Method for preparing linitinib intermediate by chemical-enzyme method

Abstract

The invention discloses a method for preparing a linitinib intermediate I by a chemical-enzyme method. According to the method disclosed by the invention, a compound II is taken as a starting material, the compound II is firstly subjected to hydrogenation reduction to form a compound III, then the compound III is converted into a compound VI under the action of monoamine oxidase, imine reductase and an amino protecting group, the compound VI is oxidized to generate a compound VII, finally, the litexitinib intermediate I is obtained under the catalytic action of transaminase, and the chiral purity of the intermediate is greater than or equal to 99%. The method disclosed by the invention is simple to operate, high in yield, free of any resolving agent, low in cost and more suitable for industrial production.

Description

Technical field:

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to a method for preparing a lecithinib intermediate by a chemical-enzymatic method. Background technology:

[0002] Ritlecitinib is a JAK3 / TEC inhibitor developed by Pfizer. It was launched in the United States on June 23, 2023 under the trade name Litfulo. It is mainly used to treat alopecia areata, vitiligo, rheumatoid arthritis, Crohn's disease and other diseases. Its chemical structure is as follows:

[0003]

[0004] Pfizer disclosed a method for preparing rituximab in patent CN106061973B, as shown in Scheme 1. The method uses compound 1 as the starting material, and obtains the key chiral intermediate rac-I through Boc protection, PtO2 reduction of pyridine, Cbz protection, hydrochloric acid deprotection and other reactions, wherein the ratio of cis-trans isomers in mixture 3 is 2:1, and its amino-protected product rac-4 needs to be separated by Chiral SFC. Subsequently, compound rac-I is subjected to nucleophilic substitution, Pd / C hydrogenation, acryloyl chloride condensation, splitting and other reactions to obtain rituximab.

[0005]

[0006]

[0007] Scheme 1

[0008] Through Scheme 1, it was found that compound I is a key chiral intermediate for the synthesis of rituxitinib. Therefore, the synthesis research of rituxitinib intermediate I has attracted attention and concern in the industry.

[0009] Pfizer disclosed an improved method for intermediate I in patent CN112888691A, as shown in Scheme 2. This method uses compound II as the starting material, uses Rh / C catalyst to reduce the pyridine ring, increases the ratio of cis-trans isomer product rac-3 to 2.8:1, and uses (R)-N-3,5-dinitrobenzoylphenylglycine as a resolving agent, but the resolving agent is expensive, has a violent exothermic phenomenon when used, has a large safety hazard, and this route still cannot avoid the production of isomers, resulting in a low yield.

[0010]

[0011] Scheme 2

[0012] Patent WO2019225267A1 discloses a new method for preparing key intermediate I, as shown in Scheme 3. The method uses compound 9 as the starting material, reduces it to compound 10 by palladium carbon, and then splits it under the action of hydrolase to obtain compound 11, whose ee value is only 91.4%, thereby obtaining intermediate I.

[0013]

[0014] Scheme 3

[0015] Patent CN112824381A discloses a method for preparing intermediate I, as shown in Scheme 4. This method uses compound 14 as a starting material, and generates compound 16 through Witting and Pd / C reduction reactions, and then compound 16 reacts with trimethylsulfoxide iodide to obtain compound 17, and then cyclizes under the catalytic action of a catalyst (1,5-cyclooctadiene) iridium chloride dimer to obtain compound 18, and finally compound 18 generates intermediate I under the action of transaminase. In this method, the cyclization reaction requires the use of precious metals, the reagents are expensive, the reaction conditions are harsh, and it is not conducive to industrial production.

[0016]

[0017] Scheme 4

[0018] In the prior art, the preparation method of rituximab intermediate I has low yield and high cost, which is not conducive to industrialization. Therefore, we need to develop a preparation method that is simple to operate, has cheap and readily available raw materials, high yield and is suitable for industrialization. Summary of the invention:

[0019] The object of the present invention is to provide a method for preparing rituximab intermediate I by an efficient chemical-enzymatic method in view of the deficiencies in the prior art.

[0020] The technical solution adopted by the present invention is shown in Scheme 5.

[0021]

[0022] Scheme 5

[0023] Wherein, R is selected from one of tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), methoxycarbonyl, ethoxycarbonyl, acetyl, benzoyl, methylsulfonyl, benzenesulfonyl, and 2-nitrobenzenesulfonyl, and preferably R is tert-butyloxycarbonyl (Boc) or benzyloxycarbonyl (Cbz).

[0024] The present invention provides a method for preparing a rituximab intermediate I, which comprises the following specific steps: using a compound II as a starting material, subjecting the compound II to a reduction reaction under the action of a catalyst to generate a compound III, subjecting the compound III to the action of a monoamine oxidase to obtain a compound IV, and then subjecting the compound IV to reduction under the action of an imine reductase to generate a chiral compound V, and then protecting the amino group of the compound V to obtain a compound VI, and then subjecting the compound VI to oxidation of a hydroxyl group to oxidation of a compound VII under the action of an oxidant, and finally subjecting the compound I to the action of a transaminase.

[0025] Furthermore, the monoamine oxidase is derived from Brevibacterium oxydans, Aspergillus niger, Pseudomonas monteilii, Pseudomonas putida, Pseudomonas aeruginosa or homologs thereof.

[0026] Furthermore, the monoamine oxidase participates in the catalytic reaction in the form of monoamine oxidase enzyme powder, monoamine oxidase enzyme solution, monoamine oxidase homogenate solution, cells containing monoamine oxidase, etc.

[0027] Furthermore, the cells containing monoamine oxidase are selected from genetically engineered Escherichia coli or yeast.

[0028] Furthermore, the concentration of the monoamine oxidase powder is 1-10 g / L or the concentration of the cells containing monoamine oxidase is 10-100 g / L.

[0029] Furthermore, the monoamine oxidase is selected from monoamine oxidases ES-MAO-101 to ES-MAO-106 sold by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

[0030] Furthermore, the imine reductase is derived from Bacillus, Pseudomonas, Streptomyces, Citrobacter amalonaticus, Sciscionella marin, Sandaracinus amylolyticus or homologs thereof.

[0031] Furthermore, the imine reductase participates in the catalytic reaction in the form of imine reductase enzyme powder, imine reductase enzyme solution, imine reductase homogenate solution, cells containing imine reductase, etc.

[0032] Furthermore, the cells containing imine reductase are selected from genetically engineered Escherichia coli or yeast.

[0033] Furthermore, the concentration of the imine reductase powder is 1-10 g / L or the concentration of the cells containing the imine reductase is 10-100 g / L.

[0034] Furthermore, the imine reductase is selected from the imine reductases ES-IRED-101 to ES-IRED-115 sold by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

[0035] Furthermore, coenzymes can be added to the reaction system to promote the reaction. When cells containing imine reductase are used, a small amount of coenzymes are contained in the cells, and the coenzymes may not be added at this time. In some cases, the prepared imine reductase powder also contains a small amount of coenzymes, and the coenzymes may not be added at this time. However, coenzymes can also be added to the reaction system to further promote the reaction. When coenzymes are added to the reaction system to promote the reaction, the coenzymes are selected from NAD + , NADH, NADP + , NADPH or a combination thereof, preferably NADP + .

[0036] Furthermore, the concentration of the added coenzyme is 0.02 to 0.4 g / L, preferably 0.1 to 0.2 g / L.

[0037] Furthermore, the coenzymes used in this technical solution are all selected from the coenzyme products sold by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

[0038] Furthermore, the transaminase is derived from Chromobaterium violaceum, Aspergillus terreus or a homolog thereof.

[0039] Furthermore, the transaminase participates in the catalytic reaction in the form of transaminase powder, transaminase solution, transaminase homogenate solution, cells containing transaminase, etc.

[0040] Furthermore, the cells containing transaminase are selected from genetically engineered Escherichia coli or yeast.

[0041] Furthermore, the concentration of the transaminase powder is 1-10 g / L or the concentration of the cells containing the transaminase is 10-100 g / L.

[0042] Furthermore, the transaminase is selected from the transaminase ES-ATA-101 to ES-ATA-166 sold by Shangke Biopharmaceutical (Shanghai) Co., Ltd.

[0043] The beneficial effect of the present invention is that the present invention provides a method for preparing the intermediate I of rituximab by chemical-enzymatic method, wherein the method uses compound II as a starting material, first reduces it to compound III, then converts it into compound VI under the action of monoamine oxidase, imine reductase and amino protecting group, then oxidizes alcohol to ketone to generate compound VII, and finally obtains compound I under the action of transaminase, and the chiral purity is ≥99%. The route is simple to operate, has high atom economy, does not require splitting, and is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] none DETAILED DESCRIPTION

[0045] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0046] Example 1 Preparation of Compound III

[0047]

[0048] Compound II (100.0 g, 1.0 eq), water (50 mL, 5 v / w) and Ru / C (5%) (10.0 g, 10% w / w) were added to the reactor, and the mixture was replaced with N2 for 3 times (0.5 MPa) and H2 for 3 times (0.9 MPa). The H2 pressure was increased to 3.50 MPa, and the temperature was increased to 110°C. The reaction was stirred for 10 hours until the reaction was completed. The reaction system was cooled to room temperature, filtered, and the filter cake was rinsed with water (50 mL, 0.5 v / w). The mixture was concentrated under reduced pressure in a 65°C water bath and distilled to obtain 90 g of compound III with a yield of 85.3%.

[0049] Example 2 Preparation of Compound IV

[0050]

[0051] To a 2L reaction bottle, add MAO enzyme solution (100.00 g, 1.0 w / w, ES-MAO-105, Shangke Biopharmaceutical (Shanghai) Co., Ltd.), pH = 7.0 phosphate buffer (700 mL, 7 v / w), and compound III (100.00 g) in sequence. After stirring evenly, adjust the pH of the reaction system to 7.4 with 3 mol / L sodium hydroxide aqueous solution, and then insert an air bubbler into the reaction system for bubbling. Stir at room temperature for 48 hours until the reaction is completed. Finally, heat to 60 ° C to inactivate MAO enzyme, stir for 3 hours, then cool to room temperature, and the reaction solution directly proceeds to the next step.

[0052] Example 3 Preparation of Compound V

[0053]

[0054] The pH of the reaction solution of compound IV obtained in Example 2 was adjusted to 7.2 with hydrochloric acid, and then 187 g of glucose (1.2 eq), 0.1 g of NADP, 20 mL of GDH enzyme solution (ES-GDH-109) and 50 g of IRED enzyme solution (ES-IRED-106) were added. The reaction was carried out at 30±2°C for 36 h. During the reaction, the pH value was adjusted to 7.2±1 with 10% sodium carbonate. The reaction was complete after monitoring by TLC, and the reaction was centrifuged and ultrafiltered. The filtrate was collected and used directly in the next reaction.

[0055] Example 4 Preparation of Compound VIa

[0056]

[0057] To the filtrate of compound V obtained in Example 3, di-tert-butyl dicarbonate (208.0 g, 1.1 eq) was added, and the reaction was stirred at room temperature for 12 h until the reaction was completed. Then, it was extracted three times with ethyl acetate, and the organic phases were combined and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized from n-heptane and ethyl acetate to precipitate a large amount of solid, which was filtered. The filter cake was washed twice with n-heptane, and the obtained solid was dried in vacuo at 50 ° C. Finally, 121 g of compound VIa was obtained, and the reaction yield was 65%, the purity was >97%, and the chiral purity was >99.0%.

[0058] Example 5 Preparation of Compound VIIa

[0059]

[0060] Into a three-necked flask, 100 g of compound VIa, 500 mL of dichloromethane, 500 mL of water, and 5 g of 2,2,6,6-tetramethylpiperidine-N-oxide were added in sequence, the pH value was adjusted to 9.0 with sodium hydroxide, and the mixture was stirred at room temperature for 12 h. During the stirring process, 380 mL of 10% sodium hypochlorite solution was slowly added dropwise. After TLC monitoring showed that the substrate disappeared, stirring was stopped, the dichloromethane layer was separated, and concentrated under reduced pressure. The obtained reactant was directly used in the next reaction.

[0061] Example 6 Preparation of Compound Ia

[0062]

[0063] Into a three-necked flask, the crude compound VIIa in Example 5, 500 mL 0.1 M phosphate buffer (pH = 7.0), 50 mL DMSO, 0.6 g pyridoxal phosphate, 192 g isopropylamine (7 eq), and 50 g ATA enzyme solution (ES-ATA-107) were added in sequence. After stirring evenly, HCl solution was slowly added dropwise to adjust the pH of the reaction solution to 7.5. The reaction was terminated after 36 h. The pH was adjusted to 2-3 with 3N hydrochloric acid solution, filtered, and the aqueous phase was adjusted to pH 9-10 with 6N sodium hydroxide solution. Ethyl acetate was added and extracted three times. The organic layers were combined and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized from n-heptane and ethyl acetate to precipitate a large amount of solid, which was filtered. The filter cake was washed twice with n-heptane and the obtained solid was dried in vacuo at 50°C to finally obtain 70 g of compound Ia with a reaction yield of 70%, a purity of >98%, and a chiral purity of >99.0%.

[0064] Example 7 Preparation of Compound VIb

[0065]

[0066] To the filtrate of compound V in Example 3, sodium bicarbonate (273.0 g, 7.0 eq) and benzyl chloroformate (118.6 g, 1.5 eq) were added and stirred at room temperature for 2 h. After the reaction was completed, the mixture was extracted three times with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized from n-heptane and ethyl acetate to precipitate a large amount of solid. The product was filtered and the filter cake was washed twice with n-heptane. The obtained solid was dried in vacuo at 50 ° C to finally obtain 151 g of compound VIb with a reaction yield of 70%, a purity of >98%, and a chiral purity of >99.3%.

[0067] Example 8 Preparation of Compound VIIb

[0068]

[0069] To a three-necked flask, 115 g of compound VIb, 500 mL of dichloromethane, 500 mL of water, and 5 g of 2,2,6,6-tetramethylpiperidine-N-oxide were added in sequence, the pH value was adjusted to 9.0 with sodium hydroxide, and the mixture was stirred at room temperature for 12 h. During the stirring process, 380 mL of 10% sodium hypochlorite solution was slowly added dropwise. After TLC monitoring showed that the substrate disappeared, stirring was stopped, the dichloromethane layer was separated, and concentrated under reduced pressure. The reactant obtained was directly used in the next reaction.

[0070] Example 9 Preparation of Compound Ib

[0071]

[0072] Into a three-necked flask, the crude compound VIIb in Example 8, 500 mL 0.1 M phosphate buffer (pH = 7.0), 50 mL DMSO, 0.6 g pyridoxal phosphate, 192 g isopropylamine (7 eq), and 5 g ATA enzyme powder (ES-ATA-153) were added in sequence. After stirring evenly, HCl solution was slowly added dropwise to adjust the pH of the reaction solution to 7.5. The reaction was terminated after 36 h. The pH was adjusted to 2-3 with 3N hydrochloric acid solution, filtered, and the aqueous phase was adjusted to pH 9-10 with 6N sodium hydroxide solution. Ethyl acetate was added and extracted three times. The organic layers were combined and concentrated under reduced pressure to obtain a crude product. The crude product was recrystallized from n-heptane and ethyl acetate to precipitate a large amount of solid, which was filtered. The filter cake was washed twice with n-heptane and the obtained solid was dried in vacuo at 50°C to finally obtain 87 g of compound Ib with a reaction yield of 75%, a purity of >97%, and a chiral purity of >99.4%.

Claims

1. The present invention discloses a method for preparing a rituximab intermediate I by a chemical-enzymatic method, characterized in that: The method uses compound II as a starting material, performs hydrogenation reduction to obtain compound III, compound III generates compound IV under the action of monoamine oxidase, and then compound IV is reduced under the action of imine reductase to obtain compound V, and then the amino group of compound V is protected to obtain compound VI, and then compound VI is oxidized to obtain compound VII by hydroxyl group under the action of an oxidant, and finally compound VII is treated with transaminase to obtain rituximab intermediate I. Wherein, R is selected from tert-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

2. The method according to claim 1, characterized in that The monoamine oxidase is derived from ES-MAO-101 to ES-MAO-106 produced by Shangke Biopharmaceuticals (Shanghai) Co., Ltd.

3. The method according to claim 1, characterized in that The imine reductase is selected from the imine reductase ES-IRED-101 to ES-IRED-115 produced by Shangke Biopharmaceutical (Shanghai) Co., Ltd.

4. The method according to claim 1, characterized in that The transaminase is derived from ES-ATA-101 to ES-ATA-166 of Shangke Biopharmaceutical (Shanghai) Co., Ltd.

5. The method according to claim 1, characterized in that The monoamine oxidase, imine reductase and transaminase all participate in the catalytic reaction in the form of enzyme powder, enzyme solution, homogenized solution, cells containing enzymes and the like.

6. The method according to claim 1, characterized in that The monoamine oxidase, imine reductase and transaminase are selected from genetically engineered Escherichia coli or yeast.

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