A method for preparing a listatin intermediate

Abstract

This invention relates to the field of organic chemical synthesis, specifically disclosing a method for preparing a ristat intermediate. The method uses 3-bromobenzaldehyde and acetylglycine as raw materials, and prepares L-3-bromophenylalanine through enzymatic resolution. The obtained L-3-bromophenylalanine is then processed to prepare 3-methylsulfonyl-L-phenylalanine benzyl ester by adjusting pH, protecting the tert-butyloxycarbonyl group, introducing a methyl sulfone group, benzyl ester protection, and deprotecting the tert-butyloxycarbonyl group. This method is simple to operate, uses inexpensive and readily available raw materials and reagents, operates under mild reaction conditions, has high conversion and high yield, can reduce production costs, improve production efficiency, and is environmentally friendly, thus enabling industrial-scale production.

Description

Technical Field

[0001] This invention belongs to the field of organic chemical synthesis, and specifically relates to a method for preparing a ristat intermediate. Background Technology

[0002] Lifitegrast is a novel small-molecule integrin inhibitor. In vitro studies have shown that lifitegrast can inhibit inflammatory factors associated with dry eye syndrome. Its specific structure is as follows:

[0003]

[0004] Litastar.

[0005] 3-Methylsulfonyl-L-phenylalanine benzyl ester is one of the key intermediates in the synthesis of lifitegrast. Various methods for preparing lifitegrast intermediates have been reported in the prior art. For example, patent CN111471003A discloses a method for preparing a lifitegrast intermediate, the synthetic route of which is as follows:

[0006] .

[0007] However, the starting materials for this route are relatively expensive, and it uses the costly transition metal palladium catalyst and ligands; the preparation of Grignard zinc reagent is difficult, requires harsh conditions, has a complex process, and has a low yield, so it is obviously not suitable for scale-up reactions.

[0008] For example, patent CN104797574A discloses two methods for synthesizing 3-methylsulfonyl-L-phenylalanine: a. Synthetic route: 3-bromo-L-phenylalanine is used as the starting material, protected by a protecting group, substituted with methanesulfonyl group, and then deprotected. This route uses 3-bromo-L-phenylalanine as the starting material. This product has not reached industrial production, and the reagent-grade raw materials are quite expensive.

[0009]

[0010] b. Starting with 3-methanesulfonylbenzaldehyde, an intermediate is obtained through a three-step reaction. This route requires noble metal catalysts and ligands for asymmetric oxidation, which is costly and has low chirality.

[0011] .

[0012] For example, patent CN106947792A discloses a method for synthesizing m-methylsulfonyl-L-phenylalanine, the synthetic route of which is as follows:

[0013] .

[0014] This method uses 3-bromobenzaldehyde as the starting material and obtains the intermediate through four steps of reaction. This route requires the use of a noble metal catalyst for hydrogenation, which is costly and not suitable for scale-up.

[0015] For example, patent CN113072471A discloses a rifastard intermediate and its preparation method, with the following synthetic route:

[0016] ,

[0017] However, this route is cumbersome to operate, has a low yield, and is not suitable for industrial production.

[0018] Therefore, there is an urgent need to develop a method for preparing 3-methylsulfonyl-L-phenylalanine benzyl ester that can simplify the synthesis steps, reduce production costs, improve production efficiency, and is suitable for large-scale production. Summary of the Invention

[0019] This invention addresses the problems existing in the prior art by providing a method for preparing ristat intermediates. The method uses 3-bromobenzaldehyde and acetylglycine as raw materials to prepare ristat intermediate 3-methylsulfonyl-L-phenylalanine benzyl ester. The reaction conditions are mild, the yield is high, the purity is good, and it is environmentally friendly, enabling industrial production.

[0020] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0021] This invention provides a method for preparing a ristat intermediate, comprising the following steps:

[0022] S1. Using 3-bromobenzaldehyde and acetylglycine as raw materials, an Erlenmeyer-Plöchl lactone synthesis reaction and hydrolysis reaction were carried out to obtain the compound shown in Formula I. ;

[0023] S2. Using the compound shown in Formula I as a substrate, the compound shown in Formula II is obtained by reduction reaction under the conditions of metal powder and acid. The reaction route is as follows:

[0024] ;

[0025] S3. The compound shown in Formula II is enzymatically dissected to obtain the compounds shown in Formula III and Formula IV, respectively. The reaction routes are as follows:

[0026] .

[0027] Specifically, the reaction route of step S3 is as follows:

[0028] ;

[0029] The compound shown in S4 and Formula III was prepared into the compound shown in Formula V by pH adjustment with alkali, tert-butyloxycarbonyl protection, and extraction. The reaction route is as follows:

[0030] ;

[0031] S5. Using the compound shown in Formula V as a substrate, a sulfone group was introduced, and the compound shown in Formula VIII was prepared by benzyl ester protection and removal of the tert-butyloxycarbonyl protecting group. The reaction route is as follows:

[0032] .

[0033] Preferably, in steps S1 and S2, in the compounds represented by Formula I and Formula II, R is a halogen atom and R2 is a C1-C6 alkyl group.

[0034] More preferably, in steps S1 and S2, in the compounds represented by Formula I and Formula II, R is F, Cl or Br, and R2 is CH3, CH2CH3, CH2CH2CH3, CH=CH2, C6H5, CH2C6H5 or COOHCH2CH2.

[0035] Preferably, the metal powder in step S2 includes at least one of iron powder, magnesium powder, and zinc powder; the acid in step S2 is a solution of the acid, which is at least one of an aqueous solution of the acid, a 1,4-dioxane solution of the acid, an ethyl hexanoate solution of the acid, a methanol solution of the acid, an ethanol solution of the acid, and an isopropanol solution of the acid.

[0036] Preferably, the molar ratio of the compound represented by Formula I, the metal powder, and the acid in step S2 is 1:1.2-2.4:1.2-6.0.

[0037] Preferably, step S2 further includes the step of adding the compound of formula I to a solvent.

[0038] Preferably, the solvent includes at least one selected from tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, acetone, acetonitrile, methanol, ethanol, and isopropanol.

[0039] Preferably, step S2 further includes an extraction step after the reduction reaction, wherein the extraction solvent includes at least one of ethyl acetate, methyl tert-butyl ether, isopropyl acetate and 2-methyltetrahydrofuran.

[0040] Preferably, the enzyme described in step S3 is a hydrolase, including lipase, protease, esterase or acylase; the concentration of the compound represented by formula II in step S3 is 50-1000 g / L.

[0041] Preferably, the lipase includes, but is not limited to, Candida antarcticis lipase, Candida columnaris lipase, Pseudomonas cepacia lipase PS, Lipozyme TL IM lipase, and koji lipase.

[0042] Preferably, the protease includes, but is not limited to, chymotrypsin, cathepsin, papain, and subtilisin.

[0043] Preferably, the esterase includes, but is not limited to, porcine liver esterase PLE, esterase RO, Aspergillus oryzae TL recombinant, Escherichia coli esterase BS1 recombinant and Escherichia coli esterase BS2 recombinant.

[0044] Preferably, the acylase includes, but is not limited to, porcine liver acylase, acylase derived from Escherichia coli, acylase derived from Aspergillus niger, acylase AmACY, and acylase AMID.

[0045] Preferably, the enzyme dissociation conditions in step S3 are: reaction temperature of 10℃-50℃, reaction time of 0.1-240 hours, and pH of 5-9; the reduction reaction temperature in step S2 is -10℃ to 10℃.

[0046] More preferably, the enzyme dissociation conditions in step S3 are: reaction temperature of 20℃-40℃, reaction time of 0.5-120 hours, and pH of 5.5-7.5.

[0047] More preferably, the enzyme dissociation conditions in step S3 are: reaction temperature of 25℃-35℃, reaction time of 1-72 hours, and pH of 5.8-7.3.

[0048] More preferably, the enzyme dissociation conditions in step S3 are: reaction temperature of 25℃-35℃, reaction time of 3-10 hours, and pH of 6.5-7.0.

[0049] Specifically, the hydrolytic enzyme is a free enzyme, an immobilized enzyme, or an enzyme in bacterial cell form.

[0050] Preferably, the alkali mentioned in step S4 is at least one selected from sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, triethylamine, and diethylamine; and the pH is 5.0-14.0.

[0051] Preferably, after extraction in step S4, an amine salt purification step or a pulping step is required.

[0052] Preferably, the amine in the amine salt purification step is at least one of triethylamine, diethylamine, benzylamine, dibenzylamine, dicyclohexylamine, and ammonia water; and the solvent for pulping is at least one of n-hexane, cyclohexane, n-heptane, n-pentane, cyclopentane, and petroleum ether.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] This invention provides a novel method for preparing 3-methylsulfonyl-L-phenylalanine benzyl ester. Using 3-bromobenzaldehyde and acetylglycine as raw materials, L-3-bromophenylalanine is prepared by enzymatic resolution (hydrolysis). The obtained L-3-bromophenylalanine compound is then protected with a tert-butyloxycarbonyl group, introduced with a methylsulfonyl group, protected with benzyl ester, and deprotected with the tert-butyloxycarbonyl group to prepare 3-methylsulfonyl-L-phenylalanine benzyl ester. This method is simple to operate, uses inexpensive and readily available raw materials and reagents, operates under mild reaction conditions, has a high conversion rate and high yield, can reduce production costs, improve production efficiency, and is environmentally friendly, thus enabling industrial-scale production. Attached Figure Description

[0055] Figure 1 The above is the H-NMR spectrum of Formula I in the embodiment of the present invention;

[0056] Figure 2 The image shown is the C-NMR spectrum of Formula I in the embodiment of the present invention;

[0057] Figure 3 This is the HRMS diagram of Formula I in the embodiment of the present invention;

[0058] Figure 4 The above is the H-NMR spectrum of Formula II in the embodiment of the present invention;

[0059] Figure 5 The C-NMR spectrum of Formula II in the embodiment of the present invention;

[0060] Figure 6 This is the HRMS diagram of Formula II in the embodiment of the present invention;

[0061] Figure 7 The above is the H-NMR spectrum of Formula III in the embodiment of the present invention;

[0062] Figure 8 The C-NMR spectrum of Formula III in the embodiment of the present invention;

[0063] Figure 9 This is the HRMS diagram of Formula III in the embodiment of the present invention;

[0064] Figure 10 The above is the H-NMR spectrum of formula V in an embodiment of the present invention;

[0065] Figure 11 The image shows the C-NMR spectrum of formula V in an embodiment of the present invention.

[0066] Figure 12 The HRMS diagram of formula V in the embodiment of the present invention;

[0067] Figure 13 This is the SCXRD diagram of formula V in an embodiment of the present invention;

[0068] Figure 14 The above is the H-NMR spectrum of formula VI in the embodiment of the present invention;

[0069] Figure 15 The image shown is the C-NMR spectrum of formula VI in the embodiment of the present invention;

[0070] Figure 16 This is the HRMS diagram of formula VI in the embodiment of the present invention;

[0071] Figure 17 This is the racemic chiral HPLC spectrum of Formula III in an embodiment of the present invention;

[0072] Figure 18 This is the chiral HPLC spectrum of Formula III in an embodiment of the present invention. Detailed Implementation

[0073] The following description of the embodiments is merely to aid in understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of the claims. The following description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not limited to the embodiments shown herein, but can be applied to a wider scope consistent with the principles and novel features disclosed herein. While any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, preferred methods and materials are listed herein.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0075] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort should fall within the scope of protection of this invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described, unless otherwise specified, are commercially available.

[0076] In this invention, "acylase" is an enzyme that can selectively catalyze the conversion of L-acetyl amino acids to L-amino acids.

[0077] In this invention, the acylase can be wild-type or mutant. Furthermore, it can be isolated or recombinant.

[0078] The amino acid sequence of the acylase in this invention is shown in SEQ ID NO:2, and its encoding gene is shown in SEQ ID NO:1. The acylases used in this invention can be from different species. Furthermore, enzymes (including enzymes from other species) that have similar activity or homology (e.g., ≥80%, preferably ≥90%, more preferably ≥95%) to the acylase of this invention are also within the scope of this invention.

[0079] Table 1. Amino acid sequence and gene sequence of the acylase of this invention.

[0080]

[0081] Due to the degeneracy of codons, the base sequence encoding the amino acid sequence shown in SEQ ID NO:2 is not limited to SEQ ID NO:1. Those skilled in the art can obtain homologues of this base sequence by appropriately introducing substitutions, deletions, alterations, insertions, or additions. This invention covers such homologues, provided that the recombinase expressed by them maintains its catalytic reduction activity against the compound of formula II. Homologues of polynucleotides in this invention can be prepared by substituting, deleting, or adding one or more bases of the base sequence SEQ ID NO:1 within the range of maintaining enzyme activity.

[0082] The acylase of the present invention also includes an amino acid sequence obtained by substituting, deleting, altering, inserting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO:2 within the range of maintaining enzyme activity.

[0083] Example 1: Synthesis of Formula I (1,3-bromoacetamidocinnamic acid)

[0084] 3-Bromobenzaldehyde (150.0 g, 0.81 mol) and acetylglycine (104.4 g, 0.89 mol) were dissolved in acetic anhydride (500 mL), heated to 70°C, and reacted for 18 hours. After cooling to room temperature, a large amount of yellow solid precipitated and was filtered. The yellow solid was dissolved in 1 L of water, heated to 90°C, and reacted for 12 hours. After cooling to room temperature, the resulting yellow solid was filtered. The conversion rate was >98%, the yield was 80%, and the purity was 98.06%.

[0085] 1H NMR (400 MHz, DMSO-d6): δ 9.49 (s, 1H), 7.62 (d, J = 7.2 Hz, 2H), 7.38 (dt, J = 21.5, 7.1 Hz, 3H), 7.24 (s, 1H), 2.00 (s, 3H). 13C NMR (100MHz, DMSO-d6): δ 169.59, 166.60, 136.71, 132.33, 132.02, 131.05, 129.43,129.06, 129.01, 122.22, 22.96. HRMS (ESI-QTOF): m / z calculated forC11H10BrNO3 [M + H]+ 283.99168, found 283.99029.

[0086] Synthesis route:

[0087]

[0088] Example 2 Synthesis of Formula II (3-bromoacetylaminophenylalanine)

[0089] Compound I (141.5 g, 0.50 mol) was added to tetrahydrofuran (710.0 mL). The reaction system was cooled to 10°C, and zinc powder (65.38 g, 1.0 mol) was added. 3N hydrochloric acid (330.0 mL, 1.0 mol) was added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 12 hours. The mixture was washed with saturated sodium chloride solution, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, and the solvent was removed by rotary evaporation to obtain a pale yellow solid with a conversion >98%, yield 92%, and purity 95.06%.

[0090] 1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 8.1 Hz, 1H), 7.43 (d, J =16.5 Hz, 2H), 7.25 (d, J = 4.9 Hz, 2H), 4.42 (td, J = 9.4, 4.9 Hz, 1H), 3.06(dd, J = 13.8, 4.9 Hz, 1H), 2.84 (dd, J = 13.8, 9.7 Hz, 1H), 1.79 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 173.32, 169.74, 141.05, 132.30, 130.74, 129.77,128.67, 121.85, 53.67, 36.69, 22.75. HRMS (ESI-QTOF): m / z calculated forC11H12BrNO3 [M + H]+ 286.00733, found 286.00614.

[0091] Synthesis route: .

[0092] Example 3 Synthesis of Formula III (L-3-bromophenylalanine)

[0093] Compound II (285.5 g, 1.0 mol) was added to phosphate buffer (4000 mL), and the pH was adjusted to 8.5 with 10% sodium hydroxide aqueous solution. Acylase was added, and the reaction was carried out at 30°C for 24 hours. The pH was then adjusted to 2.0, and the unhydrolyzed compound II, i.e., compound IV, was extracted into the organic phase with ethyl acetate and racemic recovered. The aqueous phase was not purified and was used directly in the next reaction. HPLC analysis showed a conversion rate of 40% (theoretical conversion rate 50%).

[0094] 1H NMR (600 MHz, DMSO-d6) δ 8.65 (s, 3H), 7.53 (d, J = 1.5 Hz, 1H), 7.49 – 7.44 (m, 1H), 7.30 (dt, J = 15.4, 7.7 Hz, 2H), 4.14 (t, J = 6.2 Hz,1H), 3.19 (d, J = 6.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 170.49, 138.46,132.75, 131.06, 130.51, 129.24, 122.18, 53.40, 35.40. HRMS (ESI-QTOF): m / zcalculated for C9H10BrNO2 [M + H]+ 243.99677, found 243.99568.

[0095] Synthesis route: .

[0096] The racemic process of the unhydrolyzed compound II, which is also the compound IV, is as follows:

[0097] Compound IV (28.5 g, 0.1 mol) was dissolved in acetic acid (280 mL), and 0.1 equivalent of acetic anhydride was added. The mixture was heated to 60 °C and stirred for 8 hours. After the reaction was completed, the mixture was cooled to room temperature, and 50 mL of ethanol and 280 mL of water were added to quench the reaction. The mixture was extracted three times with ethyl acetate, and the organic phases were combined and washed three times with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to give 27.06 g of a pale yellow solid with a yield of 95% and a purity of 97.86%.

[0098] 1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 8.1 Hz, 1H), 7.43 (d, J =16.5 Hz, 2H), 7.25 (d, J = 4.9 Hz, 2H), 4.42 (td, J = 9.4, 4.9 Hz, 1H), 3.06(dd, J = 13.8, 4.9 Hz, 1H), 2.84 (dd, J = 13.8, 9.7 Hz, 1H), 1.79 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 173.32, 169.74, 141.05, 132.30, 130.74, 129.77,128.67, 121.85, 53.67, 36.69, 22.75. HRMS (ESI-QTOF): m / z calculated for C11H12BrNO3 [M + H]+ 286.00733, found 286.00614.

[0099] Synthesis route: .

[0100] Example 4 Synthesis of Formula V (N-tert-Butoxycarbonyl-L-3-bromophenylalanine)

[0101] The aqueous solution obtained in the previous step was adjusted to pH 8.0 with 10% sodium hydroxide aqueous solution. Di-tert-butoxycarbonyl carbonate (218 g, 1.0 mol) was added, and the reaction was carried out at room temperature for 8 hours. After the reaction was complete, the pH was adjusted to 5.0 with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. Dicyclohexylamine (90 g, 0.5 mol) was added to the dried solution, resulting in the precipitation of a large amount of white solid. The solid was filtered and dried at 50°C. The conversion rate was >99%, the yield was 45% (theoretical yield 50%), and the purity was 99.06%. Enantiomer excess percentage: 99.99%.

[0102] 1H NMR (400 MHz, DMSO-d6) δ 7.46 (s, 1H), 7.40 (d, J = 7.0 Hz, 1H), 7.26 (d, J = 13.7 Hz, 2H), 7.11 (d, J = 8.5 Hz, 1H), 4.12 (dd, J = 10.7, 8.2Hz, 1H), 3.05 (dd, J = 13.7, 4.3 Hz, 1H), 2.82 (dd, J = 13.6, 10.7 Hz, 1H), 1.33 (s, 9H). 13 HRMS (ESI-QTOF):m / z calculated for C14H18BrNO4 [M + Na] + 366.03114, found 366.02947.

[0103] Synthesis route:

[0104]

[0105] Example 5 Synthesis of Formula VI (N-tert-Butoxycarbonyl-L-3-methylsulfonylphenylalanine)

[0106] Compound v (34.4 g, 0.1 mol) was dissolved in 340 mL of DMSO (13.2 g, 0.13 mol). Cuprous iodide (22.8 g, 0.12 mol), L-proline (15.8 g, 0.12 mol), and potassium carbonate (6.9 g, 0.05 mol) were added to the reaction solution. The mixture was heated to 120°C and reacted for 8 hours, achieving a conversion rate >99%. After the reaction was complete, the pH was adjusted to 3 with 10% citric acid solution. The resulting solution was filtered, and the filtrate was extracted three times with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The solution was then used directly in the next step.

[0107] Synthesis route: .

[0108] Example 6 Synthesis of Formula VII (N-tert-butoxycarbonyl-L-3-methylsulfonylphenylalanine benzyl ester)

[0109] The compound of formula VI obtained in the previous step was dissolved in dichloromethane (340 mL), and DMAP (2.4 g, 20.0 mmol) and benzyl alcohol (11.9 g, 0.11 mol) were added. The mixture was cooled to 0 degrees Celsius, and a dichloromethane suspension of EDC·HCl (21.1 g, 0.11 mol) was added dropwise to the reaction solution. The mixture was stirred for two hours, and the conversion rate was >99%. After the reaction was complete, the organic phase was washed with saturated sodium bicarbonate aqueous solution, and then dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain a brown oily liquid, which was used directly in the next step without purification.

[0110] Synthesis route:

[0111] .

[0112] Example 7 Synthesis of Formula VIII (L-3-methylsulfonylphenylalanine benzyl ester hydrochloride)

[0113] The compound of formula VII obtained in the previous step was dissolved in dichloromethane (340 mL), and an ethyl acetate solution of hydrogen chloride (4 M) was added to the reaction system. The mixture was stirred for 8 hours, and a large amount of white solid precipitated out. The mixture was filtered, and the filter cake was washed with a small amount of ethyl acetate. The filter cake was dried at 50 degrees Celsius to obtain 33.94 g of white solid. The conversion rate was >99%, the yield was 92%, and the purity was 99.71%.

[0114] 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 3H), 7.92 (s, 1H), 7.83 (dt, J =6.7, 1.9 Hz, 1H), 7.61 – 7.51 (m, 2H), 7.41 – 7.32 (m, 3H), 7.27 (dd, J =7.0, 2.5 Hz, 2H), 5.15 (q, J = 12.4 Hz, 2H), 4.45 (dd, J = 7.5, 6.0 Hz, 1H), 3.41 (dd, J = 14.0, 5.8 Hz, 1H), 3.29 (dd, J = 14.1, 7.7 Hz, 1H), 3.17 (s,3H). 13C NMR (100 MHz, DMSO-d6) δ 169.12, 141.46, 136.98, 135.25, 135.13,130.07, 128.88, 128.79, 128.64, 128.41, 126.21, 67.55, 53.33, 43.99, 35.81.HRMS (ESI-QTOF): m / z calculated for C17H19NO4S [M + H]+ 334.11076, found334.10928.

[0115] Synthesis route:

[0116] .

[0117] Comparative Example 1: Synthesis of Formula II (3-bromoacetylaminophenylalanine)

[0118] Compared to Examples 1-7, only the molar ratio of the compound shown in Formula I, zinc powder, and acid in Example 2 was adjusted; all other steps remained the same as in Examples 1-7. The specific adjusted steps are shown below:

[0119] Compound I (141.5 g, 0.50 mol) was added to tetrahydrofuran (710.0 mL), and the reaction system was cooled to 10°C. Zinc powder (32.69 g, 0.5 mol) was then added, followed by dropwise addition of 3N hydrochloric acid (330.0 mL, 1.0 mol). After the addition was complete, the reaction was allowed to proceed at room temperature for 12 hours. HPLC monitoring showed a conversion rate of 25%, indicating incomplete conversion.

[0120] 1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 8.1 Hz, 1H), 7.43 (d, J =16.5 Hz, 2H), 7.25 (d, J = 4.9 Hz, 2H), 4.42 (td, J = 9.4, 4.9 Hz, 1H), 3.06(dd, J = 13.8, 4.9 Hz, 1H), 2.84 (dd, J = 13.8, 9.7 Hz, 1H), 1.79 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 173.32, 169.74, 141.05, 132.30, 130.74, 129.77,128.67, 121.85, 53.67, 36.69, 22.75. HRMS (ESI-QTOF): m / z calculated forC11H12BrNO3 [M + H]+ 286.00733, found 286.00614.

[0121] Synthesis route:

[0122] Comparative Example 2: Synthesis of Formula III (L-3-bromophenylalanine)

[0123] Compared to Examples 1-7, only the pH of the enzyme dissociation in Example 3 was adjusted to 4.5; all other steps were the same as in Examples 1-7. The specific adjusted operating steps are shown below:

[0124] Compound II (285.5 g, 1.0 mol) was added to phosphate buffer (4000 mL), and the pH was adjusted to 4.5 with 10% sodium hydroxide aqueous solution. Acylase was added, and the reaction was carried out at 30°C for 24 hours. The pH was then adjusted to 2.0, and the unhydrolyzed compound II, i.e., compound IV, was extracted into the organic phase with ethyl acetate and racemic recovered. The aqueous phase was not purified and was used directly in the next reaction. HPLC analysis showed a conversion of 1.9% (theoretical conversion rate 50%). The conversion was incomplete.

[0125] 1H NMR (600 MHz, DMSO-d6) δ 8.65 (s, 3H), 7.53 (d, J = 1.5 Hz, 1H), 7.49 – 7.44 (m, 1H), 7.30 (dt, J = 15.4, 7.7 Hz, 2H), 4.14 (t, J = 6.2 Hz,1H), 3.19 (d, J = 6.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 170.49, 138.46,132.75, 131.06, 130.51, 129.24, 122.18, 53.40, 35.40. HRMS (ESI-QTOF): m / zcalculated for C9H10BrNO2 [M + H]+ 243.99677, found 243.99568.

[0126] Synthesis route: .

[0127] Synthesis of Formula III (L-3-bromophenylalanine) in Comparative Example 3

[0128] Compared to Examples 1-7, only the pH of the enzyme dissociation in Example 3 was adjusted to 9.5; all other steps were the same as in Examples 1-7. The specific adjusted steps are shown below:

[0129] Compound II (285.5 g, 1.0 mol) was added to phosphate buffer (4000 mL), and the pH was adjusted to 9.5 with 10% sodium hydroxide aqueous solution. Acylase was added, and the reaction was carried out at 30°C for 24 hours. The pH was then adjusted to 2.0, and the unhydrolyzed compound II, i.e., compound IV, was extracted into the organic phase with ethyl acetate and racemic recovered. The aqueous phase was not purified and was used directly in the next reaction. HPLC analysis showed a conversion of 3.2% (theoretical conversion rate 50%). The conversion was incomplete.

[0130] 1H NMR (600 MHz, DMSO-d6) δ 8.65 (s, 3H), 7.53 (d, J = 1.5 Hz, 1H), 7.49 – 7.44 (m, 1H), 7.30 (dt, J = 15.4, 7.7 Hz, 2H), 4.14 (t, J = 6.2 Hz,1H), 3.19 (d, J = 6.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 170.49, 138.46,132.75, 131.06, 130.51, 129.24, 122.18, 53.40, 35.40. HRMS (ESI-QTOF): m / zcalculated for C9H10BrNO2 [M + H]+ 243.99677, found 243.99568.

[0131] Synthesis route: .

[0132] The above description, in conjunction with specific embodiments, further illustrates the present invention. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention can be made without departing from the spirit and scope of the invention, and all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. A method for preparing a ristat intermediate, characterized in that: Includes the following steps: S1. Using 3-bromobenzaldehyde and acetylglycine as raw materials, an Erlenmeyer-Plöchl lactone synthesis reaction and a hydrolysis reaction were carried out to obtain the compound shown in Formula I. ; S2. Using the compound shown in Formula I as a substrate, under the conditions of metal powder and acid, a reduction reaction yields the compound shown in Formula II. The reaction route is as follows: ； S3. The compound shown in Formula II is enzymatically dissociated to obtain the compounds shown in Formula III and Formula IV, respectively. The reaction routes are as follows: ； The compound shown in S4 and Formula III was prepared into the compound shown in Formula V by pH adjustment with alkali, tert-butyloxycarbonyl protection, and extraction. Reaction route: ； S5. Using the compound shown in Formula V as a substrate, a methyl sulfone group was introduced, and the compound shown in Formula VIII was prepared by benzyl ester protection and removal of the tert-butyloxycarbonyl protecting group, namely 3-methyl sulfone-L-phenylalanine benzyl ester. The temperature of the reduction reaction in step S2 is -10℃ to -10℃; The conditions for enzyme dissociation in step S3 are: reaction temperature of 10℃-50℃, reaction time of 0.1-240 hours, and pH of 5-9. The enzyme described in step S3 is an acylase, the amino acid sequence of which is shown in SEQ ID NO:2, and its encoding gene is shown in SEQ ID NO:1; the concentration of the compound represented by formula II in step S3 is 50-1000 g / L.

2. The preparation method according to claim 1, characterized in that: The metal powder mentioned in step S2 includes at least one of iron powder, magnesium powder, and zinc powder; the acid mentioned in step S2 is a solution of the acid, which is at least one of an aqueous solution of the acid, a 1,4-dioxane solution of the acid, an ethyl hexanoate solution of the acid, a methanol solution of the acid, an ethanol solution of the acid, and an isopropanol solution of the acid.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the compound represented by Formula I, the metal powder, and the acid in step S2 is 1:1.2-2.4:1.2-6.

0.

4. The preparation method according to claim 1, characterized in that: The alkali mentioned in step S4 is at least one selected from sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, triethylamine, and diethylamine; the pH is 5.0-14.

0.

5. The preparation method according to claim 1, characterized in that: After extraction as described in step S4, an amine salt purification step or a pulping step is required.

6. The preparation method according to claim 5, characterized in that: The solvent used for pulping is at least one selected from n-hexane, cyclohexane, n-heptane, n-pentane, cyclopentane, and petroleum ether.

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