KR100848935B1 - Enantioselective hydrolysis using biocatalytic enzyme - Google Patents

Enantioselective hydrolysis using biocatalytic enzyme Download PDF

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KR100848935B1
KR100848935B1 KR1020070047746A KR20070047746A KR100848935B1 KR 100848935 B1 KR100848935 B1 KR 100848935B1 KR 1020070047746 A KR1020070047746 A KR 1020070047746A KR 20070047746 A KR20070047746 A KR 20070047746A KR 100848935 B1 KR100848935 B1 KR 100848935B1
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selective hydrolysis
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민연식
황성관
이형철
박재욱
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Abstract

An enantioselective hydrolysis method of asymmetric diester compounds is provided to improve purity and yield of a target compound and inhibit environmental pollution by using a biocatalytic enzyme, so that the method is useful for mass production of intermediates in synthesis of lercanidipine. A (4S)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)pyridine-3-carboxylic acid isomer represented by the formula(1) is prepared by enantioselective hydrolysis of methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl) pyridine-3,5-dicarboxylate represented by the formula(2) in a solvent selected from a mixture containing water and C2-C8 dialkylether by using lipase derived from Pseudomonas cepacia at 15-30 deg. C. Further, the solvent additionally comprises acetone, and the dialkylether organic solvent is dialkylether or diisopropylether.

Description

생체 촉매 효소를 이용한 광학 선택적 가수분해방법{Enantioselective hydrolysis using biocatalytic enzyme}Enantioselective hydrolysis using biocatalytic enzyme

본 발명은 생체 촉매 효소를 이용한 광학 선택적 가수분해방법에 관한 것으로, 더욱 상세하게는 하기 화학식 2로 표시되는 메틸 피발로일옥시메틸 1,4-디히드로-2,6-디메틸-4-(3-니트로페닐)피리딘-3,5-디카르복실레이트 라세미체를 생체 촉매 효소로서 슈도모나스 세페시아(Pseudomonas Cepacia)로부터 유래된 리파아제를 이용한 광학 선택적 가수분해하여, 하기 화학식 1로 표시되는 (4S)-1,4-디히드로-2,6-디메틸-5-메톡시카르보닐-4-(3-니트로페닐)피리딘-3-카르복실산 이성질체를 제조하는 방법에 관한 것이다.The present invention relates to an optical selective hydrolysis method using a biocatalyst enzyme, and more particularly, methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4- (3) -Nitrophenyl ) pyridine-3,5-dicarboxylate racemate was subjected to optical selective hydrolysis using a lipase derived from Pseudomonas Cepacia as a biocatalyst enzyme, represented by formula (1 S) ), 1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4- (3-nitrophenyl) pyridine-3-carboxylic acid isomer.

Figure 112007036145325-pat00002
Figure 112007036145325-pat00002

본 발명이 목적하는 상기 화학식 1로 표시되는 화합물은, 고혈압 치료제로 잘 알려져 있는 레르카니디핀(Lercanidipine) 의약품 합성을 위한 핵심 중간체이다.The compound represented by Chemical Formula 1, which is the object of the present invention, is a key intermediate for the synthesis of lercanidipine pharmaceuticals, which are well known as therapeutic agents for hypertension.

Figure 112007036145325-pat00003
Figure 112007036145325-pat00003

레르카니디핀(Lercanidipine) 또는 이의 염산염은 작용시간이 길고 혈관선택성이 높은 친유성이 큰 디하이드로피리딘 칼슘 길항제로서 혈관 세포막의 칼슘채널에 작용해 혈관을 확장시키고 혈압을 낮추는 역할을 하는 최신 고혈압 치료제이다. 또한, 레르카니디핀의 약제학적으로 허용 가능한 염은 L-타입의 칼슘 채널 길항물질로서 항고혈압제, 안기나(angina: 인후 편도선 염증 등), 관상동맥 질환 등의 치료에 유효한 것으로 잘 알려져 있다.Lercanidipine or its hydrochloride is a lipophilic dihydropyridine calcium antagonist with a long duration of action and high vascular selectivity. . In addition, the pharmaceutically acceptable salt of lercanidipine is an L-type calcium channel antagonist, which is well known to be effective in the treatment of antihypertensives, angina (such as throat tonsil inflammation), coronary artery disease, and the like.

레르카니디핀(Lercanidipine) 염화수소의 제조방법과 관련된 대표적인 선행기술로서 미국특허 제5,767,136호, 제4,968,832호, 제5,696,139호, 제5,912,351호, 제4,705,797호, 독일특허 제284737호 및 한국특허공보 제395441호 등이 있다. 상기 선행된 제조방법은 공통적으로 상기 화학식 1로 표시되는 화합물을 핵심 중간체로서 합성 경유하고 있다.Representative prior art related to the production method of lercanidipine hydrogen chloride, U.S. Pat. Etc. The above-mentioned manufacturing method is commonly used via the compound represented by the formula (1) as a key intermediate.

레르카니디핀의 핵심 중간체로서 상기 화학식 1로 표시되는 화합물의 합성방법으로는 염기를 이용한 가수분해 방법이 일반적으로 알려져 왔고, 또 다른 방법으로 생체 촉매 효소를 이용한 가수분해 방법이 보고되었다. 다음은 몇 가지 공 지된 예들을 하기 반응식 1과 반응식 2에 소개하였다.As a method for synthesizing the compound represented by Chemical Formula 1 as a key intermediate of lercanidipine, a hydrolysis method using a base has been generally known, and as another method, a hydrolysis method using a biocatalytic enzyme has been reported. The following are some of the known examples in Scheme 1 and Scheme 2 below.

하기 반응식 1은 t-부틸 메틸 에테르 용매하에서 리파아제를 이용한 1,4-디히드로피리딘 계열의 광학 선택적 가수분해반응의 예로서 비대칭 가수분해 및 광학 분할까지도 가능하다. (Tetrahedron: Asymmetry 6, 1995, pp. 2917-2920; Biocatalysis and Biotransformation, 22(4), 2004, pp. 231-252)Scheme 1 below is an example of optically selective hydrolysis of the 1,4-dihydropyridine family using lipase in a t -butyl methyl ether solvent, and asymmetric hydrolysis and even optical cleavage are possible. ( Tetrahedron: Asymmetry 6, 1995, pp. 2917-2920; Biocatalysis and Biotransformation , 22 (4), 2004, pp. 231-252)

Figure 112007036145325-pat00004
Figure 112007036145325-pat00004

하기 반응식 2는 세아프로제 에스(Seaprose S: Aspergillus melleus)와 같은 프로테아제를 이용한 효소 가수분해반응의 예로서 일정한 pH 범위에서 수용액 상태로 가수분해가 이루어지며, 높은 광학 순도를 보인다. (Tetrahedron Lett. 34, 1993, pp. 3441-3444, Tetrahedron: Asymmetry 4, 1993, pp. 2061-2068, Biocatalysis and Biotransformation, 22(4), 2004, pp. 231-252)Scheme 2 is an example of enzymatic hydrolysis using a protease such as Seaprose S: Aspergillus melleus , and hydrolysis is performed in an aqueous solution at a constant pH range, and shows high optical purity. ( Tetrahedron Lett. 34, 1993, pp. 3441-3444, Tetrahedron: Asymmetry 4, 1993, pp. 2061-2068, Biocatalysis and Biotransformation , 22 (4), 2004, pp. 231-252)

Figure 112007036145325-pat00005
Figure 112007036145325-pat00005

상기 반응식 1과 2에서 소개된 1,4-디히드로피리딘 계열의 생체 촉매 효소 가수분해 반응의 예들은 대부분 대칭적 구조를 갖는 1,4-디히드로피리딘 계열의 화합물을 생체 촉매 효소 가수분해 반응의 대상으로 삼았으며, 모두의 경우에서 동일한 형태의 에스테르기를 갖는 대칭적 구조의 디에스테르 화합물로서, 구조적으로 크고 상대적으로 이탈하기 쉬운 구조를 갖는 특징이 있다.Examples of the 1,4-dihydropyridine-based biocatalytic enzyme hydrolysis reactions introduced in Schemes 1 and 2 include the 1,4-dihydropyridine-based compound having a symmetrical structure. As a target, in all cases, as a diester compound having a symmetrical structure having the same type of ester group, it is characterized by having a structure that is large in structure and relatively easy to escape.

그러나, 본 발명이 광학 선택적인 가수분해 반응의 대상으로 하는 상기 화학식 2로 표시되는 1,4-디히드로피리딘 계열의 화합물은 서로 다른 형태의 에스테르기가 치환된 비대칭적 구조의 디에스테르 화합물로서, 생체 효소 촉매를 이용하여 서로 다른 형태의 에스테르기를 선택적으로 가수분해하는 방법에 대한 연구는 극히 미흡한 실정이다. However, the 1,4-dihydropyridine-based compound represented by Chemical Formula 2, which is an object of the optical selective hydrolysis reaction of the present invention, is a diester compound having an asymmetric structure substituted with ester groups of different forms. There is very little research on the method of selectively hydrolyzing different types of ester groups using enzyme catalysts.

즉, 비대칭적 구조의 디에스테르 화합물인 상기 화학식 2로 표시되는 메틸 피발로일옥시메틸 1,4-디히드로-2,6-디메틸-4-(3-니트로페닐)피리딘-3,5-디카르복실레이트를 생체 효소 촉매를 이용하여 광학 선택적으로 가수분해하는 방법은 아직까지 보고되어 있지 않다. That is, methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) pyridine-3,5-dica represented by Formula 2, which is a diester compound having an asymmetric structure The method of optically selectively hydrolyzing carboxylates using a bioenzyme catalyst has not been reported yet.

본 발명의 목적은 특정의 생체 촉매 효소를 이용하여 비대칭적 구조의 디에스테르 화합물을 선택적으로 가수분해함과 동시에 광학분리하는 일명, '광학 선택적 가수분해'하는 방법을 제공하는데 있다.SUMMARY OF THE INVENTION An object of the present invention is to provide a method of selectively hydrolyzing a diester compound having an asymmetric structure by using a specific biocatalyst and simultaneously separating optically, a method of 'optical selective hydrolysis'.

본 발명은 하기 화학식 2로 표시되는 메틸 피발로일옥시메틸 1,4-디히드로-2,6-디메틸-4-(3-니트로페닐)피리딘-3,5-디카르복실레이트 라세미체를, 생체 촉매 효소로서 슈도모나스 세페시아(Pseudomonas Cepacia; LPS-C)로부터 유래된 리파아제를 사용하는 광학 선택적 가수분해반응에 의해 하기 화학식 1로 표시되는 (4S)-1,4-디히드로-2,6-디메틸-5-메톡시카르보닐-4-(3-니트로페닐)피리딘-3-카르복실산 이성질체를 제조하는 방법을 그 특징으로 한다.The present invention is methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) pyridine-3,5-dicarboxylate racemate represented by the following formula (2) , as a biocatalyst enzyme Pseudomonas three facia (Pseudomonas Cepacia; LPS-C) (4 S) -1,4- dihydro -2 represented by the following general formula (1) by an optical selective hydrolysis reaction using a lipase derived from, It is characterized by a method for producing 6-dimethyl-5-methoxycarbonyl-4- (3-nitrophenyl) pyridine-3-carboxylic acid isomer.

Figure 112007036145325-pat00006
Figure 112007036145325-pat00006

본 발명에 따른 비대칭적 1,4-디히드로피리딘 계열의 화합물에 대한 광학 선택적인 가수분해 공정을 보다 상세히 설명하면 다음과 같다.The optical selective hydrolysis process for the asymmetric 1,4-dihydropyridine family of compounds according to the present invention will be described in more detail as follows.

본 발명에 따른 광학 선택적인 가수분해 반응은, 상기 화학식 2로 표시되는 메틸 피발로일옥시메틸 1,4-디히드로-2,6-디메틸-4-(3-니트로페닐)피리딘-3,5-디카르복실레이트를 물과 유기용매의 혼합용매에 적절히 용해 및 분산시킨 후 생체 촉 매 효소를 첨가하여 15 내지 50 ℃에서 24시간 내지 72시간 동안 교반하여 수행한다. 또한, 상기 광학 선택적인 가수분해 반응의 결과로 생성된 상기 화학식 1로 표시되는 화합물은 여과 등의 통상의 분리 방법을 통해 고체 화합물로서 쉽게 수득할 수 있다.The optically selective hydrolysis reaction according to the present invention, methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) pyridine-3,5 represented by the formula (2) The dicarboxylate is dissolved and dispersed in a mixed solvent of water and an organic solvent as appropriate, followed by addition of a biocatalyst enzyme and stirring at 15 to 50 ° C. for 24 to 72 hours. In addition, the compound represented by Chemical Formula 1 produced as a result of the optical selective hydrolysis reaction can be easily obtained as a solid compound through a conventional separation method such as filtration.

본 발명에서는 생체 촉매 효소로 슈도모나스 세페시아(Pseudomonas Cepacia)로부터 유래된 리파아제(예를 들면, 엘피에스-씨(LPS-C: Lipase from Pseudomonas Cepacia))를 사용하였다. 또한, 상기 효소는 특정 세라믹 담체에 고정시킨 고정화 효소로 사용할 수 있고, 또는 비고정화 효소로 사용할 수도 있다.In the present invention, a lipase derived from Pseudomonas Cepacia (eg, LPS-C: Lipase from Pseudomonas Cepacia ) was used as a biocatalyst . In addition, the enzyme may be used as an immobilized enzyme immobilized on a specific ceramic carrier, or may be used as an unimmobilized enzyme.

상기 생체 촉매 효소의 사용량은 상기 화학식 2로 표시되는 화합물 1 mmol을 기준으로 100 내지 2000 mg 범위로 사용하고, 바람직하기로는 200 내지 1000 mg 범위로 사용할 수 있다. 생체 촉매 효소의 사용량이 증가할수록 수득율은 향상될 수 있지만, 2000 mg을 초과하여 다량의 효소 사용은 오히려 제조 비용의 상승원인이 될 수 있으므로 바람직하지 못하다.The biocatalyst may be used in the range of 100 to 2000 mg, preferably 200 to 1000 mg, based on 1 mmol of the compound represented by Chemical Formula 2. Yield may be improved as the amount of biocatalyst enzyme used increases, but the use of a large amount of enzyme in excess of 2000 mg is rather undesirable because it may cause an increase in the manufacturing cost.

본 발명에서는 반응용매로서 물과 유기용매의 혼합용매를 사용하는데, 유기용매는 탄소수 2 내지 8의 디알킬에테르를 사용하며, 보다 구체적으로는 디에틸에테르 또는 디이소프로필에테르를 사용할 수 있으며, 디이소프로필에테르가 가장 바람직하게 사용될 수 있다. 상기 물과 유기용매의 혼합비는 적절히 조절이 가능하고, 유기용매에 물이 포화된 상태의 혼합용매를 사용하는 것이 반응물질의 원활한 용해 및 분산을 위해 보다 바람직하다. 물과 유기용매의 바람직한 혼합비는 물 0.01 ∼ 5 부피%와 유기용매 95 ∼ 99.99 부피%를 이루는 것이고, 보다 바람직하기로는 물 0.01 ∼ 2 부피%와 유기용매 98 ∼ 99.99 부피%를 이루는 것이다. 더욱 좋기로는 상기 물과 유기용매의 혼합용매에, 아세톤이 추가로 첨가된 혼합용매를 사용하는 것이다. 아세톤은 전체 혼합용매 중에 1 내지 30 부피% 범위, 바람직하기로는 1 내지 20 부피% 범위로 첨가하여, 상기 혼합용매에 아세톤이 첨가된 경우가 가장 효율적이었다. 구체적으로는 물 : 아세톤 : 디알킬에테르의 부피비가 0.01 ∼ 5 : 1 ∼ 30 : 69 ∼ 98 부피%를 이루는 혼합용매를 사용할 수 있고, 보다 바람직하기로는 0.01 ∼ 2 : 1 ∼ 20 : 79 ∼ 98 부피%를 이루는 혼합용매를 사용할 수 있다.In the present invention, a mixed solvent of water and an organic solvent is used as the reaction solvent. The organic solvent may be a dialkyl ether having 2 to 8 carbon atoms, and more specifically, diethyl ether or diisopropyl ether may be used. Isopropyl ether may be most preferably used. The mixing ratio of the water and the organic solvent can be appropriately adjusted, it is more preferable to use a mixed solvent in a water saturated state in the organic solvent for smooth dissolution and dispersion of the reactant. The preferred mixing ratio of water and organic solvent is 0.01 to 5% by volume of water and 95 to 99.99% by volume of organic solvent, and more preferably 0.01 to 2% by volume of water and 98 to 99.99% by volume of organic solvent. More preferably, a mixed solvent in which acetone is additionally added to the mixed solvent of water and an organic solvent is used. Acetone was added in the range of 1 to 30% by volume, preferably 1 to 20% by volume in the total mixed solvent, so that acetone was added to the mixed solvent most efficiently. Specifically, a mixed solvent having a volume ratio of water: acetone: dialkyl ether of 0.01 to 5: 1 to 30:69 to 98% by volume can be used, and more preferably 0.01 to 2: 1 to 20:79 to 98 Mixed solvents which constitute volume percent can be used.

광학 선택적인 가수분해 반응의 온도는 15 내지 50 ℃ 범위이며, 바람직하게는 15 내지 30 ℃ 범위이며, 가장 바람직하기로는 20 내지 25 ℃ 범위에서 수행될 수 있다.The temperature of the optically selective hydrolysis reaction is in the range from 15 to 50 ° C., preferably in the range from 15 to 30 ° C., and most preferably in the range from 20 to 25 ° C.

광학 선택적인 가수분해 반응 시간은 24 시간 내지 72 시간이며, 바람직하게는 48 시간 동안 수행될 수 있다.The optically selective hydrolysis reaction time is from 24 hours to 72 hours, preferably for 48 hours.

이상에서 설명한 바와 같은 광학 선택적인 가수분해 반응 결과로 생성된 상기 화학식 1로 표시되는 화합물은 통상적인 방법에 의해 산부가염으로 전환될 수 있다.The compound represented by Chemical Formula 1 produced as a result of the optical selective hydrolysis reaction as described above may be converted into an acid addition salt by a conventional method.

또한, 본 발명에서 제조된 상기 화학식 1로 표시되는 화합물은 수율 및 순도가 높아서 의약품 제조를 위한 제약학적으로 요구하는 최소한의 순도인 99.5 % 이상의 순도를 갖는 레르카니디핀 염산염 제조에 유용하다. In addition, the compound represented by the formula (1) prepared in the present invention is high in yield and purity is useful for the preparation of lercanidipine hydrochloride having a purity of 99.5% or more, which is the minimum purity pharmaceutically required for the manufacture of pharmaceuticals.

이상에서 설명한 바와 같은 본 발명은 다음의 실시예에 의거하여 더욱 상세히 설명하겠는 바, 본 발명이 이에 한정되는 것은 아니다.The present invention as described above will be described in more detail based on the following examples, but the present invention is not limited thereto.

실시예 1: (4S)-1,4-디히드로-2,6-디메틸-5-메톡시카르보닐-4-(3-니트로페닐)피리딘-3-카르복실산(화학식 1)의 제조Example 1: Preparation of (4 S) -1,4- dihydro-2,6-dimethyl-5-methoxycarbonyl-4- (3-nitrophenyl) pyridine-3-carboxylic acid (I)

메틸 피발로일옥시메틸 1,4-디히드로-2,6-디메틸-4-(3-니트로페닐)피리딘-3,5-디카르복실레이트 (화학식 2) 100 g과 엘피에스-씨(LPS-C) 효소 44.8 g을 물/아세톤/디이소프로필에테르(2/10/88 부피%)의 혼합용매 448 mL에 가하였다. 그리고 생성된 현탁액을 25 ℃에서 48 시간 동안 교반하였다. TLC(Thin layer chromatography)를 통해 반응 종결을 확인한 후 테트라히드로퓨란 200 mL를 첨가하고 교반하여 혼합한 후, 여과하여 효소를 제거하였다. 여액은 감압 농축하고 디클로로메탄 150 mL를 첨가하여 30분 동안 교반하여 침전을 유도하였다. 생성된 결정은 여과하고 디클로로메탄으로 세척한 후 50 ℃에서 감압 건조하여 백색 고체상의 표제화합물 33.5 g (수율: 90 %)을 수득하였다.100 g of methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) pyridine-3,5-dicarboxylate (Formula 2) and LPS-C (LPS) 44.8 g of enzyme were added to 448 mL of a mixed solvent of water / acetone / diisopropylether (2/10/88% by volume). The resulting suspension was stirred at 25 ° C. for 48 hours. After confirming the reaction termination through TLC (Thin layer chromatography), 200 mL of tetrahydrofuran was added, stirred, and mixed, followed by filtration to remove the enzyme. The filtrate was concentrated under reduced pressure and 150 mL of dichloromethane was added and stirred for 30 minutes to induce precipitation. The resulting crystals were filtered, washed with dichloromethane and dried under reduced pressure at 50 ° C. to obtain 33.5 g (yield: 90%) of the title compound as a white solid.

융점 193-195 ℃; 비선광도 [α]D 20 + 23.3 (c=0.75, 아세톤); 광학순도 99.2 %ee (HPLC); 1H NMR (DMSO-d 6 , ppm) δ 2.27 (s, 6H), 3.54 (s, 3H), 4.49 (s, 1H), 7.55 (m, 2H), 8.0 (m, 2H), 8.97 (s, 1H), 11.91(brs, 1H)Melting point 193-195 ° C .; Specific light [α] D 20 +23.3 (c = 0.75, acetone); Optical purity 99.2% ee (HPLC); 1 H NMR (DMSO- d 6, ppm) δ 2.27 (s, 6H), 3.54 (s, 3H), 4.49 (s, 1H), 7.55 (m, 2H), 8.0 (m, 2H), 8.97 (s , 1H), 11.91 (brs, 1H)

실시예 2: 광학 선택적인 가수분해 반응에 사용되는 생체 촉매 효소의 선별Example 2: Screening of Biocatalytic Enzymes Used for Optically Selective Hydrolysis Reactions

메틸 피발로일옥시메틸 1,4-디히드로-2,6-디메틸-4-(3-니트로페닐)피리딘- 3,5-디카르복실레이트(화학식 2) 100 mg과 하기 표 1에 나타낸 바와 같이 다양한 생체 촉매 효소 44.8 mg을 물/아세톤/디이소프로필에테르 (2/10/88 부피%)의 혼합용매 5 mL에 가하였다. 그리고, 생성된 현탁액을 25 ℃에서 72 시간 동안 교반하였다. TLC(Thin layer chromatography)를 통해 반응 종결을 확인한 후 테트라히드로퓨란 2 mL를 첨가하고 교반하여 혼합한 후, 여과하여 효소를 제거하였다. 여액은 감압 농축한 후 HPLC로 가수분해 생성물의 생성 비율을 측정하였다. 실험에 사용된 효소의 종류와 활성도의 결과를 하기 표 1에서 비교하였다.100 mg of methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) pyridine-3,5-dicarboxylate (Formula 2) as shown in Table 1 below. Likewise, 44.8 mg of various biocatalyst enzymes were added to 5 mL of a mixed solvent of water / acetone / diisopropylether (2/10/88% by volume). And the resulting suspension was stirred at 25 ° C. for 72 hours. After confirming the reaction termination through TLC (Thin layer chromatography), 2 mL of tetrahydrofuran was added, stirred, and mixed, followed by filtration to remove the enzyme. The filtrate was concentrated under reduced pressure and then the production rate of the hydrolysis product was measured by HPLC. The type and activity of the enzymes used in the experiments were compared in Table 1 below.

Figure 112007036145325-pat00007
Figure 112007036145325-pat00007

상기 표 1에 의하면, 상기 화학식 2로 표시되는 화합물은 엘피에스-씨(LPS-C: Lipase from Pseudomonas Cepacia)를 이용한 가수분해 반응에서 우수한 광학 선택적 가수분해의 활성을 보였다.According to Table 1, the compound represented by Formula 2 is LPS-C ( LPase : Lipase from Pseudomonas Cepacia ) showed excellent optical selective hydrolysis activity in the hydrolysis reaction.

실시예 3: 광학 선택적인 가수분해 반응에 사용되는 용매의 선별Example 3: Screening of Solvents Used in Optically Selective Hydrolysis Reactions

메틸 피발로일옥시메틸 1,4-디히드로-2,6-디메틸-4-(3-니트로페닐)피리딘-3,5-디카르복실레이트(화학식 2) 100 mg과 엘피에스-씨(LPS-C) 효소 44.8 mg을 아래 표 2에 나타낸 다양한 용매 5 mL에 가하여 생성된 현탁액을 25 ℃에서 72 시간 동안 교반하였다. TLC(Thin layer chromatography)를 통해 반응 종결을 확인한 후 테트라히드로퓨란 2 mL를 첨가하고 교반하여 혼합한 후, 여과하여 효소를 제거하였다. 여액은 감압 농축한 후 HPLC로 가수분해 생성물의 생성 비율을 측정하였다. 실험에 사용된 용매의 종류와 활성도의 결과를 하기 표 2에서 비교하였다.100 mg of methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) pyridine-3,5-dicarboxylate (Formula 2) and LPS-C (LPS) -C) 44.8 mg of enzyme was added to 5 mL of the various solvents shown in Table 2 below, and the resulting suspension was stirred at 25 ° C. for 72 hours. After confirming the reaction termination through TLC (Thin layer chromatography), 2 mL of tetrahydrofuran was added, stirred, and mixed, followed by filtration to remove the enzyme. The filtrate was concentrated under reduced pressure and then the production rate of the hydrolysis product was measured by HPLC. The type of solvent used in the experiment and the results of the activity were compared in Table 2 below.

Figure 112007036145325-pat00008
Figure 112007036145325-pat00008

상기 표 2에 의하면, 물+디이소프로필에테르의 혼합용매 또는 물+디이소프로필에테르+아세톤의 혼합용매를 사용하여 가수분해 반응을 수행하였을 때, 우수한 광학 선택적 가수분해의 활성을 보였다.According to Table 2, when the hydrolysis reaction was performed using a mixed solvent of water + diisopropyl ether or a mixed solvent of water + diisopropyl ether + acetone, it showed excellent optical selective hydrolysis activity.

실시예 4: 반응시간별 광학 선택적인 가수분해 수율 비교Example 4 Optical Selective Hydrolysis Yield Comparison by Reaction Time

상기 실시예 1과 동일한 조건으로 실시하되 시간대 별로 반응 혼합물을 취하여 효소만을 제거한 후 HPLC로 가수분해 생성물의 전환 비율을 측정하여 그 결과를 하기 표 3에서 비교하였다.The reaction was carried out under the same conditions as in Example 1, but the reaction mixture was taken at each time interval to remove only the enzyme, and then the conversion ratio of the hydrolysis product was measured by HPLC. The results are compared in Table 3 below.

Figure 112007036145325-pat00009
Figure 112007036145325-pat00009

이상에서 설명한 바와 같이, 본 발명의 제조방법은 특정의 생체 촉매 효소를 이용하여 비대칭적 구조의 디에스테르 화합물을 선택적으로 가수분해하는 방법을 특징으로 하는 발명으로, 목적하는 화합물의 순도 및 수율이 우수하다. 또한, 본 발명의 제조방법은 효소를 사용하는 친환경적인 청정 생산 공정에 해당된다.As described above, the production method of the present invention is a method of selectively hydrolyzing a diester compound having an asymmetric structure using a specific biocatalyst enzyme, and has excellent purity and yield of a desired compound. Do. In addition, the production method of the present invention corresponds to an environment-friendly clean production process using an enzyme.

따라서, 본 발명은 고순도가 요구되는 레르카니디핀 의약품의 중간체 합성을 위한 대량 생산 방법으로서 유용하다.Accordingly, the present invention is useful as a mass production method for the synthesis of intermediates of lercanidipine pharmaceuticals that require high purity.

Claims (9)

하기 화학식 2로 표시되는 메틸 피발로일옥시메틸 1,4-디히드로-2,6-디메틸-4-(3-니트로페닐)피리딘-3,5-디카르복실레이트를, 생체 촉매 효소로서 슈도모나스 세페시아(Pseudomonas Cepacia)로부터 유래된 리파아제를 사용하는 광학 선택적인 가수분해반응하여 제조하는 것을 특징으로 하는 하기 화학식 1로 표시되는 (4S)-1,4-디히드로-2,6-디메틸-5-메톡시카르보닐-4-(3-니트로페닐)피리딘-3-카르복실산 이성질체의 제조방법.Pseudomonas using methyl pivaloyloxymethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) pyridine-3,5-dicarboxylate represented by the following formula (2) as a biocatalyst enzyme: to characterized in that the three fascia prepared by reaction the optical selective hydrolysis using a lipase derived from (Pseudomonas Cepacia) represented by the general formula 1 (4 S) -1,4- dihydro-2,6-dimethyl- Process for the preparation of 5-methoxycarbonyl-4- (3-nitrophenyl) pyridine-3-carboxylic acid isomer. [화학식 2][Formula 2]
Figure 112008018882773-pat00010
Figure 112008018882773-pat00010
 [화학식 1][Formula 1]
Figure 112008018882773-pat00011
Figure 112008018882773-pat00011
 제 1 항에 있어서,The method of claim 1, 상기 광학 선택적인 가수분해반응에 사용되는 용매는 물(H2O)과, 탄소수 2 내지 8의 디알킬에테르 유기용매의 혼합용매인 것을 특징으로 하는 제조방법.The solvent used for the optically selective hydrolysis reaction is a mixed solvent of water (H 2 O) and a dialkyl ether organic solvent having 2 to 8 carbon atoms. 제 2 항에 있어서,The method of claim 2, 상기 광학 선택적인 가수분해반응에 사용되는 물(H2O)과, 탄소수 2 내지 8의 디알킬에테르 유기용매가 0.01 ∼ 5 : 95 ∼ 99.99 부피%를 이루는 혼합용매인 것을 특징으로 하는 제조방법.The water (H 2 O) used for the optically selective hydrolysis reaction and the dialkyl ether organic solvent having 2 to 8 carbon atoms are a mixed solvent of 0.01 to 5:95 to 99.99% by volume. 제 2 항 또는 제 3 항에 있어서, The method of claim 2 or 3, 상기 광학 선택적인 가수분해반응에 사용되는 용매는 추가로 아세톤이 포함된 것을 특징으로 하는 제조방법.The solvent used for the optically selective hydrolysis reaction is characterized in that it further comprises acetone. 제 4 항에 있어서, The method of claim 4, wherein 상기 광학 선택적인 가수분해반응에 사용되는 용매는 물 : 아세톤 : 디알킬에테르의 부피비가 0.01 ∼ 5 : 1 ∼ 30 : 69 ∼ 98 부피%를 이루는 혼합용매인 것을 특징으로 하는 제조방법.The solvent used for the optically selective hydrolysis reaction is a mixed solvent in which the volume ratio of water: acetone: dialkyl ether is 0.01 to 5: 1 to 30: 69 to 98% by volume. 제 2 항 또는 제 3 항에 있어서,The method of claim 2 or 3, 상기 디알킬에테르 유기용매는 디에틸에테르 또는 디이소프로필에테르인 것을 특징으로 하는 제조방법.The dialkyl ether organic solvent is diethyl ether or diisopropyl ether. 제 6 항에 있어서,The method of claim 6, 상기 디알킬에테르 유기용매는 디이소프로필에테르인 것을 특징으로 하는 제조방법.The dialkyl ether organic solvent is diisopropyl ether. 제 1 항에 있어서,The method of claim 1, 상기 광학 선택적인 가수분해반응 온도는 15 내지 30 ℃인 것을 특징으로 하는 제조방법.The optically selective hydrolysis reaction temperature is 15 to 30 ℃ manufacturing method characterized in that. 제 1 항에 있어서,The method of claim 1, 상기 가수분해반응 후에 생성된 상기 화학식 1로 표시되는 화합물은 여과하여 고체로서 수득하는 것을 특징으로 하는 제조방법.The compound represented by the formula (1) produced after the hydrolysis reaction is obtained by filtration to obtain a solid.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4705797A (en) 1984-02-14 1987-11-10 Recordati S.A., Chemical And Pharmaceutical Company N-(3,3-diphenylpropyl) aminoethyl esters of 1,4-dihydro-2,6-dimethyl-pyridine-3,5-dicarboxylic acid, compositions and use
KR20010027210A (en) * 1999-09-11 2001-04-06 박호군 Lipase Catalysed Resolutions of Verapamil Intermediate and Process for Preparing (R)- and (S)-Verapamil
KR20040016171A (en) * 2002-08-16 2004-02-21 엘지전자 주식회사 A method and a device of equalizing filter for mobile phone
KR20040056592A (en) * 2002-12-24 2004-07-01 한국과학기술연구원 Catalyzed resolution of (±)-cis-1,3-dioxolane derivatives

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4705797A (en) 1984-02-14 1987-11-10 Recordati S.A., Chemical And Pharmaceutical Company N-(3,3-diphenylpropyl) aminoethyl esters of 1,4-dihydro-2,6-dimethyl-pyridine-3,5-dicarboxylic acid, compositions and use
KR20010027210A (en) * 1999-09-11 2001-04-06 박호군 Lipase Catalysed Resolutions of Verapamil Intermediate and Process for Preparing (R)- and (S)-Verapamil
KR20040016171A (en) * 2002-08-16 2004-02-21 엘지전자 주식회사 A method and a device of equalizing filter for mobile phone
KR20040056592A (en) * 2002-12-24 2004-07-01 한국과학기술연구원 Catalyzed resolution of (±)-cis-1,3-dioxolane derivatives

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