KR19990074164A - Pyridin-2-one derivatives useful as farnesyl transferase inhibitors - Google Patents
Pyridin-2-one derivatives useful as farnesyl transferase inhibitors Download PDFInfo
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Abstract
본 발명은 파네실 전이효소 억제활성을 갖는 하기 화학식 1의 피리딘-2-온 유도체, 그의 약제학적으로 허용가능한 염, 용매화물 및 그의 제조방법에 관한 것이다 :The present invention relates to a pyridin-2-one derivative of formula (1) having a farnesyl transferase inhibitory activity, a pharmaceutically acceptable salt, a solvate thereof and a method for preparing the same:
상기식에서,In the above formula,
R1, R2, R3, R4및 n 은 명세서에서 정의한 바와 같다.R 1 , R 2 , R 3 , R 4 and n are as defined in the specification.
Description
본 발명은 파네실 전이효소 억제활성을 갖는 하기 화학식 1의 피리딘-2-온 유도체, 그의 약제학적으로 허용가능한 염 및 그의 용매화물에 관한 것이다 :The present invention relates to a pyridin-2-one derivative of formula (1) having a farnesyl transferase inhibitory activity, a pharmaceutically acceptable salt thereof and a solvate thereof:
화학식 1Formula 1
상기식에서,In the above formula,
R1은 저급알킬; 할로겐 또는 저급알킬에 의해 치환되거나 비치환된 아릴; 또는 질소 및 황원자로 구성된 그룹중에서 선택된 1종 이상의 헤테로원자를 포함하는 헤테로아릴을 나타내고,R 1 is lower alkyl; Aryl unsubstituted or substituted by halogen or lower alkyl; Or heteroaryl including one or more heteroatoms selected from the group consisting of nitrogen and sulfur atoms,
R2는 수소; 저급알킬; 또는 하기 구조식의 그룹중 선택된 어느 하나를 나타내며,R 2 is hydrogen; Lower alkyl; Or any one selected from the group of the following structural formulas,
, ,
여기에서 R5는 수소, 할로겐, 시아노, 니트로, 저급알킬, 저급알콕시, 저급알콕시카보닐, 아미노, 머캅토아미노 또는 저급알킬티오를 나타내고,R 5 here represents hydrogen, halogen, cyano, nitro, lower alkyl, lower alkoxy, lower alkoxycarbonyl, amino, mercaptoamino or lower alkylthio,
R6은 수소 또는를 나타내며, 여기에서 A 는 CH2, C=O 또는 SO2를 나타내고, B 는 수소; 페닐 또는 비페닐에 의해 치환되거나 비치환된 저급알킬; 저급알콕시; 페닐; 벤질; 벤질옥시; 또는 저급알킬, 페닐, 벤질, C3-C6사이클로알킬 또는 페녹시(저급)알킬에 의해 치환되거나 비치환된 아미노를 나타내며,R 6 is hydrogen or Wherein A represents CH 2 , C═O or SO 2 , and B is hydrogen; Lower alkyl unsubstituted or substituted by phenyl or biphenyl; Lower alkoxy; Phenyl; benzyl; Benzyloxy; Or amino substituted or unsubstituted by lower alkyl, phenyl, benzyl, C 3 -C 6 cycloalkyl or phenoxy (lower) alkyl,
R3및 R4는 각각 독립적으로 수소, 할로겐 또는 저급알킬을 나타내고,R 3 and R 4 each independently represent hydrogen, halogen or lower alkyl,
n 은 1 내지 3의 정수를 나타낸다.n represents the integer of 1-3.
본 발명에는 또한, 상기 화학식 1의 화합물을 제조하는 방법도 포함되어 있다. 따라서, 이러한 제조방법도 본 발명의 대상이다.The present invention also includes a method for preparing the compound of Formula 1. Therefore, such a manufacturing method is also the subject of this invention.
Ras 단백질은 세포의 성장과 분화에 중요한 역활을 하는 21 kda의 단백질로서 구아닌 뉴클레오타이드와 결합하며, 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 binds guanine nucleotides and hydrolyzes guanosine triphosphate (GTP) to guanosine diphosphate (GDP). 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 188개의 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 as amino acids 188 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 3 개 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 카복시 말단에 있는 CA1A2X라는 아미노산 네개의 펩타이드이며, 여기서 A1및 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) 도메인이라 불리는 여러개의 라이신이 배열된 부위를 가지고 있으며, 이를 통해 세포막내의 음이온성 지질과의 결합이 용이하게 되는 것으로 알려져 있다. Ras 단백질이 세포막내에 최적으로 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. Terminal modification is required. The first step, panesylation, is achieved by farnesyl transferase (FTase). The substrate of the farnesyl transferase is a peptide of four amino acids, CA 1 A 2 X, at the carboxy terminus of the Ras protein, where A 1 and A 2 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 a site where several lysines, called poly basic domains, are arranged in place of cysteine required for palmitoylation. It is known that binding to lipids is facilitated. All modification steps are necessary for optimal attachment of Ras protein to the cell membrane. However, the activation of Ras protein is known to be sufficient for panicylation alone. Therefore, studies are actively underway to suppress Ras carcinogenicity by mutations by blocking this panicylation. (Bus, JE et al., Chemistry & Biology, 1995, 2, 787).
그간의 연구결과, Ras로 형질전환된 세포에서 파네실 전이효소를 저해했을 때 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., 1994, 91, 9141; Kohl, N.E. et al, Nature Medicine, 1995, 1, 792). 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, 1993, 260, 1934; Graham et al., PCT/US95/12224), 페닐기로 변형된 펩타이드(참조: Sebti, S. M., J. Biol. Chem. 1995, 270, 26802), 향정신성 의약품 골격구조중 벤조디아제핀을 펩타이드의 turn 모사구조로 활용한 변형체(참조: James, G. L. et al., Science, 1993, 260, 1937), 펩타이드 구조에서 벗어나 트리사이클릭 유기 화합물을 골격으로한 저해제(참조: Bishop W. R. et al., J. Biol. Chem. 1995, 270, 30611)를 들 수 있다. 또한, 파네실 전이효소가 프레닐기를 전이시키는 작용기전이 전자 친화적 치환반응(Electrophilic Displacement)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. 1996, 118, 8761).Indeed, several inhibitors of farnesyl transferase have been shown to selectively inhibit the response of Ras oncogenic genes to intracellular prenyl groups (see Kohl, NE et al, Proc. Natl. Acad. Sci. USA., 1994, 91, 9141; Kohl, NE et al, Nature Medicine, 1995, 1, 792). The developed panesyl transferase inhibitors include peptide variants containing cysteine thiol groups and similar structures to CAAX, and inhibitors that improve them (see US Patent No. 5,141,851; Kohl, NE et al, Science, 1993, 260, 1934; Graham et al., PCT / US95 / 12224), peptides modified with phenyl groups (Sebti, SM, J. Biol. Chem. 1995, 270, 26802), benzodiazepine in the psychotropic pharmaceutical framework Variants utilized as turn simulations (James, GL et al., Science, 1993, 260, 1937), inhibitors based on tricyclic organic compounds away from peptide structures (Bishop WR et al., J) Biol. Chem. 1995, 270, 30611). In addition, since the mechanism of action by which the farnesyl transferase transfers the prenyl group is an electrophilic displacement reaction, the positive state of the transition state is connected to the prenyl group, and the positive state is connected to the prenyl group. New types of inhibitors have been proposed (Poulter, CD et al., J. Am. Chem. Soc. 1996, 118, 8761).
그러나, 많은 경우의 인체 암에서 K-Ras 활성화가 주요 원인으로 작용하며, 지금까지 개발된 대부분의 프레닐 전이효소 저해제들은 K-Ras의 활성화를 효과적으로 억제시키지 못한다. 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 cases of human cancer, and most of the prenyl transferase inhibitors developed to date do not effectively inhibit K-Ras activation. 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 피리딘-2-온 유도체를 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. A novel pyridin-2-one derivative that inhibits panesylation was synthesized and its 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, 그의 약제학적으로 허용되는 염 및 용매화물에 관한 것이다.Accordingly, the present invention relates to a compound of formula 1, pharmaceutically acceptable salts and solvates thereof having excellent anticancer effects.
본 발명은 또한, 화학식 1의 화합물을 제조하는 신규한 방법에 관한 것이다.The invention also relates to a novel process for preparing the compound of formula (1).
화학식 1Formula 1
상기식에서,In the above formula,
R1은 저급알킬; 할로겐 또는 저급알킬에 의해 치환되거나 비치환된 아릴; 또는 질소 및 황원자로 구성된 그룹중에서 선택된 1종 이상의 헤테로원자를 포함하는 헤테로아릴을 나타내고,R 1 is lower alkyl; Aryl unsubstituted or substituted by halogen or lower alkyl; Or heteroaryl including one or more heteroatoms selected from the group consisting of nitrogen and sulfur atoms,
R2는 수소; 저급알킬; 또는 하기 구조식의 그룹중 선택된 어느 하나를 나타내며,R 2 is hydrogen; Lower alkyl; Or any one selected from the group of the following structural formulas,
, ,
여기에서 R5는 수소, 할로겐, 시아노, 니트로, 저급알킬, 저급알콕시, 저급알콕시카보닐, 아미노, 머캅토아미노 또는 저급알킬티오를 나타내고,R 5 here represents hydrogen, halogen, cyano, nitro, lower alkyl, lower alkoxy, lower alkoxycarbonyl, amino, mercaptoamino or lower alkylthio,
R6은 수소 또는를 나타내며, 여기에서 A 는 CH2, C=O 또는 SO2를 나타내고, B 는 수소; 페닐 또는 비페닐에 의해 치환되거나 비치환된 저급알킬; 저급알콕시; 페닐; 벤질; 벤질옥시; 또는 저급알킬, 페닐, 벤질, C3-C6사이클로알킬 또는 페녹시(저급)알킬에 의해 치환되거나 비치환된 아미노를 나타내며,R 6 is hydrogen or Wherein A represents CH 2 , C═O or SO 2 , and B is hydrogen; Lower alkyl unsubstituted or substituted by phenyl or biphenyl; Lower alkoxy; Phenyl; benzyl; Benzyloxy; Or amino substituted or unsubstituted by lower alkyl, phenyl, benzyl, C 3 -C 6 cycloalkyl or phenoxy (lower) alkyl,
R3및 R4는 각각 독립적으로 수소, 할로겐 또는 저급알킬을 나타내고,R 3 and R 4 each independently represent hydrogen, halogen or lower alkyl,
n 은 1 내지 3의 정수를 나타낸다.n represents the integer of 1-3.
상기 화학식 1의 화합물에 대한 치환기 정의에서 용어 "저급알킬"은 메틸, 에틸, 이소프로필, 이소부틸, t-부틸 등과 같은 탄소수 1 내지 4의 직쇄 또는 측쇄알킬을 의미하며, "질소 및 황원자로 구성된 그룹중에서 선택된 1종 이상의 헤테로원자를 포함하는 헤테로아릴"은 1 또는 2개의 질소 또는 황원자가 방향족환내에 포함되어 있는 모노 또는 비사이클릭 방향족 라디칼을 의미한다.In the substituent definition for the compound of Formula 1, the term "lower alkyl" refers to a straight or branched chain alkyl having 1 to 4 carbon atoms such as methyl, ethyl, isopropyl, isobutyl, t-butyl, etc., consisting of nitrogen and sulfur atoms. Heteroaryl containing one or more heteroatoms selected from the group means a mono or bicyclic aromatic radical containing one or two nitrogen or sulfur atoms in the aromatic ring.
본 발명은 또한 상기 화학식 1의 화합물의 약제학적으로 허용되는 염 및 그의 수화물을 포함한 용매화물을 포함한다. 약제학적으로 허용되는 무독성염의 대표적인 예로는 염산, 브롬화수소산, 인산, 황산 등과 같은 무기산과의 염 또는 아세트산, 트리플루오로아세트산, 시트르산, 말레인산, 옥살산, 숙신산, 벤조산, 타타르산, 푸마르산, 만데르산, 아스코르빈산 또는 말린산과 같은 유기카르복실산 또는 메탄설폰산, 파라-톨루엔설폰산과 같은 술폰산과의 염 및 피리디놀계 화합물의 기술분야에서 공지되어 사용되고 있는 그밖의 다른 산들과의 염을 포함한다. 또한, 화학식 1의 화합물은 염기와 무독성 염을 형성할 수도 있으며, 이때 사용되는 염기로는 알칼리금속 수산화물류(예: 수산화나트륨, 수산화칼륨), 알칼리금속 탄산화물류(예: 중탄산나트륨, 중탄산칼륨, 탄산나트륨, 탄산칼륨), 알칼리토금속 탄산화물류(예: 탄산칼슘) 등을 들 수 있다. 이들 산 또는 염기부가염들은 통상의 전환공정에 의하여 제조될 수 있다.The present invention also includes solvates including pharmaceutically acceptable salts of compounds of Formula 1 and hydrates thereof. Representative examples of pharmaceutically acceptable non-toxic salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, or the like, or acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid. Salts with organic carboxylic acids, such as ascorbic acid or dried acid, or salts with sulfonic acids such as methanesulfonic acid, para-toluenesulfonic acid and other acids known and used in the art of pyridinol compounds . In addition, the compound of Formula 1 may form a non-toxic salt with a base, wherein the base used may be alkali metal hydroxides (eg, sodium hydroxide, potassium hydroxide), alkali metal carbonates (eg sodium bicarbonate, potassium bicarbonate, Sodium carbonate, potassium carbonate), alkaline earth metal carbonates such as calcium carbonate, and the like. These acid or base addition salts may be prepared by conventional conversion processes.
우수한 항암효과를 나타내는 상기 화학식 1의 화합물 중에서도 바람직한 화합물은Among the compounds of Formula 1, which show excellent anticancer effects, preferred compounds are
R1은 저급알킬에 의해 치환되거나 비치환된 아릴을 나타내고,R 1 represents aryl unsubstituted or substituted by lower alkyl,
R2는 수소; 저급알킬; 또는 하기 구조식의 그룹중 선택된 어느 하나를 나타내며,R 2 is hydrogen; Lower alkyl; Or any one selected from the group of the following structural formulas,
, ,
여기에서 R5는 수소, 할로겐, 시아노 또는 저급알콕시를 나타내고,R 5 here represents hydrogen, halogen, cyano or lower alkoxy,
R6은 수소 또는를 나타내며, 여기에서 B 는 벤질옥시; 페닐 또는 비페닐에 의해 치환되거나 비치환된 저급알킬; 또는 벤질에 의해 치환되거나 비치환된 아미노를 나타내고,R 6 is hydrogen or Wherein B is benzyloxy; Lower alkyl unsubstituted or substituted by phenyl or biphenyl; Or amino unsubstituted or substituted by benzyl,
R3및 R4는 각각 독립적으로 수소, 할로겐 또는 저급알킬을 나타내며,R 3 and R 4 each independently represent hydrogen, halogen or lower alkyl,
n 은 1인 화합물이다.n is a compound of 1.
본 발명에 따른 화학식 1 화합물의 대표적인 예는 하기 표 1a 내지 1c에 나타낸 바와 같다.Representative examples of the compound of formula 1 according to the present invention are as shown in Tables 1A to 1C.
본 발명에 따른 화학식 1의 화합물은 하기 화학식 2의 화합물을 용매중에서 염기 존재하에 하기 화학식 3의 화합물과 반응시킴을 특징으로하여 제조할 수 있으며, 이러한 화학식 1 화합물의 제조방법도 또한 본 발명의 목적이다.The compound of formula 1 according to the present invention may be prepared by reacting a compound of formula 2 with a compound of formula 3 in a solvent in the presence of a base, and a method for preparing the compound of formula 1 is also an object of the present invention. to be.
그러나, 본 발명에 따른 화합물 및 그 중간체의 제조방법이 하기에 설명하는 것으로만 한정된 것은 아니며, 본 명세서에 기재되거나 선행문헌에 개시된 여러 가지 합성방법을 임의로 조합함으로써 용이하게 제조할 수 있고, 이러한 조합은 본 발명이 속하는 기술분야의 당업자에게 범용화된 통상의 기술이다.However, the method of preparing the compound and the intermediate thereof according to the present invention is not limited only to those described below, and can be easily prepared by arbitrarily combining various synthesis methods described in this specification or disclosed in the prior literature, and such combinations. Is a common technique generalized to those skilled in the art to which the present invention belongs.
상기식에서In the above formula
R1내지 R4는 앞에서 정의한 바와 같고,R 1 to R 4 are as defined above,
X 는 반응성 이탈기, 바람직하게는 할로겐을 나타낸다.X represents a reactive leaving group, preferably halogen.
화학식 1의 화합물을 제조하는 상기 방법에서 용매로는 디메틸포름아미드, 톨루엔, 디클로로메탄, 테트라하이드로푸란, 디메틸아세트아미드, 에틸아세테이트, 디에틸에테르, 피리딘, 메탄올 및 클로로포름 중에서 선택된 1 종 이상을 사용할 수 있으며, 염기로는 수소화나트륨, 포타슘 t-부톡사이드, 소듐 비스(트리메틸실릴)아미드, 포타슘 비스(트리메틸실릴)아미드, 수산화나트륨, 탄산칼륨, 탄산나트륨 및 중탄산칼륨 중에서 선택된 1 종 이상을 사용할 수 있다. 상기 반응은 통상 상온 내지 0℃ 온도범위에서 1 내지 24 시간동안 진행시킨다.In the method for preparing the compound of Formula 1, at least one selected from dimethylformamide, toluene, dichloromethane, tetrahydrofuran, dimethylacetamide, ethyl acetate, diethyl ether, pyridine, methanol, and chloroform may be used. The base may be at least one selected from sodium hydride, potassium t-butoxide, sodium bis (trimethylsilyl) amide, potassium bis (trimethylsilyl) amide, sodium hydroxide, potassium carbonate, sodium carbonate and potassium bicarbonate. The reaction is usually carried out for 1 to 24 hours at room temperature to 0 ℃ temperature range.
화학식 1의 화합물을 제조하는 상기 방법에서 출발물질로 사용된 화학식 2의 화합물은 하기 반응식 1에 도시한 바와 같이 알데히드 화합물로부터 축합, 환원, 산화 및 N-(카바모일메틸)-피리디늄 화합물과의 반응을 거쳐 제조할 수 있다.Compounds of formula (2) used as starting materials in the process for preparing compounds of formula (1) are condensed, reduced, oxidized and reacted with N- (carbamoylmethyl) -pyridinium compounds from aldehyde compounds as shown in Scheme 1 below. It can manufacture through reaction.
상기 반응식 1에서 R1은 앞에서 정의한 바와 동일하다.R 1 in Scheme 1 is the same as defined above.
이미다졸기를 포함하고 있는 화학식 3의 화합물은 하기 반응식 2 내지 4에 도시한 바와 같이 R2치환기의 종류에 따라 여러가지 다른 방법으로 제조할 수 있으며, 이들 개시된 방법을 임의로 조합함으로써도 원하는 화합물을 제조할 수 있다.The compound of formula 3 containing an imidazole group may be prepared by various methods according to the type of R 2 substituents, as shown in Schemes 2 to 4 below, and a desired compound may be prepared by arbitrarily combining these disclosed methods. can do.
상기 반응식 2에서 R2는 앞에서 정의한 바와 동일하며, 아민유도체로부터 디하이드록시아세톤 화합물과의 축합반응, 머캅토기 제거반응 및 할로겐화 반응을 통해 화학식 3의 화합물에서 n이 1이고 R3및 R4가 각각 수소인 화합물이 제조된다.In Reaction Scheme 2, R 2 is the same as defined above, n is 1 in the compound of Formula 3 through condensation reaction with a dihydroxyacetone compound, a mercapto group removal reaction and a halogenation reaction from an amine derivative, and R 3 and R 4 are Compounds that are each hydrogen are prepared.
상기 반응식 2의 구체적인 예를 하기 반응식 3에 도시하였다.Specific examples of Scheme 2 are shown in Scheme 3 below.
상기 반응식 3에서 Cbz 는 벤질옥시카보닐을 나타내며, 이하 동일한 의미로 사용된다. 반응식 3에서는 4-아미노메틸피페리딘으로부터 보호기화, 벤질옥시카보닐화 및 탈보호기화 반응을 수행하여 1-벤질옥시카보닐-4-아미노메틸피페리딘을 제조한 다음, 이를 디하이드록시아세톤 및 포타슘티오시아네이트(KSCN)과 반응시키고 머캅토기를 제거한 후 할로겐화 반응을 수행하여 화학식 3의 화합물에서 n이 1이고 R3및 R4가 각각 수소이며, R2가 1-(벤질옥시카보닐)피페리딘메틸인 화합물을 제조한다. 또한, 상기 반응의 마지막 단계에서 할로겐화를 수행하는대신 아세틸화를 수행하고 Pd/C 존재하에 벤질옥시카보닐기를 제거한 다음 적절한 치환기를 피페리딘환의 1-번 위치에 치환시키고 할로겐화를 수행함으로써 R2가 각각 1-(3-페닐프로피오닐)피페리딘메틸 및 1-(벤질카바모일)피페리딘메틸인 화학식 3의 화합물을 제조한다.In the Scheme 3, Cbz represents benzyloxycarbonyl and is used hereinafter with the same meaning. In Scheme 3, 1-benzyloxycarbonyl-4-aminomethylpiperidine was prepared by protecting, benzyloxycarbonylation and deprotection reaction from 4-aminomethylpiperidine to prepare dihydroxyacetone. And reacting with potassium thiocyanate (KSCN), removing the mercapto group, and then performing a halogenation reaction, where n is 1 and R 3 and R 4 are each hydrogen in the compound of Formula 3, and R 2 is 1- (benzyloxycarbonyl To prepare a compound that is piperidinemethyl. Further, by carrying out acetylation, rather than performing the halogenation at the end of the reaction, and then substituting the appropriate substituent in the presence Pd / C removed the group benzyloxycarbonyl piperidine-1-ring position and performs halide R 2 Is a 1- (3-phenylpropionyl) piperidinemethyl and 1- (benzylcarbamoyl) piperidinemethyl, respectively.
상기 반응식 4에서 Tr 은 트리틸을 나타내며, 이하 동일한 의미로 사용된다. 반응식 4에서는 하이드록시메틸 이미다졸 유도체로부터 보호기화, 아세틸화, 커플링, 탈보호기화 및 할로겐화 반응을 통해 화학식 3의 화합물에서 n이 1이고 R3및 R4가 각각 수소이며, R2가 4-시아노벤질인 화합물이 제조된다.In Scheme 4, Tr represents trityl and is used hereinafter in the same sense. In Scheme 4, n is 1 and R 3 and R 4 are each hydrogen and R 2 is 4 in the compound of Formula 3 through protecting, acetylation, coupling, deprotection and halogenation from hydroxymethyl imidazole derivatives. A compound that is cyanobenzyl is prepared.
본 발명, 특히 상기 설명한 제조방법들을 하기 제조예, 실시예 및 실험예에 의거하여 보다 구체적으로 설명한다. 그러나, 이들 제조예, 실시예 및 실험예는 본 발명에 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: 1-(4-브로모벤질)-5-클로로메틸-1H-이미다졸의 합성Preparation Example 1 Synthesis of 1- (4-bromobenzyl) -5-chloromethyl-1H-imidazole
1-1) 1-(4-브로모벤질)-5-하이드록시메틸-1H-이미다졸의 제조1-1) Preparation of 1- (4-bromobenzyl) -5-hydroxymethyl-1H-imidazole
4-브로모벤질아민 하이드로클로라이드 8.9g(0.04 몰)와 아세트산 4㎖를 n-부탄올 85㎖에 녹인 후, 여기에 포타슘티오시아네이트 5.19g(0.05 몰) 및 1,3-디하이드록시아세톤 다이머 3.21g(0.02 몰)을 가하고 4일 동안 교반하였다. 석출된 고체를 감압여과하여 수득한 후, 물과 디에틸에테르로 세척해주었다. 수득된 고체를 10% 질산수용액에 가하고 3시간동안 교반하였다. 녹지않은 불순물을 감압여과하여 제거한 후, 용액을 4N 수산화나트륨 수용액으로 염기화시켜 생성물을 석출시켰다. 고체를 물로 수회 세척해준 다음, 진공하에 건조시켜 표제화합물 6.7g(0.024몰, 수율 60%)을 수득하였다.8.9 g (0.04 mol) of 4-bromobenzylamine hydrochloride and 4 ml of acetic acid were dissolved in 85 ml of n-butanol, followed by 5.19 g (0.05 mol) of potassium thiocyanate and 1,3-dihydroxyacetone dimer. 3.21 g (0.02 mol) was added and stirred for 4 days. The precipitated solid was obtained by filtration under reduced pressure and then washed with water and diethyl ether. The obtained solid was added to an aqueous 10% nitric acid solution and stirred for 3 hours. After insoluble impurities were removed by filtration under reduced pressure, the solution was basified with 4N aqueous sodium hydroxide solution to precipitate the product. The solid was washed several times with water and then dried in vacuo to yield 6.7 g (0.024 moles, yield 60%) of the title compound.
1H NMR(CDCl3) δ(ppm) 4.45(s, 2H), 5.20(s, 2H), 6.94(s, 1H), 7.03(d, 2H), 7.18(m, 4H) 1 H NMR (CDCl 3 ) δ (ppm) 4.45 (s, 2H), 5.20 (s, 2H), 6.94 (s, 1H), 7.03 (d, 2H), 7.18 (m, 4H)
FAB Mass 267 (M+H)FAB Mass 267 (M + H)
1-2) 1-(4-브로모벤질)-5-클로로메틸-1H-이미다졸의 제조1-2) Preparation of 1- (4-bromobenzyl) -5-chloromethyl-1H-imidazole
상기 1-1)에서 수득한 화합물 6.7g(24 밀리몰)을 클로로포름 20㎖에 녹인 후 티오닐클로라이드 7.1g(60 밀리몰)을 0℃에서 천천히 적가하고 상온에서 2시간동안 교반하였다. 감압증류하에 용매를 제거한 후, 잔존 하이드로클로라이드를 진공하에 제거하여 표제화합물의 하이드로클로라이드 염 7.3g(22.8 밀리몰, 수율 95%)을 수득하였다. 수득된 화합물은 정제과정없이 후속반응에 사용하였다.6.7 g (24 mmol) of the compound obtained in 1-1) was dissolved in 20 ml of chloroform, and then 7.1 g (60 mmol) of thionyl chloride was slowly added dropwise at 0 ° C, and stirred at room temperature for 2 hours. After removal of the solvent under reduced pressure distillation, the remaining hydrochloride was removed under vacuum to give 7.3 g (22.8 mmol, 95% yield) of the hydrochloride salt of the title compound. The obtained compound was used for the subsequent reaction without purification.
제조예 2: 1-(4-시아노벤질)-5-클로로메틸-1H-이미다졸의 합성Preparation Example 2 Synthesis of 1- (4-cyanobenzyl) -5-chloromethyl-1H-imidazole
2-1) 4-하이드록시메틸-1-트리틸-1H-이미다졸의 제조2-1) Preparation of 4-hydroxymethyl-1-trityl-1H-imidazole
4-하이드록시메틸-이미다졸 하이드로클로라이드 3.99g(29.6 밀리몰)을 디메틸포름아미드 30㎖와 트리에틸아민 10㎖에 녹인 후, 여기에 트리페닐메틸 클로라이드 9.35g(33.5 밀리몰)의 디메틸포름아미드 110㎖ 용액을 서서히 가하였다. 2시간이 경과한 후에 반응액에 얼음물 500㎖를 가하여 생성된 고체를 디옥산으로 재결정하여 표제화합물 8.82g(수율 87%)을 수득하였다.3.99 g (29.6 mmol) of 4-hydroxymethyl-imidazole hydrochloride was dissolved in 30 ml of dimethylformamide and 10 ml of triethylamine, followed by 110 ml of 9.35 g (33.5 mmol) 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 recrystallized from dioxane to obtain 8.82 g (yield 87%) of the title compound.
융점: 227-229℃Melting Point: 227-229 ℃
2-2) 4-아세톡시메틸-1-트리틸-1H-이미다졸의 제조2-2) Preparation of 4-acetoxymethyl-1-trityl-1H-imidazole
피리딘 100㎖에 상기 2-1)에서 수득한 화합물 5.00g(14.7 밀리몰)을 가하고, 여기에 아세트산무수물 1.65g(16.2 밀리몰)을 가한 다음 상온에서 24시간동안 교반하였다. 반응액을 감압증류하여 피리딘을 제거하고 잔류물을 에틸아세테이트 200㎖에 녹인 다음, 포화 염화나트륨 수용액 100㎖로 세척해주었다. 유기용매를 감압 증류하여 제거하고 실리카겔 칼럼 크로마토그래피(용리제: 디클로로메탄/메탄올=97/3, v/v)를 수행하여 표제화합물 5.22g(13.7 밀리몰, 수율 93%)을 수득하였다.5.00 g (14.7 mmol) of the compound obtained in 2-1) was added to 100 ml of pyridine, and 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 and washed with 100 ml of saturated aqueous sodium chloride solution. The organic solvent was removed by distillation under reduced pressure and silica gel column chromatography (eluent: dichloromethane / methanol = 97/3, v / v) was carried out to give 5.22 g (13.7 mmol, 93% yield) of the title compound.
1H NMR(CDCl3) δ(ppm): 2.01(3H, s), 4.95(2H, s), 6.88(1H, s), 7.08(5H, s), 7.27(10H, s), 7.45(1H, s) 1 H NMR (CDCl 3 ) δ (ppm): 2.01 (3H, s), 4.95 (2H, s), 6.88 (1H, s), 7.08 (5H, s), 7.27 (10H, s), 7.45 (1H , s)
2-3) 4-아세톡시메틸-3-(4-시아노벤질)-1-트리틸-1H-이미다졸 브로마이드의 제조2-3) Preparation of 4-acetoxymethyl-3- (4-cyanobenzyl) -1-trityl-1H-imidazole bromide
상기 2-2)에서 수득한 화합물 5.00g(13.1 밀리몰)을 디클로로메탄 20㎖에 녹이고, 4-시아노벤질브로마이드 2.82g(14.4 밀리몰)을 가한 다음 상온에서 60시간 동안 교반하였다. 감압증류하여 유기용매를 제거하고 실리카겔 칼럼 크로마토그래피(용리제: 디클로로메탄/메탄올=93/7, v/v)를 수행하여 표제화합물 5.31g(9.17 밀리몰, 수율 70%)을 수득하였다.5.00 g (13.1 mmol) of the compound obtained in 2-2) was dissolved in 20 mL of dichloromethane, and 2.82 g (14.4 mmol) of 4-cyanobenzyl bromide was added thereto, followed by stirring at room temperature for 60 hours. The organic solvent was removed by distillation under reduced pressure and silica gel column chromatography (eluent: dichloromethane / methanol = 93/7, v / v) was carried out to obtain 5.31 g (9.17 mmol, yield 70%) of the title compound.
1H NMR(CDCl3/CD3OD) δ(ppm): 1.95(3H, s), 4.95(2H, s), 5.45(2H, s), 7.11-7.40(18H, m), 7.65(2H, d), 8.21(1H, s) 1 H NMR (CDCl 3 / CD 3 OD) δ (ppm): 1.95 (3H, s), 4.95 (2H, s), 5.45 (2H, s), 7.11-7.40 (18H, m), 7.65 (2H, d), 8.21 (1 H, s)
2-4) 4-아세톡시메틸-3-(4-시아노벤질)-3H-이미다졸의 제조2-4) Preparation of 4-acetoxymethyl-3- (4-cyanobenzyl) -3H-imidazole
상기 2-3)에서 수득한 화합물 9.10g(15.7 밀리몰)을 디클로로메탄 500㎖에 녹인 후, 0℃에서 트리플루오로아세트산 6.06㎖(78.7 밀리몰)과 트리에틸실란 12.5 ㎖(78.7 밀리몰)를 서서히 가한 다음 상온에서 1시간동안 교반하였다. 유기용매를 감압증류하여 제거하고, 포화 탄산칼륨 수용액을 사용하여 pH를 10으로 조정한 다음 에틸아세테이트 300㎖로 추출하였다. 유기용매를 감압증류하여 제거하고 실리카겔 칼럼 크로마토그래피(용리제: 에틸아세테이트)를 수행하여 표제화합물 3.60g (14.1 밀리몰, 수율 90%)을 수득하였다.9.10 g (15.7 mmol) of the compound obtained in the above 2-3) was 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. Then stirred at room temperature for 1 hour. The organic solvent was removed by distillation under reduced pressure, the pH was adjusted to 10 using a saturated aqueous potassium carbonate solution and then extracted with 300 ml of ethyl acetate. The organic solvent was removed by distillation under reduced pressure and silica gel column chromatography (eluent: ethyl acetate) was carried out to obtain 3.60 g (14.1 mmol, 90% yield) of the title compound.
1H NMR(CDCl3) δ(ppm): 1.90(3H, s), 4.97(2H, s), 5.25(2H, s), 7.14(2H, d), 7.21(1H, d), 7.67(1H, s), 7.75(2H, d) 1 H NMR (CDCl 3 ) δ (ppm): 1.90 (3H, s), 4.97 (2H, s), 5.25 (2H, s), 7.14 (2H, d), 7.21 (1H, d), 7.67 (1H , s), 7.75 (2H, d)
2-5) 1-(4-시아노벤질)-5-하이드록시메틸-1H-이미다졸의 제조2-5) Preparation of 1- (4-cyanobenzyl) -5-hydroxymethyl-1H-imidazole
상기 2-4)에서 수득한 화합물 4.20g(16.5 밀리몰)을 메탄올 200㎖에 녹인 후, 탄산칼륨 4.50g(32.9 밀리몰)을 가하고 상온에서 20분 동안 교반하였다. 감압증류하여 유기용매를 제거하고 에틸아세테이트 300㎖로 추출한 다음, 실리카겔 칼럼 크로마토그래피(용리제: 디클로로메탄/메탄올=93/7, 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 2-4) was dissolved in 200 mL of methanol, and then 4.50 g (32.9 mmol) of potassium carbonate was added thereto, followed by stirring at room temperature for 20 minutes. After distillation under reduced pressure to remove the organic solvent and extraction with 300 ml of ethyl acetate, silica gel column chromatography (eluent: dichloromethane / methanol = 93/7, v / v) was carried out to give 3.19 g (15.0 mmol, yield 91) of the title compound. %) Was obtained.
1H NMR (CDCl3/CD3OD) δ(ppm): 4.28(2H, s), 5.18(2H, s), 6.84(1H, s), 7.12(2H, d), 7.42(1H, s), 7.55(2H, d) 1 H NMR (CDCl 3 / CD 3 OD) δ (ppm): 4.28 (2H, s), 5.18 (2H, s), 6.84 (1H, s), 7.12 (2H, d), 7.42 (1H, s) , 7.55 (2H, d)
2-6) 5-클로로메틸-1-(4-시아노벤질)-1H-이미다졸의 제조2-6) Preparation of 5-chloromethyl-1- (4-cyanobenzyl) -1H-imidazole
상기 2-5)에서 수득한 화합물 3.00g(14.1 밀리몰)을 클로로포름 40㎖에 녹인 후, 0℃에서 티오닐클로라이드 5.02㎖(70.5 밀리몰)를 서서히 가하고 상온에서 2시간 동안 교반하였다. 감압증류하여 유기용매를 제거하고, 에틸아세테이트 50㎖에 용해시키고, 포화 중탄산나트륨 수용액으로 세척해준 다음 유기용매를 감압증류하여 표제화합물 2.91g(12.5 밀리몰, 수율 89%)을 수득하였다. 수득된 화합물은 정제하지 않고 바로 후속 반응에 사용하였다.3.00 g (14.1 mmol) of the compound obtained in 2-5) 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 at room temperature for 2 hours. The organic solvent was removed by distillation under reduced pressure, dissolved in 50 ml of ethyl acetate, washed with saturated aqueous sodium bicarbonate solution, and the organic solvent was distilled under reduced pressure to obtain 2.91 g (12.5 mmol, 89% yield) of the title compound. The obtained compound was used directly in the subsequent reaction without purification.
제조예 3: 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸의 합성Preparation Example 3 Synthesis of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -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 250㎖에 녹인 후, 벤즈알데히드 21.2g(0.2 몰)을 가하였다. 반응물을 딘스탁하에서 3시간 동안 환류시킨 다음, 0℃로 냉각시키고 벤질클로로포르메이트 34.2g(0.2 몰)을 교반하면서 적가하였다. 3시간 동안 반응물을 교반한 후 상온에서 1N KHSO4수용액 220㎖를 가하였다. 반응물을 디에틸에테르 200㎖로 3회 추출한 후 수용액층을 포화 수산화나트륨 수용액으로 염기화하였다. 수용액을 포화 염화나트륨 수용액으로 처리하고 디클로로메탄 100㎖로 3회 추출하였다. 디클로로메탄 용액을 무수 마그네슘설페이트로 건조시킨 다음 감압증류하여 표제화합물 38g(0.15몰, 수율 91%)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 Deanstock for 3 hours, then cooled to 0 ° C. and 34.2 g (0.2 mol) of benzylchloroformate was added dropwise with stirring. After stirring the reaction for 3 hours, 220 ml of 1N KHSO 4 aqueous solution was added at room temperature. The reaction was extracted three times with 200 ml of diethyl ether and the aqueous layer was basified with saturated aqueous sodium hydroxide solution. The aqueous solution was treated with saturated aqueous sodium chloride solution and extracted three times with 100 mL of dichloromethane. The dichloromethane solution was dried over anhydrous magnesium sulfate and distilled under reduced pressure to obtain 38 g (0.15 mol, 91% yield) of the title compound.
1H NMR(CDCl3) δ(ppm): 1.11(s, 2H), 1.49(s, 3H), 1.70(d, 2H), 2.57(d, 2H), 2.78(s, 2H), 4.20(s, 2H), 5.12(s, 2H), 7.34-7.35(m, 5H) 1 H NMR (CDCl 3 ) δ (ppm): 1.11 (s, 2H), 1.49 (s, 3H), 1.70 (d, 2H), 2.57 (d, 2H), 2.78 (s, 2H), 4.20 (s , 2H), 5.12 (s, 2H), 7.34-7.35 (m, 5H)
FAB Mass 249 (M+H)FAB Mass 249 (M + H)
3-2) 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-하이드록시메틸-2-머캅토-1H-이미다졸의 제조3-2) Preparation of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-hydroxymethyl-2-mercapto-1H-imidazole
상기 3-1)에서 수득한 화합물 24.8g(0.1 몰)을 아세트산 6.0g(0.1 몰)과 함께 n-부탄올 50㎖에 녹인 후, 이를 포타슘티오시아네이트 12.6g(0.13 몰), 1.3-디하이드록시아세톤 다이머 15.2g(0.1 몰) 및 아세트산 10.0g(0.17 몰)이 n-부탄올 50㎖에 녹아있는 용액에 가하고 48시간 동안 교반하였다. 감압증류하여 용매를 제거한 후 에틸아세테이트 200㎖를 첨가하고 에틸아세테이트 층을 물 100㎖로 3회 세척해주었다. 유기층을 무수 마그네슘설페이트로 건조시킨 후 감압증류로 용매를 제거하여 표제화합물 27g(75 밀리몰, 수율 75%)을 수득하였다.24.8 g (0.1 mol) of the compound obtained in 3-1) was dissolved in 50 ml of n-butanol together with 6.0 g (0.1 mol) of acetic acid, which was then dissolved in 12.6 g (0.13 mol) of potassium thiocyanate and 1.3-dihydride. 15.2 g (0.1 mol) of roxiacetone 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 for 48 hours. After distillation under reduced pressure to remove the solvent, ethyl acetate 200ml was added, and the ethyl acetate layer was washed three times with 100ml of water. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was removed by distillation under reduced pressure to obtain 27 g (75 mmol, 75% yield) of the title compound.
1H NMR(CDCl3) δ(ppm): 1.22(d, 2H), 1.57(d, 2H), 2.30(s, 1H), 2.72 (s, 2H), 3.96(s, 2H), 4.15(d, 2H), 4.46(s, 2H), 5.10(s, 2H), 6.62(s, 1H), 7.26-7.37(m, 5H) 1 H NMR (CDCl 3 ) δ (ppm): 1.22 (d, 2H), 1.57 (d, 2H), 2.30 (s, 1H), 2.72 (s, 2H), 3.96 (s, 2H), 4.15 (d , 2H), 4.46 (s, 2H), 5.10 (s, 2H), 6.62 (s, 1H), 7.26-7.37 (m, 5H)
FAB Mass 362 (M+H)FAB Mass 362 (M + H)
3-3) 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-하이드록시메틸-1H-이미다졸의 제조3-3) Preparation of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-hydroxymethyl-1H-imidazole
상기 3-2)에서 수득한 화합물 18.05g(50 밀리몰)을 10% 질산 수용액 100㎖ 및 에틸아세테이트 10㎖의 혼합용액에 0℃에서 가한 후, 상온에서 3시간 동안 교반하였다. 반응물을 4N 수산화나트륨 수용액으로 염기화한 후, 에틸아세테이트 100 ㎖로 2회 추출하였다. 추출된 유기용액을 무수 마그네슘설페이트로 건조시킨 후 감압증류하여 표제화합물 12.3g(38 밀리몰, 수율 75%)을 수득하였다.18.05 g (50 mmol) of the compound obtained in 3-2) was added to a mixed solution of 100 ml of 10% nitric acid solution and 10 ml of ethyl acetate at 0 ° C., followed by stirring 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 anhydrous magnesium sulfate and distilled under reduced pressure to obtain 12.3 g (38 mmol, yield 75%) of the title compound.
1H NMR(CDCl3) δ(ppm): 1.16(d, 2H), 1.56(d, 2H), 1.98(s, 1H), 2.70(s, 2H), 3.88(d, 2H), 4.18(s, 2H), 4.49(s, 1H), 4.56(s, 3H), 5.10(s, 2H), 6.82 (s, 1H), 7.27-7.40(m, 5H) 1 H NMR (CDCl 3 ) δ (ppm): 1.16 (d, 2H), 1.56 (d, 2H), 1.98 (s, 1H), 2.70 (s, 2H), 3.88 (d, 2H), 4.18 (s , 2H), 4.49 (s, 1H), 4.56 (s, 3H), 5.10 (s, 2H), 6.82 (s, 1H), 7.27-7.40 (m, 5H)
FAB Mass 330 (M+H)FAB Mass 330 (M + H)
3-4) 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸의 제조3-4) Preparation of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole
상기 3-3)에서 수득한 화합물 9.9g(30 밀리몰)을 클로로포름 50㎖에 녹인 후 티오닐클로라이드 7.1g(60 밀리몰)을 0℃에서 천천히 적가하고 상온에서 2시간 동안 교반하였다. 감압증류하여 용매를 제거한 후, 잔존 하이드로클로라이드를 진공하에서 제거하여 표제화합물의 하이드로클로라이드염 9.9g(28.5 밀리몰, 수율 95%)을 수득하였다. 수득된 화합물은 정제없이 후속 반응에 사용하였다.9.9 g (30 mmol) of the compound obtained in 3-3) was dissolved in 50 mL of chloroform, and then 7.1 g (60 mmol) of thionyl chloride was slowly added dropwise at 0 ° C., and stirred at room temperature for 2 hours. After distillation under reduced pressure to remove the solvent, the remaining hydrochloride was removed in vacuo to give 9.9 g (28.5 mmol, 95% yield) of the hydrochloride salt of the title compound. The obtained compound was used for the subsequent reaction without purification.
1H NMR(CDCl3) δ(ppm): 1.12(d, 2H), 1.53(d, 2H), 2.65(s, 2H), 3.82 (d, 2H), 4.22(s, 2H), 4.42(s, 1H), 4.49(s, 3H), 5.12(s, 2H), 6.60(s, 1H), 7.30-7.41(m, 5H) 1 H NMR (CDCl 3 ) δ (ppm): 1.12 (d, 2H), 1.53 (d, 2H), 2.65 (s, 2H), 3.82 (d, 2H), 4.22 (s, 2H), 4.42 (s , 1H), 4.49 (s, 3H), 5.12 (s, 2H), 6.60 (s, 1H), 7.30-7.41 (m, 5H)
FAB Mass 349 (M+H)FAB Mass 349 (M + H)
실시예 1: 1-[1-(1-벤질옥시카보닐-피페리딘-4-일메틸)-1H-이미다졸-5-일메틸]-4-(나프탈렌-1-일)-피리딘-2-온(11)의 합성Example 1: 1- [1- (1-Benzyloxycarbonyl-piperidin-4-ylmethyl) -1H-imidazol-5-ylmethyl] -4- (naphthalen-1-yl) -pyridine- Synthesis of 2-one (11)
1-1) 3-(나프탈렌-1-일)-아크릴산 에틸에스테르의 제조1-1) Preparation of 3- (naphthalen-1-yl) -acrylic acid ethyl ester
트리에틸포스포노아세테이트 22.4g(0.10 몰)을 테트라하이드로푸란 500㎖에 녹인 후, 여기에 포타슘 t-부톡사이드 12.4g(1.1 몰)을 서서히 첨가하였다. 생성된 용액에 1-나프탈데히드 15.6g(0.10 몰)을 테트라하이드로푸란 20㎖에 녹여 서서히 가하고 8시간 동안 교반하였다. 감압증류하여 유기용매를 제거한 후 에틸아세테이트에 녹이고 물로 2회 세척해주었다. 무수 마그네슘설페이트로 건조시키고 농축시킨 후 실리카겔 칼럼 크로마토그라피(용리제: 헥산/에틸아세테이트=95/5, v/v)를 수행하여 표제화합물 20.3g(0.090 몰, 수율 90%)을 수득하였다.22.4 g (0.10 mol) of triethylphosphonoacetate was dissolved in 500 ml of tetrahydrofuran, and then 12.4 g (1.1 mol) of potassium t-butoxide was slowly added thereto. 15.6 g (0.10 mol) of 1-naphthalaldehyde was dissolved in 20 ml of tetrahydrofuran and slowly added to the resulting solution, followed by stirring for 8 hours. After distillation under reduced pressure to remove the organic solvent, it was dissolved in ethyl acetate and washed twice with water. After drying over anhydrous magnesium sulfate and concentration, silica gel column chromatography (eluent: hexane / ethyl acetate = 95/5, v / v) was carried out to give 20.3 g (0.090 mol, 90% yield) of the title compound.
1H NMR(CDCl3) δ(ppm): 1.33(t, 3H), 4.10(q, 2H), 6.75(q, 1H), 7.50(m, 3H), 7.73(d, 1H), 7.85(m, 2H), 8.10(d, 1H), 8.21(d, 1H) 1 H NMR (CDCl 3 ) δ (ppm): 1.33 (t, 3H), 4.10 (q, 2H), 6.75 (q, 1H), 7.50 (m, 3H), 7.73 (d, 1H), 7.85 (m , 2H), 8.10 (d, 1H), 8.21 (d, 1H)
FAB Mass 227 (M+H)FAB Mass 227 (M + H)
1-2) 3-(나프탈렌-1-일)-프로프-2-엔-1-올의 제조1-2) Preparation of 3- (naphthalen-1-yl) -prop-2-en-1-ol
상기 1-1)에서 수득한 화합물 2.26g(10 밀리몰)을 디클로로메탄 20㎖에 녹인 후 -78℃에서 1N 디이소부틸알루미늄하이드리드 톨루엔 용액 12㎖를 천천히 적가하고 -78℃에서 2시간동안 교반하였다. 반응액에 6N 염산 수용액 5㎖와 포화 암모늄클로라이드 수용액 3㎖를 가하여 반응을 종결시켰다. 디클로로메탄 용액을 포화 염화나트륨 수용액 5㎖로 2회 세척하고 무수 마그네슘설페이트로 건조시킨 후 감압증류로 용매를 제거하여 표제화합물 1.47g(8 밀리몰, 수율 80%)을 수득하였다.2.26 g (10 mmol) of the compound obtained in 1-1) was dissolved in 20 ml of dichloromethane, and 12 ml of 1N diisobutylaluminum hydride toluene solution was slowly added dropwise at -78 ° C, and stirred at -78 ° C for 2 hours. It was. 5 ml of 6N aqueous hydrochloric acid solution and 3 ml of saturated ammonium chloride aqueous solution were added to the reaction solution to terminate the reaction. The dichloromethane solution was washed twice with 5 ml of saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was removed by distillation under reduced pressure to obtain 1.47 g (8 mmol, 80% yield) of the title compound.
1H NMR(CDCl3) δ(ppm): 3.14(d, 2H), 6.83(m, 1H), 7.51(m, 3H), 7.74(d, 1H), 7.91(m, 2H), 8.11(d, 1H), 8.31(d, 1H) 1 H NMR (CDCl 3 ) δ (ppm): 3.14 (d, 2H), 6.83 (m, 1H), 7.51 (m, 3H), 7.74 (d, 1H), 7.91 (m, 2H), 8.11 (d , 1H), 8.31 (d, 1H)
FAB Mass 187 (M+H)FAB Mass 187 (M + H)
1-3) 3-(나프탈렌-1-일)-프로프-2-에날의 제조1-3) Preparation of 3- (naphthalen-1-yl) -prop-2-enal
상기 1-2)에서 수득한 화합물 920mg(5 밀리몰)을 디클로로메탄 10㎖에 녹인 후, 피리디늄클로로크로메이트 1.72g(8.5 밀리몰)을 가하고 16시간동안 교반하였다. 반응혼합물을 여과하여 얻은 여액을 농축시킨 후, 실리카겔 칼럼 크로마토그래피(용리제: 에틸아세테이트/헥산=1/3, v/v)를 수행하여 표제화합물 773mg(4.25 밀리몰, 수율 85%)을 수득하였다.920 mg (5 mmol) of the compound obtained in the above 1-2) was dissolved in 10 ml of dichloromethane, and then 1.72 g (8.5 mmol) of pyridinium chlorochromate was added and stirred for 16 hours. The reaction mixture was filtered and the filtrate was concentrated, and then silica gel column chromatography (eluent: ethyl acetate / hexane = 1/3, v / v) was carried out to give 773 mg (4.25 mmol, yield 85%) of the title compound. .
1H NMR(CDCl3) δ(ppm): 6.75(m, 1H), 7.54(m, 3H), 7.70(d, 1H), 7.82(m, 2H), 8.09(d, 1H), 8.20(d, 1H), 9.77(d, 1H) 1 H NMR (CDCl 3 ) δ (ppm): 6.75 (m, 1H), 7.54 (m, 3H), 7.70 (d, 1H), 7.82 (m, 2H), 8.09 (d, 1H), 8.20 (d , 1H), 9.77 (d, 1H)
FAB Mass 183 (M+H)FAB Mass 183 (M + H)
1-4) 4-(나프탈렌-1-일)-피리딘-2-온의 제조1-4) Preparation of 4- (naphthalen-1-yl) -pyridin-2-one
상기 1-3)에서 수득한 화합물 728mg(4 밀리몰)을 n-부탄올 35㎖에 녹인 후, N-(카바모일메틸)피리디늄클로라이드 2.61g(12 밀리몰)을 첨가하였다. 반응액에 피페리딘 680mg 및 아세트산 480mg을 가하고 18시간동안 교반하였다. 감압증류로 용매를 제거하고, 에틸아세테이트 60㎖를 가한 다음 물 20㎖로 2회 세척하고, 무수 마그네슘설페이트로 건조시키고 감압증류하여 표제화합물 265mg(1.2 밀리몰, 수율 30%)을 수득하였다.728 mg (4 mmol) of the compound obtained in the above 1-3) was dissolved in 35 ml of n-butanol, and 2.61 g (12 mmol) of N- (carbamoylmethyl) pyridinium chloride was added thereto. 680 mg of piperidine and 480 mg of acetic acid were added to the reaction solution, and the mixture was stirred for 18 hours. The solvent was removed by distillation under reduced pressure, 60 ml of ethyl acetate was added, followed by washing twice with 20 ml of water, dried over anhydrous magnesium sulfate, and distilled under reduced pressure to obtain 265 mg (1.2 mmol, 30% yield) of the title compound.
1H NMR(CDCl3) δ(ppm): 6.60(s, 1H), 6.80(s, 1H), 7.52(m, 5H), 7.92(m, 3H) 1 H NMR (CDCl 3 ) δ (ppm): 6.60 (s, 1H), 6.80 (s, 1H), 7.52 (m, 5H), 7.92 (m, 3H)
FAB Mass 222 (M+H)FAB Mass 222 (M + H)
1-5) 1-[1-(1-벤질옥시카보닐-피페리딘-4-일메틸)-1H-이미다졸-5-일메틸]-4- (나프탈렌-1-일)-피리딘-2-온의 제조1-5) 1- [1- (1-Benzyloxycarbonyl-piperidin-4-ylmethyl) -1H-imidazol-5-ylmethyl] -4- (naphthalen-1-yl) -pyridine- Preparation of 2-one
상기 1-4)에서 수득한 화합물 100mg(0.45 밀리몰)을 디메틸포름아미드 10㎖에 녹인 후 수소화나트륨 180mg(4.5 밀리몰)을 가하고 10분간 교반하였다. 반응액에 제조예 3-4)에서 수득한 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 208mg(0.54 밀리몰)을 가하고 1시간 동안 교반하였다. 감압증류로 디메틸포름아미드를 제거한 후, 에틸아세테이트 20㎖를 가하고 물(20㎖x3)과 포화 염화나트륨 수용액(10㎖)으로 세척해주었다. 용매를 감압증류로 제거한 후 실리카겔 칼럼 크로마토그래피(용리제: 메탄올/디클로로메탄=7/93, v/v)를 수행하여 표제화합물 0.22mg(0.41 밀리몰, 수율 90%)을 수득하였다.100 mg (0.45 mmol) of the compound obtained in 1-4) was dissolved in 10 ml of dimethylformamide, followed by 180 mg (4.5 mmol) of sodium hydride, followed by stirring for 10 minutes. 208 mg (0.54 mmol) of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole obtained in Preparation Example 3-4) were added to the reaction solution, Stir for hours. After removing dimethylformamide by distillation under reduced pressure, 20 ml of ethyl acetate was added and washed with water (20 ml × 3) and saturated aqueous sodium chloride solution (10 ml). After distilling off the solvent under reduced pressure, silica gel column chromatography (eluent: methanol / dichloromethane = 7/93, v / v) was performed to obtain 0.22 mg (0.41 mmol, 90% yield) of the title compound.
1H NMR(CDCl3) δ(ppm): 1.25(1H, m), 1.70(1H, m), 2.68(1H, s), 4.06(2H, d), 4.21(1H, s), 5.09(2H, s), 5.29(2H, s), 6.40(1H, q), 6.73(1H, m), 7.25- 7.54(12H, m), 7.89(3H, m), 8.01(1H, s) 1 H NMR (CDCl 3 ) δ (ppm): 1.25 (1H, m), 1.70 (1H, m), 2.68 (1H, s), 4.06 (2H, d), 4.21 (1H, s), 5.09 (2H , s), 5.29 (2H, s), 6.40 (1H, q), 6.73 (1H, m), 7.25- 7.54 (12H, m), 7.89 (3H, m), 8.01 (1H, s)
FAB Mass 532 (M+H)FAB Mass 532 (M + H)
실시예 2: 1-(1H-이미다졸-5-일메틸)-4-페닐-피리딘-2-온(1)의 합성Example 2: Synthesis of 1- (1H-imidazol-5-ylmethyl) -4-phenyl-pyridin-2-one (1)
실시예 1의 단계 1-1)에서 1-나프탈데히드 대신에 벤즈알데히드를 사용하고, 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 4-클로로메틸-1-트리페닐메틸-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행한 다음, 트리페닐메틸기를 트리플루오로아세트산으로 제거하여 표제화합물을 수득하였다(단계 5에서의 수율 85%).Benzaldehyde instead of 1-naphthalaldehyde in step 1-1) of Example 1, and 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5 in step 1-5) The same procedure as in Example 1 was carried out except that 4-chloromethyl-1-triphenylmethyl-1H-imidazole was used instead of -chloromethyl-1H-imidazole, and then a triphenylmethyl group Removal with roacetic acid afforded the title compound (yield 85% in step 5).
1H NMR (CDCl3) δ(ppm): 5.16(2H, s), 6.50(1H, s), 6.81(1H, d), 7.03(1H, s), 7.45(1H, d), 7.56-7.85(6H, m) 1 H NMR (CDCl 3 ) δ (ppm): 5.16 (2H, s), 6.50 (1H, s), 6.81 (1H, d), 7.03 (1H, s), 7.45 (1H, d), 7.56-7.85 (6H, m)
FAB Mass (M+H): 252FAB Mass (M + H): 252
실시예 3: 1-(1-메틸-1H-이미다졸-5-일메틸)-4-페닐-피리딘-2-온(2)의 합성Example 3: Synthesis of 1- (1-methyl-1H-imidazol-5-ylmethyl) -4-phenyl-pyridin-2-one (2)
실시예 1의 단계 1-1)에서 1-나프탈데히드 대신에 벤즈알데히드를 사용하고, 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 5-클로로메틸-1-메틸-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 80%).Benzaldehyde instead of 1-naphthalaldehyde in step 1-1) of Example 1, and 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5 in step 1-5) The title compound was obtained in the same manner as in Example 1 except that 5-chloromethyl-1-methyl-1H-imidazole was used instead of -chloromethyl-1H-imidazole. Yield 80%).
1H NMR (CDCl3) δ(ppm): 3.57(3H, s), 5.16(2H, s), 6.50(1H, s), 6.81(1H, d), 7.03(1H, s), 7.45(1H, d), 7.56-7.85(6H, m) 1 H NMR (CDCl 3 ) δ (ppm): 3.57 (3H, s), 5.16 (2H, s), 6.50 (1H, s), 6.81 (1H, d), 7.03 (1H, s), 7.45 (1H , d), 7.56-7.85 (6H, m)
FAB Mass (M+H): 266FAB Mass (M + H): 266
실시예 4: 1-[1-(4-시아노벤질)-1H-이미다졸-5-일메틸]-4-페닐-피리딘-2-온(3)의 합성Example 4: Synthesis of 1- [1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl] -4-phenyl-pyridin-2-one (3)
실시예 1의 단계 1-1)에서 1-나프탈데히드 대신에 벤즈알데히드를 사용하고, 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 5-클로로메틸-1-(4-시아노벤질)-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 88%).Benzaldehyde instead of 1-naphthalaldehyde in step 1-1) of Example 1, and 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5 in step 1-5) The title compound is obtained in the same manner as in Example 1, except that 5-chloromethyl-1- (4-cyanobenzyl) -1H-imidazole is used instead of -chloromethyl-1H-imidazole. (88% yield in step 5).
1H NMR (CDCl3) δ(ppm): 5.16(2H, s), 5.56(2H, s), 6.36(1H, d), 6.54(1H, s), 7.12(2H, d), 7.27(1H, s), 7.30(1H, d), 7.40-7.62(7H, m), 8.28(1H, s) 1 H NMR (CDCl 3 ) δ (ppm): 5.16 (2H, s), 5.56 (2H, s), 6.36 (1H, d), 6.54 (1H, s), 7.12 (2H, d), 7.27 (1H , s), 7.30 (1H, d), 7.40-7.62 (7H, m), 8.28 (1H, s)
FAB Mass (M+H) 367FAB Mass (M + H) 367
실시예 5: 1-[1-(4-브로모벤질)-1H-이미다졸-5-일메틸]-4-페닐-피리딘-2-온(4)의 합성Example 5: Synthesis of 1- [1- (4-bromobenzyl) -1H-imidazol-5-ylmethyl] -4-phenyl-pyridin-2-one (4)
실시예 1의 단계 1-1)에서 1-나프탈데히드 대신에 벤즈알데히드를 사용하고, 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 1-(4-브로모벤질)-5-클로로메틸-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 90%).Benzaldehyde instead of 1-naphthalaldehyde in step 1-1) of Example 1, and 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5 in step 1-5) The title compound was obtained in the same manner as in Example 1, except that 1- (4-bromobenzyl) -5-chloromethyl-1H-imidazole was used instead of -chloromethyl-1H-imidazole. (90% yield in step 5).
1H NMR (CDCl3) δ(ppm): 5.22(2H, s), 5.36(2H, s), 6.30(1H, d), 6.56(1H, s), 6.92(2H, d), 7.26(1H, s), 7.32(1H, d), 7.39-7.64(8H, m) 1 H NMR (CDCl 3 ) δ (ppm): 5.22 (2H, s), 5.36 (2H, s), 6.30 (1H, d), 6.56 (1H, s), 6.92 (2H, d), 7.26 (1H , s), 7.32 (1 H, d), 7.39-7.64 (8 H, m)
FAB Mass (M+H) 420FAB Mass (M + H) 420
실시예 6: 1-[1-(1-벤질옥시카보닐-피페리딘-4-일메틸)-1H-이미다졸-5-일메틸]-4-페닐-피리딘-2-온(5)의 합성Example 6: 1- [1- (1-Benzyloxycarbonyl-piperidin-4-ylmethyl) -1H-imidazol-5-ylmethyl] -4-phenyl-pyridin-2-one (5) Synthesis of
실시예 1의 단계 1-1)에서 1-나프탈데히드 대신에 벤즈알데히드를 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 79%).The title compound was obtained in the same manner as in Example 1 except that benzaldehyde was used instead of 1-naphthalaldehyde in Step 1-1) of Example 1 (yield 79% in step 5).
1H NMR (CDCl3) δ(ppm): 1.27(2H, m), 1.72(2H, m), 2.65(1H, s), 4.10(2H, d), 4.22(1H, s), 5.11(2H, s), 5.31(2H, s), 6.35(1H, q), 6.70(1H, m), 7.20- 7.51(12H, m), 7.84(3H, m), 8.00(1H, s) 1 H NMR (CDCl 3 ) δ (ppm): 1.27 (2H, m), 1.72 (2H, m), 2.65 (1H, s), 4.10 (2H, d), 4.22 (1H, s), 5.11 (2H , s), 5.31 (2H, s), 6.35 (1H, q), 6.70 (1H, m), 7.20- 7.51 (12H, m), 7.84 (3H, m), 8.00 (1H, s)
FAB Mass (M+H) 483FAB Mass (M + H) 483
실시예 7: 1-(1-메틸-1H-이미다졸-5-일메틸)-4-(나프탈렌-1-일)-피리딘-2-온(6)의 합성Example 7: Synthesis of 1- (1-methyl-1H-imidazol-5-ylmethyl) -4- (naphthalen-1-yl) -pyridin-2-one (6)
실시예 1의 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 5-클로로메틸-1-메틸-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 70%).5-chloromethyl-1-methyl instead of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole in step 1-5) of Example 1 The title compound was obtained in the same manner as in Example 1 except for using -1H-imidazole (yield 70% in step 5).
1H NMR (CDCl3) δ(ppm): 3.57(3H, s), 5.16(2H, s), 6.50(1H, s), 6.62(1H, d), 6.80(1H, s), 7.03(1H, s), 7.45(1H, d), 7.48-7.60(4H, m), 7.83-8.02(3H, m) 1 H NMR (CDCl 3 ) δ (ppm): 3.57 (3H, s), 5.16 (2H, s), 6.50 (1H, s), 6.62 (1H, d), 6.80 (1H, s), 7.03 (1H , s), 7.45 (1H, d), 7.48-7.60 (4H, m), 7.83-8.02 (3H, m)
FAB Mass (M+H) 316FAB Mass (M + H) 316
실시예 8: 1-(1-벤질-1H-이미다졸-5-일메틸)-4-(나프탈렌-1-일)-피리딘-2-온(7)의 합성Example 8: Synthesis of 1- (1-benzyl-1H-imidazol-5-ylmethyl) -4- (naphthalen-1-yl) -pyridin-2-one (7)
실시예 1의 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 1-벤질-5-클로로메틸-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 92%).1-benzyl-5-chloromethyl instead of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole in Example 1 step 1-5) The title compound was obtained in the same manner as in Example 1 except for using −1H-imidazole (yield 92% in step 5).
1H NMR (CDCl3) δ(ppm): 5.18(2H, s), 5. 29(2H, s), 6.30(1H, d), 6.50 (1H, s), 6.92(2H, d), 7.03-7.60(9H, m), 7.83-8.02(4H, m) 1 H NMR (CDCl 3 ) δ (ppm): 5.18 (2H, s), 5. 29 (2H, s), 6.30 (1H, d), 6.50 (1H, s), 6.92 (2H, d), 7.03 -7.60 (9H, m), 7.83-8.02 (4H, m)
FAB Mass (M+H) 392FAB Mass (M + H) 392
실시예 9: 1-[1-(4-시아노벤질)-1H-이미다졸-5-일메틸]-4-(나프탈렌-1-일)-피리딘-2-온(8)의 합성Example 9: Synthesis of 1- [1- (4-cyanobenzyl) -1H-imidazol-5-ylmethyl] -4- (naphthalen-1-yl) -pyridin-2-one (8)
실시예 1의 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 5-클로로메틸-1-(4-시아노벤질)-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 67%).In Example 1 step 1-5) 5-chloromethyl-1- (instead of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole The title compound was obtained in the same manner as in Example 1 except that 4-cyanobenzyl) -1H-imidazole was used (yield 67% in step 5).
1H NMR (CDCl3) δ(ppm): 5.20(2H, s), 5.49(2H, s), 6.29(1H, d), 6.50 (1H, s), 7.08(2H, d), 7.20-7.40(4H, m), 7.45-7.60(4H, m), 7.65(1H, s), 7.77(1H, d), 7.90(2H, d) 1 H NMR (CDCl 3 ) δ (ppm): 5.20 (2H, s), 5.49 (2H, s), 6.29 (1H, d), 6.50 (1H, s), 7.08 (2H, d), 7.20-7.40 (4H, m), 7.45-7.60 (4H, m), 7.65 (1H, s), 7.77 (1H, d), 7.90 (2H, d)
FAB Mass (M+H) 417FAB Mass (M + H) 417
실시예 10: 1-[1-(4-브로모벤질)-1H-이미다졸-5-일메틸]-4-(나프탈렌-1-일)-피리딘-2-온(9)의 합성Example 10 Synthesis of 1- [1- (4-Bromobenzyl) -1H-imidazol-5-ylmethyl] -4- (naphthalen-1-yl) -pyridin-2-one (9)
실시예 1의 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 1-(4-브로모벤질)-5-클로로메틸-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 90%).Example 1- (1-bromobenzyl instead of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole in Example 1-5) The title compound was obtained in the same manner as in Example 1 except for using) -5-chloromethyl-1H-imidazole (yield 90% in step 5).
1H NMR (CDCl3) δ(ppm): 5.22(2H, s), 5.36(2H, s), 6.30(1H, d), 6.56(1H, s), 6.92(2H, d), 7.22-7.60(8H, m), 7.81-8.08(4H, m) 1 H NMR (CDCl 3 ) δ (ppm): 5.22 (2H, s), 5.36 (2H, s), 6.30 (1H, d), 6.56 (1H, s), 6.92 (2H, d), 7.22-7.60 (8H, m), 7.81-8.08 (4H, m)
FAB Mass (M+H) 470FAB Mass (M + H) 470
실시예 11: 1-[1-(4-메톡시벤질)-1H-이미다졸-5-일메틸]-4-(나프탈렌-1-일)-피리딘-2-온(10)의 합성Example 11: Synthesis of 1- [1- (4-methoxybenzyl) -1H-imidazol-5-ylmethyl] -4- (naphthalen-1-yl) -pyridin-2-one (10)
실시예 1의 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 5-클로로메틸-1-(4-메톡시벤질)-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 87%).In Example 1 step 1-5) 5-chloromethyl-1- (instead of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole The title compound was obtained in the same manner as in Example 1 except that 4-methoxybenzyl) -1H-imidazole was used (yield 87% in step 5).
1H NMR (CDCl3) δ(ppm): 3.79(3H, s), 5.22(2H, s), 5.36(2H, s), 6.30(1H, d), 6.56(1H, s), 6.92(2H, d), 7.22-7.60(8H, m), 7.81-8.08(4H, m) 1 H NMR (CDCl 3 ) δ (ppm): 3.79 (3H, s), 5.22 (2H, s), 5.36 (2H, s), 6.30 (1H, d), 6.56 (1H, s), 6.92 (2H , d), 7.22-7.60 (8H, m), 7.81-8.08 (4H, m)
FAB Mass (M+H) 422FAB Mass (M + H) 422
실시예 12: 4-(나프탈렌-1-일)-1-{1-[1-(3-페닐프로피오닐)-피페리딘-4-일메틸]-1H-이미다졸-5-일메틸}-피리딘-2-온(12)의 합성Example 12: 4- (naphthalen-1-yl) -1- {1- [1- (3-phenylpropionyl) -piperidin-4-ylmethyl] -1H-imidazol-5-ylmethyl} Synthesis of Pyridin-2-one (12)
실시예 1의 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 5-클로로메틸-1-[1-(3-페닐프로피오닐)피페리딘-4-일메틸]-1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 86%).5-chloromethyl-1- [instead of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole in step 1-5) of Example 1 The title compound was obtained in the same manner as in Example 1 except for using 1- (3-phenylpropionyl) piperidin-4-ylmethyl] -1H-imidazole (in Step 5). Yield 86%).
1H NMR(CDCl3) δ(ppm): 1.25(1H, m), 1.70(1H, m), 2.65(2H, t), 2.68(1H, s), 2.97(2H, t), 4.06(2H, d), 4.21(1H, s), 5.29(2H, s), 6.40(1H, q), 6.73 (1H, m), 7.25-7.54(12H, m), 7.89(3H, m), 8.01(1H, s) 1 H NMR (CDCl 3 ) δ (ppm): 1.25 (1H, m), 1.70 (1H, m), 2.65 (2H, t), 2.68 (1H, s), 2.97 (2H, t), 4.06 (2H , d), 4.21 (1H, s), 5.29 (2H, s), 6.40 (1H, q), 6.73 (1H, m), 7.25-7.54 (12H, m), 7.89 (3H, m), 8.01 ( 1H, s)
FAB Mass (M+H) 531FAB Mass (M + H) 531
실시예 13: 1-[1-(1-벤질카바모일-피페리딘-4-일메틸)-1H-이미다졸-5-일메틸]-4-(나프탈렌-1-일)-피리딘-2-온(13)의 합성Example 13: 1- [1- (1-Benzylcarbamoyl-piperidin-4-ylmethyl) -1H-imidazol-5-ylmethyl] -4- (naphthalen-1-yl) -pyridine-2 Synthesis of On- (13)
실시예 1의 단계 1-5)에서 1-[1-(벤질옥시카보닐)피페리딘-4-일메틸]-5-클로로메틸-1H-이미다졸 대신에 5-클로로메틸-1-[1-(벤질카바모일)피페리딘-4-일메틸]- 1H-이미다졸을 사용하는 점을 제외하고는 실시예 1에서와 동일하게 수행하여 표제화합물을 수득하였다(단계 5에서의 수율 30%).5-chloromethyl-1- [instead of 1- [1- (benzyloxycarbonyl) piperidin-4-ylmethyl] -5-chloromethyl-1H-imidazole in step 1-5) of Example 1 The title compound was obtained in the same manner as in Example 1 except for using 1- (benzylcarbamoyl) piperidin-4-ylmethyl] -1H-imidazole (yield 30 in step 5). %).
1H NMR(CDCl3) δ(ppm): 1.25(1H, m), 1.70(1H, m), 2.68(1H, s), 4.06(2H, d), 4.21(1H, s), 4.28(2H, d), 5.29(2H, s), 6.40(1H, q), 6.73(1H, m), 7.25- 7.54(12H, m), 7.89(3H, m), 8.01(1H, s) 1 H NMR (CDCl 3 ) δ (ppm): 1.25 (1H, m), 1.70 (1H, m), 2.68 (1H, s), 4.06 (2H, d), 4.21 (1H, s), 4.28 (2H , d), 5.29 (2H, s), 6.40 (1H, q), 6.73 (1H, m), 7.25- 7.54 (12H, m), 7.89 (3H, m), 8.01 (1H, s)
FAB Mass (M+H) 532FAB Mass (M + H) 532
본 발명에 따른 화합물의 파네실 전이효소 억제능을 확인하기 위하여 다음과 같은 실험을 실시하였다.In order to confirm panesyl transferase inhibitory ability of the compound according to the present invention was carried out the following experiment.
실험예 1: Ras 파네실 전이효소 억제능 분석Experimental Example 1: Analysis of Ras farnesyl transferase inhibitory activity
본 실험에서는 폼프리아노 등의 방법(참조: Pompliano et al., Biochemistry , 1992, 31, 3800)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., Bichimica et Biophysica Acta, 1992, 278, 1129)에 따라 정제하여 사용하였다.In this experiment, an improved method of Pompriano et al. (Pompliano et al., Biochemistry, 1992, 31, 3800) 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 (Korean Patent Application No. 97-14409) was purified and used according to the previously reported method (Chung et al., Bichimica et Biophysica Acta, 1992, 278, 1129).
효소 반응은 염화칼륨 25mM, 염화마그네슘 25mM, 디티티(DTT) 10mM 및 염화아연 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 1㎖를 첨가하여 반응을 정지시켰다. 생성된 침전물을 필터바인딩을 위한 호퍼 하베스터(호퍼 #FH 225V)를 사용하여 GF/B 필터에 흡착시킨 후, 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: Schaber et al. J. Biol Chem., 1990, 265, 14701)을 변형하여 소뇌로부터 정제하여 사용하였으며, 파네실 전이효소 반응과 유사한 조건에서 제라닐제라닐 전이효소의 특이적 기질인 제라닐 제라닐 피로 포스페이트와 Ras-CVIL 기질 단백질을 사용하여 실험을 수행하였다. 실험결과는 하기 표 2에 나타내었다.Enzyme 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. The reaction was stopped by the addition of farnesyl transferase and the reaction was continued at 37 ° C. for 30 minutes, followed by the addition of 1 ml of ethanol solution containing 1 M hydrochloric acid. 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 the state of substrate unsaturation where the concentration of Ras substrate protein and panesyl enzyme showed a quantitative titer relationship. 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 ability of the test compound was modified by the method of Shaver et al. (Schaber et al. J. Biol Chem., 1990, 265, 14701) and purified from cerebellum. Experiments were performed using geranyl geranyl 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 제97-14409 호)를 사용하였으며, 실험은 드크루 등의 방법(참조: Declue. J. E. et al., Cancer Research, 1991, 51, 712)을 변형시켜 다음과 같이 수행하였다.In this experiment, a rat2 cell line expressing a C-Harvey-Ras protein having a transforming activity by mutation and a Rat2 cell line transformed with an 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, 1991, 51, 712) as follows.
형질전환된 Rat2 섬유아세포(fibroblast) 세포주를 60㎜ 세포배양 디쉬에 디쉬당 3x105세포의 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 1㎖당 150μCi의 방사성동위원소[35S]로 표지된 메티오닌을 첨가하고 20시간동안 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) 1㎖를 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., 1982, 43, 294)를 가하고 4℃에서 15시간 동안 반응시켰다. 이 용액에 다시 고트에서 유래된 쥐의 면역글로블린에 대한 항체가 결합된 Protein A-아가로즈 현탁액을 가하여 1시간 동안 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 완충액에 가하여 끓인 후 13.5%의 SDS 폴리아크릴아마이드 겔을 사용하여 전기영동을 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(CIC50)로 나타내었다. 실험을 수행한 결과 본 발명에 따른 대표적인 화합물들의 IC50(실험예 1)은 10μM 이하이고 CIC50(실험예 2)은 100μM 이하인 것으로 나타났다.The transformed rat2 fibroblast cell line was seeded 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 in a 37 ° C. cell culture medium, 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 and incubated 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 μM Sodium Hippies buffer solution 2 μM) After the addition of the cells to lysate, 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., 1982, 43, 294), 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 the immunoglobulin antibody of rats derived from Goth, and reacted at 4 ° C for 1 hour, and then buffered (sodium chloride) to remove nonspecific binding from the immunoreaction precipitate. 50 mM, sodium dioxycholate 0.5%, NP40 0.5% and Tris chloride 50 mM buffer containing 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 fixed, dried, and then subjected to photo printing by exposing to an X-ray film. The inhibitory effect of intracellular Ras farnesyl transferase was expressed as the concentration of the test compound (CIC 50 ) in which the farnesyl binding was inhibited by measuring the intensity of the non-bound bands of the farnesyl bound band of the Ras protein. As a result of the experiment, the IC 50 (Experimental Example 1) of the representative compounds according to the present invention was found to be 10 μM or less and CIC 50 (Experimental Example 2) was 100 μM or less.
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