CN116239513B - Preparation method of MMAE key intermediate, preparation method of MMAE and antibody coupling drug - Google Patents

Preparation method of MMAE key intermediate, preparation method of MMAE and antibody coupling drug Download PDF

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CN116239513B
CN116239513B CN202310494927.4A CN202310494927A CN116239513B CN 116239513 B CN116239513 B CN 116239513B CN 202310494927 A CN202310494927 A CN 202310494927A CN 116239513 B CN116239513 B CN 116239513B
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CN116239513A (en
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李九远
董博
李常峰
王金超
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Tianjin Asymchem Pharmaceutical Co Ltd
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D207/00Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
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    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
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    • A61K47/6801Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/55Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The invention provides a preparation method of an MMAE key intermediate, a preparation method of the MMAE and an antibody coupling drug. The preparation method of the MMAE key intermediate comprises the following steps: step S1, carrying out condensation reaction on a D-alanine compound and N, O-dialkylhydroxylamine salt in an alkaline environment to obtain a first product; step S2, R on the first product 1 Removing to obtain a second product; step S3, performing condensation coupling reaction on the second product and a pyridine alkyl carboxylic acid compound to obtain a third product; step S4, coupling reaction is carried out on the third product and the phenyl compound, so as to obtain a fourth product; s5, selectively reducing the fourth product to obtain an MMAE key intermediate; by applying the technical scheme of the invention, the key intermediate of the MMAE is synthesized by adopting cheap and easily available commercial raw materials as the initial raw materials, thereby avoiding the direct participation of the norephedrine in the reaction and providing a novel synthetic method for the MMAE.

Description

Preparation method of MMAE key intermediate, preparation method of MMAE and antibody coupling drug
Technical Field
The invention relates to the technical field of pharmaceutical chemistry, in particular to a preparation method of an MMAE key intermediate, a preparation method of the MMAE and an antibody coupling drug.
Background
The Antibody conjugated drug anti-drug Conjugate (ADC) is formed by coupling a chemotherapeutic drug with strong cytotoxicity with monoclonal Antibody through a linker, and has strong killing power of a small molecular drug and high targeting of pure monoclonal Antibody, so that the Antibody conjugated drug is a research and development hot spot of tumor targeted therapy. The ADC drug is characterized in that: the treatment efficacy is strong; the tumor cell specificity is high, and the false killing rate is low; the immunogenicity is weak, and drug resistance is not easy to generate; the circulation time in serum is long; has weak cytotoxicity to non-target cells.
The antibody class (macromolecular fraction) of ADC drugs is unlimited, whereas the class (small fraction) of toxins is limited, monomethyl auristatin E Monomethyl auristation E (MMAE; SGD-1010; CAS: 474645-27-7) being the most commonly used class of toxin compounds. MMAE is a synthetic derivative of dolastatin 10, is a novel, synthetic, antimitotic/antimitotic protein capable of blocking tubulin polymerization, shows strong activity against a range of lymphomas, leukemias and solid tumors in preclinical studies in vitro and in vivo, has extremely strong antitumor effects, belongs to the class of antimicrotubules as vinblastine, but has toxicity approximately equal to 200 times that of vinblastine. However, due to its high toxicity, it cannot be used as a drug directly. MMAE is widely used as a cytotoxic ingredient in the manufacture of antibody-conjugated drugs to treat cancer.
The existing disclosed synthetic methods of MMAE all adopt norephedrine as a starting material, but the norephedrine belongs to a class of easy-to-poison compounds in China and cannot be widely applied. Thus, there is a need in the art to develop a synthetic method of MMAE that does not require the use of norephedrine as a starting material.
Disclosure of Invention
The invention mainly aims to provide a preparation method of an MMAE key intermediate, a preparation method of the MMAE and an antibody coupling drug, so as to solve the problem that the synthesis method of the MMAE in the prior art needs to adopt an easily-toxic compound, namely, norephedrine, as a starting material, so that the MMAE cannot be widely applied.
In order to achieve the above object, according to a first aspect of the present invention, there is provided a process for preparing an MMAE-critical intermediate having a structure corresponding to the following formula (i):
formula (I);
the preparation method comprises the following steps: step S1, carrying out condensation reaction on D-alanine compounds and N, O-dialkylhydroxylamine salts in an alkaline environment to obtain a first product system,purifying the first product system to obtain a first product; the D-alanine compound has a structure shown in the following formula (II), the N, O-dialkylhydroxylamine salt has a structure shown in the following formula (III), and the first product has a structure shown in the following formula (IV); step S2, R on the first product 1 Removing to obtain a second product system, and purifying the second product system to obtain a second product, wherein the second product has a structure shown in the following formula (V); step S3, performing condensation coupling reaction on the second product and a pyridine alkyl carboxylic acid compound to obtain a third product system, and purifying the third product system to obtain a third product, wherein the pyrrole alkyl carboxylic acid compound has a structure shown in the following formula (VI), and the third product has a structure shown in the following formula (VI)) The structure is shown; step S4, coupling reaction is carried out on the third product and a phenyl compound to obtain a fourth product system, and the fourth product system is purified to obtain a fourth product, wherein the phenyl compound has the following formula (the formula:)>) The structure shown, the fourth product has the formula (++>) The structure is shown; s5, selectively reducing the fourth product to obtain a fifth product system, and purifying the fifth product system to obtain an MMAE key intermediate;
formula (II); />Formula (III); />Formula (IV);
formula (V);/>(-)>);
(-)>);
Wherein R and R 1 Each independently represents a protecting group comprising at least one of an alkoxycarbonyl group, an acyl group or an alkyl group; z represents hydrochloric acid, nitric acid or sulfuric acid; r is R a Is methyl; r is R 2 And R is 3 Each independently represents a C1 to C6 alkyl group; m represents a metal or a halogenated metal.
Further, R and R 1 Each independently represents benzyloxycarbonyl, t-butyloxycarbonyl, fluorenylmethoxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, benzoyl, phthaloyl, p-toluenesulfonyl, trifluoroacetyl, trityl, 2,4 dimethoxybenzyl, p-methoxybenzyl or benzyl; and/or R 2 And R is 3 Each independently represents a C1 to C4 alkyl group; and/or M represents lithium, sodium, potassium, magnesium bromide, magnesium chloride or magnesium iodide.
Further, R and R 1 Each independently represents a benzyloxycarbonyl group, a t-butyloxycarbonyl group, a fluorenylmethoxycarbonyl group or a benzoyl group; and/or, Z represents hydrochloric acid; and/or R 2 And R is 3 Each independently represents methyl; and/or M represents lithium or magnesium bromide.
Further, in the step S1, the pH value of the alkaline environment is 8.0-11.0.
Further, in the above step S1, the condensation reaction is performed in an alkaline environment formed by a first solvent including at least one of dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran, and N, N-dimethylformamide.
Further, the temperature of the condensation reaction is 0-10 ℃, and the time of the condensation reaction is 4.0-24.0 h.
Further, the purification of the first product system includes quenching, extraction and concentration, which are performed sequentially.
Further, in the step S2, R is performed on the first product and the hydrogen chloride solution in a second solvent 1 The removal reaction is carried out, and the hydrogen chloride solution is preferably at least one of a hydrogen chloride methanol solution, a hydrogen chloride dioxane solution, a hydrogen chloride ethanol solution or an isopropyl hydrogen acetate solution.
Further, the second solvent includes at least one of methanol, ethanol, dioxane or isopropyl acetate.
Further, R 1 The temperature of the removal reaction is 0-10 ℃, and the time of the removal reaction is 2.0-8.0 h.
Further, the purification of the second product system comprises solid-liquid separation, extraction and concentration, which are performed sequentially.
Further, in the step S3, the second product and the pyrrolidinyl carboxylic acid compound are subjected to condensation coupling reaction under the action of a condensation reagent, wherein the condensation reagent is preferably at least one of HOBT/EDCI, HATU, HOBT/HBTU or T3P.
Further, the second product is subjected to a condensation coupling reaction with a pyrrolidinecarboxylic acid compound in a third solvent, preferably at least one of N, N-dimethylformamide, methylene chloride, tetrahydrofuran and 2-methyltetrahydrofuran.
Further, the temperature of the condensation coupling reaction is 15-25 ℃, and the time of the condensation coupling reaction is 1.0-8.0 h.
Further, the purification of the third product system comprises quenching, crystallization, recrystallization or beating, which are performed sequentially.
Further, in the step S5, the fourth product is selectively reduced by a reducing agent, and the reducing agent preferably includes at least one of aluminum isopropoxide, tributyl lithium hydride or lithium aluminum hydride.
Further, the fourth product and the reducing agent are selectively reduced in a fifth agent, which is preferably at least one of toluene, an ether solvent or an alcohol solvent.
Further, the temperature of the selective reduction is 40-55 ℃, and the time of the selective reduction is 1.0-8.0 h.
Further, the purification of the above substrate product system includes quenching, extraction, quenching, concentration, crystallization and beating, which are sequentially performed.
In order to achieve the above object, according to a second aspect of the present invention, there is also provided a process for producing MMAE, the process comprising a process for producing MMAE-critical intermediate, the process for producing MMAE-critical intermediate being any one of the processes for producing MMAE-critical intermediate provided in the first aspect.
According to a third aspect of the present application, there is also provided an antibody-conjugated drug, the raw material of which comprises an antibody and the MMAE key intermediate obtained by any one of the preparation methods provided in the first aspect.
By using the technical scheme of the application, the MMAE key intermediate with the structure shown in the formula (I) is synthesized by adopting cheap and easily available commercial raw materials as starting materials, thereby avoiding the direct participation of norephedrine in the reaction and providing a new synthesis method for the MMAE. In addition, the preparation method of the MMAE key intermediate provided by the application has mild and controllable reaction conditions, fully considers the atom economy of the reaction, effectively improves the yield of the product, and simultaneously has the advantages of green chemistry concept, low cost of the reaction solvent, environmental friendliness and good cost advantage and commercialization basis.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application.
In the drawings:
FIG. 1 shows a nuclear magnetic spectrum of a key intermediate of MMAE obtained by the preparation method provided in example 9 of the present application;
Fig. 2 shows a high performance liquid chromatogram of the MMAE key intermediate obtained according to the preparation method provided in example 9 of the present application.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.
As analyzed in the background of the application, in the existing MMAE synthesis methods, norephedrine is required to be used as a starting material, but the norephedrine belongs to a class of easily toxic compounds in China, so that the norephedrine cannot be widely applied. In order to solve the technical problem, the application provides a preparation method of an MMAE key intermediate, a preparation method of the MMAE and an antibody coupling drug.
In one exemplary embodiment of the present application, a process is provided for the preparation of an MMAE key intermediate having the structure of formula (i):
formula (I);
the preparation method of the MMAE key intermediate with the structure shown in the formula (I) comprises the following steps: step S1, carrying out condensation reaction on a D-alanine compound and N, O-dialkylhydroxylamine salt in an alkaline environment to obtain a first product system, and purifying the first product system to obtain a first product; the D-alanine compound has a structure shown in the following formula (II), the N, O-dialkylhydroxylamine salt has a structure shown in the following formula (III), and the first product has a structure shown in the following formula (IV);
Formula (II); />Formula (III); />Formula (IV);
step S2, R on the first product 1 Removing to obtain a second product system, and purifying the second product system to obtain a second product, wherein the second product has a structure shown in the following formula (V);
formula (V);
step S3, performing condensation coupling reaction on the second product and the pyrrolidinyl carboxylic acid compound to obtain a third product system, and purifying the third product system to obtain a third product;
formula (VI); />(-)>);
The pyrrolidinyl carboxylic acid compound has the structure shown in the following formula (VI), and the third product has the structure shown in the following formula) The structure is shown; step S4, coupling reaction is carried out on the third product and phenyl compound to obtain a fourth product system, and the fourth product system is purified to obtain a fourth product, wherein the phenyl compound has the following formula ()>) The structure shown, the fourth product has the formula (++>) The structure is shown;
(-)>);/>(-)>);
And S5, selectively reducing the fourth product to obtain a fifth product system, and purifying the fifth product system to obtain the MMAE key intermediate.
The formula (I) to (II)) Wherein R and R 1 Each independently represents a protecting group comprising at least one of an alkoxycarbonyl group, an acyl group or an alkyl group; z represents hydrochloric acid, nitric acid or sulfuric acid; r is R a Is methyl; r is R 2 And R is 3 Each independently represents a C1 to C6 alkyl group; m represents a metal or a halogenated metal.
The above alkoxycarbonyl group includes, but is not limited to, any one of benzyloxycarbonyl, t-butyloxycarbonyl, fluorenylmethoxycarbonyl, or allyloxycarbonyl, and the above acyl group includes, but is not limited to, phthaloyl or p-toluenesulfonyl; the above alkyl group includes, but is not limited to, any of trityl, 2,4 dimethoxybenzyl, p-methoxybenzyl or benzyl.
In order to further increase the efficiency of the preparation of the above-mentioned MMAE key intermediates, R and R are preferably selected 1 Each independently represents any one of benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), benzoyl (Bz), phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl (Tfa), trityl (Trt), 2,4 Dimethoxybenzyl (DMB), p-methoxybenzyl (Pmb) or benzyl (Bn); and/or, preferably R 2 And R is 3 Each independently represents a C1-C4 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or the like; and/or M represents any one of lithium, sodium, potassium, magnesium bromide, magnesium chloride or magnesium iodide.
To further reduce the controllability of the reaction conditions and to further increase the purity and yield of MMAE key intermediates, R and R are preferredR 1 Each independently represents benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc) or benzylformyl (Bz); and/or, Z represents hydrochloric acid; and/or R 2 And R is 3 Each independently represents methyl; and/or M represents lithium or magnesium bromide.
In some embodiments of the present application, in the step S1, in order to further improve the efficiency of the condensation reaction, the pH of the alkaline environment is preferably 8 to 11.0.
In order to further accelerate the rate of the condensation reaction, it is preferable that the above condensation reaction is carried out in an alkaline environment formed by a first solvent including, but not limited to, a mixed solvent formed by any one or more of methylene chloride, tetrahydrofuran, 2-methyltetrahydrofuran, and N, N-dimethylformamide.
In order to further promote the condensation reaction to proceed more completely and to further enhance the efficiency of the first product production, it is preferable that the temperature of the condensation reaction is 0 to 10 ℃ and the time of the condensation reaction is 4.0 to 24.0 hours.
Typically, but not by way of limitation, the pH of the alkaline environment is, for example, 8.0, 8.2, 8.5, 9.0, 9.4, 9.8, 10.0, 10.5, 11.0 or any two values in the range; the temperature of the condensation reaction is, for example, 0 ℃,1 ℃, 2 ℃, 5 ℃, 8 ℃, 10 ℃ or a range value consisting of any two values; the time of the above-mentioned condensation reaction is, for example, 4h, 8h, 12h,15h, 18h, 20h, 24h or a range of values consisting of any two values.
The above-mentioned method for purifying the first product system is not limited, and any purification method capable of separating the first product from the first product system may be used. To further increase the yield of the first product system, it is preferred that the purification of the first product system comprises quenching, extraction and concentration, which are performed sequentially.
From the standpoint of environmental protection and cost reduction, the first product system is preferably quenched by an aqueous solution of sodium bicarbonate, and the mass concentration of the sodium bicarbonate is preferably 10% -15%. In order to further improve the yield of the first product, the quenched first product system is preferably extracted by an organic solvent, so as to obtain a first product extraction phase, the type of the organic solvent is not limited, any organic solvent capable of extracting the first product can be used, and from the aspects of environmental protection and cost reduction, the organic solvent is preferably a mixed solvent formed by any one or more of dichloromethane, tetrahydrofuran and 2-methyltetrahydrofuran. The way of concentrating the first product extract phase described above is not limited, and includes, but is not limited to, evaporation, membrane concentration, and the like.
In the step S2, in order to further improve the R1 removal reaction efficiency, it is preferable to mix the first product with a hydrogen chloride solution in a second solvent to perform the R1 removal reaction. The above hydrogen chloride solution includes, but is not limited to, at least one of a hydrogen chloride methanol solution, a hydrogen chloride dioxane solution, a hydrogen chloride ethanol solution, or an isopropyl hydrogen acetate solution; from the standpoint of environmental protection and cost reduction, it is preferable that the second solvent is a mixed solvent formed of any one or more of methanol, ethanol, dioxane or isopropyl acetate.
In order to further promote the removal of R1 more completely and improve the efficiency of the R1 removal reaction, the temperature of the R1 removal reaction is preferably 0-10 ℃, and the time of the R1 removal reaction is preferably 2.0-8.0 h.
Typically, but not by way of limitation, the temperature of the R1 removal reaction is, for example, 0 ℃, 5 ℃, 8 ℃, 10 ℃ or any two values, and the time of the R1 removal reaction is, for example, 2 hours, 4 hours, 6 hours, 8 hours or any two values.
The above-mentioned method for purifying the second product system is not limited, and any purification method capable of separating the second product from the second product system may be used. In order to further increase the yield of the second product system, it is preferred that the purification of the second product system comprises solid-liquid separation, extraction and concentration, which are performed sequentially. The solid-liquid separation mode is not limited, and includes, but is not limited to, filtration, centrifugation, etc., and the organic solvent used for the extraction is not limited, and any organic solvent capable of extracting the second product can be used, including, but not limited to, a mixed solvent formed by any one or more of methanol, ethanol, dioxane or isopropyl acetate; the manner of concentration is not limited and includes, but is not limited to, evaporation and the like.
In the above step S3, in order to further enhance the efficiency of the condensation coupling reaction, it is preferable that the second product is subjected to the above condensation coupling reaction with the pyrrolidinylcarboxylic acid compound under the action of a condensing agent, the type of the above condensing agent is not limited, and any agent capable of promoting the condensation coupling reaction of an amino group with a carboxyl group may be used, including, but not limited to, a mixed agent formed of any one or more of HOBT (1-hydroxybenzotriazole)/EDCI (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride), HATU (2- (7-azabenzotriazole) -N, N '-tetramethylurea hexafluorophosphate), HOBT (1-hydroxybenzotriazole)/HBTU (benzotriazole-N, N' -tetramethylurea hexafluorophosphate) or T3P (1-propylphosphoric anhydride).
In order to further improve the yield and production efficiency of the third product, it is preferable that the second product and the pyrrolidinyl carboxylic acid compound are subjected to the condensation coupling reaction in a third solvent, and from the viewpoints of green chemistry and cost reduction, the third solvent is preferably a mixed solvent formed of any one or more of N, N-dimethylformamide, dichloromethane, tetrahydrofuran and 2-methyltetrahydrofuran.
In order to further promote the progress of the condensation coupling reaction more sufficiently and to further increase the yield of the third product, it is preferable that the temperature of the condensation coupling reaction is 15 to 25℃and the time of the condensation coupling reaction is 1.0 to 8.0 hours.
Typically, but not by way of limitation, the temperature of the condensation coupling reaction is, for example, 15 ℃, 18 ℃, 22 ℃, 25 ℃ or any two values, and the time of the condensation coupling reaction is, for example, 1h, 3h, 5h, 6h, 8h or any two values.
The above-mentioned method for purifying the third product system is not limited, and any purification method capable of separating the third product from the third product system may be used. To further increase the yield of the third product system, it is preferred that the purification of the third product system comprises quenching, crystallization, beating or recrystallization, which are performed sequentially. From the viewpoints of cost saving and environmental protection, it is preferable to dropwise add an acidic aqueous solution, an alkaline aqueous solution or an aqueous solution to the third product system for quenching treatment, wherein the acidic aqueous solution includes, but is not limited to, an aqueous solution of citric acid or an aqueous solution of hydrochloric acid; the above-mentioned alkaline aqueous solution includes, but is not limited to, an aqueous solution of sodium bicarbonate or an aqueous solution of sodium carbonate; the crystallization mode is not limited, and the quenched third product system is preferably subjected to crystallization. In order to further improve the purity of the third product, it is preferable to subject the crude third product obtained by the crystallization treatment to beating or recrystallization treatment.
In step S4, in order to further enhance the coupling reaction efficiency, it is preferable that the coupling reaction between the third product and the phenyl compound is performed in a fourth solvent, and from the viewpoints of green chemistry and cost reduction, the fourth solvent is preferably a mixed solvent of any one or more of tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether and diethyl ether.
In order to further promote the progress of the coupling reaction more completely and further improve the efficiency of the coupling reaction, it is preferable that the temperature of the coupling reaction is 15 to 25℃and the time of the coupling reaction is 4 to 24 h.
Typically, but not by way of limitation, the temperature of the coupling reaction is, for example, 15 ℃, 18 ℃, 22 ℃, 25 ℃ or any two values, and the time of the coupling reaction is, for example, 4 hours, 10 hours, 12 hours, 16 hours, 20 hours, 24 h or any two values.
The above-mentioned method for purifying the fourth product system is not limited, and any purification method capable of separating the fourth product from the fourth product system may be used. To further increase the yield of the fourth product system, it is preferred that the purification of the fourth product system comprises quenching, extraction, concentration, crystallization and beating, which are performed sequentially. From the viewpoints of cost saving and environmental protection, the fourth product system is preferably added with an ammonium chloride solution dropwise for quenching treatment, and the mass concentration of the ammonium chloride solution is preferably 2-4% to further improve the quenching efficiency. The extraction is performed by using an organic solvent, the type of the organic solvent used for extraction is not limited, and any organic solvent capable of extracting the fourth product can be used, including but not limited to a mixed solvent formed by any one or more of tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether or diethyl ether; the manner of concentration is not limited and includes, but is not limited to, evaporation and the like. The crystallization mode is not limited, and preferably, the concentrated fourth product system is dropwise added into a small polar solvent for crystallization to obtain solid crystals. The small polar solvent is, for example, a mixed polar solvent formed by any one or more of methyl tertiary butyl ether, n-heptane, n-hexane and isopropyl ether. In order to further improve the purity of the fourth product, it is preferable to obtain the fourth product by subjecting the solid crystals obtained by the crystallization to a pulping treatment.
In step S5, in order to further increase the efficiency of the selective reduction reaction, it is preferable that the fourth product is selectively reduced by the reducing agent. In order to further promote the selective reduction reaction more efficiently, the reducing agent is preferably selected from the group consisting of aluminum isopropoxide, tributyl lithium hydride and lithium aluminum hydride.
In order to further promote the selective reduction of the fourth product by the reducing agent in step S5, the fourth product is preferably dissolved in a fifth solvent, and then the reducing agent is added to the fifth solvent to perform the selective reduction. The type of the fifth solvent is not limited, and any solvent can be used to dissolve the fourth product. From the viewpoint of green chemistry and cost reduction, the fifth solvent is preferably toluene, an ether solvent or an alcohol solvent. The above ether solvents include, but are not limited to, mixed solvents of any several or more of methyl tertiary butyl ether, isopropyl ether, or diethyl ether; the above alcohol solvents include, but are not limited to, mixed solvents formed of any several or more of isopropanol, methanol, ethanol or n-butanol.
In order to further promote the selective reduction reaction to be more complete and further improve the preparation efficiency of the MMAE key intermediate, the temperature of the selective reduction is preferably 40-55 ℃, and the time of the selective reduction is preferably 1-8 hours.
Typically, but not by way of limitation, the temperature of the selective reduction is, for example, 40, 45, 50 or 55℃and the time of the coupling reaction is, for example, 2, 4, 6 or 8h.
The above-mentioned method for purifying the fifth product system is not limited, and any purification method capable of separating the fifth product from the fifth product system may be used. To further increase the yield of the fifth product system, it is preferred that the purification of the fifth product system comprises quenching, extraction, quenching, concentration, crystallization and beating, which are performed sequentially. From the viewpoints of cost saving and environmental protection, the quenching treatment is preferably performed by dropwise adding an ammonium chloride solution into the fifth product system, and the mass concentration of the ammonium chloride solution is preferably 2-4% so as to further improve the quenching efficiency. The extraction is performed by using an organic solvent, the type of the organic solvent used for extraction is not limited, and any organic solvent capable of extracting the fifth product can be used, including but not limited to a mixed solvent formed by any one or more of ethyl acetate, isopropyl acetate, dichloromethane and 2-methyltetrahydrofuran; the manner of the above-mentioned quenching is not limited, and it is preferable to conduct the quenching with saturated brine. The manner of concentration is not limited and includes, but is not limited to, evaporation and the like. The crystallization mode is not limited, and preferably, the concentrated fifth product system is dropwise added into a small polar solvent for crystallization to obtain solid crystals. And a mixed solvent formed by any one or more of the small polar solvents such as methyl tertiary butyl ether, n-heptane and n-hexane. In order to further improve the purity of the MMAE key intermediate, it is preferable to perform pulping treatment on the solid crystal obtained by crystallization to obtain the MMAE key intermediate.
In some embodiments of the present application, the process scheme of the preparation method of the MMAE key intermediate is shown below:
the Me represents methyl, and the R and R 1 The same meaning as in any of the above preparation methods is used, wherein Me represents methyl, and step represents "step".
In another exemplary embodiment of the present application, there is also provided a process for the preparation of MMAE, the process comprising a process for the preparation of MMAE key intermediates, the process for the preparation of MMA key intermediates being any of the processes provided in the first exemplary embodiment above.
By applying the technical scheme of the application, the MMAE key intermediate is prepared by adopting the preparation method of the MMAE key intermediate, and then the MMAE is prepared by utilizing the MMAE key intermediate, so that the use of norephedrine as a starting material is avoided, a novel synthetic method is provided for the MMAE, and the mass production is more facilitated.
In a third exemplary embodiment of the present application, there is also provided an antibody-conjugated drug, the raw materials of which include an antibody and an MMAE-key intermediate prepared by any one of the preparation methods provided in the first exemplary embodiment above.
By applying the technical scheme of the application, the antibody coupling drug provided by the application adopts the MMAE key intermediate and the antibody obtained by any one of the preparation methods provided by the first exemplary embodiment of the application as raw materials, avoids the use of norephedrine as a starting material, provides a novel synthesis method for the antibody coupling drug, and has wide application prospect.
The advantageous effects of the present application will be further described below with reference to examples.
In the examples below, vol refers to the volume ratio, i.e., the ratio of the volume of the solvent added to the mass of the reference material, eq refers to the equivalent ratio, i.e., the ratio of the molar ratio of the material added to the reference material to which 1.0eq of the main material was added per operation.
Example 1
The embodiment provides a preparation method of an MMAE key intermediate, wherein the MMAE key intermediate has a structure shown in the following formula (I); wherein R is tert-butoxycarbonyl (Boc), R a Methyl (Me).
Formula (I);
the preparation method of the MMAE key intermediate comprises the following steps of (1) mixing 150g (0.79 mol,1.0 eq) of Boc-D-acrylic acid with a protecting group Boc on the amino group and 96.6g (0.97 mol,1.22 eq) of N, O-dimethylhydroxylamine hydrochloride in 1500mL (10 Vol) of methylene chloride solvent, then adding 387g (1.02 mol,1.29 eq) of HATU (2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate), using 257.4g (2.54 eq) of triethylamine to adjust the pH value of the system to 10.5, carrying out condensation reaction on the Boc-D-acrylic acid and N, O-dimethylhydroxylamine hydrochloride at the temperature of 15-25 ℃ for 14h, and ending the reaction to obtain a first product system. Adding 1500mL of purified water into the first product system for quenching treatment, and separating to obtain an organic phase of a first product; the organic phase of the first product is heated for 5 hours at the temperature of T less than or equal to 45 ℃ to be concentrated, 750mL of ethyl acetate solvent is adopted to be concentrated until no solvent exists, then 750mL of ethyl acetate is added into the system, pulping is carried out for 2 hours at the temperature of 15-25 ℃, 157g of the first product is obtained through filtration, and the first product is white-like solid with the yield of 85.5%.
(2) Mixing the first product with 628mL (4 Vol) of 4M (mol/L) 1, 4-dioxane solution of hydrogen chloride in 314mL (2 Vol) 1, 4-dioxane solvent, and carrying out R of the first product at 15-25 DEG C 1 And (3) carrying out radical removal reaction, wherein the reaction lasts for 6 hours, and a second product system is obtained after the reaction is finished. Concentrating the second product system at the temperature of T less than or equal to 40 ℃, and carrying out four times of concentration treatment by using 235mL of n-heptane to obtain a second product, wherein the second product is a light yellow oily matter, and the yield is 95.0%.
(3) 115.9g (0.69 mol,1.1 eq) of the second product and 180g (0.63 mol,1.0 eq) of a pyrrolidinylcarboxylic acid compound with a Boc protecting group are mixed in 1440mL (8 Vol) of dichloromethane solvent, then 325.1g (2.5 mol,4 eq) of N, N-diisopropylethylamine, 127g (0.94 mol,1.5 eq) of HOBT and 180.2g (0.94 mol,1.5 eq) of EDCI condensing reagent are added to the solvent at a temperature of 0-10 ℃ for 2h, and a condensation coupling reaction is carried out at 15-25 ℃ to obtain a third product system after the reaction is finished. And (3) dropwise adding 1800mL of citric acid aqueous solution with the mass concentration of 10% into the third product system for quenching treatment, separating the obtained quenching system of the third product to obtain an organic phase, sequentially using 1800mL of purified water and 1800mL of 10% sodium bicarbonate aqueous solution for extraction and washing, concentrating the organic phase at the temperature of less than or equal to 40 ℃ to obtain a third product crude product, adding 900mL of methyl tertiary butyl ether reagent into the third product crude product, pulping the third product crude product, and filtering to obtain 230g of the third product with the yield of 91.5%.
(4) 230g (0.57 mol,1.0 eq) of the third product is dissolved in 2760mL (12 vol) of tetrahydrofuran solvent, the system is cooled to 10 ℃, 1140mL (1.14 mol,2.0 eq) of 1M phenylmagnesium bromide tetrahydrofuran solution is added dropwise into the system to be mixed with the third product for coupling reaction, cooling liquid is removed after the addition, the reaction lasts for 13h at 15-25 ℃ and a fourth product system is obtained after the reaction is finished. And (3) dropwise adding 1150mL of ammonium chloride solution with the mass concentration of 3.7% into the fourth product system for quenching treatment to obtain a quenching system of the fourth product. Extracting a quenching system of a fourth product by adopting 1150mL of ethyl acetate solvent, continuously extracting an aqueous phase once by adopting 1150mL of ethyl acetate, merging organic phases to obtain a fourth product extract phase, concentrating the fourth product extract phase to 460mL, then dropwise adding the fourth product extract phase into 4600mL of n-heptane solvent for crystallization treatment, crystallizing and separating out a crude product of the fourth product, adding 2300mL of n-heptane into the crude product of the fourth product for pulping treatment to obtain 225g of the fourth product which is a white solid, and obtaining the yield of 93.7%.
(5) 225g (0.54 mol,1.0 eq) of the fourth product was dissolved in a mixed solvent of 810mL (3.6 Vol) toluene and 450mL (2 Vol) isopropanol, and then 198.5. 198.5 g aluminum isopropoxide (0.97 mol,1.8 eq) was added to the system three times, and the reaction was continued at 40-55℃for 5 hours, and the reaction was completed to obtain a fifth product system. And (3) dropwise adding 1125mL of ammonium chloride solution with the mass concentration of 3.7% into a fifth product system for quenching treatment, separating out solids in the system, filtering, extracting filtrate once by using 1125mL of ethyl acetate, extracting water phase once by using 1125mL of ethyl acetate after liquid separation, extracting and washing organic phases once by using 1125mL of saturated sodium chloride solution after merging, and then decompressing and desolventizing at the temperature T being less than or equal to 40 ℃ and concentrating to 2 Vol. And (3) obtaining a concentrated system of a fifth product, dropwise adding the concentrated system of the fifth product into a mixed solvent formed by 1125mL of methyl tertiary butyl ether and 1575mL of n-heptane to perform crystallization treatment, crystallizing and separating out a crude product of the fifth product, and adding 675mL of n-heptane into the crude product of the fifth product to perform pulping treatment to obtain 214.8g of the MMAE key intermediate with the yield of 95%.
The preparation process of the MMAE intermediate is schematically shown as follows:
in the above process scheme, R 1 And R are each independently a Boc protecting group, me is methyl.
Example 2
This example differs from example 1 in that the MMAE key intermediate has the structure shown in formula (i) below; wherein R is fluorenylmethoxycarbonyl (Fmoc)),R a Methyl (Me).
Formula (I);
the preparation process differs from example 1 in that in step (3) the protecting group of the pyrrolidinyl carboxylic acid compound used is Fmoc and the amount of the pyrrolidinyl carboxylic acid compound used is 0.63mol.
Example 3
This example differs from example 1 in that the MMAE key intermediate has the structure shown in formula (i) below; wherein R is carbobenzoxy (Cbz), R a Methyl (Me).
Formula (I);
the preparation process differs from example 1 in that in step (3) the protecting group of the pyrrolidinyl carboxylic acid compound used is Cbz and the amount of the pyrrolidinyl carboxylic acid compound used is 0.63mol.
Example 4
This example differs from example 1 in that the MMAE key intermediate has the structure shown in formula (i) below; wherein R is benzoyl (Bz), R a Methyl (Me).
Formula (I);
the preparation process differs from example 1 in that in step (3) the protecting group of the pyrrolidinyl carboxylic acid compound used is Bz and the amount of the pyrrolidinyl carboxylic acid compound used is 0.63mol.
Example 5
This example differs from example 1 in that in step (4), phenylmagnesium bromide is replaced with phenyllithium as the formative reagent, and the amount of the substance of phenyllithium is the same as phenylmagnesium bromide.
Example 6
This example differs from example 1 in that in step (5), aluminum isopropoxide is replaced with tributyl lithium hydride, and the amount of the substance of tributyl lithium hydride is the same as that of aluminum isopropoxide.
Example 7
This example differs from example 1 in that in step (1), N, O-dimethylhydroxylamine nitrate was used instead of N, O-dimethylhydroxylamine hydrochloride, and the amount of the substance of N, O-dimethylhydroxylamine nitrate was the same as that of N, O-dimethylhydroxylamine hydrochloride.
Example 8
This example differs from example 1 in that in step (1), N, O-dimethylhydroxylamine sulfate was used instead of N, O-dimethylhydroxylamine hydrochloride, and the amount of the substance of N, O-dimethylhydroxylamine sulfate was the same as that of N, O-dimethylhydroxylamine hydrochloride.
Example 9
The present example provides a kg-grade preparation method of the MMAE key intermediate provided in example 1 above, which is performed according to the following steps:
(1) Boc-D-acrylic acid with 3.00 kg (15.8 mol,1.0 eq) amino group as Boc and 3.20 kg (19.0 mol,1.2 eq) N, O-dimethylhydroxylamine hydrochloride are mixed in 30.0L (10 Vol) methylene chloride solvent, HATU 7.81 Kg (20.5 mol,1.3 eq) is added, the pH value of the system is adjusted to 10.4 by using 5.11kg (50.6 mol,3.2 eq) triethylamine, condensation reaction is carried out at 15-25 ℃ for 12h, and the reaction is ended to obtain a first product system. Adding 30.0L of purified water into the first product system for quenching treatment, and separating to obtain an organic phase of a first product; the organic phase is heated for 5 hours at the temperature of less than or equal to 45 ℃ for concentration, 15.0L of ethyl acetate solvent is adopted for shrinking until the solvent is basically absent, then 15.0L of ethyl acetate is added into the system for pulping for 2 hours at the temperature of 15-25 ℃, the first product 3.28 and kg is obtained after filtration, and the first product is off-white solid with the yield of 89.4 percent.
(2) Mixing 3.28kg of the first product with 13.1L (4 Vol) of 4M hydrogen chloride 1, 4-dioxane solution in 6.6L (2 Vol) of 1, 4-dioxane solvent, and carrying out R of the first product at 15-25 DEG C 1 And (3) carrying out radical removal reaction, wherein the reaction lasts for 7 hours, and a second product system is obtained after the reaction is finished. Concentrating the second product system at a temperature of less than or equal to 40 ℃, and concentrating 4 times by using 4.9L of n-heptane to obtain a second product, wherein the second product is a light yellow oily matter, and the yield is 99.0%.
(3) 2.38Kg (14.1 mol,1.1 eq) of the second product and 3.68Kg (12.8 mol,1.0 eq) of a pyrrolidinylcarboxylic acid compound having a protecting group Boc were mixed in 29.5L (8 Vol) of methylene chloride solvent, then 6.60Kg (51.2 mol,4 eq) of N, N-diisopropylethylamine, 2.59Kg (19.2 mol,1.5 eq) of HOBT,3.68Kg (19.2 mol,1.5 eq) of EDCI condensing reagent were added to the solvent at a controlled temperature of 0 to 10℃for 2 hours to obtain a third product system. Dropwise adding 36.8L of 10% citric acid aqueous solution in mass concentration into a third product system for quenching treatment to obtain a quenching system of the third product, separating the quenching system to obtain an organic phase, sequentially using 1800ml of purified water and 36.8ml of 10% sodium bicarbonate aqueous solution for extraction and washing, then concentrating the organic phase at the temperature of less than or equal to 40 ℃ to obtain a third product crude product, adding 18.4L of methyl tertiary butyl ether reagent into the third product crude product, pulping the third product crude product, filtering to obtain 4.75kg of the third product, and obtaining 92.4% of yield.
(4) And (3) dissolving 4.75kg (11.8 mol,1.0 eq) of a third product in 57.0L (12 vol) tetrahydrofuran solvent, cooling the system to 10 ℃, dropwise adding 23.6L (mol/L) of 1M (mol/L) phenylmagnesium bromide tetrahydrofuran solution (23.6 mol,2.0 eq) into the system, mixing with the third product for coupling reaction, removing cooling liquid after the addition, continuing the reaction at 15-25 ℃ for 13h, and obtaining a fourth product system after the reaction is finished. And (3) dropwise adding 23.8L of ammonium chloride solution with the mass concentration of 3.7% into the fourth product system for quenching treatment to obtain a quenching system of the fourth product. Extracting a quenching system of a fourth product by using 23.8L of ethyl acetate solvent, continuously extracting a water phase by using 23.8L of ethyl acetate for one time, merging organic phases to obtain a fourth product extract phase, concentrating the fourth product extract phase to 9.5L, then dropwise adding the fourth product extract phase into 95L of n-heptane solvent for crystallization treatment, crystallizing and separating out a fourth product crude product, adding 47.5L of n-heptane into the fourth product crude product for pulping treatment to obtain a fourth product of 4.67kg, wherein the fourth product is a white solid product, and the yield is 94.4%.
(5) The fourth product 4.67Kg (11.2 mol,1.0 eq) was dissolved in 16.8L (3.6 Vol) toluene and 9.3L (2 Vol) isopropanol, then 4.12Kg aluminum isopropoxide (20.2 mol,1.8 eq) was added to the system in five portions, and the reaction was continued at 40-55℃for 6 hours, and the reaction was completed to obtain a fifth product system. And (3) dropwise adding 23.5L of ammonium chloride solution with the mass concentration of 3.7% into a fifth product system for quenching treatment, separating out solids in the system, filtering, extracting filtrate with 23.5L of ethyl acetate once, separating liquid, extracting water phase with 23.5L of ethyl acetate once, merging organic phases, extracting and washing the organic phases with 23.5L of saturated sodium chloride solution once, then carrying out reduced pressure desolventizing at the temperature T of less than or equal to 40 ℃, concentrating to 10L to obtain a concentrated system of a fifth product, dropwise adding the concentrated system of the fifth product into a mixed solvent formed by 23.5L of methyl tertiary butyl ether and 33.0L of n-heptane for crystallization treatment, crystallizing and separating out crude products of the fifth product, adding 14.0L of n-heptane into the crude products of the fifth product for pulping treatment, thus obtaining 4.48kg of the key intermediate of the MMAE, and the yield of 95.5%.
Test example 1
The nuclear magnetic resonance test and the high performance liquid phase test are performed on the MMAE key intermediate prepared in the embodiment 9, and the results are shown in fig. 1 and 2, and it can be seen from fig. 1 that the MMAE key intermediate prepared in the embodiment 9 is consistent with the expected structure of the molecule through nuclear magnetic resonance identification.
As can be seen from fig. 2, the purity of the MMAE key intermediate prepared in example 9 is 99.43%, and the maximum unknown single impurity rrt=1.04 is 0.26%, so that the requirement of downstream production of the intermediate can be met, and the requirement meets expectations.
Test example 2
The purity and yield of the first, second, third, fourth and MMAE key intermediates prepared in examples 1 to 9 were measured, respectively, and the results are shown in table 1 below.
Wherein the purity is measured by high performance liquid chromatography.
The calculation method of the yield comprises the following steps:
theoretical yield per step was calculated: theoretical yield = (starting material charge/starting material molecular weight) ×intermediate molecular weight
Actual yield calculation method per step, yield= (crude yield/theoretical yield) ×100%
TABLE 1
From the above description, it can be seen that the above embodiments of the present application achieve the following technical effects:
the method adopts cheap and easily available commercial raw materials as the initial raw materials to synthesize the MMAE key intermediate, avoids the direct participation of the norephedrine in the reaction, provides a novel synthetic method for the MMAE, and solves the problem that the synthetic method of the MMAE in the prior art needs to adopt the easily-toxic compound of the norephedrine as the initial raw material, so that the method cannot be widely applied.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (7)

1. The preparation method of the MMAE key intermediate is characterized in that the MMAE key intermediate has a structure shown in the following formula (I);
formula (I);
the preparation method comprises the following steps:
step S1, carrying out condensation reaction on a D-alanine compound and N, O-dialkylhydroxylamine salt in an alkaline environment to obtain a first product system, and purifying the first product system to obtain a first product; the D-alanine compound has a structure shown in the following formula (II), the N, O-dialkylhydroxylamine salt has a structure shown in the following formula (III), and the first product has a structure shown in the following formula (IV);
formula (II); />Formula (III); />Formula (IV);
step S2, R on the first product 1 Removing to obtain a second product system, and purifying the second product system to obtain a second product; the second product has a structure shown in the following formula (V);
Formula (V);
step S3, performing condensation coupling reaction on the second product and a pyrrolidinyl carboxylic acid compound to obtain a third product system, and purifying the third product system to obtain a third product; the pyrrolidinyl carboxylic acid compound has a structure shown in the following formula (VI), and the third product has a structure shown in the following formula (VII);
formula (VI); />Formula (VII);
step S4, performing a coupling reaction on the third product and a phenyl compound to obtain a fourth product system, and purifying the fourth product system to obtain a fourth product; the phenyl compound has a structure shown in the following formula (VIII), and the fourth product has a structure shown in the following formula (IX);
formula (VIII); />Formula (IX);
s5, selectively reducing the fourth product to obtain a fifth product system, and purifying the fifth product system to obtain the MMAE key intermediate;
wherein R and R 1 Each independently represents a protecting group selected from at least one of benzyloxycarbonyl, t-butyloxycarbonyl, fluorenylmethoxycarbonyl, or benzoyl; z represents hydrochloric acid; r is R a Is methyl; r is R 2 And R is 3 Each independently represents methyl; m represents at least one of lithium or magnesium bromide.
2. The preparation method according to claim 1, wherein in the step S1, the pH value of the alkaline environment is 8.0 to 11.0;
and/or the condensation reaction is carried out in an alkaline environment formed by a first solvent, wherein the first solvent is selected from at least one of dichloromethane, tetrahydrofuran, 2-methyltetrahydrofuran and N, N-dimethylformamide;
and/or the temperature of the condensation reaction is 0-10 ℃, and the time of the condensation reaction is 4.0-24.0 h;
and/or, the purification of the first product system comprises quenching, extraction and concentration performed sequentially.
3. The method according to claim 1 or 2, wherein the step S2 is performed by mixing the first product with a hydrogen chloride solution in a second solvent to perform the R 1 Removing reaction;
the second solvent is selected from at least one of methanol, ethanol, dioxane or isopropyl acetate;
and/or, the R 1 The temperature of the removal reaction is 0-10 ℃ and the time is 2.0-8.0 h;
and/or, the purification of the second product system comprises solid-liquid separation, extraction and concentration which are sequentially carried out.
4. The process according to claim 1 or 2, wherein in step S3, the second product is subjected to the condensation coupling reaction with the pyrrolidinylcarboxylic acid compound under the action of a condensing agent selected from HOBT/EDCI, HATU, HOBT/HBTU or T 3 At least one of P;
and/or, the second product and the pyrrolidinyl carboxylic acid compound are subjected to the condensation coupling reaction in a third solvent, wherein the third solvent is selected from at least one of N, N-dimethylformamide, dichloromethane, tetrahydrofuran and 2-methyltetrahydrofuran;
and/or the temperature of the condensation coupling reaction is 15-25 ℃ and the time is 1.0-8.0 h;
and/or the purification of the third product system comprises quenching, crystallization, recrystallization or beating, which are performed sequentially.
5. The preparation method according to claim 1 or 2, wherein in the step S4, the coupling reaction of the third product and the phenyl compound is performed in a fourth solvent, and the fourth solvent is at least one selected from tetrahydrofuran, 2-methyltetrahydrofuran, cyclopentyl methyl ether and diethyl ether;
and/or the temperature of the coupling reaction is 15-25 ℃ and the time is 4-24 hours;
and/or, the purification of the fourth product system comprises quenching, extraction, concentration, crystallization and beating which are sequentially carried out.
6. The preparation method according to claim 1 or 2, wherein in the step S5, the fourth product is subjected to the selective reduction under the action of a reducing agent selected from at least one of aluminum isopropoxide or lithium aluminum tetrahydroide;
And/or, the fourth product and the reducing agent are subjected to the selective reduction in a fifth solvent, the fifth solvent being selected from at least one of toluene, an ether solvent or an alcohol solvent;
and/or the temperature of the selective reduction is 40-55 ℃ and the time is 1.0-8.0 h;
and/or, the purification of the fifth product system comprises quenching, extraction, quenching, concentration, crystallization and beating which are sequentially carried out.
7. A process for the preparation of MMAE, comprising a process for the preparation of a MMAE-key intermediate according to any one of claims 1 to 6.
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