KR100388789B1 - Method for manufacturing farnesyl transferase inhibitor having pyrrole structure - Google Patents
Method for manufacturing farnesyl transferase inhibitor having pyrrole structure Download PDFInfo
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Abstract
Description
본 발명은 피롤구조를 포함하며 파네실 전이효소를 억제할 수 있는 하기 화학식 1의 화합물 또는 그의 약제학적으로 허용되는 염에 관한 것이다.The present invention relates to a compound of formula (1) or a pharmaceutically acceptable salt thereof that includes a pyrrole structure and is capable of inhibiting farnesyl transferase.
화학식 1Formula 1
상기식에서In the above formula
A 는 수소 또는 저급알킬을 나타내거나, 하기 구조식의 그룹중 선택된 어느 하나를 나타내고,A represents hydrogen or lower alkyl, or represents any one selected from the group of the following structural formulas,
여기에서 R1은 수소, 할로겐, 시아노, 니트로, 하이드록시카보닐, 아미노카보닐, 아미노티오카보닐, 저급알콕시, 페녹시, 페닐 또는 저급알킬을 나타내며, R2는 수소, 저급알킬, 저급알카노일, 또는 아릴에 의해 치환되거나 비치환된 저급알콕시카도닐을 나타내고,Wherein R 1 represents hydrogen, halogen, cyano, nitro, hydroxycarbonyl, aminocarbonyl, aminothiocarbonyl, lower alkoxy, phenoxy, phenyl or lower alkyl, R 2 represents hydrogen, lower alkyl, lower Lower alkoxycardonyl substituted or unsubstituted by alkanoyl or aryl,
B 는 하기 구조식의 그룹중 선택된 어느 하나를 나타내며,B represents any one selected from the group of the following structural formulas,
여기에서 R3및 R4는 각각 독립적으로 수소, 저급알킬, 저급알콕시, 할로겐, 시아노, 하이드록시카보닐, 아미노카보닐, 아미노티오카보닐, 하이드록시, 페닐 또는 페녹시를 나타내고,Wherein R 3 and R 4 each independently represent hydrogen, lower alkyl, lower alkoxy, halogen, cyano, hydroxycarbonyl, aminocarbonyl, aminothiocarbonyl, hydroxy, phenyl or phenoxy,
또는 S 를 나타내며, n 은 2 내지 4의 정수를 나타내고, R7은 저급알킬을 나타낸다.Or S, n represents an integer of 2 to 4, and R 7 represents lower alkyl.
특히, 본 발명에 따른 화합물은 지금까지 알려진 파네실 전이효소 억제제와는 상이한 구조를 갖고 있을 뿐아니라 티올기도 전혀 포함하고 있지 않다.In particular, the compound according to the present invention not only has a structure different from the farnesyl transferase inhibitors known to date, but also contains no thiol groups at all.
본 발명에는 또한, 상기 화학식 1의 화합물을 제조하는 방법도 포함되어 있다. 따라서, 그 제조 방법도 본 발명의 대상이다.The present invention also includes a method for preparing the compound of Formula 1. Therefore, the manufacturing method is also the subject of this invention.
Ras 단백질은 세포의 성장과 분화에 중요한 역활을 하는 21 kda의 단백질로서 구아닌 뉴클레오타이드와 결합하면, 구아노신 트리포스페이트(GTP)를 구아노신 디포스페이트(GDP)로 가수분해하는 효소로서 세포내에서 특이적인 GTPase 회로를 조절하는 분자스위치로 작용하는 것으로 알려져있다(참조: Bourne, H.R., Sanders, D.A., McCormick, F., Nature 1991, 349, 117).Ras protein is a 21 kda protein that plays an important role in cell growth and differentiation. It is an enzyme that hydrolyzes guanosine triphosphate (GTP) to guanosine diphosphate (GDP) when combined with guanine nucleotides. It is known to act as a molecular switch that regulates the GTPase circuit (Bourne, HR, Sanders, DA, McCormick, F., Nature 1991, 349, 117).
Ras 단백질은 포유동물세포에서 3 가지의 Ras 유전자에 의해 아미노산 188개와 K-Ras-4B 또는 189개의 H-Ras, K-ras-4A 및 N-Ras로 생성된다. 이 단백질의 12, 13, 61번 위치에 있는 아미노산들은 GTP의 인산기와 근접하여 있어, 이 아미노산 잔기들은 GTP의 가수분해에 관여하는 물분자의 공간적 위치에 영향을 미침으로써 GTPase 효소 활성을 저해한다. 인체에서 암이 발생한 경우, 이 위치의 아미노산에 돌연변이가 관찰되는데, 이 돌연변이가 결국 Ras 단백질 고유의 GTPase 활성을 저해하여 GTP 결합상태를 지속시킴으로써 비정상적인 성장신호를 지속적으로 전달하여 발암성을 나타내는 것으로 알려져 있다. 이와 같이 발암성인 Ras 유전자는 특이적으로 췌장암, 방광암, 폐암 및 피부암등에 밀접한 관련이 있는 것으로 알려져 있다(참조: Bos, J. L., Cancer Res., 1989, 49, 4682).Ras protein is produced by mammalian cells by three Ras genes, 188 amino acids and K-Ras-4B or 189 H-Ras, K-ras-4A and N-Ras. The amino acids at positions 12, 13 and 61 of the protein are in close proximity to the phosphate groups of GTP, and these amino acid residues inhibit GTPase enzyme activity by affecting the spatial position of water molecules involved in the hydrolysis of GTP. When cancer occurs in the human body, mutations in amino acids at this position are observed, which eventually inhibits Ras protein's intrinsic GTPase activity and maintains GTP binding status, which is known to show carcinogenicity by continuously transmitting abnormal growth signals. have. Such a carcinogenic Ras gene is specifically known to be closely related to pancreatic cancer, bladder cancer, lung cancer and skin cancer (see Bos, J. L., Cancer Res., 1989, 49, 4682).
Ras 단백질이 생물학적으로 활성화 상태에 있기 위해서는 세포막 내에 부착되어야 하는데 이를 위해서는 Ras 파네실 전이효소, Ras 단백질 카복시 말단의 3개 AAX 펩타이드 절단 효소, 메틸 전이효소 및 팔미토일 전이효소에 의한, 단백질 전이 후의 탄소말단의 변형이 요구된다. 이중 첫번째 단계인 파네실화는 파네실 전이효소(FTase)에 의해 이루어진다. 파네실 전이효소의 기질은 Ras 단백질의 카복시말단에 있는 CA1A2X라는 네개의 펩타이드이며, 여기서 A1 및 A2는 전기적 부하를 띄지 않는 지방족 아미노산이고 X는 메티오닌, 알라닌 또는 세린 등이다. 파네실 반응은 시스테인에 일어나 황에테르 결합을 형성시키며, H-Ras와 N-Ras의 경우에는 카복시 말단 근처의 또 다른 시스테인에 팔미토일화가 일어난다. 파네실화의 결과로 Ras 단백질은 친소성이 증가되어 세포막 내에 부착되게되며, 파네실화된 Ras 단백질은 카복시 말단으로부터 연이어 3개의 AAX 펩타이드가 절단효소에 의해 제거되고 메틸화되어 파네실기가 세포막 내의 지질층 또는 다른 수용체와 결합하는 것을 용이하게 해주는 것으로 알려져 있다. 한편, K-Ras-4B는 H-Ras, N-Ras와는 달리 팔미토일화에 필요한 시스테인 대신 폴리베이직(Poly basic) 도매인이라 불리는 여러개의 라이신이 배열된 부위를 가지고 있으며, 이를 통해 세포막 내의 음이온성 지질과의 결합이 용이하게 되는 것으로 알려져 있다. Ras 단백질이 세포막내에 잘 부착하기 위해서는 모든 변형 단계가 필요하나, Ras 단백질의 활성화에는 파네실화만으로도 충분한 것으로 알려져 있으므로 이 파네실화를 차단함으로써 돌연변이에 의한 Ras 발암성을 억제하기 위한 연구가 활발히 진행되고 있다(참조: Buss, J.E. et al., Chemistry & Biology, 1995, 2, 787).In order for the Ras protein to be biologically active, it must be attached to the cell membrane, which requires carbon after protein transfer by Ras farnesyl transferase, three AAX peptide cleavage enzymes at the Ras protein carboxy terminus, methyl transferase and palmitoyl transferase. Terminal modification is required. The first step, panesylation, is achieved by farnesyl transferase (FTase). The substrate of the farnesyl transferase is four peptides, CA1A2X, at the carboxy terminus of the Ras protein, where A1 and A2 are aliphatic amino acids with no electrical load and X is methionine, alanine or serine. The farnesyl reaction occurs on cysteine to form sulfur ether bonds, and in the case of H-Ras and N-Ras, palmitoylation occurs at another cysteine near the carboxy terminus. As a result of the panesylation, the Ras protein is increased in affinity to adhere to the cell membrane, and the panesylated Ras protein is subsequently removed from the carboxy terminus by three AAX peptides removed and methylated so that the panesyl group is in the lipid layer or other in the cell membrane. It is known to facilitate binding with receptors. On the other hand, unlike H-Ras and N-Ras, K-Ras-4B has several lysine sites called poly basic wholesalers instead of cysteine required for palmitoylation. It is known that binding to lipids is facilitated. In order for Ras protein to adhere well to the cell membrane, all modification steps are required. However, the activation of Ras protein is known to be sufficient for panicylation alone. Therefore, studies are being actively conducted to inhibit Ras carcinogenicity by mutation by blocking this panicylation. (Bus, JE et al., Chemistry & Biology, 1995, 2, 787).
그간의 연구결과, Ras로 형질전환된 세포에서 파네실 전이효소를 저해했을때 세포의 성장이 저해되는 것으로 관찰되었으며, 또한 Ras에 의해 변형된 세포형질을 개선하는 것으로 나타났다.Previous studies have shown that inhibition of farnesyl transferase in Ras-transformed cells inhibits cell growth and improves the cell morphology modified by Ras.
실제로 파네실 전이효소의 몇몇 저해제들은 Ras 발암성 유전인자의 세포내 프레닐기에 의한 반응을 선택적으로 저해하는 것으로 밝혀졌다(참조: Kohl, N. E.et al., Proc. Natl. Acad. Sci. USA., 91, 9141(1994); Kohl, N.E.et al, Nature Medicine, 1, 792(1995)). 개발된 파네실 전이효소 저해제로는 시스테인 티올(thiol)기를 함유하며 CAAX와 유사한 구조를 갖는 펩타이드 변형체 및 이를 개선한 저해제(참조: US Patent No. 5,141,851 호; Kohl, N. E. et al, Science, 260, 1934(1993); Graham et al., PCT/US95/12224), 페닐기로 변형된 펩타이드(참조: Sebti, S. M.,J. Biol. Chem. 270, 26802, 1995), 향정신성 의약품 골격구조중 벤조디아제핀을 펩타이드의 turn 모사구조로 활용한 변형체(참조: James, G. L. et al., Science, 260, 1937,1993), 펩타이드 구조에서 벗어나 트리사이클릭 유기 화합물을 골격으로한 저해제(참조: Bishop W. R.et al., J. Biol. Chem. 270, 30611, 1995)를 들 수 있다. 또한, 파네실 전이효소가 프레닐기를 전이시키는 작용기전이 전자 친화적 치환반응(ElectrophilicDisplacement)이므로 반응의 트랜지션 상태(transition state)에 양성부하를 요구함에 착안하여 프레닐기에 트랜지션 상태의 양성부하를 연결시킨 새로운 형태의 저해제가 제시되었다(참조: Poulter, C. D.et al., J. Am. Chem. Soc. 118, 8761, 1996).Indeed, several inhibitors of farnesyl transferase have been found to selectively inhibit the response of Ras oncogenic genes to intracellular prenyl groups (see Kohl, NE et al., Proc. Natl. Acad. Sci. USA . 91, 9141 (1994); Kohl, NE et al, Nature Medicine , 1, 792 (1995). The developed panesyl transferase inhibitors include a peptide variant containing a cysteine thiol group and having a structure similar to that of CAAX, and an inhibitor improving the same (see US Patent No. 5,141,851; Kohl, NE et al, Science, 260, 1934 (1993); Graham et al., PCT / US95 / 12224), peptides modified with phenyl groups (Sebti, SM, J. Biol. Chem . 270, 26802, 1995), benzodiazepines in the psychotropic pharmaceutical framework Transformation (James, GL et al., Science, 260, 1937,1993), which is used as a turn simulation structure of an inhibitor, and a inhibitor based on a tricyclic organic compound away from the peptide structure (Bishop WR et al., J. Biol. Chem . 270, 30611, 1995). In addition, since the mechanism by which the farnesyl transferase transfers the prenyl group is an electrophilic displacement reaction, it is considered that a positive load is required in the transition state of the reaction, thereby linking the positive load of the transition state to the prenyl group. New forms of inhibitors have been proposed (Poulter, CD et al., J. Am. Chem. Soc . 118, 8761, 1996).
그러나, 많은 경우의 인체 암에서 K-Ras 활성화가 주요 원인으로 작용하며, 지금까지 개발된 대부분의 프레닐 전이효소 저해제들은 K-Ras를 활성화시킨다. 따라서, K-Ras에 의해 형질전환된 세포의 성장저해 정도가 H-Ras, N-Ras에 의해 형질전환된 세포의 성장저해에 비해 떨어지므로 K-Ras 활성을 효과적으로 저해할 수 있는 새로운 저해제의 연구가 주목을 받고 있다.However, K-Ras activation is a major cause in many human cancers, and most of the prenyl transferase inhibitors developed to date activate K-Ras. Therefore, the inhibition of growth of cells transformed by K-Ras is lower than the growth inhibition of cells transformed by H-Ras and N-Ras, so the study of novel inhibitors that can effectively inhibit K-Ras activity Is getting attention.
이에 본 발명자들은 K-Ras 기질에 대한 효소활성 저해능 및 세포내 K-Ras 프레닐화 저해능을 평가할 수 있는 새로운 평가체계를 확립하여 이를 활용함으로써, K-Ras 뿐만 아니라 H-Ras, N-Ras 기질의 파네실화를 저해하는 신규한 화합물을 합성하고 그 저해능을 평가하였다. 그 결과, 본 발명자들은 상기 화학식 1의 화합물이 본 발명의 소기 목적에 부합되어 이들이 항암제로서 유용하게 사용될 수 있음을 발견하고 본 발명을 완성하게 되었다.Accordingly, the present inventors have established a new evaluation system for evaluating the enzyme activity inhibitory ability and intracellular K-Ras prenylation inhibitory activity against K-Ras substrates, and utilizing them, thereby utilizing not only K-Ras but also H-Ras and N-Ras substrates. Novel compounds that inhibit farnesylation were synthesized and their inhibitory ability was evaluated. As a result, the present inventors have found that the compound of Formula 1 meets the intended purpose of the present invention and that they can be usefully used as an anticancer agent.
따라서, 본 발명은 우수한 항암효과를 갖는 화학식 1의 화합물 및 그의 제조 방법에 관한 것이다.Accordingly, the present invention relates to a compound of formula 1 having a good anticancer effect and a method for producing the same.
본 발명은 피롤구조를 포함하며 파네실 전이효소를 억제할 수 있는 하기 화학식 1의 화합물 또는 그의 약제학적으로 허용되는 염에 관한 것이다 :The present invention relates to a compound of formula (1) or a pharmaceutically acceptable salt thereof, which comprises a pyrrole structure and is capable of inhibiting farnesyl transferase:
화학식 1Formula 1
상기식에서In the above formula
A 는 수소 또는 저급알킬을 나타내거나, 하기 구조식의 그룹중 신택된 어느하나를 나타내고,A represents hydrogen or lower alkyl, or any of those selected from the group of the following structural formulas,
여기에서 R1은 수소, 할로겐, 시아노, 니트로, 하이드록시카보닐, 아미노카보닐, 아미노티오카보닐, 저급알콕시, 페녹시, 페닐 또는 저급알킬을 나타내며, R2는 수소, 저급알킬, 저급알카노일, 또는 아릴에 의해 치환되거나 비치환된 저급알콕시카보닐을 나타내고,Wherein R 1 represents hydrogen, halogen, cyano, nitro, hydroxycarbonyl, aminocarbonyl, aminothiocarbonyl, lower alkoxy, phenoxy, phenyl or lower alkyl, R 2 represents hydrogen, lower alkyl, lower Lower alkoxycarbonyl unsubstituted or substituted by alkanoyl, or aryl,
B 는 하기 구조식의 그룹중 선택된 어느 하나를 나타내며,B represents any one selected from the group of the following structural formulas,
여기에서 R3및 R4는 각각 독립적으로 수소, 저급알킬, 저급알콕시, 할로겐, 시아노, 하이드록시카보닐, 아미노카보닐, 아미노티오카보닐, 하이드록시, 페닐 또는 페녹시를 나타내고,Wherein R 3 and R 4 each independently represent hydrogen, lower alkyl, lower alkoxy, halogen, cyano, hydroxycarbonyl, aminocarbonyl, aminothiocarbonyl, hydroxy, phenyl or phenoxy,
또는 S 를 나타내며, n 은 2 내지 4의 정수를 나타내고, R7은 저급알킬을 나타낸다.Or S, n represents an integer of 2 to 4, and R 7 represents lower alkyl.
상기 화학식 1의 화합물에 대한 치환기 정의에서 용어 "저급알킬"은 메틸, 에틸, 이소프로필, 이소부틸, t-부틸 등과 같은 탄소수 1 내지 4의 직쇄 또는 측쇄 알킬을 의미하며, 용어 "저급알카노일"은 포르밀, 아세틸, 프로피오닐 등과 같은 탄소수 1 내지 4의 알킬카보닐을 의미한다.In the substituent definition for the compound of Formula 1, the term "lower alkyl" means straight or branched chain alkyl having 1 to 4 carbon atoms, such as methyl, ethyl, isopropyl, isobutyl, t-butyl, etc. Means alkylcarbonyl having 1 to 4 carbon atoms such as formyl, acetyl, propionyl and the like.
우수한 항암효과를 나타내는 상기 화학식 1의 화합물 중에서도 바람직한 화합물은Among the compounds of Formula 1, which show excellent anticancer effects, preferred compounds are
A는 하기 구조식의 그룹중 선택된 어느 하나를 나타내고,A represents any one selected from the group of the following structural formulas,
여기에서 R1은 수소, 할로겐 또는 시아노를 나타내며, R2는 저급알카노일을 나타내거나, 아릴에 의해 치환되거나 비치환된 저급알콕시카보닐을 나타내고,Wherein R 1 represents hydrogen, halogen or cyano, R 2 represents lower alkanoyl or lower alkoxycarbonyl unsubstituted or substituted by aryl,
B 는 할로겐, 저급알킬 또는 저급알콕시에 의해 치환되거나 비치환된 나프틸을 나타내며,B represents naphthyl unsubstituted or substituted by halogen, lower alkyl or lower alkoxy,
저급알킬을 나타내며, R7은 저급알킬을 나타내는 화합물이다.Lower alkyl and R 7 is lower alkyl.
본 발명에 따른 화학식 1 화합물의 대표적인 예는 하기 표 1에 나타낸 바와 같다.Representative examples of the compound of formula 1 according to the present invention are as shown in Table 1 below.
[표 1]TABLE 1
본 발명에 따른 화학식 1의 화합물은 (a) 하기 화학식 2의 화합물을 용매중에서 염기의 존재하에 하기 화학식 3의 화합물과 반응시켜 제조하거나, (b) 하기 화학식 1a의 화합물을 용매중에서 하기 화학식 4의 화합물과 커플링시켜 하기 화학식 1b의 화합물을 제조함을 특징으로하여 제조할 수 있으며, 이러한 화학식 1 화합물의 제조방법도 또한 본 발명의 목적이다.The compound of formula 1 according to the present invention is prepared by (a) reacting a compound of formula 2 with a compound of formula 3 in the presence of a base in a solvent, or (b) a compound of formula 1a The compound of Formula 1b may be prepared by coupling with a compound, and the method for preparing the compound of Formula 1 is also an object of the present invention.
그러나, 본 발명에 따른 화합물의 제조방법이 하기에 설명하는 것으로만 한정된 것은 아니며, 본 명세서에 기재되거나 선행문헌에 개시된 여러가지 합성방법을 임의로 조합함으로써 용이하게 제조할 수 있고, 이러한 조합은 본 발명이 속하는 기술 분야의 당업자에게 범용화된 통상의 기술이다.However, the method for preparing the compound according to the present invention is not limited only to the following description, and can be easily prepared by arbitrarily combining various synthetic methods described in the present specification or disclosed in the prior literature, and such a combination can be prepared by the present invention. It is a common technique generalized to those skilled in the art.
[화학식 2][Formula 2]
[화학식 3][Formula 3]
[화학식 1a][Formula 1a]
[화학식 4][Formula 4]
[화학식 1b][Formula 1b]
상기식에서In the above formula
A, B, C 및 R2는 앞에서 정의한 바와 같고,A, B, C and R 2 are as defined above,
X는 하이드록시 또는 반응성 이탈기, 바람직하게는 할로겐을 나타낸다.X represents a hydroxy or reactive leaving group, preferably halogen.
본 발명에 따른 화학식 1의 화합물을 제조하는 상기방법 (a) 및 (b)에서 용매로는 반응에 불활성인 용매중 이느 것이라도 사용할 수 있으나, 바람직하게는 디메틸포름아미드, 디메틸아세트아미드 및 테트라하이드로푸란 중에서 선택된 1 종 이상을 사용한다. 또한, 방법 (a)에서 염기로는 수소화나트륨, 포타슘 t-부톡사이드, 소듐 비스(트리메틸실릴)아미드 및 포타슘 비스(트리메틸실릴)아미드 중에서 선택된 1 종 이상을 사용할 수 있다. 방법 (b)에서 출발물질로 사용하는 화학식 1a의 화합물은 피페리딘의 1번 위치에 벤질옥시카보닐기가 치환되어있는 화합물을 탈보호기화시켜 제조할 수 있다. 탈보호기화 반응은 통상의 반응조건을 적용하여 수행할 수 있으나, 바람직하게는 알콜 용매중에서 수소대기하에 Pd(OH)2/C 또는 Pd/C 를 사용하여 수행한다. 이렇게 생성된 화학식 1a의 화합물을 상기 언급된 불활성용매중에서 임의로 3차아민 염기 존재하에 저급알킬할라이드, 저급알카노일 할라이드또는 저급알킬클로로포르메이트와 커플링시키면 화학식 1b의 화합물이 수득된다. 또는, 커플링제의 존재하에 화학식 1a의 화합물을 카복실산 유도체(X=OH)와 반응시켜 아미드 형태의 화학식 1b 화합물을 제조할 수도 있다. 이때 커플링제로는 디사이클로헥실카르보디이미드(DCC) , 1-에틸-3-(3-디메틸아미노프로필)카르보디이미드(EDC), 1,1'-디카보닐디이미다졸(CDI) 등의 카르보이미드류를 1-하이드록시벤조트리아졸(HOBT)과 혼합된 상태로 사용할 수 있다.In the above methods (a) and (b) of preparing the compound of formula 1 according to the present invention, any solvent may be used as the solvent, but is preferably dimethylformamide, dimethylacetamide and tetrahydro. Use at least one selected from furan. In the method (a), at least one selected from sodium hydride, potassium t-butoxide, sodium bis (trimethylsilyl) amide, and potassium bis (trimethylsilyl) amide can be used. The compound of Formula 1a, which is used as a starting material in method (b), may be prepared by deprotecting a compound having a benzyloxycarbonyl group substituted at position 1 of piperidine. The deprotection gasification reaction may be carried out by applying the usual reaction conditions, but is preferably performed using Pd (OH) 2 / C or Pd / C under hydrogen atmosphere in an alcohol solvent. The compound of formula 1a thus produced is coupled with a lower alkyl halide, lower alkanoyl halide or lower alkyl chloroformate in the above-mentioned inert solvent, optionally in the presence of a tertiary amine base, to give a compound of formula 1b. Alternatively, the compound of formula 1a may be reacted with a carboxylic acid derivative (X = OH) in the presence of a coupling agent to prepare a compound of formula 1b in amide form. In this case, as the coupling agent, dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1,1'-dicarbonyldiimidazole (CDI), or the like Carbodiimides can be used in a mixed state with 1-hydroxybenzotriazole (HOBT).
본 발명에 따른 방법 (a) 내지 (b)에서 출발물질로 사용되는 화합물들은 하기 반응식 1 내지 5에 도시된 방법에 따라 제조할 수 있다.The compounds used as starting materials in the methods (a) to (b) according to the present invention can be prepared according to the methods shown in Schemes 1 to 5 below.
먼저, 화학식 2의 화합물은 치환기 A의 종류에 따라 4-하이드록시메틸이미다졸 하이드로클로라이드를 출발물질로 하여 반응식 1에 도시한 바에 따라 보호기화, 아세틸화, 커플링, 탈보호기화 및 할로겐화 반응을 거쳐 합성하거나, 1차 아민을 출발물질로 하여 반응식 2에 도시한 바에 따라 폐환, 탈황 및 할로겐화 반응을 수행하여 합성할 수 있다.First, the compound of formula (2) according to the type of substituent A as a starting material 4-hydroxymethylimidazole hydrochloride as shown in Scheme 1, the protection group, acetylation, coupling, deprotection and halogenation reaction Synthesis may be carried out via a ring closure, desulfurization and halogenation as shown in Scheme 2 using primary amine as a starting material.
[반응식 1]Scheme 1
[반응식 2]Scheme 2
상기 반응식 1 및 2에서In Schemes 1 and 2
A 는 앞에서 정의한 바와 같고,A is as defined above,
Tr 은 트리틸을 나타내며, 이하 동일한 의미로 사용된다.Tr represents trityl and is used by the same meaning below.
화학식 2의 화합물에서 A가 1-(벤질옥시카보닐)피페리딘-4일메틸인 화합물은 4-아비노메틸피페리딘을 출발물질로 하여 반응식 3에 도시한 바에 따라 보호기화,벤질옥시카보닐화, 탈보호기화 반응을 거쳐 아민을 제조한 후, 이를 이용하여 반응식 2에 따라 합성할 수 있다.In the compound represented by Chemical Formula 2, A is 1- (benzyloxycarbonyl) piperidin-4ylmethyl, and the protecting group, benzyloxycarbo, as shown in Scheme 3 using 4-avinomethylpiperidine as starting material After preparing an amine through a nitrification and deprotection reaction, it can be synthesized according to Scheme 2.
[반응식 3]Scheme 3
상기 반응식 3에서 Cbz 는 벤질옥시카보닐을 나타내고, CbzCl은 벤질클로로 포르메이트를 나타내며, 이하 동일한 의미로 사용된다.In Scheme 3, Cbz represents benzyloxycarbonyl, and CbzCl represents benzylchloro formate, which is used hereinafter in the same sense.
한편, 화학식 3의 화합물에서 C가 수소이고 B가 1-나프틸인 화합물은 1-나프토산을 출발물질로 하여 하기 반응식 4에 도시한 바에 따라 합성할 수 있으며, C가 치환된 카바모일기이고 B가 1-나프틸인 화합물은 하기 반응식 5에 도시한 바에 따라 합성할 수 있다.Meanwhile, in the compound of Formula 3, C is hydrogen and B is 1-naphthyl can be synthesized as shown in Scheme 4 using 1-naphthoic acid as a starting material, and C is a carbamoyl group. Compounds in which B is 1-naphthyl can be synthesized as shown in Scheme 5 below.
[반응식 4]Scheme 4
[반응식 5]Scheme 5
상기 반응식 4 및 5에서In Schemes 4 and 5 above
TosMIC 는 토실메틸이소시아나이드를 나타내며, 이하 동일한 의미로 사용된다.TosMIC represents tosylmethyl isocyanide and is used hereinafter with the same meaning.
본 발명, 특히 상기 설명한 제조방법들을 하기 제조예, 실시예 및 실험예에 의거하여 보다 구체적으로 설명한다. 그러나, 이들 제조예, 실시예 및 실험예는 본발명에 대한 이해를 돕기위한 것일 뿐, 어떤 의미로든 본 발명의 범위가 이들에 의해 한정되는 것은 아니다.The present invention, in particular the production methods described above will be described in more detail based on the following Preparation Examples, Examples and Experimental Examples. However, these preparation examples, examples and experimental examples are only intended to help the understanding of the present invention, and the scope of the present invention in any sense is not limited thereto.
제조예 1: 4-(5-클로로메틸-1H-이미다졸-1-일메틸)벤조니트릴 하이드로클로라이드의 합성Preparation Example 1 Synthesis of 4- (5-chloromethyl-1H-imidazol-1-ylmethyl) benzonitrile Hydrochloride
1-1) 4-하이드록시메틸-1-트리틸-1H-이미다졸의 제조1-1) Preparation of 4-hydroxymethyl-1-trityl-1H-imidazole
하이드록시메틸이미다졸 하이드로클로라이드 3.99g(29.6 밀리몰)을 디메틸포름아미드 30mℓ와 트리에틸아민 10mℓ의 혼합물에 녹인 후, 여기에 트리페닐메틸 클로라이드 9.35g(33.5 밀리몰)을 디메틸포름아미드 110mℓ에 용해시킨 용액을 서서히 가하였다. 2시간 후, 반응액에 얼음물 500mℓ를 가하고, 생성된 고체를 수득하였다. 이 고체를 디옥산으로 재결정하여 표제화합물 8.82g(수율 87%)을 수득하였다.3.99 g (29.6 mmol) of hydroxymethylimidazole hydrochloride was dissolved in a mixture of 30 ml of dimethylformamide and 10 ml of triethylamine, and then 9.35 g (33.5 mmol) of triphenylmethyl chloride was dissolved in 110 ml of dimethylformamide. The solution was added slowly. After 2 hours, 500 ml of ice water was added to the reaction solution, and the resulting solid was obtained. This solid was recrystallized from dioxane to give 8.82 g (87% yield) of the title compound.
융점 : 227-229℃Melting Point: 227-229 ℃
1-2) 4-아세톡시메틸-1-트리틸-1H-이미다졸의 제조1-2) Preparation of 4-acetoxymethyl-1-trityl-1H-imidazole
피리딘 100mℓ에 제조예 1-1)에서 수득한 화합물 5.00g(14.7 밀리몰)을 가하고 아세트산무수물 1.65g(16.2 밀리몰)을 가한 다음, 상온에서 24시간동안 교반하였다. 반응액을 감압증류하여 피리딘을 제거하고 잔류물을 에틸아세테이트 200mℓ에 녹인 다음, 소금물 100mℓ로 세척해주었다. 유기용매를 감압증류하여 제거한 후, 칼럼 크로마토그래피(용리제: 디클로로메탄/메탄올=20/l, v/v)를 실시하여 표제화합물 5.22g(13.7 밀리몰, 수율 93%)을 수득하였다.To 100 ml of pyridine was added 5.00 g (14.7 mmol) of the compound obtained in Preparation Example 1-1, 1.65 g (16.2 mmol) of acetic anhydride was added thereto, followed by stirring at room temperature for 24 hours. The reaction solution was distilled under reduced pressure to remove pyridine, and the residue was dissolved in 200 ml of ethyl acetate, followed by washing with 100 ml of brine. After distilling off the organic solvent under reduced pressure, column chromatography (eluent: dichloromethane / methanol = 20 / l, v / v) was carried out to obtain 5.22 g (13.7 mmol, 93% yield) of the title compound.
1-3) 4-(4-아세톡시메틸-1-트리틸-1H-이미다졸-3-일메틸)벤조니트릴 브로마이드의 제조1-3) Preparation of 4- (4-acetoxymethyl-1-trityl-1H-imidazol-3-ylmethyl) benzonitrile bromide
제조예 1-2)에서 수득한 화합물 5.00g(13.1 밀리몰)을 디클로로메탄 20mℓ에 녹이고, 4-시아노벤질브로마이드 2.82g(14.4 밀리몰)을 가한 다음, 상온에서 60 시간동안 교반하였다. 유기용매를 감압증류하여 제거하고 잔류물에 대해 칼럼 크로마토그래피(용리제: 디클로로메탄/메탄올=5/1, v/v)를 실시하여 표제화합물 5.31g(9.17 밀리몰, 수율 70%)을 수득하였다.5.00 g (13.1 mmol) of the compound obtained in Preparation Example 1-2) was dissolved in 20 mL of dichloromethane, and 2.82 g (14.4 mmol) of 4-cyanobenzylbromide was added thereto, followed by stirring at room temperature for 60 hours. The organic solvent was removed by distillation under reduced pressure and the residue was subjected to column chromatography (eluent: dichloromethane / methanol = 5/1, v / v) to give 5.31 g (9.17 mmol, yield 70%) of the title compound. .
1-4) 4-(5-아세톡시메틸-1H-이미다졸-1-일메틸)벤조니트릴의 제조1-4) Preparation of 4- (5-acetoxymethyl-1H-imidazol-1-ylmethyl) benzonitrile
제조예 1-3)에서 수득한 화합물 9.10g(15.7 밀리몰)을 디클로로메탄 500mℓ에 녹인 후, 0℃에서 트리플루오로아세트산 6.06mℓ(78.7 밀리몰) 및 트리에틸실란 12.5mℓ(78.7 밀리몰)를 서서히 가하고 상온에서 1시간동안 교반하였다. 유기용매를 감압증류하여 제거하고, 포화 K2CO3수용액으로 pH를 10으로 맞춘 다음, 에틸아세테이트 300mℓ로 추출하였다. 유기용매를 감압증류하여 제거하고 잔류물에 대해 에틸아세테이트를 용리제로 하는 칼럼 크로마토그래피를 실시하여 표제화합물 3.60g(14.1 밀리몰, 수율 90%)을 수득하였다.9.10 g (15.7 mmol) of the compound obtained in Preparation Example 1-3) were dissolved in 500 ml of dichloromethane, and then 6.06 ml (78.7 mmol) of trifluoroacetic acid and 12.5 ml (78.7 mmol) of triethylsilane were slowly added at 0 ° C. Stirred at room temperature for 1 hour. The organic solvent was removed by distillation under reduced pressure, the pH was adjusted to 10 with saturated K 2 CO 3 aqueous solution, and extracted with 300 ml of ethyl acetate. The organic solvent was removed by distillation under reduced pressure, and the residue was subjected to column chromatography using ethyl acetate as an eluent to obtain 3.60 g (14.1 mmol, 90% yield) of the title compound.
1-5) 4-(5-하이드록시메틸-1H-이미다졸-1-일메틸)벤조니트릴의 제조1-5) Preparation of 4- (5-hydroxymethyl-1H-imidazol-1-ylmethyl) benzonitrile
제조예 1-4)에서 수득한 화합물 4.20g(16.5 밀리몰)을 메탄올 200mℓ에 녹인 후, K2CO34.50g(32.9 밀리몰)을 가한 다음, 상온에서 20분동안 교반하였다. 상온에서 유기 용매를 감압증류하여 제거하고 에틸아세테이트 300mℓ로 추출한 다음, 칼럼 크로마토그래피(용리제: 디클로로메탄/메탄올=10/1, v/v)를 수행하여 표제화합물 3.19g (15.0 밀리몰, 수율 91%)을 수득하였다.4.20 g (16.5 mmol) of the compound obtained in Preparation Example 1-4) was dissolved in 200 mL of methanol, and then 4.50 g (32.9 mmol) of K 2 CO 3 was added thereto, followed by stirring at room temperature for 20 minutes. The organic solvent was removed by distillation under reduced pressure at room temperature, extracted with 300 ml of ethyl acetate, and then subjected to column chromatography (eluent: dichloromethane / methanol = 10/1, v / v) to give 3.19 g (15.0 mmol, yield 91) of the title compound. %) Was obtained.
1-6) 4-(5-클로로메틸-1H-이미다졸-1-일메틸)벤조니트릴 하이드로클로라이드의 제조1-6) Preparation of 4- (5-chloromethyl-1H-imidazol-1-ylmethyl) benzonitrile hydrochloride
제조예 1-5)에서 수득한 화합물 3.00g(14.1 밀리몰)을 클로로포름 40mℓ에 녹인 후, 0℃에서 티오닐클로라이드 5.02mℓ(70.5 밀리몰)를 서서히 가하고 상온에서 2시간동안 교반하였다. 유기용매를 감압하에 제거하여 표제화합물 3.50g(13.1 밀리몰, 수율 93%)을 수득하였다. 이 화합물은 정제하지 않고 바로 반응에 사용하였다.3.00 g (14.1 mmol) of the compound obtained in Preparation Example 1-5) was dissolved in 40 mL of chloroform, and then 5.02 mL (70.5 mmol) of thionyl chloride was slowly added at 0 ° C, and stirred for 2 hours at room temperature. The organic solvent was removed under reduced pressure to yield 3.50 g (13.1 mmol, yield 93%) of the title compound. This compound was used directly in the reaction without purification.
제조예 2: 1-(4-브로모벤질)-5-클로로메틸-1H-이미다졸 하이드로클로라이드의 합성Preparation Example 2 Synthesis of 1- (4-bromobenzyl) -5-chloromethyl-1H-imidazole hydrochloride
2-1) 1-(4-브로모벤질)-5-하이드록시메틸-1H-이미다졸의 제조2-1) Preparation of 1- (4-bromobenzyl) -5-hydroxymethyl-1H-imidazole
디하이드록시아세톤 다이머와 4-브로모벤질아민 하이드로클로라이드를 출발 물질로 하여 문헌(참조: J.M. Dener, L-H Zhang, H.Rapoport,J. Org. Chem., 1993, 58, 1159)에 기재된 방법에 따라 실시함으로써 표제화합물을 50% 수율로 수득하였다.The method described in the literature (JM Dener, LH Zhang, H. Rapoport, J. Org. Chem. , 1993, 58, 1159) using dihydroxyacetone dimer and 4-bromobenzylamine hydrochloride as starting materials . According to the title compound in 50% yield.
2-2) 1-(4-브로모벤질)-5-클로로메틸-1H-이미다졸 하이드로클로라이드의 제조2-2) Preparation of 1- (4-bromobenzyl) -5-chloromethyl-1H-imidazole hydrochloride
제조예 2-1)에서 수득한 화합물을 출발물질로 사용하는 점을 제외하고는 제조예 1-6)에서와 유사하게 실시하여 표제화합물을 96% 수율로 수득하였다. 수득된 화합물은 정제하지 않고 바로 반응에 사용하였다.The title compound was obtained in 96% yield by the same procedure as in Preparation Example 1-6) except that the compound obtained in Preparation Example 2-1) was used as a starting material. The obtained compound was used directly for the reaction without purification.
제조예 3 : 1-[1-(벤질옥시카보닐)피페리딘-4-일]메틸-5-클로로메틸-1H-이미다졸의 합성Preparation Example 3 Synthesis of 1- [1- (benzyloxycarbonyl) piperidin-4-yl] methyl-5-chloromethyl-1H-imidazole
3-1) 4-아미노메틸-1-(벤질옥시카보닐)피페리딘의 제조3-1) Preparation of 4-aminomethyl-1- (benzyloxycarbonyl) piperidine
4-아미노메틸피페리딘 22.2g(0.2 몰)을 톨루엔 250mℓ에 녹인 후, 벤즈알데히드 21.2g(0.2 몰)을 가하였다. 반응물을 딘스탁 장치 하에서 3시간 동안 환류하여 물을 제거한 후, 반응물을 0℃로 냉각시키고 교반하면서 벤질클로로포르메이트34.2g(0.2 몰)을 서서히 가하였다. 상온에서 3시간동안 교반한 다음 1N KHSO4수용액 220mℓ를 가하였다. 디에틸에테르 200mℓ로 반응물을 3회에 걸쳐 추출한 후 수용액층을 1N 수산화나트륨 수용액으로 염기화시키고 염화나트륨으로 포화시켰다. 수용액층을 디클로로메탄 100mℓ로 3회 추출하고, 무수 마그네슘설페이트로 건조시킨 후 감압증류하여 표제화합물 38g (수율 91%, 분자량 248)을 수득하였다.22.2 g (0.2 mol) of 4-aminomethylpiperidine was dissolved in 250 ml of toluene, and then 21.2 g (0.2 mol) of benzaldehyde was added. The reaction was refluxed under a Deanstock apparatus for 3 hours to remove water, and then 34.2 g (0.2 mol) of benzylchloroformate was slowly added while cooling to 0 ° C. and stirring. After stirring at room temperature for 3 hours, 220 ml of 1N KHSO 4 aqueous solution was added thereto. After the reaction was extracted three times with 200 ml of diethyl ether, the aqueous layer was basified with 1N aqueous sodium hydroxide solution and saturated with sodium chloride. The aqueous layer was extracted three times with 100 mL of dichloromethane, dried over anhydrous magnesium sulfate, and then distilled under reduced pressure to obtain 38 g of the title compound (yield 91%, molecular weight 248).
3-2) 1-[1-(벤질옥시카보닐)피페리딘-4-일]메틸-5-하이드록시메틸-2-머캅토-1H-이미다졸의 제조3-2) Preparation of 1- [1- (benzyloxycarbonyl) piperidin-4-yl] methyl-5-hydroxymethyl-2-mercapto-1H-imidazole
제조예 3-1)에서 수득한 화합물 24.8g(0.1 몰)을 아세트산 6.0g(0.1 몰)과 함께 n-부탄올 50mℓ에 녹인 후, 포타슘티오시아네이트(KSCN) 12.6g(0.13 몰), 1.3-디하이드록시아세톤 다이머 15.2g(0.1 몰) 및 아세트산 10.0g(0.17 몰)을 n-부탄올 50mℓ에 용해시킨 용액에 가하고 상온에서 교반하였다. 48시간 후 용매를 감압증류하여 제거하고 잔류물을 에틸아세테이트 200mℓ에 녹인 후 물 100mℓ로 3회 세척하였다. 유기층을 무수 마그네슘설페이트로 건조시킨 후 농축시켜 표제화합물27g(75 밀리몰; 수율 75%)을 수득하였다.24.8 g (0.1 mol) of the compound obtained in Preparation Example 3-1) was dissolved in 50 ml of n-butanol with 6.0 g (0.1 mol) of acetic acid, and then 12.6 g (0.13 mol) of potassium thiocyanate (KSCN), 1.3- 15.2 g (0.1 mol) of dihydroxyacetone dimer and 10.0 g (0.17 mol) of acetic acid were added to a solution dissolved in 50 ml of n-butanol and stirred at room temperature. After 48 hours, the solvent was distilled off under reduced pressure, and the residue was dissolved in 200 ml of ethyl acetate and washed three times with 100 ml of water. The organic layer was dried over anhydrous magnesium sulfate and concentrated to give 27 g (75 mmol; 75% yield) of the title compound.
3-3) 1-[1-(벤질옥시카보닐)피페리딘-4-일]메틸-5-하이드록시메틸-1H-이미다졸의 제조3-3) Preparation of 1- [1- (benzyloxycarbonyl) piperidin-4-yl] methyl-5-hydroxymethyl-1H-imidazole
제조예 3-2)에서 수득한 화합물 18.05g(50 밀리몰)을 10% 질산 100mℓ 및 에틸아세테이트 10mℓ의 혼합용액에 가한 후 반응물을 차가운 얼음물로 냉각시키고 상온에서 3시간동안 교반하였다. 반응물을 4N 수산화나트륨 수용액으로 염기화시킨 다음 에틸아세테이트 100mℓ로 2회 추출하였다. 추출유기용액을 마그네슘설페이트로 건조시키고 감압증류하여 표제화합물 12.3g(38 밀리몰, 수율 75%)를 수득하였다.18.05 g (50 mmol) of the compound obtained in Preparation Example 3-2) was added to a mixed solution of 10 ml of 10% nitric acid and 10 ml of ethyl acetate, and the reaction was cooled with cold ice water and stirred at room temperature for 3 hours. The reaction was basified with 4N aqueous sodium hydroxide solution and then extracted twice with 100 mL of ethyl acetate. The extracted organic solution was dried over magnesium sulfate and distilled under reduced pressure to obtain 12.3 g (38 mmol, yield 75%) of the title compound.
3-4) 1-[1-(벤질옥시카보닐)피페리딘-4-일]메틸-5-클로로메틸-1H-이미다졸의 제조3-4) Preparation of 1- [1- (benzyloxycarbonyl) piperidin-4-yl] methyl-5-chloromethyl-1H-imidazole
제조예 3-3)에서 수득한 화합물 9.9g(30 밀리몰)을 클로로포름 50mℓ에 녹인 후 0℃에서 티오닐클로라이드 7.1g(60 밀리몰)을 천천히 가하였다. 반응액을 2시간동안 교반한 후 용매를 감압증류하여 제거함으로써 표제화합물의 하이드로클로라이드염 9.9g(수율 95 %; 분자량 347.5)을 수득하였다. 이 화합물은 정제하지 않고 바로 반응에 사용하였다.9.9 g (30 mmol) of the compound obtained in Preparation Example 3-3) was dissolved in 50 mL of chloroform, and then 7.1 g (60 mmol) of thionyl chloride was slowly added at 0 ° C. After stirring the reaction solution for 2 hours, the solvent was distilled off under reduced pressure to obtain 9.9 g (yield 95%; molecular weight 347.5) of the hydrochloride salt of the title compound. This compound was used directly in the reaction without purification.
제조예 4 : 3-(나프탈렌-1-일)카보닐-1H-피롤의 합성Preparation Example 4 Synthesis of 3- (naphthalen-1-yl) carbonyl-1H-pyrrole
4-1) 메틸 N-메틸-1-나프탈렌 하이드록사메이트의 제조4-1) Preparation of Methyl N-methyl-1-naphthalene Hydroxamate
1-나프토산 3.44g(20 밀리몰)을 디메틸포름아미드 20mℓ에 녹이고 EDC 4.6g(24 밀리몰), 트리에틸아민 2.02g(20 밀리몰) 및 HOBT 3.24g(24 밀리몰)을 가한 다음 0℃에서 5분간 교반하였다. 여기에 N,O-디메틸하이드록실아민 하이드로클로라이드 1.85mg(20 밀리몰)을 가하고 상온에서 5시간동안 교반하였다. 용매를 감압하에 제거하고 포화 K2CO3수용액 100mℓ를 가한 다음 에틸아세테이트로 추출하였다. 유기층을 1N 염산 수용액, 소금물, 물로 차례로 세척하고 무수 소듐설페이트로 건조시킨 후 농축시켜 표제화합물 3.04g(1.50 밀리몰)을 수득하였다.3.44 g (20 mmol) of 1-naphthoic acid was dissolved in 20 ml of dimethylformamide, 4.6 g (24 mmol) of EDC, 2.02 g (20 mmol) of triethylamine and 3.24 g (24 mmol) of HOBT were added, followed by 5 minutes at 0 ° C. Stirred. 1.85 mg (20 mmol) of N, O-dimethylhydroxylamine hydrochloride was added thereto, followed by stirring at room temperature for 5 hours. The solvent was removed under reduced pressure, 100 ml of saturated K 2 CO 3 aqueous solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed successively with 1N aqueous hydrochloric acid solution, brine, water, dried over anhydrous sodium sulfate, and concentrated to give 3.04 g (1.50 mmol) of the title compound.
FAB 216 (M+H)FAB 216 (M + H)
4-2) 1-(나프탈렌-1-일)-프로프-2-엔-1-온의 제조4-2) Preparation of 1- (naphthalen-1-yl) -prop-2-en-1-one
제조예 4-1)에서 수득한 화합물 2.03g(9.4 밀리몰)을 무수 테트라하이드로푸란 20mℓ에 녹인 후, 여기에 1N 비닐마그네슘브로마이드 테트라하이드로푸란 용액 20mℓ를 0℃에서 서서히 가하였다. 상온에서 30분간 교반한 후 1N 염산 20mℓ를 첨가하고 에틸아세테이트 50mℓ로 추출하였다. 유기층을 무수 마그네슘설페이트로 건조시킨 후 감압하에 용매를 제거하여 표제화합물 1.63g(9 밀리몰; 수율 96%)을 수득하였다.2.03 g (9.4 mmol) of the compound obtained in Preparation Example 4-1) was dissolved in 20 ml of anhydrous tetrahydrofuran, and 20 ml of 1N vinylmagnesium bromide tetrahydrofuran solution was slowly added thereto at 0 ° C. After 30 minutes of stirring at room temperature, 20 ml of 1N hydrochloric acid was added and extracted with 50 ml of ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and then the solvent was removed under reduced pressure to yield 1.63 g (9 mmol; 96% yield) of the title compound.
4-3) 3-(나프탈렌-1-일)카보닐-1H-피롤의 제조4-3) Preparation of 3- (naphthalen-1-yl) carbonyl-1H-pyrrole
제조예 4-2)에서 수득한 화합물 901mg(5 밀리몰)과 토실메틸이소시아나이드 1.01g(5.5 밀리몰)을 테트라하이드로푸란 10mℓ에 용해시킨 후, 여기에 포타슘 t-부톡사이드 555mg(5.5 밀리몰)의 테트라하이드로푸란(10mℓ) 용액을 천천히 첨가하고 30분간 교반하였다. 반응액에 물 10mℓ를 가하여 반응을 중지시키고 감압하에 용매를 제거하였다. 잔류물에 물 20mℓ를 가하고 에틸아세테이트로 추출한 다음, 소금물로 세척하고 무수 마그네슘설페이트로 건조시켰다. 용매를 감압하에 제거하고 칼럼 크로마토그래피(용리제: 에틸아세테이트/헥산=1/3, v/v)를 실시하여 표제 화합물 884mg(4 밀리몰; 수율 80%)을 수득하였다.901 mg (5 mmol) of the compound obtained in Production Example 4-2) and 1.01 g (5.5 mmol) of tosylmethyl isocyanide were dissolved in 10 mL of tetrahydrofuran, and then 555 mg (5.5 mmol) of potassium t-butoxide was added thereto. Tetrahydrofuran (10 mL) solution was added slowly and stirred for 30 minutes. 10 ml of water was added to the reaction mixture to stop the reaction, and the solvent was removed under reduced pressure. To the residue was added 20 ml of water, extracted with ethyl acetate, washed with brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure and column chromatography (eluent: ethyl acetate / hexane = 1/3, v / v) afforded 884 mg (4 mmol; yield 80%) of the title compound.
제조예 5 : 4-(나프탈렌-1-일)카보닐-3-[N-(2-메톡시에틸)-N-메틸카바모일]-1H-피롤의 합성Preparation Example 5 Synthesis of 4- (naphthalen-1-yl) carbonyl-3- [N- (2-methoxyethyl) -N-methylcarbamoyl] -1 H-pyrrole
5-1) 4-(나프탈렌-1-일)-4-옥소-2-부테노산의 제조5-1) Preparation of 4- (naphthalen-1-yl) -4-oxo-2-butenoic acid
무수말레산 5.88g(60 밀리몰)을 무수 테트라하이드로푸란 100mℓ에 녹이고 78℃로 냉각시킨 용액을 준비하였다. 1-브로모나프탈렌 4.14g(20 밀리몰)을 무수 테트라하이드로푸란 100mℓ에 녹이고 78℃에서 1.6N n-부틸리튬 헥산용액 13.8mℓ를 가하였다. 이 반응액을 5분동안 교반한 후 캐눌라를 이용하여 미리 준비된 무수 말레산 용액에 가하였다. 생성된 혼합액을 10분간 교반한 후 물을 가하여 반응을 중지시키고 감압하에 용매를 제거하였다. 잔류물을 1N 염산 수용액으로 처리하여 산성화하고 에틸아세테이트로 추출하였다. 유기층을 물과 소금물로 세척한 후 무수 마그네슘설페이트로 건조시켰다. 감압하에 농축시키고 칼럼 크로마토그래피(용리제: 에틸아세테이트/헥산=2/1, v/v)로 정제하여 표제화합물 1.35g(6.0 밀리몰; 수율 30%)을 수득하였다.5.88 g (60 mmol) of maleic anhydride was dissolved in 100 ml of anhydrous tetrahydrofuran, and a solution cooled to 78 ° C was prepared. 4.14 g (20 mmol) of 1-bromonaphthalene was dissolved in 100 ml of anhydrous tetrahydrofuran and 13.8 ml of 1.6N n-butyllithium hexane solution was added at 78 ° C. The reaction solution was stirred for 5 minutes and then added to a maleic anhydride solution prepared in advance using a cannula. After stirring the resulting mixture for 10 minutes, water was added to stop the reaction, and the solvent was removed under reduced pressure. The residue was acidified by treatment with 1N aqueous hydrochloric acid solution and extracted with ethyl acetate. The organic layer was washed with water and brine and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification by column chromatography (eluent: ethyl acetate / hexane = 2/1, v / v) gave 1.35 g (6.0 mmol; yield 30%) of the title compound.
5-2) N-(2-메톡시에틸)-N-메틸-4-(나프탈렌-1-일)-4-옥소-2-부테노아미드의 제조5-2) Preparation of N- (2-methoxyethyl) -N-methyl-4- (naphthalen-1-yl) -4-oxo-2-butenoamide
제조예 5-1)에서 수득한 화합물 1.3g(5.9 밀리몰)을 디메틸포름아미드 10mℓ에 용해시키고 0℃에서 EDC 1.7g(8.9 밀리몰) 과 HOBT 1.2g(8.9 밀리몰)을 첨가한 후 5분간 교반하였다. 반응액에 N-(2-메톡시에틸)-N-메틸아민 530mg(5.9 밀리몰)과 트리에틸아민 1.2mℓ(8.9 밀리몰)를 첨가하고 상온에서 2시간동안 교반하였다. 감압하에 용매를 제거한 후 잔류물에 물 50mℓ를 가하고 에틸아세테이트로 추출하였다. 유기층을 소금물로 세척하고, 무수 마그네슘설페이트로 건조시킨 후 감압하에서 농축시켰다. 잔류물을 칼럼 크로마토그래피(용리제: 에틸아세테이트/헥산=1/1, v/v)로 정제하여 표제화합물 1.4g(4.7 밀리몰, 수율 80%)을 수득하였다.1.3 g (5.9 mmol) of the compound obtained in Preparation Example 5-1) were dissolved in 10 ml of dimethylformamide, and 1.7 g (8.9 mmol) of EDC and 1.2 g (8.9 mmol) of HOBT were added at 0 ° C., followed by stirring for 5 minutes. . 530 mg (5.9 mmol) of N- (2-methoxyethyl) -N-methylamine and 1.2 mmol (8.9 mmol) of triethylamine were added to the reaction solution, and the mixture was stirred at room temperature for 2 hours. After removing the solvent under reduced pressure, 50 ml of water was added to the residue, followed by extraction with ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (eluent: ethyl acetate / hexane = 1/1, v / v) to give 1.4 g (4.7 mmol, 80% yield) of the title compound.
5-3) 3-[N-(2-메톡시에틸)-N-메틸]카바모일-4-(나프탈렌-1-일)카보닐-1H-피롤의 제조5-3) Preparation of 3- [N- (2-methoxyethyl) -N-methyl] carbamoyl-4- (naphthalen-1-yl) carbonyl-1H-pyrrole
제조예 5-2)에서 수득한 화합물 1.4g(4.7 밀리몰)과 토실메틸이소시아나이드 1.0g(5.1 밀리몰)을 테트라하이드로푸란 20mℓ에 용해시킨 후 포타슘 t-부톡사이드 790mg(7.0 밀리몰)을 첨가하고 상온에서 3시간동안 교반하였다. 반응액에 물 2mℓ를 가하여 반응을 중지시키고 감압하에 용매를 제거하였다. 잔류물을 에틸아세테이트로 추출하고 소금물로 세척해준 후 무수 마그네슘설페이트로 건조시켰다. 용매를 감압하에 제거하고, 잔류물을 칼럼 크로마토그래피(용리제: 에틸아세테이트/헥산=2/3, v/v)로 정제하여 표제화합물 1.2g(3.6 밀리몰; 수율 76%)을 수득하였다.1.4 g (4.7 mmol) of the compound obtained in Preparation Example 5-2) and 1.0 g (5.1 mmol) of tosylmethyl isocyanide were dissolved in 20 ml of tetrahydrofuran, followed by addition of 790 mg (7.0 mmol) of potassium t-butoxide. Stir at room temperature for 3 hours. 2 ml of water was added to the reaction mixture to stop the reaction, and the solvent was removed under reduced pressure. The residue was extracted with ethyl acetate, washed with brine and dried over anhydrous magnesium sulfate. The solvent was removed under reduced pressure and the residue was purified by column chromatography (eluent: ethyl acetate / hexane = 2/3, v / v) to give 1.2 g (3.6 mmol; yield 76%) of the title compound.
실시예 1: 1-{1-[1-(벤질옥시카보닐)피페리딘-4-일]메틸-1H-이미다졸-5-일}메틸-3-(나프탈렌-1-일)카보닐-1H-피롤의 합성Example 1: 1- {1- [1- (benzyloxycarbonyl) piperidin-4-yl] methyl-1H-imidazol-5-yl} methyl-3- (naphthalen-1-yl) carbonyl Synthesis of -1H-pyrrole
제조예 4-3)에서 수득한 화합물 62.6mg(0.28 밀리몰)을 디메틸포름아미드 1 mℓ에 용해시키고 수소화나트륨 60mg(1.5 밀리몰)을 가하였다. 상온에서 30분간 교반한 다음, 제조예 3-4)에서 수득한 화합물 115mg(0.30 밀리몰)을 가하였다. 1시간동안 교반한 후 용매를 감압하에 제거하고 포화 NaHCO3수용액 5mℓ를 가한다음 에틸아세테이트 20mℓ로 추출하였다. 유기층을 소금물로 세척하고 무수 마그네슘설페이트로 건조시킨 후 감압하에 농축시켰다. 잔류물을 칼럼 크로마토그래피(용리제: 디클로로메탄/메탄올=95/5, v/v)로 정제하여 표제화합물 110mg(0.21 밀리몰; 수율 75%)을 수득하였다.62.6 mg (0.28 mmol) of the compound obtained in Preparation Example 4-3) were dissolved in 1 ml of dimethylformamide, and 60 mg (1.5 mmol) of sodium hydride were added thereto. After stirring at room temperature for 30 minutes, 115 mg (0.30 mmol) of the compound obtained in Preparation Example 3-4) were added thereto. After stirring for 1 hour, the solvent was removed under reduced pressure, 5 ml of saturated aqueous NaHCO 3 solution was added, and 20 ml of ethyl acetate was extracted. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane / methanol = 95/5, v / v) to give 110 mg (0.21 mmol; yield 75%) of the title compound.
FAB : 533 (M+H)FAB: 533 (M + H)
실시예 2 : 1-[1-(4-시아노벤질)-1H-이미다졸-5-일]메틸-3-(나프탈렌-1-일)카보닐-1H-피롤의 합성Example 2 Synthesis of 1- [1- (4-cyanobenzyl) -1H-imidazol-5-yl] methyl-3- (naphthalen-1-yl) carbonyl-1H-pyrrole
제조예 1-6)에서 수득한 화합물과 제조예 4-3)에서 수득한 화합물을 사용하는 점을 제외하고는 실시예 1에서와 유사하게 수행하여 표제화합물을 35%, 수율로수득하였다.The title compound was obtained in 35% and yield in the same manner as in Example 1 except for using the compound obtained in Preparation Example 1-6) and the compound obtained in Preparation Example 4-3).
FAB : 417 (M+1)FAB: 417 (M + 1)
실시예 3 : 1-[1-(4-브로모벤질)-1H-이미다졸-5-일]메틸-3-(나프탈렌-1-일)카보닐-1H-피롤의 합성Example 3: Synthesis of 1- [1- (4-bromobenzyl) -1H-imidazol-5-yl] methyl-3- (naphthalen-1-yl) carbonyl-1H-pyrrole
제조예 2-2)에서 수득한 화합물과 제조예 4-3)에서 수득한 화합물을 사용하는 점을 제외하고는 실시예 1에서와 유사하게 수행하여 표제화합물을 20% 수율로 수득하였다.The title compound was obtained in 20% yield by the same procedure as in Example 1 except for using the compound obtained in Preparation Example 2-2) and the compound obtained in Preparation Example 4-3).
FAB : 470 (M+1)FAB: 470 (M + 1)
실시예 4 : 1-[1-(4-브로모벤질)-1H-이미다졸-5-일]메틸-3-[N-(2-메톡시에틸)-N-메틸]카바모일-4-(나프탈렌-1-일)카보닐-1H-피롤의 합성Example 4: 1- [1- (4-Bromobenzyl) -1H-imidazol-5-yl] methyl-3- [N- (2-methoxyethyl) -N-methyl] carbamoyl-4- Synthesis of (naphthalen-1-yl) carbonyl-1H-pyrrole
제조예 2-2)에서 수득한 화합물과 제조예 5-3)에서 수득한 화합물을 사용하는 점을 제외하고는 실시예 1에서와 유사하게 수행하여 표제화합물을 81% 수율로수득하였다.The title compound was obtained in 81% yield in the same manner as in Example 1 except for using the compound obtained in Preparation Example 2-2) and the compound obtained in Preparation Example 5-3).
FAB : 585 (M+1)FAB: 585 (M + 1)
실시예 5 : 1-[1-(1-아세틸피페리딘-4-일)메틸-1H-이미다졸-5-일]메틸-3-(나프탈렌-1-일)카보닐-1H-피롤의 합성Example 5 of 1- [1- (1-acetylpiperidin-4-yl) methyl-1H-imidazol-5-yl] methyl-3- (naphthalen-1-yl) carbonyl-1H-pyrrole synthesis
실시예 1에서 수득한 화합물 110mg(0.211 밀리몰)을 메탄올 10mℓ에 용해시키고, Pd(OH)/C 20mg을 가한 후 수소대기하에 3시간 교반하여 벤질옥시카보닐기를 제거하였다. 반응물을 셀라이트로 여과하여 촉매를 제거하고 감압하에 용매를 제거하였다. 잔류물을 정제하지 않은채 디메틸포름아미드 5mℓ에 용해시킨 후 아세틸클로라이드 16.5mℓ(0.232 밀리몰)를 첨가하였다. 반응액을 상온에서 30분간 교반하고 감압하에 용매를 제거하였다. 잔류물에 포화 NaHCO3수용액 5mℓ를 가한 후 에틸아세테이트 20mℓ로 추출하였다. 유기층을 소금물로 세척하고 무수 마그네슘설페이트로 건조시킨 다음 감압하에 농축시켰다. 잔류물을 칼럼 크로마토그래피(용리제: 디클로로메탄/메탄올=9/1, v/v)로 정제하여 표제화합물 20.3mg(0.046 밀리몰; 수율22%)을 수득하였다.110 mg (0.211 mmol) of the compound obtained in Example 1 was dissolved in 10 ml of methanol, and 20 mg of Pd (OH) / C was added, followed by stirring for 3 hours under hydrogen atmosphere to remove the benzyloxycarbonyl group. The reaction was filtered through celite to remove the catalyst and the solvent was removed under reduced pressure. The residue was dissolved in 5 mL of dimethylformamide without purification and 16.5 mL (0.232 mmol) of acetylchloride was added. The reaction solution was stirred at room temperature for 30 minutes and the solvent was removed under reduced pressure. 5 ml of saturated NaHCO 3 aqueous solution was added to the residue, followed by extraction with 20 ml of ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (eluent: dichloromethane / methanol = 9/1, v / v) to give 20.3 mg (0.046 mmol; yield 22%) of the title compound.
FAB : 441 (M+1)FAB: 441 (M + 1)
실험예 1: Ras 파네실 전이효소 억제능 분석Experimental Example 1: Analysis of Ras farnesyl transferase inhibitory activity
본 실험에서는 폼프리아노 등의 방법(참조: Pomplianoet al., Biochemistry31, 3800, 1992)의 개선된 방법을 이용하였다. 즉, 유전자 재조합 기술에 의해 제조된 Ras 파네실 전이효소를 사용하였으며, 기질로는 H-Ras(H-Ras-CVLS)와 K-Ras의 카복시말단에 존재하는 다염기성 라이신 도메인을 치환시킨 H-Ras와의 결합단백질(참조: 대한민국 특허출원 제 97-14409 호)을 기보고된 방법(참조: Chung et al.,Bichimical et Biophysica Acta1129, 278, 1992)에 따라 정제하여 사용하였다.In this experiment, the improved method of Pompiano et al. (Pompliano et al., Biochemistry 31, 3800, 1992) was used. In other words, Ras farnesyl transferase prepared by genetic recombination technology was used, and H-Ras (H-Ras-CVLS) as a substrate and H- which substituted the polybasic lysine domain present at the carboxy terminus of K-Ras. The binding protein with Ras (see Korean Patent Application No. 97-14409) was purified and used according to the previously reported method (Chung et al., Bichimical et Biophysica Acta 1129, 278, 1992).
효소 반응은 염화칼륨 25mM, 염화마그네슘 25mM, 디티티(DTT) 10mM 및 염화 아연 50μM을 함유하는 50㎕의 50mM 소듐히피스 완충용액중에서 수행하였으며, Ras 기질 단백질 1.5μM, 트리튬-파네실 피로 포스페이트 0.15μM 및 파네실 전이효소 4.5nM이 사용되었다. 파네실 전이효소를 첨가하고 37℃에서 30분간 반응을 지속시킨 후 1M 염산을 함유한 에탄올 용액 1mℓ를 첨가하여 반응을 정지시켰다. 생성된침전물을 필터바인딩을 위한 호퍼 하베스터(호퍼 #FH 225V)를 사용하여 GF/B 필터에 흡착시킨후, 에탄올을 사용하여 세척하고, 건조시킨 필터의 방사능을 LKB 베타 카운터를 사용하여 측정하였다. 효소의 역가검정은 Ras 기질 단백질과 파네실 효소의 농도가 정량적 역가관계를 나타내는 기질 불포화 상태에서 측정되었으며, 본 발명에 따라 합성된 화합물은 디메틸설폭사이드(DMSO) 용매에 용해시켜 전체 반응액의 5% 이내로 첨가하여 효소 저해능을 평가하였다. 효소 저해능은 시험화합물이 없는 상태에서 Ras 기질 단백질에 도입된 파네실량에 대해 시험화합물 존재하에 측정된 파네실 도입량을 백분율로 표시하였으며, 50%의 효소활성을 저해하는 농도를 각 시험화합물의 IC50으로 결정하였다. 시험화합물의 선택적 저해능을 평가하기 위한 제라닐제라닐 전이효소는 샤버 등의 방법(참조: Schaberet al. J. Biol Chem., 265, 14701, 1990)을 변형하여 소뇌로부터 정제하여 사용하였으며, 파네실 전이효소 반응과 유사한 조건에서 제라닐제라닐 전이효소의 특이적 기질인 제라닐제라닐 피로 포스페이트와 Ras-CVIL 기질 단백질을 사용하여 실험을 수행하였다. 실험결과는 하기 표 2에 나타내었다.Enzymatic reactions were carried out in 50 μl of 50 mM sodium hippies buffer containing 25 mM potassium chloride, 25 mM magnesium chloride, 10 mM Diti (DTT) and 50 μM zinc chloride, 1.5 μM Ras substrate protein, 0.15 μM tritium-panesyl pyrophosphate And farnesyl transferase 4.5 nM was used. Panesyl transferase was added and the reaction was continued at 37 DEG C for 30 minutes, and then 1 ml of ethanol solution containing 1 M hydrochloric acid was added to stop the reaction. The resulting precipitate was adsorbed onto a GF / B filter using a hopper harvester (hopper #FH 225V) for filter binding, washed with ethanol, and the radioactivity of the dried filter was measured using an LKB beta counter. The enzyme titer was measured in a substrate unsaturated state in which the concentration of Ras substrate protein and panesyl enzyme showed a quantitative titer relationship, and the compound synthesized according to the present invention was dissolved in dimethyl sulfoxide (DMSO) solvent to obtain 5 The enzyme inhibition was assessed by addition within%. The enzyme inhibitory activity was expressed as a percentage of the amount of farnesyl introduced in the presence of the test compound relative to the amount of farnesyl introduced into the Ras substrate protein in the absence of the test compound, and the concentration that inhibited the enzyme activity of 50% was determined by IC 50 of each test compound. Determined. Geranylgeranyl transferase for evaluating the selective inhibitory activity of the test compound was purified from the cerebellum by modifying the method of Shaver et al. (Schaber et al. J. Biol Chem ., 265, 14701, 1990). Experiments were carried out using geranylgeranyl pyrophosphate and Ras-CVIL substrate protein, which are specific substrates of geranylgeranyl transferase under conditions similar to the real transferase reaction. The experimental results are shown in Table 2 below.
실험예 2: 세포내 Ras 파네실 전이효소의 억제효능 분석Experimental Example 2 Analysis of Inhibitory Effect of Intracellular Ras Farnesyl Transferase
본 실험에서는 돌연변이에 의해 형질전환 활성을 갖는 C-Harvey-Ras 단백질을 발현하는 Rat2 세포주 및 K-Ras 카복시 말단의 다염기성 라이신 도메인으로 치환된 H-Ras 결합 단백질로 형질전환된 Rat2 세포주(참조: 대한민국 특허출원 제 97-14409 호)를 사용하였으며, 실험은 드크루 등의 방법(참조: Declue J. E. etal., Cancer Research, 51, 712, 1991)을 변형시켜 다음과 같이 수행하였다.In this experiment, a rat2 cell line expressing a C-Harvey-Ras protein having a transgenic activity by mutation and a H-Ras binding protein transformed with a H-Ras binding protein substituted with a polybasic lysine domain at the K-Ras carboxy terminus (see: Korean Patent Application No. 97-14409) was used, and the experiment was performed by modifying the method of Decrue et al. (See Declue JE et al., Cancer Research, 51, 712, 1991) as follows.
형질전환된 Rat2 섬유아세포(fibroblast) 세포주를 60mm 세포배양 디쉬에 디쉬당 3x105 세포의 밀도로 분주하여 37℃ 세포배양기에서 48시간동안 배양함으로써 50%이상의 밀도로 성장시킨 후 시험화합물로 처리하였다. 이때 시험화합물의 용매로는 디메틸설폭사이드(DMSO)를 사용하였으며, 대조군과 시험군 모두 디메틸설폭사이드 농도를 1%로 사용하였다. 시험화합물로 처리한 지 4시간이 경과한 후에 배지 1mℓ 당 150μCi의 방사성동위원소[35S]로 표지된 메티오닌을 첨가하고 20시간 동안 배양한 후 생리적 식염수로 세포를 세척하였다. 냉각된 세포 용해 완충용액(염화마그네슘 5mM, 디티티 1mM, NP40 1%, EDTA 1mM, PMSF 1mM, 루펩틴 2μM, 펩스타틴에이 2μM 및 안티페인 2μM을 포함하는 소듐히피스 완충용액 50mM) 1mℓ를 가하여 세포를 용해시킨 후, 세포가 용해되어있는 상등액을 고속원심분리(12,000g x 5분)에 의해 수득하였다. 상등액의 방사성 동위원소 표지량을 측정하여 면역침전 반응시 정량적 결과를 얻을 수 있도록 표준화하였다. 그 후, 반응액에 Ras 단백질에 특이적 결합을 하는 단일클론항체, Y13-259(참조: Furth, M.E.et al., J. Virol., 43, 294, 1982)를 가하고 4℃에서 15시간동안 반응시켰다. 이 용액에 다시 고트에서 유래된 쥐의 면역글로블린에 대한 항체가 결합된 Protein A-아가로즈 현탁액을 가하여 1시간동안 4℃에서 반응시킨 후 면역반응 침전물로부터 비특이적 결합물을 제거하기 위해 완충용액(염화나트륨 50mM, 소듐 디옥시콜레이트 0.5%, NP40 0.5% 및 SDS 0.1%를 포함하는 트리스 클로라이드 50mM 완충용액)으로 세척하였다. 전기영동 방법을 사용하여 침전물을 분석하기 위해, 침전물을 전기영동 시료 완충액에가하여 끓인 후 13.5%의 SDS 폴리아크릴아마이드 겔을 사용하여 전기 영동을 수행하였다. 전기영동후 겔을 고정시키고 건조시킨 후 X-ray 필름에 감광시켜 현상인화하였다.. 세포내 Ras 파네실 전이효소의 억제효능은 Ras 단백질의 파네실이 결합된 밴드와 결합되지 않은 밴드의 강도를 측정하여 파네실 결합이 50% 저해된 시험화합물의 농도(IC50)로 나타내었다. 하기 표 2에는 본 발명에 따른 대표적인 화합물들의 억제효능을 나타내었다. 여기서 IC50은 실험예 1을 수행한 결과 얻어진 데이터이고 CIC50은 실험예 2를 수행한 결과 얻어진 데이터이다.The transformed Rat2 fibroblast cell line was dispensed at a density of 3 × 10 5 cells per dish in a 60 mm cell culture dish, grown to a density of 50% or more by incubating for 48 hours at 37 ° C., and then treated with a test compound. At this time, dimethyl sulfoxide (DMSO) was used as a solvent of the test compound, and dimethyl sulfoxide concentration was used as 1% in both the control group and the test group. After 4 hours of treatment with the test compound, methionine labeled with 150 μCi of radioisotope [35S] per 1 ml of medium was added thereto, and cultured for 20 hours, and the cells were washed with physiological saline. 1 ml of cooled cell lysis buffer (Magnesium chloride 5 mM, Dity 1 mM, NP40 1%, EDTA 1 mM, PMSF 1 mM, Lupetin 2 µM, Peptstatin A 2 µM and 50 mM Sodium Hippies buffer containing 2 µM) was added After lysing the cells, the supernatant in which the cells were lysed was obtained by high-speed centrifugation (12,000 gx 5 minutes). The radioisotope labeling amount of the supernatant was measured and normalized to obtain quantitative results in the immunoprecipitation reaction. Subsequently, a monoclonal antibody, Y13-259 (see Furth, ME et al., J. Virol ., 43, 294, 1982), which specifically binds to Ras protein, was added to the reaction solution for 15 hours at 4 ° C. Reacted. To this solution was added a protein A-agarose suspension bound to an immunoglobulin antibody from a goth-derived mouse and reacted at 4 ° C for 1 hour, followed by a buffer solution (sodium chloride) to remove the nonspecific binding substance from the immunoreactive precipitate. 50 mM, sodium dioxycholate 0.5%, NP40 0.5% and SDS 0.1% SDS). In order to analyze the precipitate using the electrophoretic method, the precipitate was added to the electrophoretic sample buffer and boiled, followed by electrophoresis using 13.5% SDS polyacrylamide gel. After electrophoresis, the gel was immobilized, dried, and then photosensitive on an X-ray film. The inhibitory effect of intracellular Ras farnesyl transferase showed the intensity of the unbound band with the farnesyl bound band of Ras protein. It was measured and expressed as the concentration of the test compound (IC 50 ) 50% inhibition of farnesyl binding. Table 2 shows the inhibitory effect of the representative compounds according to the present invention. Here, IC 50 is data obtained by performing Experimental Example 1 and CIC 50 is data obtained by performing Experimental Example 2.
[표 2]TABLE 2
Claims (4)
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| AT99901979T ATE229017T1 (en) | 1998-02-02 | 1999-02-01 | FARNESYLTRANSFERASE INHIBITORS WITH PIPERIDE INSTRUCTIONS AND METHOD FOR THE PRODUCTION THEREOF |
| US09/601,426 US6436960B1 (en) | 1998-02-02 | 1999-02-01 | Farnesyl transferase inhibitors having a piperidine structure and process for preparation thereof |
| AU21886/99A AU745855B2 (en) | 1998-02-02 | 1999-02-01 | Farnesyl transferase inhibitors having a piperidine structure and process for preparation thereof |
| JP2000529330A JP3283032B2 (en) | 1998-02-02 | 1999-02-01 | Funesyltransferase inhibitor having piperidine structure and method for producing the same |
| DE69904302T DE69904302T2 (en) | 1998-02-02 | 1999-02-01 | FARNESYL TRANSFERASE INHIBITORS WITH PIPERID STRUCTURE AND METHOD FOR THE PRODUCTION THEREOF |
| PCT/KR1999/000051 WO1999038862A1 (en) | 1998-02-02 | 1999-02-01 | Farnesyl transferase inhibitors having a piperidine structure and process for preparation thereof |
| CA002320233A CA2320233C (en) | 1998-02-02 | 1999-02-01 | Farnesyl transferase inhibitors having a piperidine structure and process for preparation thereof |
| EP99901979A EP1058683B1 (en) | 1998-02-02 | 1999-02-01 | Farnesyl transferase inhibitors having a piperidine structure and process for preparation thereof |
| CNB99802581XA CN1158277C (en) | 1998-02-02 | 1999-02-01 | Farnesyl transferase inhibitors having a piperidine structure and process for preparation thereof |
| ES99901979T ES2185307T3 (en) | 1998-02-02 | 1999-02-01 | FARNESIL TRANSFERASA INHIBITORS THAT HAVE A PIPERIDINIC STRUCTURE AND PROCEDURE FOR THEIR PREPARATION. |
| BR9908545-3A BR9908545A (en) | 1998-02-02 | 1999-02-01 | Piperidine derivative, process for preparing it, compound, process for preparing it, and pharmaceutical composition |
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| US4259345A (en) * | 1979-02-24 | 1981-03-31 | Pfizer Inc. | 2-(Imidazol-1-ylmethyl)pyrroles |
| JPS62240679A (en) * | 1986-04-11 | 1987-10-21 | Mitsubishi Chem Ind Ltd | Pyrrole compound |
| JPH03284676A (en) * | 1990-03-30 | 1991-12-16 | Kumiai Chem Ind Co Ltd | Pyrrole derivative and herbicide |
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| JPS62240679A (en) * | 1986-04-11 | 1987-10-21 | Mitsubishi Chem Ind Ltd | Pyrrole compound |
| JPH03284676A (en) * | 1990-03-30 | 1991-12-16 | Kumiai Chem Ind Co Ltd | Pyrrole derivative and herbicide |
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