阅读说明：本技术 一种化学酶法合成n-[(3r,6r)-6-甲基哌啶-3-基]氨基甲酸叔丁酯的方法 (Method for synthesizing N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester by chemoenzymatic method ) 是由 李彤 李杉 张雷 于 2021-01-18 设计创作，主要内容包括：本发明涉及一种化学酶法合成N-[(3R,6R)-6-甲基哌啶-3-基]氨基甲酸叔丁酯的方法,包括以下步骤：S1、以Boc-D-焦谷氨酸乙酯为底物,在-10～0℃下与甲基卤化镁反应得到酶催化底物；S2、将酶催化底物加入含有氨基供体的磷酸钾缓冲液中,再加入磷酸吡哆醛和转氨酶,通过自发原位环化反应得到双手性中心的内酰胺；S3、使内酰胺发生还原反应,得到N-[(3R,6R)-6-甲基哌啶-3-基]氨基甲酸叔丁酯；本发明以Boc-D-焦谷氨酸乙酯为原料,减少了反应步骤,反应条件更加温和,无需昂贵的金属催化剂和高压条件,反应过程更加安全,同时避免了复杂的消旋体拆分工艺过程,降低了生产成本。(The invention relates to a method for synthesizing N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester by a chemoenzymatic method, which comprises the following steps: s1, reacting Boc-D-pyroglutamic acid ethyl ester serving as a substrate with methyl magnesium halide at the temperature of-10-0 ℃ to obtain an enzyme catalysis substrate; s2, adding an enzyme catalysis substrate into potassium phosphate buffer solution containing an amino donor, adding pyridoxal phosphate and transaminase, and performing spontaneous in-situ cyclization reaction to obtain the lactam of the bimanual center; s3, carrying out reduction reaction on lactam to obtain N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester; the invention takes Boc-D-pyroglutamic acid ethyl ester as a raw material, reduces reaction steps, has milder reaction conditions, does not need expensive metal catalysts and high pressure conditions, has safer reaction process, avoids complex racemate resolution process and reduces production cost.)

1. A method for synthesizing N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester by a chemoenzymatic method is characterized by comprising the following steps:

s1, reacting Boc-D-pyroglutamic acid ethyl ester serving as a substrate with methyl magnesium halide at the temperature of-10-0 ℃ to obtain an enzyme catalysis substrate;

s2, adding an enzyme catalysis substrate into potassium phosphate buffer solution containing an amino donor, adding pyridoxal phosphate and transaminase, and performing spontaneous in-situ cyclization reaction to obtain the lactam of the bimanual center;

s3, carrying out reduction reaction on lactam to obtain N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester;

wherein the structural formula of the enzyme catalysis substrate isThe structural formula of the lactam is

2. The chemoenzymatic synthesis method of tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate according to claim 1, wherein the step S1 comprises the following steps: adding Boc-D-pyroglutamic acid ethyl ester into a reaction vessel filled with a first solvent, introducing inert gas for protection, adding methyl magnesium halide at the temperature of-10-0 ℃, uniformly stirring, heating to 5-10 ℃, quenching with a quenching agent after complete reaction, adding an extracting agent for extraction, and pulping with n-hexane to obtain the enzyme catalysis substrate.

3. The chemoenzymatic synthesis method of tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate according to claim 2, wherein the first solvent is at least one of dimethyltetrahydrofuran, tetrahydrofuran and anhydrous tetrahydrofuran.

4. The chemoenzymatic synthesis method of N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamic acid tert-butyl ester according to claim 1 or 2, wherein the feeding molar ratio of Boc-D-pyroglutamic acid ethyl ester to methylmagnesium halide is 1: 1.3-1.8; the methyl magnesium halide is at least one of methyl magnesium bromide, methyl magnesium chloride and methyl magnesium iodide.

5. The chemoenzymatic synthesis method of tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate according to claim 1, wherein in step S2, the reaction temperature is 35-50 ℃ and the reaction time is 12-16 h.

6. The chemoenzymatic synthesis method of tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate according to claim 1, wherein in step S2, the transaminase is SCUT-ATA-01, and the amino donor is isopropylamine; the content of isopropylamine in the potassium phosphate buffer is 1M.

7. The chemoenzymatic synthesis method of tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate according to claim 1, wherein the reduction reaction in step S3 comprises the following steps: dissolving lactam in a second solvent, adding sodium borohydride in batches at 0-10 ℃, adding a boron trifluoride tetrahydrofuran solution, heating to 20-25 ℃, quenching with a quenching agent after reaction, heating for refluxing, concentrating under reduced pressure, extracting with an extractant, and spin-drying an organic phase to obtain the N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamic acid tert-butyl ester.

8. The chemoenzymatic synthesis method of N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamic acid tert-butyl ester according to claim 7, wherein the feeding molar ratio of the lactam, the sodium borohydride and the boron trifluoride tetrahydrofuran is 1: 2-3.5: 1 to 3.5.

9. The chemoenzymatic synthesis method of tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate according to claim 7, wherein the second solvent is anhydrous tetrahydrofuran.

10. The chemoenzymatic synthesis method of tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate according to claim 2 or 7, wherein the quenching agent is at least one of methanol and a saturated ammonium chloride solution, and the extraction agent is ethyl acetate.

Technical Field

The invention relates to the technical field of chiral drug intermediate synthesis, in particular to a method for synthesizing N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester by a chemoenzymatic method.

Background

Orexin receptor antagonists are useful for the treatment of adult insomnia (insomnia), and IRAK4 inhibitors can be useful for the treatment of various inflammation-related diseases; and chiral piperidine amine compound: the N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamic acid tert-butyl ester has important production significance as an important intermediate for synthesizing an Orexin (Orexin) receptor antagonist and an IRAK4 inhibitor.

The currently available synthetic routes for tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate include the following:

patent WO2010048012A discloses a method for synthesizing N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester, and the specific synthetic route is as follows:

the route takes 5-amino-2-methylpyridine as a raw material, protects amino through Boc anhydride, reduces pyridine ring through rhodium catalysis, protects amino through benzyl, separates diastereoisomers through column chromatography, and finally uses palladium hydroxide and hydrogen as catalysts to remove benzyl to obtain a target product. However, the above synthetic route requires an expensive rhodium catalyst and high pressure conditions for hydrogenation, which is costly, and the yield of enantiomer separated by column chromatography is low and cannot be scaled up for industrial production.

Patent WO2015095442A discloses a method for synthesizing N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester, and the specific synthetic route is as follows:

according to the route, (2R,5S) -5-hydroxy-2-methylpiperidine-1-benzyl carboxylate is used as a raw material, ester is formed with methylsulfonyl chloride firstly, sodium azide is used for substitution to generate configuration inversion, then trimethylphosphine is used for reduction to obtain amine, Boc anhydride is used for protecting amino, and palladium carbon and hydrogen are used for removing a protecting group Cbz to obtain a target product. The synthetic route has long steps, needs to use sodium azide, is extremely toxic, has explosive danger, is difficult to synthesize the initial raw materials, needs to be synthesized in multiple steps and is obtained by utilizing chiral SFC for resolution, and has high production cost.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a method for synthesizing N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester by a chemoenzymatic method, which takes Boc-D-pyroglutamic acid ethyl ester as a raw material, reduces reaction steps, has milder reaction conditions, does not need expensive metal catalysts and high pressure conditions, has safer reaction process, avoids complex racemate resolution process and reduces production cost.

The technical purpose of the invention is realized by the following technical scheme: a method for synthesizing N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamic acid tert-butyl ester by a chemoenzymatic method comprises the following steps:

s1, reacting Boc-D-pyroglutamic acid ethyl ester serving as a substrate with methyl magnesium halide at the temperature of-10-0 ℃ to obtain an enzyme catalysis substrate;

s2, adding an enzyme catalysis substrate into potassium phosphate buffer solution containing an amino donor, adding pyridoxal phosphate and transaminase, and performing spontaneous in-situ cyclization reaction to obtain the lactam of the bimanual center;

s3, carrying out reduction reaction on lactam to obtain N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester;

wherein the structural formula of the enzyme catalysis substrate isThe structural formula of the lactam is

Further, the step S1 includes the following steps: adding Boc-D-pyroglutamic acid ethyl ester into a reaction vessel filled with a first solvent, introducing inert gas for protection, adding methyl magnesium halide at the temperature of-10-0 ℃, uniformly stirring, heating to 5-10 ℃, quenching with a quenching agent after complete reaction, adding an extracting agent for extraction, and pulping with n-hexane to obtain the enzyme catalysis substrate.

Further, the first solvent is at least one of dimethyltetrahydrofuran, tetrahydrofuran and anhydrous tetrahydrofuran.

Further, the first solvent is anhydrous tetrahydrofuran.

Further, the feeding molar ratio of the Boc-D-pyroglutamic acid ethyl ester to the methyl magnesium halide is 1: 1.3-1.8; the methyl magnesium halide is at least one of methyl magnesium bromide, methyl magnesium chloride and methyl magnesium iodide.

Further, in the step S2, the reaction temperature is 35-50 ℃, and the reaction time is 12-16 h.

Further, in step S2, the transaminase is SCUT-ATA-01, and the amino donor is isopropylamine; the content of isopropylamine in the potassium phosphate buffer is 1M.

Further, in step S3, the reduction reaction includes the following steps: dissolving lactam in a second solvent, adding sodium borohydride in batches at 0-10 ℃, adding a boron trifluoride tetrahydrofuran solution, heating to 20-25 ℃, quenching with a quenching agent after reaction, heating for refluxing, concentrating under reduced pressure, extracting with an extractant, and spin-drying an organic phase to obtain the N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamic acid tert-butyl ester.

Further, the feeding molar ratio of the lactam to the sodium borohydride to the boron trifluoride tetrahydrofuran is 1: 2-3.5: 1 to 3.5.

Further, the inert gas is nitrogen.

Further, the second solvent is anhydrous tetrahydrofuran.

Further, the quenching agent is at least one of methanol and saturated ammonium chloride solution, and the extracting agent is ethyl acetate.

In conclusion, the invention has the following beneficial effects:

1. according to the synthesis method, Boc-D-pyroglutamic acid ethyl ester is used as a raw material, reaction steps are reduced, the reaction condition is mild, high-pressure hydrogenation and expensive metal catalysts are not needed, the safety of the reaction process is high, the N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester with higher purity and ee value of more than 99.9% can be obtained without a complex racemate resolution process, and the synthesis method has a good industrial synthesis application prospect.

2. In step S2, the transaminase firstly converts the ketocarbonyl of the enzyme catalysis substrate into chiral amino, and the generated amino intermediate undergoes intramolecular cyclization to generate the target product lactam; meanwhile, the investment of reaction equipment is reduced, and the production cost is reduced.

3. Because the reaction of the sodium borohydride is more violent, the reaction temperature can be easily controlled and the phenomenon of material flushing can be prevented by adding the sodium borohydride in batches, and the safety performance is higher.

Drawings

FIG. 1 is a graph showing the relationship between the content of isopropylamine in potassium phosphate buffer and the conversion of lactam;

FIG. 2 is a graph of the catalytic reaction time of the transaminase as a function of the conversion of lactam.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

Unless defined otherwise, 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 belongs, and the terms used herein in the specification of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention.

Example 1:

the method for synthesizing tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate by using the chemoenzymatic method provided in this embodiment comprises the following steps:

s1, preparation of enzyme catalysis substrate: dissolving 60g of Boc-D-pyroglutamic acid ethyl ester in 1000mL of anhydrous tetrahydrofuran, replacing 3 times with nitrogen, then cooling to-10-0 ℃, dropwise adding 300mL of methyl magnesium halide tetrahydrofuran solution, continuously stirring for 1h at-10-0 ℃ after dropwise adding, and then heating to 5-10 ℃ for continuous reaction for 6 h. Quenching with saturated ammonium chloride solution after reaction, extracting with ethyl acetate for 3 times, and mixingDrying the organic phase by using anhydrous sodium sulfate, concentrating the organic phase to obtain a crude product, pulping the crude product by using n-hexane, filtering and drying to obtain 54.17g of enzyme catalysis substrate; wherein the structural formula of the enzyme catalysis substrate isIn step S1, the resulting enzyme-catalyzed substrate has a purity of 99.99% and a yield of about 85%.

S2, preparing lactam by transaminase catalysis: preparing 10mL of potassium phosphate buffer solution containing 1M isopropylamine, adjusting the pH value of the potassium phosphate buffer solution to 11 by using a hydrochloric acid aqueous solution, then adding 1g of enzyme catalysis substrate, adding 500mg of transaminase powder and 20mg of pyridoxal phosphate, stirring and reacting at the temperature of 35-50 ℃ for 12-16 h, filtering after the reaction is finished, extracting the mother solution by using ethyl acetate for 3 times, combining organic phases, concentrating and drying to obtain a product lactam; wherein the structural formula of the lactam is shown asIn step S2, the lactam conversion was 98%, the ee value was 99.9%, and the yield was about 90%.

Preparation of S3, tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate: dissolving 0.5g of lactam in anhydrous tetrahydrofuran, adding sodium borohydride in batches for multiple times at 0-10 ℃, stirring and reacting for 0.5h, dropwise adding a boron trifluoride tetrahydrofuran solution, heating to 20-25 ℃ after dropwise adding, stirring for 12h, quenching with methanol after reaction, heating and refluxing for 1-2 h, concentrating under reduced pressure, extracting for 3 times with ethyl acetate, and spin-drying an organic phase to obtain N- [ (3R,6R) -6-methylpiperidin-3-yl ] tert-butyl carbamate; of these, tert-butyl N- [ (3R,6R) -6-methylpiperidin-3-yl ] carbamate was obtained in a yield of about 87% and an ee value of 99.9%.

The synthetic route of this example is as follows:

in example 1, the methyl magnesium halide may also be one of methyl magnesium chloride or methyl magnesium iodide, or a mixture of any two or any three of methyl magnesium bromide, methyl magnesium chloride and methyl magnesium iodide.

Examples 2 to 6:

to further verify the influence of the type of the tryptophan on the conversion rate of lactam and the ee value in step S2, the synthesis method of example 1 was repeated according to the type of the tryptophan in Table 1, and the components, contents and reaction conditions not shown were exactly the same as those in example 1, to obtain the corresponding lactams, respectively, and the conversion rate of lactam and the ee value were calculated.

Table 1:

as can be seen from the data in Table 1, the conversion and ee value of the lactam obtained by using different types of trans-amino acids are different, wherein when the trans-amino acid is SCUT-ATA-01, the conversion of the lactam is as high as 99.91%, and the ee value is more than 99.9%.

In order to further verify the content of isopropylamine in potassium phosphate buffer and the optimal choice of reaction time in the transaminase catalytic reaction, the invention also provides the following experiments:

the synthesis process of example 2 was repeated according to the isopropylamine content in potassium phosphate buffer in Table 2, and the other non-listed components, contents and reaction conditions were exactly the same as those of example 2, to obtain lactams under different reaction conditions and calculate the corresponding conversion, the results of which are shown in FIG. 1.

Table 2:

content (M) of isopropylamine in Potassium phosphate buffer

0.5

1.0

1.5

2.0

The synthesis process of example 2 was repeated according to the reaction time of the catalytic reaction in Table 3, and other ingredients, contents and reaction conditions not listed were exactly the same as those of example 2, to obtain lactams under different reaction conditions and calculate the corresponding conversion, and the results are shown in FIG. 2.

Table 3:

reaction time (h)

2

4

6

8

10

16

24

From the results of FIGS. 1 and 2, it can be seen that the content of isopropylamine in the potassium phosphate buffer and the reaction time of the catalytic reaction have various effects on the conversion of lactam. Wherein, when the content of the isopropylamine in the potassium phosphate buffer solution is 1M, the conversion rate of the lactam is the highest, and the experimental result has no regular change. The reaction time of the catalytic reaction is 8h, namely the reaction is basically completed.

In conclusion, the Boc-D-pyroglutamic acid ethyl ester is adopted as the raw material, so that the reaction steps are reduced, compared with the prior art, the reaction conditions are mild, high-pressure hydrogenation and expensive metal catalysts are not needed, the safety of the reaction process is high, and the N- [ (3R,6R) -6-methylpiperidine-3-yl ] carbamic acid tert-butyl ester with higher purity and ee value of over 99.9 percent can be obtained without a complex racemate resolution process, wherein in the transaminase catalytic reaction, the transaminase can firstly convert the ketocarbonyl of an enzymatic substrate into chiral amino, and the generated amino intermediate can generate the target product lactam by self-intramolecular cyclization; meanwhile, the investment of reaction equipment is reduced, and the production cost is reduced.

The technical contents of the present invention are further illustrated by the examples, so as to facilitate the understanding of the reader, but the embodiments of the present invention are not limited thereto, and any technical extension or re-creation based on the present invention is protected by the present invention.