Synthesis method of 2-aminoacyl substituted L-phenylalanine

Abstract

The invention relates to a synthesis method of 2-aminoacyl substituted L-phenylalanine, which comprises the following steps: adopting a chiral raw material, activating with iodide to prepare a zinc reagent, coupling, removing a protecting group, and adding the protecting group to obtain the 2-aminoacyl substituted L-phenylalanine. According to the synthesis method of the 2-aminoacyl substituted L-phenylalanine, by optimizing a synthesis route and parameter conditions, an inner ring closing side reaction possibly occurring in the synthesis process is avoided, and the product yield is increased; meanwhile, the chiral optical purity and quality of a final product are ensured by adopting mild conditions, and the medication safety is improved.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing substituted phenylalanine, in particular to a method for synthesizing 2-aminoacyl substituted L-phenylalanine. Background Art

[0002] Unnatural amino acids and functionalized peptides are important components in modern drug discovery. 2-, 3- or 4-aminoacyl-substituted L-phenylalanine and its derivatives combine the properties of phenylalanine and glutamine, have anti-aging effects, and can be used to treat chronic pain caused by diseases such as rheumatoid arthritis, myalgia and osteoporosis, as well as being key intermediates for amino acid-based cancer drugs.

[0003] The Journal of the American Chemical Society (Journal of the American Chemical Society, 2024, vol.146, # 9, p. 6307 - 6316.) reported a method for preparing 2-aminoacyl-substituted L-phenylalanine. This method uses N-fluoro-N-alkyl-2-methylbenzamide and N-8-quinolyl-glycine tert-butyl ester as the main raw materials, uses trifluoromethanesulfonate as a catalyst, and the chiral ligand provides chiral selectivity, so that a single chiral amino acid can be selectively synthesized. Then, quinoline is oxidized and removed by cerium ammonium nitrate to obtain an amino acid with exposed amino groups, and finally the amino group is protected to obtain a protected amino acid. In addition to the complex synthesis process of N-fluoro-N-alkyl-2-methylbenzamide, this method also requires the use of expensive chiral ligands for coupling reactions, and the chiral purity of the amino acid obtained is not ideal (ee value 9-62%). Summary of the invention

[0004] The purpose of the present invention is to provide a method for synthesizing 2-aminoacyl-substituted L-phenylalanine, which mainly solves the technical problems of complex existing synthesis process, expensive raw materials and unsatisfactory chiral purity of finished products.

[0005] The technical scheme of the present invention is as follows: A method for synthesizing 2-aminoacyl-substituted L-phenylalanine comprises the following steps: (1) Compound A-1 and thionyl chloride are refluxed, and then dissolved in an organic solvent after being spin-dried. A base and an alkylamine or its hydrochloride are then added to react, and the compound A-2 is obtained by treatment; (2) (R)-2-iodo-Boc-serine methyl ester and zinc powder react in an organic solvent to generate a zinc reagent, and then compound A-2 and a palladium catalyst and a ligand are added to react, and the compound A-3 is obtained after treatment and purification; (3) Compound A-3 reacts with a base in an organic solvent and water, and then is treated to obtain compound A-4; (4) Compound A-4 is dissolved in an organic solvent, 8N hydrochloric acid gas is added to react, and then the mixture is dried to obtain compound A-5; (5) Compound A-5 is dissolved in an organic solvent and water, and sodium bicarbonate and R-OSu or R-Cl are added to react. The resulting crude product is treated and subjected to column chromatography to obtain the target product 2-aminoacyl-substituted L-phenylalanine.

[0006] Wherein, the structure of the 2-aminoacyl-substituted L-phenylalanine is shown in the following formula A:

[0007] Formula A The synthetic route is as follows:

[0008] Where X is a halogen; R 1 , R 2 is hydrogen or alkyl; R is an amino protecting group; Further, the halogen is one of chlorine, bromine or iodine; Further, the alkyl group is a C1-C6 straight chain or branched alkyl group; Furthermore, the amino protecting group is an alkoxycarbonyl group.

[0009] Furthermore, the R 1 or R 2 is one of hydrogen, methyl, ethyl, propyl or isopropyl; preferably hydrogen or methyl; Furthermore, R is one of tert-butyloxycarbonyl, fluorenylmethoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl, preferably fluorenylmethoxycarbonyl.

[0010] Furthermore, in step 1, the organic solvent is one of dichloromethane, tetrahydrofuran, dichloroethane or N,N-dimethylformamide; preferably dichloromethane; Furthermore, in step 1, the base is one of triethylamine, diisopropylethylamine, N-methylmorpholine, sodium bicarbonate, sodium carbonate or potassium carbonate; preferably triethylamine; Furthermore, in the step 1, the molar ratio of the compound A-1 to the alkylamine is 1:1.2 to 1:1.5; Furthermore, in the step 1, the molar ratio of the compound A-1 to the base is 1:3 to 1:5; Furthermore, in the step 1, the weight-to-volume ratio (g:ml) of the compound A-1 to the organic solvent is 1:3-1:5.

[0011] Furthermore, in step 2, the organic solvent is one of tetrahydrofuran, ethylene glycol dimethyl ether or N,N-dimethylformamide; preferably tetrahydrofuran; Furthermore, in step 2, the palladium catalyst is Pd(dppf)Cl 2 、Pd(PPh 3 ) 4 、Pd(PPh 3 ) 2 Cl 2 , Pd 2 (dba) 3 One or more of; preferably Pd (PPh 3 ) 4 or Pd 2 (dba) 3 ; Furthermore, in the step 2, the molar ratio of the compound A-2 to the palladium catalyst is 1:0.1 to 1:0.3; Further, in the step 2, the molar ratio of the compound A-2 to (R)-2-iodo-Boc-serine methyl ester is 1:3 to 1:5; Furthermore, in step 2, the molar ratio of compound A-2 to zinc is 1:3 to 1:6; Furthermore, in the step 2, the weight-to-volume ratio (g:ml) of the compound A-2 to the organic solvent is 1:5-1:10.

[0012] Furthermore, in step 3, the organic solvent is one or more of tetrahydrofuran, dioxane, ethanol, methanol, isopropanol, and tert-butanol; preferably tetrahydrofuran; Furthermore, in step 3, the base is one of lithium hydroxide monohydrate, sodium hydroxide, calcium hydroxide or potassium hydroxide; preferably lithium hydroxide monohydrate.

[0013] Furthermore, in step 4, the organic solvent is one of dichloromethane, dioxane, tetrahydrofuran or ethyl acetate, preferably dioxane.

[0014] Furthermore, in step 5, the organic solvent is one of dioxane, tetrahydrofuran, ethanol, methanol or isopropanol, preferably tetrahydrofuran.

[0015] According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 1, the molar ratio of the compound A-1 to the alkylamine is 1:1.2; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 1, the molar ratio of the compound A-1 to the alkylamine is 1:1.5; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 1, the molar ratio of the compound A-1 to the base is 1:3; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 1, the molar ratio of the compound A-1 to the base is 1:5; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 1, the weight-to-volume ratio (g:ml) of the compound A-1 to the organic solvent is 1:3; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 1, the weight-to-volume ratio (g:ml) of the compound A-1 to the organic solvent is 1:5; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the molar ratio of the compound A-2 to the palladium catalyst is 1:0.1; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the molar ratio of the compound A-2 to the palladium catalyst is 1:0.2; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the molar ratio of the compound A-2 to the palladium catalyst is 1:0.3; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the molar ratio of the compound A-2 to (R)-2-iodo-Boc-serine methyl ester is 1:3; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the molar ratio of the compound A-2 to (R)-2-iodo-Boc-serine methyl ester is 1:4; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the molar ratio of the compound A-2 to zinc is 1:3; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the molar ratio of the compound A-2 to zinc is 1:6; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the weight-to-volume ratio (g:ml) of the compound A-2 to the organic solvent is 1:5; According to a preferred embodiment of the method for synthesizing 2-aminoacyl-substituted L-phenylalanine of the present invention, in step 2, the weight-to-volume ratio (g:ml) of the compound A-2 to the organic solvent is 1:10; In the above operations, the treatment includes but is not limited to stirring, extraction, filtration, water washing, alkali washing, acid washing, pH adjustment, concentration, drying, recrystallization, freeze drying and the like operations or a combination of one or more of them.

[0016] Beneficial effects of the present invention: The present invention uses 2-halogenated benzamide derivatives and iodoserine as raw materials to couple and synthesize 2-aminoacyl-substituted L-phenylalanine, and studies and compares the reactivity of different halides at different temperatures and in different solvents. The study found that when the halogen is chlorine, bromine or iodine, there are different degrees of reaction, and the reactivity and reaction yield of I are the best. When X=Cl, most of the compound A-2 does not participate in the reaction, when X=Br, the compound A-2 has a remainder and one of the main impurities is the debromination byproduct N-alkylbenzamide, and when X=I, the compound A-2 participates in the reaction, and one of the main impurities is the deiodination byproduct N-alkylbenzamide. The present invention effectively synthesizes high optical purity 2-aminoacyl-substituted L-phenylalanine through five steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The nuclear magnetic resonance spectrum of the product of the present invention, N',N'-dimethyl Fmoc-2-formamidophenylalanine.

[0018] Figure 2 This is the chiral HPLC purity spectrum of the product N',N'-dimethyl Fmoc-2-formamidophenylalanine of the present invention.

[0019] Figure 3 The nuclear magnetic resonance spectrum of the product of the present invention, N'-methyl Fmoc-2-formamidophenylalanine.

[0020] Figure 4 The chiral HPLC purity spectrum of the product N'-methyl Fmoc-2-formamidophenylalanine of the present invention is shown in FIG. DETAILED DESCRIPTION

[0021] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0022] Any feature disclosed in this specification, unless otherwise specified, can be replaced by other equivalent or alternative features with similar purposes. That is, unless otherwise specified, each feature is only an example of a series of equivalent or similar features.

[0023] The synthetic method of 2-aminoacyl-substituted L-phenylalanine of the present invention will be further described below in conjunction with specific examples.

[0024] Embodiment 1:

[0025] Step 1: Place 100 g of compound A-1 in a 1L single-mouth bottle, add 300 ml of thionyl chloride, and reflux for 2 hours. Cool the reaction system to room temperature, concentrate under reduced pressure to remove most of the solvent, and then add dichloromethane and concentrate under reduced pressure twice. Dissolve the acyl chloride in 500 ml of dichloromethane, drop to zero degrees Celsius under nitrogen protection, add 203.6 g of triethylamine, and then add 49.3 g of dimethylamine hydrochloride in batches. Warm to room temperature and react for 2 hours. Pour the reaction system into 500 ml of water, separate the liquids, and extract the aqueous phase twice with dichloromethane. Combine the dichloromethane phases, wash with 2% HCl and 2% sodium bicarbonate aqueous solution by mass percentage, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound A-2a (106.5 g, 96% yield).

[0026] Step 2: Place 141.8 g of zinc powder in a 5L three-necked flask, add 500 ml of tetrahydrofuran, and replace nitrogen twice. Add 9.2 g of iodine and stir for 30 minutes. Then dropwise add 478.6 g of (R)-2-iodo-Boc-serine methyl ester in tetrahydrofuran (300 ml), and control the temperature between 30-50 degrees Celsius. Stir at room temperature for 1 hour, then add 100 g of compound A-2 in tetrahydrofuran (200 ml), 33.3 g of Pd 2 (dba) 3 , 29.8 g of SPhos. Heat to 50 degrees Celsius and stir to react overnight. Cool the reaction system to room temperature, add 1000 ml of ethyl acetate to dilute and filter, wash the filter cake with ethyl acetate and filter. Combine the filtrate, add 2000 ml of water, stir and separate, and extract the aqueous phase twice with ethyl acetate. Combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude compound A-3a (110.8 g, 87% yield).

[0027] Step 3: Dissolve 110 g of crude compound A-3a in tetrahydrofuran (200 ml) and water (200 ml), and add 39.6 g of lithium hydroxide monohydrate. Stir at room temperature for 2 hours. Adjust the pH to 7-8 with 6N hydrochloric acid aqueous solution, concentrate under reduced pressure to remove most of the tetrahydrofuran, and back-extract with petroleum ether / ethyl acetate (volume ratio 3:1) 3 times. The aqueous phase is further adjusted to pH 4-5 with 6N hydrochloric acid, and then extracted three times with ethyl acetate. Combine the ethyl acetate phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude compound A-4a (93 g, 88% yield).

[0028] Step 4: Dissolve 93 g of compound A-4a in 200 ml of dioxane, and dropwise add 100 ml of 8N HCl / dioxane solution. Stir and react for 2 hours at room temperature. Concentrate under reduced pressure to remove dioxane, and then rinse with dichloromethane twice to obtain a crude compound A-5a (85 g, 100% yield).

[0029] Step 5: 85 g of crude compound A-5a was dissolved in tetrahydrofuran (200 ml) and water (200 ml), and 69.7 g of sodium bicarbonate was added. 74.5 g of Fmoc-OSu was added in batches at room temperature. Then the reaction was stirred at room temperature for 3 hours. Fmoc-OSu was completely reacted. 9.3 g of Fmoc-OSu was added, and stirring was continued for 1 hour. It was found that there was still a small amount of Fmoc-OSu remaining, indicating that all the amino raw materials had reacted completely. Petroleum ether (600 ml) was added to the system and stirred for separation, and the aqueous phase was washed twice with petroleum ether / ethyl acetate solution (200 ml, volume ratio 3:1). Then the aqueous phase was adjusted to pH 3-4 with 1N dilute hydrochloric acid, and ethyl acetate (300 ml) was added for extraction three times. The ethyl acetate phase obtained by the three separations was combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness. The crude product was purified by dichloromethane / methanol column chromatography to obtain N',N'-dimethyl Fmoc-2-formamidophenylalanine A (81 g, 87% yield). Figure 1 , HPLC spectrum see Figure 2 .

[0030] Embodiment 2:

[0031] Step 1: Place 50 grams of compound A-1 in a 1L single-mouth bottle, add 150 milliliters of thionyl chloride, and reflux for 2 hours. Cool the reaction system to room temperature, concentrate under reduced pressure to remove most of the solvent, and then add dichloromethane and concentrate under reduced pressure twice. Dissolve the acyl chloride in 200 milliliters of dichloromethane, drop to zero degrees Celsius under nitrogen protection, add 125.6 grams of triethylamine, and then add 25.2 grams of methylamine hydrochloride in batches. Warm to room temperature and react for 2 hours. Pour the reaction system into 500 milliliters of water, separate the liquids, and extract the aqueous phase twice with dichloromethane. Combine the dichloromethane phases, wash with 2% HCl and 2% sodium bicarbonate aqueous solution by mass percentage, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain compound A-2a (50.6 grams, 95% yield).

[0032] Step 2: Place 75.9 g of zinc powder in a 2L three-necked flask, add 250 ml of tetrahydrofuran, and replace nitrogen twice. Add 5.9 g of iodine and stir for 30 minutes. Then dropwise add 384.4 g of (R)-2-iodo-Boc-serine methyl ester in tetrahydrofuran (150 ml), and control the temperature between 30-50 degrees Celsius. Stir at room temperature for 1 hour, then add 50 g of compound A-2 in tetrahydrofuran (100 ml), 27 g of Pd (PPh 3 ) 4 . Raise the temperature to 60 degrees Celsius and stir the reaction overnight. Cool the reaction system to room temperature, add 500 ml of ethyl acetate to dilute and filter, wash the filter cake with ethyl acetate and filter. Combine the filtrate, add 1000 ml of water, stir and separate, and extract the aqueous phase twice with ethyl acetate. Combine the organic phases, wash twice with saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude compound A-3a (61.3 g, 78% yield).

[0033] Step 3: 61 g of crude compound A-3a was dissolved in tetrahydrofuran (120 ml) and water (120 ml), and 22.8 g of lithium hydroxide monohydrate was added. Stir at room temperature for 2 hours. The pH was adjusted to 7-8 with 6N hydrochloric acid aqueous solution, and most of the tetrahydrofuran was removed by concentration under reduced pressure, and then back-extracted 3 times with petroleum ether / ethyl acetate (volume ratio 3:1). The aqueous phase was further adjusted to pH 4-5 with 6N hydrochloric acid, and then extracted three times with ethyl acetate. The ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude compound A-4a (38 g, 65% yield).

[0034] Step 4: Dissolve 38 g of compound A-4a in 100 ml of dioxane, and dropwise add 60 ml of 8N HCl / dioxane solution. Stir and react for 2 hours at room temperature. Concentrate under reduced pressure to remove dioxane, and then rinse with dichloromethane twice to obtain a crude product of compound A-5a (37 g, 100% yield).

[0035] Step 5: 37 g of crude compound A-5a was dissolved in tetrahydrofuran (100 ml) and water (100 ml), and 39.6 g of sodium bicarbonate was added. 27.8 g of Fmoc-OSu was added in batches at room temperature. Then the reaction was stirred at room temperature for 3 hours, and a small amount of Fmoc-OSu was found to remain, indicating that all the amino raw materials had reacted completely. Petroleum ether (300 ml) was added to the system and stirred for separation, and the aqueous phase was washed twice with petroleum ether / ethyl acetate solution (100 ml, volume ratio 3:1). Then the aqueous phase was adjusted to pH 3-4 with 1N dilute hydrochloric acid, and ethyl acetate (150 ml) was added and extracted three times. The ethyl acetate phase obtained from the three separations was combined, washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness. The crude product was purified by dichloromethane / methanol column chromatography to obtain N'-methyl Fmoc-2-formamidophenylalanine A (29 g, 79% yield). NMR spectrum is shown in Figure 3 , HPLC spectrum see Figure 4 .

Claims

1. A method for synthesizing 2-aminoacyl-substituted L-phenylalanine, characterized in that: The following steps are involved: (1) Compound A-1 and thionyl chloride are refluxed, and then dissolved in an organic solvent after being spin-dried. A base and an alkylamine or its hydrochloride are then added to react, and the compound A-2 is obtained by treatment; (2) (R)-2-iodo-Boc-serine methyl ester and zinc powder react in an organic solvent to generate a zinc reagent, and then compound A-2 and a palladium catalyst and a ligand are added to react, and the compound A-3 is obtained after treatment and purification; (3) Compound A-3 reacts with a base in an organic solvent and water, and then is treated to obtain compound A-4; (4) Compound A-4 is dissolved in an organic solvent, 8N hydrochloric acid gas is added to react, and then the mixture is dried to obtain compound A-5; (5) Compound A-5 is dissolved in an organic solvent and water, and sodium bicarbonate and R-OSu or R-Cl are added to react. The resulting crude product is subjected to column chromatography to obtain the target product 2-aminoacyl-substituted L-phenylalanine; Wherein, the structure of the 2-aminoacyl-substituted L-phenylalanine is shown in the following formula A: Formula A The synthetic route is as follows: ， Wherein, X is a halogen, R1 and R2 are hydrogen or an alkyl group, and R is an amino protecting group.

2. The method according to claim 1, characterized in that: The halogen is one of chlorine, bromine or iodine; the alkyl is a C1-C6 straight chain or branched alkyl; and the amino protecting group is an alkoxycarbonyl group.

3. The method according to claim 2, characterized in that: The C1-C6 straight-chain or branched alkyl group is one of methyl, ethyl, propyl or isopropyl; the amino protecting group is one of tert-butyloxycarbonyl, fluorenylmethoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl.

4. The method according to claim 1, characterized in that: In the step 1, the organic solvent is one of dichloromethane, tetrahydrofuran, dichloroethane or N,N-dimethylformamide; the base is one of triethylamine, diisopropylethylamine, N-methylmorpholine, sodium bicarbonate, sodium carbonate or potassium carbonate; the molar ratio of the compound A-1 to the alkylamine is 1:1.2~1:1.5; the molar ratio of the compound A-1 to the base is 1:3~1:5; the weight volume ratio of the compound A-1 to the organic solvent is 1:3~1:5 in g / ml.

5. The method according to claim 1, characterized in that: In the step 2, the organic solvent is one of tetrahydrofuran, ethylene glycol dimethyl ether or N,N-dimethylformamide; the palladium catalyst is one or more of Pd(dppf)Cl2, Pd(PPh3)4, Pd(PPh3)2Cl2, Pd2(dba)3; the molar ratio of the compound A-2 to the palladium catalyst is 1:0.1~1:0.3; the molar ratio of the compound A-2 to (R)-2-iodo-Boc-serine methyl ester is 1:3~1:5; the molar ratio of the compound A-2 to zinc is 1:5~1:10; the weight volume ratio of the compound A-2 to the organic solvent is 1:5~1:10 in grams per milliliter.

6. The method according to claim 1, characterized in that: In step 3, the organic solvent is one or more of tetrahydrofuran, dioxane, ethanol, methanol, isopropanol, and tert-butanol; and the base is one of lithium hydroxide, sodium hydroxide, calcium hydroxide, or potassium hydroxide.

7. The method according to claim 1, characterized in that: In step 4, the organic solvent is one of dichloromethane, dioxane, tetrahydrofuran or ethyl acetate.

8. The method according to claim 1, characterized in that: In step 5, the organic solvent is one of dioxane, tetrahydrofuran, ethanol, methanol or isopropanol.

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