A chemo-enzymatic combined process for the preparation of (r)-4-amino-2-ethylisoxazolidin-3-one
By employing Oxa-Michael addition and enzymatic resolution techniques, the problems of unstable raw materials and high costs in the synthesis of 4-amino-2-ethylisoxazolidine-3-one were solved, resulting in an efficient and low-cost preparation method that achieves high chiral purity and high yield.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TAIZHOU VOCATIONAL & TECHN COLLEGE
- Filing Date
- 2026-03-27
- Publication Date
- 2026-06-23
AI Technical Summary
In the existing technology, the synthesis of 4-amino-2-ethylisoxazolidine-3-one has problems such as unstable raw materials, many reaction byproducts, and high cost, making it difficult to achieve large-scale industrial application.
A ring system was constructed using methyl 2-acetamidoacrylate via Oxa-Michael addition, and enzymatic resolution technology was used to avoid the use of haloalkanes. Acylases were used to selectively resolve the racemic mixture, and unreacted isomers were recovered by racemization, achieving efficient and green preparation.
The preparation of (R)-4-amino-2-ethylisoxazolidin-3-one with high chirality, purity and high yield was achieved, avoiding highly toxic reagents, simplifying the operation process and reducing costs.
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Figure CN122256453A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical chemical synthesis, specifically to a green synthesis method for a key intermediate and derivative of an anti-tuberculosis drug, (R)-4-amino-2-ethylisoxazolidin-3-one. Background Technology
[0002] 4-Amino-2-ethylisoxazolidine-3-one is an important derivative of cyclic serine with potential biological activity. In the prior art (such as patent WO 2015 / 166094), its synthesis mainly depends on the nucleophilic substitution reaction between D-serine-derived β-chloroalanine methyl ester and N-ethylhydroxylamine.
[0003] However, existing technologies have the following significant drawbacks: 1. Unstable raw materials: β-chloroalanine methyl ester is extremely unstable, prone to self-dimerization, and has strong skin toxicity, making it unsuitable for large-scale storage and transportation.
[0004] 2. Mechanism limitations: Traditional nucleophilic substitution reactions are prone to producing O-alkylation byproducts, which leads to a decrease in yield.
[0005] 3. High cost: If chiral raw materials (D-serine) are used, the cost is high; if chemical resolution is used, the resolving agent is difficult to recover and the yield is limited (maximum 50%).
[0006] Therefore, developing a preparation method with inexpensive and stable raw materials, a novel reaction route, and the ability to achieve novice recycling has significant industrial application value. Summary of the Invention
[0007] The purpose of this invention is to provide a method for preparing (R)-4-amino-2-ethylisoxazolidin-3-one. This method is based on a novel reaction mechanism, utilizing the inexpensive bulk chemical methyl 2-acetamidoacrylate to construct a ring system via Oxa-Michael addition, and combining it with a highly selective enzymatic resolution technology to achieve efficient and green preparation of the target product.
[0008] The technical solution of the present invention is as follows: Step 1: Oxa-Michael addition-cyclization tandem reaction; utilizing N-ethylhydroxylamine to nucleophilically attack the double bond of an electron-deficient alkene (2-acetamidoacrylate) under basic conditions (Oxa-Michael addition). Due to the nucleophilicity of the oxygen atom and steric hindrance, the reaction preferentially generates the O-addition intermediate, followed by intramolecular amino group attacking the ester group to close the ring. This step avoids the use of haloalkanes, and the starting materials are all stable solids or general-purpose reagents; Step 2: Enzymatic dynamic separation; Acylase is used to specifically hydrolyze the racemic acetyl intermediate obtained in Step 1. This enzyme recognizes only the (R)-configuration amide bond, hydrolyzing it to a free amino group (i.e., the precursor of the target product), while the (S)-configuration remains in the amide form. Separation can be easily achieved by utilizing the significant differences in solubility or acid-base properties between the two (the product is readily soluble in water / acids, while the precursor is readily soluble in organic solvents). Step 3: Racemization of waste; the separated (S)-isomers can be rapidly racemized under alkaline conditions through an enolization mechanism and reintroduced into the enzymatic hydrolysis vessel, thereby breaking the 50% theoretical yield limit and significantly reducing costs.
[0009] Preferably, in the 2-acylaminoacrylate derivative (Formula II), R is selected from acetyl, trifluoroacetyl, phenylacetyl, or benzoyl; R' is selected from methyl, ethyl, or tert-butyl. Methyl 2-acetaminoacrylate is preferred.
[0010] Preferably, the alkaline reagent is selected from one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, triethylamine, and DBU (1,8-diazabicycloundec-7-ene); the reaction solvent is selected from methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, or dichloromethane.
[0011] Preferably, the biocatalyst is an immobilized or non-immobilized acylase, preferably selected from: penicillin G acylase (PGA), acylase I from Aspergillus melleus, acylase I from Porcine Kidney, or genetically engineered mutants of the above enzymes.
[0012] Preferably, the pH value of the enzymatic hydrolysis reaction system in step (2) is controlled between 6.0 and 8.5, and the reaction temperature is between 20℃ and 50℃.
[0013] Preferably, the mother liquor recycling step involves: converting the unreacted (S)-configuration III compound separated in step (2) into a racemic III compound under strongly alkaline conditions or by heating, and then returning it to step (2) for recycling.
[0014] Preferably, the key intermediate in the method is racemic 4-acetamido-2-ethylisoxazolidin-3-one.
[0015] Preferably, the yield of the racemization treatment is greater than 80%, so that the theoretical atom utilization rate of the entire process approaches 100%.
[0016] The beneficial effects of this invention are: 1. Avoiding patent barriers: The "serine-halogenation" pathway has been completely abandoned, and the "dehydroalanine-addition" pathway has been adopted, which has independent intellectual property rights.
[0017] 2. Extremely high chiral purity: The ee value of enzyme-catalyzed products is usually >99%, eliminating the need for multiple recrystallizations.
[0018] 3. High safety: The reaction process does not involve highly toxic or explosive reagents and is easy to operate. Attached Figure Description
[0019] Figure 1 The synthetic route of (R)-4-amino-2-ethylisoxazolidin-3-one provided in the embodiments of the present invention. Specific Implementation The preparation method of the present invention will be further illustrated by specific embodiments below, but the present invention is not limited to these embodiments.
[0021] Example 1: Preparation of racemic 4-acetamido-2-ethylisoxazolidin-3-one 100 mL of methanol was added to a reaction flask, followed by methyl 2-acetamidoacrylate (14.3 g, 0.1 mol). Under ice bath cooling, N-ethylhydroxylamine (6.7 g, 0.11 mol) and sodium methoxide (0.54 g, 0.01 mol) were added dropwise. After the addition was complete, the mixture was heated to reflux and reacted for 3 hours. TLC analysis showed the starting material had disappeared. The reaction solution was concentrated under reduced pressure, and the residue was recrystallized from ethyl acetate to give 15.5 g of a white solid product, with a yield of 90%.
[0022] Example 2: Enzymatic preparation of (R)-4-amino-2-ethylisoxazolidin-3-one The racemic mixture (10 g) obtained in Example 1 was suspended in phosphate buffer (pH 7.5, 100 mL), and immobilized penicillin G acylase (1 g) was added. The reaction was stirred at 37°C, and the pH was maintained by automatically adding dilute NaOH solution. The reaction was stopped when the amount of alkali consumed reached 50% of the theoretical amount. The enzyme was recovered by filtration. The filtrate was extracted three times with dichloromethane (the extract phase contained the S-isomer, which was reserved for recovery). The aqueous phase was concentrated under reduced pressure, dissolved in ethanol, and acidified by adding concentrated hydrochloric acid, resulting in the precipitation of white crystals, which were (R)-4-amino-2-ethylisoxazolidine-3-one hydrochloride. After drying, 3.8 g of product was obtained (single-pass yield ~76% based on R-type), and the ee value determined by chiral HPLC was >99.5%.
[0023] Example 3: Racemization and Cycling of (S)-Isomers The dichloromethane extract from Example 2 was concentrated to obtain (S)-4-acetamido-2-ethylisoxazolidin-3-one. This was dissolved in a small amount of ethanol, and a catalytic amount of sodium ethoxide was added. The mixture was heated under reflux for 1 hour. After cooling, the solution was evaporated to dryness. HPLC analysis showed that it had been converted to a racemic mixture. This material can be directly incorporated into the next batch of feed from Example 2.
[0024] Example 4 Methyl 2-phenylacetamidoacrylate (0.1 mol) was used as the starting material, replacing the methyl 2-acetamidoacrylate in Example 1. N-ethylhydroxylamine was reacted with DBU (1,8-diazabicycloundec-7-ene, 0.05 mol) as a base in tetrahydrofuran (THF) solvent. The reaction was carried out at 40°C for 4 hours, and post-treatment yielded racemic-4-phenylacetamido-2-ethylisoxazolidin-3-one in 88% yield with 98% HPLC purity.
[0025] The above intermediate was added to a buffer solution containing immobilized penicillin G acylase (PGA). Since phenylacetyl is a specific substrate for PGA, the reaction rate was extremely fast (3-5 times faster than acetyl). The reaction was completed after 2 hours, and the target product (R)-4-amino-2-ethylisoxazolidin-3-one was isolated with an ee value of 99.8%.
[0026] Example 5 Potassium carbonate (K₂CO₃) powder (1.5 eq) was added to ethanol. Methyl 2-acetamidoacrylate and N-ethylhydroxylamine hydrochloride were then added. The reaction was carried out under reflux for 6 hours. A racemic intermediate was given in 82% yield.
[0027] Example 6 The racemic 4-acetamido-2-ethylisoxazolidin-3-one prepared in Example 1 was hydrolyzed using Aspergillus melleus acylase. The reaction was carried out at pH 7.0, 30°C, with CoCl2 (0.5 mM) as a cofactor. After 24 hours of reaction, the conversion rate was 49.5%, and the product ee value was >99%.
[0028] Example 7 A 100g scale-up experiment was conducted. After the first round of enzymatic resolution, the (R)-product (yield 46%) and the (S)-acetyl residue were separated. The (S)-residue was racemiced by refluxing in sodium ethoxide / ethanol for 30 minutes, with a recovery rate of 95%, yielding the racemic mixture. The recovered racemic mixture was mixed with fresh starting material at a 1:1 ratio for the second round of enzymatic resolution. After three consecutive cycles, the overall yield (based on the starting material) reached 85%, and the ee value of the product remained above 99% throughout.
[0029] Comparative Example 1 Using β-chloro-D,L-alanine methyl ester hydrochloride as a starting material, N-ethylhydroxylamine and sodium hydroxide were added to methanol and reacted at the same temperature. TLC monitoring showed disordered reaction spots. HPLC analysis revealed approximately 15-20% O-alkylation byproducts in addition to the target product (yield only 45%). The starting material was unstable and partially decomposed after being left overnight.
[0030] Comparative Example 2 The racemic 4-amino-2-ethylisoxazolidine-3-one prepared according to this invention (after first hydrolyzing to remove the acetyl group) was crystallized and resolved using the classic resolving agent L-tartaric acid. Repeated recrystallization three times was required to achieve an ee value of 95%. The single-pass yield was only 25%. Furthermore, due to the extremely high water solubility of amines, a large amount of product was lost from the mother liquor, making effective recovery impossible.
Claims
1. A method for preparing (R)-4-amino-2-ethylisoxazolidin-3-one (Formula I), characterized in that, Includes the following steps: (1) Skeletonization: The 2-acylamino acrylate derivative shown in Formula II is reacted with N-ethylhydroxylamine or its salt in the presence of a basic reagent by an addition-cyclization tandem reaction to obtain the racemic intermediate compound of Formula III. (2) Enzymatic resolution: In a solvent, using acylase or esterase as a biocatalyst, the compound of formula III obtained in step (1) is stereoselectively hydrolyzed to obtain the (R)-configuration compound of formula IV and the unreacted (S)-configuration compound of formula III. (3) Conversion to salt: Separate the compound of formula IV, and deprotect it (if necessary) or directly acidify it to obtain the target product of formula I; Formula II is CH2=C(NHR)-COOR'; Formula III is 4-acylamino-2-ethylisoxazolidin-3-one.
2. The method according to claim 1, characterized in that, In the 2-acylaminoacrylate derivative (Formula II) described in step (1), R is selected from acetyl, trifluoroacetyl, phenylacetyl or benzoyl; R' is selected from methyl, ethyl or tert-butyl; preferably methyl 2-acetaminoacrylate.
3. The method according to claim 1, characterized in that, The alkaline reagent mentioned in step (1) is selected from one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydroxide, potassium hydroxide, potassium carbonate, triethylamine, and DBU (1,8-diazabicycloundec-7-ene); the reaction solvent is selected from methanol, ethanol, isopropanol, tetrahydrofuran, acetonitrile, or dichloromethane.
4. The method according to claim 1, characterized in that, The biocatalyst mentioned in step (2) is an immobilized or non-immobilized acylase, preferably selected from: penicillin G acylase (PGA), acylase I from Aspergillus melleus, acylase I from Porcine Kidney, or genetically engineered mutants of the above enzymes.
5. The method according to claim 1, characterized in that, The pH value of the enzymatic hydrolysis reaction system in step (2) is controlled between 6.0 and 8.5, and the reaction temperature is 20℃-50℃.
6. The method according to claim 1, characterized in that, It also includes a mother liquor recycling step: the unreacted (S)-configuration III compound separated in step (2) is racemized under strongly alkaline conditions or by heating to become a racemic III compound, and then returned to step (2) for recycling.
7. A key intermediate in the method of claim 1: racemic 4-acetamido-2-ethylisoxazolidin-3-one.
8. The method according to claim 6, characterized in that, The yield of the racemization treatment is greater than 80%, which makes the theoretical atom utilization rate of the entire process approach 100%.