KR20040006307A - Process for preparing cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-13-dioxolane-4-methyl alcohol of high optical selectivity - Google Patents

Process for preparing cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-13-dioxolane-4-methyl alcohol of high optical selectivity Download PDF

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KR20040006307A
KR20040006307A KR1020020040325A KR20020040325A KR20040006307A KR 20040006307 A KR20040006307 A KR 20040006307A KR 1020020040325 A KR1020020040325 A KR 1020020040325A KR 20020040325 A KR20020040325 A KR 20020040325A KR 20040006307 A KR20040006307 A KR 20040006307A
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정찬성
한예선
윤문영
조현성
이병석
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한국과학기술연구원
한올제약주식회사
경동제약 주식회사
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Abstract

PURPOSE: A process for preparing cis-2- (bromomethyl)-2- (2,4-dichlorophenyl)- 13-dioxolane- 4-methyl alcohol having high optical selectivity is provided. A stereo-selective enzyme maintains its maximum activity because the reaction product is rapidly removed from the reaction solution during the reaction in order not to inhibit the activity of the enzyme. CONSTITUTION: A process for preparing cis-2- (bromomethyl)-2- (2,4-dichlorophenyl)- 13-dioxolane- 4-methyl alcohol having high optical selectivity comprises hydrolysis of cis-2- (bromomethyl)-2- (2,4-dichlorophenyl)- 1,3-dioxolane- 4-methyl acetate using lipase produced from Acinetobacter sp. SY-01(KFCC-11111), and recovering an alcohol product as a reaction product from the reaction solution during the reaction, wherein the concentration of the alcohol product is 0.1 to 300 mg/l; the reaction product is recovered when the conversion rate of the hydrolysis reaction is 40 to 60%; the hydrolysis reaction is carried out under conditions of 40 to 60 deg. C, pH 7.0 to 9.0 and 100 to 300 rpm; and the reaction product is recovered by column chromatography.

Description

고 광학선택성을 가지는 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸알콜의 제조방법{Process for preparing cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-13-dioxolane-4-methyl alcohol of high optical selectivity}Process for preparing cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl alcohol having high optical selectivity (Process for preparing cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl alcohol of high optical selectivity}

본 발명은 고 광학선택성을 가지는 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸알콜의 제조방법에 관한 것으로서, 더욱 상세하게는 이트라코나졸의 전구체로서 라세믹 혼합물인 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸 아세테이트를 가수분해하여 고 광학순도를 가지는 이의 시스형 알콜화합물을 제조함에 있어 악시네토박터(Acinetobacter sp.) SY-01(기탁번호 KFCC-11111)로부터 생산되는 리파제를 이용한 입체 선택적 가수분해 반응을 수행하되 생성되는 알콜화합물에 의한 리파제 효소의 억제활성이 심화되는 시점(전환율 40 ∼ 60%)에서 반응생성물을 일단 분리한 후에 다시 가수분해 반응을 수행하여 반응생성물의 광학선택성을 극대화하는 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸알콜의 제조방법에 관한 것이다.The present invention relates to a method for preparing cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl alcohol having high optical selectivity, and more particularly. As a precursor of itraconazole, a cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl acetate as a racemic mixture is hydrolyzed to give its cis having high optical purity In the preparation of alcoholic compounds, a stereoselective hydrolysis reaction using a lipase produced from Acinetobacter sp . Cis-2- (bromomethyl) -2- (2, 2), which maximizes the optical selectivity of the reaction product by separating the reaction product once and then performing a hydrolysis reaction at the time of enhanced activity (conversion rate of 40 to 60%). For the preparation of 4-dichlorophenyl) -13-dioxolane-4-methyl alcohol Will.

일반적으로 라세믹 의약품에 비하여 단일 이성질체 의약품이 생체내 약리활성이 우수하고 생체내 부작용이 적은 것으로 알려져 있다. 항균제 또는 소염제 등으로 사용되는 이트라코나졸(Itraconazole) 역시 시스 형태의 이성질체가 라세믹화합물에 비교하여 항균활성이 우수하다. 라세믹 형태의 이트라코나졸은 간에 독성이 있고, 부정맥, 메스꺼움, 구토, 과민증, 두드러기, 복통, 두통, 현기증 등의 부작용을 유발할 수 있으므로 현재 화학적 합성기법을 사용하여 광학적으로 순수한 시스 이성질체를 제조하려는 연구가 시도되고 있는 실정이다[미국특허 제5,952,502호, 제5,4749,97호].In general, single isomeric drugs are known to have superior pharmacological activity and less side effects in vivo than racemic drugs. Itraconazole, which is used as an antibacterial or anti-inflammatory agent, also has excellent antibacterial activity compared to racemic compounds in the cis-type isomer. The racemic form of itraconazole is toxic to the liver and can cause side effects such as arrhythmia, nausea, vomiting, hypersensitivity, urticaria, abdominal pain, headache, and dizziness, so researchers currently try to produce optically pure cis isomers using chemical synthesis techniques. Attempts have been made (US Pat. Nos. 5,952,502, 5,4749,97).

일반적으로 이트라코나졸은 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸 아세테이트를 중간체로 하여 화학적으로 합성되고 있으나, 이 중간체가 라세믹 혼합물 형태로 제조되므로 이후의 단계가 복잡하고 또한 최종적으로 얻어지는 산물이 라세믹 형태로 제공되므로 여러 가지 부작용의 우려 및 생물학적 활성이 낮은 등의 문제가 있을 수 있어 단일 이성체 형태로 중간체를 합성하고자 하는 시도가 있었다. 그러나, 화학적 합성법에 의해 합성된 라세믹 혼합물을 광학분할하기 위해서는 별도의 추가적 공정이 수행되므로 상용화하는데 많은 어려움이 있다. 이에, 화학적 합성법을 대신에 생촉매를 이용하여 광학분할을 수행한 후 단일 이성질체를 분리하여 이후의 반응을 수행함으로써 제조공정의 간편화, 제조원가의 감소, 생물학적 활성의 증가 및 부작용의 최소화를 도모하고자 하는 노력이 있어 왔다. 이러한 시도의 일환으로서, 본 발명자들은 이트라코나졸 전구체에 대한 광학선택성을 갖은 리파제 생산균주를 분리하여 특허출원한 바도 있다[한국특허공개 제2001-109632호]. 그러나, 생촉매를 이용한 광학분할 반응은 대체로 광학선택도가 낮은 문제를 발생시키는데, 본 발명자들이 분리한 균주가 생산하는 리파제는 광학선택성의 지표인 %ee값이 96% 정도 도달하지만, 기질농도가 증가함에 따라 광학선택도가 급격히 감소하는 현상을 나타내었다.In general, itraconazole has been chemically synthesized using cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl acetate as an intermediate, but this intermediate is racemic Since it is prepared in the form of a mixture, the subsequent steps are complicated, and the final product is provided in racemic form, so there may be problems such as various side effects and low biological activity, and thus attempts to synthesize intermediates in the form of single isomers. There was. However, in order to optically split the racemic mixture synthesized by chemical synthesis, a separate additional process is performed, which makes it difficult to commercialize. Therefore, instead of chemical synthesis, optical splitting is performed using a biocatalyst, and a single isomer is separated to carry out a subsequent reaction, thereby simplifying the manufacturing process, reducing manufacturing cost, increasing biological activity, and minimizing side effects. There has been effort. As part of such an attempt, the present inventors have filed a patent application for separating a lipase producing strain having optical selectivity for the itraconazole precursor (Korean Patent Publication No. 2001-109632). However, the optical splitting reaction using a biocatalyst generally causes a problem of low optical selectivity. The lipase produced by the strains isolated by the present inventors reaches about 96% of% ee, which is an index of optical selectivity, As the increase, optical selectivity decreases rapidly.

이러한 생촉매를 이용한 광학분할공정에서 광학선택성이 낮은 문제가 많이 발생하므로 이것에 대한 문제를 해결하려는 많은 연구가 진행되고 있는데, 아직까지 반응생성물의 분리 회수에 의한 광학선택성을 증가시키는 사례는 보고된 바가 없다.In the optical splitting process using a biocatalyst, many problems have been developed to solve the problem of low optical selectivity, and many studies have been conducted to solve the problem. However, there have been reported cases of increasing optical selectivity by separation and recovery of reaction products. There is no bar.

본 발명은 악시네토박터(Acinetobacter sp.) SY-01로부터 생산되는 광학선택성을 갖는 리파제를 이용하여 에스테르형 라세믹 혼합물을 입체선택적으로 가수분해하여 시스-알콜화합물을 합성하는 과정을 수행하던 중에, 반응생성물 즉, 시스-알콜화합물의 농도가 증가되면서 급격히 리파제 효소의 활성이 억제되는 경향을 알게되었고, 효소활성의 억제가 반응생성물의 농도와 직접적인 관련이 있음을 밝힘으로써 본 발명을 완성하게 되었다.The present invention is a process for synthesizing cis-alcohol compounds by stereoselectively hydrolyzing ester type racemic mixtures using a lipase having optical selectivity produced from Acinetobacter sp. SY-01. As the concentration of the reaction product, i.e., the cis-alcohol compound, increased, the activity of the lipase enzyme was rapidly suppressed, and the present invention was completed by revealing that the inhibition of the enzyme activity is directly related to the concentration of the reaction product. .

따라서, 본 발명은 라세믹 혼합물로 존재하는 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸 아세테이트를 입체선택적 가수분해하여 고 광학순도의 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸알콜을 고수율로 합성하는 방법을 제공하는데 그 목적이 있다.Accordingly, the present invention provides high optical performance by stereoselective hydrolysis of cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl acetate present in racemic mixtures. It is an object of the present invention to provide a method for synthesizing pure cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl alcohol in high yield.

도 1은 기질농도를 변화시키면서 가수분해반응을 수행하여 전환율에 대한 광학선택성의 변화를 나타낸 그래프이다.1 is a graph showing a change in optical selectivity to conversion by performing a hydrolysis reaction while changing the substrate concentration.

도 2는 생성물을 농도별로 첨가하면서 가수분해반응을 수행하여 전환율에 대한 광학선택성의 변화를 나타낸 그래프이다.Figure 2 is a graph showing the change in optical selectivity to the conversion by performing a hydrolysis reaction with the addition of the product by concentration.

도 3은 생성물을 분리 제거하면서 가수분해반응을 수행한 것과 생성물을 분리하지 않고 가수분해반응을 수행한 경우 전환율에 대한 광학선택성의 변화를 비교한 그래프이다.Figure 3 is a graph comparing the change in the optical selectivity to the conversion when the hydrolysis reaction was performed without separating the product and the hydrolysis reaction without separating the product.

도 4는 에스터 형태의 기질과 알콜 형태의 생성물을 실리카겔 컬럼 크로마토그라피를 이용하여 분리한 분획번호에 따른 에스터 또는 알콜 화합물의 상대적 함량을 나타낸 그래프이다.Figure 4 is a graph showing the relative content of the ester or alcohol compound according to the fraction number separated from the substrate in the ester form and the product in the alcohol form using silica gel column chromatography.

본 발명은 라세믹 혼합물인 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-1,3-디옥소란-4-메틸 아세테이트를 기질로 하여 효소 가수분해하는 방법에 있어, 상기 가수분해는 악시네토박터(Acinetobacter sp.) SY-01 균주(KFCC-11111)로부터 생산된 리파제를 사용하여 수행하며, 상기 가수분해 반응중에 생성되는 알콜 생성물은 반응 도중에 반응물로부터 분리 회수하여 고 광학선택성을 가지는 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸알콜을 제조하는 방법을 그 특징으로 한다.The present invention relates to a method of enzymatic hydrolysis using a racemic mixture cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl acetate as a substrate. The hydrolysis is carried out using a lipase produced from Acinetobacter sp. SY-01 strain (KFCC-11111), wherein the alcohol product produced during the hydrolysis reaction is separated and recovered from the reactants during the reaction. To prepare cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl alcohol having high optical selectivity.

이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.

본 발명은 악시네토박터(Acinetobacter sp.) SY-01 균주(KFCC-11111)가 생산하는 리파제를 이용하여 라세믹 혼합물로 존재하는 이트라코나졸 전구체를 입체선택적 가수분해하는 과정에서 생성되는 알콜 형태의 반응 생성물을 적정시기에 분리 회수해냄으로써 목적하는 알콜 화합물에 대한 광학선택성을 증가시키는 방법에 관한 것이다.The present invention relates to alcohol-type reactions produced by stereoselective hydrolysis of itraconazole precursors present in racemic mixtures using lipases produced by Acinetobacter sp. SY-01 strain (KFCC-11111). The present invention relates to a method for increasing the optical selectivity for the desired alcohol compound by separating and recovering the product in a timely manner.

악시네토박터(Acinetobacter sp.) SY-01 균주가 생산하는 리파제는 이트라코나졸의 전구체인 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸 아세테이트의 라세믹 혼합물에 대한 입체 선택적 가수분해하는 활성을 가지고 있다[한국특허공개 제2001-109632호]. 따라서, 상기한 리파제를 가수분해제겸 광학분할제로 사용하여 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-1,3-디옥소란-4-메틸알콜의 단일 이성체를 합성할 수 있으므로 이 단일 이성체를 중간체로 사용하여 단일 광학이성질체 의약품을 합성하는데도 이용할 수 있을 것으로 기대된다. 그러나, 상기한 리파제는 반응생성물인 시스-2-(브로모메틸)-2-(2,4-디클로로 페닐)-1,3-디옥소란-4-메틸알콜에 의하여 효소활성이 억제되는 특성이 있는 바, 그 농도가 증가함에 따라 효소활성 저하는 현격하다.Lipase produced by the Acinetobacter sp. SY-01 strain is cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4, a precursor of itraconazole It has the activity of stereoselective hydrolysis of racemic mixtures of -methyl acetate (Korean Patent Publication No. 2001-109632). Thus, using the above-mentioned lipase as a hydrolyzing agent and an optical splitting agent, a single of cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl alcohol Because isomers can be synthesized, it is expected that this single isomer can also be used to synthesize single optical isomeric drugs. However, the lipase is characterized in that the enzyme activity is inhibited by cis-2- (bromomethyl) -2- (2,4-dichloro phenyl) -1,3-dioxolane-4-methyl alcohol as a reaction product. As such, as the concentration increases, the enzyme activity decreases significantly.

본 발명자들은 이 문제에 대한 해결책을 모색하던 중 가수분해 과정 중에 생성되는 알콜 형태의 생성물이 광학선택도(%ee 값)를 감소시킨다는 사실을 알게되었고, 이 알콜 형태의 생성물을 적정 시기에 분리 회수하면서 가수분해반응을 진행하여 광학선택성을 증가시킬 수 있었던 것이다.The inventors found that while in the search for a solution to this problem, the alcohol form product produced during the hydrolysis process reduced the optical selectivity (% ee value), and the alcohol form product was recovered and recovered in a timely manner. While proceeding with the hydrolysis reaction was able to increase the optical selectivity.

즉, 악시네토박터(Acinetobacter sp.) SY-01 균주(기탁번호 KFCC-11111호)가 생산하는 조효소액을 사용하고, 라세믹 혼합물인 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸 아세테이트를 기질로 하여 40 ∼ 60 ℃ 온도, pH 7.0 ∼ 9.0 및 회전속도 100 ∼ 300 rpm 조건에서 효소 가수분해반응을 수행한다. 다만, 상기 효소 가수분해반응을 수행함에 있어 가수분해 반응물 중에 포함되는 알콜 생성물의 농도가 0.1 ∼ 300 mg/L 범위를 유지하도록 조절하는 것이 주요한 바, 그 농도가 300 mg/L를 초과하여 고농도를 유지하면 효소의 활성이 저하되어 광학선택성이 크게 저하되는 결과를 초래한다. 이에 본 발명에서는 상기한 효소 가수분해 반응의 전환율이 40 ∼ 60%에 이르렀을 때, 또는 전환율 40 ∼ 60%와 70 ∼ 80%에 각각 이르렀을 때 반응물로부터 알콜 생성물을 분리 회수한 후에 재차 상기와 동일한 조건에서 가수분해반응을 수행한다. 반응물로부터 알콜 생성물을 분리 회수는 크로마토그래피 등을 비롯한 통상의 방법에 의해 수행한다. 본 발명자들의 실험 결과에 의하면, 가수분해반응 중에 반응생성물의 분리 회수공정없이 반응을 수행하여 완료하였을 때 광학선택도가 50 %ee 정도로서 의약품의 중간체로사용하는데 문제가 있는데 반하여, 가수분해반응 중에 반응생성물의 분리 회수공정을을 수행하였을 때는 광학선택도가 80 %ee 이상으로 현저하게 상승되는 결과를 얻을 수 있었다.That is, using a coenzyme solution produced by Acinetobacter sp. SY-01 strain (Accession No. KFCC-11111), cis-2- (bromomethyl) -2- (2) which is a racemic mixture Enzymatic hydrolysis is carried out using, 4-dichlorophenyl) -13-dioxolane-4-methyl acetate as a substrate at 40-60 ° C. temperature, pH 7.0-9.0 and rotational speed 100-300 rpm. However, in performing the enzymatic hydrolysis reaction, it is important to adjust the concentration of the alcohol product contained in the hydrolyzate to maintain a range of 0.1 to 300 mg / L, and the concentration exceeds 300 mg / L to maintain a high concentration. Maintaining it lowers the activity of the enzyme, resulting in a significant decrease in optical selectivity. Therefore, in the present invention, when the conversion rate of the enzyme hydrolysis reaction reaches 40 to 60%, or when the conversion rate reaches 40 to 60% and 70 to 80%, respectively, the alcohol product is separated and recovered from the reactants. The hydrolysis reaction is carried out under the same conditions. Separation and recovery of the alcohol product from the reaction is carried out by conventional methods including chromatography and the like. According to the experimental results of the present inventors, the optical selectivity is about 50% ee when the reaction is completed without performing the separation recovery process of the reaction product during the hydrolysis reaction. When the separation recovery process of the product was carried out, the optical selectivity was significantly increased to 80% ee or more.

따라서, 본 발명에 따라 악시네토박터(Acinetobacter sp.) SY-01이 생산하는 발효액 또는 조효소액을 이용하여 라세믹 혼합물인 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸 아세테이트를 가수분해하는 과정에서 발생되는 알콜 형태의 생성물을 분리 회수하면서 광학분할을 수행하게 되면, 목적물에 대한 광학선택성을 현격히 증가시키는 효과를 얻을 수 있고, 이러한 본원발명의 제조방법은 광학순도가 높은 순수 이성체를 분리하는데 이용할 수 있으며, 이 순수 이성체를 전구체로 이용하여 단일 입체이성질체 의약품을 합성하는데 유용하다.Therefore, cis-2- (bromomethyl) -2- (2,4-dichloro is a racemic mixture using a fermentation broth or coenzyme solution produced by Acinetobacter sp. SY-01 according to the present invention . When optical splitting is performed while separating and recovering the alcohol form product generated in the process of hydrolyzing phenyl) -13-dioxolane-4-methyl acetate, an effect of significantly increasing optical selectivity for the target product can be obtained. The preparation method of the present invention can be used to separate pure isomers having high optical purity, and is useful for synthesizing a single stereoisomer medicine using the pure isomers as a precursor.

광학선택도(enantiomeric excess, ee)는 가수분해에 의해 생성되는 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸 아세테이트와 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-1,3-디옥소란-4-메틸 알콜을 분석하므로써 계산할 수 있다. 전환율(Conversion)과 광학선택성의 정도를 나타내는 지표인 광학선택도(ee)값은 다음과 같이 계산하였다.Optical selectivity (enantiomeric excess, ee) is obtained by hydrolysis of cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl acetate and cis- It can be calculated by analyzing 2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl alcohol. The optical selectivity (ee), which is an index indicating the degree of conversion and optical selectivity, was calculated as follows.

분할(Resolution)공정에서 두 가지 반응을 다음과 같이 묘사할 수 있다.The two reactions in the resolution process can be described as follows:

A → C (1)A → C (1)

B → D (2)B → D (2)

상기에서, A와 B는 각각 기질(substrate)을 나타내고, C와 D는 각각 생성물(product)을 나타낸다.In the above, A and B each represent a substrate and C and D each represent a product.

이때, 전환율(Conversion, c)은 ([C]+[D])/([A]+[B]+[C]+[D])로 나타낼 수 있고, B가 A보다 반응속도가 느리다면, 기질에 대한 광학선택도(ees)는([B]-[A])/([A]+[B])로 나타낼 수 있고, 생성물에 대한 광학선택도(eep)는 ([C]-[D])/([C]+[D])로 나타낼 수 있다.At this time, the conversion rate (Conversion, c) can be expressed as ([C] + [D]) / ([A] + [B] + [C] + [D]), if B is slower than A , The optical selectivity (ee s ) for the substrate can be expressed as ([B]-[A]) / ([A] + [B]), and the optical selectivity (ee p ) for the product is ([C ]-[D]) / ([C] + [D]).

이와 같은 본 발명은 다음의 실시예에 의거하여 더욱 구체적으로 설명하겠는 바, 본 발명이 다음의 실시예에 의해 한정되는 것은 아니다Such a present invention will be described in more detail based on the following examples, but the present invention is not limited by the following examples.

[실시예 1] : 조효소액 준비Example 1 Preparation of Coenzyme Solution

올리브유 0.5%, 트립톤 0.5%, 효모추출물 0.5%, 덱스트로스 0.1%, K2HPO40.1%를 함유한 배지용액 3 L를 pH 7.0으로 조절한 후, 5 L 배양기(jar fermenter, 한국 발효기)에 넣고 121 ℃에서 15분동안 멸균하였다. 500 mL 삼각플라스크에서 악시네토박터(Acinetobacter sp.) SY-01 균주(KFCC-11111)를 미리 배양한 배양액 100 mL을 5 L 배양기(jar fermenter, 한국 발효기)에 접종한 후 500 rpm에서 24시간 호기적으로 배양하였다. 원심분리기를 이용하여 13,000 rpm에서 10분 원심분리하여 배양 상등액을 회수하여 이것을 조효소액으로 사용하였다. 본 조효소액의 활성측정은 Tietz와 Fiereck의 방법[N.W. Tietz, E.A. Fiereck, A specific method for serum lipase determination.Clin. Chim. Acta. 13, 352, 1966]에 기초를 둔 Sigma Bulletin No. 800 방법에 따라 분석하였다. Sigma Lipase Substrate(Olive Oil Emulsion) 3 mL, 200 mM TRIZMA 완충액 1 mL(pH 8.0), 배양 상등액 1 mL을 혼합하여 50 ℃에서 3시간 반응시켰다. 반응완료 후 즉시 95% 에탄올 3 mL을 넣고 반응을 정지시킨 후 배양과정에서 유리된 지방산을 50 mM NaOH 수용액을 사용하여 적정하였고, 이때 지시약으로 티몰프탈레인(thymolphthalein, 95% 에탄올에 녹인 0.9% 용액)을 사용하였다. 대조구(Blank) 실험은 배양 상등액을 넣지 않고 같은 방법으로 수행하였다. 리파제 1 unit은 상기 조건에서 1분 동안에 1 μmol의 유리 지방산을 생산하는 효소의 양으로 정의하였고, 다음 수학식 1에 의해 계산하였다.After adjusting 3 L of the medium solution containing olive oil 0.5%, tryptone 0.5%, yeast extract 0.5%, dextrose 0.1%, K 2 HPO 4 0.1% to pH 7.0, 5 L incubator (jar fermenter, Korean fermenter) And sterilized for 15 minutes at 121 ℃. Incubate 100 mL of the culture medium pre-incubated with Acinetobacter sp. Culture was aerobic. The culture supernatant was recovered by centrifugation at 13,000 rpm for 10 minutes using a centrifuge, and this was used as a crude enzyme solution. The activity of this coenzyme solution was measured by Tietz and Fiereck [NW Tietz, EA Fiereck, A specific method for serum lipase determination. Clin. Chim. Acta . 13, 352, 1966, based on Sigma Bulletin No. The analysis was carried out according to the 800 method. 3 mL of Sigma Lipase Substrate (Olive Oil Emulsion), 1 mL of 200 mM TRIZMA buffer (pH 8.0), and 1 mL of the culture supernatant were mixed and reacted at 50 ° C. for 3 hours. Immediately after completion of the reaction, 3 mL of 95% ethanol was added and the reaction was stopped, and the fatty acid liberated during the culturing was titrated with 50 mM NaOH aqueous solution. ) Was used. Control experiments were performed in the same manner without adding the culture supernatant. One unit of lipase was defined as the amount of enzyme that produced 1 μmol of free fatty acid in 1 minute under the above conditions, and was calculated by the following equation.

상기 수학식 1에서, T는 샘플(sample) 적정치이고, Tb는 대조구(Blank)의 적정치이다.In Equation 1, T is a sample titration and T b is a titration of the control.

[실시예 2] : 라세믹 기질의 농도에 따른 광학선택성의 변화 측정Example 2 Measurement of Change in Optical Selectivity According to Concentration of Racemic Substrate

라세믹 기질농도에 따른 광학선택성의 변화를 측정하기 위하여, 다음 표 1에나타낸 것과 같이 500 mL 용량의 삼각 플라스크에 상기 실시예 1의 방법에 따라 준비한 조효소 용액을 기질농도 100, 200, 500, 1000 mg/L에 따라 각각 10 mL(7.4unit), 10 mL(7.4unit), 30 mL(12.2unit), 50 mL(20.2unit)을 투입하고, Trizma 완충액(pH 8.0)으로 0.1M 농도로 맞춘 다음 총 부피가 100 mL이 되도록 반응액을 준비한 후 진탕배양기(shaking incubator)에서 50 ℃ 및 200 rpm의 조건으로 가수분해 반응을 수행하였다.In order to measure the change in optical selectivity according to racemic substrate concentration, the coenzyme solution prepared according to the method of Example 1 was prepared in a 500 mL Erlenmeyer flask as shown in Table 1 below, and the substrate concentration was 100, 200, 500, 1000. Add 10 mL (7.4 unit), 10 mL (7.4 unit), 30 mL (12.2 unit), 50 mL (20.2 unit), respectively, according to mg / L, adjust the concentration to 0.1 M with Trizma buffer (pH 8.0). After the reaction solution was prepared to have a total volume of 100 mL, a hydrolysis reaction was performed at 50 ° C. and 200 rpm in a shaking incubator.

기질농도Substrate concentration 100 mg/L100 mg / L 200 mg/L200 mg / L 500 mg/L500 mg / L 1000 mg/L1000 mg / L 조효소액Coenzyme solution 10 mL(7.4unit)10 mL (7.4 unit) 10 mL(7.4unit)10 mL (7.4 unit) 30 mL(12.2unit)30 mL (12.2 units) 50 mL(20.2unit)50 mL (20.2 units) pHpH 8.0(pH 8.0 Trizma buffer 0.1M)8.0 (pH 8.0 Trizma buffer 0.1M) 회전수Revolutions 200 rpm200 rpm 온도Temperature 50 ℃50 ℃ 총부피Total volume 100 mL100 mL

가수분해반응 진행과정에서 광학선택도(ee값)를 측정하기 위하여 시간별로 1 mL씩 샘플링을 하여 n-헥산으로 기질과 생성물을 추출 및 회수한 후 HPLC(영린기기)를 이용하여 측정하였다. HPLC 분석조건은 Chiral Cel OD 컬럼(Diacel)을 사용하였고, 이동상은 n-헥산과 이소프로필알콜을 95:5의 비율로 흘려주었고, 이동속도(flow rate)를 1.0 mL/min로 하고, 검출은 UV 230 nm에서 수행하였다.In order to measure the optical selectivity (ee value) in the course of the hydrolysis reaction was sampled by 1 mL by time and extracted and recovered the substrate and product with n-hexane and then measured by HPLC (lighting machine). HPLC analysis was carried out using a Chiral Cel OD column (Diacel), the mobile phase flowed n-hexane and isopropyl alcohol at a ratio of 95: 5, the flow rate (1.0 mL / min), the detection was UV 230 nm was performed.

도 1 은 여러 가지 기질농도에서 전환율의 변화에 따른 라세믹 기질의 광학선택성을 나타내는 그래프인데, 기질농도가 증가할 수록 광학선택성이 감소하는 것을 알 수 있었다.Figure 1 is a graph showing the optical selectivity of the racemic substrate with the change in conversion at various substrate concentrations, it was found that the optical selectivity decreases as the substrate concentration increases.

[실시예 3] 반응생성물이 광학선택성에 미치는 영향Example 3 Effect of Reaction Products on Optical Selectivity

기질농도가 증가할 수록 광학선택성이 감소하는 이유가 가수분해 반응중에 발생하는 생성물 때문인지를 알아보기 위하여 생성물 농도를 증가시키면서 광학선택성의 변화를 조사하여 보았다. 라세믹 기질을 가수분해하여 모두 알콜 형태의 생성물로 만든 다음 기질농도 100 mg/L로 준비된 반응액에 각각을 100, 300, 500 mg/L 되도록 조절 투입하여 가수분해반응을 수행하여 광학선택성의 변화를 측정하였다. 가수분해반응은 상기 실시예 2와 동일하게 수행하였다.In order to determine whether the optical selectivity decreases as the substrate concentration increases, the change in the optical selectivity was investigated while increasing the product concentration. Hydrolysis of the racemic substrate to make the product in the form of alcohol, and then to the reaction solution prepared at a substrate concentration of 100 mg / L to each of 100, 300, 500 mg / L to control the hydrolysis reaction to change the optical selectivity Was measured. Hydrolysis reaction was carried out in the same manner as in Example 2.

도 2는 알콜 형태의 생성물의 농도변화에 따른 전환율과 광학선택도(ee)의 관계를 나타낸 그래프인데, 생성물의 농도가 증가할 수록 광학선택성이 감소하는 경향을 나타내었다. 이로써, 알콜 형태의 반응생성물이 광학선택도(%ee)를 감소시킨다는 것을 확인할 수 있었다.Figure 2 is a graph showing the relationship between the conversion rate and the optical selectivity (ee) according to the concentration change of the product in the alcohol form, the optical selectivity decreased as the concentration of the product increases. As a result, it was confirmed that the reaction product in the alcohol form reduces the optical selectivity (% ee).

[실시예 4] 반응생성물 제거에 의한 가수분해반응과 반응생성물을 포함한 가수분해반응에서 광학선택도의 변화 비교Example 4 Comparison of Changes in Optical Selectivity in Hydrolysis Reactions by Removing Reaction Products and Hydrolysis Reactions Including Reaction Products

라세믹 기질 농도를 1000 mg/L로 조절한 후 상기 실시예 2와 같은 방법으로 가수분해반응을 수행하면서 전환율이 53% 정도 되었을 때 반응을 중단하고 반응 기질과 생성물을 n-헥산으로 추출 회수한 후 실리카 겔이 함유된 컬럼을 이용하여 기질과 생성물을 분리하였고, 생성물은 제거하고 기질만을 다시 가수분해 반응을 수행하여 전환율이 약 74% 되었을 때 반응을 중단하고 상기와 같은 방법으로 생성물은 다시 제거하였고 기질만을 분리하여 최종 가수분해반응을 수행하였다. 한편, 비교군으로서 상기와 동일 조건으로 가수분해 반응을 수행하되 반응 중간에 생성물을 분리하지 않고 가수분해 반응을 수행하였다.After adjusting the racemic substrate concentration to 1000 mg / L and performing a hydrolysis reaction in the same manner as in Example 2, the reaction was stopped when the conversion was about 53%, and the reaction substrate and the product were extracted and recovered with n-hexane. Subsequently, the substrate and the product were separated using a column containing silica gel. The product was removed, and only the substrate was subjected to hydrolysis to stop the reaction when the conversion was about 74%. The product was removed again in the same manner as described above. Only the substrate was separated and the final hydrolysis reaction was performed. Meanwhile, as a comparative group, the hydrolysis reaction was carried out under the same conditions as above, but the hydrolysis reaction was performed without separating the product in the middle of the reaction.

도 3에서 보는 바와 같이 반응 생성물을 분리 제거하면서 가수분해반응을 진행한 경우가 그렇지 않은 경우에 비하여 광학선택도(%ee)가 증가함을 확인하였다.As shown in FIG. 3, it was confirmed that the optical selectivity (% ee) was increased compared to the case where the hydrolysis reaction was performed while separating and removing the reaction product.

[실시예 5] 실리카겔 컬럼 크로마토그라피를 통한 기질과 생성물의 분리Example 5 Separation of Substrate and Product by Silica Gel Column Chromatography

크로마토그라피용 컬럼에 실리카겔(Sigma Cat. No. S-9258)을 10 g 충진한 후 상기 실시예 2와 같이 가수분해반응을 수행하여 n-헥산을 이용하여 회수한 에스터 형태의 기질과 알콜 형태의 생성물 용액을 컬럼을 통과시켜 실리카겔에 흡착 시켰다. 에틸 아세테이트와 n-헥산을 1:1로 혼합한 용액을 계속 흘리면서 Fraction collector(Bio-Rad Model 2110)를 이용하여 5 mL씩 회수한 후 분획 번호(Fraction No.) 별로 회수한 용액을 HPLC를 이용하여 분석하여 기질만이 함유된 분획 만을 모아 다시 가수분해 반응에 사용하였다.After filling 10 g of silica gel (Sigma Cat. No. S-9258) in the column for chromatography, hydrolysis was carried out as in Example 2, and the ester-type substrate and alcohol were recovered using n-hexane. The product solution was adsorbed onto silica gel by passing through a column. The solution was collected by fraction number (Fraction No.) using HPLC using Fraction collector (Bio-Rad Model 2110) while continuously flowing a solution of ethyl acetate and n-hexane mixed 1: 1. Analysis was performed to collect only the fraction containing only the substrate and used for hydrolysis again.

도 4에서 나타낸 바와 같이 분획번호(Fraction No.)에 따라 에스터 형태의 기질과 알콜 형태의 생성물이 분리됨을 확인할 수 있었다.As shown in FIG. 4, the ester-type substrate and the alcohol-type product were separated according to the fraction number (Fraction No.).

악시네토박터(Acinetobacter sp.) SY-01 균주로부터 생산된 광학선택성 리파제를 이용하여 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-1,3-디옥소란-4-메틸 아세테이트를 기질로 하는 효소반응에 의하여 광학분할을 수행하는 과정에서 발생되는 알콜 형태의 생성물을 분리하면서 가수분해 반응을 수행하므로써 광학선택성을증가시킬 수 있으며, 광학순도가 높은 단일 이성체를 제조할 수 있고, 이 단일 이성체를 이용하여 여러 가지 단일 이성체 의약품을 합성하는데 이용할 수 있다.Cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane using optically selective lipase produced from Acinetobacter sp. SY-01 strain The optical selectivity can be increased by performing the hydrolysis reaction while separating the alcohol form product generated during the optical splitting by the enzymatic reaction using -4-methyl acetate as a substrate, and a single isomer having high optical purity can be obtained. It can be prepared and used to synthesize a variety of single isomeric drugs using this single isomer.

Claims (6)

라세믹 혼합물인 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-1,3-디옥소란-4-메틸 아세테이트를 기질로 하여 효소 가수분해하는 방법에 있어, 상기 가수분해는 악시네토박터(Acinetobacter sp.) SY-01 균주(KFCC-11111)로부터 생산된 리파제를 사용하여 수행하며, 상기 가수분해 반응중에 생성되는 알콜 생성물은 반응 도중에 반응물로부터 분리 회수하는 것을 특징으로 하는 고 광학선택성을 가지는 시스-2-(브로모메틸)-2-(2,4-디클로로페닐)-13-디옥소란-4-메틸알콜의 제조방법.In the method of enzymatic hydrolysis using cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -1,3-dioxolane-4-methyl acetate as a substrate as the substrate, Hydrolysis is carried out using a lipase produced from Acinetobacter sp. SY-01 strain (KFCC-11111), wherein the alcohol product produced during the hydrolysis reaction is separated and recovered from the reactants during the reaction. A process for producing cis-2- (bromomethyl) -2- (2,4-dichlorophenyl) -13-dioxolane-4-methyl alcohol having high optical selectivity. 제 1 항에 있어서, 상기 가수분해 반응물 중에 포함되는 알콜 생성물의 농도가 0.1 ∼ 300 mg/L를 유지하도록 하는 것을 특징으로 하는 제조방법.The method according to claim 1, wherein the concentration of the alcohol product contained in the hydrolysis reactant is maintained at 0.1 to 300 mg / L. 제 1 항에 있어서, 상기 가수분해 반응의 전환율이 40 ∼ 60% 범위일 때 반응생성물을 분리 회수한 후에 재차 가수분해 반응을 수행하는 것을 특징으로 하는 제조방법.The method according to claim 1, wherein the hydrolysis reaction is performed again after the reaction product is separated and recovered when the conversion rate of the hydrolysis reaction is in the range of 40 to 60%. 제 1 항에 있어서, 상기 가수분해 반응의 전환율이 40 ∼ 60%와 70 ∼ 90%범위일 때 각각 2회에 걸쳐 반응생성물을 분리 회수한 후에 재차 가수분해 반응을 수행하는 것을 특징으로 하는 제조방법.The method according to claim 1, wherein when the conversion rate of the hydrolysis reaction is in the range of 40 to 60% and 70 to 90%, the reaction product is separated and recovered twice, and the hydrolysis reaction is performed again. . 제 1 항에 있어서, 상기 가수분해 반응은 40 ∼ 60 ℃ 온도, pH 7.0 ∼ 9.0 및 회전속도 100 ∼ 300 rpm 조건에서 수행하는 것을 특징으로 하는 제조방법.The method according to claim 1, wherein the hydrolysis reaction is performed at a temperature of 40 to 60 ° C, pH 7.0 to 9.0, and a rotational speed of 100 to 300 rpm. 제 1 항에 있어서, 상기 반응생성물은 컬럼 크로마토그래피법으로 분리 회수하는 것을 특징으로 하는 제조방법.The method of claim 1, wherein the reaction product is separated and recovered by column chromatography.
KR10-2002-0040325A 2002-07-11 2002-07-11 Process for preparing cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-13-dioxolane-4-methyl alcohol of high optical selectivity KR100446256B1 (en)

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