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CN-122010814-A - (2S, 4R) -1-Fmoc-4- (O-allyloxycarbonyl methyl) -proline and synthesis method thereof

CN122010814ACN 122010814 ACN122010814 ACN 122010814ACN-122010814-A

Abstract

The application relates to the technical field of organic synthesis and medical intermediates, in particular to (2S, 4R) -1-Fmoc-4- (O-allyloxycarbonyl methyl) -proline and a synthesis method thereof. The method comprises the steps of carrying out silicon-based protection on (2S, 4R) -4-hydroxypyrrolidine-2-carboxylic acid, carrying out reflux dehydration on the product and alkyl aldehyde to construct a tetrahydropyrrolooxazolone bicyclo intermediate, carrying out etherification on the product and 2-halogenated allyl acetate under strong alkali after silicon-based removal to introduce a side chain, and finally carrying out acidic ring-opening hydrolysis and Fmoc protection to obtain a target product. The application carries out in-situ protection on amino and carboxyl by constructing a double-ring structure, introduces a sensitive allyloxycarbonyl methyl side chain by using an anhydrous nonaqueous phase system, and effectively avoids the hydrolysis of a side chain ester bond. The product prepared by the method has high purity and good stereoselectivity, and provides an efficient and low-cost industrialized way for synthesizing high-performance polypeptide building blocks.

Inventors

  • DU QIANGQIANG
  • YIN HONGFEI
  • CHEN ZHANGTAO
  • XU YINGFAN
  • WANG ZHENXUAN
  • ZHANG QI
  • Liao Huitian

Assignees

  • 杭州澳赛诺医药技术有限公司

Dates

Publication Date
20260512
Application Date
20260415

Claims (10)

  1. 1. A method for synthesizing (2 s,4 r) -1-Fmoc-4- (O-allyloxycarbonylmethyl) -proline, comprising the steps of: s10, dissolving (2S, 4R) -4-hydroxypyrrolidine-2-carboxylic acid shown in a formula (I) in a solvent, adding a silicon-based protecting group reagent and alkali, reacting at-10-100 ℃ until the reaction is completed, concentrating and crystallizing a reaction solution to obtain (2S, 4R) -4- (silicon-based oxy) pyrrolidine-2-carboxylic acid shown in a formula (II); S20, dissolving (2S, 4R) -4- (silyloxy) pyrrolidine-2-carboxylic acid shown in a formula (II) in a solvent, adding alkyl aldehyde or substituted alkyl aldehyde, heating, refluxing and water diversion until the reaction is completed, and then washing, concentrating and crystallizing a reaction solution to obtain (6R, 7 aS) -6- (silyloxy) -3- (substituted alkyl) tetrahydro-1H, 3H-pyrrolo [1,2-c ] oxazol-1-one shown in a formula (III); S30, dissolving (6R, 7 aS) -6- (silyloxy) -3- (substituted alkyl) tetrahydro-1H, 3H-pyrrolo [1,2-C ] oxazol-1-one shown in a formula (III) in a solvent, adding a fluoride ion deprotection reagent to react to remove a silicon-based protecting group, then adding alkali and 2-halogenated acetic acid allyl ester to react at a temperature of between 20 ℃ below zero and 50 ℃ below zero until the reaction is completed, and extracting, washing, concentrating and crystallizing the reaction liquid to obtain (6R, 7 aS) -6- (2-allyloxycarbonyl methoxy) -3-trichloromethyl tetrahydropyrroloxazolone shown in a formula (IV); S40, dissolving (6R, 7 aS) -6- (2-allyloxycarbonyl methoxy) -3-trichloromethyl tetrahydropyrroloxazolone shown in a formula (IV) in a solvent, adding acid, reacting at 0-100 ℃ until the reaction is completed, adjusting the pH of a reaction liquid, adding Fmoc protecting group reagent, stirring until the reaction is completed, and extracting, washing, concentrating and crystallizing the reaction liquid to obtain (2S, 4R) -1-Fmoc-4- (O-allyloxycarbonyl methyl) -proline shown in the formula (V); Wherein the compound shown in the formula (I) is: , The compound shown in the formula (II) is: , the compound shown in the formula (III) is: , a compound of formula (IV): ; the compound represented by formula (V) is: 。
  2. 2. The method according to claim 1, wherein in the step S10, the silicon-based protecting group reagent comprises one or more of trimethylchlorosilane, t-butyldimethylsilyl chloride, t-butyldiphenylchlorosilane, t-butyldimethylsilyl triflate, and triisopropylchlorosilane, and the base comprises one or more of imidazole, N-diisopropylethylamine, triethylamine, and pyridine.
  3. 3. The method of claim 2, wherein in step S10, the molar ratio of (2S, 4 r) -4-hydroxypyrrolidine-2-carboxylic acid, silicon-based protecting group reagent to base is 1:1.1-1.5:1.2-1.8.
  4. 4. The method according to claim 1, wherein in the step S20, the alkyl aldehyde or the substituted alkyl aldehyde includes one or more of chloral and pivalol.
  5. 5. The method according to claim 4, wherein in the step S20, the molar ratio of (2S, 4R) -4- (silyloxy) pyrrolidine-2-carboxylic acid of formula (II) to the alkylaldehyde or substituted alkylaldehyde is 1:1.05-1.2.
  6. 6. The method according to claim 1, wherein in the step S20, the solvent used is chloroform, toluene or methylene chloride.
  7. 7. The method according to claim 1, wherein in the step S30, the fluoride ion deprotection reagent comprises one or more of tetrabutylammonium fluoride, a hydrogen fluoride pyridine complex, cesium fluoride, potassium fluoride and tetramethyl ammonium fluoride, the base comprises one or more of sodium hydride, 1, 8-diaza-bis-spiro [5.4.0] undec-7-ene and (tert-butylimino) tris (pyrrolidine) phosphine, and the 2-halogenoacetate is one or more of 2-chloroacetic acid allyl ester, 2-bromoacetic acid allyl ester and 2-iodoacetic acid allyl ester.
  8. 8. The method according to claim 7, wherein in the step S30, the molar ratio of (6 r,7 as) -6- (silyloxy) -3- (substituted alkyl) tetrahydro-1 h,3 h-pyrrolo [1,2-c ] oxazol-1-one, fluoride ion deprotecting reagent, base to allyl 2-haloacetate of formula (III) is 1:1.0-1.1:1.3-1.6:1.1-1.3.
  9. 9. The method according to claim 1, wherein in the step S40, the acid is selected from one or more of hydrochloric acid, sulfuric acid, trifluoroacetic acid, p-toluenesulfonic acid, ferric trichloride and trimethylsilyl triflate, the base is any inorganic base or organic base, the Fmoc-protecting agent comprises one or more of 9-fluorenylmethyl chloroformate, 9-fluorenylmethyl-N-succinimidyl carbonate and N- (9-fluorenylmethoxycarbonyl) benzotriazole ester, and the molar ratio of (6R, 7 aS) -6- (2-allyloxycarbonyl methoxy) -3-trichloromethyl tetrahydropyrroloxazolone of the formula (IV) to the Fmoc-protecting agent is 1:1.05-1.2.
  10. 10. A (2 s,4 r) -1-Fmoc-4- (O-allyloxycarbonylmethyl) -proline prepared according to the process of any one of claims 1 to 9.

Description

(2S, 4R) -1-Fmoc-4- (O-allyloxycarbonyl methyl) -proline and synthesis method thereof Technical Field The invention belongs to the technical field of organic synthesis and medical intermediates, and particularly relates to (2S, 4R) -1-Fmoc-4- (O-allyloxycarbonyl methyl) -proline and a synthesis method thereof. Background With the rapid development of polypeptide drugs, protein engineering and bioconjugation technologies, there is an increasing demand for polypeptides having complex structures and specific functions. Traditional 20 natural amino acids have certain limitations in meeting the need for precise chemical modification, conformational fixation, or introduction of functional attachment points to polypeptides. Therefore, the design and synthesis of unnatural amino acids with orthogonal reactive side chains is one of the core technologies that motivate the development of this field. Hydroxyproline is a key component of biomacromolecules such as collagen, and the 4-hydroxy group of the hydroxyproline provides a natural site for chemical modification. By carrying out specific derivatization on the hydroxyl, a programmable chemical handle can be introduced into the polypeptide skeleton, so that the post-modification such as cyclization, labeling, carrier connection or functional molecule coupling is realized on the premise of not influencing the main sequence of the peptide chain. Among them, allyl ester protecting genes are favored for their excellent orthogonal reactivity, which remain stable under both acidic (e.g., TFA) and basic (e.g., piperidine) conditions of polypeptide synthesis, but can be selectively removed by mild palladium-catalyzed or nucleophile conditions. Fmoc-4- (O-allyloxycarbonyl methyl) hydroxyproline is a high-grade polypeptide synthetic building block designed based on the concept. The molecular structure integrates Fmoc protecting group, proline rigid skeleton and 4-position (O-allyloxycarbonyl methyl) ether side chain. The compounds show great potential for use in the synthesis of complex polypeptides (e.g., about binding peptides, cell penetrating peptides, antibody drug conjugate linkers) and 3CLpro inhibitors. However, since allyl ester side chains are relatively sensitive to alkaline environments, the use of conventional ester derivative synthesis strategies presents challenges in controlling side reactions and maintaining side chain stability. Patent CN110551178a discloses a method for synthesizing proline-containing head-to-tail cyclic peptides by coupling Fmoc-3-carboxy-Pro-OAll and preparing cyclic peptides according to the Fmoc solid phase synthesis strategy. Although this technical scheme involves the use of Fmoc protection strategies and allyl protecting groups, its modification site is located at the 3-position of proline and focuses on the cyclization process on solid supports. For the non-natural amino acid building block requiring the introduction of a specific ether bond side chain (O-allyloxycarbonyl methyl) at the 4-position, the molecular structure provided by the scheme is limited in reference, and the problem of how to accurately construct a 4-position complex side chain and give consideration to the stereoselectivity of a product in the liquid phase synthesis process is not solved. Patent CN102336697a discloses a synthesis method of (2 s,4 s) -4-hydroxyproline, which realizes configuration conversion by performing steps of carboxyl esterification, nitrogen protection, hydroxyl esterification and the like on (2 s,4 r) -4-hydroxyproline. The scheme is mainly aimed at isomerisation and basic protection of hydroxyproline, and lacks a targeted reaction design in the aspect of introducing a (O-allyloxycarbonyl methyl) functional side chain with high orthogonalization activity. In particular, when treating allyloxycarbonyl side chains containing very alkaline hydrolysis, the conventional hydrolysis and purification conditions employed in the process tend to result in impaired side chain stability, and there is room for optimization in the purity control and overall yield improvement of the product. Therefore, the development of the synthetic method which is simple and convenient to operate, mild in condition, high in stereoselectivity, ideal in total yield and capable of effectively protecting the sensitive side chain has important value for promoting the popularization and application of the high-performance polypeptide building blocks. Disclosure of Invention The application provides (2S, 4R) -1-Fmoc-4- (O-allyloxycarbonyl methyl) -proline and a synthesis method thereof, which aim to pretreat (2S, 4R) -4-hydroxypyrrolidine-2-carboxylic acid by adopting a silicon-based protecting group, construct a rigid intermediate of tetrahydropyrroloxazone by utilizing an alkylaldehyde reagent, introduce allyloxycarbonyl methyl into a proline side chain under specific fluoride ion deprotection and etherification reaction conditions, and finally obtain a target product through