A method for synthesizing Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester)

Abstract

This invention relates to a method for synthesizing Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester). It primarily addresses the technical problems of existing synthesis methods, such as demanding conditions, cumbersome operations, low yields, and unsuitability for industrial production. The preparation steps of this invention are as follows: (1) Synthesizing Cbz-(S)-2-formylalanine methyl ester according to literature methods; (2) Reaction of Cbz-(S)-2-formylalanine methyl ester with (tert-butyloxycarbonylmethylene)triphenyl via Wittig reaction; (3) Selective hydrolysis of the methyl ester; (4) Hydrogenation of the double bond and the Cbz protecting group to obtain α-methyl-L-glutamic acid (5-tert-butyl ester); (5) Reaction of α-methyl-L-glutamic acid (5-tert-butyl ester) with Fmoc-OSu to obtain the final product. This invention has the advantages of high yield, low cost, simple operation and purification, good economic benefits, and is more suitable for industrial production.

Description

Technical Field

[0001] This invention relates to a method for synthesizing Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester). Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester) is a raw material used in the synthesis of polypeptide drugs. Background Technology

[0002] A-methyl amino acids with a single chiral configuration are important raw materials for polypeptide drugs. Due to their metabolic stability and structural rigidity, they are often introduced into cyclic peptide drugs to modify the peptide chain structure, thereby reducing conformational freedom and changing their biological properties. The synthesis technology of Fmoc-a-methyl-L-glutamic acid (5-tert-butyl ester) with a single chiral configuration has been reported in the literature: for example: (1) Journal of Pharmaceutical Chemistry (J. Med. Chem. 2008, 51, P6371–6380) Ya-Qiu Long et al. used tert-butyl bromoacetate as an electrophilic reagent and lithium diisopropylamino (LDA) as a base to alkylate Cbz-(2R,4S)-2-phenyl-4-methyloxazolidinone at -78℃ to obtain Cbz-(2R,4S)-4-disubstituted oxazolidinone, and then obtained chiral pure Fmoc-a-methyl-L-glutamic acid (5-tert-butyl ester) through several steps. (2) Literature: Journal of Computer-Aided Molecular Design, 2013, 27, P31–43. David Zanuy et al. used tert-butyl 3-bromoallyl as an electrophile and lithium bis(trimethylsilyl)amino (LiHMDS) as a base to alkylate Cbz-(2R,4S)-2-phenyl-4-methyloxazolidinone at -78°C to obtain Cbz-(2R,4S)-4-disubstituted oxazolidinone. Then, through several steps, chiral pure Fmoc-a-methyl-L-glutamic acid (5-tert-butyl ester) was synthesized. Both methods are performed under deep cryogenic conditions using the strong organic base lithium diisopropylamino (LDA) or lithium bis(trimethylsilyl)amino (LiHMDS). The reaction conditions are harsh, the operation is cumbersome, the yield is low, and it is not suitable for industrial production. Summary of the Invention

[0003] The purpose of this invention is to provide a method for synthesizing Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester), which mainly solves the technical problems of existing synthesis methods, such as harsh conditions, cumbersome operation, low yield, and unsuitability for industrial production.

[0004] The present invention uses α-methyl-L-serine methyl ester hydrochloride as a raw material, first preparing Cbz-(S)-2-formylalanine methyl ester b according to the literature Tetrahedron Asymmetry (2008, 19, 247-257), then proceeding with Wittig reaction, selective hydrolysis and hydrogenation, and finally Fmoc protection to prepare Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester).

[0005] The technical solution of this invention is: a method for synthesizing Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester), comprising the following steps:

[0006] (1) Preparation of Cbz-(S)-α-methylserine methyl ester

[0007] (S)-a-methyl-L-serine methyl ester hydrochloride was added to dichloromethane, followed by the addition of triethylamine in an amount of 2.2 to 2.5 times that of (S)-a-methyl-L-serine methyl ester hydrochloride, and 0.95 to 0.98 times that of Cbz-OSu. The reaction was carried out at room temperature for 6 hours. After the reaction was completed, the reaction solution was washed with hydrochloric acid and brine, respectively, and then concentrated to obtain Cbz-(S)-a-methylserine methyl ester a.

[0008] (2) Oxidation

[0009] Cbz-(S)-a-methylserine methyl ester a, 4A molecular sieve, and pyridinium chlorochromate (PCC) oxidant were added to dichloromethane. The reaction was carried out for 2 hours, and pyridinium chlorochromate (PCC) oxidant was added to continue the reaction until complete. The reaction mixture was filtered through a vacuum funnel with silica gel padding. The filter cake was washed with dichloromethane, and the filtrate was concentrated to obtain Cbz-(S)-2-formylalanine methyl ester b.

[0010] (3) Wittig reaction

[0011] Cbz-(S)-2-formylalanine methyl ester b, (tert-butoxycarbonylmethylene)triphenyl was added to dichloromethane. After the reaction was completed, the mixture was concentrated, and isopropyl ether was added and stirred before filtration. The filter cake was washed with isopropyl ether, and the filtrate was concentrated to obtain compound c.

[0012] (4) Selective hydrolysis

[0013] Compound c was added to methanol, and lithium hydroxide aqueous solution was added dropwise under ice bath. After the addition was complete, the temperature was controlled at 5-10℃ and the reaction continued until no raw material was produced. The pH of the reaction solution was adjusted to 9 with 3N hydrochloric acid, and methanol was removed by vacuum evaporation at room temperature. The remaining aqueous phase was washed with methyl tert-butyl ether, the pH was adjusted to 2-3, and the mixture was extracted twice with ethyl acetate. The combined organic phases were washed with brine and concentrated, and then crystallized with isopropyl ether to obtain compound d; (5) Hydrogenation

[0014] Compound d and 10% palladium on carbon were added to methanol and hydrogenated until no raw material was available. The reaction solution was filtered, the filtrate was concentrated, and crystallized with tert-methyl ether to obtain α-methyl-L-glutamic acid (5-tert-butyl ester) e.

[0015] (6) Fmoc protection

[0016] Add α-methyl-L-glutamic acid (5-tert-butyl ester) e to water, adjust the pH with an inorganic base and stir until dissolved. Add an organic solvent and 0.95-0.98 equivalents of Fmoc-OSu, adjust the pH to 9-11 with an inorganic base, and react at room temperature until no raw material remains. Filter the reaction solution. Wash the filtrate with a mixed solvent to remove impurities, then adjust the pH to 3-4 with acid. Extract twice with ethyl acetate. Wash the combined organic phases with brine and concentrate to remove the solvent to obtain Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester).

[0017] The reaction formula is as follows:

[0018] .

[0019] In the above reaction, the preferred concentration of the lithium hydroxide aqueous solution in step (4) is 2.0~2.5 M. In step (6), the pH value is adjusted to 9.5~10.5 with an inorganic base, which is one of sodium hydroxide, lithium hydroxide, sodium carbonate, or potassium carbonate, preferably sodium carbonate. The equivalent of Fmoc-OSu is preferably 0.96-0.98 times that of α-methyl-L-glutamic acid (5-tert-butyl ester). The organic solvent is one of tetrahydrofuran, 1,4-dioxane, or acetone. Acetone is preferred as the solvent, and hydrochloric acid is preferred. The mixed solvent is ethyl acetate and n-hexane (v / v = 1:5).

[0020] The beneficial effects of this invention are as follows: This invention uses α-methyl-L-serine methyl ester hydrochloride as a raw material, which undergoes oxidation, Wittig reaction, hydrolysis, and hydrogenation, and finally Fmoc protection to synthesize Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester). This method has mild reaction conditions, is simple, efficient, and has a high yield and quality, making it economical and suitable for large-scale industrial production. Detailed Implementation

[0021] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention.

[0022] Example 1:

[0023] (1) Preparation of Cbz-(S)-α-methylserine methyl ester

[0024] Add (S)-α-methylserine methyl ester hydrochloride (160 g, 1.06 mol) and dichloromethane (2 L) to a 5 L three-necked flask. Add triethylamine (257 g, 2.55 mol) at room temperature. Cool to 5-10 °C in an ice bath, and add dropwise a dichloromethane solution (1 L) of Cbz-OSu (350 g, 1.04 mol). After the addition is complete, warm to room temperature. After reacting for 16 hours, add 1 L of water to the reaction mixture, and allow it to separate into layers. Wash the organic phase with 0.5 N hydrochloric acid (3 x 1 L), then with saturated brine, and dry to anhydrous sodium sulfate. Filter, and evaporate the filtrate directly to dryness to obtain Cbz-(S)-α-methylserine methyl ester a (247 g, yield 89.0%).

[0025] (2) Oxidation of Cbz-(S)-α-methylserine methyl ester

[0026] Activated 4A molecular sieve (600 g) and PCC (242 g, 1.12 mol) were sequentially added to a dichloromethane (3 L) solution of Cbz-(S)-α-methylserine methyl ester (200 g, 0.72 mol). The resulting brown suspension was stirred vigorously for 2 hours. Then, PCC (77.6 g, 0.36 mol) and 4A molecular sieve (100 g) were added. After stirring for another 1.5 hours, the reaction mixture was filtered through a silica gel-lined suction funnel. The filter cake was first carefully washed with isopropyl ether (2 L), and then carefully washed with dichloromethane until a clear, colorless filtrate was obtained. The solvent was evaporated under reduced pressure at room temperature to give Cbz-(S)-2-formylalanine methyl ester b (169 g). This was used directly in the next step without further purification.

[0027] (3) Wittig reaction

[0028] Add 150 g (0.57 mol) of Cbz-(S)-2-formylalanine methyl ester b and 900 mL of dichloromethane to a 3 L three-necked flask. Add (tert-butyloxycarbonylmethylene)triphenyl (319 g, 0.85 mol) at room temperature and stir for 16 hours. Concentrate the reaction solution to dryness, add isopropyl ether, stir, and filter. Wash the filter cake with isopropyl ether, concentrate the filtrate to give compound c (206 g), which can be used directly in the next step.

[0029] (4) Selective hydrolysis

[0030] Compound c (206 g, 0.57 mol) and methanol (2 L) were added to a 5 L three-necked flask and cooled to 5-10°C in an ice bath. A 2.5 M aqueous solution of lithium hydroxide (680 mL, 1.7 mol) was added dropwise. After the addition was complete, the temperature was maintained at 5-10°C, and the reaction was allowed to proceed for 2 hours. The pH of the reaction mixture was adjusted to 9 with 3N hydrochloric acid, and methanol was removed by evaporation under reduced pressure at room temperature. The remaining aqueous phase was washed with methyl tert-butyl ether, the pH was adjusted to 3-4, and the mixture was extracted with ethyl acetate (2 x 800 mL). The combined ethyl acetate phases were washed with brine (800 mL) and dried over anhydrous sodium sulfate. The mixture was filtered, and the filtrate was directly evaporated to dryness. The filtrate was then crystallized from isopropyl ether to give compound d (138 g, yield 69.7%).

[0031] (5) Hydrogenation

[0032] Compound d (101 g, 0.29 mol) and methanol (4 L) were added to a 5 L three-necked flask. 10% palladium on carbon (containing 55% water, 20 g) was added. The reaction was hydrogenated at 40°C under normal pressure for 24 hours. The reaction solution was filtered, the filtrate was concentrated, and crystallized with methyl tert-butyl ether to obtain α-methyl-L-glutamic acid (5-tert-butyl ester) e (62 g, yield 98.7%).

[0033] (6) Fmoc protection

[0034] Add α-methyl-L-glutamic acid (5-tert-butyl ester) e (62 g, 0.285 mol, 1 eq.) and water (465 mL) to a 2 L three-necked flask. Adjust the pH to 9-11 with sodium carbonate, and add acetone (465 mL) and Fmoc-OSu (92.3 g, 0.274 mol, 0.96 eq.) at room temperature. After the addition is complete, maintain the pH at 9-11 and react for 16 hours. Filter the reaction mixture, wash the filtrate with ethyl acetate and n-hexane (v / v = 1:5) (3 x 600 mL), adjust the pH to 3-4 with 3 N hydrochloric acid, extract with ethyl acetate (2 x 500 mL), wash the combined ethyl acetate phases with brine (500 mL), and dry with anhydrous sodium sulfate. Filter the solution and evaporate the filtrate directly to dryness to obtain Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester) g (98.6 g, yield 79.5%).

[0035] 1 H NMR (DMSO, d 6): d 12.49 (brs, 1H), 7.91-7.89 (m, 2H), 7.74-7.72 (m,2H), 7.55 (s, 1H), 7.44-7.40 (m, 2H), 7.35-7.32 (m, 2H), 4.26-4.22 (m, 3H), 2.23-2.06 (3H), 1.95-1.92 (m, 1H), 1.40 (s, 9H), 1.30 (s, 3H).

[0036] In Example 2, the concentration of the lithium hydroxide aqueous solution in step (4) is 2.4 M, and the rest is the same as in Example 1.

[0037] Example 3, step (6) adjusts pH to 9.5-10.5, the organic solvent is tetrahydrofuran, the equivalent of Fmoc-OSu is 0.97 times that of α-methyl-L-glutamic acid (5-tert-butyl ester), and the rest is the same as in Example 1.

[0038] In Example 4, step (6) involves adjusting the pH to 9.5-10.5 with potassium carbonate, using 1,4-dioxane as the organic solvent, and using Fmoc-OSu equivalents that of α-methyl-L-glutamic acid (5-tert-butyl ester) at 0.98 times the amount of α-methyl-L-glutamic acid (5-tert-butyl ester). The rest is the same as in Example 1.

Claims

1. A method for synthesizing Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester), characterized in that: Including the following step: (1): Cbz-(S)-2-formylalanine methyl ester b, (tert-butoxycarbonylmethylene)triphenyl was added to dichloromethane. After the reaction was completed, the mixture was concentrated, and isopropyl ether was added and stirred before filtration. The filter cake was washed with isopropyl ether, and the filtrate was concentrated to obtain compound c. (2): Compound c was added to methanol, and a 2.2-2.5 M lithium hydroxide aqueous solution was added dropwise under an ice bath. After the addition was complete, the temperature was controlled at 5-10℃ and the reaction was continued until no raw material was produced. The reaction solution was washed with methyl tert-butyl ether, the pH of the aqueous phase was adjusted to 2-3, and the organic solvent was extracted twice. The combined organic phases were washed with brine and concentrated, and then crystallized with isopropyl ether to obtain compound d. (3): Compound d and 10% palladium on carbon were added to methanol and hydrogenated until no raw material was available. The reaction solution was filtered, the filtrate was concentrated, and crystallized with methyl tert-butyl ether to obtain α-methyl-L-glutamic acid (5-tert-butyl ester) e. (4): Add α-methyl-L-glutamic acid (5-tert-butyl ester) to water, adjust the pH to 9.5-10.5 with sodium carbonate and stir until dissolved. Add acetone and 0.96-0.98 times the amount of Fmoc-OSu, adjust the pH to 9-11 with sodium carbonate, and react until no raw material is produced. Filter the reaction solution. Wash the filtrate with a mixed solvent of ethyl acetate and n-hexane (volume ratio of ethyl acetate to n-hexane 1:5). Then adjust the pH to 3-4 with hydrochloric acid. Extract twice with ethyl acetate. Wash the combined organic phases with brine and concentrate to remove the solvent to obtain Fmoc-α-methyl-L-glutamic acid (5-tert-butyl ester). The reaction formula is as follows: 。

Images