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KR0161141B1 - Irreversible hiv protease inhibitors with anti-aids effect, processes for the preparation thereof and compositions containing the same - Google Patents

Irreversible hiv protease inhibitors with anti-aids effect, processes for the preparation thereof and compositions containing the same Download PDF

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KR0161141B1
KR0161141B1 KR1019950013366A KR19950013366A KR0161141B1 KR 0161141 B1 KR0161141 B1 KR 0161141B1 KR 1019950013366 A KR1019950013366 A KR 1019950013366A KR 19950013366 A KR19950013366 A KR 19950013366A KR 0161141 B1 KR0161141 B1 KR 0161141B1
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hiv
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윤홍식
문광률
최낙현
정원희
손영찬
박지효
최호일
김성천
김충렬
이태규
권영도
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성재갑
주식회사엘지화학
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Abstract

본 발명은 HIV 프로테아제의 억제제로서 유용한 신규 화합물 및 그의 제조방법 및 이를 포함하는 조성물에 관한 것으로서, 하기의 일반식(Ⅰ)의 시스-에폭사이드 구조를 갖는 화합물 및 그의 약학적으로 허용되는 염, 수화물 또는 용매화물은, HIV 프로테아제의 비가역적 억제제로서 억제 효과는 높은 반면 그 세포독성은 낮기 때문에, 에이즈 또는 HIV 감염의 치료 또는 예방용 제제로서 유용하게 사용될 수 있다.The present invention relates to a novel compound useful as an inhibitor of HIV protease, a method for preparing the same, and a composition comprising the same, the compound having a cis-epoxide structure of the following general formula (I) and a pharmaceutically acceptable salt and hydrate thereof Alternatively, solvates can be usefully used as agents for the treatment or prevention of HIV or HIV infection because of their high inhibitory effect and low cytotoxicity as irreversible inhibitors of HIV proteases.

상기식에서, R1, R2, R3및 R4는 명세서 중에서 정의한 바와 같다.In the above formula, R 1 , R 2 , R 3 and R 4 are as defined in the specification.

Description

항 에이즈(AIDS)효과를 갖는 비가역적 HIV 프로테아제 억제제, 이의 제조방법 및 이를 포함하는 조성물Irreversible HIV Protease Inhibitor with Anti-Aids (AIDS) Effect, Method of Preparation and Composition Comprising the Same

본 발명은 인간 면역결핍 바이러스(Human immunodeficiency virus, HIV)프로테아제 억제 화합물, 이의 제조방법 및 이를 포함하는 조성물에 관한 것으로, 보다 상세하게는 HIV 프로테아제를 비가역적으로 억제하는 신규의 시스-에폭시 화합물과 이의 제조방법, 그리고 이 화합물을 유효성분으로 포함하여 HIV 감염으로 발생되는 후천성 면역 결핍증(Aquired Immune Deficiency Syndrome, AIDS, 이하 에이즈로 칭한다)의 치료 및 예방을 위한 조성물에 관한 것이다.The present invention relates to a human immunodeficiency virus (HIV) protease inhibitory compound, a method for preparing the same, and a composition comprising the same, and more particularly to a novel cis-epoxy compound that irreversibly inhibits HIV protease and its The present invention relates to a preparation method and a composition for the treatment and prevention of acquired immune deficiency syndrome (AIDS, hereinafter referred to as AIDS) caused by HIV infection, including the compound as an active ingredient.

HIV는 에이즈 및 에이즈 증후군을 일으키는 레트로 바이러스이다. 레트로 바이러스는 유전정보 물질로 RNA를 갖는데, 숙주를 감염시킨 후 역전사효소(reverse transcriptase)에 의해 바이러스 RNA로 부터 이중 나선의 DNA를 만들게 된다. 이렇게 만들어진 이중나선 DNA는 인테그라제(integrase)에 의해 숙주의 염색체에 접목되어, 감염된 새로운 바이러스 RNA 뿐 아니라, 숙주의 효소적 기작을 이용하여 바이러스의 단백질도 만들 수 있다. 생성된 다단백질(polyprotein)은 새로운 바이러스를 형성하기 위하여 변형되어야 하는데, 이러한 변형은 숙주의 효소 또는 바이러스가 갖고 있는 호소에 의해서 이루어진다. 이들 효소중에서 가장 중요한 것은 하나의 프로테아제(protease)로서 이 효소는 다단백질을 바이러스 형성에 필요한 구조단백질 및 효소로 분해시키는 효소이다.HIV is a retrovirus that causes AIDS and AIDS syndrome. Retroviruses contain RNA as a genetic information material, which infects the host and creates double-stranded DNA from viral RNA by reverse transcriptase. This double-stranded DNA is integrated into the host's chromosome by integrase, making it possible not only to infect new viral RNA, but also to produce viral proteins using the host's enzymatic mechanism. The resulting polyprotein must be modified to form a new virus, which is made by the host's enzyme or the appeal of the virus. The most important of these enzymes is a protease, an enzyme that breaks down polyproteins into structural proteins and enzymes necessary for virus formation.

레트로 바이러스의 프로테아제 중에서는 HIV 프로테아제가 가장 집중적인 연구 대상이 되어 왔다. HIV는 껍질 단백질 및 필요한 효소를 만들 때 이들을 mRNA로부터 각각 합성해 내는 것이 아니라, 일단 껍질 단백질과 효소 등이 다 함께 결합되어 있는 긴 형태의 다단백질 gag-단백질(p55) 또는 gag-pol 단백질(p165)을 먼저 만든 다음이 다단백질이 자신의 내부에 있는 프로테아제에 의하여 껍질 단백질과 역전사효소, 인테그라제 등 바이러스 형성에 필요한 효소들로 분해되게 된다. 이러한 분해 과정을 담당하는 프로테아제를 억제하면 바이러스의 복제가 중단되는데, 이러한 원리에 기초한 것이 프로테아제 억제제이다.Of the retrovirus proteases, HIV proteases have been the most intensive study subjects. HIV does not synthesize shell proteins and enzymes from mRNA when they are produced, but rather the long form of the multiprotein gag-protein (p55) or gag-pol protein (p165). ), Then the polyprotein is broken down by the proteases inside it into enzymes necessary for virus formation, such as shell proteins, reverse transcriptase, and integrase. Inhibiting the protease responsible for this degradation process stops the replication of the virus. Protease inhibitors are based on this principle.

돌연변이 실험을 통하여 프로테아제 기능이 없는 바이러스는 감염성이 없는 것으로 보고되었다[Kohl et al., Proc. Nat. Acad. Sci., USA, 85, 4686-4960 (1988); and Peng et al., J Virol., 63, 2550(1989)]. 따라서, HIV 프로테아제의 기능을 저해하는 억제제는 HIV 감염에 의해 발생되는 질환의 치료제로서의 가능성이 제시되어 오고 있다.Mutation experiments have reported that viruses without protease function are not infectious [Kohl et al., Proc. Nat. Acad. Sci., USA, 85, 4686-4960 (1988); and Peng et al., J Virol., 63, 2550 (1989). Thus, inhibitors that inhibit the function of HIV proteases have been suggested as potential therapeutic agents for diseases caused by HIV infection.

HIV 프로테아제는 99개의 아미노산으로 구성되어 있으며, 그의 구조는 X-선 구조 결정에 의해 결정되었다[Navia, et al., Nature 337, 615-620(1989); Wlodawer, et al., Science, 245, 616-621(1989); and Miller, et al., Science, 246, 1149-1152(1989)]. HIV 프로테아제는 두개의 동일한 단위체가 이량체로 존재하고 있는데, 각 단위체의 분자량은 10,793이다. 이러한 HIV 프로테아제는 반응 부위에 아스파르테이트-트레오닌-글리신의 배열을 갖는 전형적인 아스파르틱 프로테아제이다.The HIV protease consists of 99 amino acids and its structure was determined by X-ray structural determination [Navia, et al., Nature 337, 615-620 (1989); Wlodawer, et al., Science, 245, 616-621 (1989); and Miller, et al., Science, 246, 1149-1152 (1989). The HIV protease contains two identical units as dimers, each of which has a molecular weight of 10,793. Such HIV proteases are typical aspartic proteases with an arrangement of aspartate-threonine-glycine at the reaction site.

HIV 프로테아제 억제제의 개발은 다른 종류의 아스파르틱 프로테아제(특히 레닌)의 억제제 개발 동향에 따르고 있다. 그 기본적인 접근 방식은 효소의 전이상태(transition state)와 유사한 화합물(transition state analogue, TSA)을 개발하여 효소의 친화도를 높임으로써 효소에 대한 결합력을 높이는데 있다. 이러한 방식으로 개발한 HIV 프로테아제 억제제는 여러 문헌에 개시되어 있다(Roberts, et al., Science, 248, 358(1990); 유럽 특허 공개 제 0337714 호; 제 0346847 호; 제 356223 호; 제 352000 호; 제 357332 호; 제 362002 호; 및 제 361341 호; Bone et al., JACS, 113, 9382(1991)). 그러나, 상기와 같은 공지의 HIV 프로테아제 억제제들은 모두 효소의 친화도를 높인 가역적 억제제들로서, 실제 치료제를 사용하기에는 부족함이 있었다.The development of HIV protease inhibitors follows the trend of developing inhibitors of other types of aspartic proteases (particularly Lenin). The basic approach is to develop a compound similar to the enzyme's transition state (TSA) to increase the affinity of the enzyme, thus enhancing its binding to the enzyme. HIV protease inhibitors developed in this manner are disclosed in several documents (Roberts, et al., Science, 248, 358 (1990); European Patent Publication No. 0337714; 0346847; No. 356223; 352000; 357332; 362002; and 361341; Bone et al., JACS, 113, 9382 (1991). However, all of these known HIV protease inhibitors are reversible inhibitors that increase the affinity of the enzyme, which is insufficient to use the actual therapeutic agent.

이에, 본 발명자들은 전술한 바와 같이 전이상태를 모방한 가역적인 억제제보다 강력한 억제효과를 얻기 위하여, 전이상태와는 다른 시스-에폭사이드 구조를 도입한 비가역적인 억제제를 개발하여 출원 한 바 있으며(1993년 6월 14일자 대한민국 특허출원 제 93-10811호 참조), 본 발명에 이르러서는 시스-에폭사이드 구조에 L-아미노산이 아닌 알킬아민을 C-말단에 도입하고, N-말단에는 페니실아민으로부터 제조된 β-메탄설포닐-L-우레아 카바메이트 유도체를 도입함으로써 보다 강력한 비가역적 HIV 바이러스 증식 억제 효과를 갖는 신규한 (4R, 3S)-시스 에폭사이드 화합물을 개발하게 되었다.Accordingly, the present inventors have developed and applied an irreversible inhibitor incorporating a cis-epoxide structure different from the transition state in order to obtain a stronger inhibitory effect than the reversible inhibitor that mimics the transition state as described above (1993). Korean Patent Application No. 93-10811 dated June 14, 2012). According to the present invention, an alkylamine other than L-amino acid is introduced into the C-terminus in the cis-epoxide structure, and at the N-terminus, By introducing the prepared β-methanesulfonyl-L-urea carbamate derivative, a novel (4R, 3S) -cis epoxide compound having a more potent irreversible HIV virus growth inhibition effect was developed.

따라서, 본 발명의 목적은 보다 향상된 인간 면역결핍 바이러스(HIV) 억제 활성을 갖는 신규 화합물 및 그의 제조방법을 제공하는 것이다.Accordingly, it is an object of the present invention to provide novel compounds with improved human immunodeficiency virus (HIV) inhibitory activity and methods for their preparation.

본 발명의 또다른 목적은 상기 화합물을 유효 성분으로 포함하는, 후천성 면역결핍증 또는 HIV감염의 예방 또는 치료에 유용한 조성물을 제공하는 것이다.It is another object of the present invention to provide a composition useful for the prevention or treatment of acquired immunodeficiency syndrome or HIV infection, comprising the compound as an active ingredient.

상기 목적을 달성하기 위하여 본 발명에서는 하기 일반식(I)의 시스-에폭사이드 구조를 갖는 화합물 그의 약학적으로 허용되는 염, 수화물 또는 용매화물을 제공한다;In order to achieve the above object, the present invention provides a compound having a cis-epoxide structure of the general formula (I): a pharmaceutically acceptable salt, hydrate or solvate thereof;

상기식에서,In the above formula,

R1은 탄소수 1 내지 8의 직쇄 또는 측쇄 알킬, 사이클릭 알킬 라디칼 또는 사이클릭 알킬 라디칼로 치환된 저급알킬이고,R 1 is lower alkyl substituted with straight or branched chain alkyl, cyclic alkyl radical or cyclic alkyl radical having 1 to 8 carbon atoms,

R2및 R3는 서로 독립적으로 저급알킬, 방향족 라디칼로 치환된 저급 알킬, 탄소수 3 내지 8의 사이클릭 알킬, 사이클릭 알킬 라디칼로 치환된 저급 알킬 또는 방향족 라디칼이며,R 2 and R 3 are independently of each other lower alkyl, lower alkyl substituted by aromatic radicals, cyclic alkyl having 3 to 8 carbon atoms, lower alkyl substituted by cyclic alkyl radicals or aromatic radicals,

R4는 수소 또는 저급알킬이다.R 4 is hydrogen or lower alkyl.

이하 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.

본 명세서에서 사용된 용어중 저급알킬은 메틸, 에틸, 이소프로필, 이소부틸, t-부틸을 포함하는 탄소수 1 내지 4의 직쇄 또는 측쇄 알킬을 의미한다.As used herein, lower alkyl refers to straight or branched chain alkyl of 1 to 4 carbon atoms including methyl, ethyl, isopropyl, isobutyl, t-butyl.

본 명세서에서 사용된 아미노산에 관한 약어들은 아미노산 및 펩타이드에 대한 생화학적 명명법에 관한 IUPAC-IUB(International Union of Pure and Applied Chemistry-International Union of Biochemistry) 합동회의에 따른 것이다[Eur. J. Biochem. 158, 9-31(1984)].Abbreviations for amino acids as used herein are in accordance with the International Union of Pure and Applied Chemistry-International Union of Biochemistry (IUPAC-IUB) Joint Conference on Biochemical Nomenclature for Amino Acids and Peptides [Eur. J. Biochem. 158, 9-31 (1984).

본 발명에 따른 화합물은 또한 비대칭 탄소 중심을 가질 수 있으며, 라세미체, 라세미 화합물, 부분입체이성체 혼합물 및 개개인의 부분입체이성체로서 존재할 수 있으며, 이들 모든 형태의 이성체도 본 발명에 포함된다.The compounds according to the invention may also have asymmetric carbon centers and may exist as racemates, racemic compounds, diastereomeric mixtures and individual diastereomers, all of which are in the form of isomers.

본 발명의 바람직한 화합물은 R1이 2-푸라닐메틸 또는 벤질이고, R2가 수소 또는 메틸이며, R3가 이소프로필 이고, R4가 벤질인 일반식(I)의 화합물이다.Preferred compounds of the invention are compounds of formula (I) wherein R 1 is 2-furanylmethyl or benzyl, R 2 is hydrogen or methyl, R 3 is isopropyl and R 4 is benzyl.

본 발명의 대표적인 화합물의 구체적인 예를 들어 다음과 같다:Specific examples of representative compounds of the present invention are as follows:

화합물 1: [(5S)-[[[N,N-사이클로프로필메틸)아미노카보닐]-β-메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드Compound 1: [(5S)-[[[N, N-cyclopropylmethyl) aminocarbonyl] -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-1- Hexanoyl]-[(2S)-[1-phenyl-3-methyl] butyl] amide

화합물 2: [(5S)-[[[(N,N-메틸부틸)아미노카보닐]-β-메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드Compound 2: [(5S)-[[[((N, N-methylbutyl) aminocarbonyl] -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl- 1-hexanoyl]-[(2S)-[1-phenyl-3-methyl] butyl] amide

화합물 3: [(5S)-[[(N-벤질아미노카보닐)-β-메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드Compound 3: [(5S)-[[((N-benzylaminocarbonyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl] -[(2S)-[1-phenyl-3-methyl] butyl] amide

화합물 4: [(5S)-[[(N-2-푸라닐메틸아미노카보닐)-β-메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드Compound 4: [(5S)-[[(N-2-furanylmethylaminocarbonyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1 -Hexanoyl]-[(2S)-[1-phenyl-3-methyl] butyl] amide

본 발명에 따른 일반식(Ⅰ)의 화합물은 하기 반응도식 1에 도시된 바와 같이 제조할 수 있다.Compounds of formula (I) according to the invention can be prepared as shown in Scheme 1 below.

여기서에서, R1, R2및 R3및 R4는 상기에서 정의한 바와 같고, P는 아미노기 보호그룹이다.Wherein R 1, R 2 and R 3 and R 4 are as defined above and P is an amino group protecting group.

상기 반응도식 1에 따르면, 먼저 일반식(Ⅱ)의 화합물을 일반식(Ⅲ)의 화합물과 커플링 반응시킨 후 mCPBA(m-chloroperbenzoic acid, 메타클로로퍼옥시벤조산)으로 산화반응시켜 일반식(Ⅳ)의 화합물을 얻는다. 이로부터 벤질옥시카보닐 보호그룹을 제거하고 일반식(Ⅴ)의 화합물을 커플링시켜 일반식(Ⅵ)의 화합물을 얻은 후, 상기와 같이 산화반응시켜 목적하는 일반식(Ⅰ)의 화합물을 얻는다.According to Scheme 1, first, a compound of Formula (II) is reacted with a compound of Formula (III), followed by oxidation with mCPBA (m-chloroperbenzoic acid, metachloroperoxybenzoic acid) to give a general formula (IV). To a compound. From this, the benzyloxycarbonyl protecting group is removed and the compound of formula (V) is coupled to obtain a compound of formula (VI), and then oxidized as described above to obtain a compound of the general formula (I). .

상기 커플링 반응에는 커플링 시약으로서 예를 들면, 디사이클로헥실 카보디이미드, 3-에틸-3'-(디메틸아미노)-프로필카보디이미드(3-ethy1-3'-(dimethylamino)-propylcarbodiimide, EDC), 비스-(2-옥소-3-옥사졸리디닐)-포스핀산 클로라이드, 디페닐포스포릴아지드 등을 사용할 수 있는데, 이들로서만 한정되는 것은 아니다. 상기 공정에 사용된 카볼실산은 산 할라이드 또는 기타 활성 에스테르 유도체로 전환시킨 후 커플링 반응시킬 수도 있다. 상기 산 할라이드 유도체로는 산 클로라이드가 포함되며, 활성 에스테르 유도체는 아민과의 커플링 반응에 의해 아미드 결합을 형성하게 하거나 알콜과의 커플링 반응에 의해 에스테르 결합을 형성하도록 하기 위해 카복실산 그룹을 활성화시키는데 통상적으로 사용되는 것들로서, 예를 들면, 메록시카보닐 클로라이드, 이소부록시카보닐 클로라이드 등과 같은 알콕시카보닐 할라이드와, 커플링 시약으로부터 유도된 카복실산 무수물, N-하이드록시벤조트리아졸 유도된 에스테르, N-하이드록시 프탈이미드 유도된 에스테르, N-하이드록시숙신이미드 유도된 에스테르, N-하이드록시-5-노르보넨-2',3'-디카복스아미드 유도된 에스테르, 2,4,5-트리클로로페놀 유도된 에스테르 등이 포함되나, 이들로서만 한정되는 것은 아니다.The coupling reaction includes, for example, dicyclohexyl carbodiimide, 3-ethyl-3 '-(dimethylamino) -propylcarbodiimide (3-ethy1-3'-(dimethylamino) -propylcarbodiimide, EDC), bis- (2-oxo-3-oxazolidinyl) -phosphinic chloride, diphenylphosphoryl azide and the like can be used, but are not limited thereto. The carbolic acid used in the process may be converted to an acid halide or other active ester derivative and then subjected to a coupling reaction. The acid halide derivatives include acid chlorides, and active ester derivatives are used to activate carboxylic acid groups to form amide bonds by coupling reactions with amines or to form ester bonds by coupling reactions with alcohols. Commonly used ones include, for example, alkoxycarbonyl halides such as methoxycarbonyl chloride, isobutoxycarbonyl chloride, and the like, carboxylic acid anhydrides derived from coupling reagents, N-hydroxybenzotriazole derived esters, N-hydroxy phthalimide derived ester, N-hydroxysuccinimide derived ester, N-hydroxy-5-norbornene-2 ', 3'-dicarboxamide derived ester, 2,4,5 Trichlorophenol derived esters and the like, but are not limited thereto.

탈보호 반응, 예를 들어 벤질옥시카보닐기는 통상의 방법에 의하여, 예를 들면 Pd/C 촉매 존재하에서 수조가압하에 반응시켜 제거할 수 있다.Deprotection reactions, for example benzyloxycarbonyl groups, can be removed by conventional methods, for example by reaction under water bath pressure in the presence of a Pd / C catalyst.

상기 반응도식 1에서 일반식(Ⅱ)의 화합물은 하기 반응도식 2에 도시된 바와 같이 제조될 수 있다.Compounds of formula (II) in Scheme 1 may be prepared as shown in Scheme 2 below.

여기에서, X는 할로겐원자, 예를 들면 Br 또는 I이고, ⓟ는 아미노기보호그룹이다.Here, X is a halogen atom, for example Br or I, ⓟ is an amino group protecting group.

상기 반응도식 2는 문헌[Keinan et al., Tetrahedron, 47, 4631-4638(1991); and Corey Shimaji, J.Am. Chem. Soc., 105, 1662-1664(1983)]에 기술되어 있는 방법에 의거한 시스-β, γ-올레핀 산의 제조공정을 나타내는 것이다. 일반식(Ⅶ)의 화합물을 비티그 반응시켜 일반식(Ⅷ)의 화합물을 수득한 다음, 이를 산 촉매 존재하에 t-부탄올중에서 용매의 환류온도로 가열 교반함으로써 목적하는 일반식(Ⅱ)의 화합물을 제조할 수 있다. 여기에서, 상기 논문에 기재된 방법에 따라 일반식(Ⅷ)의 오르토에스테르를 산처리하여 일반식(Ⅸ)의 화합물을 만든 후 염기 처리하여 보호 그룹을 제거하는 경우에는 β, γ-올레핀 산에서 α, β-올레핀 산으로 50%이상 전환되어 목적 화합물의 수율이 매우 낮아질 뿐 아니라 이의 분리에도 어려움이 따르기 때문에, 본 발명에서는 일반식(Ⅷ)의 오르토에스테르를 산촉매의 존재하에 t-부탄올을 용매로 하여 용매의 환류온도로 가열 교반함으로써 α, β-올레핀 산으로 전환되지 않은 β, γ-올레핀 산 화합물(Ⅱ)를 80 내지 90%수율로 얻을 수 있었다.Scheme 2 is described by Keinan et al., Tetrahedron, 47, 4631-4638 (1991); and Corey Shimaji, J. Am. Chem. Soc., 105, 1662-1664 (1983), shows a process for producing cis-β and γ-olefin acids according to the method described in the above. The compound of formula (II) was obtained by subjecting the compound of formula (IV) to bitig reaction to obtain a compound of formula (IV), which was then stirred by heating to a reflux temperature of the solvent in t-butanol in the presence of an acid catalyst. Can be prepared. Here, the acid treatment of the orthoester of general formula (IV) to prepare a compound of general formula (IV) followed by base treatment to remove the protecting group according to the method described in the above paper. In the present invention, the orthoester of general formula (t) is converted to t-butanol in the presence of an acid catalyst because the yield of the target compound is very low and the separation thereof becomes difficult. By heating and stirring at the reflux temperature of the solvent, β, γ-olefin acid compound (II) which was not converted into α, β-olefin acid was obtained in 80 to 90% yield.

상기 반응도식 1에서 일반식(Ⅲ)의 그룹을 제공하는 작용화된 아민은 하기 반응도식 3에서 도시된 바와 같이, 문헌[Weinreb et al., Tetrahedron Lett., 22, 3815(1981)]에 기술된 방법으로 제조한 N, O-디메틸아미드를 그리나드 반응시킨 후, 비티그 반응 및 수소화 반응을 통해 제조할 수 있다.Functionalized amines providing groups of general formula (III) in Scheme 1 are described in Weinreb et al., Tetrahedron Lett., 22, 3815 (1981), as shown in Scheme 3 below. N, O-dimethylamide prepared by the conventional method may be prepared by Grignard reaction, followed by a bitig reaction and a hydrogenation reaction.

또한, 반응도식 1에서 사용된 일반식(Ⅴ)의 화합물은 하기 반응도식 4에 도시된 바와 같이, L-페니실아민으로부터 염기의 존재하에서 티올기를 메틸화하고 t-부톡시카보닐기로 아민을 보호한 후, 산 부분을 벤질 에스테르로 전환시키고 탈보호 반응을 수행한 다음, 우레아기룰 도입하고 탈보호 반응을 수행함으로써 제조할 수 있다.In addition, the compound of formula (V) used in Scheme 1 methylates a thiol group in the presence of a base from L-penicylamine and protects the amine with t-butoxycarbonyl as shown in Scheme 4 below. The acid moiety can then be prepared by converting the acid moiety to benzyl ester and carrying out the deprotection reaction followed by the introduction of urea group and carrying out the deprotection reaction.

여기에서, R1및 R4는 상기에서 정의한 바와 같다.Here, R 1 and R 4 are as defined above.

본 발명의 일반식(Ⅰ)의 화합물은 HIV 프로테아제 억제 활성을 가지므로 에이즈 또는 HIV 감염의 치료 또는 예방을 위한 제제로서 사용될 수 있다. 이러한 목적으로 1일 체중 1㎏당 5 내지 30㎎의 양을 한번에 또는 수회에 걸쳐 숙주에 투여할 수 있으며, 특정 환자에 대한 투여 수준은 사용될 특정 화합물, 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율, 약제혼합 및 질환의 중증도에 따라 변환될 수 있다.Since the compound of general formula (I) of the present invention has HIV protease inhibitory activity, it can be used as an agent for treating or preventing AIDS or HIV infection. For this purpose, an amount of 5 to 30 mg / kg body weight per day may be administered to the host at one time or several times, and the dosage level for a particular patient may be determined by the specific compound to be used, weight, age, sex, health condition, diet, It can be converted according to the time of administration, the method of administration, the rate of excretion, drug mixture and the severity of the disease.

또한 본 발명의 화합물은 목적하는 바에 따라 경구 또는 비경구 투여할 수 있다. 경구투여용 제형으로는 캅셀제, 정제, 환제, 산제 및 과립제가 가능한데, 화합물 특성을 고려한다면 캅셀제가 특히 바람직하고, 정제로 할 경우에는 장용성 제제로 하는 것이 바람직하다. 경구 투여형 제형으로 제조할 때에는 본 발명의 활성화합물을 수크로오즈, 락 토오즈 또는 전분과 같은 적어도 하나의 불활성 희석제, 그리고 스테아린산 마그네슘과 같은 적어도 하나의 활제를 포함할 수 있다. 주사용 제제로 제조할 경우에는 공지된 기술에 따라, 예를 들어 멸균 주사용 수성 또는 유성 현탁액과 적절한 분산제, 수화제 또는 현탁화제와 함께 사용하여 제조할 수 있는데, 허용 가능한 용매로서는 예를 들어 폴리에틸렌글리클, 에틸렌글리콜, 폴리프로필렌글리콜, 에탄올 등이 있다.The compounds of the invention can also be administered orally or parenterally as desired. Capsules, tablets, pills, powders and granules may be used as oral dosage forms. In view of compound properties, capsules are particularly preferred, and tablets are preferably enteric preparations. When prepared in oral dosage form, the active compounds of the present invention may comprise at least one inert diluent such as sucrose, lactose or starch, and at least one lubricant such as magnesium stearate. When prepared in injectable preparations can be prepared according to known techniques, for example, in combination with sterile injectable aqueous or oily suspensions and with suitable dispersing, wetting or suspending agents. Reckle, ethylene glycol, polypropylene glycol, ethanol and the like.

한편, 본 발명의 일반식(Ⅰ)의 화합물은 하나이상의 다른 에이즈치료제, 면역조절제 등과 병행하여 투여될 수 있으며, HIV 감염의 치료 및 예방에 유용한 제제라면, 상기에 언급한 제제 이외에도 어떠한 것이라도 사용될 수 있다.On the other hand, the compound of formula (I) of the present invention may be administered in parallel with one or more other AIDS treatments, immunomodulators and the like, and any agent other than the above-mentioned formulations can be used as long as it is a useful agent for the treatment and prevention of HIV infection. Can be.

이하 본 발명을 다음의 제조예 및 실시예를 통하여 보다 구체적으로 설명한다. 단 하기의 실시예는 본 발명에 따른 신규한 화합물의 제조 방법에 대한 이해를 돕기 위한 것일 뿐, 본 발명이 이로서 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following Preparation Examples and Examples. However, the following examples are only for the understanding of the preparation method of the novel compounds according to the present invention, but the present invention is not limited thereto.

[제조예 1][Production Example 1]

[ (S)-2-아미노-3-메틸-1-페닐 부탄의 제조][Production of (S) -2-amino-3-methyl-1-phenylbutane]

(1-1) L-N-벤질옥시카보닐-N'-메톡시-N'-메틸-페닐알라닌 아미드의 제조(1-1) Preparation of L-N-benzyloxycarbonyl-N'-methoxy-N'-methyl-phenylalanine amide

25g(0.084 몰)의 N-벤질옥시카보닐-L-페닐알라닌과 18.4㎖(0.167 몰)의 N-메틸모르폴린을 500㎖의 디클로로메탄에 용해시킨 후 -15℃에서 10.8㎖ (83.6 밀리몰)의 이소부틸 클로로포르메이트를 가하였다. 15분후에 1.2당량의 N, O-디메틸하이드록실아민을 -15℃에서 가하고 동일한 온도에서 1시간 동안 교반한 다음 상온에서 3시간 동안 교반하였다. 500㎖의 포화 염화나트륨 용액으로 2회 세척하고 유기층을 무수 MgSO4로 건조시킨 다음 셀라이트에 통과시켜 유기용매를 제거하여 표제화합물 26.7g(수율 93%)을 얻었다.25 g (0.084 mol) of N-benzyloxycarbonyl-L-phenylalanine and 18.4 ml (0.167 mol) of N-methylmorpholine were dissolved in 500 ml of dichloromethane and then 10.8 ml (83.6 mmol) at -15 ° C. Isobutyl chloroformate was added. After 15 minutes, 1.2 equivalents of N, O-dimethylhydroxylamine were added at -15 ° C, stirred for 1 hour at the same temperature, and then stirred for 3 hours at room temperature. The organic layer was washed twice with 500 mL of saturated sodium chloride solution, dried over anhydrous MgSO 4 , and passed through celite to remove the organic solvent to give 26.7 g (yield 93%) of the title compound.

1H NMR(CDCl3) δ 2.87(m, 1H), 3.05(m, 1H), 3.17(s, 3H), 3.66(s, 3H), 4.96(m, 1H), 5.07(s, 2H), 5.23(d, 1H), 7.12-7.34(m, 10H) 1 H NMR (CDCl 3 ) δ 2.87 (m, 1H), 3.05 (m, 1H), 3.17 (s, 3H), 3.66 (s, 3H), 4.96 (m, 1H), 5.07 (s, 2H), 5.23 (d, 1 H), 7.12-7.34 (m, 10 H)

(1-2) (S)-3-[(N-벤질옥시카보닐)아미노]-1-메틸-4-페닐-2-부탄온의 제조(1-2) Preparation of (S) -3-[(N-benzyloxycarbonyl) amino] -1-methyl-4-phenyl-2-butanone

상기(1-1)에서 제조한 화합물 6.84g(0.02 몰)을 60㎖의 테트라하이드로푸란에 용해시키고 3M 메틸 마그네슘 브로마이드 에테르용액 30㎖(0.09 몰)을 0 ℃에서 가한 후 상온에서 3시간 동안 교반하였다. 0 ℃에서 물을 가하여 반응을 종결시키고 용매를 감압 증류하여 제거한 다음 300㎖의 에테르를 가하여 희석하고 200㎖의 염화 암모늄용액으로 3회 세척하였다. 이어서, 무수 MgSO4로 건조시키고 감압 증류한 다음 컬럼 크로마토그래피(헥산:에틸아세테이트=8:2)를 실시하여 표제 화합물 5.11g(수율 86%)을 얻었다.6.84 g (0.02 mol) of the compound prepared in (1-1) was dissolved in 60 ml of tetrahydrofuran, and 30 ml (0.09 mol) of 3M methyl magnesium bromide ether solution was added at 0 ° C., followed by stirring at room temperature for 3 hours. It was. The reaction was terminated by addition of water at 0 ° C., the solvent was distilled off under reduced pressure, diluted with 300 ml of ether, and washed three times with 200 ml of ammonium chloride solution. Subsequently, the mixture was dried over anhydrous MgSO 4 , distilled under reduced pressure, and subjected to column chromatography (hexane: ethyl acetate = 8: 2) to obtain 5.11 g (yield 86%) of the title compound.

1H NMR(CDCl3) δ 2.13(s, 3H), 3.04(m, 2H), 4.57(m ,1H), 5.08(s, 2H), 5.17(d, 1H), 7.12-7.34(m, 10H) 1 H NMR (CDCl 3 ) δ 2.13 (s, 3H), 3.04 (m, 2H), 4.57 (m, 1H), 5.08 (s, 2H), 5.17 (d, 1H), 7.12-7.34 (m, 10H )

(1-3) (S)-3-[(N-벤질옥시카보닐)아미노]-2-메틸-4-페닐-1-부텐의 제조(1-3) Preparation of (S) -3-[(N-benzyloxycarbonyl) amino] -2-methyl-4-phenyl-1-butene

8.57g(0.024 몰)의 메틸트리페닐포스포늄 브로마이드를 60㎖의 무수 톨루엔에 용해시키고 44㎖(0.022 몰)의 0.5M 칼륨 비스(트리메틸실릴)아미드 톨루엔 용액을 -20℃에서 가한 후 동일한 온도에서 1시간 동안 교반하였다. 생성된 용액에 상기 (1-2)에서 제조한 화합물 5.94g(0.02 몰)을 50㎖의 무수 톨루엔에 용해시킨 용액을 -20℃에서 10분간 가한 다음 동일한 온도에서 1시간 동안 교반하였다. 물을 가하여 반응을 종결시키고 용매를 감압 증류하여 제거한 다음 200㎖의 디클로로메탄을 가하여 희석하고 1N 염산으로 세척하였다. 무수 MgSO4로 건조시키고 감압 증류하여 유기 용매를 제거한 다음 컬럼 크로마토그래피(헥산:에틸아세테이트=9:1)를 실시하여 표제 화합물 5.25g(수율 89%)을 얻었다.8.57 g (0.024 mol) of methyltriphenylphosphonium bromide was dissolved in 60 ml of anhydrous toluene and 44 ml (0.022 mol) of 0.5M potassium bis (trimethylsilyl) amide toluene solution was added at -20 ° C and then at the same temperature. Stir for 1 hour. To the resulting solution, a solution obtained by dissolving 5.94 g (0.02 mol) of the compound prepared in the above (1-2) in 50 ml of anhydrous toluene was added at -20 ° C for 10 minutes and then stirred at the same temperature for 1 hour. The reaction was terminated by addition of water, the solvent was distilled off under reduced pressure, and then diluted with 200 ml of dichloromethane and washed with 1N hydrochloric acid. After drying over anhydrous MgSO 4 and distillation under reduced pressure to remove an organic solvent, column chromatography (hexane: ethyl acetate = 9: 1) was carried out to obtain 5.25 g (yield 89%) of the title compound.

1H NMR(CDCl3) δ 1.79(s, 3H), 2.84(m, 2H), 4.31(m, 1H), 4.52(bs, 1H), 4.81(d, 2H), 5.07(s, 2H), 7.13-7.35(m, 10H) 1 H NMR (CDCl 3 ) δ 1.79 (s, 3H), 2.84 (m, 2H), 4.31 (m, 1H), 4.52 (bs, 1H), 4.81 (d, 2H), 5.07 (s, 2H), 7.13-7.35 (m, 10H)

(1-4)(S)-2-아미노-3-메틸-1-페닐 부탄의 제조(1-4) Preparation of (S) -2-amino-3-methyl-1-phenyl butane

상기 (1-3)에서 제조한 화합물 2.95g(0.01 몰)을 30㎖의 메탄올에 용해시키고 200㎎의 10% Pd/C를 가한 후 1기압의 수소 조건하에서 4시간 동안 교반하였다. 이어서 셀라이트를 통과시켜 무기물을 제거한 다음 유기용매를 감압 증류하여 표제 화합물 1.63g을 정량적으로 얻었다.2.95 g (0.01 mol) of the compound prepared in (1-3) was dissolved in 30 ml of methanol, 200 mg of 10% Pd / C was added, followed by stirring for 4 hours under 1 atmosphere of hydrogen. Subsequently, the inorganic material was removed by passing through celite, and the organic solvent was distilled off under reduced pressure to quantitatively obtain 1.63 g of the title compound.

1H NMR(CDCl3) δ 0.94(m, 6H), 1.11(bs, 2H), 1.65(m, 1H), 2.39(m, 1H), 2.82(m, 2H), 7.13-7.32(m, 5H) 1 H NMR (CDCl 3 ) δ 0.94 (m, 6H), 1.11 (bs, 2H), 1.65 (m, 1H), 2.39 (m, 1H), 2.82 (m, 2H), 7.13-7.32 (m, 5H )

[α]D=37.4(C=0.12, 디클로로메탄)[α] D = 37.4 (C = 0.12, dichloromethane)

e. e. =37.4/-38.1=98%e. e. = 37.4 / -38.1 = 98%

[제조예 2][Production Example 2]

[[S-메틸-O-벤질페니실아민]-[메틸-n-부틸아민]우레아의 제조][Preparation of [S-methyl-O-benzylphenicylamine]-[methyl-n-butylamine] urea]

(2-1) (N-t-부톡시카보닐)아미노-S-메틸-L-페닐실아민의 제조(2-1) Preparation of (N-t-butoxycarbonyl) amino-S-methyl-L-phenylsilamine

8.9g(0.06 몰)의 L-페니실아민을 디옥산 120㎖와 물 40㎖에 가하여 0℃로 냉각시킨 다음 20㎖의 6N 수산화나트륨 수용액을 첨가하여 용해시켰다. 생성된 용액에 9.24g(0.066 몰)의 요오드화메탄을 가하고 플라스크 마개를 막은 다음 0℃에서 3시간, 이어서 상온에서 2시간 동안 반응시켰다. 반응물을 0℃로 냉각시키고 12.18g(0.07 몰)의 t-부톡시디카보네이트를 서서히 가한 다음, 0℃에서 1시간, 이어서 상온에서 2시간 동안 교반하여 반응을 종결시켰다. 용매를 감압 증류하여 제거한 후, 미반응 벤질클로로포르메이트를 제거하기 위해 물과 에테르 20㎖를 가한 다음 유기층을 제거하였다. 수용액층에 300㎖의 에틸 아세테이트를 가하고 6N 염산으로 pH를 3으로 맞추었다. 유기층을 분리한 후 무수 MgSO4로 건조시키고 용매를 감압 증류하여 표제 화합물 12.62g(수율 80%)을 얻었다.8.9 g (0.06 mol) of L-phenicylamine was added to 120 ml of dioxane and 40 ml of water, cooled to 0 ° C, and dissolved by adding 20 ml of 6N aqueous sodium hydroxide solution. 9.24 g (0.066 mol) of methane iodide was added to the resulting solution, and the flask was capped and then reacted at 0 ° C. for 3 hours and then at room temperature for 2 hours. The reaction was cooled to 0 ° C. and 12.18 g (0.07 mol) of t-butoxydicarbonate was added slowly, followed by stirring at 0 ° C. for 1 hour and then at room temperature for 2 hours to terminate the reaction. After distilling off the solvent under reduced pressure, 20 ml of water and ether were added to remove unreacted benzylchloroformate, and then the organic layer was removed. 300 ml of ethyl acetate was added to the aqueous layer and the pH was adjusted to 3 with 6N hydrochloric acid. The organic layer was separated, dried over anhydrous MgSO 4 , and the solvent was distilled off under reduced pressure to obtain 12.62 g (yield 80%) of the title compound.

1H NMR(CDCl3) δ 1.38(s, 3H), 1.41(s, 3H), 1.46(s, 9H), 2.06(s, 3H), 4.35(d, 1H), 5.68(d, 1H) 1 H NMR (CDCl 3 ) δ 1.38 (s, 3H), 1.41 (s, 3H), 1.46 (s, 9H), 2.06 (s, 3H), 4.35 (d, 1H), 5.68 (d, 1H)

(2-2) S-메틸-O-벤질 페니실아민의 제조(2-2) Preparation of S-methyl-O-benzyl penicylamine

50㎖의 디메틸포름아미드에 상기 (2-1)에서 제조한 화합물 2.63g(0.01 몰)을 용해시키고 2 당량의 탄산칼륨과 1 당량의 브롬화 벤질을 가한 다음 상온에서 3시간 동안 반응시켰다. 용매를 감압 증류하여 제거한 후 에틸아세테이트 100㎖와 물 200㎖를 가하였다. 유기층을 분리한 후 무수 Na2SO4로 건조시키고 용매를 감압 증류한 다음 20㎖의 디클로로메탄에 용해시키고 10㎖의 CF3COOH를 가한 후 1시간 동안 교반하였다. 용매를 감압 증류하여 제거한 다음 에틸아세테이트 100㎖와 NaHCO3포화용액 200㎖를 가하였다. 유기층을 분리한 후 무수 Na2SO4로 건조시키고 용매를 감압 증류하여 표제 화합물 2.33g(수율 92%)을 얻었다.2.63 g (0.01 mol) of the compound prepared in (2-1) was dissolved in 50 ml of dimethylformamide, 2 equivalents of potassium carbonate and 1 equivalent of benzyl bromide were added, and then reacted at room temperature for 3 hours. After distilling off the solvent under reduced pressure, 100 ml of ethyl acetate and 200 ml of water were added thereto. The organic layer was separated, dried over anhydrous Na 2 SO 4 , the solvent was distilled under reduced pressure, dissolved in 20 ml of dichloromethane, 10 ml of CF 3 COOH was added, and the mixture was stirred for 1 hour. The solvent was distilled off under reduced pressure, and then 100 ml of ethyl acetate and 200 ml of saturated NaHCO 3 solution were added. The organic layer was separated, dried over anhydrous Na 2 SO 4 , and the solvent was distilled off under reduced pressure to obtain 2.33 g (yield 92%) of the title compound.

1H NMR(CDCl3) δ 1.26(s, 3H), 1.34(s, 3H), 2.02(s, 3H), 3.52(s, 1H), 5.17(s, 2H), 7.37-7.43(m, 5H) 1 H NMR (CDCl 3 ) δ 1.26 (s, 3H), 1.34 (s, 3H), 2.02 (s, 3H), 3.52 (s, 1H), 5.17 (s, 2H), 7.37-7.43 (m, 5H )

(2-3) (N-p-니트로페닐옥시카보닐-S-메틸-O-벤질 페니실아민 제조(2-3) Preparation of (N-p-nitrophenyloxycarbonyl-S-methyl-O-benzyl penicylamine

50㎖의 아세토니트릴로 희석시킨 p-니트로페틸클로로포르메이트 2.1g(10.5 밀리 몰)에 NaHCO31.5 당량과 상기 (2-2)에서 제조한 화합물 1당량을 0℃에서 가한 다음 상온에서 1시간 동안 교반하였다. 용매를 감압 증류하여 제거하고 에틸아세테이트에 용해시킨 다음 K2CO3용액으로 세척하였다. 무수MgSO4로 건조시키고 용매를 제거하여 표제 화합물 3.84g(수율 92%)을 얻었다.To 2.1 g (10.5 mmol) of p-nitropetylchloroformate diluted with 50 ml of acetonitrile, 1.5 equivalents of NaHCO 3 and 1 equivalent of the compound prepared in (2-2) were added at 0 ° C., followed by 1 hour at room temperature. Was stirred. The solvent was distilled off under reduced pressure, dissolved in ethyl acetate, and washed with K 2 CO 3 solution. Drying with anhydrous MgSO 4 and removal of solvent gave 3.84 g (yield 92%) of the title compound.

1H NMR(CDCl3) δ 1.40(s, 3H), 1.42(s, 3H), 2.03(s, 3H), 4.43(d, 1H), 5.21(dd, 2H), 6.03(d, H), 7.32-7.73(m, 7H), 8.23(d, 2H) 1 H NMR (CDCl 3 ) δ 1.40 (s, 3H), 1.42 (s, 3H), 2.03 (s, 3H), 4.43 (d, 1H), 5.21 (dd, 2H), 6.03 (d, H), 7.32-7.73 (m, 7H), 8.23 (d, 2H)

(2-4) (S-메틸-O-벤질-페니실아민]-[메틸-n-부틸아민]우레아의 제조(2-4) Preparation of (S-methyl-O-benzyl-phenylamine]-[methyl-n-butylamine] urea

메틸-n-부틸아민(0.12g, 10 밀리몰)과 1.1 당량의 트리에틸아민을 10㎖의 아세토니트릴에 용해시킨 다음, 상온에서 (2-3)의 반응용액에 가하였다. 10분후에 용매를 감압 증류하여 제거하고 에틸아세테이트에 용해시킨 후 1N HCI 용액과 K2CO3용액으로 세척하였다. 무수 MgSO4로 건조시킨 다음 용매를 제거하고 컬럼크로마토그래피(헥산:에틸아세테이트=8:2)를 실시하여 표제 화합물 3.09g(수율 92%)을 얻었다.Methyl-n-butylamine (0.12 g, 10 mmol) and 1.1 equivalents of triethylamine were dissolved in 10 ml of acetonitrile and then added to a reaction solution of (2-3) at room temperature. After 10 minutes, the solvent was distilled off under reduced pressure, dissolved in ethyl acetate, and washed with 1N HCI solution and K 2 CO 3 solution. After drying over anhydrous MgSO 4 , the solvent was removed, and column chromatography (hexane: ethyl acetate = 8: 2) was carried out to obtain 3.09 g (yield 92%) of the title compound.

1H NMR(CDCl3) δ 0.82(t, 3H), 1.18(m, 8H), 1.35(m, 2H), 1.98(s, 3H), 2.81(s, 3H), 3.12(t, 2H), 4.32(d, 1H), 5.05(dd, 2H), 5.2(d, 1H), 7.24(m, 5H) 1 H NMR (CDCl 3 ) δ 0.82 (t, 3H), 1.18 (m, 8H), 1.35 (m, 2H), 1.98 (s, 3H), 2.81 (s, 3H), 3.12 (t, 2H), 4.32 (d, 1H), 5.05 (dd, 2H), 5.2 (d, 1H), 7.24 (m, 5H)

[제조예 3][Manufacture example 3]

[(S)-5-[(N-벤질옥시카보닐)아미노]-6-페닐-헥스-3-(시스)-엔-1-카보닐산의 제조][Preparation of (S) -5-[(N-benzyloxycarbonyl) amino] -6-phenyl-hex-3- (cis) -ene-1-carbonyl acid]

(3-1) 5-L-(N-벤질옥시카보닐)아미노-6-페닐-헥스-3-(시스)-엔일-4'-메틸-2', 6', 7'-트리옥사-비사이클로-[2', 2', 2']-옥세탄의 제조(3-1) 5-L- (N-benzyloxycarbonyl) amino-6-phenyl-hex-3- (cis) -enyl-4'-methyl-2 ', 6', 7'-trioxa- Preparation of Bicyclo- [2 ', 2', 2 ']-oxetane

문헌[Keinan et al., Tetrahedron, 26, 4631-4638(1991)에 기술된 방법에 따라 합성된 1-(2-트리페닐포스포늄-메틸)-4'-메틸-2', 6', 7'-트리옥사-비사이클로-[2', 2', 2']-옥세탄 브로마이드 60.89 g(0.12 몰)을 400㎖의 테트라하이드로푸란에 용해시키고 -78℃에서 교반하였다. 이어서 220㎖(0.11 몰)의 0.5M 칼륨헥사메틸 디실라잔 용액을 -78℃에서 1시간 동안 냉각시킨 다음 상기 용액에 20분 동안 서서히 가하고 -78℃에서 1시간, 이어서 상온에서 1시간 동안 교반한 뒤 물을 가하여 반응을 종결시켰다. 용매를 제거한 후 에틸아세테이트에 용해시키고 NaHCO3포화용액 및 물로 세척하였다. 유기층을 무수 MgSO4로 건조시키고 감압 증류한 다음 컬럼 크로마토그래피(헥산:에틸아세테이트:트리에틸아민=70:30:5)를 실시하여 표제 화합물 36.5(수율 84%)을 얻었다.1- (2-triphenylphosphonium-methyl) -4'-methyl-2 ', 6', 7 synthesized according to the method described in Keinan et al., Tetrahedron, 26, 4631-4638 (1991). 60.89 g (0.12 mol) of '-trioxa-bicyclo- [2', 2 ', 2']-oxetane bromide was dissolved in 400 ml of tetrahydrofuran and stirred at -78 ° C. Subsequently, 220 ml (0.11 mol) of 0.5 M potassium hexamethyl disilazane solution was cooled at -78 ° C for 1 hour, and then slowly added to the solution for 20 minutes, and stirred at -78 ° C for 1 hour, and then at room temperature for 1 hour. The reaction was then terminated by adding water. After removing the solvent, it was dissolved in ethyl acetate and washed with saturated NaHCO 3 solution and water. The organic layer was dried over anhydrous MgSO 4 , distilled under reduced pressure, and subjected to column chromatography (hexane: ethyl acetate: triethylamine = 70: 30: 5) to give the title compound 36.5 (yield 84%).

1H NMR(CDCl3) δ 0.8(s, 3H), 2.2-3.0(m, 4H), 3.9(s, 6H), 4.6(m, 1H), 4.8(br, 1H), 5.05(s, 2H), 5.4-5.6(m, 2H), 7.1-7.5[m, 10H) 1 H NMR (CDCl 3 ) δ 0.8 (s, 3H), 2.2-3.0 (m, 4H), 3.9 (s, 6H), 4.6 (m, 1H), 4.8 (br, 1H), 5.05 (s, 2H ), 5.4-5.6 (m, 2H), 7.1-7.5 (m, 10H)

(3-2) (S)-[(N-벤질옥시카보닐)아미노]-6-페닐-헥스-3-(시스)-엔-1-카보닐산의 제조(3-2) Preparation of (S)-[(N-benzyloxycarbonyl) amino] -6-phenyl-hex-3- (cis) -ene-1-carbonyl acid

상기 (3-1)에서 제조한 화합물 2.5g(6 밀리몰)을 1% 미만의 진한 염산이 포함된 t-부탄올과 물의 혼합용액에 용해시켜 반응용매의 환류온도로 약 20시간 동안 교반한 뒤, 반응용매를 증류감압하여 제거하고 K3CO3용액을 가하여 pH를 9이상으로 조정한 다음 에틸아세테이트로 세척하였다. HC1로 수용액층의 pH를 2로 맞추고 에틸아세테이트로 추출한 다음 MgSO4로 건조시키고 유기용매를 제거하여 1.62g(수율 80%)의 표제 화합물을 얻었다.2.5 g (6 mmol) of the compound prepared in (3-1) was dissolved in a mixed solution of t-butanol and water containing less than 1% concentrated hydrochloric acid and stirred at a reflux temperature of the reaction solvent for about 20 hours. The reaction solvent was removed by distillation under reduced pressure, and the pH was adjusted to 9 or more by adding K 3 CO 3 solution, and then washed with ethyl acetate. The pH of the aqueous layer was adjusted to 2 with HC1, extracted with ethyl acetate, dried over MgSO 4 , and the organic solvent was removed to obtain 1.62 g (yield 80%) of the title compound.

1H NMR(CDCl3) δ 2.7-3.3(m, 4H), 4.6(m, 1H), 4.8(br, 1H), 5.05(s, 2H), 5.4(t, 1H), 5.6(m, 1H), 7.1-7.5[m, 10H) 1 H NMR (CDCl 3 ) δ 2.7-3.3 (m, 4H), 4.6 (m, 1H), 4.8 (br, 1H), 5.05 (s, 2H), 5.4 (t, 1H), 5.6 (m, 1H ), 7.1-7.5 (m, 10H)

MS(FAB, m/e) 340(M+1), [α]D=+25.2(C=0.50, 메탄올)MS (FAB, m / e) 340 (M + l), [α] D = + 25.2 (C = 0.50, methanol)

[실시예 1]Example 1

[[5S)-[[[(N,N-메틸부틸)아미노카보닐]-β-메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[2S)-[1-페닐-3-메틸]부틸]아미드(화합물 2)의 제조][[5S)-[[[((N, N-methylbutyl) aminocarbonyl] -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexa Noyl]-[2S)-[1-phenyl-3-methyl] butyl] amide (Compound 2)]

(1-1)[(5S)-(N-벤질옥시카보닐)아미노]-3-(시스)-엔-6-페닐-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드의 제조(1-1) [(5S)-(N-benzyloxycarbonyl) amino] -3- (cis) -ene-6-phenyl-1-hexanoyl]-[(2S)-[1-phenyl-3 Preparation of -methyl] butyl] amide

상기 제조예 3에서 제조한 화합물 0.339g(1 밀리몰)을 각각 1.5 당량의 EDC, 1-하이드록시-벤조트리아졸 하이드레이트(1-hydroxy-benzotriazol hydrate, HOBT) 및 트리에틸아민과 혼합하여 20㎖의 디메틸포름아미드에 용해시킨 다음 제조예 1의 생성물 0.179g(1 밀리몰)을 0℃에서 가하고 상온에서 12시간 동안 교반하였다. 용매를 감압 증류에서 제거한 후 에틸아세테이트에 용해시키고 1N 염산과 NaHCO3포화용액으로 세척하였다. 유기층을 무수 MgSO4로 건조시키고 감압 증류하여 용매를 제거한 후 컬럼 크로마토그래피(에틸아세테이트:헥산=1:1)를 실시하여 표제 화합물 0.44g(수율 87.5%)을 얻었다.0.339 g (1 mmol) of the compound prepared in Preparation Example 3 was mixed with 1.5 equivalents of EDC, 1-hydroxy-benzotriazol hydrate (HOBT), and triethylamine, respectively. After dissolving in dimethylformamide, 0.179 g (1 mmol) of the product of Preparation Example 1 was added at 0 ° C. and stirred at room temperature for 12 hours. The solvent was removed by distillation under reduced pressure, dissolved in ethyl acetate, and washed with 1N hydrochloric acid and saturated NaHCO 3 solution. The organic layer was dried over anhydrous MgSO 4 , distilled under reduced pressure to remove the solvent, and column chromatography (ethyl acetate: hexane = 1: 1) was carried out to obtain 0.44 g (yield 87.5%) of the title compound.

1H NMR(CDCl3) δ 1.01(m, 6H), 1.78(m, 1H), 2.42-3.25(m, 6H), 4.05(m, 1H), 4.58(m, 1H), 4.95(bs, 1H), 5.08(s, 2H), 5.35(m, 1H), 7.09-7.41(m, 16H) 1 H NMR (CDCl 3 ) δ 1.01 (m, 6H), 1.78 (m, 1H), 2.42-3.25 (m, 6H), 4.05 (m, 1H), 4.58 (m, 1H), 4.95 (bs, 1H ), 5.08 (s, 2H), 5.35 (m, 1H), 7.09-7.41 (m, 16H)

(1-2) [(5S)-[(N-벤질옥시카보닐)아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[2S)-[1-페닐-3-메틸]부틸]아미드의 제조(1-2) [(5S)-[(N-benzyloxycarbonyl) amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]-[2S)-[1-phenyl-3 Preparation of -methyl] butyl] amide

상기 (1-1)에서 제조한 화합물 339㎎(0.7 밀리몰)을 20㎖의 디클로로메탄에 용해시키고 3 당량의 메타클로로퍼옥시벤조산을 가한 다음 상온에서 24시간 동안 교반하였다. 30㎖의 10% Na2S2O3용액을 가하고 30분간 교반한 후 유기층을 NaHCO3포화용액으로 세척하였다. 유기층을 무수 MgSO4로 건조시키고 유기용매를 제거하여 표제화합물 278㎎(수율 82%)을 얻었다.339 mg (0.7 mmol) of the compound prepared in (1-1) was dissolved in 20 ml of dichloromethane, 3 equivalents of metachloroperoxybenzoic acid was added, followed by stirring at room temperature for 24 hours. 30 ml of 10% Na 2 S 2 O 3 solution was added thereto, stirred for 30 minutes, and the organic layer was washed with saturated NaHCO 3 solution. The organic layer was dried over anhydrous MgSO 4 , and the organic solvent was removed to give 278 mg (yield 82%) of the title compound.

1H NMR(CDCl3) δ 0.83-0.99(m, 6H), 1.77(m, 1H), 2.61-3.24(m, 8H), 3.74(m, 1H), 4.11(m, 1H), 4.95(d, 1H), 5.10(s, 2H), 7.21-7.50[m, 15H) 1 H NMR (CDCl 3 ) δ 0.83-0.99 (m, 6H), 1.77 (m, 1H), 2.61-3.24 (m, 8H), 3.74 (m, 1H), 4.11 (m, 1H), 4.95 (d , 1H), 5.10 (s, 2H), 7.21-7.50 [m, 15H)

(1-3) [(5S)-[[[(N,N-메틸부틸)아미노카보닐)-β-메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[2S)-[1-페닐-3-메틸]부틸]아미드의 제조(1-3) [(5S)-[[[((N, N-methylbutyl) aminocarbonyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6- Preparation of Phenyl-1-hexanoyl]-[2S)-[1-phenyl-3-methyl] butyl] amide

상기 (1-2)에서 제조한 화합물 250㎎(0.5 밀리몰)을 20㎖의 메탄올에 용해시킨 후 10 중량 %의 Pd/C와 혼합하여 수소풍선 조건에서 3시간동안 교반하였다. 반응 용액을 셀라이트에 통과시켜 무기 촉매를 제거한 다음 반응 용매를 감압 증류하여 제거하였다. 상기 제조예 2의 생성물을 LiOH로 처리하여 생성된 산 0.5 밀리몰과 각각 1.5 당량의 EDC, HOBT 및 트리에틸아민을 10㎖의 디메틸포롬아미드에 용해시킨 다음 상기에서 얻은 아민을 0℃에서 가하고 상온에서 3시간 동안 교반하였다. 용매를 감압 증류하여 제거하고 에틸아세테이트에 용해시킨 후 NaHCO3포화용액으로 세척하였다. 유기층을 20㎖의 디클로로메탄에 용해시킨 후 3 당량의 메타클로로퍼옥시벤조산을 가하고 상온에서 24시간 동안 교반하였다. 30㎖의 10% Na2S2O3용액을 가하고 30분간 교반한 후 유기층을 HaHC3포화용액으로 세척하였다. 유기층을 무수 MgSO4로 건조시키고 유기 용매를 감압 증류하여 제거한 후 컬럼 크로마토그래피(디클로로메탄:메탄올=20:1)를 실시하여 표제 화합물 122㎎(수율 68%)을 얻었다.250 mg (0.5 mmol) of the compound prepared in (1-2) was dissolved in 20 ml of methanol, mixed with 10 wt% of Pd / C, and stirred for 3 hours under hydrogen balloon conditions. The reaction solution was passed through celite to remove the inorganic catalyst, and then the reaction solvent was removed by distillation under reduced pressure. 0.5 mmol of the acid produced by treating the product of Preparation Example 2 and 1.5 equivalents of EDC, HOBT and triethylamine, respectively, were dissolved in 10 ml of dimethylformromamide, and the amine obtained above was added at 0 ° C. Stir for 3 hours. The solvent was distilled off under reduced pressure, dissolved in ethyl acetate, and washed with saturated NaHCO 3 solution. The organic layer was dissolved in 20 ml of dichloromethane and then 3 equivalents of metachloroperoxybenzoic acid was added and stirred at room temperature for 24 hours. 30 ml of 10% Na 2 S 2 O 3 solution was added and stirred for 30 minutes, and then the organic layer was washed with saturated HaHC 3 solution. The organic layer was dried over anhydrous MgSO 4 , the organic solvent was distilled off under reduced pressure, and then subjected to column chromatography (dichloromethane: methanol = 20: 1) to give 122 mg (yield 68%) of the title compound.

1H NMR(CDCl3) δ 0.92(m, 9H), 1.21-2.22(m, 10H), 2.64-3.32(m, 14H), 4.02(m, 2H), 4.82(d, 1H), 6.2(bs, 2H), 7.12-7.33[m, 11H) 1 H NMR (CDCl 3 ) δ 0.92 (m, 9H), 1.21-2.22 (m, 10H), 2.64-3.32 (m, 14H), 4.02 (m, 2H), 4.82 (d, 1H), 6.2 (bs , 2H), 7.12-7.33 (m, 11H)

FAB MS: 657(M+1)FAB MS: 657 (M + 1)

[실시예 2]Example 2

제조예(2-4)에서 메틸-n-부틸아민 대신 사이클로프로메틸아민을 사용하는 것을 제외하고는 실시예 1과 동일하게 실시하여 하기 표1에 열거한 화합물 1을 수득하였다.Except for using cyclopromethylamine instead of methyl-n-butylamine in Preparation Example (2-4) was carried out in the same manner as in Example 1 to obtain a compound 1 listed in Table 1.

[실시예 3]Example 3

제조예 (2-4)에서 메틸-n-부틸아민 대신 벤질아민을 사용하는 것을 제외하고는 실시예 1과 동일하게 실시하여 하기 표1에 열거한 화합물 3로 수득하였다.Except for using benzylamine instead of methyl-n-butylamine in Preparation Example (2-4) was carried out in the same manner as in Example 1 to obtain a compound 3 listed in Table 1 below.

[실시예 4]Example 4

제조예(2-4)에서 메틸-n-부틸아민 대신 2-부라닐메틸아민을 사용하는 것을 제외하고는 실시예 1과 동일하게 실시하여 하기 표1에 열거한 화합물 4을 수득하였다.Compound 4 listed in Table 1 was obtained in the same manner as in Example 1, except that 2-buranylmethylamine was used instead of methyl-n-butylamine in Preparation Example (2-4).

[HIV 프로테아제의 억제 효능 분석][Inhibitory Effect Analysis of HIV Protease]

본 발명의 화합물들의 HIV 프로테아제에 대한 억제 효능을 다음의 방법을 사용하여 확인하였다.The inhibitory efficacy of the compounds of the present invention against HIV protease was confirmed using the following method.

먼저 반응기질이 없는 상태에서 HIV 프로테아제 효소와 본 발명의 화합물을 혼합한 다음(배양전 용액), 반응용액의 일부를 취하여 과량의 반응기질이 있는 용액(분석 용액)에 가하여 효소 활성의 감소정도를 남아있는 활성의 정도로서 측정하였다. 배양전 용액은 50mM 아세트산 나트륨, pH 5.5, 1mM 디티오트레이톨(dithiotreitol, DTT), 1mM 에틸렌디아민테트라아세테이트(ethylenediaminetetraacetate, EDTA), 0.75M 황산암모늄, 0.1% NP 40(Noniget 40, Sigma Chemical Co.)과, 본 발명의 화합물이 여러 농도로 포함되어 있다. 억제 반응은 2.6 nM 의 HIV-1 프로테아제를 가함으로써 개시하였다. 일정시간마다 10㎕씩 취하여, 상기와 동일한 완층용액에 100μM의 반응기질이 포함되어 있는 80㎕의 용액에 가하여 남아있는 효소 활성을 검정하였다. 이때, 반응기질로는 H-His-Lys-Ala-Arg-Val-Leu-(p-니트로)-Phe -Glu-Ala-Ile-Ser-NH의 11개 아미노산으로 이루어진 올리고펩티드를 사용하였는데, 이 기질은 HIV 프로테아제에 의해 (p-니트로)-Phe 과 Leu 사이의 아미드 결합이 끊어지게 된다. 반응속도는 (p-니트로)-Phe 의 280nm에서의 강한 흡광도를 이용하여, 반응전의 기질과 반응후의 생성물을 고성능 액체 크로카토그래피(High Performance Liquid Chromatography, HPLC)로 분리하여 생성물의 상대적 양을 측정함으로써 결정하였다. 시간에 따른 효소 활성의 감소량을 구하고 감소량의 자연로그값(1n)을 시간에 대하여 그래프로 나타낸 직선의 기울기로부터 k를 구하였다. 억제 상수는 하기식에 의해 구하였다.First, the HIV protease enzyme is mixed with the compound of the present invention in the absence of the reactive material (pre-culture solution), and then a part of the reaction solution is taken and added to the excess reactive solution (analytical solution) to reduce the enzyme activity. It was measured as the degree of remaining activity. The pre-culture solution was 50 mM sodium acetate, pH 5.5, 1 mM dithiotreitol (DTT), 1 mM ethylenediaminetetraacetate (EDTA), 0.75 M ammonium sulfate, 0.1% NP 40 (Noniget 40, Sigma Chemical Co. ) And the compound of the present invention in various concentrations. Inhibition was initiated by adding 2.6 nM of HIV-1 protease. 10 μl each was taken at regular intervals, and the remaining enzyme activity was assayed by adding 80 μl of the solution containing 100 μM of reactive mass to the same complete solution. At this time, the oligopeptide consisting of 11 amino acids of H-His-Lys-Ala-Arg-Val-Leu- (p-nitro) -Phe-Glu-Ala-Ile-Ser-NH was used. The substrate breaks down the amide bond between (p-nitro) -Phe and Leu by HIV protease. The reaction rate is determined by using a high absorbance at 280 nm of (p-nitro) -Phe to separate the substrate before the reaction and the product after the reaction by High Performance Liquid Chromatography (HPLC) to measure the relative amount of the product. Determined by. The amount of decrease in enzyme activity over time was determined, and k was calculated from the slope of the straight line graph of the decrease in natural log value (1n) versus time. The inhibition constant was calculated by the following formula.

여기에서, kobs는 일정한 농도의 제제 존재하에서 시간에 따라 효소활성이 감소되는 정도를 나타내는 속도상수이고, kina는 억제제와 효소간의 미카엘리스-멘텐(Michaelis-Menten)결합체로부터 공유결합이 형성되는 화학반응이 일어나는 속도상수이고, K1는 억제제와 효소간의 미카엘리스-맨텐 해리상수(Inhibition Constant)이고, [I]는 억제제의 농도이다.Here, k obs is a rate constant indicating the degree to which the enzyme activity decreases with time in the presence of a constant concentration of the agent, k ina is a covalent bond is formed from the Michaelis-Menten conjugate between the inhibitor and the enzyme The rate constant at which the chemical reaction occurs, K 1 is the Michaelis-Manten Inhibition Constant between the inhibitor and the enzyme, and [I] is the concentration of the inhibitor.

상기식은 억제제 농도가 효소의 농도보다 월등히 높을 경우(정상 상태 조건)에 적용이 가능하다. 그러나, 억제제 농도가 고활성이어서 억제제의 농도와 효소의 농도가 비슷한 조건에서 실험을 행하여야 할 경우에는 상기식보다(E: 유리 효소, I; 유리 억제제, EI: 미카엘리스-멘텐 결합체, EI': 효소와 억제제의 공유 결합물)의 반응식을 사용하여 정상상태 가정을 하지 않은 각 시간마다 살아있는 효소의 상대적 농도 [E]/([E)] +[EI] +[EI'])의 값을 KINSIM/FITSIM 프로그램(Willams and Morrison; Zimmerle and Frieden)에 입력하여 계산함으로써 억제상수 ki와 kina및 2차 억제상수(second order rate constant)인 kina/KI를 구하여 하기 표2에 나타내었다. 본 발명의 화합물의 경우, 어느 것이나 이차억제상수 kina /KI의 값이 충분히 크므로, 효소와 억제제 간에 비가역적인 반응이 일어났음을 알 수 있다.The above formula is applicable when the inhibitor concentration is much higher than the enzyme concentration (steady state conditions). However, if the inhibitor concentration is high activity and the experiment should be conducted under similar conditions of inhibitor concentration and enzyme concentration, (E: free enzyme, I; free inhibitor, EI: Michaelis-Mentene conjugate, EI ': covalent combination of enzyme and inhibitor) using the relative concentration of live enzyme at each time without steady-state assumption [ E] / ([E)] + [EI] + [EI ']) is calculated by entering the KINSIM / FITSIM program (Willams and Morrison; Zimmerle and Frieden) to calculate the inhibitory constants k i and k ina and secondary suppression. To obtain a constant (second order rate constant) k ina / K I is shown in Table 2 below. In the case of the compound of the present invention, since the value of the second inhibitory constant k ina / K I is sufficiently large, it can be seen that an irreversible reaction occurred between the enzyme and the inhibitor.

[항 HIV-1 바이러스 활성 및 세포독성 측정][Anti HIV-1 Virus Activity and Cytotoxicity Measurement]

본 발명의 화합물의 항 바이러스 효과는 HIV-1 바이러스의 양을 50%로 저지하는 화합물 농도(50% Inhibitory Concentration, IC50)를 결정하여 평가하였다.The antiviral effect of the compounds of the present invention was evaluated by determining the compound concentration (50% Inhibitory Concentration, IC 50 ) that inhibits the amount of HIV-1 virus by 50%.

실험에 사용할 HIV-1 바이러스는 다음과 같이 배양하여 얻었다. 먼저, COS 세포[시미안(Simian) 바이러스 40으로 형질전환된 아프리카 녹색 원숭이의 신장 세포, 입수처: 미국 타입 컬쳐 콜렉션(American Type Culture Collection, ATCC) Cat# CRL-1651]를 DME 배지(Dulbecco's modified Eagle's medium)에서 배양한 후 이 COS 세포 배양액 1 ㎖(107세포)를 일렉트로포레이션(electroporation) 용기에 넣고 여기에 20㎕의 HIV-1 유전자를 함유한 플라스미드(pNL 4-3, 입수처: 미국 AIDS Research and Reference Reagent Program Cat. #114)를 가하여 섞어준 다음, 20% 태아 소 혈청(Fetal Bovine Serum, FBS)을 함유한 RPMI 1640 배지(Gibco-RRL사)중에서, 셀-포레이터(Cell-Porator, BRL)를 이용하여 800 μF, 250 V 조건으로 일렉트로포레이션시켰다. 이어서, 일렉트로포레이션된 세포액을 얼음중에서 10분간 방치한 후 15㎖의 DME 배지(10% 태아 소 혈청 함유)를 들어있는 T75 플라스크에 넣고 CO2배양기에서 배양하였다. 24시간 후에 10㎖의 동일한 새 배지로 바꾸어 주고, 제 2일째에 배양액을 모아서 원심 분리한 후 상층액을 0.22mm 필터에 통과시켜 HIV-1바이러스를 얻고, 이를 분배하여 -70℃에서 보관하였다. 그런다음, 얻어진 HIV-1 바이러스를 MT-2 세포주(입수처: 미국 AIDS Research and Reference Reagent Program Cat. # 237)에 감염시켜 바이러스 양을 증폭시켰다. 즉, 4 x 106세포의 MT-2 세포주 1500 TCID50(50% 세포감염농도, tissue culture infectious dose)의 HIV-1 바이러스와 함께 섞어주어 감염시킨 뒤 배양하였다. 감염된 MT-2 세포가 융합되어 풍선모양의 신키티움(Syncytium)을 많이 형성하는 7일경에 다시 5배의 감염되지 않은 MT-2 세포를 넣고 배양하였다. 이틀 후부터 3일동안 매일 바이러스 배양액을 취하여 원심분리한 후 상층액을 0.22mm 필터에 통과시켜 중폭된 HIV-1 바이러스를 얻고, 이를 분배시켜 -70℃에서 보관하였다. 그 결과 HIV-1 바이러스의 구조단백질인 p24의 양이 131 ng/㎖이고, 바이러스 양이 4 x 104TCID50/㎖ 이며, 역전사효소 활성이 3.8 x 105cpm/㎖ 인 HIV-1 바이러스를 얻었다.The HIV-1 virus to be used in the experiment was obtained by culturing as follows. First, COS cells (renal cells of African green monkeys transformed with Simian virus 40, source: American Type Culture Collection (ATCC) Cat # CRL-1651) were added to DME medium (Dulbecco's modified). After culturing in Eagle's medium, 1 ml (10 7 cells) of this COS cell culture solution was placed in an electroporation vessel, where a plasmid containing 20 μl of HIV-1 gene (pNL 4-3, obtained from: After the addition of US AIDS Research and Reference Reagent Program Cat. # 114, the mixture is mixed and then cell-porated in RPMI 1640 medium (Gibco-RRL) containing 20% Fetal Bovine Serum (FBS). -Porator, BRL) was electroporated at 800 μF, 250 V conditions. The electroporated cell solution was then left in ice for 10 minutes and then placed in a T75 flask containing 15 ml of DME medium (containing 10% fetal bovine serum) and incubated in a CO 2 incubator. After 24 hours, the cells were replaced with 10 ml of the same fresh medium. On the second day, the culture solution was collected and centrifuged, and the supernatant was passed through a 0.22 mm filter to obtain HIV-1 virus, which was distributed and stored at -70 ° C. The resulting HIV-1 virus was then infected with MT-2 cell line (obtained from US AIDS Research and Reference Reagent Program Cat. # 237) to amplify the amount of virus. That is, 4 x 10 6 cells of MT-2 cell line 1500 TCID 50 (50% cell culture infection, tissue culture infectious dose) and mixed with HIV-1 virus and then cultured. Infected MT-2 cells were fused to form a balloon-like synthium (Syncytium) around 7 days again 5 times uninfected MT-2 cells were incubated. After two days, virus cultures were taken daily for 3 days, centrifuged, and the supernatants were passed through a 0.22 mm filter to obtain amplified HIV-1 virus, which was distributed and stored at -70 ° C. As a result, the HIV-1 virus, a structural protein of p24, was 131 ng / ml, the virus amount was 4 x 10 4 TCID 50 / ml, and the reverse transcriptase activity was 3.8 x 10 5 cpm / ml. Got it.

본 발명의 화합물의 HIV-1 증식 억제 효과를 알아보기 위해 다음과 같은 실험을 행하였다. 대전 혈액원으로부터 입수한 혈액에서 파이콜-하이팩(Ficoll-Hypaque, Pharmacia)을 이용하여 PBMC(Peripheral Blood Mononuclear Cells, 백혈구 농축액)를 분리한 다음, 여기에 5㎕/㎖ 의 PHA-P(phytohemagglutinin-P, 식물성 적혈구 응집소 -P, Sigma사)를 가하여 배양하였다. 상기 배양은 20% 태아 소혈청, 인터루킨-2(interleukin-2, IL-2, 20 Units/㎖), 페니실린(100 Units/㎖), 스트렙토마이신(100 ㎕/㎖) 및 암포테리신 B(0.25 ㎕/㎖)를 함유하는 RPMI 1640 배지를 사용하여 수행하였다. 3일 후에 RPMI 1640 배지로 세포를 세척한 후 1.2 x 107세포를 12,000 TCID50의 HIV-1 바이러스와 37℃ 에서 2시간 동안 섞어주어 감염시켰다. 원심분리 후 얻은 감염된 세포를 RPMI 1640 배지로 2회 세척한 후 세포수를 2 x 106세포/㎖ 로 조정하였다. 측정하고자 하는 HIV-1 억제 화합물을 디멜틸설폭사이드(dimethylsufoxide, DMSO)로 희석시켜 200, 63.2, 20, 6.32, 3.56, 2, 0.632, 0.2, 0.0632 및 0 μM의 용액(각각 200㎕)을 준비하였다. RPMI 1640 배지 495 ㎕를 웰 배양 평판에 가하고, 여기에 5 ㎕의 HIV-1 억제 화합물을 첨가한 다음 500 ㎕의 HIV-1 바이러스에 감염된 세포(1 x 106세포)를 가하고 배양하였다. 따라서 화합물의 최종 농도는 1000, 316, 100, 31.6, 17.78, 10, 3.16, 1, 0.316 및 0 nM 이다. 4일후에 750㎕의 배지를 따라 버리고 화합물을 함유한 동일 배지 1㎖를 새로 첨가하고 배양하였다. 7일 후에 배양액중의 단백질 p24 양 또는 역전사효소 활성을 측정하여, HIV-1 바이러스의 양을 50%로, 억제하는 화합물의 농도인 IC50을 측정하였다.In order to examine the effects of inhibiting the proliferation of HIV-1 of the compound of the present invention, the following experiment was conducted. Peripheral Blood Mononuclear Cells (PBMC) were isolated from Fibro-Hypaque (Pharmacia) using blood obtained from Daejeon blood sources, and then 5 μl / ml PHA-P (phytohemagglutinin-P) was added thereto. , Plant erythrocyte aggregate -P, Sigma) was added and cultured. The culture consisted of 20% fetal bovine serum, interleukin-2 (IL-2, 20 Units / ml), penicillin (100 Units / ml), streptomycin (100 μl / ml) and amphotericin B (0.25 And RPMI 1640 medium containing [mu] l / ml). After 3 days, the cells were washed with RPMI 1640 medium, and then 1.2 × 10 7 cells were infected by mixing 12,000 TCID 50 HIV-1 virus at 37 ° C. for 2 hours. Infected cells obtained after centrifugation were washed twice with RPMI 1640 medium and the cell number was adjusted to 2 × 10 6 cells / ml. Dilute the HIV-1 inhibitory compound to be measured with dimethylsufoxide (DMSO) to prepare 200, 63.2, 20, 6.32, 3.56, 2, 0.632, 0.2, 0.0632 and 0 μM solutions (200 μl each). It was. 495 μl of RPMI 1640 medium was added to the well culture plate, to which 5 μl of HIV-1 inhibitory compound was added, followed by 500 μl of HIV-1 virus infected cells (1 × 10 6 cells) and cultured. The final concentrations of the compounds are therefore 1000, 316, 100, 31.6, 17.78, 10, 3.16, 1, 0.316 and 0 nM. After 4 days, 750 µl of medium was discarded and 1 ml of the same medium containing the compound was added and cultured. After 7 days, the amount of protein p24 or reverse transcriptase activity in the culture was measured to determine the amount of the compound that inhibits the amount of the HIV-1 virus at 50% and IC 50 .

단백질 p24 양은 듀퐁(Du Pont)사의 HIV-1 p24 핵 프로필 엘리자[Core Profile ELISA(Enzyme Linked Immunosorbent Assay)](NEN Cat # NEK-060) 또는 셀룰라 프로덕츠 인코포레이티드(Cellular Products Inc.)사의 Retro-tekTM HIV-1 p24 하원 엘리자(CPI Cat # 0801111)을 이용하여 측정하였다.Protein p24 amount was determined by Du Pont's HIV-1 p24 nuclear profile ELISA (Enzyme Linked Immunosorbent Assay) (NEN Cat # NEK-060) or Cellular Products Inc. Retro. -tekTM HIV-1 p24 House Eliza (CPI Cat # 0801111) was used to measure.

역전사효소 활성은 다음과 같이 측정하였다. 먼저 800㎕의 상기 배양액을 400 ㎕의 폴리에틸렌글리콜(polyethylene glycol, PEG) 용액(30% PEG 8000, 0.4 M NaC1)과 혼합한 다음 4 ℃에서 보관하였다. 다음날 에펜도르프 원심분리기로 15,000 rpm에서 20분간 원심분리시켜 HIV-1 바이러스를 얻고, 40㎕의 용액 1(25 mM 트리스-염산, pH 7.8, 0.25mM EDTA, 0.025 % 트리톤 X-100, 50 % 글리세롤, 10 mM DTT 및 100 mM KC1)과 20㎕의 용액 2(0.9 % 트리톤 X-100, 43.7 mM KC1)에 용해시킨 다음, 이중 10㎕를 취하여 90㎕의 역전사효소 활성 측정액[46.5 mM 트리스-염산, pH 7.8, 8.89 mM DTT, 11.1 mM MgC12, 3.75 mM NaC1, 2.5 μCi3H-TTP(methy1-3H-thymidine-5-triphosphate)(NEN-Du Pont, 10-25 Ci/밀리몰), 0.556 μ/㎖ 폴리(rA):올리고(dT)(파마시아(Pharmacia사))]과 혼합하고 37℃에서 1시간 동안 반응시켰다. 반응후 9 ㎕의 0.25 M EDTA를 첨가하여 반응을 중단시킨 다음, 이반응액을 DEAE(diethylaminoethyl, 디에틸아미노에틸) 필터(DE 81, Whatmann Cat # 3658N323)에 옮겨 건조시키고 2 x SSC 완충용액(0.3M NaC1, 0.03M Na3Citrate.2H20 pH 7.0)으로 세척하였다. 필터를 건조시킨 다음 5㎖의 액체섬광 용액을 첨가하고 액체섬광계수기를 이용하여 정량하였다.Reverse transcriptase activity was measured as follows. First, 800 µl of the culture solution was mixed with 400 µl of polyethylene glycol (PEG) solution (30% PEG 8000, 0.4 M NaC1) and then stored at 4 ° C. The following day centrifuged for 20 minutes at 15,000 rpm with an Eppendorf centrifuge to obtain HIV-1 virus, 40 μl of Solution 1 (25 mM Tris-HCl, pH 7.8, 0.25 mM EDTA, 0.025% Triton X-100, 50% Glycerol) , 10 mM DTT and 100 mM KC1) and 20 μl of solution 2 (0.9% Triton X-100, 43.7 mM KC1), and then 10 μl of the 90 μl reverse transcriptase activity assay [46.5 mM Tris- Hydrochloric acid, pH 7.8, 8.89 mM DTT, 11.1 mM MgC1 2 , 3.75 mM NaC1, 2.5 μCi 3 H-TTP (methy1- 3 H-thymidine-5-triphosphate) (NEN-Du Pont, 10-25 Ci / mmolmol), 0.556 μ / ml poly (rA): oligo (dT) (Pharmacia)] was reacted at 37 ° C. for 1 hour. After the reaction, 9 µl of 0.25 M EDTA was added to stop the reaction, and the reaction solution was transferred to a DEAE (diethylaminoethyl, diethylaminoethyl) filter (DE 81, Whatmann Cat # 3658N323), dried, and 2 x SSC buffer (0.3 M NaCl, 0.03 M Na 3 Citrate. 2 H 2 0 pH 7.0). After the filter was dried, 5 ml of liquid scintillation solution was added and quantified using a liquid scintillation counter.

항에이즈제의 최대 허용치를 결정하기 위한 세포 독성을 검사하기 위하여, PHA-P로 자극시킨 1 x 106PBMC 세포를 24 웰 배양 평판에 가하고 0.1 내지 100 μM 범위의 본 발명의 화합물을 첨가한 다음 RPMI 1640 배지에서 37℃로 배양하고 4일간격으로 배지를 바꾸어 주면서 세포의 증식 정도를 트리판 블루다이 익스크루전 방법(Trypanblue dye exclusion technique)으로 혈구 계산판에서 2주간 관찰하여 세포독성 CT50(세포의 50%가 죽는 값)을 결정하였다. 측정된 항바이러스 활성 및 세포 독성의 결과를 하기 표2에서 나타내었다. 하기 표2에서 보듯이, 본 발명의 화합물들은 모두 HIV 프로테아제 억제 효과가 높으면서도 세포독성은 낮다는 것을 알 수 있다.To test the cytotoxicity to determine the maximum tolerated anti-Aid agent, 1 x 10 6 PBMC cells stimulated with PHA-P were added to a 24-well culture plate and the compounds of the invention in the range of 0.1-100 μM were added RPMI cultured in 1640 medium with 37 ℃ and 4 days changing the medium at intervals while the proliferation of the cells trypan blue die IX crew former method (Trypanblue dye exclusion technique) as observed two weeks in blood calculation plate cytotoxic CT 50 ( 50% of cells die). The results of the measured antiviral activity and cytotoxicity are shown in Table 2 below. As shown in Table 2, it can be seen that the compounds of the present invention are all low in cytotoxicity while having a high HIV protease inhibitory effect.

이상에서 살펴본 바와 같이, 본 발명의 일반식(Ⅰ)의 혼합물은 HIV 프로테아제의 비가역적 억제제로서 억제효과는 높은 반면 세포독성은 낮기 때문에, 에이즈 또는 HIV 감염의 치료 또는 예방을 위한 약학적 조성물로서 유용하게 사용될 수 있다.As described above, the mixture of the formula (I) of the present invention is useful as a pharmaceutical composition for the treatment or prevention of HIV or HIV infection because of its high inhibitory effect and low cytotoxicity as an irreversible inhibitor of HIV protease. Can be used.

Claims (6)

하기 일반식(Ⅰ)의 시스-에폭사이드 구조를 갖는 화합물 또는 그의 약제학적으로 허용되는 염, 수화물 또는 용매화물:Compounds having a cis-epoxide structure of formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof: 상기식에서, R1은 탄소수 1 내지 8의 직쇄 또는 측쇄 알킬, 사이클릭 알킬 라디칼 또는 사이클릭 알킬 라디칼로 치환된 저급알킬이고, R2및 R3는 서로 독립적으로 저급알킬, 방향족 라디칼로 치환된 저급 알킬, 탄소수 3 내지 8의 사이클릭 알킬, 사이클릭 알킬 라디칼로 치환된 저급 알킬 또는 방향족 라디칼이며, R4수소 또는 저급알킬이다.Wherein R 1 is lower alkyl substituted with straight or branched chain alkyl, cyclic alkyl radical or cyclic alkyl radical having 1 to 8 carbon atoms, and R 2 and R 3 are independently substituted with lower alkyl, aromatic radical Alkyl, cyclic alkyl having 3 to 8 carbon atoms, lower alkyl or aromatic radical substituted by cyclic alkyl radical, and R 4 hydrogen or lower alkyl. 제1항에 있어서, R1이 2-푸라닐메틸 또는 벤질이고, R2가 수소 또는 메틸이며, R3가 이소프로필이고, R4가 벤질인 화합물.The compound of claim 1, wherein R 1 is 2-furanylmethyl or benzyl, R 2 is hydrogen or methyl, R 3 is isopropyl, and R 4 is benzyl. 제1항에 있어서, [(5S)-[[[(N,N-사이클로프로필메틸)아미노카보닐]-β메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드; [(5S)-[[[(N,N-메틸부틸)아미노카보닐]-β-메탄설포닐-L-발리닐]아미노](4R,3S)-에폭시-6-페닐-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드; [(5S)-[[(N-벤질아미노카보닐)-β-메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드; [(5S)-[[(N-2-푸라닐메틸아미노카보닐)-β-메탄설포닐-L-발리닐]아미노]-(4R,3S)-에폭시-6-페닐-1-헥사노일]-[(2S)-[1-페닐-3-메틸]부틸]아미드; 및 이들의 약학적으로 허용되는 염, 수화물 및 용매화물 중에서 선택된 화합물.The compound of claim 1, wherein [(5S)-[[[((N, N-cyclopropylmethyl) aminocarbonyl] -βmethanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6 -Phenyl-1-hexanoyl]-[(2S)-[1-phenyl-3-methyl] butyl] amide; [(5S)-[[[((N, N-methylbutyl) aminocarbonyl] -β-methanesulfonyl-L-valinyl] amino] (4R, 3S) -epoxy-6-phenyl-1-hexanoyl ]-[(2S)-[1-phenyl-3-methyl] butyl] amide; [(5S)-[[(N-benzylaminocarbonyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl]-[( 2S)-[1-phenyl-3-methyl] butyl] amide; [(5S)-[[(N-2-furanylmethylaminocarbonyl) -β-methanesulfonyl-L-valinyl] amino]-(4R, 3S) -epoxy-6-phenyl-1-hexanoyl ]-[(2S)-[1-phenyl-3-methyl] butyl] amide; And pharmaceutically acceptable salts, hydrates, and solvates thereof. 일반식(Ⅱ)의 화합물을 일반식(Ⅲ)의 화합물과 커플링 반응시킨 후 산화시켜 일반식(Ⅳ)의 화합물을 얻고; 일반식(Ⅳ)의 화합물을 탈보호시킨 후 일반식(Ⅴ)의 화합물을 커플링시켜 일반식(Ⅵ)의 화합물을 얻은 후 이를 산화시키는 것을 특징으로 하는, 일반식(Ⅰ)의 화합물의 제조방법.Coupling the compound of formula (II) with the compound of formula (III) and then oxidizing to obtain a compound of formula (IV); Preparation of a compound of formula (I), characterized by deprotecting the compound of formula (IV) and then coupling the compound of formula (V) to obtain a compound of formula (VI) and then oxidizing it. Way. 상기에서, R1, R2, R3및 R4는 제1항에서 정의한 바와 같고, ⓟ는 아미노기 보호그룹이다.In the above, R 1 , R 2 , R 3 and R 4 are as defined in claim 1, ⓟ is an amino group protecting group. 제4항에 있어서, 일반식(Ⅶ)의 화합물을 비티그 반응시켜 일반식(Ⅷ)의 화합물을 수득한 다음, 이를 산 촉매 존재하에 t-부탄올중에서 용매의 환류온도로 가열하는 것에 의해 상기 일반식(Ⅱ)의 화합물을 제조하는 단계를 더욱 포함하는 것을 특징으로 하는 방법.The method of claim 4, wherein the compound of general formula (VIII) is subjected to a vitig reaction to obtain a compound of general formula (IV), which is then heated to the reflux temperature of the solvent in t-butanol in the presence of an acid catalyst. Further comprising preparing a compound of formula (II). 상기에서, X는 할로겐 원자이고, ⓟ는 아미노기 보호그룹이다.In the above, X is a halogen atom, ⓟ is an amino group protecting group. 제1항 내지 제3항중 어느 한 항의 화합물 또는 그의 약학적으로 허용되는 염, 수화물 또는 용매화물을 유효성분으로 포함하는, 후천성 면역결핍증 또는 인간 면역결핍 바이러스(HIV) 감염의 예방 또는 치료에 유용한 조성물.A composition useful for the prevention or treatment of acquired immunodeficiency or human immunodeficiency virus (HIV) infection, comprising the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt, hydrate or solvate thereof as an active ingredient. .
KR1019950013366A 1995-05-26 1995-05-26 Irreversible hiv protease inhibitors with anti-aids effect, processes for the preparation thereof and compositions containing the same KR0161141B1 (en)

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