug
conjugates Time to Generic name Company market Target Linker Drug
moiety Brentuximab Seattle, 2011 CD30 Mc-Val-Cit-PAB MMAE vedotin
Takeda Trastuzumab Genentech 2013 Her2 MCC (thioether linker) DM1
emtansine Inotuzumab Pfizer 2017 CD22 4-(4-acetylphenoxy)
Ozogamicin ozogamicin butanoic acid Gemtuzumab Pfizer 2017 CD33
4-(4-acetylphenoxy) Ozogamicin ozogamicin butanoic acid Polatuzumab
Genentech 2019 CD79b Mc-Val-Cit-PAB MMAE vedotin-piiq
[0004] There are currently three main methods for synthesizing
oligopeptide linkers (Reference 2: Mondal D, Ford J, Pinney K G.
Improved Methodology for the Synthesis of a Cathepsin B Cleavable
Dipeptide Linker, Widely Used in Antibody-Drug Conjugate Research
[J]. Tetrahedron letters, 2018, 59 (40): 3594-3599.), in which the
first method uses Fmoc protecting group, the second uses Cbz
protecting group, and the third uses Boc protecting group.
[0005] The above three preparation processes are described below
using valine-citrulline dipeptide linkers as examples:
[0006] (1) Using Fmoc Protecting Group
##STR00001##
[0007] In this method, Fmoc-Val is first condensed with Cit to
obtain Fmoc-Val-Cit, which then is condensed with p-aminobenzyl
alcohol to obtain Fmoc-Val-Cit-PAB, and then Fmoc is removed to
give Val-Cit-PAB. However, lower amines are usually used in this
method to remove the Fmoc protecting group, so that byproducts are
easily formed and hard to completely remove. In addition, Fmoc has
strong UV absorption, and trace residues have a great impact on the
purity test of the product.
[0008] (2) Using Cbz Protecting Group
##STR00002##
[0009] In this method, Cbz-Val is first condensed with Cit to
obtain Cbz-Val-Cit, which then is condensed with p-aminobenzyl
alcohol to obtain Cbz-Val-Cit-PAB, and then Val-Cit-PAB is obtained
by removing Cbz. However, transition metal, usually Pd, is required
as the catalyst when removing the Cbz protecting group in this
method, which is harmful to the human body by resulting in heavy
metal residues which is hard to remove. In addition, a hydrogen
source is required, usually hydrogen gas, which is extremely
explosive, leading to high safety risks and unsuitable for
large-scale use. Although other hydrogen sources may be used, some
hydrogen gas is also released during use, bringing safety
risks.
[0010] (3) Using Boc Protecting Group
##STR00003##
[0011] In this method, Boc-Val is first condensed with Cit to
obtain Boc-Val-Cit, which then is condensed with p-aminobenzyl
alcohol to obtain Boc-Val-Cit-PAB, and then Val-Cit-PAB is obtained
by removing Boc. This method uses strong acid, usually HCl solution
and trifluoroacetic acid, to remove the Boc protecting group. When
using HCl, the alcoholic hydroxyl at the benzyl position of the
Boc-Val-Cit-PAB molecule will be replaced by chlorine to form the
corresponding benzyl chloride, which would affects product quality.
When using trifluoroacetic acid, the alcohol at the benzyl position
will undergo an esterification reaction to give the corresponding
trifluoroacetate, therefore an additional step of hydrolysis
reaction is required to obtain Val-Cit-PAB. In addition, the basic
hydrolysis has the risk of racemizing the chiral center of the
amino acid units.
SUMMARY OF THE INVENTION
[0012] In order to solve the above problems, the present invention
provides a new method for preparing oligopeptide linkers. The
method for preparing Val-Cit-PAB using Teoc as the protecting group
provided by the present invention is easily carried out under mild
reaction conditions, and since almost no side reactions occur in
the reaction, the method produces a high-purity product with fewer
impurities and easy to be purified, achieving unexpected technical
effects.
[0013] Specifically, the present invention provides an oligopeptide
linker intermediate, with the structure as shown in formulas
(1)-(4):
##STR00004##
[0014] wherein, the AA.sub.1, AA.sub.2, AA.sub.3 and AA.sub.4 are
any amino acid.
[0015] Further, the AA.sub.1, AA.sub.2, AA.sub.3, and AA.sub.4 are
independently selected from the group consisting of -valine-
(-Val-), -citrulline -(-Cit-), -alanine- (-Ala-), -lysine-(-Lys-),
-lysine(trityl)- (-Lys(Trt)-), -lysine(monomethoxytrityl)-
(-Lys(Mmt)-), -lysine(fluorenylmethyloxycarbonyl)- (-Lys(Fmoc)-),
-arginine- (-Arg-), -phenylalanine- (-Phe-), -glycine- (-Gly-),
-leucine- (-Leu-), and -isoleucine- (-Ile-).
[0016] Furthermore, the -AA.sub.1-AA.sub.2- is selected from the
group consisting of -valine-citrulline- (-Val-Cit-),
-valine-alanine- (-Val-Ala-), -valine-lysine- (-Val-Lys-),
-valine-lysine(trityl)- (-Val-Lys(Trt)-),
-valine-lysine(monomethoxytrityl)-(-Val-Lys(Mmt)-),
-valine-lysine(fluorenylmethyloxycarbonyl)- (-Val-Lys(Fmoc)-),
-valine-arginine- (-Val-Arg-), -phenylalanine-citrulline-
(-Phe-Cit-), -phenylpropyl-lysine- (-Phe-Lys-),
-phenylalanine-lysine(trityl)- (-Phe-Lys(Trt)-),
-phenylalanine-lysine(monomethoxytrityl)- (-Phe-Lys (Mmt)-),
-phenylalanine-lysine (fluorenylmethyloxycarbonyl)-
(-Phe-Lys(Fmoc)-), leucine-citrulline- (-Leu-Cit-),
isoleucine-citrulline- (-Ile-Cit-) and -phenylalanine-arginine-
(-Phe-Arg-); the -AA.sub.1-AA.sub.2-AA.sub.3- is selected from
-phenylalanine-arginine-arginine-(-Ala-Arg-Arg-); the
-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is selected from the group
consisting of -glycine-glycine-phenylalanine-glycine-
(-Gly-Gly-Phe-Gly-), -glycine-phenylalanine-leucine-glycine-
(-Gly-Phe-Leu-Gly-), and -alanine-leucine-alanine-leucine
(-Ala-Leu-Ala-Leu-).
[0017] Preferably, the oligopeptide linker intermediate includes
the following structure:
##STR00005## ##STR00006## ##STR00007## ##STR00008##
##STR00009##
[0018] The present invention also provides a method for preparing
the oligopeptide linker intermediate of formulae (1) to (3),
wherein the reaction route of the method is as follows: [0019] 1)
performing a condensation reaction between a carbonylation reagent,
2-(trimethylsilyl)ethanol and amino acid AA.sub.1, and then amino
acid AA.sub.2, or amino acid AA.sub.1, amino acid AA.sub.2 and
amino acid AA.sub.3 in sequence, or amino acid AA.sub.1, amino acid
AA.sub.2, amino acid AA.sub.3 and amino acid AA.sub.4 in sequence,
to obtain a 2-(trimethylsilyl)ethoxycarbonyl-oligopeptide
condensate; [0020] 2) reacting the resulting
2-(trimethylsilyl)ethoxycarbonyl-oligopeptide condensate and
p-aminobenzyl alcohol to obtain a
2-(trimethylsilyl)ethoxycarbonyl-oligopeptide-p-aminobenzyl alcohol
condensate, [0021] wherein the carbonylation reagent is any
compound containing a carbonyl group.
[0022] Further, the oligopeptide linker intermediate of formula (1)
is obtained via the following reaction route:
##STR00010## [0023] wherein the preparation method comprises the
following steps: [0024] 1) performing a condensation reaction
between a carbonylation reagent, 2-(trimethylsilyl)ethanol and
amino acid AA.sub.1 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate; [0025] 2)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino
acid AA.sub.2 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; and [0026]
3) performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and
p-aminobenzyl alcohol to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide-p-aminobenzyl alcohol
condensate.
[0027] Further, the oligopeptide linker intermediate of formula (2)
is obtained via the following reaction route:
##STR00011## [0028] wherein the preparation method comprises the
following steps: [0029] 1) performing a condensation reaction
between a carbonylation reagent, 2-(trimethylsilyl)ethanol and
amino acid AA.sub.1 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate; [0030] 2)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino
acid AA.sub.2 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; [0031] 3)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino
acid AA.sub.3 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; and [0032]
4) performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and
p-aminobenzyl alcohol to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tripeptide-p-aminobenzyl alcohol
condensate.
[0033] Further, the oligopeptide linker intermediate of formula (3)
is obtained via the following reaction route:
##STR00012## [0034] wherein the preparation method comprises the
following steps: [0035] 1) performing a condensation reaction
between a carbonylation reagent, 2-(trimethylsilyl)ethanol and
amino acid AA.sub.1 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate; [0036] 2)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino
acid AA.sub.2 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; [0037] 3)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino
acid AA.sub.3 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; [0038] 4)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and amino
acid AA.sub.4 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate; and
[0039] 5) performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate and
p-aminobenzyl alcohol to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide-p-aminobenzyl alcohol
condensate.
[0040] The present invention further provides a method for
preparing the oligopeptide linker intermediate of formulae (4),
wherein the reaction route of the method is as follows: performing
a condensation reaction between a carbonylation reagent,
2-(trimethylsilyl)ethanol and amino acid AA.sub.1, amino acid
AA.sub.2, amino acid AA.sub.3 and amino acid AA.sub.4 in sequence,
to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide
condensate, wherein the carbonylation reagent is any compound
containing a carbonyl group.
[0041] Further, the oligopeptide linker intermediate of formula (4)
is obtained via the following reaction route:
##STR00013## [0042] 1) performing a condensation reaction between a
carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid
AA.sub.1 to obtain a 2-(trimethylsilyl)ethoxy carbonyl-amino acid
condensate; [0043] 2) performing a condensation reaction between
the 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and
amino acid AA.sub.2 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; [0044] 3)
performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino
acid AA.sub.3 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; and [0045]
4) performing a condensation reaction between the
2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and amino
acid AA.sub.4 to obtain a
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate.
[0046] Further, the carbonylation reagent has a structure of
formula (5):
##STR00014## [0047] wherein: [0048] the R.sub.1 and R.sub.2 are
independently selected from the group consisting of:
##STR00015##
[0049] Furthermore, the carbonylation reagent is selected from the
group consisting of:
##STR00016## ##STR00017##
[0050] Further, the AA.sub.1, AA.sub.2, AA.sub.3, and AA.sub.4 are
independently selected from the group consisting of -valine-
(-Val-), -citrulline -(-Cit-), -alanine- (-Ala-), -lysine-(-Lys-),
-lysine(trityl)- (-Lys(Trt)-), -lysine(monomethoxytrityl)-
(-Lys(Mmt)-), -lysine(fluorenylmethyloxycarbonyl)- (-Lys(Fmoc)-),
-arginine- (-Arg-), -phenylalanine- (-Phe-), -glycine- (-Gly-),
-leucine- (-Leu-), and -isoleucine- (-Ile-).
[0051] Furthermore, the -AA.sub.1-AA.sub.2- is selected from
-valine-citrulline- (-Val-Cit-), -valine-alanine- (-Val-Ala-),
-valine-lysine- (-Val-Lys-),
-valine-lysine(trityl)-(-Val-Lys(Trt)-),
-valine-lysine(monomethoxytrityl)- (-Val-Lys(Mmt)-),
-valine-lysine(fluorenylmethyloxycarbonyl)- (-Val-Lys(Fmoc)-),
-valine-arginine-(-Val-Arg-), -phenylalanine-citrulline-
(-Phe-Cit-), -phenylpropyl-lysine- (-Phe-Lys-),
-phenylalanine-lysine(trityl)- (-Phe-Lys(Trt)-),
-phenylalanine-lysine(monomethoxytrityl)- (-Phe-Lys (Mmt)-),
-phenylalanine-lysine (fluorenylmethyloxycarbonyl)-
(-Phe-Lys(Fmoc)-), leucine-citrulline- (-Leu-Cit-),
isoleucine-citrulline- (-Ile-Cit-) and -phenylalanine-arginine-
(-Phe-Arg-); the -AA.sub.1-AA.sub.2-AA.sub.3- is selected from
-phenylalanine-arginine-arginine-(-Ala-Arg-Arg-); the
-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is selected from the group
consisting of -glycine-glycine-phenylalanine-glycine-
(-Gly-Gly-Phe-Gly-), -glycine-phenylalanine-leucine-glycine-
(-Gly-Phe-Leu-Gly-), and -alanine-leucine-alanine-leucine
(-Ala-Leu-Ala-Leu-).
[0052] Preferably, the -AA.sub.1-AA.sub.2- is selected from the
following structures:
##STR00018## ##STR00019## ##STR00020##
[0053] Preferably, the -AA.sub.1-AA.sub.2-AA.sub.3- is selected
from the following structures:
##STR00021##
[0054] Preferably, the -AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is
selected from the following structures:
##STR00022##
[0055] The present invention further provides the use the
intermediate according to any one of the foregoing contents in the
preparation of an antibody drug conjugate.
[0056] Further, the solvent used in the condensation reaction
described above is any polar solvent or non-polar solvent;
preferably, the solvent is one or more selected from the group
consisting of tetrahydrofuran, dioxane, acetonitrile, DMF, DMSO,
DMAc, DMPU, HMPA, ethylene glycol dimethyl ether, diethyl ether,
tert-butyl methyl ether, tert-butanol, water, ethyl acetate,
methanol, ethanol, isopropanol, dichloromethane, chloroform, and
carbon tetrachloride; more preferably, the solvent is one or more
selected from the group consisting of tetrahydrofuran, dioxane,
acetonitrile, DMF, DMSO, water, methanol, dichloromethane,
chloroform, carbon tetrachloride and ethanol.
[0057] In some specific embodiments, when performing a condensation
reaction between an carbonylation reagent with
2-(trimethylsilyl)ethanol and amino acid AA.sub.1, the reagent used
in the condensation reaction is preferably one or more selected
from the group consisting of tetrahydrofuran, dioxane,
acetonitrile, DMF, DMSO, DMAc, DMPU, HMPA, ethylene glycol dimethyl
ether, diethyl ether, tert-butyl methyl ether, tert-butanol, water
and ethyl acetate; more preferably, the reagent is one or more
selected from the group consisting of tetrahydrofuran, dioxane,
acetonitrile, DMF, DMSO and water.
[0058] In another specific embodiments, after the reaction of the
carbonylation reagent with 2-(trimethylsilyl)ethanol and amino acid
AA.sub.1, the reagent used in the condensation reaction of the
2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and
AA.sub.2, and further with AA.sub.3 and/or AA.sub.4 in another
embodiments, is one or more selected from the group consisting of
tetrahydrofuran, dioxane, acetonitrile, DMF, DMSO, DMAc, DMPU,
HMPA, methanol, ethanol, isopropanol, ethylene glycol dimethyl
ether, diethyl ether, tert-butyl methyl ether, tert-butanol, water
and ethyl acetate; more preferably, the reagent is one or more
selected from the group consisting of tetrahydrofuran, dioxane,
acetonitrile, DMF, DMSO, water and methanol.
[0059] In another specific embodiments, after the above reactions,
when performing a condensation reaction between the polypeptide
condensate (i.e., 2-(trimethylsilyl)ethoxycarbonyl-dipeptide
condensate, 2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate,
2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate) with
p-aminobenzyl alcohol, the reagent used in the condensation
reaction is preferably one or more selected from the group
consisting of dichloromethane, chloroform, carbon tetrachloride,
methanol, ethanol, isopropanol, tetrahydrofuran, dioxane,
acetonitrile, DMF, DMSO, DMAc, DMPU, HMPA, ethylene glycol dimethyl
ether, diethyl ether, tert-butyl methyl ether, tert-butanol and
ethyl acetate; more preferably, the reagent is one or more selected
from the group consisting of dichloromethane, chloroform, carbon
tetrachloride, methanol, ethanol and DMF.
[0060] The present invention also provides a use of the method
according to any one of the foregoing contents in the preparation
of an antibody-drug conjugate.
[0061] The preparation of antibody-drug conjugates further
comprises the process of removing Teoc protection:
##STR00023##
[0062] The product obtained by removing Teoc protection as
described above is often used as part of the linker to be
covalently linked to the drug moiety in the preparation of
antibody-drug conjugate. In order to achieve covalent linking to
the antibody moiety, the said product is often coupled with a
connector via the following specific reaction routes:
##STR00024##
wherein, L and L' are connectors, and can be any linking group.
[0063] In some embodiments, the connector L is selected from:
##STR00025## ##STR00026## ##STR00027##
[0064] Accordingly, the linking group L' is selected from:
##STR00028## ##STR00029## ##STR00030##
[0065] In a specific embodiment, the oligopeptide linker is
selected from:
##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036## ##STR00037## ##STR00038##
BRIEF DESCRIPTION OF THE FIGURE
[0066] FIGS. 1-A to 1-F show the relevant LC-MS diagrams of the
Val-Cit-PAB prepared by Teoc protecting group method, in which FIG.
1-A is a mass spectrum of the blank control, F1-B is a liquid phase
spectrum of the blank control, FIG. 1-C is a mass spectrum of the
Val-Cit-PAB prepared by Teoc protecting group method, FIG. 1-D is a
liquid phase spectrum of the product, FIG. 1-E is a positive ion
mass spectrum of the product, and FIG. 1-F is a negative ion mass
spectrum of the product.
[0067] FIG. 2-A to FIG. 2-H show relevant LC-MS diagrams of the
Val-Cit-PAB prepared by Cbz protecting group method, in which FIG.
2-A is a mass spectrum of the blank control, FIG. 2-B is a liquid
phase spectrum of the blank control, FIG. 2-C is a mass spectrum of
the Val-Cit-PAB prepared by Cbz protecting group method, FIG. 2-D
is a liquid phase spectrum of the product, FIG. 2-E is a positive
ion mass spectrum of the product, FIG. 2-F is a negative ion mass
spectrum of the product, FIG. 2-G is a ion mass spectrum of the
impurity, and FIG. 2-H is a negative ion mass spectrum of the
impurity.
[0068] FIG. 3-A to FIG. 3-D show relevant LC-MS diagrams of the
Val-Cit-PAB prepared by Boc protecting group method (hydrochloric
acid deprotection method), in which FIG. 3-A is a mass spectrum of
the Val-Cit-PAB prepared by Boc protecting group method, FIG. 3-B
is a liquid phase spectrum of the product, FIG. 3-C is a positive
ion spectrum, and FIG. 3-D is a negative ion spectrum.
[0069] FIG. 4-A to FIG. 4-D show relevant LC-MS diagrams of the
Val-Cit-PAB prepared by Boc protecting group method
(trifluoroacetic acid deprotection method), in which FIG. 4-A is a
mass spectrum of the Val-Cit-PAB prepared by Boc protecting group
method, FIG. 4-B is a liquid phase spectrum of the product, FIG.
4-C is a positive ion spectrum, and FIG. 4-D is a negative ion
spectrum.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviation
[0070] Unless otherwise stated, all abbreviations used in the
present invention have the same meaning as understood by those of
ordinary skill in the art. As used in the present invention, the
common abbreviations and their definitions are as follows:
TABLE-US-00002 Abbreviation Definition Fmoc
fluorenylmethyloxycarbonyl Cbz carbobenzyloxy Boc
tert-butoxycarbonyl Teoc 2-(trimethylsilyl)ethoxycarbonyl Val
valine Cit citrulline Ala alanine Lys lysine Arg arginine Phe
phenylalanine
Definition
[0071] Various terms related to various aspects of the
specification are used throughout the specification and claims.
Unless otherwise indicated, such terms are given their ordinary
meaning in the art. Other specifically defined terms should be
understood in a manner consistent with the definitions provided
herein.
[0072] As used herein, the terms "a" and "an" and "the" are used in
accordance with standard practice and mean one or more, unless the
context indicates otherwise. Thus, for example, reference to "an
antibody drug conjugate" includes a combination of two or more
antibody drug conjugates and the like.
[0073] It should be understood that wherever an aspect is described
herein with the word "comprising", it also provides similar aspects
described with "consisting of" and/or "substantially consisting
of".
[0074] Although the numerical ranges and parameter approximations
shown in the broad scope of the present invention, the numerical
values shown in the specific examples are described as accurately
as possible. However, any numerical value inherently must contain a
certain amount of error, which is caused by the standard deviation
present in their respective measurements. In addition, all ranges
disclosed herein are understood to cover any and all subranges
contained therein. For example, a recorded range of "1 to 10"
should be considered to include any and all subranges between a
minimum of 1 and a maximum of 10 (inclusive); that is, all
subranges beginning 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 referred to as "incorporated
herein" is to be understood as being incorporated in its
entirety.
[0075] As used in the present invention, "" means that the group
containing "" is connected to another group through a chemical bond
here.
[0076] The term "linking group" used in the present invention
refers to a bifunctional or multifunctional molecule, which can
react with a protein/antibody molecule and a dipeptide linker,
respectively, and thus function as a "bridge" to link the
protein/antibody with the dipeptide linker.
[0077] The term "oligopeptide linker" used in the present invention
generally refers to a linking structure containing two or more
amino acid residues, which further comprises a p-benzyl alcohol
residue. The two amino acids are linked together by means of
dehydration condensation. The amino acids referred to herein
generally refer to those organic compounds containing both amino
and carboxyl groups, including all essential amino acids and
non-essential amino acids. Preferably, the amino acid includes, but
is not limited to, -valine- (-Val-), -citrulline -(-Cit-),
-alanine-(-Ala-), -lysine- (-Lys-), -lysine(trityl)- (-Lys(Trt)-),
-lysine(monomethoxytrityl)-(-Lys(Mmt)-),
-lysine(fluorenylmethyloxycarbonyl)- (-Lys(Fmoc)-),
-arginine-(-Arg-), -phenylalanine- (-Phe-), -glycine- (-Gly-),
-leucine- (-Leu-), and -isoleucine-(-Ile-).
SPECIFIC EXAMPLES
[0078] The following further describes the present invention in
combination with specific examples. It should be understood that
these examples are only used to illustrate the present invention
and not to limit the scope of the present invention. The
experimental methods without specific conditions in the following
examples are generally performed under conventional conditions or
conditions recommended by the manufacturer. The reagents without
specific sources are conventional reagents purchased on the market.
Unless otherwise stated, all percentages, ratios, proportions, or
parts are by weight.
[0079] The units in weight-volume percentage in the present
invention are well known to those skilled in the art, and for
example, refer to the weight of a solute in 100 ml of a
solution.
[0080] Unless otherwise defined, all specialties and sciences used
herein are used in the same sense as those familiar to those
skilled in the art. In addition, any method or material similar or
equal to the content described can be used in the method of the
present invention. The preferred embodiments and materials
described herein are for illustration purposes only.
Example 1 Preparation of Val-Cit-PAB by Teoc Protecting Group
Method
[0081] (1) Preparation of Teoc-Val-Cit
##STR00039##
[0082] 14.16 g of 2-(trimethylsilyl)ethanol (120 mmol), 38.7 g of
N,N-diisopropylethylamine (300 mmol), 30.4 g of
bis(4-nitrophenyl)carbonate (100 mmol), and 400 mL of acetonitrile
were sequentially added into a 500 mL single-neck flask. After the
addition, the mixture was stirred at room temperature for 16 h to
form reaction solution 1. 14.08 g of L-valine (120 mmol) and 25.8 g
of N,N-diisopropylethylamine (200 mmol) were added into a 1 L
single-neck flask, and dissolved in 400 mL of water to form
reaction solution 2. The reaction solution 1 was added to the
reaction solution 2 with stirring, and after stirring at room
temperature for 16 h, the completion of the reaction was detected
by LC-MS method. After passing the detection, the reaction solution
was rotary evaporated to remove the solvent. After evaporation, 500
mL of water was added, and 1 mol/L hydrochloric acid was added
dropwise with stirring to adjust the pH to 1. Then, the aqueous
phase was extracted twice, each with 300 mL of ethyl acetate. The
extracts were combined and washed twice, each with 300 mL of water.
After washing with water, the extract was dried by adding anhydrous
magnesium sulfate, and the solvent was rotary evaporated to obtain
54 g of a pale yellow viscous solid product (i.e.,
2-(trimethylsilyl)ethoxycarbonyl-L-valyl mixture containing
4-nitrophenol (simply referred to as
2-(trimethylsilyl)ethoxycarbonyl-L-valyl mixture)). LC-MS (M-H)-:
260.1. The 2-(trimethylsilyl)ethoxycarbonyl-L-valyl mixture was
used in the next reaction without purification.
[0083] 10.5 g of the 2-(trimethylsilyl)ethoxycarbonyl-L-valyl
mixture (40 mmol) as prepared above, 24.3 g of
N,N,N',N'-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (80
mmol), 25.8 g N,N-diisopropylethylamine (200 mmol), and 200 mL
N,N-dimethylformamide were sequentially added into a 500 mL
single-necked flask. After the addition, the mixture was stirred at
room temperature for 6 h to form reaction solution 3. 21 g of
L-citrulline (120 mmol) was added into a 1 L single-neck flask, and
completely dissolved under stirring with the addition of 400 mL of
water, to form a reaction solution 4. 15.5 g of
N,N-diisopropylethylamine (120 mmol) and 200 mL of
N,N-dimethylformamide were added into a 500 mL beaker, to form a
reaction solution 5. The reaction solution 5 was added to the
reaction solution 4, then transferred to a low-temperature bath,
and stirred at -10.degree. C. for 0.5 h. Then, the reaction
solution 3 was slowly added dropwise to the above reaction system
under stirring at -10.degree. C., which was controlled to be
completed in about 2 h. After the completion of the addition, the
mixture was kept at -10.degree. C. for 16 h under stirring. Then,
the completion of the reaction was detected by LC-MS method. After
passing the detection, the system was rotary evaporated to remove
the solvent, and after rotary evaporation, 500 mL of 0.1 mol/L
hydrochloric acid was added and stirred well. The aqueous phase was
extracted twice, each with 300 mL of ethyl acetate. The extracts
were combined and washed twice, each with 300 mL 0.1 mol/L
hydrochloric acid, and then washed twice with 300 mL of water.
After washing with water, the extract was dried by adding anhydrous
magnesium sulfate, and the solvent was rotary evaporated before 100
mL of ethyl acetate and 1000 mL of toluene were added with stirring
for 16 h and then filtered. The filter cake was dissolved in 50 mL
of THF, and 1 L of methyl tert-butyl ether: n-hexane=1:1 was added
to prepare a solution. The mixture was stirred at room temperature
for 16 hours and then filtered. The filter cake was dissolved in 50
mL of THF before 1 L of toluene was added. After stirring at room
temperature for 6 h, the mixture was filtered and the filter cake
was dried to obtain 11.1 g of an off-white powdery solid product
with a yield of 68%. LC-MS(M+H).sup.+: 418.7, LC-MS(M-H).sup.-:
416.7.
[0084] (2) Preparation of Teoc-Val-Cit-PAB
##STR00040##
[0085] 7.5 g of
2-(trimethylsilyl)ethoxycarbonyl-L-valyl-L-citrulline (18 mmol), 9
g of N-ethoxyacyl-2-ethoxy-1,2-dihydroquinoline (36 mmol), 4.5 g of
p-aminobenzyl alcohol (36 mmol), 150 mL of dichloromethane and 75
mL of methanol were sequentially added into a 500 mL single-neck
flask. After the addition, the mixture was stirred at room
temperature for 16 h. Then, the completion of the reaction was
detected by LC-MS method. After passing the detection, the reaction
solution was dried by rotary evaporation, and after evaporation,
200 mL of dichloromethane and 200 mL of ethyl acetate were added
and stirred for 16 h, and then filtered. To the filter cake, 100 mL
of dichloromethane, 100 mL of ethyl acetate, and 200 mL of n-hexane
were added with stirring for 16 h, and then filtered. The filter
cake was dried to obtain 7.5 g of an off-white powdery solid
product (the product was
2-(trimethylsilyl)ethoxycarbonyl-L-valyl-L-citrull amido-p-benzyl
alcohol, namely Teoc-Val-Cit- PAB) with a yield of 80%.
LC-MS(M+H).sup.+: 523.5, LC-MS(M-H).sup.-: 522.
[0086] (3) Preparation of Val-Cit-PAB (Relative Molecular Weight:
379.46)
##STR00041##
[0087] 3.15 g of 2-(trimethylsilyl)ethoxycarbonyl-L-valyl-L-citrull
amido-p-benzyl alcohol (6.2 mmol), 2.16 g of potassium fluoride
(37.2 mmol), 12.4 mL of a 1.0 mol/L tetrabutylammonium fluoride
solution in tetrahydrofuran, and 100 mL of N,N-dimethylformamide
were sequentially added into a 500 mL single-necked flask. After
the addition, the mixture was stirred at room temperature for 24 h
and then the completion of the reaction was detected by LC-MS
method. After the completion of the reaction, the reaction solution
was filtered. After rotatory evaporation of the solvent of the
filtrate, 100 mL of anhydrous ethanol was added to fully dissolve
the residue. 18 mL of a 1 mol/L dilute hydrochloric acid solution
was added and stirred well before the mixture was dried by adding
anhydrous magnesium sulfate and then filtered. The filtrate was
dried by rotary evaporation, and then 30 mL of ethanol was added to
completely dissolve the residue. Under stirring at room
temperature, a mixed solution of 200 mL of methyl tert-butyl ether
and 200 mL of dichloromethane was added and stirred for 1 h before
filtered. The filter cake was dried to obtain 1.83 g of an
off-white powdery solid product (the product was L-valyl-L-citrull
amido-p-benzyl alcohol, that is, Val-Cit-PAB) with a yield of
80%.
[0088] In the LC-MS detection, FIG. 1-A is a mass spectrum of the
blank control, F1-B is a liquid phase spectrum of the blank
control, FIG. 1-C is a mass spectrum of the Val-Cit-PAB prepared by
Teoc protecting group method (the product in this example), FIG.
1-D is a liquid phase spectrum of the product, FIG. 1-E is a
positive ion mass spectrum of the product, and FIG. 1-F is a
negative ion mass spectrum of the product. By comparing FIG. 1-A
with FIG. 1-C, it can be seen that the peak at the time point of
2.87 min is the product ion peak. By comparing FIG. 1-B with FIG.
1-D, it can be seen that the product has a strong HPLC signal, and
by combining with FIG. 1-A and FIG. 1-C, it can be seen that the
Val-Cit-PAB prepared by the Teoc protecting group method has fewer
impurities. By analyzing the positive ion detection spectrum (2.849
min) and negative ion detection spectrum (2.866 min) at 2.87 min,
it can be known that: LC-MS(M+H).sup.+: 379.6, LC-MS(M-H).sup.-:
378.0.
Example 2 Preparation of Val-Cit-PAB by Cbz Protecting Group
Method
[0089] (1) Preparation of Cbz-Val-Cit
##STR00042##
[0090] 10 g of N-benzyloxycarbonyl-L-valine (i.e., Cbz-Val) (40
mmol), 18.2 g of N,N,N',N'-tetramethyl-O-(N-succinimide)urea
tetrafluoroborate (60 mmol), 15.5 g of N,N-diisopropylethylamine
(120 mmol), and 300 mL of acetonitrile were sequentially added into
a 1 L single-neck flask. After the addition, the mixture was
stirred at room temperature for 4 h. After dissolving 7.7 g of
L-citrulline (44 mmol) in 300 ml of water, the mixture was added to
the above reaction system and stirred at room temperature for 16 h.
Samples were taken for detection by LC-MS, and then the reaction
was completed. Under stirring, 0.1 mol/L hydrochloric acid was
added dropwise to adjust the pH to 1. After the solvent was dried
by rotary evaporation, 800 mL of water was added, stirred at room
temperature for 16 h, and filtered. After the filter cake was dried
under vacuum, 400 mL of ethyl acetate and 400 mL of dichloromethane
were added and stirred for 16 h before filtered. After the filter
cake was dried under vacuum, 200 mL of ethyl acetate, 200 mL of
dichloromethane, and 400 mL of n-hexane were added, and the mixture
was stirred for 16 hours and then filtered. The filter cake was
dried to obtain 12.65 g of an off-white powdery solid product
(i.e., N-benzyloxycarbonyl-L-valyl-L-citrulline) with a yield of
77%. LC-MS(M+H).sup.+: 408.7, LC-MS(M-H).sup.-: 406.9.
[0091] (2) Preparation of Cbz-Val-Cit-PAB
##STR00043##
[0092] 12.65 g of N-benzyloxycarbonyl-L-valyl-L-citrulline (31
mmol), 15.74 g of N-ethoxyacyl-2-ethoxy-1,2-dihydroquinoline (62
mmol), 7.53 g of p-aminobenzyl alcohol (62 mmol), 200 mL of
dichloromethane, and 100 mL of methanol were sequentially added
into a 500 mL single-neck flask. After stirring at room temperature
for 16 h, samples were taken for detection by LC-MS, and then the
reaction was completed. After the reaction solution was dried by
rotary evaporation, 200 mL of dichloromethane and 200 mL of ethyl
acetate were added, stirred for 16 h, and filtered. 100 mL of
dichloromethane, 100 mL of ethyl acetate, and 200 mL of n-hexane
were added to the filter cake, stirred for 16 h and filtered. The
filter cake was dried to obtain 9.1 g of an off-white powdery solid
product with a yield of 60%. LC-MS(M+H)+.sup.+: 513.6.
[0093] (3) Preparation of Val-Cit-PAB (Relative Molecular Weight:
379.46)
##STR00044##
[0094] 1.12 g of N-benzyloxycarbonyl-L-valyl-L-citrull
amido-p-benzyl alcohol (2 mmol), 0.3 g of palladium on carbon, and
100 mL of methanol were sequentially added into a 250 mL
single-neck flask and stirred at 10.degree. C. The reaction system
was covered with a balloon filled with hydrogen gas, and the
mixture was stirred at 10.degree. C. for 16 h. Samples were taken
for detection by LC-MS, and then the reaction was completed. The
reaction solution was filtered, and the filtrate was dried by
rotary evaporation. 400 mL of n-hexane was added, stirred for 16 h,
and then filtered. The filter cake was dried to obtain 0.69 g of an
off-white powdery solid product with a yield of 91%.
[0095] In the LC-MS detection, FIG. 2-A is a mass spectrum of the
blank control, FIG. 2-B is a liquid phase spectrum of the blank
control, FIG. 2-C is a mass spectrum of the Val-Cit-PAB prepared by
Cbz protecting group method (the product in this example), FIG. 2-D
is a liquid phase spectrum of the product, FIG. 2-E is a positive
ion mass spectrum of the product, FIG. 2-F is a negative ion mass
spectrum of the product, FIG. 2-G is a ion mass spectrum of the
impurity, and FIG. 2-H is a negative ion mass spectrum of the
impurity. By Comparing FIG. 2-A with FIG. 2-C, it can be seen that
there are two stronger ion peaks: at 4.33 min and at 6.54 min. By
comparing FIG. 2-B and 2-D, it can be seen that there are two peaks
with strong HPLC signals at 4.12 min and at 5.39 min, and combining
with FIG. 2-A and FIG. 2-C, it can be inferred that one of the two
peaks is a product peak and one is an impurity peak, and the
content of impurities is higher. By analyzing the positive ion mass
spectrum and negative ion mass spectrum at 4.33 min and at 5.54
min, it can be found that the peak at 4.33 min is the product peak
(LC-MS(M+H).sup.+: 379.6, LC-MS(M-H).sup.-: 378.0), and the peak at
5.54 min is the impurity peak (LC-MS(M+H).sup.+: 363.6), which is
speculated to be a by-product of Val-Cit-PAB dehydroxylation (with
a molecular weight difference of only 16), and its content is
higher. Since the chemical properties of the by-product are very
similar to the product, the by-product is very hard to be removed,
and has a great effect on the subsequent reactions and product
quality. In addition, hydrogen gas is used as a reactant in the
deprotection process, which poses a safety risk.
Example 3 Preparation of Phe-Cit-PAB by Boc Protecting Group
Method
[0096] (1) Preparation of Boc-Phe-Cit
##STR00045##
[0097] 2.65 g of N-(tert-butoxycarbonyl)-L-phenylalanine (10 mmol)
(i.e., Boc-Phe), 3.61 g of
N,N,N',N'-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (12
mmol), 3.87 g of N,N-diisopropylethylamine (30 mmol), and 50 mL of
N,N-dimethylformamide were sequentially added into a 250 mL
single-neck flask, and stirred at room temperature for 4 h.
[0098] 1.75 g of L-citrulline (10 mmol) was dissolved in 50 ml of
water, which is added to the above reaction system and stirred at
room temperature for 16 h. Samples were taken for detection by
LC-MS, and then the reaction was completed. Under stirring, 0.1
mol/L hydrochloric acid was added dropwise to adjust the pH to 1.
After the system was dried by rotary evaporation, 200 mL of water
was added, stirred at room temperature for 16 h, and filtered.
After the filter cake was dried, 100 mL of ethyl acetate and 100 mL
of dichloromethane were added, stirred for 16 h and filtered. The
filter cake was dried to obtain 3 g of a yellow powdery solid
product (that is,
N-(tert-butoxycarbonyl)-L-phenylalanyl-L-citrulline, Boc-Phe-Cit)
with a yield of 71%. LC-MS(M-H).sup.-: 421.4.
[0099] (2) Preparation of Boc-Phe-Cit-PAB
##STR00046##
[0100] 3 g of N-(tert-butoxycarbonyl)-L-phenylalanyl-L-citrulline
(7.1 mmol) (i.e., Boc-Phe-Cit), 3.5 g of
N-ethoxyacyl-2-ethoxy-1,2-dihydroquinoline (14.2 mmol), 1.75 g of
p-aminobenzyl alcohol (14.2 mmol), 60 mL of dichloromethane, and 30
mL of methanol were sequentially added into a 250 mL single-neck
flask, and stirred at room temperature for 16 h. Then samples were
taken for detection by LC-MS, and then the reaction was completed.
After the reaction solution was dried by rotary evaporation, 100 mL
of dichloromethane and 100 mL of ethyl acetate were added, stirred
for 16 h, and centrifuged. 100 mL of n-hexane was added to the
white solid in the lower layer, stirred for 2 h and centrifuged.
The white solid in the lower layer was dried to obtain 2.3 g of a
off-white powdery solid product (that is,
N-(tert-butoxycarbonyl)-L-phenylalanyl-L-citrull amido-p-benzyl
alcohol, Boc-Phe-Cit-PAB) with a yield of 62%. LC-MS(M-H)
526.2.
[0101] (3) Preparation of Phe-Cit-PAB (Relative Molecular Weight:
427.51)
[0102] The Boc protecting group is removed by hydrochloric acid
deprotection and trifluoroacetic acid deprotection, respectively,
shown as follows.
##STR00047##
[0103] (a) Hydrochloric Acid Deprotection Method
[0104] 0.5 g of N-(tert-butoxycarbonyl)-L-phenylalanyl-L-citrull
amido-p-benzyl alcohol (1 mmol) (i.e., Boc-Phe-Cit-PAB), 3 mL of 4
mol/L hydrogen chloride solution in dioxane and 3 mL of dioxane
were sequentially added into a 10 mL single-neck flask. After the
addition, the mixture was stirred at room temperature for 3 h.
Samples were taken for detection by LC-MS, and then the reaction
was completed.
[0105] In the LC-MS detection, FIG. 3-A is a mass spectrum of the
Phe-Cit-PAB prepared by Boc protecting group method (the product in
this example), FIG. 3-B is a liquid phase spectrum of the product,
FIG. 3-C is a positive ion spectrum, and FIG. 3-D is a negative ion
spectrum. It can be seen from FIG. 3-A that there is a strong ion
peak at 5.39 min. By analyzing the positive ion mass spectrum
(5.390 min) and negative ion mass spectrum (5.403 min) at this
position, the value of LC-MS(M+H).sup.+: 446.2, LC-MS(M-H).sup.-:
444.1 was obtained. It is speculated that most of the products
prepared by this method are by-products in which the hydroxyl group
at the benzyl position is replaced by chlorine.
[0106] (b) Trifluoroacetic Acid Deprotection Method
[0107] 0.5 g of N-(tert-butoxycarbonyl)-L-phenylalanyl-L-citrull
amido-p-benzyl alcohol (1 mmol) and 3 mL of trifluoroacetic acid
were sequentially added into a 10 mL single-neck flask. After the
addition, the mixture was stirred at room temperature for 2 h and
the completion of the reaction was detected by LC-MS.
[0108] In the LC-MS detection, FIG. 4-A is a mass spectrum of the
Phe-Cit-PAB prepared by Boc protecting group method (the product in
this example), FIG. 4-B is a liquid phase spectrum of the product,
FIG. 4-C is a positive ion spectrum, and FIG. 4-D is a negative ion
spectrum. It can be seen from FIG. 4-A that there is a strong ion
peak at 5.77 min. By analyzing the positive ion mass spectrum
(5.768 min) and negative ion mass spectrum (5.781 min) at this
position, the value of LC-MS(M+H).sup.+: 524.4, LC-MS(M-H) 522.1
was obtained. It is speculated that most of the products prepared
by this method are by-products in which the hydroxyl group at the
benzyl position forms trifluoroacetate.
[0109] In summary, the method for preparing Val-Cit-PAB using Teoc
as a protecting group provided by the present invention is easily
carried out under mild reaction conditions, and since almost no
side reactions occur in the reaction, the method produces a
high-purity product with fewer impurities and easy to be purified.
The method for preparing Val-Cit-PAB using Cbz as a protecting
group produces large amounts of by-products from dehydroxylation.
Because the chemical properties of the by-products are very similar
to the products, they are very difficult to be removed, greatly
affecting the subsequent reactions and product quality. In
addition, hydrogen gas is used as a reactant in the deprotection
process, which poses a safety risk. The method of preparing
Val-Cit-PAB by using Boc as a protecting group has the problems
that the hydroxyl group at the benzyl position is replaced by
chlorine, and forms trifluoroacetate in a high conversion rate.
Therefore, compared with the other two methods, the Teoc method
provided by the present invention can be easily carried out and
produces few impurities, thus achieving unexpected technical
effects.
TABLE-US-00003 Easy or Protecting difficult to group By-products
purify Reaction conditions Teoc -- very few easy mild Cbz
By-products from more difficult Use of hydrogen poses safety
dehydroxylation of risks Val-Cit-PAB Boc By-products in which most
difficult Use of large amounts of acid the hydroxyl group at
presents safety risks and is not the benzyl position of
environmentally friendly. Val-Cit-PAB is replaced by chlorine
By-products in which most difficult the hydroxyl group at the
benzyl position of Val-Cit-PAB forms trifluoroacetate
[0110] The invention has been exemplified by various specific
embodiments. However, those of ordinary skill in the art can
understand that the present invention is not limited to the
specific embodiments. Those of ordinary skill in the art can make
various changes or modifications within the scope of the present
invention, and various technical features mentioned in various
places in this specification can be combined with each other
without departing from the spirit and scope of the present
invention. Such modifications and variations are all within the
scope of the present invention.
* * * * *