CN113930404B - Method for synthesizing chiral tofacitinib citrate intermediate by enzymatic method - Google Patents
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Abstract
The invention discloses a method for synthesizing chiral tofacitinib citrate intermediate by an enzymatic method, which takes 4-methyl-1- (phenylmethyl) -3-piperidone (TOF 15) as a raw material, chiral intermediate TOF20-A is obtained through biocatalysis, and methylation reaction is carried out to obtain intermediate TOF25-A, namely free (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine, wherein the intermediate TOF25-A can be further subjected to hydrochloride formation to obtain (3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride (TOF 30). The invention is a synthesis technology with low cost, high yield and environmental friendliness, is suitable for large-scale industrial production, and has huge market application value.
Description
Technical Field
The invention relates to the technical field of enzyme catalysis, in particular to a transaminase catalyst, and also relates to a method for synthesizing a chiral tofacitinib citrate intermediate ((3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride) by an enzyme method and a production method of the chiral tofacitinib citrate intermediate ((3R, 4R) -1-benzyl-N, 4-dimethylpiperidine-3-amine dihydrochloride).
Background
(3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride is an important pharmaceutical and organic synthetic intermediate, in particular for synthesizing tofacitinib citrate.
Tofacitinib citrate, developed by the american pyrotechnical company, is a JAK pathway inhibitor of the first mechanism of action, and is a novel oral protein tyrosine kinase inhibitor. The product was first approved for sale in the united states in 2012, and has been approved for sale in more than 50 countries and regions worldwide, such as the united states, japan, china, russia, australia, canada, etc.
The existing synthetic methods of (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride are chemical synthesis methods, TOF20 is obtained from 4-methyl-1- (phenylmethyl) -3-piperidone (TOF 15) through aminomethylation, TOF20 is a mixed rotator, chiral TOF25 can be obtained through L-DTTA salifying resolution, and TOF25 is free and salified, so that (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride (TOF 30) is obtained through the following synthetic route:
however, the existing chemical synthesis method involves more reagents, is complex to operate, has complicated procedures, particularly, 50% of the final resolved product is discarded, resulting in low yield and particularly poor atomic economy, thus having high cost and failing to obtain the target product with high optical purity.
Disclosure of Invention
Aiming at various technical defects existing in the prior art, the invention aims to provide a brand-new enzymatic synthesis route which can efficiently prepare a target product- (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride (TOF 30), and the ee value of the product is up to more than 99.7%.
In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:
the first aspect of the invention provides a transaminase catalyst, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of which is shown in SEQ ID NO.2, see in particular Table 1 below:
TABLE 1 nucleotide and amino acid sequences of transaminase catalysts
In a second aspect, the invention provides a method for enzymatic synthesis of (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine or dihydrochloride thereof (TOF 30):
1) In the presence of a transaminase catalyst, the reaction substrate TOF15 reacts with an amino donor to generate chiral TOF20-A,
2) The TOF20-A is subjected to methylation reaction with a methylation reagent, and an acid binding agent is added to obtain TOF25-A, namely free (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine, or TOF25-A is further subjected to hydrochloride formation to obtain (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride (TOF 30);
the nucleotide sequence of the aminotransferase catalyst is shown as SEQ ID NO.1, and the amino acid sequence of the aminotransferase catalyst is shown as SEQ ID NO. 2.
The reaction equation is as follows:
preferably, in the above method for synthesizing TOF20-A by enzymatic method, the amino donor is selected from any one of isopropylamine, tert-butylamine, alanine and leucine; further preferably, the amino donor is isopropylamine. The solvent is a mixed solvent of water and an organic solvent or water, the organic solvent is dimethyl sulfoxide, tetrahydrofuran or DMF, the pH value of the transaminase reaction is 7-10, and the reaction temperature of the transaminase reaction is 10-50 ℃; further preferably, the solvent is a mixed solvent of water and dimethyl sulfoxide, the pH value of the transaminase reaction is 7-8, and the reaction temperature of the transaminase reaction is 15-25 ℃.
In the synthetic method of TOF20-A to TOF25-A, the methylation reagent used in the methylation reaction is selected from any one of methyl iodide and dimethyl sulfate; the acid binding agent is selected from any one of potassium carbonate, sodium carbonate and sodium bicarbonate; the reaction solvent is selected from any one of dichloromethane, ethyl acetate, ethanol and methanol; further preferably, the methylation reagent is methyl iodide, the acid binding agent is potassium carbonate, the reaction solvent is methylene dichloride, and the reaction temperature is 15-25 ℃.
The invention takes 4-methyl-1- (phenylmethyl) -3-piperidone (TOF 15) as a raw material, chiral intermediate TOF20-A is obtained through biocatalysis, and methylation reaction is carried out to obtain intermediate TOF25-A, namely free (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine, or further hydrochloride is formed to obtain (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride (TOF 30). The invention is a synthesis technology with low cost, high yield and environmental friendliness, is suitable for large-scale industrial production, and has huge market application value.
Detailed Description
The invention is further illustrated and described below in connection with specific embodiments. The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict.
EXAMPLE one preparation of transaminase bacteria lyophilized powder
The synthesized aminotransferase catalyst gene DNA fragment is digested for 6 hours with restriction enzymes NdeI and avrII at 35-37 ℃, purified by agarose gel electrophoresis, and the target fragment (SEQ ID NO. 1) is recovered by using an agarose gel DNA recovery kit; then, the target fragment is connected with plasmid pACYCDuet-B which is also digested by NdeI and EcoRI under the action of T4DNA ligase, and the temperature is 25-27 ℃ overnight to obtain recombinant expression plasmid; transforming the recombinant expression plasmid into competent cells of Escherichia coli under the following conditions: and (3) performing heat shock for 80-90 seconds at the temperature of 40-45 ℃, screening positive recombinant on a resistance plate containing chloramphenicol, selecting monoclonal, culturing recombinant bacteria, extracting plasmids after plasmid amplification, re-transforming the plasmids into competent cells, coating a transforming solution on the LB plate containing chloramphenicol resistance, and culturing overnight at the temperature of 35-37 ℃ to obtain the positive recombinant transformant (namely recombinant escherichia coli). Inoculating the recombinant escherichia coli into LB solid medium containing chloramphenicol resistance, and culturing at 35-37 ℃ for 18-20h; single colony is selected and inoculated in 100mL LB liquid culture medium containing chloramphenicol resistance, shake culture is carried out for 18-20h, bacterial liquid is removed and inoculated in 500mL TB liquid culture medium after culture is finished, 0.1mM IPTG is added to induce protein expression after 2.5h of culture, shake culture is carried out for 18-20h at 28-30 ℃, thalli is collected by centrifugation at 10000rpm, and then the thalli is frozen and dried to obtain thalli freeze-dried powder containing transaminase, and the thalli is preserved at-70 ℃ for standby.
EXAMPLE two TOF20-A preparation
100g of isopropylamine is dissolved in 100ml of water, the pH value is regulated to 7.0-8.0 by using hydrochloric acid aqueous solution under ice water bath cooling, 10ml of dimethyl sulfoxide is added, then the mixture is diluted to 700ml by using 0.1M Tris-HCl buffer solution, the mixture is preheated to 30 ℃, then 100ml of dimethyl sulfoxide solution containing 50g of 4-methyl-1- (phenylmethyl) -3-piperidone (TOF 15) is added, finally 1g of transaminase bacterial lyophilized powder and 0.8g of PLP (pyridoxal phosphate) are added, the pH value is controlled to 7.0-8.0 by using 20% isopropylamine aqueous solution, the reaction is converted for more than 12 hours at 15-25 ℃, and TLC monitors the completion of the reaction. The solids were removed by filtration, the mother liquor was extracted 3 times with dichloromethane and the combined organic phases were dried over anhydrous sodium sulfate and concentrated to give TOF 20-A45 g as an oil in a yield of about 90%.
Example preparation of tris (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine (TOF 25-A)
TOF 20-A10 g (about 50 mmol) as an oil in example one was dissolved in 100ml of methylene chloride, 7g (about 50 mmol) of powdered potassium carbonate was added, the temperature was lowered to 0-5℃under ice-water bath cooling, methyl iodide (7.1 g, about 50 mmol) was added dropwise, and after completion of the dropwise addition, the temperature was slowly raised to 15-25℃and the reaction was allowed to stand for 2 hours, whereupon TLC was monitored to complete the reaction. The solid was removed by filtration, the dichloromethane layer was washed 3 times with water, the dichloromethane phase was dried over anhydrous sodium sulfate, and concentrated to give TOF 25-A10 g as an oil, the yield was about 95%.
Example preparation of tetrakis (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride (TOF 30)
10g of TOF25-A as oily matter in the first embodiment is dissolved in 20ml of absolute ethyl alcohol, 10ml of 30% hydrochloric acid ethanol solution is started to be added dropwise at room temperature, white solid is separated out after the dropwise addition is completed, the mixture is stirred for 1 hour at 0-5 ℃, the white solid is obtained after filtration, and about 12g of (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride TOF30 is obtained after drying, and the yield is about 90% and the ee value is 99.8%.
1 H-NMR(400M,D 2 O)δ：7.53(m,5H),4.45(s,2H),3.67(m,1H),3.36-3.67(m,2H), 3.23-3.36(m,2H),2.76(s,3H),2.58(m,1H),1.99(m,2H),1.12(d,3H)。
HRMS(ESI + )m/z calcd for 219.1856；found：[M+H] + 219.1859。
Comprehensive analysis of the experimental results shows that the transaminase catalyst used by the invention has higher catalytic activity, can generate (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride with extremely high optical purity, and is environment-friendly. Therefore, the production method is favorable for realizing the large-scale industrial production of the chiral tofacitinib citrate intermediate, and has huge market application value.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.
Sequence listing
<110> Zhejiang Lepu pharmaceutical Co., ltd
<120> method for synthesizing chiral tofacitinib citrate intermediate by enzymatic method
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 622
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atggcttcga tggacaaagt cttctcaggt tactacgccc gtcaaaaact gctggaacgc 60
tcagataatc cgttctcaaa aggtattgcc tatgtcgaag gctgccgagt aaatatcctg 120
tcatgcgata aaatgcgtct gaaatttccg ctggcactga gctctgtcaa gtaaactggt 180
ctacgacgtt gcagaaatgg tggctaaaag cggcattcgt tcgaagtgat cgttacccgc 240
gatgcgcgca ttccgctgct ggacgaaggc tttatgcata gtgatctgac gacgctttcg 300
ctggtgctgc cgtatatctg ccggaaaatc agctgcatgg cattctggaa cggtgaagct 360
ggcctgacgg ctctaaaccg gaagatctgt ataacaataa catttacctg atctccgtct 420
gggacggccg tttctttcgc ctggatgacc acctgcagcg gtgttcgtgg gctgatggca 480
attatcaccc gtacggtgcg tcgcaccccg ccgggtgcct ttgatccgac gatcaaaaac 540
taccaagaaa atttgggatg gctattggga aatgcactac aacccggctt attcgtttcc 600
ggtggattat ggcagcggtt ag 622
<210> 2
<211> 62
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr Tyr Ala Arg Gln Lys
1 5 10 15
Leu Leu Gln Arg Ser Asp Asn Pro Phe Ser Lys Gly Ala Tyr Val Glu
20 25 30
Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Gly Arg Phe Phe Asn Asp
35 40 45
Gly Gln Val Gly Tyr Ser Phe Pro Val Asp Tyr Gly Ser Gly
50 55 60
Claims (8)
1. A method for synthesizing chiral tofacitinib citrate intermediate by using an enzymatic method is characterized in that:
1) In the presence of a transaminase catalyst, the reaction substrate TOF15 reacts with an amino donor to generate chiral TOF20-A,
2) The TOF20-A is subjected to methylation reaction with a methylation reagent, and an acid binding agent is added to obtain TOF25-A, namely free (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine, or TOF25-A is further subjected to hydrochloride formation to obtain (3R, 4R) -1-benzyl-N, 4-dimethylpiperidin-3-amine dihydrochloride (TOF 30);
wherein the nucleotide sequence of the aminotransferase catalyst is shown as SEQ ID NO.1, and the amino acid sequence of the aminotransferase catalyst is shown as SEQ ID NO. 2;
the reaction equation is as follows:
。
2. the method according to claim 1, characterized in that: the amino donor is one or more of isopropylamine, tert-butylamine, alanine or butylamine.
3. The method according to claim 1, characterized in that: in the step 1), the reaction is carried out in a solvent, wherein the solvent is water, an organic solvent or a mixed solvent of water and a co-solvent, and the organic solvent is one or more of dimethyl sulfoxide, tetrahydrofuran or DMF.
4. The method according to claim 1, characterized in that: the pH value of the reaction system in the step 1) is 7-10, and the reaction temperature is 10-50 ℃.
5. A method according to claim 3, characterized in that: in the step 1), the solvent is a mixed solvent of water and dimethyl sulfoxide, the pH value of a reaction system is 7-8, and the reaction temperature is 15-25 ℃.
6. The method according to claim 1, characterized in that: in the step 2), the methylation reagent used in the methylation reaction is selected from methyl iodide or dimethyl sulfate, and the acid binding agent is one or more of potassium carbonate, sodium carbonate or sodium bicarbonate; the reaction of the step 2) is carried out in a solvent which is one or more of dichloromethane, ethyl acetate, ethanol and methanol.
7. The method according to claim 6, wherein: in the step 2), the methylating agent is methyl iodide, the acid binding agent is potassium carbonate, and the reaction solvent is dichloromethane.
8. The method according to claim 1, characterized in that: in step 2), the methylation reaction temperature is 15-25 ℃.
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| HPLC法枸橼酸托法替布的含量;陈娇婷等;《中国民族民间医药》;第23-24页 * |
| Tofacitinib citrate for ulcerative keratitis in a patient with rheumatoid arthritis;Philip B.Meadow et al.;《Case Rep Rheumatol》;第1-3页 * |
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