Ketoreductase with stereoselective reduction activity and application thereof

Abstract

The invention discloses ketoreductase with stereoselective reduction activity and application of the ketoreductase, and belongs to the technical field of medicines, the amino acid sequence of the ketoreductase is shown as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; according to the method, racemic nebivolol chloroketone is taken as an initiator, an R-configuration ketone substrate is recognized through the ketoreductase and is reduced into optically pure alcohol (R, R-2), meanwhile, a configuration S-configuration substrate is split, and a simple and efficient preparation method with high atom economy is provided for an optically pure intermediate necessary for nebivolol drug synthesis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the synthesis of chiral intermediates for the synthesis of nebivolol raw materials. Specifically, it relates to a method for preparing optically pure 2-chloro-1S-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl) ethyl ketone (S-1) using an enzymatic method, which is then used in the synthesis of nebivolol hydrochloride. Background Technology

[0002] Nebivolol, developed by Johnson & Johnson (USA), was first launched in Germany in May 1997, and subsequently in the UK and several other countries in May 1999. It was approved for marketing in the US on December 17, 2007. Nebivolol is a potent and selective third-generation β-adrenergic receptor blocker, with a blocking power of 290 times that of β2 receptors against β1 receptors. Comparing its selectivity, bisoprolol is 26 times more selective, atenolol 15 times more selective, and propranolol 1.9 times more selective. Therefore, nebivolol exhibits higher selectivity, does not cause bronchial or vascular smooth muscle contraction, and has no endogenous sympathomimetic activity. The chemical structure of nebivolol is as follows:

[0003]

[0004] According to literature [1] The report states that the final three steps of the nebirol synthesis process are shown in the figure below:

[0005]

[0006]

[0007] As can be seen from the above synthetic route, nebivolol is synthesized from four different stereoconfigurations of nebivolol. Therefore, how to obtain optically pure nebivolol with multiple configurations has become the focus of nebivolol synthesis. The preparation of chiral nebivolol has been attempted in various ways over the past ten years. The main research results in recent years are the preparation of chiral 6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid (nebivolol acid) and chiral 2-chloro-1-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)ethyl-1-one (1).

[0008]

[0009] Zhang Fangjiang et al. [2]A chiral source synthesis method for the asymmetric synthesis of R or S-6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid is reported. The reaction of L-malic acid with methanesulfonyl chloride yields an anhydride, which is then reacted with p-fluorophenol in the presence of Lewis acid. Following this, an intramolecular SN2 substitution reaction occurs in the presence of sodium hydroxide to give chiral cyclized product 3. The carbonyl group in compound 3 is reduced under catalytic hydrogenation to finally obtain the optically pure target product R-6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid, i.e., (R)-nebirolic acid.

[0010]

[0011] Professor Wei Dongzhi's research group at East China University of Science and Technology [3] Based on two esterases, EstS and EstR, isolated from *Geobacillus thermocatenulatus*, a practical enzymatic resolution method for methyl 6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid was proposed. In a water-toluene two-phase system, racemic methyl 6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid was used as a substrate, and (S)- and (R)-6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid were generated by EstS and EstR catalysis, respectively, with ee% >99.0% and 95.0-96.0%, respectively.

[0012] From the perspective of the economy and controllability of pharmaceutical processes, the later the chirality is introduced into the compound structure, the fewer impurities of the corresponding variants are generated during synthesis, and the higher the economy. This invention can support the achievement of this objective, namely, using racemic chloroketone (1) as raw material and utilizing a stereoselective ketone reductase to achieve the resolution of racemic nebirolol chloroketone.

[0013] Summary of the Invention

[0014] In order to overcome the problems existing in the prior art, the present invention provides a method for resolving racemic nebirolol chloroketone (1) using a stereoselective ketone reductase as raw material.

[0015] The present invention provides a ketone reductase, the amino acid sequence of which is shown in SEQ ID NO:1 (ketone reductase 1), SEQ ID NO:2 (ketone reductase 2) or SEQ ID NO:3 (ketone reductase 3).

[0016] Furthermore, the present invention provides that the products of the ketone reductase catalyzed reaction are separated by crystallization and chromatography to obtain optically pure chloroketone (S-1) and optically pure alcohol (R, R-2).

[0017] The synthetic route for the reaction is as follows:

[0018]

[0019] Starting with racemic nebirolol chloroketone (1), nebirolol chloroketone (R-1) was selectively reduced by a ketoreductase that stereoselectively recognizes one of the R-configuration ketone substrates to generate an optically pure alcohol (R,R-2), while 2-chloro-1S-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl) ethyl ketone (S-1) did not react, resulting in resolution.

[0020] Beneficial technical effects of the present invention:

[0021] This invention screened a ketone reductase with high stereoselectivity using racemic nebivolol chloroketone (1). Using this enzyme as a biocatalyst, the R-configuration nebivolol chloroketone can be reduced with high stereoselectivity using racemic nebivolol chloroketone (1) as a substrate, thereby achieving the resolution of the S-configuration nebivolol chloroketone. This provides a new route for the preparation of chiral alcohols, which are essential in the synthesis of nebivolol. Attached Figure Description

[0022] Figure 1 Identification of reactant and product peaks;

[0023] Figure 2 Results of the reaction between crude ketone reductase 1 enzyme solution and racemic chloroketone;

[0024] Figure 3 Results of the reaction between crude ketone reductase 1 and chloroketone (R)-1;

[0025] Figure 4 Results of the reaction between crude ketone reductase 2 solution and racemic chloroketone;

[0026] Figure 5 Results of the reaction between crude ketone reductase 2 solution and chloroketone (R-1);

[0027] Figure 6 Results of the reaction between crude ketone reductase 3 enzyme solution and racemic chloroketone;

[0028] Figure 7 Results of the reaction between crude ketone reductase 3 solution and chloroketone (R-1) ketone;

[0029] Figure 8 The proton NMR spectrum of chloroketone S-1;

[0030] Figure 9 Hydrogen spectroscopy of chloroalcohols R and R-2. Detailed Implementation

[0031] The present invention will be further described below with reference to the embodiments. It should be noted that the following embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0032] Table 1. Enzyme Reaction System

[0033]

[0034] Ethyl acetate or DMSO can be used as a co-solvent.

[0035] Table 2. Chiral HPLC conditions

[0036]

[0037]

[0038] Standardization of reactant and product peaks, as follows: Figure 1 As shown.

[0039] Example 1

[0040] Ketoreductase 1 reaction

[0041] Using the above-described enzyme reaction system, the crude enzyme solution of ketoreductase 1 was reacted with cyclopentyl chloride ketone and R-configuration chloroketone (R-1), respectively, with a conversion rate greater than 95%. The liquid phase results are as follows. Figure 2-3 .

[0042] Example 2

[0043] Ketoreductase 2 reaction

[0044] Using the above-described enzyme reaction system, the crude enzyme solution of ketoreductase 2 was reacted with cyclopentolol chloroketone and R-configuration chloroketone (R-1), respectively, with a conversion rate greater than 98%. The liquid phase results are as follows. Figure 4-5 .

[0045] Example 3

[0046] Ketoreductase 3 reaction

[0047] Using the above-described enzyme reaction system, the crude enzyme solution of ketoreductase 3 was reacted with racemic nebivolol chloroketone and R-configuration chloroketone (R-1), respectively, with a conversion rate greater than 93%. The liquid phase results are as follows. Figure 6-7 .

[0048] Example 4

[0049] After the racemic nebirolol chloroketone substrate in Example 1 above reacted completely (detected by chiral HPLC), the reaction mixture was extracted with ethyl acetate. The extract was concentrated under reduced pressure to obtain a yellow oil. The residue was purified by rapid column chromatography (n-hexane:ethyl acetate = 20:1) to give compound (S-1) as a white solid with a yield of 39.7%, ee > 99%, [α]. 2 D 0 = +22.3° (c = 0.1 in MeOH); Compound (R, R-2) is a pale yellow oil with a yield of 38.4% and ee > 99%.

[0050] S-1: 1 H-NMR (400MHz, CDCl3) δ6.84 (dd, J=6.1, 1.5Hz, 2H), 6.80–6.74 (m, 1H), 4.70–

[0051] 4.65 (m, 1H), 4.65–4.43 (m, 2H), 2.90–2.81 (m, 1H), 2.75 (dt, J = 16.9, 5.4 Hz, 1H), 2.30 (dddd, J = 14.1, 6.1, 5.1, 3.2 Hz, 1H), 2.07 (dtd, J = 13.7, 9.2, 5.7 Hz, 1H). See detailed NMR results. Figure 8 .

[0052] R,R-2: 1 H NMR (400MHz, CDCl3) δ6.87–6.64 (m, 3H), 3.99 (ddd, J = 9.8, 7.1, 2.3Hz, 1H),

[0053] 3.94–3.87 (m, 2H), 3.86–3.79 (m, 1H), 2.81 (qdd, J = 16.8, 6.3, 4.5 Hz, 2H), 2.25 (dddd, J = 13.7, 6.0, 3.6, 2.3 Hz, 1H), 1.83 (dtd, J = 13.6, 10.5, 6.0 Hz, 1H). See detailed NMR results. Figure 9 .

[0054] References

[0055] [1] Luo Kaiwei. Study on the synthesis process of nebivolol hydrochloride. Master's thesis, Jilin University, 2015.

[0056] [2] Zhang Fangjiang, Gan Jiangang. Synthesis of (R) or (S)-6-fluoro-3,4-dihydro-2H-1-benzopyran-2-carboxylic acid [P]. China. CN 102408402A, 2012.04.11.

[0057] [3] Gao Bei, Wei Dongzhi, Wang Fengqing, Jiang Min. A genetically engineered Escherichia coli expressing esterase EstR and its application [P]. China. CN 114149956 A, 2024.04.09.

Claims

1. A ketone reductase with stereoselective reducing activity, characterized in that... The amino acid sequence is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

2. The ketone reductase with selective reducing activity according to claim 1, characterized in that... The ketone reductase exhibits selective reducing activity for 2-chloro-1R-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)ethyl ketone.

3. The ketone reductase with stereoselective reducing activity according to claim 2, characterized in that... The ketone reductase, or the enzyme-containing cell or carrier, uses nebivolol racemic chloroketone as a starting material. It stereoselectively recognizes the R-configuration ketone substrate and reduces it to optically pure alcohol R,R-2, while the S-configuration ketone substrate does not react, resulting in resolution.

4. The ketone reductase with stereoselective reducing activity according to claim 3, characterized in that... After the reaction was completed, the reaction mixture was extracted with ethyl acetate. The extract was concentrated and then subjected to crystallization or silica gel column chromatography to obtain optically pure compounds S-1 and R,R-2, respectively.

5. The use of the ketone reductase with stereoselective reducing activity as described in any one of claims 1-4 in the resolution of racemic nebirolol chloroone.

Images