KR20130108876A - Composition for specifically inhibiting shp-2 activity and method the same - Google Patents
Composition for specifically inhibiting shp-2 activity and method the same Download PDFInfo
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- KR20130108876A KR20130108876A KR1020120030692A KR20120030692A KR20130108876A KR 20130108876 A KR20130108876 A KR 20130108876A KR 1020120030692 A KR1020120030692 A KR 1020120030692A KR 20120030692 A KR20120030692 A KR 20120030692A KR 20130108876 A KR20130108876 A KR 20130108876A
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- C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
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- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
Abstract
Description
본 발명은 SHP-2의 활성을 특이적으로 저해하는 조성물 및 이의 제조방법에 관한 것으로, 보다 구체적으로 유방암, 폐암, 흑색종(melanoma), 신경아세포종(neuroblastoma), 급성 골수성 백혈병(acute myeloid leukemia)레오파드 증후군 타입1(LEOPARD syndrome type1), 누난 증후군(Noonan syndrome) 또는 색소 융모 결절성 활막염 질환 등에 대한 치료용 약학적 조성물 및 이의 제조방법에 관한 것이다.The present invention relates to a composition that specifically inhibits the activity of SHP-2 and a method of preparing the same, and more particularly to breast cancer, lung cancer, melanoma, neuroblastoma, acute myeloid leukemia The present invention relates to a pharmaceutical composition for treating Leopard
단백질 인산화 및 탈인산화는 세포 기능의 여러 단계 가운데 세포에 의한 신호를 유도하기 위하여 활용되는 중요한 메카니즘으로서 널리 알려져 있다. 세포에 의한 신호는 주로 키나제가 관여하는 인산화 및 단백질 포스파타제가 관여하는 탈인산화 과정을 통해 전달된다. 특히, 상기 탈인산화 과정에 작용하는 단백질 티로신 포스파타제(PTP, protein tyrosine phosphatase)는 생체 내에서 이들의 독특한 활성으로 인해 대사, 성장, 증식 및 분화에 포함된 기본적인 세포 신호화 메카니즘의 세포 내 조정 및 조절에 중요한 역할을 담당하는 것으로 알려져 있다. Protein phosphorylation and dephosphorylation are well known as important mechanisms utilized to induce signaling by cells among the various stages of cellular function. Signals by the cells are primarily transmitted through phosphorylation involving kinases and dephosphorylation involving protein phosphatase. In particular, protein tyrosine phosphatase (PTP), which acts on the dephosphorylation process, due to its unique activity in vivo, modulates and regulates the basic cellular signaling mechanisms involved in metabolism, growth, proliferation and differentiation. It is known to play an important role.
단백질 티로신 포스파타제의 활성 조절이 다양한 세포 생물학적 기능과 질병에 관여하고 있다는 연구결과를 보이고 있다. 예를 들면, 마우스에서의 PTP1B 유전자의 돌연변이는 인슐린 신호전달에, PTPRC는 많은 경화증에 관여한다는 것이다. 따라서, 이들은 암과 당뇨병을 포함한 많은 질병에 대한 치료학적 개입을 위한 중요한 표적으로 가능성이 높다.It has been shown that the regulation of protein tyrosine phosphatase activity is involved in various cellular biological functions and diseases. For example, mutations in the PTP1B gene in mice are responsible for insulin signaling and PTPRC is involved in many sclerosis. Thus, they are likely to be important targets for therapeutic intervention in many diseases, including cancer and diabetes.
SHP-2는 PTPN11 유전자에 의해 인코딩되며 N-SH2와 C-SH2의 2개의 도메인을 가진 단백질 티로신 포스파타제이다. SHP-2는 EGF, hepatocyte growth factor 및 interleukin-6과 같은 세포 신호전달 성장인자 및 싸이토카인에 의해 매개되는 세포 신호전달을 조절한다. 또한 SHP-2는 EGF에 의한 Erk 1/2 MAP kinase의 활성에도 관여한다. SHP-2 돌연변이는 아동발달장애, 누난증후군(Noonan syndrome) 및 인간의 다양한 악성종양을 일으킨다. 따라서, SHP-2는 누난증후군(Noonan syndrome) 및 SHP-2와 관련된 암 등에 대한 치료제로서 중요한 타깃이 되고 있다.SHP-2 is a protein tyrosine phosphatase encoded by the PTPN11 gene and has two domains, N-SH2 and C-SH2. SHP-2 modulates cell signaling mediated by cytokine and cellular signaling growth factors such as EGF, hepatocyte growth factor and interleukin-6. SHP-2 is also involved in
이와 관련, SHP-2 활성을 조절하는 저해제에 관한 연구가 있으나, 이는 SHP-2를 포함한 다른 단백질 티로신 포스파타제의 활성에도 관여하는 문제점을 보여왔다.(Liwei Chen et al., Mol Pharmacol 80:562-570,2006)In this regard, there have been studies of inhibitors that modulate SHP-2 activity, but this has been shown to be involved in the activity of other protein tyrosine phosphatase, including SHP-2 (Liwei Chen et al., Mol Pharmacol 80: 562-). 570,2006)
본 발명은 상기와 같은 종래 기술의 문제점을 해결하기 위하여, 단백질 티로신 포스파타제중 SHP-2의 활성을 특이적으로 저해 시키는 치료용 약학적 조성물 및 이의 제공방법을 제공하는 것을 목적으로 한다..In order to solve the problems of the prior art as described above, an object of the present invention is to provide a therapeutic pharmaceutical composition and method for providing the same specifically inhibiting the activity of SHP-2 in protein tyrosine phosphatase.
상기의 목적을 달성하기 위하여, In order to achieve the above object,
본 발명은 MLS-1 화합물을 제공한다.The present invention provides MLS-1 compounds.
또한, 본 발명은 MLS-1을 유효성분으로 포함하는 유방암, 폐암, 흑색종(melanoma), 신경아세포종(neuroblastoma), 급성 골수성 백혈병(acute myeloid leukemia), 레오파드 증후군 타입1(LEOPARD syndrome type1), 누난 증후군(Noonan syndrome) 또는 색소 융모 결절성 활막염 질환 치료용 약학적 조성물을 제공한다.In addition, the present invention is a breast cancer, lung cancer, melanoma (melanoma), neuroblastoma (neuroblastoma), acute myeloid leukemia (LEOPARD syndrome type 1), nunan comprising MLS-1 as an active ingredient Provided is a pharmaceutical composition for treating Noonan syndrome or pigmented chorionic nodular synoviitis disease.
또한. 본 발명은 상기 치료용 약학적 조성물은 SHP-2(Src homology 2 (SH2)-containing protein tyrosine phosphatase)를 특이적으로 억제하는, 치료용 약학적 조성물을 제공한다Also. The present invention provides a therapeutic pharmaceutical composition, the therapeutic pharmaceutical composition specifically inhibits SHP-2 (SH2) -containing protein tyrosine phosphatase (SHP-2).
또한, 본 발명은 하기단계를 포함하는, In addition, the present invention includes the following steps,
1) 벤즈이미다졸 및 소듐하이드라드를 THF(Tetrahydrofuran)에 녹인 후, 반응 혼합물에 메틸브로모아세테이트를 첨가하고, 반응 혼합물을 에틸아세테이트로 희석 후, 물 및 브린(brine)으로 세척하여 무수 마그네슘설페이트으로 건조한 후에 여액을 감압 농축하여 얻은 잔사를 컬럼크로마트그래피하여 메틸 2-(1에이치-벤조[d]이미다졸-1-일)아세테이트를 제조하는 단계;1) After dissolving benzimidazole and sodium hydride in THF (Tetrahydrofuran), methylbromoacetate is added to the reaction mixture, the reaction mixture is diluted with ethyl acetate, washed with water and brine, and dried over anhydrous magnesium sulfate. Preparing a methyl 2- (1-H-benzo [d] imidazol-1-yl) acetate by column chromatography on a residue obtained by concentrating the filtrate under reduced pressure and drying the mixture;
2) 상기 메틸 2-(1에이치-벤조[d]이미다졸-1-일)아세테이트를 알코올에 녹인후 알코올에 녹인 하이드라진 하이드레이트를 첨가 후, 반응 혼합물을 환류하고, 반응 종결 후 재결정하여 2-(1에이치-벤조[d]이미다졸-l)아세토하이드라자이드를 제조하는 단계;2) After dissolving the methyl 2- (1-H-benzo [d] imidazol-1-yl) acetate in alcohol and adding hydrazine hydrate dissolved in alcohol, the reaction mixture was refluxed and recrystallized after completion of the reaction to give 2- ( Preparing 1-benzo [d] imidazole-1) acetohydrazide;
3) 알코올에 수산화칼륨을 녹이고 상기 2-(1에이치-벤조[d]이미다졸-일)아세토하이드라자이드 및 카본 디설파이드를 첨가 후, 에테르로 희석 후 떨어진 침전물을 모으고, 에테르로 한번 더 세척 후 건조시켜 물에 녹이고 하이드라진 하이드레이트을 첨가하여 환류 후, 산성조건에서 얻어진 침전물을 건조하여 5-((1에이치-벤조[d]이미다졸-1-일)메틸)-4-아미노-4에이치-1,2,4-트리아졸-3-티올을 제조하는 단계; 및3) After dissolving potassium hydroxide in alcohol and adding the 2- (1-H-benzo [d] imidazol-yl) acetohydrazide and carbon disulfide, the precipitates separated after dilution with ether are collected and washed once more with ether. After drying, it was dissolved in water, hydrazine hydrate was added to reflux, and the precipitate obtained under acidic condition was dried to give 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1, Preparing 2,4-triazole-3-thiol; And
4) 상기 5-((1에이치-벤조[d]이미다졸-1-일)메틸)-4-아미노-4에이치-1,2,4-트리아졸-3-티올, 피-톨루익 산 및 포스포릴 클로라이드를 넣고 환류하고서 침전물을 생성하고 건조하여 MLS-1을 제조하는 단계를 제공한다.4) the 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol, py-toluic acid and Phosphoryl chloride is added and refluxed to form a precipitate and dried to provide a step for preparing MLS-1.
본 발명은 단백질 티로신 포스파타제중 SHP-2의 활성을 특이적으로 저해 시킴으로서, 유방암, 폐암, 흑색종(melanoma), 신경아세포종(neuroblastoma), 급성 골수성 백혈병(acute myeloid leukemia), 레오파드 증후군 타입1(LEOPARD syndrome type1), 누난 증후군(Noonan syndrome) 또는 색소 융모 결절성 활막염 질환 등 SHP-2와 연관된 질병의 연구와 SHP-2의 기작의 연구에 이용될 수 있을 것으로 기대된다.The present invention specifically inhibits the activity of SHP-2 in protein tyrosine phosphatase, such as breast cancer, lung cancer, melanoma, neuroblastoma, acute myeloid leukemia, leopard syndrome type 1 (LEOPARD) It is expected to be used for the study of SHP-2-related diseases such as syndrome type1, Nuonan syndrome or pigmented chorio nodular synovial disease, and the mechanism of SHP-2.
도 1은 MLS-1의 농도에 따른 SHP-2의 저해효과를 나타낸 결과이다.
도 2은 SHP-2에 대한 MLS-1의 kinectic analysis를 나타낸 결과이다.
도 3은 MLS-1의 세포투과성과 세포 내 발현된 SHP-2 억제효과를 나타낸 결과이다.1 is a result showing the inhibitory effect of SHP-2 according to the concentration of MLS-1.
2 shows the results of kinectic analysis of MLS-1 on SHP-2.
3 is a result showing the cell permeability of MLS-1 and the SHP-2 inhibitory effect expressed in cells.
본 발명자들은 단백질 티로신 포스파타제 중 SHP-2의 활성을 특이적으로 저해하는 저해제를 개발하기 위하여 노력한 결과, MLS-1인 신규화합물 및 이의 제조방법을 완성하게 되었다.The present inventors have endeavored to develop an inhibitor that specifically inhibits the activity of SHP-2 in protein tyrosine phosphatase. As a result, a novel compound of MLS-1 and a method for preparing the same have been completed.
본 발명은 하기 화학식 1의 화합물 또는 이의 약학적으로 허용가능한 염을 제공한다.The present invention provides a compound of
[화학식 1][Formula 1]
구체적으로 MLS-1을 유효성분으로 포함하는 유방암, 폐암, 흑색종(melanoma), 신경아세포종(neuroblastoma), 급성 골수성 백혈병(acute myeloid leukemia), 레오파드 증후군 타입1(LEOPARD syndrome type1), 누난 증후군(Noonan syndrome) 또는 색소 융모 결절성 활막염 질환, 치료용 약학적 조성물을 제공하며, SHP-2(Src homology 2 (SH2)-containing protein tyrosine phosphatase)를 특이적으로 억제하는, 치료용 약학적 조성물을 제공한다.Specifically, breast cancer, lung cancer, melanoma, neuroblastoma, acute myeloid leukemia, LEOPARD
본 발명에서는 MLS-1은 상기 화학식 1의 화합물을 말한다.
In the present invention, MLS-1 refers to the compound of Formula 1.
발명의 치료용 약학적 조성물은 기존 치료 활성 성분, 기타 보조제, 약제학적으로 허용 가능한 담체를 포함할 수 있다. 상기 약제학적으로 허용 가능한 담체는 이러한 조성물에 포함될 수 있는 적합한 담체, 부형제 및 희석제의 예로는 락토오스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 비정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 들 수 있다. 상기 조성물은 약제화하는 경우, 통상의 충진제, 증량제, 결합제, 붕해제, 계면활성제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있으며, 이에 한정되지는 않는다.The therapeutic pharmaceutical compositions of the invention may comprise existing therapeutically active ingredients, other adjuvants, pharmaceutically acceptable carriers. Examples of suitable carriers, excipients and diluents which may be included in such compositions include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When formulated, the composition may further include, but is not limited to, conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like.
본 발명의 치료용 약학적 조성물은 일반적인 의약품 제제의 형태로 사용될 수 있으며, 제제화 할 경우에는 약제학적으로 허용 가능한 희석제 또는 부형제 등을 사용할 수 있다. 본 발명의 조성물은 각각의 사용 목적에 맞게 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁제, 에멀젼, 시럽, 에어로졸 등의 경구 제형, 멸균 주사용액의 형태 등 다양한 형태로 제형화 하여 사용할 수 있으며, 경구투여하거나 정맥내, 복강내, 피하, 직장, 국소 투여 등을 포함한 다양한 경로를 통해 투여될 수 있다.The therapeutic pharmaceutical composition of the present invention may be used in the form of a general pharmaceutical formulation, and when formulated, a pharmaceutically acceptable diluent or excipient may be used. The composition of the present invention may be formulated into various forms such as powders, granules, tablets, capsules, oral formulations such as suspensions, emulsions, syrups and aerosols, and sterilized injection solutions according to conventional methods And can be administered via various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.
경구투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 조성물에 적어도 하나 이상의 부형제, 예를 들면 전분, 탄산칼슘, 수크로스, 락토오스, 젤라틴 등을 섞어 제형화 한다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크와 같은 윤활제들도 사용된다.Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin and the like. Mix and formulate. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
경구용 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 액체 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin.
비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 주사제의 기제로는 용해제, 등장화제, 현탁화제, 유화제, 안정화제 및 방부제와 같은 종래의 첨가제를 포함할 수 있다.Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Base materials for injections may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.
본 발명에 있어서 투여는 임의의 적절한 방법으로 환자에게 소정의 물질을 제공하는 것을 의미하며, 본 발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 일반적인 모든 경로를 통하여 경구 또는 비경구 투여될 수 있다. 또한, 조성물은 활성물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.Administration in the present invention means providing the desired material to the patient by any suitable method, and the administration route of the composition of the present invention may be administered orally or parenterally through any general route as long as it can reach the target tissue . In addition, the composition may be administered by any device in which the active agent may migrate to the target cell.
본 발명의 조성물은 약제학적으로 유효한 양으로 투여한다.The composition of the present invention is administered in a pharmaceutically effective amount.
본 발명에 있어서 용어, 약제학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.As used herein, the term pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug, the drug. Sensitivity to, time of administration, route of administration and rate of administration, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
또한, 본 발명에 따른 화합물의 투여량은 체내 흡수도, 체중, 환자의 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율, 질환의 중증도 등에 따라 변화될 수 있다. 투여는 하루에 한번 투여할 수도 있고, 여러번 나누어 투여할 수 있다.
In addition, the dosage of the compound according to the present invention may be changed according to body absorption, weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, and the like. Administration may be administered once a day or may be divided several times.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
[실시예][Example]
실시예Example 1 One : : MLSMLS -1 합성법-1 synthesis
1. One. MethylMethyl 2-(1H- 2- (1H- benzobenzo [d]imidazol-1-[d] imidazol-1- ylyl )) acetateacetate 의 합성Synthesis of
Benzimidazole (300mg, 2.54mmol)및 NaH (91mg, 3.75mmol)을 THF(5ml) 에 녹인후 상온에서 교반하였다. 30 분 후 0℃에서 반응 혼합물에 Methyl Bromo Acetate (388mg, 2.5mmol)을 첨가하고 실온에서 3시간 동안 교반하였다. 반응 혼합물을 EtOAc로 희석한 후에 물과 brine으로 세척하였다. 무수 MgSO4으로 건조한 후에 여액을 감압 농축하여 얻은 잔사를 column chromatography (MC:MeOH=10:1)하여 Methyl 2-(1H-benzo[d]imidazol-1-yl)acetate(83mg,0.41mmol,16%)을 합성하였다. Benzimidazole (300 mg, 2.54 mmol) and NaH (91 mg, 3.75 mmol) were dissolved in THF (5 ml) and stirred at room temperature. After 30 minutes, Methyl Bromo Acetate (388mg, 2.5mmol) was added to the reaction mixture at 0 ° C, and stirred for 3 hours at room temperature. The reaction mixture was diluted with EtOAc and washed with water and brine. After drying over anhydrous MgSO 4 , the filtrate was concentrated under reduced pressure. The residue obtained by column chromatography (MC: MeOH = 10: 1) was purified by Methyl 2- (1H-benzo [d] imidazol-1-yl) acetate (83mg, 0.41mmol, 16). %) Was synthesized.
1H-NMR(DMSO-d6,300MHz): δ 8.20 (s, 1H), 7.66 (dd, 1H, J=6Hz,1.5Hz),7.55(dd,1H,J=6Hz,1.5Hz),7.21~7.26(m,2H),5.27(s,2H),3.71(s,3H). 1 H-NMR (DMSO-d 6 , 300 MHz): δ 8.20 (s, 1H), 7.66 (dd, 1H, J = 6Hz, 1.5Hz), 7.55 (dd, 1H, J = 6Hz, 1.5Hz), 7.21 7.26 (m, 2H), 5.27 (s, 2H), 3.71 (s, 3H).
2. 2-(1H-2. 2- (1H- benzobenzo [d]imidazol-1-[d] imidazol-1- ylyl )) acetohydrazideacetohydrazide 의 합성Synthesis of
상기 Methyl 2-(1H-benzo[d]imidazol-1-yl)acetate(83mg, 0.41mmol)을 EtOH 10ml에 녹인후 EtOH(1ml)에 녹인 Hydrazine hydrate(40mg, 0.81mmol)을 한방울씩 첨가하면서 교반하였다. 반응 혼합물을 14시간 환류하였다. 반응 종결 후 재결정하여 2-(1H-benzo[d]imidazol-1-yl)acetohydrazide(20mg, 0.36mmol, 89%)을 합성하였다. The Methyl 2- (1H-benzo [d] imidazol-1-yl) acetate (83mg, 0.41mmol) was dissolved in 10ml of EtOH and stirred while adding Hydrazine hydrate (40mg, 0.81mmol) dissolved in EtOH (1ml) drop by drop. It was. The reaction mixture was refluxed for 14 hours. After completion of the reaction, recrystallization was performed to synthesize 2- (1H-benzo [d] imidazol-1-yl) acetohydrazide (20mg, 0.36mmol, 89%).
1H-NMR (DMSO-d6,300MHz): δ 9.55 (s, 1H), 8.15 (s, 1H), 7.65 (d, 1H, J=8.1Hz),7.47(d,1H,J=7.5Hz),7.18~7.27(m,2H),4.88(s,2H),4.45(br,2H).1 H-NMR (DMSO-d 6,300 MHz): δ 9.55 (s, 1H), 8.15 (s, 1H), 7.65 (d, 1H, J = 8.1 Hz), 7.47 (d, 1H, J = 7.5 Hz), 7.18-7.27 (m, 2H), 4.88 (s, 2H), 4.45 (br, 2H).
3. 5-((1H-benzo[d]imidazol-1-yl)methyl)-4-amino-4H-1,2,4-triazole-3-thiol의 합성3. Synthesis of 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol
Absolute ethanol(1ml)에 KOH(20mg, 0.36mmol)를 녹이고 0℃에서 2-(1H-benzo[d]imidazol-1-yl)acetohydrazide(20mg, 0.36mmol) 및 carbon disulfide (27mg, 0.36mmol) 를 첨가하고 1시간 동안 교반하였다. Ether로 희석한 후 떨어진 침전물을 모았다. Ether로 한번 더 씻어 준 후 건조시켜서 다음 반응을 진행 시켰다. 침전물을 물(2ml)에 녹이고 물(1ml)에 녹인 Hydrazine hydrate(12mg, 0.36mmol)을 한방울씩 반응 플라스크에 첨가하여 주었다. 반응 혼합물을 12시간 환류하였다. 반응 종결 후 산성조건에서 얻어진 침전물을 건조하여 5-((1H-benzo[d]imidazol-1-yl)methyl)-4-amino-4H-1,2,4-triazole-3-thiol(20mg, 0.08mmol, 30%)을 합성하였다. Dissolve KOH (20mg, 0.36mmol) in Absolute ethanol (1ml) and 2- (1H-benzo [d] imidazol-1-yl) acetohydrazide (20mg, 0.36mmol) and carbon disulfide (27mg, 0.36mmol) at 0 ℃ Add and stir for 1 hour. After dilution with Ether collected the sediment that fell. Washed once more with ether and dried to proceed to the next reaction. The precipitate was dissolved in water (2ml) and Hydrazine hydrate (12mg, 0.36mmol) dissolved in water (1ml) was added dropwise to the reaction flask. The reaction mixture was refluxed for 12 hours. After completion of the reaction, the precipitate obtained under acidic condition was dried to give 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol (20mg, 0.08 mmol, 30%).
1H-NMR (DMSO-d6,300MHz): δ 8.28 (s, 1H), 7.65 (d, 1H, J=6Hz),7.57(d,1H,J=6Hz),7.21~7.26(m,2H),5.66(s,2H),5.59(s,2H).1 H-NMR (DMSO-d 6,300 MHz): δ 8.28 (s, 1H), 7.65 (d, 1H, J = 6 Hz), 7.57 (d, 1H, J = 6 Hz), 7.71-7.26 (m, 2H), 5.66 (s, 2 H), 5.59 (s, 2 H).
4. 3-((1H- benzo [d]imidazol-1- yl ) methyl )-6-p- tolyl -[1,2,4] triazolo [3,4-b][1,3,4] t hiadiazole(MLS-001)의 합성 4. 3 - ((1H- benzo [ d] imidazol-1- yl) methyl) -6-p- tolyl - [1,2,4] triazolo [3,4-b] [1,3,4] t Synthesis of hiadiazole (MLS-001)
반응 플라스크에 5-((1H-benzo[d]imidazol-1-yl)methyl)-4-amino-4H-1,2,4-triazole-3-thiol(20mg,0.08mmol), p-toluic acid (16mg, 0.12mmol)및 POCl3 (2ml)를 넣고 환류하였다. pH 8 이 되도록 조절하여 침전물을 생성하고 건조하여 3-((1H-benzo[d]imidazol-1-yl)methyl)-6-p-tolyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole(MLS-001)(20mg, 0.06mmol, 72%)를 얻었다. 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol (20mg, 0.08mmol), p-toluic acid in the reaction flask (16 mg, 0.12 mmol) and POCl 3 (2 ml) were added and refluxed. pH is adjusted to 8 to form a precipitate and dried to 3-((1H-benzo [d] imidazol-1-yl) methyl) -6-p-tolyl- [1,2,4] triazolo [3,4- b] [1,3,4] thiadiazole (MLS-001) (20 mg, 0.06 mmol, 72%) was obtained.
1H-NMR (DMSO-d6,300MHz): δ 8.44 (s, 1H), 7.76 (d, 2H, J=5.1Hz),7.65(t,2H,J=4.5Hz),7.36(d,2H,J=5.1Hz),7.19~7.25(m,2H),6.04(s,2H),2.39(s,3H).1 H-NMR (DMSO-d 6,300 MHz): δ 8.44 (s, 1H), 7.76 (d, 2H, J = 5.1 Hz), 7.65 (t, 2H, J = 4.5 Hz), 7.36 (d, 2H, J = 5.1 Hz), 7.19-7.25 (m, 2H), 6.04 (s, 2H), 2.39 (s, 3H).
[반응식][Reaction Scheme]
실시예Example 2 2 : 6x : 6x HisHis -- taggedtagged 포스파타제Phosphatase 단백질의 정제 Purification of proteins
PTP(protein tyrosine phosphatase) 발현 플라스미드는 pET28a (+)(Novagen, Darmstadt)를 사용하였으며, BL21 (DE3)-RIL E. coli에 형질전환하였다. 재조합된 단백질은 E.coli를 37, 30 또는 22 ℃에서 3시간 내지 16시간동안 1mM isopropyl β-D-1-thiogalactopyranoside로 처리하여 발현이 유도되었다. 세포를 배양한 후, 50 mM Tris-HCl (pH 8), 300 mM NaCl, 1% Tergitol-type NP-40, 1 mM phenylmethylsulphonyl fluoride (PMSF) 조건에서 음파처리(sonication)하였다. 용해물은 4℃에 30분간 10000rpm으로 처리하였다. 그 다음, 상등액을 Ni-NTA resin (PEPTRON, Daejon) 칼럼에 통과시켰다. resin을 20 mM Tris-HCl (pH 8), 500 mM NaCl, 50 mM imidazole 하에 세척하였으며, 20 mM Tris-HCl (pH 8), 500 mM NaCl, 200 - 300 mM imidazole 하에 분리하였다. 분리된 단백질은 - 80℃ 상태에서 저장하기 전, 20 mM Tris-HCl (pH 8), 100 mM NaCl, 30% glycerol, 0.5 mM PMSF 하에 하룻밤동안 투석하였다.
PTP (protein tyrosine phosphatase) expression plasmid was used pET28a (+) (Novagen, Darmstadt) and transformed to BL21 (DE3) -RIL E. coli. The recombinant protein was induced by treatment of E. coli with 1 mM isopropyl β-D-1-thiogalactopyranoside at 37, 30 or 22 ° C. for 3 to 16 hours. The cells were cultured and then sonicated in 50 mM Tris-HCl (pH 8), 300 mM NaCl, 1% Tergitol-type NP-40, and 1 mM phenylmethylsulphonyl fluoride (PMSF). The lysate was treated at 10000 rpm at 4 ° C. for 30 minutes. The supernatant was then passed through a Ni-NTA resin (PEPTRON, Daejon) column. The resin was washed under 20 mM Tris-HCl (pH 8), 500 mM NaCl, 50 mM imidazole and separated under 20 mM Tris-HCl (pH 8), 500 mM NaCl, 200-300 mM imidazole. The isolated protein was dialyzed overnight under 20 mM Tris-HCl (pH 8), 100 mM NaCl, 30% glycerol, 0.5 mM PMSF before storage at -80 ° C.
실시예 3 : 생체외(In vitro)에서의 phosphatase assays 및 kinetic analysis Example 3 : In vitro phosphatase assays and kinetic 분석
PTP의 활성은 96-well microtiter plate assay에서 3-Omethylfluorescein phosphate (OMFP; Sigma, St. Louis, MO)을 이용하여 측정하였다. MLS-1 및 OMFP는 DMSO에서 가용하였으며, 모든 반응에 있어 최종농도는 1% DMSO에서 수행하였다. 최종 배양 혼합물(100μL)은 효소활성을 위해 최적화 되었으며, 30 mM Tris-HCl (pH 7), 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.33% bovine serum albumin (BSA), 100 nM of PTP 및 저해제들로 구성되었다. 반응은 OMFP를 추가하면서 시작되었고, 37 ℃에서 30분간 배양되었다. 산물로부터 형광배출은 multiwell plate reader (synergy H1 (BioTek); excitation filter, 485 nm; emission filter, 535 nm)로 측정되었다. 실험 시간에 따라 직접적으로 효소와 기질의 농도가 비례하였다. Half-maximal inhibition constant (IC50)은 PTP의 활성을 50%정도 감소시키는 농도를 의미한다. Half-maximal inhibition constant(IC50) 및 Lineweaver-Burk plots의 커브피트(Curve fit)은 curve fitting program Prism 3.0 (GraphPad Software)을 이용하여 결정되었다. 모든 실험은 세번에 걸쳐 수행되었다.
PTP activity was measured using 3-Omethylfluorescein phosphate (OMFP; Sigma, St. Louis, MO) in a 96-well microtiter plate assay. MLS-1 and OMFP were available in DMSO and final concentrations were performed in 1% DMSO for all reactions. The final culture mixture (100 μL) was optimized for enzymatic activity, 30 mM Tris-HCl (pH 7), 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.33% bovine serum albumin (BSA), 100 nM of PTP and Consist of inhibitors. The reaction started with the addition of OMFP and incubated at 37 ° C. for 30 minutes. Fluorescence emission from the product was measured with a multiwell plate reader (synergy H1 (BioTek); excitation filter, 485 nm; emission filter, 535 nm). The concentration of enzyme and substrate was directly proportional to the experiment time. Half-maximal inhibition constant (IC 50 ) is the concentration that reduces PTP activity by 50%. The curve fit of the half-maximal inhibition constant (IC 50 ) and Lineweaver-Burk plots was determined using the curve fitting program Prism 3.0 (GraphPad Software). All experiments were performed three times.
실시예Example 4 4 : 저해 연구 : Inhibition studies
PTP에 대한 저해제의 저해 상수(inhibition constant, Ki)는 각각의 고정된 저해제 농도와 여러가지 OMFP의 초기 속도 측정에 의해 결정되었다. 아래의 식에 따라 경쟁적 저해제의 저해상수를 얻을 수 있다. 기울기는 저해제 농도에 따라 표시되었다. Ki값은 기울기에 의해 얻어진다.Inhibition constants (K i ) of inhibitors against PTP were determined by measuring the respective fixed inhibitor concentrations and the initial rate of various OMFPs. The inhibition constant of the competitive inhibitor can be obtained by the following equation. The slope was expressed according to the inhibitor concentration. K i values are obtained by slope.
1 / V = Km (1 + [I] / Ki) Vmax [S] + 1 / Vmax
1 / V = Km (1 + [I] / K i ) V max [S] + 1 / V max
실시예Example 5 5 : : MLSMLS -1의 -1 of 세포내에서의Intracellular SHPSHP -2 -2 포스파타제Phosphatase 활성 확인 실험 Activity Verification Experiment
HEK293 세포를 FLAG-SHP-2 포스파타제가 발현된 플라스미드를 이용하여 형질전환하였다. 전환 48시간 후, MLS-1 (20 μM)를 3시간 전처리 하였다. 세포는 phosphate buffered saline (PBS) buffer로 2번 세척하였고, 4℃에 30분간 PTP lysis buffer(0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% glycerol, 1 mM PMSF, and 1 μg/ml aprotinin)로 용해시켰다. 원심분리기로부터의 세포용해물을 세척된 FLAG M2-agarose (Sigma-Aldrich, St.Louis, MO)와 혼합하였고, 4℃에 1시동안 회전장치를 이용하여 배양하였다. 배양후, FLAG M2-agarose는 PTP lysis buffer에 의해 3번 세척하였으며, 포스파타제의 활성을 측정하였다.
HEK293 cells were transformed using a plasmid expressing FLAG-SHP-2 phosphatase. 48 hours after conversion, MLS-1 (20 μΜ) was pretreated for 3 hours. Cells were washed twice with phosphate buffered saline (PBS) buffer and PTP lysis buffer (0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0) for 30 minutes at 4 ° C. , 1 mM EDTA, 1% glycerol, 1 mM PMSF, and 1 μg / ml aprotinin). Cell lysates from the centrifuge were mixed with washed FLAG M2-agarose (Sigma-Aldrich, St. Louis, MO) and incubated using a rotator at 4 ° C. for 1 hour. After incubation, FLAG M2-agarose was washed three times with PTP lysis buffer, and phosphatase activity was measured.
실시예 6 : 세포내 ERK 인산화에서의 MLS -1의 효과 확인 실험 Example 6 : Intracellular Experiment confirming the effect of MLS- 1 on ERK phosphorylation
혈청이 없는 배지에서 배양한 BOSC 세포를 MLS-1 (0, 20 μM)에서 3시간 전처리하였고, EGF (1ng/ml, 5min)로 자극을 주었다. 세포는 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 및 1 mM NaF을 포함하는 lysis buffer에서 용해시켰다. 이러한 샘플들은 SDS-PAGE에 의해 분리되었고, 웨스턴블로팅을 수행하였다.
BOSC cells cultured in serum-free medium were pretreated for 3 hours in MLS-1 (0, 20 μM) and stimulated with EGF (1 ng / ml, 5 min). The cells comprise 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 mM Na 3 VO 4 , and 1 mM NaF. It was dissolved in lysis buffer. These samples were separated by SDS-PAGE and western blotting was performed.
실시예 7 : 웨스턴 블로팅 분석 Example 7 : Western Blotting analysis
샘플은 SDS-12% polyacrylamide gel에 통과시켰으며, 나이트로셀룰로스막에 전달하였다. 막을 5% 저지방 탈지유로 차단하였고, 적절한 항체로 배양하였으며, 겨자무과산화효소에 2차 항체를 결합하여 배양하였다. immunoreactive band는 ECL system(Younginfrontier)을 이용하여 관찰하였다.
Samples were passed through SDS-12% polyacrylamide gel and delivered to nitrocellulose membrane. Membranes were blocked with 5% low fat skim milk, incubated with appropriate antibodies, and cultured by binding a secondary antibody to mustard peroxidase. Immunoreactive bands were observed using an ECL system (Younginfrontier).
실시예Example
8 8
: :
SHPSHP
-2의 특이적인 활성 저해제의 확인 : Identification of specific activity inhibitors of -2:
MLSMLS
-1-One
1. MLS-1에 의해 저해되는 PTPs의 선별1. Screening of PTPs Inhibited by MLS-1
실험을 통해 12가지 재조합 단백질에서의 MLS-1의 저해 효과를 측정하였다. 구체적으로 ACP-1, cdc25A, DUSP3, DUSP14, DUSP18, DUSP22, DUSP23, DUSP26, PTPN6, PTPn2, SSh3, SHP-2를 포함한 PTPs의 MLS-1에 의한 활성저해를 측정하였다. 각 실험은 3번에 걸쳐 진행되었다. PTPs를 MLS-1의 다양한 농도로 처리하였을 때(0 - 50 μM), 오직 SHP-2의 활성만이 MLS-1의 용량의존적으로 감소하였다(표 1). SHP-2의 저해 곡선을 나타내었고 IC50 값을 계산하였다. IC50 값은 4.49±0.34 μM(n=3)을 보였다(도 2).Experiments have determined the inhibitory effects of MLS-1 on 12 recombinant proteins. Specifically, MLS-1 inhibition of PTPs including ACP-1, cdc25A, DUSP3, DUSP14, DUSP18, DUSP22, DUSP23, DUSP26, PTPN6, PTPn2, SSh3 and SHP-2 was measured. Each experiment was conducted three times. When PTPs were treated at various concentrations of MLS-1 (0-50 μM), only SHP-2 activity decreased dose dependently of MLS-1 (Table 1). Inhibition curves of SHP-2 were shown and IC 50 values were calculated. IC 50 values showed 4.49 ± 0.34 μM (n = 3) (FIG. 2).
2. Kinetic assay2. Kinetic assay
SHP-2와 MLS-1의 결합 기작을 알기 위해, SHP-2와 MLS-1과의 kinetic assay를 수행하였다. kinetic assay를 통해 SHP-2와 MLS-1은 경쟁적 저해라는 것을 알게 되었다. 3-Omethylfluorescein phosphate (OMFP)의 SHP-2 phsophatase의 KM 값은 3.41 μM 이었다. Lineweaver-Burk plot은 Ki가 3.477 μM인 것을 보여 주었다(도 3). 이러한 결과는 MLS-1이 SHP-2를 경쟁적으로 저해하며, MLS-1이 SHP-2의 활성부위에 결합한다는 것을 제시한다.
To determine the binding mechanism of SHP-2 and MLS-1, kinetic assay of SHP-2 and MLS-1 was performed. The kinetic assay revealed that SHP-2 and MLS-1 are competitive inhibitions. The K M value of SHP-2 phsophatase of 3-Omethylfluorescein phosphate (OMFP) was 3.41 μM. Lineweaver-Burk plot showed that K i was 3.477 μM (FIG. 3). These results suggest that MLS-1 competitively inhibits SHP-2 and that MLS-1 binds to the active site of SHP-2.
3. MLS-1의 세포내에서의 SHP-2 포스파타제 활성 저해 효과3. Inhibitory Effect of MLS-1 on SHP-2 Phosphatase Activity in Cells
MLS-1이 동물세포속으로 들어가 SHP-2 작용을 억제하는지를 보여주는 실험을 실시하였다. 다음 실험을 통해, MLS-1은 세포를 관통한다는 것을 알게 되었다. HEK 293 세포에 FLAG-SHP-2가 발현된 플라스미드로 형질전환하였고, MLS-1을 20 μM로 3시간 전처리하였으며, 그 다음으로 HepG2 세포를 용해하였고, anti FLAG M2-agarose를 이용하여 면역침강을 수행하였다. SHP-2 면역침강의 포스파타제 활성은 OMFP를 이용하여 측정하였다(도 4). 구체적으로, 면역침강된 SHP-2은 OMFP와 37℃에서 30분간 배양되었다. MLS-1은 SHP-2 세포에서 포스파타제 활성을 저해하였다. 이러한 결과는 MLS-1은 세포내로 이용하여 SHP-2의 활성을 저해한다는 것을 의미한다.
Experiments were conducted to show whether MLS-1 enters animal cells and inhibits SHP-2 action. In the next experiment, we found that MLS-1 penetrated the cells. HEK 293 cells were transformed with plasmids expressing FLAG-SHP-2, MLS-1 was pretreated at 20 μM for 3 hours, HepG2 cells were lysed, and immunoprecipitation was performed using anti FLAG M2-agarose. Was performed. Phosphatase activity of SHP-2 immunoprecipitation was measured using OMFP (FIG. 4). Specifically, immunoprecipitated SHP-2 was incubated with OMFP at 37 ° C. for 30 minutes. MLS-1 inhibited phosphatase activity in SHP-2 cells. These results indicate that MLS-1 inhibits the activity of SHP-2 intracellularly.
4. 세포내 ERK 인산화에서의 MLS-1의 효과4. Effect of MLS-1 on Intracellular ERK Phosphorylation
MLS-1의 세포내에서의 SHP-2활성은 내생의 ERK(extracellular signal regulated kinase)의 인산화 상태를 측정함에 따라 알 수 있었다. BOSC 세포를 20 μM으로 3시간 전처리 하였으며, EGF(1ng/ml, 5min)로 자극을 주었다. phospho-ERK의 수준은 웨스턴 블로팅(Western blotting)을 통해 알게 되었다. EGF가 매개하는 ERK의 인산화는 MLS-1에 의해 현저하게 저해되었다(도9). 이러한 결과는 MLS-1이 SHP-2 활성을 저해함에 따라 SHP-2가 매개하는 ERK인산화 과정이 저해됨을 나타낸다.Intracellular SHP-2 activity of MLS-1 was determined by measuring the phosphorylation status of endogenous extracellular signal regulated kinase (ERK). BOSC cells were pretreated with 20 μM for 3 hours and stimulated with EGF (1ng / ml, 5min). The level of phospho-ERK was determined by Western blotting. EGF-mediated phosphorylation of ERK was markedly inhibited by MLS-1 (FIG. 9). These results indicate that as MLS-1 inhibits SHP-2 activity, SHP-2 mediated ERK phosphorylation process is inhibited.
Claims (4)
[화학식 1]
[Formula 1]
1) 벤즈이미다졸 및 소듐하이드라드를 THF(Tetrahydrofuran)에 녹인 후, 반응 혼합물에 메틸브로모아세테이트를 첨가하고, 반응 혼합물을 에틸아세테이트로 희석 후, 물 및 브린(brine)으로 세척하여 무수 마그네슘설페이트으로 건조한 후에 여액을 감압 농축하여 얻은 잔사를 컬럼크로마트그래피하여 메틸 2-(1에이치-벤조[d]이미다졸-1-일)아세테이트를 제조하는 단계;
2) 상기 메틸 2-(1에이치-벤조[d]이미다졸-1-일)아세테이트를 알코올에 녹인후 알코올에 녹인 하이드라진 하이드레이트를 첨가 후, 반응 혼합물을 환류하고, 반응 종결 후 재결정하여 2-(1에이치-벤조[d]이미다졸-l)아세토하이드라자이드를 제조하는 단계;
3) 알코올에 수산화칼륨을 녹이고 상기 2-(1에이치-벤조[d]이미다졸-일)아세토하이드라자이드 및 카본 디설파이드를 첨가 후, 에테르로 희석 후 떨어진 침전물을 모으고, 에테르로 한번 더 세척 후 건조시켜 물에 녹이고 하이드라진 하이드레이트을 첨가하여 환류 후, 산성조건에서 얻어진 침전물을 건조하여 5-((1에이치-벤조[d]이미다졸-1-일)메틸)-4-아미노-4에이치-1,2,4-트리아졸-3-티올을 제조하는 단계; 및
4) 상기 5-((1에이치-벤조[d]이미다졸-1-일)메틸)-4-아미노-4에이치-1,2,4-트리아졸-3-티올, 피-톨루익 산 및 포스포릴 클로라이드를 넣고 환류하고서 침전물을 생성하고 건조하여 상기 화학식 1을 제조하는 단계.
Including the following steps, the preparation method of Chemical Formula 1.
1) After dissolving benzimidazole and sodium hydride in THF (Tetrahydrofuran), methylbromoacetate was added to the reaction mixture, the reaction mixture was diluted with ethyl acetate, washed with water and brine, and dried over anhydrous magnesium sulfate. Preparing a methyl 2- (1-H-benzo [d] imidazol-1-yl) acetate by column chromatography on a residue obtained by concentrating the filtrate under reduced pressure and drying the mixture;
2) After dissolving the methyl 2- (1-H-benzo [d] imidazol-1-yl) acetate in alcohol and adding hydrazine hydrate dissolved in alcohol, the reaction mixture was refluxed and recrystallized after completion of the reaction to give 2- ( Preparing 1-benzo [d] imidazole-1) acetohydrazide;
3) After dissolving potassium hydroxide in alcohol and adding the 2- (1-H-benzo [d] imidazol-yl) acetohydrazide and carbon disulfide, the precipitates separated after dilution with ether are collected and washed once more with ether. After drying, it was dissolved in water, hydrazine hydrate was added to reflux, and the precipitate obtained under acidic condition was dried to give 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1, Preparing 2,4-triazole-3-thiol; And
4) the 5-((1H-benzo [d] imidazol-1-yl) methyl) -4-amino-4H-1,2,4-triazole-3-thiol, py-toluic acid and Phosphoryl chloride was added to reflux to form a precipitate and dried to prepare the formula (1).
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| WO2008060578A2 (en) * | 2006-11-15 | 2008-05-22 | Cytovia, Inc. | 3-aryl-6-aryl-[1,2,4] triazolo[3,4-b][1,3,4] thiadiazoles and related compounds as activators of caspases and inducers of apoptosis and the use thereof |
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| CN111295384A (en) * | 2018-10-10 | 2020-06-16 | 江苏豪森药业集团有限公司 | Bicyclic derivative inhibitor, preparation method and application thereof |
| CN111295384B (en) * | 2018-10-10 | 2022-08-12 | 江苏豪森药业集团有限公司 | Bicyclic derivative inhibitor, preparation method and application thereof |
| CN114920759A (en) * | 2022-05-18 | 2022-08-19 | 江南大学 | Heterocyclic-triazole thiadiazole heterocyclic series compound, synthesis method, pharmaceutical composition and application |
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