CN110141665A - A kind of method of aziridine in the application modification of antibody coupling drug-linker - Google Patents
A kind of method of aziridine in the application modification of antibody coupling drug-linker Download PDFInfo
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- CN110141665A CN110141665A CN201910432284.4A CN201910432284A CN110141665A CN 110141665 A CN110141665 A CN 110141665A CN 201910432284 A CN201910432284 A CN 201910432284A CN 110141665 A CN110141665 A CN 110141665A
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- aziridine
- reaction
- linker
- antibody coupling
- polypeptide chain
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- 238000000034 method Methods 0.000 title claims abstract description 21
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 238000010168 coupling process Methods 0.000 title claims abstract description 18
- 230000008878 coupling Effects 0.000 title claims abstract description 17
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 17
- 230000004048 modification Effects 0.000 title claims description 15
- 238000012986 modification Methods 0.000 title claims description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 46
- 229920001184 polypeptide Polymers 0.000 claims abstract description 23
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 23
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 23
- 239000007853 buffer solution Substances 0.000 claims abstract description 21
- -1 aziridine compound Chemical class 0.000 claims abstract description 20
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000006471 dimerization reaction Methods 0.000 claims abstract description 4
- 239000002904 solvent Substances 0.000 claims abstract description 4
- 150000003555 thioacetals Chemical class 0.000 claims abstract description 4
- 238000004262 preparative liquid chromatography Methods 0.000 claims abstract 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- 125000003854 p-chlorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1Cl 0.000 claims description 2
- 239000007789 gas Substances 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 24
- 229940079593 drug Drugs 0.000 abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 11
- 238000001228 spectrum Methods 0.000 abstract description 4
- 239000012299 nitrogen atmosphere Substances 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 239000003863 metallic catalyst Substances 0.000 abstract description 2
- 238000006011 modification reaction Methods 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 239000012074 organic phase Substances 0.000 description 14
- 238000001819 mass spectrum Methods 0.000 description 12
- 235000018417 cysteine Nutrition 0.000 description 10
- 150000001408 amides Chemical class 0.000 description 9
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- 201000011510 cancer Diseases 0.000 description 7
- 238000004440 column chromatography Methods 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000003810 ethyl acetate extraction Methods 0.000 description 6
- 238000003809 water extraction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 101100454807 Caenorhabditis elegans lgg-1 gene Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- VPMIAOSOTOODMY-KJAPKAAFSA-N (4r)-6-[(e)-2-[6-tert-butyl-4-(4-fluorophenyl)-2-propan-2-ylpyridin-3-yl]ethenyl]-4-hydroxyoxan-2-one Chemical compound C([C@H](O)C1)C(=O)OC1/C=C/C=1C(C(C)C)=NC(C(C)(C)C)=CC=1C1=CC=C(F)C=C1 VPMIAOSOTOODMY-KJAPKAAFSA-N 0.000 description 1
- BIKSKRPHKQWJCW-UHFFFAOYSA-N 3,4-dibromopyrrole-2,5-dione Chemical compound BrC1=C(Br)C(=O)NC1=O BIKSKRPHKQWJCW-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229940122277 RNA polymerase inhibitor Drugs 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 150000001541 aziridines Chemical class 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of methods that aziridine is modified in the application of antibody coupling drug-linker, method includes the following steps: aziridine compound is added into the Tris-HCl buffer solution that pH value is 7.0~7.6, polypeptide chain is added under room temperature, nitrogen atmosphere environment to be reacted, solvent is spin-dried for after reaction, by the Thioacetal of the isolated dimerization of preparative liquid chromatography.The method of the present invention can directly react in water phase, and without metallic catalyst, reaction condition is simple, mild, and substrate spectrum is extensive, and reaction yield is high；Product prepared by the present invention is obtained by modification reaction of the 2H- aziridine to polypeptide chain, this kind of product provides certain technical support for application of this reaction in antibody coupling drug in the future, so that this reaction is possible to be applied among the synthesis of targeted drug.
Description
Technical field
The invention belongs to Bioorganic Chemistry technical fields more particularly to a kind of aziridine to connect in antibody coupling drug
The method of the application modification of junctor.
Background technique
Cancer is one of the principal disease of current puzzlement aging of population.Namely change for the common treatment means of cancer
Learning therapy, radiotherapy, operation excision and targeted therapy, one of the most common is chemotherapy.But since chemotherapeutics cannot
Cancer cell and normal cell are efficiently differentiated, so this kind of chemotherapeutics is there are serious side effect and can reduce the life of patient
Bioplasm amount.In addition, the exploitation time-consuming of small-molecule drug is too long, and risk is larger.Therefore, it needs to develop and can target elimination
New type antineoplastic medicine of the cancer cell without influencing normal cell.And antibody coupling drug (ADC) is exactly a kind of reply cancer
Novel form of therapy, it is thin that it can realize that selective be transferred to effective drug molecule targets cancer on molecular level
Born of the same parents, generation effect, to improve therapeutic effect, reduce drug to the toxicity of people's whole body.This treatment means are obvious
Better than current classic chemotherapy mode.
ADC is made of three main components, comprising: (1) antibody, usually m protein matter；(2) by antibody and effectively
Load the connector of connection；(3) with the payload of high-drug-effect.ADC mechanism of drug action: when ADC molecule is released to blood
In liquid, the antibody of molecular surface can accurately identify the antigen part of target cancer cell, be made by the endocytosis of ADC and antigenic compound
With being brought into inside antigen, drug molecule is processed in lysosome and discharged to subsequent this compound, effectively destroys DNA chain
Or apply RNA polymerase inhibitor, lead to the death of cell.In view of such mechanism of action, ideal antibody is needed enough
Strong antigen affinity and specificity.But the presently found antibody for having high antigen affinity penetrated in actual clinical it is swollen
The efficiency of tumor is very low, so the efficient antigen affinity of ADC drug can not necessarily bring efficient clinical efficacy.
Inside ADC drug coupling method, amide coupling is that a kind of effectively method utilizes lysine residue and activation
Carboxylic acid connector afterwards generates antibody acid esters.Carboxylic acid molecules using a molecule amide and a molecule activation are in current organic synthesis
It is most reliable, most efficient chemical transformation.But usually have about 80 lysine residues on an antibody molecule, but only
About 10 residues are available.Therefore this obtained DAR value of connection form is changeable, and link position is different.?
In maytansinoid type ADC drug, DAR value average out to 3.5~4 is distributed between 0~7.Thus, DAR value and its distribution
Range greatly affects the efficiency and cytotoxicity of ADC drug.In addition, some lysine residues are in the effect of antibody antigen
Due to interaction, cause to reduce with the affinity of connector, therefore will lead to the complexity of ADC drug system, therapeutic index is poor
The case where.So the connection reaction based on lysine residue needs to control in reaction process DAR value and distribution (usually 3
Between~4).
The cysteine residues and a molecule that connection reaction based on cysteine depends in specific antibodies connect medicine
The reaction of the sulfhydryl compound of object load.Usually, without containing free sulfydryl in antibody, and all cysteines are residual
Base is presented all in the form of disulfide bond, in the lgG1 antibody of the mankind, this be generally also modern times ADC drug in study at most
Antibody contains 4 interchains, the disulfide bond in 12 chains.The disulfide bond of 4 interchains is not usually the pass for determining lgG1 antibody stabilization
Key factor selective reduction can obtain 2,4,6,8 free sulfydryls while guarantee two in 12 chains under mild conditions
Sulfide linkage it is complete.Due to the limitation of connection site quantity, and the specificity of reaction, the connection reaction based on cysteine exists
Conjugation in DAR value and heterogeneity is better than the connection reaction of lysine.But there is room for improvement with reality for this connection method
Now better DAR and heterogeneity.Junutula and colleague describe selectivity of two new cysteine residues for antibody
Modification, and this technology study in vivo in have been achieved for good curative effect and treat window.Dibromo-maleimide, two
Bromine Pyridazindione and based on bis- (p-toluenesulfonyl) the propane base class of 1,3- can receive from interchain disulfide bond reduction two points
Sub- cysteine derivative is to generate the antibody of bridge joint.Theoretically, stabilization of the combination of these specific sites in structure
Property, there can be all well and good performance in homogeney and the DAR value that can well control, consequently, it is possible to improving corresponding ADC drug
Therapeutic efficiency.
In view of ADC drug, clinically huge application potential, all kinds of novel connectors of design just seem increasingly important.
Relative to traditional small-molecule chemical therapy, well-designed connector is advantageous in the clinical medical of ADC drug, disease
Treatment window it is extensive, the customization of molecule has flexibility.ADC drug is expected to the main flow direction as recent anticancer therapy.To the greatest extent
It manages it to have a high potential, but also needs us in biochemistry, pharmacology to preferably designing and developing efficient ADC drug
It learns, the multiple fields such as molecules continue to have breakthrough.The especially stability of connector and the heterogeneity of reaction (point of DAR value
Cloth range) often the curative effect of ADC drug is played a crucial role.Atom economy simultaneously, efficient chemo-selective
And there are also many shortcomings in terms of mild reaction condition.So the stability side of design and reaction in connector
There are also the work of many urgently final results in face.
Summary of the invention
The purpose of the present invention is to provide a kind of aziridines in the side of the application modification of antibody coupling drug-linker
Method.
The invention is realized in this way a kind of aziridine is in the side of the application modification of antibody coupling drug-linker
Method, method includes the following steps: aziridine chemical combination is added into the Tris-HCl buffer solution that pH value is 7.0~7.6
Object is added polypeptide chain under room temperature, nitrogen atmosphere environment and is reacted, be after reaction spin-dried for solvent, by preparing liquid phase
Chromatographic isolation obtains the Thioacetal of dimerization.
Preferably, the structural formula of the aziridine compound are as follows:
In formula, R1For Ph, 4-OMe-Ph or 4-Cl-Ph；R2For COOtBu、COONH2Or H.
Preferably, the polypeptide chain is CSL-NH2、CTWSL-NH2、RCLITCK-NH2、CKAR GGC-NH2、
RACKGGCG、CPCRAGKG-NH2、CRAKPALGKGKC-NH2In any one.
Preferably, the Molar ratio of the buffer solution, aziridine compound and polypeptide chain is 1mL:(0.05
~0.2) mmol:(0.3~1.2) mmol.
Preferably, the Molar ratio of the buffer solution, aziridine compound and polypeptide chain is 1mL:
0.05mmol:0.4mmol.
Preferably, the pH value of Tris-HCl buffer solution is 7.40.
The present invention overcomes the deficiencies of the prior art and provide a kind of aziridine in the application of antibody coupling drug-linker
The method of modification, using aziridine compound as raw material, under conditions of nitrogen protection, in buffer solution system, benefit
With the amino acid containing sulfydryl, the carbon-to-nitrogen double bon of polypeptide chain attack aziridine compound, so that aziridine compound
The selective modification to sulfydryl on amino acid or polypeptide chain is completed in direct open loop.
When reacting under the conditions of standard reaction with 2H- aziridine using cysteine, institute can be successfully obtained
The product needed, yield 42%, is illustrated in fig. 1 shown below.
In Fig. 1, a is unless otherwise stated, all reactions are all using cysteine (1.6mmol, 8.0equiv.)
With 2H- aziridine (0.2mmol, 1.0equiv.) in nitrogen atmosphere, 20h is reacted in 37 DEG C of PBS buffer solution, is produced
Rate is nuclear-magnetism yield, and b separates yield.
This reaction can react realization in the pure water phase system for not needing organic solvent, and yield can reach 65%.?
In bioconversion, pure water phase solvent is essential a part.The present invention finally has studied with methoxyl group, chloro and cyano
The response situation of the different 2H- aziridines of functional group, finds in experimental result, to the 2H- aziridine of methoxyl group
Can with converted completely in the reacting of cysteine, and reached very high yield (separation yield has reached 87%).
The present invention attempted a plurality of different polypeptide chain containing free sulfhydryl groups with react performance it is optimal to methoxyl group 2H-
Aziridine 2g reaction, as shown in Figure 2.It, can be with 2H- aziridine with the cysteine there are two free sulfhydryl groups in Fig. 2
Reaction generates cricoid thioacetal compound (entry 4~6).Still further aspect, the cysteine with single free sulfhydryl groups
The polypeptide chain of residue can react the Thioacetal (entry 1~3) for generating dimerization with 2H- aziridine.In both reactions
In the case of, reaction can react in aqueous solution at room temperature, and amino other for amino, hydroxyl, carboxyl etc.
Sour residue has good tolerance.Then, the present invention continues the application that protein is extended to from polypeptide chain, such as bovine serum albumin
White, mAbs etc. can also obtain very high yield.
Compared with the prior art the shortcomings that and deficiency, the invention has the following advantages:
(1) the method for the present invention can directly react in water phase, and without metallic catalyst, reaction condition is simple, mild,
Substrate spectrum is extensive, and reaction yield is high；
(2) product prepared by the present invention is obtained by modification reaction of the 2H- aziridine to polypeptide chain, this kind of production
Object provides certain technical support for application of this reaction in antibody coupling drug in the future, so that this reaction is possible to be answered
It uses among the synthesis of targeted drug.
Detailed description of the invention
Fig. 1 is reaction result of cysteine under the conditions of standard reaction with 2H- aziridine；In figure, EtOH is second
Alcohol, Buffer are buffer solution, and Buffer solution is buffer solution；
Fig. 2 is a plurality of different polypeptide chain containing free sulfhydryl groups and to react performance optimal to methoxyl group 2H- azacyclo-
The reaction result of propylene；
Fig. 3 is the nuclear magnetic spectrogram hydrogen spectrum characterization result of the product of products therefrom in embodiment 1；
Fig. 4 is the high resolution mass spectrum data of the P-1 of products therefrom in embodiment 2；
Fig. 5 is the high resolution mass spectrum data of the P-2 of products therefrom in embodiment 3；
Fig. 6 is the high resolution mass spectrum data of the P-3 of products therefrom in embodiment 4；
Fig. 7 is the high resolution mass spectrum data of the P-4 of products therefrom in embodiment 5；
Fig. 8 is the high resolution mass spectrum data of the P-5 of products therefrom in embodiment 6；
Fig. 9 is the high resolution mass spectrum data of the P-6 of products therefrom in embodiment 7.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
Embodiment 1
The aziridine compound 2g with amide of 1.0equiv. (0.05mmol) is added into Shrek pipe,
The buffer solution (Tris-HCl) that 1.0mLpH value is 7.40, vacuumizes, and is added dropwise under conditions of logical nitrogen into Shrek pipe
The cysteine of 6.0equiv. reacts 10min under the conditions of 25 DEG C.Reaction terminates to put reaction system on a rotary evaporator
It is spin-dried for, extracts organic phase three times with water and ethyl acetate, organic phase is dried with anhydrous sodium sulfate, then be spin-dried for, by further column
Chromatography (methylene chloride/methanol) is to get product, and the nuclear magnetic spectrogram hydrogen spectrum characterization result of the product is as shown in figure 3, product
Yield is 87%.
Embodiment 2
The aziridine chemical combination with amide in the embodiment 1 of 1.0equiv. (0.2mmol) is added into Shrek pipe
Object, the buffer solution (Tris-HCl) that 1.0mLpH value is 7.40, vacuumizes, and drips under conditions of logical nitrogen into Shrek pipe
The polypeptide chain P-1 for adding 6.0equiv. reacts 10min under the conditions of 25 DEG C.Reaction terminates reaction system being placed on Rotary Evaporators
On be spin-dried for, three times with water and ethyl acetate extraction organic phase, with the dry organic phase of anhydrous sodium sulfate, then be spin-dried for, by further
For column chromatography for separation (methylene chloride/methanol) to get product, the high resolution mass spectrum data of the product is as shown in Figure 4.
Embodiment 3
The aziridine chemical combination with amide in the embodiment 1 of 1.0equiv. (0.2mmol) is added into Shrek pipe
Object, the buffer solution (Tris-HCl) that 1.0mLpH value is 7.40, vacuumizes, and drips under conditions of logical nitrogen into Shrek pipe
The polypeptide chain P-2 for adding 6.0equiv. reacts 10min under the conditions of 25 DEG C.Reaction terminates reaction system being placed on Rotary Evaporators
On be spin-dried for, three times with water and ethyl acetate extraction organic phase, with the dry organic phase of anhydrous sodium sulfate, then be spin-dried for, by further
For column chromatography for separation (methylene chloride/methanol) to get product, the high resolution mass spectrum data of the product is as shown in Figure 5.
Embodiment 4
The aziridine chemical combination with amide in the embodiment 1 of 1.0equiv. (0.2mmol) is added into Shrek pipe
Object, the buffer solution (Tris-HCl) that 1.0mLpH value is 7.40, vacuumizes, and drips under conditions of logical nitrogen into Shrek pipe
The polypeptide chain P-3 for adding 6.0equiv. reacts 10min under the conditions of 25 DEG C.Reaction terminates reaction system being placed on Rotary Evaporators
On be spin-dried for, three times with water and ethyl acetate extraction organic phase, with the dry organic phase of anhydrous sodium sulfate, then be spin-dried for, by further
For column chromatography for separation (methylene chloride/methanol) to get product, the high resolution mass spectrum data of the product is as shown in Figure 6.
Embodiment 5
The aziridine chemical combination with amide in the embodiment 1 of 1.0equiv. (0.2mmol) is added into Shrek pipe
Object, the buffer solution (Tris-HCl) that 1.0mLpH value is 7.40, vacuumizes, and drips under conditions of logical nitrogen into Shrek pipe
The polypeptide chain P-4 for adding 6.0equiv. reacts 10min under the conditions of 25 DEG C.Reaction terminates reaction system being placed on Rotary Evaporators
On be spin-dried for, three times with water and ethyl acetate extraction organic phase, with the dry organic phase of anhydrous sodium sulfate, then be spin-dried for, by further
For column chromatography for separation (methylene chloride/methanol) to get product, the high resolution mass spectrum data of the product is as shown in Figure 7.
Embodiment 6
The aziridine chemical combination with amide in the embodiment 1 of 1.0equiv. (0.2mmol) is added into Shrek pipe
Object, the buffer solution (Tris-HCl) that 1.0mLpH value is 7.60, vacuumizes, and drips under conditions of logical nitrogen into Shrek pipe
The polypeptide chain P-5 for adding 6.0equiv. reacts 10min under the conditions of 25 DEG C.Reaction terminates reaction system being placed on Rotary Evaporators
On be spin-dried for, three times with water and ethyl acetate extraction organic phase, with the dry organic phase of anhydrous sodium sulfate, then be spin-dried for, by further
For column chromatography for separation (methylene chloride/methanol) to get product, the high resolution mass spectrum data of the product is as shown in Figure 8.
Embodiment 7
The aziridine chemical combination with amide in the embodiment 1 of 1.0equiv. (0.2mmol) is added into Shrek pipe
Object, the buffer solution (Tris-HCl) that 1.0mLpH value is 7.00, vacuumizes, and drips under conditions of logical nitrogen into Shrek pipe
The polypeptide chain P-6 for adding 6.0equiv. reacts 10min under the conditions of 25 DEG C.Reaction terminates reaction system being placed on Rotary Evaporators
On be spin-dried for, three times with water and ethyl acetate extraction organic phase, with the dry organic phase of anhydrous sodium sulfate, then be spin-dried for, by further
For column chromatography for separation (methylene chloride/methanol) to get product, the high resolution mass spectrum data of the product is as shown in Figure 9.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. a kind of aziridine is in the method for the application modification of antibody coupling drug-linker, which is characterized in that this method packet
It includes following steps: aziridine compound being added into the Tris-HCl buffer solution that pH value is 7.0~7.6, in room temperature, nitrogen
Polypeptide chain is added under gas atmosphere environment to be reacted, is after reaction spin-dried for solvent, it is isolated by preparative liquid chromatography
The Thioacetal of dimerization.
2. aziridine as described in claim 1 is in the method for the application modification of antibody coupling drug-linker, feature
It is, the structural formula of the aziridine compound are as follows:
In formula, R1For Ph, 4-OMe-Ph or 4-Cl-Ph；R2For COOtBu、COONH2Or H.
3. aziridine as described in claim 1 is in the method for the application modification of antibody coupling drug-linker, feature
It is, the polypeptide chain is CSL-NH2、CTWSL-NH2、RCLITCK-NH2、CKARGGC-NH2、RACKGGCG、CPCRAGKG-
NH2、CRAKPALGKGKC-NH2In any one.
4. aziridine as described in claim 1 is in the method for the application modification of antibody coupling drug-linker, feature
It is, the Molar ratio of the buffer solution, aziridine compound and polypeptide chain is 1mL:(0.05~0.2)
Mmol:(0.3~1.2) mmol.
5. aziridine as claimed in claim 4 is in the method for the application modification of antibody coupling drug-linker, feature
It is, the Molar ratio of the buffer solution, aziridine compound and polypeptide chain is 1mL:0.05mmol:
0.4mmol。
6. aziridine as described in claim 1 is in the method for the application modification of antibody coupling drug-linker, feature
It is, the pH value of Tris-HCl buffer solution is 7.40.
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| CN101575332A (en) * | 2009-05-31 | 2009-11-11 | 北京欧凯纳斯科技有限公司 | Diazacyclo propenyl-contained non-natural nucleoside, preparation method and applications thereof |
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| CN101575332A (en) * | 2009-05-31 | 2009-11-11 | 北京欧凯纳斯科技有限公司 | Diazacyclo propenyl-contained non-natural nucleoside, preparation method and applications thereof |
Non-Patent Citations (1)
| Title |
|---|
| R. S. EL"KINSON ET AL.: "Reactions of azirines with sulfur nucleophiles. 2. Reactions of 2H-azirine with β,γ-dihydroxy thiols and β-dithiols. a new route to the synthesis of cyclic sulfides", 《CHEM HETEROCYCL COMPD》 * |
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