KR20220007447A - Compounds as a TRAP1 selective inhibitor, and composition for preventing or treating cancer comprising the same - Google Patents

Compounds as a TRAP1 selective inhibitor, and composition for preventing or treating cancer comprising the same Download PDF

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KR20220007447A
KR20220007447A KR1020200085681A KR20200085681A KR20220007447A KR 20220007447 A KR20220007447 A KR 20220007447A KR 1020200085681 A KR1020200085681 A KR 1020200085681A KR 20200085681 A KR20200085681 A KR 20200085681A KR 20220007447 A KR20220007447 A KR 20220007447A
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강병헌
윤남구
양수재
박은선
강수성
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이화여자대학교 산학협력단
울산과학기술원
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    • C—CHEMISTRY; METALLURGY
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    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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Abstract

Provided are a compound as a selective TRAP1 inhibitor, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition for preventing or treating cancer comprising the same. The novel compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof according to an aspect has TRAP1 selective inhibitory activity, and thus can be used to prevent or treat various types of cancer associated with TRAP1.

Description

TRAP1 선택적 억제제로서의 화합물 및 이를 포함하는 암 예방 또는 치료용 조성물{Compounds as a TRAP1 selective inhibitor, and composition for preventing or treating cancer comprising the same}Compounds as a TRAP1 selective inhibitor, and a composition for preventing or treating cancer comprising the same

TRAP1 선택적 억제제로서의 화합물, 및 이를 포함하는 암 예방 또는 치료용 약학적 조성물에 관한 것이다.It relates to a compound as a selective inhibitor of TRAP1, and to a pharmaceutical composition for preventing or treating cancer comprising the same.

Hsp90 (heat shock protein 90) 패밀리에 속하는 TRAP1 (TNF Receptor-Associated Protein 1)은 다른 단백질이 올바르게 접히는 것을 돕는 미토콘드리아 분자 샤페론이다. TRAP1은 정상 세포에서 거의 검출되지 않지만, 유방암, 전립선암, 췌장암, 폐암 및 결장암과 같은 다양한 암에서 과발현되며, 암 세포가 가혹한 종양 환경에 적응할 수 있도록 세포 대사를 재프로그래밍한다. TRAP1의 불활성화는 in vitro 및 in vivo 암 세포에서 대량의 세포사멸 (apoptosis)을 유도하므로, TRAP1을 표적으로 하는 억제제가 개발되고 있다(Kang, B. H., TRAP1 regulation of mitochondrial life or death decision in cancer cells and mi-tochondria-targeted TRAP1 inhibitors. BMB Rep. 2012, 45, 1-6).TNF Receptor-Associated Protein 1 (TRAP1), a member of the heat shock protein 90 (Hsp90) family, is a mitochondrial molecular chaperone that helps other proteins fold correctly. Although rarely detected in normal cells, TRAP1 is overexpressed in various cancers such as breast, prostate, pancreatic, lung and colon cancers, reprogramming cellular metabolism to allow cancer cells to adapt to the harsh tumor environment. Since inactivation of TRAP1 induces massive apoptosis in cancer cells in vitro and in vivo , inhibitors targeting TRAP1 have been developed (Kang, BH, TRAP1 regulation of mitochondrial life or death decision in cancer cells). and mi-tochondria-targeted TRAP1 inhibitors. BMB Rep. 2012, 45, 1-6).

Hsp90 패밀리는 Hsp90α, Hsp90β, Grp94 및 TRAP1을 포함하는 4 개의 서로 다른 파라로그 (paralog)로 구별된다. Hsp90 패밀리는 세포에서 서로 달리 위치하고 발현되지만, 이들은 모두 호모다이머로서 존재한다. 각 프로토머 (protomer)에는 세 가지 특징적인 도메인이 있다: N-말단 도메인 (NTD), 중간 도메인 (M-도메인) 및 C-말단 도메인 (CTD). 호모다이머 상의 각 프로토머의 CTD는 1차 이량체화를 제공하기 위해 상호 작용한다. M-도메인은 다양한 클라이언트 단백질과 상호 작용할 수 있는 넓은 표면을 갖는다. NTD는 결합된 ATP가 ADP로 가수분해되는 ATPase 촉매 부위를 갖는다. 이 ATP 결합 부위는 약물 결합 부위로도 알려져 있다. ATP의 가수분해는 프로토머 이량체화를 용이하게 하기 위해 전체 단백질 구조의 형태적 재배열을 담당한다. NTD 입체형태 변화의 주요 원인은 ATP-lid의 이동이며, 이는 NTD 이량체화에 필수적인 소수성 표면을 제공하기 위해 ATP 결합 부위에 걸쳐 폐쇄되어야 한다. ATP-lid의 힌지 운동을 위해, 여러 유연한 서열이 ATP-lid의 적절한 위치에 위치되어 있다. 이는 약물-결합 부위가 다양한 억제제 스캐폴드에 대한 다양한 유도-적합에 적응할 수 있게 한다. Hsp90 및 Grp94의 공-결정 구조는 결합 포켓의 구조적 다양성을 나타내며, ATP 또는 ADP에 의해 결합 될 때 Hsp90의 부분 개방/폐쇄 추정 중간 형태를 보여준다. Hsp90 패밀리 단백질에 대한 몇몇 억제제들은 NTD 상의 ATP 결합 부위를 타겟으로 하여 개발되었다(Jhaveria, k. et al., Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2012, 1823, 742-755.). Hsp90 억제제로는 PU-H71 (1), BIIB021 (2), SNX-0723 (3) 등이 공개되어 있다(Immormino, R. M. et al., Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors. J. Med. Chem. 2006, 49, 4953-4960.; Lundgren, K. et al., BIIB021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein Hsp90. Mol. Cancer. Ther. 2009, 8, 921-929.; Ernst, J. T. et al., Identification of Novel HSP90α/β Isoform Selective Inhibitors Using Structure-Based Drug Design. Demonstration of Potential Utility in Treating CNS Disorders such as Huntington's Disease J. Med. Chem. 2014, 57, 3382-3400.).The Hsp90 family is distinguished into four different paralogs including Hsp90α, Hsp90β, Grp94 and TRAP1. Although the Hsp90 family is located and expressed differently in cells, they all exist as homodimers. Each protomer has three characteristic domains: an N-terminal domain (NTD), an intermediate domain (M-domain) and a C-terminal domain (CTD). The CTD of each protomer on the homodimer interacts to provide primary dimerization. The M-domain has a large surface that can interact with various client proteins. NTDs have an ATPase catalytic site where bound ATP is hydrolyzed to ADP. This ATP binding site is also known as the drug binding site. Hydrolysis of ATP is responsible for the conformational rearrangement of the overall protein structure to facilitate protomeric dimerization. A major cause of NTD conformational changes is the migration of ATP-lid, which must be occluded across the ATP binding site to provide a hydrophobic surface essential for NTD dimerization. For the hinge movement of the ATP-lid, several flexible sequences are located in the proper position of the ATP-lid. This allows the drug-binding site to adapt to different induction-fits to different inhibitor scaffolds. The co-crystal structures of Hsp90 and Grp94 reveal a structural diversity of the binding pocket, showing a partially open/closed putative intermediate form of Hsp90 when bound by ATP or ADP. Several inhibitors of Hsp90 family proteins have been developed by targeting the ATP binding site on NTD (Jhaveria, k. et al., Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers. Biochimica et Biophysica Acta ( BBA) - Molecular Cell Research . 2012, 1823 , 742-755.). As Hsp90 inhibitors, PU-H71 ( 1 ), BIIB021 ( 2 ), SNX-0723 ( 3 ), etc. have been published (Immormino, RM et al., Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors) J. Med. Chem . 2006, 49, 4953-4960.; Lundgren, K. et al., BIIB021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein Hsp90. Mol. Cancer. Ther. 2009 , 8, 921-929.; Ernst, JT et al., Identification of Novel HSP90α/β Isoform Selective Inhibitors Using Structure-Based Drug Design. Demonstration of Potential Utility in Treating CNS Disorders such as Huntington's Disease J. Med. Chem . 2014 , 57, 3382-3400.).

Figure pat00001
Figure pat00001

또한, 본 발명자들은 최근 Hsp90 및 TRAP1과 같은 Hsp90 상동단백질을 동시에 저해하는 화합물인 DN401을 개발하였으며, 이는 한국 공개특허공보 제10-2018-0045781호로 공개되어 있다.In addition, the present inventors have recently developed DN401, a compound that simultaneously inhibits Hsp90 and Hsp90 homologous proteins such as TRAP1, which is disclosed as Korean Patent Application Laid-Open No. 10-2018-0045781.

그러나, Hsp90은 저해하지 않으면서 Hsp90의 파라로그인 TRAP1을 선택적으로 저해하는 TRAP1 선택적 억제제의 개발이 필요하다.However, it is necessary to develop a TRAP1-selective inhibitor that selectively inhibits TRAP1, a paralog of Hsp90, without inhibiting Hsp90.

일 양상은 하기 화학식 1로 표시되는 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 제공한다.One aspect provides a compound represented by the following Chemical Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

다른 양상은 상기 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 포함하는, TRAP1의 활성과 연관된 질환의 예방 또는 치료용 약학적 조성물을 제공한다.Another aspect provides a pharmaceutical composition for preventing or treating a disease associated with TRAP1 activity, comprising the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

다른 양상은 상기 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다.Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

다른 양상은 상기 약학적 조성물을 개체에게 투여하는 단계를 포함하는 암을 예방 또는 치료하는 방법을 제공한다.Another aspect provides a method for preventing or treating cancer, comprising administering the pharmaceutical composition to a subject.

다른 양상은 암 예방 또는 치료용 의약의 제조에 사용하기 위한 상기 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염의 용도를 제공한다.Another aspect provides the use of the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the prevention or treatment of cancer.

일 양상은 하기 화학식 1로 표시되는 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 제공한다:One aspect provides a compound represented by the following formula (1), a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:

[화학식 1][Formula 1]

Figure pat00002
Figure pat00002

상기 화학식 1에서,In Formula 1,

X1은 OH, NH2, SH, 또는 O이고;X1 is OH, NH 2 , SH, or O;

X2는 N, NH 또는 CH2이고;X2 is N, NH or CH 2 ;

a 및 b 중 어느 하나는 단일 결합이고, 다른 하나는 이중 결합이고;one of a and b is a single bond and the other is a double bond;

R1, R2, R3, R4 및 R5는 각각 독립적으로, H, 할로, C1-6 알킬, C2-6 알케닐, 시아노, OR7, SR7 또는 NHR7이거나,R1, R2, R3, R4 and R5 are each independently H, halo, C 1-6 alkyl, C 2-6 alkenyl, cyano, OR7, SR7 or NHR7;

R1, R4 및 R5는 각각 독립적으로, H, 할로, C1-6 알킬, C2-6 알케닐, 시아노, OR7, SR7 또는 NHR7이고, R2 및 R3은 함께 N, O 및 S로 이루어진 군으로부터 각각 독립적으로 선택되는 1개 또는 2개의 헤테로원자를 포함하는 4원 내지 7원의 헤테로고리를 형성하고;R1, R4 and R5 are each independently H, halo, C 1-6 alkyl, C 2-6 alkenyl, cyano, OR7, SR7 or NHR7, and R2 and R3 together are N, O and S to form a 4- to 7-membered heterocycle containing 1 or 2 heteroatoms each independently selected from;

R6은 H, 할로 또는 C1-6 알킬이고;R6 is H, halo or C 1-6 alkyl;

상기 R7은 H 또는 C1-6 알킬이고;wherein R7 is H or C 1-6 alkyl;

상기 C1-6 알킬 또는 C2-6 알케닐은 선택적으로 할로 또는 C1-6 알킬로 치환될 수 있다.The C 1-6 alkyl or C 2-6 alkenyl may be optionally substituted with halo or C 1-6 alkyl.

상기 화학식 1로 표시되는 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염은 TRAP1 선택적 억제제로 사용 가능하다. 본 발명자들은 TRAP1 구조를 기초로 일련의 퓨린-8-온 및 피롤로[2,3-d]피리미딘 유도체를 설계하고 합성하였으며, 합성된 화합물들은 in vitro에서 파라로그성(paralogous) 효소 Hsp90 및 Grp94에 비해 TRAP1에 대해 매우 선택적인 것으로 확인되었다. ATP-lid의 Asn171 잔기의 이용을 통한 TRAP1 선택적 억제 전략은 X-선 결정학 및 MD 시뮬레이션 연구에 기초하여 조사되었다. 다양한 합성된 강력한 TRAP1 억제제 중에서, 일 실시예에 따른 화합물 5j는 Hsp90에 비해 65 배 선택성 및 Grp94에 비해 13 배 선택성을 가졌다. 또한, 일 실시예에 따른 화합물 6j의 TRAP1에 대한 IC50은 63.5 nM이었고 Hsp90 및 Grp94에 비해 각각 78 배 및 30 배 선택성을 가졌다. 따라서, 일 양상에 따른 화합물들은 우수한 TRAP1 선택적 억제 활성을 가진다.The compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof can be used as a selective TRAP1 inhibitor. The present inventors designed and synthesized a series of purin-8-one and pyrrolo[2,3- d ]pyrimidine derivatives based on the TRAP1 structure, and the synthesized compounds were in vitro paralogous enzymes Hsp90 and It was found to be highly selective for TRAP1 compared to Grp94. A strategy for selective inhibition of TRAP1 through the use of the Asn171 residue of ATP-lid was investigated based on X-ray crystallography and MD simulation studies. Among the various synthesized potent TRAP1 inhibitors, compound 5j according to one embodiment had 65-fold selectivity over Hsp90 and 13-fold selectivity over Grp94. In addition, the IC 50 for TRAP1 of compound 6j according to an embodiment was 63.5 nM, and had 78-fold and 30-fold selectivity compared to Hsp90 and Grp94, respectively. Accordingly, the compounds according to one aspect have excellent TRAP1 selective inhibitory activity.

용어 “치환”은 유기 화합물 중의 하나 이상의 수소 원자를 다른 원자단으로 치환하여 유도체를 형성한 경우 수소 원자 대신에 도입되는 것을 말하고, 치환기는 도입된 원자단을 말한다.The term “substitution” refers to introduced instead of a hydrogen atom when a derivative is formed by substituting one or more hydrogen atoms in an organic compound with another atomic group, and the substituent refers to an introduced atomic group.

용어 “할로 (halo)”는 “할로겐 (halogen)”과 상호교환적으로 사용된다. 상기 할로는 예를 들어, F, Cl, Br, 또는 I 등이다.The term “halo” is used interchangeably with “halogen”. The halo is, for example, F, Cl, Br, or I and the like.

용어 “알킬 (alkyl)”은 완전 포화된 분지형 또는 비분지형 (또는, 직쇄 또는 선형) 탄화수소 라디칼을 말한다. 상기 알킬은 C1 내지 C10, 또는 C1 내지 C6인 알킬기일 수 있다. 상기 알킬은 예를 들어, 메틸, 에틸, n-프로필, 이소프로필, n-부틸, 이소부틸, 2차 부틸(sec-butyl), n-펜틸, 이소펜틸, 네오펜틸, iso-아밀, n-헥실, 3-메틸헥실, 2,2-디메틸펜틸, 2,3-디메틸펜틸, n-헵틸, n-옥틸, n-노닐, 또는 n-데실이다.The term “alkyl” refers to a fully saturated branched or unbranched (or straight-chain or linear) hydrocarbon radical. The alkyl may be a C 1 to C 10 , or a C 1 to C 6 alkyl group. The alkyl is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, iso-amyl, n- hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.

용어 “알케닐 (alkenyl)”은 적어도 하나 이상의 탄소-탄소 이중결합(C=C)을 갖는 불포화된 탄화수소 라디칼을 말한다. 상기 알케닐은 C2 내지 C10, 또는 C2 내지 C6인 알케닐기일 수 있다. 상기 알케닐은 예를 들어, 비닐, 프로페닐, 부테닐, 펜테닐, 또는 헥세닐이다.The term “alkenyl” refers to an unsaturated hydrocarbon radical having at least one carbon-carbon double bond (C=C). The alkenyl may be a C 2 to C 10 , or C 2 to C 6 alkenyl group. The alkenyl is, for example, vinyl, propenyl, butenyl, pentenyl, or hexenyl.

용어 “헤테로고리”는 N, O 및 S로부터 독립적으로 선택된 1 내지 4개의 헤테로원자를 함유하는 포화 또는 부분적으로 불포화된 4-, 5-, 6-, 및 7-원의 헤테로고리를 말한다. 용어, “헤테로고리”, “헤테로시클릴”, “헤테로시클릴 라디칼”은 상호교환적으로 사용된다. 상기 헤테로고리는 예를 들어, 테트라히드로퓨라닐, 테트라히드로티에닐, 테트라히드로피라닐, 피롤리디닐, 피롤리도닐, 피페리디닐, 옥세타닐, 옥사졸리디닐, 피페라지닐, 디옥사닐, 디옥소라닐, 모르폴리닐, 디히드로퓨라닐, 디히드로피라닐, 디히드로피리디닐, 테트라히드로피리디닐, 디히드로피리미디닐, 또는 테트라히드로피리미디닐이다. 상기 헤테로고리는 모노시클릭 또는 바이시클릭일 수 있다.The term “heterocycle” refers to a saturated or partially unsaturated 4-, 5-, 6-, and 7-membered heterocycle containing 1 to 4 heteroatoms independently selected from N, O and S. The terms “heterocycle”, “heterocyclyl” and “heterocyclyl radical” are used interchangeably. The heterocycle is, for example, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, oxetanyl, oxazolidinyl, piperazinyl, dioxa nyl, dioxolanyl, morpholinyl, dihydrofuranyl, dihydropyranyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl, or tetrahydropyrimidinyl. The heterocycle may be monocyclic or bicyclic.

상기 화학식 1에서, 상기 X1은 OH, NH2 또는 SH이고, 상기 X2는 N이고, 상기 a는 단일 결합이고, 상기 b는 이중 결합일 수 있다. 구체적으로, 상기 X1은 OH이고, 상기 X2는 N이고, 상기 a는 단일 결합이고, 상기 b는 이중 결합이거나; 상기 X1은 NH2이고, 상기 X2는 N이고, 상기 a는 단일 결합이고, 상기 b는 이중 결합이거나; 상기 X1은 SH이고, 상기 X2는 N이고, 상기 a는 단일 결합이고, 상기 b는 이중 결합일 수 있다.In Formula 1, X1 may be OH, NH 2 or SH, X2 may be N, a may be a single bond, and b may be a double bond. Specifically, X1 is OH, X2 is N, a is a single bond, and b is a double bond; wherein X1 is NH 2 , X2 is N, a is a single bond, and b is a double bond; X1 may be SH, X2 may be N, a may be a single bond, and b may be a double bond.

상기 화학식 1에서, 상기 X1은 O이고, 상기 X2는 NH 또는 CH2이고, 상기 a는 이중 결합이고, 상기 b는 단일 결합일 수 있다. 구체적으로, 상기 X1은 O이고, 상기 X2는 NH이고, 상기 a는 이중 결합이고, 상기 b는 단일 결합이거나; 상기 X1은 O이고, 상기 X2는 CH2이고, 상기 a는 이중 결합이고, 상기 b는 단일 결합일 수 있다.In Formula 1, X1 may be O, X2 may be NH or CH 2 , a may be a double bond, and b may be a single bond. Specifically, X1 is O, X2 is NH, a is a double bond, and b is a single bond; Wherein X1 is O, X2 is CH 2 , wherein a is a double bond, and b may be a single bond.

상기 화학식 1에서, 상기 R1, R2, R3, R4 및 R5는 각각 독립적으로, H, F, Cl, Br, I, C1-4 알킬, C2-4 알케닐, 시아노, OR7, SR7 또는 NHR7이거나,In Formula 1, R1, R2, R3, R4 and R5 are each independently H, F, Cl, Br, I, C 1-4 alkyl, C 2-4 alkenyl, cyano, OR7, SR7 or or NHR7;

R1, R4 및 R5는 각각 독립적으로, H, F, Cl, Br, I, C1-4 알킬, C2-4 알케닐, 시아노, OR7, SR7 또는 NHR7이고, R2 및 R3은 함께 N, O 및 S로 이루어진 군으로부터 각각 독립적으로 선택되는 1개 또는 2개의 헤테로원자를 포함하는 5원 내지 6원의 헤테로고리를 형성하고;R1, R4 and R5 are each independently H, F, Cl, Br, I, C 1-4 alkyl, C 2-4 alkenyl, cyano, OR7, SR7 or NHR7, R2 and R3 together are N, Forming a 5- to 6-membered heterocycle containing 1 or 2 heteroatoms each independently selected from the group consisting of O and S;

R6은 H, 할로 또는 C1-4 알킬이고;R6 is H, halo or C 1-4 alkyl;

상기 R7은 H 또는 C1-4 알킬이고;wherein R7 is H or C 1-4 alkyl;

상기 C1-4 알킬 또는 C2-4 알케닐은 선택적으로 F, Cl, Br 또는 메틸로 치환될 수 있다.The C 1-4 alkyl or C 2-4 alkenyl may be optionally substituted with F, Cl, Br or methyl.

일 구체예에서, 상기 화학식 1로 표시되는 화합물은 화학식 1a로 표시되는 것일 수 있다:In one embodiment, the compound represented by Formula 1 may be one represented by Formula 1a:

[화학식 1a][Formula 1a]

Figure pat00003
Figure pat00003

X1, X2, a, b 및 R6은 상기에서 정의된 바와 같고;X1, X2, a, b and R6 are as defined above;

R은 하기로부터 선택된다:R is selected from:

Figure pat00004
.
Figure pat00004
.

일 구체예에서, 상기 화학식 1의 화합물은 하기 화합물로 이루어진 군으로부터 선택될 수 있다:In one embodiment, the compound of Formula 1 may be selected from the group consisting of the following compounds:

2-아미노-9-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-6-클로로-9H-퓨린-8-올 (화합물 5a);2-Amino-9 - ((6-bromo-benzo [d] [1,3] dioxol-5-yl) methyl) -6-chloro -9 H-purine-8-ol (compound 5a);

2-아미노-9-(4-브로모-2-플루오로벤질)-6-클로로-9H-퓨린-8-올 (화합물 5b);2-amino-9- (4-benzyl-Mo-2-fluorobenzyl) -6-chloro -9 H - purin-8-ol (compound 5b);

2-아미노-6-클로로-9-(2-메톡시-4-(트리플루오로메틸)벤질)-9H-퓨린-8-올 (화합물 5c);2-amino-6-chloro-9- (2-methoxy-4- (trifluoromethyl) benzyl) -9 H - purin-8-ol (compound 5c);

2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9H-퓨린-8-올 (화합물 5d);2-Amino-6-chloro-9-(3-chloro-4-fluorobenzyl)-9 H -purin-8-ol (Compound 5d);

2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9H-퓨린-8-올 (화합물 5e);2-Amino-6-chloro-9-(3-chloro-4-fluorobenzyl)-9 H -purin-8-ol (Compound 5e);

2-아미노-6-클로로-9-(4-(트리플루오로메틸)벤질)-9H-퓨린-8-올 (화합물 5f);2-amino-6-chloro-9- (4- (trifluoromethyl) benzyl) -9 H - purin-8-ol (compound 5f);

2-아미노-6-클로로-9-(5-클로로-2-플루오로벤질)-9H-퓨린-8-올 (화합물 5g);2-amino-6-chloro-9- (benzyl-5-chloro-2-fluorophenyl) -9 H - purin-8-ol (compound 5g);

2-아미노-6-클로로-9-(4-클로로-3-(트리플루오로메틸)벤질)-9H-퓨린-8-올 (화합물 5h);2-amino-6-chloro-9- (4-chloro-3- (trifluoromethyl) benzyl) -9 H - purin-8-ol (Compound 5h);

2-아미노-6-클로로-9-(2-클로로-5-(트리플루오로메틸)벤질)-9H-퓨린-8-올 (화합물 5i);2-amino-6-chloro-9- (benzyl 2-chloro-5- (trifluoromethyl)) -9 H - purin-8-ol (Compound 5i);

2-아미노-6-클로로-9-(3,4-디클로로벤질)-9H-퓨린-8-올 (화합물 5j);2-amino-6-chloro-9- (3,4-dichlorobenzyl) -9 H - purin-8-ol (compound 5j);

2-아미노-6-클로로-9-(2,6-디플루오로-4-메톡시벤질)-9H-퓨린-8-올 (화합물 5k);2-Amino-6-chloro-9-(2,6-difluoro-4-methoxybenzyl)-9 H -purin-8-ol (Compound 5k);

2-아미노-6-클로로-9-(2-플루오로-4-메틸벤질)-9H-퓨린-8-올 (화합물 5l);2-amino-6-chloro-9- (4-methyl-2-fluorobenzyl) -9 H - purin-8-ol (Compound 5l);

2-아미노-6-클로로-9-(2,3,6-트리플루오로벤질)-9H-퓨린-8-올 (화합물 5m);2-Amino-6-chloro-9-(2,3,6-trifluorobenzyl)-9 H -purin-8-ol (Compound 5m);

2-아미노-9-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-6-클로로-9H-퓨린-8-티올 (화합물 6a);2-Amino-9 - ((6-bromo-benzo [d] [1,3] dioxol-5-yl) methyl) -6-chloro -9 H-purine-8-thiol (compound 6a);

2-아미노-9-(4-브로모-2-플루오로벤질)-6-클로로-9H-퓨린-8-티올 (화합물 6b);2-amino-9- (4-benzyl-Mo-2-fluorobenzyl) -6-chloro -9 H - purine-8-thiol (compound 6b);

2-아미노-6-클로로-9-(2-메톡시-4-(트리플루오로메틸)벤질)-9H-퓨린-8-티올 (화합물 6c);2-Amino-6-chloro-9-(2-methoxy-4-(trifluoromethyl)benzyl)-9 H -purine-8-thiol (Compound 6c);

2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9H-퓨린-8-티올 (화합물 6d);2-Amino-6-chloro-9-(3-chloro-4-fluorobenzyl)-9 H -purine-8-thiol (Compound 6d);

2-아미노-6-클로로-9-(3,4-디플루오로벤질)-9H-퓨린-8-티올 (화합물 6e);2-Amino-6-chloro-9-(3,4-difluorobenzyl)-9 H -purine-8-thiol (Compound 6e);

2-아미노-6-클로로-9-(4-(트리플루오로메틸)벤질)-9H-퓨린-8-티올 (화합물 6f);2-amino-6-chloro-9- (4- (trifluoromethyl) benzyl) -9 H - purine-8-thiol (compound 6f);

2-아미노-6-클로로-9-(2-클로로-5-(트리플루오로메틸)벤질)-9H-퓨린-8-티올 (화합물 6i);2-amino-6-chloro-9- (2-chloro-5- (trifluoromethyl) benzyl) -9 H - purine-8-thiol (compound 6i);

2-아미노-6-클로로-9-(3,4-디클로로벤질)-9H-퓨린-8-티올 (화합물 6j);2-Amino-6-chloro-9-(3,4-dichlorobenzyl)-9 H -purine-8-thiol (Compound 6j);

2-아미노-6-클로로-9-(2,6-디플루오로-4-메톡시벤질)-9H-퓨린-8-티올 (화합물 6k);2-Amino-6-chloro-9-(2,6-difluoro-4-methoxybenzyl)-9 H -purine-8-thiol (Compound 6k);

2-아미노-6-클로로-9-(2-플루오로-4-메틸벤질)-9H-퓨린-8-티올 (화합물 6l);2-Amino-6-chloro-9-(2-fluoro-4-methylbenzyl)-9 H -purine-8-thiol (Compound 6l);

2-아미노-6-클로로-9-(2,3,6-트리플루오로벤질)-9H-퓨린-8-티올 (화합물 6m);2-Amino-6-chloro-9-(2,3,6-trifluorobenzyl)-9 H -purine-8-thiol (Compound 6m);

9-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-6-클로로-9H-퓨린-2,8-디아민 (화합물 7a);9 - ((6-bromo-benzo [d] [1,3] dioxol-5-yl) methyl) -6-chloro -9 H-purine-2,8-diamine (compound 7a);

9-(4-브로모-2-플루오로벤질)-6-클로로-9H-퓨린-2,8-디아민 (화합물 7b);9- (4-benzyl-Mo-2-fluorobenzyl) -6-chloro -9 H - purine-2,8-diamine (Compound 7b);

6-클로로-9-(3-클로로-4-플루오로벤질)-9H-퓨린-2,8-디아민 (화합물 7d);6-Chloro-9-(3-chloro-4-fluorobenzyl)-9 H -purine-2,8-diamine (Compound 7d);

6-클로로-9-(3,4-디플루오로벤질)-9H-퓨린-2,8-디아민 (화합물 7e);6-Chloro-9-(3,4-difluorobenzyl)-9 H -purine-2,8-diamine (Compound 7e);

2-아미노-7-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-4-클로로-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11a);2-amino-7 - ((6-bromo-benzo [d] [1,3] dioxol-5-yl) methyl) -4-chloro-5,7-dihydro -6 H-pyrrolo [2, 3- d ]pyrimidin-6-one (Compound 11a);

2-아미노-4-클로로-7-(4-(트리플루오로메틸)벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11f);2-amino-4-chloro-7- (4- (trifluoromethyl) benzyl) - 5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6-one (compound 11f );

2-아미노-7-(4-브로모-2-플루오로벤질)-4-클로로-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11g);2-amino-7- (4-benzyl-Mo-2-fluorobenzyl) -4-chloro-5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6-one (Compound 11 g);

2-아미노-4-클로로-7-(2-클로로-5-(트리플루오로메틸)벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11i);2-amino-4-chloro-7- (2-chloro-5- (trifluoromethyl) benzyl) -5,7-dihydro-6 H -pyrrolo [2,3- d ] pyrimidine-6- on (compound 11i);

2-아미노-4-클로로-7-(3,4-디클로로벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11j);2-amino-4-chloro-7- (3,4-dichlorobenzyl) -5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6-one (Compound 11j);

2-아미노-4-클로로-7-(2-플루오로-4-메틸벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11l);2-Amino-4-chloro-7-(2-fluoro-4-methylbenzyl)-5,7-dihydro-6H-pyrrolo[2,3- d ]pyrimidin-6-one (Compound 11l) ;

2-아미노-7-(벤조[d][1,3]디옥솔-5-일메틸)-4-클로로-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11n);2-amino-7- (benzo [d] [1,3] dioxol-5-ylmethyl) -4-chloro-5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidine -6-one (compound 11n);

2-아미노-4-클로로-7-(2,3-디메톡시벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11o);2-amino-4-chloro-7- (2,3-dimethoxybenzyl) -5,7-dihydro -6 H - pyrrolo [2,3-d] pyrimidin-6-one (Compound 11o);

2-아미노-4-클로로-7-(2,6-디플루오로-3-메톡시벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 11p);2-amino-4-chloro-7- (2, 6-Difluoro-3-methoxy-benzyl) - 5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6 on (compound 11p);

2-아미노-7-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-4-메틸-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 15a); 및2-amino-7-((6-bromobenzo[d][1,3]dioxol-5-yl)methyl)-4-methyl-5,7-dihydro-6H-pyrrolo[2,3 - d ]pyrimidin-6-one (compound 15a); and

2-아미노-7-(4-브로모-2-플루오로벤질)-4-메틸-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온 (화합물 15b).2-amino-7- (4-benzyl-Mo-2-fluorobenzyl) -4-methyl-5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6-one (Compound 15b).

용어 "입체이성질체"의 "이성질체(isomer)"는 분자식은 같지만 분자 내에 있는 구성 원자의 연결 방식이나 공간 배열이 동일하지 않은 화합물을 말한다. 이성질체는 예를 들면, 구조 이성질체(structural isomers), 및 입체이성질체(stereoisomer)를 포함한다. 상기 입체이성질체는 부분입체 이성질체(diasteromer) 또는 거울상 이성질체(enantiomer)일 수 있다. 거울상이성질체는 왼손과 오른손의 관계처럼 그 거울상과 겹쳐지지 않는 이성질체를 말하고, 광학 이성질체(optical isomer)라고도 한다. 거울상 이성질체는 키랄 중심 탄소에 4개 이상의 치환기가 서로 다른 경우 R(Rectus: 시계방향) 및 S(Sinister: 반시계 방향)로 구분한다. 부분입체이성질체는 거울상 관계가 아닌 입체 이성질체를 말하고, 원자의 공간 배열이 달라 생기 시스(cis)-트랜스(trans) 이성질체로 나뉠 수 있다.The term "isomer" of the term "stereoisomer" refers to a compound that has the same molecular formula but does not have the same spatial arrangement or connection mode of constituent atoms in the molecule. Isomers include, for example, structural isomers, and stereoisomers. The stereoisomer may be a diasteromer or an enantiomer. An enantiomer refers to an isomer that does not overlap the mirror image as in the relationship between the left hand and the right hand, and is also called an optical isomer. Enantiomers are divided into R (Rectus: clockwise) and S (Sinister: counterclockwise) when 4 or more substituents differ from each other at the chiral central carbon. Diastereomers refer to stereoisomers that are not mirror images, and can be divided into cis-trans isomers due to the spatial arrangement of atoms.

용어 "용매화물(solvate)"는 유기 또는 무기 용매에 용매화된 화합물을 말한다. 상기 용매화물은 예를 들어 수화물이다.The term “solvate” refers to a compound solvated in an organic or inorganic solvent. The solvate is, for example, a hydrate.

용어 "염(salt)"은 화합물의 무기 및 유기산 부가염을 말한다. 상기 약학적으로 허용가능한 염은 화합물이 투여되는 유기체에 심각한 자극을 유발하지 않고 화합물의 생물학적 활성과 물성들을 손상시키지 않는 염일 수 있다. 상기 무기산염은 염산염, 브롬산염, 인산염, 황산염, 또는 이황산염일 수 있다. 상기 유기산염은 포름산염, 아세트산염, 프로피온산염, 젖산염, 옥살산염, 주석산염, 말산염, 말레인산염, 구연산염, 푸마르산염, 베실산염, 캠실산염, 에디실염, 트리클로로아세트산, 트리플루오로아세트산염, 벤조산염, 글루콘산염, 메탄술폰산염, 글리콜산염, 숙신산염, 4-톨루엔술폰산염, 갈룩투론산염, 엠본산염, 글루탐산염, 에탄술폰산염, 벤젠술폰산염, p-톨루엔술폰산염, 또는 아스파르트산염일 수 있다. 상기 금속염은 칼슘염, 나트륨염, 마그네슘염, 스트론튬염, 또는 칼륨염일 수 있다.The term "salt" refers to the inorganic and organic acid addition salts of compounds. The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and properties of the compound. The inorganic acid salt may be hydrochloride, bromate, phosphate, sulfate, or disulfate. The organic acid salts include formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edicyl salt, trichloroacetic acid, trifluoroacetate , benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or aspartate It may be an acid. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt.

다른 양상은 일 양상에 따른 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 포함하는, TRAP1의 활성과 연관된 질환의 예방 또는 치료용 약학적 조성물을 제공한다.Another aspect provides a pharmaceutical composition for preventing or treating a disease associated with the activity of TRAP1, comprising the compound according to the aspect, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

다른 양상은 일 양상에 따른 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다.Another aspect provides a pharmaceutical composition for preventing or treating cancer, comprising the compound according to the aspect, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

화합물, 입체이성질체, 용매화물, 및 약학적으로 허용가능한 염은 전술한 바와 같다.Compounds, stereoisomers, solvates, and pharmaceutically acceptable salts are as described above.

용어 "예방"은 조성물의 투여에 의해 질병을 억제시키거나 발병을 지연시키는 모든 행위를 말한다. 용어 "치료"는 조성물의 투여에 의해 질병의 증세가 호전되거나 이롭게 변경하는 모든 행위를 말한다.The term "prevention" refers to any action that suppresses or delays the onset of a disease by administration of the composition. The term "treatment" refers to any act of improving or beneficially changing the symptoms of a disease by administering the composition.

상기 약학 조성물에 있어서, 상기 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염은, Hsp90 (Heat shock protein 90) 또는 Grp94 (Glucose-regulated protein 94)에 비해 TRAP1 (TNF receptor-Associated Protein 1)를 선택적으로 억제하는 것일 수 있다. 예를 들어, 화학식 1로 표시되는 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염은 암세포의 세포자살 (apoptosis)을 유도하는 것일 수 있다.In the pharmaceutical composition, the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is, compared to Hsp90 (Heat shock protein 90) or Grp94 (Glucose-regulated protein 94) TRAP1 (TNF receptor-Associated Protein) 1) may be selectively suppressed. For example, the compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof may induce apoptosis in cancer cells.

상기 암은 TRAP1의 활성과 연관된 암일 수 있다. TRAP1의 활성과 연관된 암은, 예를 들어, 유방암, 췌장암, 대장암, 폐암 (Kang, Byoung Heon, et al. "Regulation of tumor cell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone network."　Cell　131.2 (2007): 257-270; Costantino, Eleonora, et al. "TRAP1, a novel mitochondrial chaperone responsible for multi-drug resistance and protection from apoptotis in human colorectal carcinoma cells."　Cancer letters　279.1 (2009): 39-46); 전립선암, 자궁경부암, 자궁내막암, 신경교종, 간암 (Park, Hye-Kyung, et al. "Unleashing the full potential of Hsp90 inhibitors as cancer therapeutics through simultaneous inactivation of Hsp90, Grp94, and TRAP1."　Experimental & molecular medicine　(2020): 1-13); 난소암 (Aust, Stefanie, et al. "Role of TRAP1 and estrogen receptor alpha in patients with ovarian cancer-a study of the OVCAD consortium."　Molecular cancer　11.1 (2012): 69); 뇌암 (Park, Hye-Kyung, et al. "Interplay between TRAP1 and sirtuin-3 modulates mitochondrial respiration and oxidative stress to maintain stemness of glioma stem cells."　Cancer research　79.7 (2019): 1369-1382); 방광암, 신장암 (Yoshida, Soichiro, et al. "Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis."　Proceedings of the National Academy of Sciences　110.17 (2013): E1604-E1612; Si, Tong, et al. "Expression of tumor necrosis factor receptor-associated protein 1 and its clinical significance in kidney cancer."　International journal of clinical and experimental pathology　8.10 (2015): 13090.); 신경아세포종 (Ramkumar, Balaji, et al. "Mitochondrial chaperone, TRAP1 as a potential pharmacological target to combat cancer metabolism."　Mitochondrion　50 (2020): 42-50); 구강암 (Landriscina, Matteo, et al. "Mitochondrial chaperone Trap1 and the calcium binding protein Sorcin interact and protect cells against apoptosis induced by antiblastic agents."　Cancer research　70.16 (2010): 6577-6586); 위암 (Han, Ping, Qing-Ling Wang, and Xia Zhang. "Expression of TRAP1 in gastric cancer tissue and its correlation with malignant biology."　Asian Pacific Journal of Tropical Medicine　9.1 (2016): 67-71); 골암 (Gesualdi, N. Montesano, et al. "Tumor necrosis factor-associated protein 1 (TRAP-1) protects cells from oxidative stress and apoptosis."　Stress　10.4 (2007): 342-350); 직장암 (Chen, Ru, et al. "Up-regulation of mitochondrial chaperone TRAP1 in ulcerative colitis associated colorectal cancer."　World Journal of Gastroenterology: WJG　20.45 (2014): 17037); 갑상선암 (Palladino, Giuseppe, et al. "TRAP1 regulates cell cycle and apoptosis in thyroid carcinoma cells. "　Endocrine-related cancer　23.9 (2016): 699-709); 피부암, 흑색종 (Basit, Farhan, et al. "Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells."　Cell death & disease　8.3 (2017): e2716-e2716); 혈액암, 림프종, 췌장신경내분비종양 (Hu, Jiangting, et al. "TRAP1 is involved in cell cycle regulated by retinoblastoma susceptibility gene (RB1) in early hypoxia and has variable expression patterns in human tumors."　Journal of Cancer Research Updates　2.3 (2013): 194-210) 등이 있으나, 이에 제한되지 않는다.The cancer may be a cancer associated with the activity of TRAP1. Cancers associated with TRAP1 activity are, for example, breast cancer, pancreatic cancer, colorectal cancer, and lung cancer (Kang, Byoung Heon, et al. "Regulation of tumor cell mitochondrial homeostasis by an organelle-specific Hsp90 chaperone network." Cell 131.2 (2007) ): 257-270; Costantino, Eleonora, et al. "TRAP1, a novel mitochondrial chaperone responsible for multi-drug resistance and protection from apoptotis in human colorectal carcinoma cells." Cancer letters 279.1 (2009): 39-46); Prostate cancer, cervical cancer, endometrial cancer, glioma, liver cancer (Park, Hye-Kyung, et al. "Unleashing the full potential of Hsp90 inhibitors as cancer therapeutics through simultaneous inactivation of Hsp90, Grp94, and TRAP1." Experimental & molecular medicine (2020): 1-13); ovarian cancer (Aust, Stefanie, et al. "Role of TRAP1 and estrogen receptor alpha in patients with ovarian cancer-a study of the OVCAD consortium." Molecular cancer 11.1 (2012): 69); brain cancer (Park, Hye-Kyung, et al. "Interplay between TRAP1 and sirtuin-3 modulates mitochondrial respiration and oxidative stress to maintain stemness of glioma stem cells." Cancer research 79.7 (2019): 1369-1382); Bladder cancer, kidney cancer (Yoshida, Soichiro, et al. "Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis." Proceedings of the National Academy of Sciences 110.17 (2013): E1604-E1612; Si, Tong, et al. "Expression of tumor necrosis factor receptor-associated protein 1 and its clinical significance in kidney cancer." International journal of clinical and experimental pathology 8.10 (2015): 13090.); neuroblastoma (Ramkumar, Balaji, et al. "Mitochondrial chaperone, TRAP1 as a potential pharmacological target to combat cancer metabolism." Mitochondrion 50 (2020): 42-50); oral cancer (Landriscina, Matteo, et al. "Mitochondrial chaperone Trap1 and the calcium binding protein Sorcin interact and protect cells against apoptosis induced by antiblastic agents." Cancer research 70.16 (2010): 6577-6586); gastric cancer (Han, Ping, Qing-Ling Wang, and Xia Zhang. "Expression of TRAP1 in gastric cancer tissue and its correlation with malignant biology." Asian Pacific Journal of Tropical Medicine 9.1 (2016): 67-71); bone cancer (Gesualdi, N. Montesano, et al. "Tumor necrosis factor-associated protein 1 (TRAP-1) protects cells from oxidative stress and apoptosis." Stress 10.4 (2007): 342-350); rectal cancer (Chen, Ru, et al. "Up-regulation of mitochondrial chaperone TRAP1 in ulcerative colitis associated colorectal cancer." World Journal of Gastroenterology: WJG 20.45 (2014): 17037); thyroid cancer (Palladino, Giuseppe, et al. "TRAP1 regulates cell cycle and apoptosis in thyroid carcinoma cells." Endocrine-related cancer 23.9 (2016): 699-709); skin cancer, melanoma (Basit, Farhan, et al. "Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells." Cell death & disease 8.3 (2017): e2716-e2716); Hematological cancer, lymphoma, pancreatic neuroendocrine tumor (Hu, Jiangting, et al. "TRAP1 is involved in cell cycle regulated by retinoblastoma susceptibility gene (RB1) in early hypoxia and has variable expression patterns in human tumors." Journal of Cancer Research Updates 2.3 (2013): 194-210), but is not limited thereto.

일 구체예에서, 상기 암은 유방암, 췌장암, 대장암, 폐암, 전립선암, 자궁경부암, 자궁내막암, 신경교종, 간암, 난소암, 뇌암, 방광암, 신장암, 신경아세포종, 구강암, 위암, 골암, 직장암, 갑상선암, 피부암, 흑색종, 혈액암, 림프종, 및 췌장신경내분비종양으로 이루어진 군으로부터 선택되는 것일 수 있으나, 이에 제한되지 않는다.In one embodiment, the cancer is breast cancer, pancreatic cancer, colorectal cancer, lung cancer, prostate cancer, cervical cancer, endometrial cancer, glioma, liver cancer, ovarian cancer, brain cancer, bladder cancer, kidney cancer, neuroblastoma, oral cancer, stomach cancer, bone cancer , rectal cancer, thyroid cancer, skin cancer, melanoma, blood cancer, lymphoma, and may be selected from the group consisting of pancreatic neuroendocrine tumor, but is not limited thereto.

상기 약학적 조성물은 항암 활성을 갖는 공지의 유효성분을 더 포함할 수 있다. 항암 활성을 갖는 공지의 유효성분은 항암제일 수 있다. 상기 화학식 1로 표시되는 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염과 상기 항암제는 동시 또는 순차 투여를 위한 단일 또는 개별 조성물일 수 있다.The pharmaceutical composition may further include a known active ingredient having anticancer activity. A known active ingredient having anticancer activity may be an anticancer agent. The compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof and the anticancer agent may be a single or separate composition for simultaneous or sequential administration.

상기 약학적 조성물은 담체, 부형제 또는 희석제를 더 포함할 수 있다. 담체, 부형제 및 희석제는 예를 들면, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로스, 미정질 셀 룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트, 또는 광물유를 포함할 수 있다.The pharmaceutical composition may further include a carrier, excipient or diluent. Carriers, excipients and diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

상기 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화될 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다.The pharmaceutical composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

상기 약학적 조성물에 있어서, 경구 투여를 위한 고형 제제는 정제, 환제, 산제, 과립제, 또는 캡슐제일 수 있다. 상기 고형 제제는 부형제를 더 포함할 수 있다. 부형제는 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose), 락토오스(lactose), 또는 젤라틴일 수 있다. 또한, 상기 고형 제제는 마그네슘 스테아레이트, 또는 탈크와 같은 윤활제를 더 포함할 수 있다. 상기 약학적 조성물에 있어서, 경구를 위한 액상 제제는 현탁제, 내용액제, 유제, 또는 시럽제일 수 있다. 상기 액상 제제는 물, 또는 리퀴드 파라핀을 포함할 수 있다. 상기 액상 제제는 부형제, 예를 들면 습윤제, 감미제, 방향제, 또는 보존제를 포함할 수 있다. 상기 약학적 조성물에 있어서, 비경구 투여를 위한 제제는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 또는 및 좌제일 수 있다. 비수성용제 또는 현탁제는 식물성 기름 또는 에스테르를 포함할 수 있다. 식물성 기름은 예를 들면, 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 또는 올리브 오일일 수 있다. 에스테르는 예를 들면 에틸올레이트일 수 있다. 좌제의 기제는 위텝솔 (witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 또는 글리세로젤라틴 일 수 있다.In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid formulation may further include an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid formulation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the oral liquid formulation may be a suspension, an oral solution, an emulsion, or a syrup. The liquid formulation may contain water or liquid paraffin. The liquid formulation may contain excipients, for example, wetting agents, sweetening agents, perfuming agents, or preservatives. In the pharmaceutical composition, the preparation for parenteral administration may be a sterile aqueous solution, non-aqueous solution, suspension, emulsion, freeze-dried or suppository. The non-aqueous solvent or suspending agent may include vegetable oil or ester. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin, or glycerogelatin.

상기 약학적 조성물은 화학식 1로 표시되는 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 유효한 양으로 포함할 수 있다. 용어 "유효한 양"은 예방 또는 치료를 필요로 하는 개체에게 투여되는 경우 예방 또는 치료의 효과를 나타내기에 충분한 양을 말한다. 상기 유효한 양은 당업자가 선택되는 세포 또는 개체에 따라 적절하게 선택할 수 있다. 상기 약학적 조성물의 바람직한 투여량은 개체의 상태 및 체중, 질병의 정도, 약물 형태, 투여 경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나, 상기 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염은 예를 들면, 약 0.0001 ㎎/㎏ 내지 약 100 ㎎/㎏, 또는 약 0.001 ㎎/㎏ 내지 약 100 ㎎/㎏의 양을 1일 1회 내지 24회, 2일 내지 1주에 1 내지 7회, 또는 1개월 내지 12개월에 1 내지 24회로 나누어 투여할 수 있다. 상기 약학적 조성물에서 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염은 전체 조성물 총 중량에 대하여 약 0.0001 중량% 내지 약 10 중량%, 또는 약 0.001 중량% 내지 약 1 중량%로 포함될 수 있다.The pharmaceutical composition may include the compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof in an effective amount. The term "effective amount" refers to an amount sufficient to exhibit the effect of prophylaxis or treatment when administered to a subject in need thereof. The effective amount can be appropriately selected by those skilled in the art depending on the cell or individual to be selected. The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the individual, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, the compound, a stereoisomer, solvate, or pharmaceutically acceptable salt thereof may be, for example, in an amount from about 0.0001 mg/kg to about 100 mg/kg, or from about 0.001 mg/kg to about 100 mg/kg. may be administered in divided doses 1 to 24 times a day, 1 to 7 times a week for 2 days to 1 week, or 1 to 24 times a month to 12 months. In the pharmaceutical composition, the compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof is included in an amount of from about 0.0001% to about 10% by weight, or from about 0.001% to about 1% by weight, based on the total weight of the composition. can

투여 방법은 경구, 또는 비경구 투여일 수 있다. 투여 방법은 예를 들어, 경구, 경피, 피하, 직장, 정맥내, 동맥내, 복강내, 근육내, 흉골내, 국소, 코안(intranasal), 기관내(intratracheal), 또는 피내 경로일 수 있다. 상기 조성물은 전신적으로 또는 국부적으로 투여될 수 있고, 단독으로 또는 다른 약학적 활성 화합물과 함께 투여될 수 있다.The administration method may be oral or parenteral administration. The method of administration can be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal routes. The compositions may be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.

다른 양상은 일 양상에 따른 약학적 조성물을 개체에게 투여하는 단계를 포함하는 암을 예방 또는 치료하는 방법을 제공한다.Another aspect provides a method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition according to an aspect.

상기 약학적 조성물, 암, 예방, 및 치료는 전술한 바와 같다.The pharmaceutical composition, cancer, prevention, and treatment are the same as described above.

상기 개체는 포유동물, 예를 들면, 인간, 마우스, 래트, 소, 말, 돼지, 개, 원숭이, 양, 염소 또는 고양이일 수 있다. 상기 개체는 암과 연관된 증상을 앓고 있거나, 앓을 가능성이 큰 개체일 수 있다.The subject may be a mammal, such as a human, mouse, rat, cow, horse, pig, dog, monkey, sheep, goat or cat. The subject may be suffering from, or likely to suffer from, symptoms associated with cancer.

상기 방법은 상기 개체에 항암제를 투여하는 단계를 더 포함할 수 있다. 상기 항암제는 화학식 1로 표시되는 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염과 동시, 개별, 또는 순차로 상기 개체에 투여될 수 있다.The method may further comprise administering an anticancer agent to the subject. The anticancer agent may be administered to the subject simultaneously, individually, or sequentially with the compound represented by Formula 1, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof.

투여 방법은 경구, 또는 비경구 투여일 수 있다. 투여 방법은 예를 들어, 경구, 경피, 피하, 직장, 정맥내, 동맥내, 복강내, 근육내, 흉골내, 국소, 코안(intranasal), 기관내(intratracheal), 또는 피내 경로일 수 있다. 상기 약학적 조성물은 전신적으로 또는 국부적으로 투여될 수 있고, 단독으로 또는 다른 약학적 활성 화합물과 함께 투여될 수 있다.The administration method may be oral or parenteral administration. The method of administration can be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal routes. The pharmaceutical composition may be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.

상기 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여 경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 상기 투여량은 예를 들어, 성인 기준으로 약 0.001 ㎎/kg 내지 약 100 ㎎/kg, 약 0.01 ㎎/kg 내지 약 10 ㎎/kg, 또는 약 0.1 ㎎/kg 내지 약 1 ㎎/kg의 범위 내 일 수 있다. 상기 투여는 1일 1회, 1일 다회, 또는 1주일에 1회, 2주일에 1회, 3주일에 1회, 또는 4주일에 1회 내지 1년에 1회 투여될 수 있다.The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. The dosage is, for example, in the range of from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, or from about 0.1 mg/kg to about 1 mg/kg on an adult basis. can be The administration may be administered once a day, multiple times a day, or once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks to once a year.

다른 양상은 암 예방 또는 치료용 의약의 제조에 사용하기 위한 일 양상에 따른 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염의 용도를 제공한다.Another aspect provides the use of a compound according to one aspect, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the prevention or treatment of cancer.

일 양상에 따른 신규 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염은 TRAP1 선택적 억제 활성을 가지므로, TRAP1과 연관된 다양한 종류의 암을 예방 또는 치료하는데 사용될 수 있다.Since the novel compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof according to an aspect has TRAP1 selective inhibitory activity, it can be used to prevent or treat various types of cancer associated with TRAP1.

도 1은 (A) TRAP1 NTD에서 화합물 5a의 X-선 공-결정 구조, (B) Hsp90-NTD에서 PU-H71의 X-선 공-결정 구조, (C) TRAP1 NM 도메인에서 화합물 5b의 X-선 공-결정 구조를 나타낸 것이다. 골드색은 ATP-lid를 나타낸다.
도 2는 (A) DN401-TRAP1 NTD 구조, (B) 화합물 5a-TRAP1 NTD 구조를 나타낸 X-선 결정학 결과이다. 물은 붉은색 구체이다.
도 3은 TRAP1-5a 복합체의 MD 시뮬레이션 (300ns)을 나타낸 것이다. (A) 30 ns 간격에서 화합물 5a의 결합 방식의 중첩. (B) TRAP1 잔기와의 화합물 5a 상호 작용의 상세한 원자의 개략도. 선택한 궤적 (0-300 ns)에서 30% 이상의 시뮬레이션 시간에 발생하는 상호 작용이 표시된다. (C) 300 ns MD 궤적에 따른 TRAP1 NTD의 RMSD 변화. (D) 5a-TRAP1 상호 작용 분획 누적 막대 차트; 수소 결합 (녹색), 소수성 결합 (회색), 이온성 결합 (빨간색) 및 물 연결 (파란색).
도 4는 화합물 5a 또는 DN401과 Hsp90의 도킹된 복합체의 MD 시뮬레이션을 나타낸 것이다. (A) 30 ns 간격에서 Hsp90-5a 복합체의 결합 방식의 중첩. (B) 30 ns 간격에서 Hsp90-DN401 복합체의 결합 방식의 중첩. (C) 300 ns MD 궤적에 따른 Hsp90 NTD의 RMSD 변화. (D) 300 ns MD 궤적에 따른 Hsp90 NTD의 RMSD 변화.
도 5는 TRAP1 NTD 및 Hsp90 NTD와 다양한 억제제들의 RMSF 값을 나타낸 것이다.
도 6은 300 ns MD 시뮬레이션의 30 ns 간격에서 (A) BIIB021, (B) SNX0723 및 (C) PUH71의 Hsp90에 대한 결합 방식의 중첩을 나타낸 것이다.
도 7은 Hsp90 (청록색) MD 시뮬레이션의 궤적 (90 ns)과 함께 TRAP1-5a 복합체 (TRAP1: 흰색, 5a: 녹색)를 오버레이한 것이다.1 shows (A) X-ray co-crystal structure of compound 5a in TRAP1 NTD, (B) X-ray co-crystal structure of PU-H71 in Hsp90-NTD, (C) X-ray co-crystal structure of compound 5b in TRAP1 NM domain. The -line co-crystal structure is shown. Gold color represents ATP-lid.
2 is an X-ray crystallography result showing the structure of (A) DN401-TRAP1 NTD and (B) compound 5a-TRAP1 NTD. Water is a red sphere.
Figure 3 shows the MD simulation (300ns) of the TRAP1-5a complex. (A) Superimposition of the binding mode of compound 5a at 30 ns intervals. (B) Schematic of detailed atoms of compound 5a interaction with TRAP1 moieties. Interactions occurring over 30% of the simulation time on the selected trajectory (0-300 ns) are shown. (C) RMSD change of TRAP1 NTD along 300 ns MD trajectory. (D) 5a-TRAP1 interaction fraction stacked bar chart; Hydrogen bonds (green), hydrophobic bonds (grey), ionic bonds (red) and water bonds (blue).
Figure 4 shows the MD simulation of compound 5a or the docked complex of DN401 and Hsp90. (A) Superimposition of the binding mode of the Hsp90-5a complex at 30 ns intervals. (B) Overlapping mode of binding of Hsp90-DN401 complexes at 30 ns intervals. (C) RMSD changes of Hsp90 NTDs along the 300 ns MD trajectory. (D) RMSD changes of Hsp90 NTDs along the 300 ns MD trajectory.
5 shows the RMSF values of TRAP1 NTD and Hsp90 NTD and various inhibitors.
Figure 6 shows the overlap of the binding mode of (A) BIIB021, (B) SNX0723 and (C) PUH71 to Hsp90 at 30 ns interval of 300 ns MD simulation.
7 is an overlay of the TRAP1-5a complex (TRAP1: white, 5a: green) with the trajectories (90 ns) of Hsp90 (cyan) MD simulations.

이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

합성 방법synthesis method

출발 물질은 Sigma-Aldrich, TCI, Alfa-Aesar 및 Combi-Blocks에서 구입하였다. 모든 물질은 추가 정제 없이 사용하였다. Starting materials were purchased from Sigma-Aldrich, TCI, Alfa-Aesar and Combi-Blocks. All materials were used without further purification.

마이크로웨이브 보조 반응은 바이오티지 이니시에이터 마이크로웨이브 시스템을 사용하였다. The microwave assisted reaction was performed using a Biotage initiator microwave system.

반응은 Merch 사전 코팅된 실리카 겔 60 F254 플레이트에서 수행된 박막 크로마토그래피에 의해 모니터링되었다. The reaction was monitored by thin layer chromatography performed on Merch pre-coated silica gel 60 F254 plates.

다양한 사전 패킹된 실리카 겔 카트리지를 갖는 Teledyne ISCO 플래시 정제 시스템을 플래시 컬럼 크로마토그래피에 사용하였다. A Teledyne ISCO flash purification system with various prepacked silica gel cartridges was used for flash column chromatography.

1H 및 13C NMR 스펙트럼은 Varian Unity 400 기기 (1H 및 13C의 경우 각각 400 및 101 MHz) 분광계를 사용하여 지시된 용매에서 기록하였다. 1 H and 13 C NMR spectra were recorded in the indicated solvents using a Varian Unity 400 instrument ( 400 and 101 MHz for 1 H and 13 C, respectively) spectrometer.

화학적 이동은 DMSO-d6 또는 CDCl3의 내부 표준으로서 TMS (0.0)에서 백만 다운필드 당 델타 값으로 보고하였다. Chemical shifts were reported as delta values per million downfields in TMS (0.0) as internal standards for DMSO- d6 or CDCl 3 .

질량분석(Mass Spectrometry, MS)은 Agilent 6230B TOF (Time of Flight) 액체 크로마토 그래피-질량 분석계에서 전자 분무 이온화 (ESI) 소스로 구성된 시스템에서 수행되었다. Mass Spectrometry (MS) was performed on an Agilent 6230B Time of Flight (TOF) liquid chromatography-mass spectrometer in a system configured with an Electrospray Ionization (ESI) source.

생물학적 분석을 거친 모든 화합물의 순도는 >95%이었다.All compounds subjected to biological analysis were >95% pure.

[반응도식 1][Reaction Scheme 1]

Figure pat00005
Figure pat00005

a 시약 및 조건: (a) 4,6-디클로로피리미딘-2,5-디아민, DIEA, n-BuOH, 170 ℃, 2.5시간, 마이크로웨이브, 50~85%; (b) 1,1'-카르보닐디이미다졸, 실온, 3시간, 15~65%; (c) 1,1'-티오카르보닐디이미다졸, 실온, 3시간, 23%; (d) 시아노겐 브로마이드, EtOH, 밤새 환류, 15%. a Reagents and conditions: (a) 4,6-dichloropyrimidine-2,5-diamine, DIEA, n-BuOH, 170° C., 2.5 hours, microwave, 50-85%; (b) 1,1'-carbonyldiimidazole, room temperature, 3 hours, 15-65%; (c) 1,1'-thiocarbonyldiimidazole, room temperature, 3 hours, 23%; (d) Cyanogen bromide, EtOH, reflux overnight, 15%.

제조예 1: 중간체 4a 내지 4m의 합성Preparation Example 1: Synthesis of intermediates 4a to 4m

n-부탄올 (1.5 mL) 중 벤질 아민 (8 mmol), 4,6-디클로로피리미딘-2,5-디아민 (8 mmol) 및 N,N-디이소프로필에틸아민 (10.4 mmol)을 170 ℃에서 2.5 시간 동안 마이크로웨이브 반응기에서 교반 하였다. 실온으로 냉각 한 후, 진공 하에서 용매를 제거하였다. 잔류물을 실리카 겔 컬럼 크로마토그래피 (EtOAc/Hex)로 정제하여 목적 화합물 4a 내지 4m을 수득하였다.Benzyl amine (8 mmol), 4,6-dichloropyrimidine-2,5-diamine (8 mmol) and N,N-diisopropylethylamine (10.4 mmol) in n-butanol (1.5 mL) at 170 °C It was stirred in a microwave reactor for 2.5 h. After cooling to room temperature, the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (EtOAc/Hex) to obtain the target compounds 4a to 4m .

제조예 1-1:Preparation 1-1: NN 44 -((6-브로모벤조[-((6-bromobenzo[ dd ][1,3]디옥솔-5-일)메틸)-6-클로로피리미딘-2,4,5-트리아민 (4a)][1,3]dioxol-5-yl)methyl)-6-chloropyrimidine-2,4,5-triamine (4a)

수율: 36%; 1H NMR (400 MHz, DMSO-d 6 ) δ 7.21 (s, 1H), 6.93 (s, 1H), 6.04 (s, 2H), 6.03 (s, 1H), 5.68 (s, 2H), 4.47 (2H, s), 3.95 (s, 2H); MS (ESI, m/z) C12H11BrClN5O2 [M+H]+ 계산치 372.98, 실측치 372.95.Yield: 36%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.21 (s, 1H), 6.93 (s, 1H), 6.04 (s, 2H), 6.03 (s, 1H), 5.68 (s, 2H), 4.47 ( 2H, s), 3.95 (s, 2H); MS (ESI, m/z) C 12 H 11 BrClN 5 O 2 [M+H] + calculated 372.98, found 372.95.

제조예 1-2: N 4 -(4-브로모-2-플루오로벤질)-6-클로로피리미딘-2,4,5-트리아민 (4b). 수율: 27%; 1H NMR (400 MHz, CDCl3) δ 7.26 - 7.22 (m, 3H), 5.73 (s, 1H), 4.61 (dd, J = 6.2, 1.1 Hz, 4H), 2.70 (s, 2H); MS (ESI, m/z) C11H10BrClFN5 [M+H]+ 계산치 346.98, 실측치 346.90. Preparation 1-2: N 4 -(4-bromo-2-fluorobenzyl)-6-chloropyrimidine-2,4,5-triamine (4b). Yield: 27%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.26 - 7.22 (m, 3H), 5.73 (s, 1H), 4.61 (dd, J = 6.2, 1.1 Hz, 4H), 2.70 (s, 2H); MS (ESI, m/z) C 11 H 10 BrClFN 5 [M+H] + cal 346.98, found 346.90.

제조예 1-3: 6-클로로- N 4 -(2-메톡시-4-(트리플루오로메틸)벤질)피리미딘-2,4,5-트리아민 (4c). 수율: 34%; 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 7.8 Hz, 1H), 7.21 - 7.17 (m, 1H), 7.08 (d, J = 1.6 Hz, 1H), 5.84 (s, 1H), 4.64 (d, J = 6.2 Hz, 2H), 4.58 (s, 2H), 2.73 (d, J = 16.8 Hz, 2H); MS (ESI, m/z) C13H13ClF3N5O [M+H]+ 계산치 349.07, 실측치 349.05. Preparation 1-3: 6-chloro- N 4 -(2-methoxy-4-(trifluoromethyl)benzyl)pyrimidine-2,4,5-triamine (4c). Yield: 34%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.39 (d, J = 7.8 Hz, 1H), 7.21 - 7.17 (m, 1H), 7.08 (d, J = 1.6 Hz, 1H), 5.84 (s, 1H) , 4.64 (d, J = 6.2 Hz, 2H), 4.58 (s, 2H), 2.73 (d, J = 16.8 Hz, 2H); MS (ESI, m/z) C 13 H 13 ClF 3 N 5 O [M+H] + cal 349.07, found 349.05.

제조예 1-4: 6-클로로- N 4 -(3-클로로-4-플루오로벤질)피리미딘-2,4,5-트리아민 (4d). 수율: 68%; 1H NMR (400 MHz, DMSO-d 6) δ 7.54 - 7.49 (m, 1H), 7.39 - 7.30 (m, 2H), 7.09 (t, J = 6.0 Hz, 1H), 5.67 (s, 2H), 4.52 (d, J = 5.9 Hz, 2H), 3.90 (s, 2H); MS (ESI, m/z) C13H13ClF3N5O [M+H]+ 계산치 349.07, 실측치 349.05. Preparation 1-4: 6-chloro- N 4 -(3-chloro-4-fluorobenzyl)pyrimidine-2,4,5-triamine (4d). Yield: 68%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.54 - 7.49 (m, 1H), 7.39 - 7.30 (m, 2H), 7.09 (t, J = 6.0 Hz, 1H), 5.67 (s, 2H), 4.52 (d, J = 5.9 Hz, 2H), 3.90 (s, 2H); MS (ESI, m/z) C 13 H 13 ClF 3 N 5 O [M+H] + cal 349.07, found 349.05.

제조예 1-5: 6-클로로- N 4 -(3,4-디플루오로벤질)피리미딘-2,4,5-트리아민 (4e). 수율: 84%; 1H NMR (400 MHz, DMSO-d 6) δ 7.42 - 7.32 (m, 2H), 7.17 (ddt, J = 7.5, 5.3, 2.3 Hz, 1H), 7.09 (t, J = 6.0 Hz, 1H), 5.67 (s, 2H), 4.52 (d, J = 5.9 Hz, 2H), 3.90 (s, 2H); MS (ESI, m/z) C11H10ClF2N5 [M+H]+ 계산치 287.06, 실측치 287.00. Preparation 1-5: 6-chloro- N 4 -(3,4-difluorobenzyl)pyrimidine-2,4,5-triamine (4e). Yield: 84%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.42 - 7.32 (m, 2H), 7.17 (ddt, J = 7.5, 5.3, 2.3 Hz, 1H), 7.09 (t, J = 6.0 Hz, 1H), 5.67 (s, 2H), 4.52 (d, J = 5.9 Hz, 2H), 3.90 (s, 2H); MS (ESI, m/z) C 11 H 10 ClF 2 N 5 [M+H] + calculated 287.06, found 287.00.

제조예 1-6: 6-클로로- N 4 -(4-(트리플루오로메틸)벤질)피리미딘-2,4,5-트리아민 (4f). 수율: 53%; 1H NMR (400 MHz, CDCl3) δ 7.62 - 7.58 (m, 2H), 7.43 (dp, J = 8.1, 0.8 Hz, 2H), 5.82 (s, 1H), 4.67 (d, J = 6.0 Hz, 2H), 4.64 (s, 2H), 2.73 (s, 2H); MS (ESI, m/z) C12H11ClF3N5 [M+H]+ 계산치 319.06, 실측치 319.00. Preparation 1-6: 6-chloro- N 4 -(4-(trifluoromethyl)benzyl)pyrimidine-2,4,5-triamine (4f). Yield: 53%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.62 - 7.58 (m, 2H), 7.43 (dp, J = 8.1, 0.8 Hz, 2H), 5.82 (s, 1H), 4.67 (d, J = 6.0 Hz, 2H), 4.64 (s, 2H), 2.73 (s, 2H); MS (ESI, m/z) C 12 H 11 ClF 3 N 5 [M+H] + calculated 319.06, found 319.00.

제조예 1-7: 6-클로로- N 4 -(5-클로로-2-플루오로벤질)피리미딘-2,4,5-트리아민 (4g). 수율: 32%; 1H NMR (400 MHz, CDCl3) δ 7.33 (dd, J = 6.4, 2.7 Hz, 1H), 7.21 (ddd, J = 8.7, 4.5, 2.7 Hz, 1H), 7.03 - 6.97 (m, 1H), 5.75 (s, 1H), 4.64 (d, J = 1.1 Hz, 2H), 4.61 (s, 2H), 2.72 (s, 2H); MS (ESI, m/z) C11H10Cl2FN5 [M+H]+ 계산치 303.03, 실측치 302.95. Preparation 1-7: 6-chloro- N 4 -(5-chloro-2-fluorobenzyl)pyrimidine-2,4,5-triamine (4g). Yield: 32%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.33 (dd, J = 6.4, 2.7 Hz, 1H), 7.21 (ddd, J = 8.7, 4.5, 2.7 Hz, 1H), 7.03 - 6.97 (m, 1H), 5.75 (s, 1H), 4.64 (d, J = 1.1 Hz, 2H), 4.61 (s, 2H), 2.72 (s, 2H); MS (ESI, m/z) C 11 H 10 Cl 2 FN 5 [M+H] + cal 303.03, found 302.95.

제조예 1-8: 6-클로로- N 4 -(4-클로로-3-(트리플루오로메틸)벤질)피리미딘-2,4,5-트리아민 (4h). 수율: 30%; 1H NMR (400 MHz, CDCl3) δ 7.65 (dq, J = 2.1, 0.7 Hz, 1H), 7.48 - 7.41 (m, 2H), 5.81 (s, 1H), 4.62 (t, J = 5.2 Hz, 4H), 2.72 (s, 2H); MS (ESI, m/z) C12H11Cl2F3N5 [M+H]+ 계산치 352.03, 실측치 352.50. Preparation 1-8: 6-chloro- N 4 -(4-chloro-3-(trifluoromethyl)benzyl)pyrimidine-2,4,5-triamine (4h). Yield: 30%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.65 (dq, J = 2.1, 0.7 Hz, 1H), 7.48 - 7.41 (m, 2H), 5.81 (s, 1H), 4.62 (t, J = 5.2 Hz, 4H), 2.72 (s, 2H); MS (ESI, m/z) C 12 H 11 Cl 2 F 3 N 5 [M+H] + calculated 352.03, found 352.50.

제조예 1-9: 6-클로로- N 4 -(2-클로로-5-(트리플루오로메틸)벤질)피리미딘-2,4,5-트리아민 (4i). 수율: 25%; 1H NMR (400 MHz, CDCl3) δ 7.27 (d, J = 0.6 Hz, 1H), 7.25 (t, J = 0.6 Hz, 1H), 6.90 - 6.85 (m, 2H), 5.57 (s, 1H), 4.60 (s, 2H), 4.52 (d, J = 5.6 Hz, 2H), 3.81 (s, 3H), 2.69 (s, 2H); MS (ESI, m/z) C12H11Cl2F3N5 [M+H]+ 계산치 352.03, 실측치 352.35. Preparation 1-9: 6-chloro- N 4 -(2-chloro-5-(trifluoromethyl)benzyl)pyrimidine-2,4,5-triamine (4i). Yield: 25%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.27 (d, J = 0.6 Hz, 1H), 7.25 (t, J = 0.6 Hz, 1H), 6.90 - 6.85 (m, 2H), 5.57 (s, 1H) , 4.60 (s, 2H), 4.52 (d, J = 5.6 Hz, 2H), 3.81 (s, 3H), 2.69 (s, 2H); MS (ESI, m/z) C 12 H 11 Cl 2 F 3 N 5 [M+H] + calculated 352.03, found 352.35.

제조예 1-10: 6-클로로- N 4 -(3,4-디클로로벤질)피리미딘-2,4,5-트리아민 (4j). 수율: 73%; 1H NMR (400 MHz, DMSO-d 6) δ 7.59 - 7.55 (m, 2H), 7.31 (dd, J = 8.3, 2.1 Hz, 1H), 7.12 (t, J = 6.0 Hz, 1H), 5.67 (s, 2H), 4.53 (d, J = 5.9 Hz, 2H), 3.90 (s, 2H); MS (ESI, m/z) C11H11Cl3N5 [M+H]+ 계산치 318.01, 실측치 318.95. Preparation 1-10: 6-chloro- N 4 -(3,4-dichlorobenzyl)pyrimidine-2,4,5-triamine (4j). Yield: 73%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.59 - 7.55 (m, 2H), 7.31 (dd, J = 8.3, 2.1 Hz, 1H), 7.12 (t, J = 6.0 Hz, 1H), 5.67 ( s, 2H), 4.53 (d, J = 5.9 Hz, 2H), 3.90 (s, 2H); MS (ESI, m/z) C 11 H 11 Cl 3 N 5 [M+H] + cal 318.01, found 318.95.

제조예 1-11: 6-클로로- N 4 -(5-플루오로-4-메톡시-2-메틸벤질)피리미딘-2,4,5-트리아민 (4k). 1H NMR (400 MHz, CDCl3) δ 6.50 - 6.41 (m, 2H), 5.55 (s, 1H), 4.62 (s, 2H), 4.59 (dd, J = 5.6, 1.0 Hz, 2H), 3.78 (s, 3H), 2.69 (s, 2H); MS (ESI, m/z) C12H13ClF2N5O [M+H]+ 계산치 316.08, 실측치 317.00. Preparation 1-11: 6-chloro- N 4 -(5-fluoro-4-methoxy-2-methylbenzyl)pyrimidine-2,4,5-triamine (4k). 1 H NMR (400 MHz, CDCl 3 ) δ 6.50 - 6.41 (m, 2H), 5.55 (s, 1H), 4.62 (s, 2H), 4.59 (dd, J = 5.6, 1.0 Hz, 2H), 3.78 ( s, 3H), 2.69 (s, 2H); MS (ESI, m/z) C 12 H 13 ClF 2 N 5 O [M+H] + cal 316.08, found 317.00.

제조예 1-12: 6-클로로- N 4 -(2-플루오로-4-메틸벤질)피리미딘-2,4,5-트리아민 (4l). 수율: 66%; 1H NMR (400 MHz, CDCl3) δ 7.23 (t, J = 7.8 Hz, 1H), 6.93 - 6.85 (m, 2H), 5.65 (s, 1H), 4.61 (d, J = 5.9 Hz, 4H), 2.70 (s, 2H); MS (ESI, m/z) C12H14ClFN5 [M+H]+ 계산치 282.09, 실측치 282.05. Preparation 1-12: 6-chloro- N 4 -(2-fluoro-4-methylbenzyl)pyrimidine-2,4,5-triamine (41). Yield: 66%; 1 H NMR (400 MHz, CDCl 3 ) δ 7.23 (t, J = 7.8 Hz, 1H), 6.93 - 6.85 (m, 2H), 5.65 (s, 1H), 4.61 (d, J = 5.9 Hz, 4H) , 2.70 (s, 2H); MS (ESI, m/z) C 12 H 14 ClFN 5 [M+H] + calculated 282.09, found 282.05.

제조예 1-13: 6-클로로- N 4 -(2,3,6-트리플루오로벤질)피리미딘-2,4,5-트리아민 (4m). 수율: 80%; 1H NMR (400 MHz, DMSO-d 6) δ 7.47 (dtd, J = 10.1, 9.2, 5.1 Hz, 1H), 7.15 (tdd, J = 9.2, 3.9, 2.2 Hz, 1H), 6.82 (t, J = 4.9 Hz, 1H), 5.61 (s, 2H), 4.58 (d, J = 4.8 Hz, 2H), 3.94 (s, 2H); MS (ESI, m/z) C11H9ClF3N5 [M+H]+ 계산치 304.06, 실측치 305.00. Preparation 1-13: 6-chloro- N 4 -(2,3,6-trifluorobenzyl)pyrimidine-2,4,5-triamine (4m). Yield: 80%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.47 (dtd, J = 10.1, 9.2, 5.1 Hz, 1H), 7.15 (tdd, J = 9.2, 3.9, 2.2 Hz, 1H), 6.82 (t, J) = 4.9 Hz, 1H), 5.61 (s, 2H), 4.58 (d, J = 4.8 Hz, 2H), 3.94 (s, 2H); MS (ESI, m/z) C 11 H 9 ClF 3 N 5 [M+H] + cal 304.06, found 305.00.

실시예 1: 화합물 5a 내지 5o의 합성Example 1: Synthesis of compounds 5a to 5o

무수 DMF (3 mL) 중 화합물 4 (0.4 mmol) 및 카르보닐디이미다졸 (0.48 mmol)을 질소 대기 하에 실온에서 3-4 시간 동안 교반하였다. 물 (20 mL) 첨가 후, 유기 물질을 EtOAc (20 mL x 3)로 추출하고, MgSO4로 건조시키고 여과하였다. 여과물을 진공하에 농축시키고 잔류물을 실리카 겔 컬럼 크로마토그래피 (EtOAc/Hex)로 정제하여 화합물 5a 내지 5o를 수득하였다. Compound 4 (0.4 mmol) and carbonyldiimidazole (0.48 mmol) in anhydrous DMF (3 mL) were stirred under nitrogen atmosphere at room temperature for 3-4 h. After addition of water (20 mL), the organics were extracted with EtOAc (20 mL×3), dried over MgSO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by silica gel column chromatography (EtOAc/Hex) to give compounds 5a to 5o .

실시예 1-1: 2-아미노-9-((6-브로모벤조[Example 1-1: 2-amino-9-((6-bromobenzo[ dd ][1,3]디옥솔-5-일)메틸)-6-클로로-9][1,3]dioxol-5-yl)methyl)-6-chloro-9 HH -퓨린-8-올 (5a)-purine-8-ol (5a)

1H NMR (400 MHz, DMSO-d 6) δ 11.38 (s, 1H), 7.26 (s, 1H), 6.58 (s, 2H), 6.04 (s, 2H), 5.75 (s, 1H), 4.77 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 158.6, 152.9, 152.5, 147.5, 147.5, 135.8, 128.1, 112.5, 111.7, 110.5, 107.5, 102.0, 43.0; MS (ESI, m/z) C13H9BrClN5O3 [M+H]+ 계산치 399.9628, 실측치 399.9631. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.38 (s, 1H), 7.26 (s, 1H), 6.58 (s, 2H), 6.04 (s, 2H), 5.75 (s, 1H), 4.77 ( s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 158.6, 152.9, 152.5, 147.5, 147.5, 135.8, 128.1, 112.5, 111.7, 110.5, 107.5, 102.0, 43.0; MS (ESI, m/z) C 13 H 9 BrClN 5 O 3 [M+H] + cal 399.9628, found 399.9631.

실시예 1-2: 2-아미노-9-(4-브로모-2-플루오로벤질)-6-클로로-9Example 1-2: 2-Amino-9- (4-bromo-2-fluorobenzyl) -6-chloro-9 HH -퓨린-8-올 (5b)-purine-8-ol (5b)

1H NMR (400 MHz, DMSO-d 6) δ 11.40 (s, 1H), 7.57 (dd, J = 9.8, 1.9 Hz, 1H), 7.46 - 7.31 (m, 1H), 7.16 (t, J = 8.3 Hz, 1H), 6.63 (s, 2H), 4.89 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 159.6 (d, J = 250.5 Hz), 158.6, 152.8, 152.3, 135.9, 130.7 (d, J = 4.5 Hz), 127.8 (d, J = 3.3 Hz), 122.9 (d, J = 14.4 Hz), 120.9 (d, J = 9.6 Hz), 118.8 (d, J = 24.5 Hz), 110.2, 36.4 (d, J = 5.1 Hz); HRMS (ESI, m/z) C12H8BrClFN5O [M+H]+ 계산치 373.9636, 실측치 373.9637. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.40 (s, 1H), 7.57 (dd, J = 9.8, 1.9 Hz, 1H), 7.46 - 7.31 (m, 1H), 7.16 (t, J = 8.3) Hz, 1H), 6.63 (s, 2H), 4.89 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 159.6 (d, J = 250.5 Hz), 158.6, 152.8, 152.3, 135.9, 130.7 (d, J = 4.5 Hz), 127.8 (d, J = 3.3 Hz) , 122.9 (d, J = 14.4 Hz), 120.9 (d, J = 9.6 Hz), 118.8 (d, J = 24.5 Hz), 110.2, 36.4 (d, J = 5.1 Hz); HRMS (ESI, m/z) C 12 H 8 BrClFN 5 O [M+H] + calculated 373.9636, found 373.9637.

실시예 1-3: 2-아미노-6-클로로-9-(2-메톡시-4-(트리플루오로메틸)벤질)-9Example 1-3: 2-Amino-6-chloro-9- (2-methoxy-4- (trifluoromethyl) benzyl) -9 HH -퓨린-8-올 (5c)-purine-8-ol (5c)

1H NMR (400 MHz, DMSO-d 6) δ 11.42 (s, 1H), 7.30 (d, J = 1.7 Hz, 1H), 7.22 (dd, J = 7.9, 1.6 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H), 6.59 (s, 2H), 4.87 (s, 2H), 3.94 (s, 3H); HRMS (ESI, m/z) C14H11ClF3N5O2 [M+H]+ 계산치 374.0626, 실측치 374.0630. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.42 (s, 1H), 7.30 (d, J = 1.7 Hz, 1H), 7.22 (dd, J = 7.9, 1.6 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H), 6.59 (s, 2H), 4.87 (s, 2H), 3.94 (s, 3H); HRMS (ESI, m/z) C 14 H 11 ClF 3 N 5 O 2 [M+H] + cal 374.0626, found 374.0630.

실시예 1-4: 2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9Example 1-4: 2-Amino-6-chloro-9- (3-chloro-4-fluorobenzyl)-9 HH -퓨린-8-올 (5d)-purine-8-ol (5d)

1H NMR (400 MHz, DMSO-d 6) δ 11.38 (s, 1H), 7.52 (dd, J = 7.1, 2.2 Hz, 1H), 7.38 (dd, J = 9.4, 8.5 Hz, 1H), 7.28 (ddd, J = 8.5, 4.7, 2.2 Hz, 1H), 6.63 (s, 2H), 4.87 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 158.6, 156.6 (d, J = 246.0 Hz), 152.8, 152.3, 135.8, 134.4 (d, J = 3.6 Hz), 129.3, 127.9 (d, J = 7.6 Hz), 119.5 (d, J = 17.8 Hz), 117.1 (d, J = 21.1 Hz), 110.2, 41.5; HRMS (ESI, m/z) C12H8Cl2FN5O [M+H]+ 계산치 328.0163, 실측치 328.0166. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.38 (s, 1H), 7.52 (dd, J = 7.1, 2.2 Hz, 1H), 7.38 (dd, J = 9.4, 8.5 Hz, 1H), 7.28 ( ddd, J = 8.5, 4.7, 2.2 Hz, 1H), 6.63 (s, 2H), 4.87 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 158.6, 156.6 (d, J = 246.0 Hz), 152.8, 152.3, 135.8, 134.4 (d, J = 3.6 Hz), 129.3, 127.9 (d, J = 7.6) Hz), 119.5 (d, J = 17.8 Hz), 117.1 (d, J = 21.1 Hz), 110.2, 41.5; HRMS (ESI, m/z) C 12 H 8 Cl 2 FN 5 O [M+H] + calculated 328.0163, found 328.0166.

실시예 1-5: 2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9Example 1-5: 2-Amino-6-chloro-9- (3-chloro-4-fluorobenzyl) -9 HH -퓨린-8-올 (5e)-purine-8-ol (5e)

1H NMR (400 MHz, DMSO-d 6) δ 11.36 (s, 1H), 7.44 - 7.33 (m, 2H), 7.13 - 7.07 (m, 1H), 6.62 (s, 2H), 4.86 (s, 2H); HRMS (ESI, m/z) C12H8ClF2N5O [M+H]+ 계산치 312.0461, 실측치 312.0458. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.36 (s, 1H), 7.44 - 7.33 (m, 2H), 7.13 - 7.07 (m, 1H), 6.62 (s, 2H), 4.86 (s, 2H) ); HRMS (ESI, m/z) C 12 H 8 ClF 2 N 5 O [M+H] + cal 312.0461, found 312.0458.

실시예 1-6: 2-아미노-6-클로로-9-(4-(트리플루오로메틸)벤질)-9Example 1-6: 2-Amino-6-chloro-9-(4-(trifluoromethyl)benzyl)-9 HH -퓨린-8-올 (5f)-purine-8-ol (5f)

1H NMR (400 MHz, DMSO-d 6) δ 11.41 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 6.64 (s, 2H), 4.98 (s, 2H); HRMS (ESI, m/z) C13H9ClF3N5O [M+H]+ 계산치 344.0520, 실측치 344.0525. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.41 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 6.64 (s, 2H) , 4.98 (s, 2H); HRMS (ESI, m/z) C 13 H 9 ClF 3 N 5 O [M+H] + calculated 344.0520, found 344.0525.

실시예 1-7: 2-아미노-6-클로로-9-(5-클로로-2-플루오로벤질)-9Example 1-7: 2-Amino-6-chloro-9- (5-chloro-2-fluorobenzyl) -9 HH -퓨린-8-올 (5g)-Purine-8-ol (5g)

수율: 30%, 흰색 고체 1H NMR (400 MHz, DMSO-d 6) δ 11.38 (s, 1H), 7.46 - 7.37 (m, 1H), 7.34 - 7.23 (m, 2H), 6.61 (s, 2H), 4.91 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 158.6, 158.4 (d, J = 246.0 Hz), 152.8, 152.3, 129.4 (d, J = 8.5 Hz), 128.6 (d, J = 4.2 Hz), 128.4 (d, J = 3.2 Hz), 125.5 (d, J = 16.3 Hz), 117.4 (d, J = 23.3 Hz), 110.4, 36.5 (d, J = 4.7 Hz); HRMS (ESI, m/z) C12H8Cl2FN5O [M+H]+ 계산치 328.0163, 실측치 328.0164.Yield: 30%, white solid 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.38 (s, 1H), 7.46 - 7.37 (m, 1H), 7.34 - 7.23 (m, 2H), 6.61 (s, 2H) ), 4.91 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 158.6, 158.4 (d, J = 246.0 Hz), 152.8, 152.3, 129.4 (d, J = 8.5 Hz), 128.6 (d, J = 4.2 Hz), 128.4 (d, J = 3.2 Hz), 125.5 (d, J = 16.3 Hz), 117.4 (d, J = 23.3 Hz), 110.4, 36.5 (d, J = 4.7 Hz); HRMS (ESI, m/z) C 12 H 8 Cl 2 FN 5 O [M+H] + calculated 328.0163, found 328.0164.

실시예 1-8: 2-아미노-6-클로로-9-(4-클로로-3-(트리플루오로메틸)벤질)-9Example 1-8: 2-Amino-6-chloro-9- (4-chloro-3- (trifluoromethyl) benzyl) -9 HH -퓨린-8-올 (5h)-purine-8-ol (5h)

1H NMR (400 MHz, DMSO-d 6) δ 11.40 (s, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.54 (dd, J = 8.4, 2.1 Hz, 1H), 6.60 (s, 2H), 4.95 (s, 2H); HRMS (ESI, m/z) C13H8Cl2F3N5O [M+H]+ 계산치 378.0131, 실측치 378.0133. 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.40 (s, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.54 (dd, J = 8.4, 2.1 Hz, 1H), 6.60 (s, 2H), 4.95 (s, 2H); HRMS (ESI, m/z) C 13 H 8 C l2 F 3 N 5 O [M+H] + calculated 378.0131, found 378.0133.

실시예 1-9: 2-아미노-6-클로로-9-(2-클로로-5-(트리플루오로메틸)벤질)-9Example 1-9: 2-Amino-6-chloro-9- (2-chloro-5- (trifluoromethyl) benzyl) -9 HH -퓨린-8-올 (5i)-purine-8-ol (5i)

수율: 52%; 1H NMR (400 MHz, DMSO-d 6) δ 11.43 (s, 1H), 7.83 - 7.68 (m, 2H), 7.44 (d, J = 2.2 Hz, 1H), 6.63 (s, 2H), 5.01 (s, 2H); 13C NMR (101 MHz, DMSO- d 6) δ 158.6, 152.9, 152.5, 136.0, 135.9, 135.0, 130.7, 128.1 (q, J = 32.3 Hz), 126.1 (q, J = 3.7 Hz), 124.7 (q, J = 3.6 Hz), 123.6 (q, J = 272.3 Hz), 110.4, 40.7; HRMS (ESI, m/z) C13H8Cl2F3N5O [M+H]+ 계산치 378.0131, 실측치 378.0134.Yield: 52%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.43 (s, 1H), 7.83 - 7.68 (m, 2H), 7.44 (d, J = 2.2 Hz, 1H), 6.63 (s, 2H), 5.01 ( s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 158.6, 152.9, 152.5, 136.0, 135.9, 135.0, 130.7, 128.1 (q, J = 32.3 Hz), 126.1 (q, J = 3.7 Hz), 124.7 (q , J = 3.6 Hz), 123.6 (q, J = 272.3 Hz), 110.4, 40.7; HRMS (ESI, m/z) C 13 H 8 C 12 F 3 N 5 O [M+H] + calc. 378.0131, found 378.0134.

실시예 1-10: 2-아미노-6-클로로-9-(3,4-디클로로벤질)-9Example 1-10: 2-Amino-6-chloro-9- (3,4-dichlorobenzyl) -9 HH -퓨린-8-올 (5j)-purine-8-ol (5j)

수율: 60%; 1H NMR (400 MHz, DMSO-d 6) δ 11.38 (s, 1H), 7.64 - 7.53 (m, 2H), 7.24 (dd, J = 8.3, 2.1 Hz, 1H), 6.62 (s, 2H), 4.88 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 158.6, 152.8, 152.3, 137.6, 135.8, 131.1, 130.8, 130.1, 129.2, 127.4, 110.2, 41.5; HRMS (ESI, m/z) C12H8Cl3N5O [M+H]+ 계산치 343.9867, 실측치 343.9872.Yield: 60%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.38 (s, 1H), 7.64 - 7.53 (m, 2H), 7.24 (dd, J = 8.3, 2.1 Hz, 1H), 6.62 (s, 2H), 4.88 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 158.6, 152.8, 152.3, 137.6, 135.8, 131.1, 130.8, 130.1, 129.2, 127.4, 110.2, 41.5; HRMS (ESI, m/z) C 12 H 8 Cl 3 N 5 O [M+H] + calculated 343.9867, found 343.9872.

실시예 1-11: 2-아미노-6-클로로-9-(2,6-디플루오로-4-메톡시벤질)-9Example 1-11: 2-Amino-6-chloro-9-(2,6-difluoro-4-methoxybenzyl)-9 HH -퓨린-8-올 (5k)-purine-8-ol (5k)

수율: 20%; 1H NMR (400 MHz, DMSO-d 6) δ 11.26 (s, 1H), 6.70 (s, 1H), 6.68 (s, 1H), 4.84 (s, 1H), 3.76 (s, 1H) ; 13C NMR (101 MHz, DMSO-d 6) δ 161.5 (dd, J = 253.5 Hz), 160.4 (t, 14.6 Hz), 158.5, 152.2, 152.1, 135.5, 110.0, 103.4 (t, J = 19.0 Hz), 98.2 (d, J = 28.4 Hz), 56.0, 31.8-31.5 (t, J = 3.0 Hz); HRMS (ESI, m/z) C13H10ClF2N5O2 [M+H]+ 계산치 342.0564, 실측치 342.0567.Yield: 20%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.26 (s, 1H), 6.70 (s, 1H), 6.68 (s, 1H), 4.84 (s, 1H), 3.76 (s, 1H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 161.5 (dd, J = 253.5 Hz), 160.4 (t, 14.6 Hz), 158.5, 152.2, 152.1, 135.5, 110.0, 103.4 (t, J = 19.0 Hz) , 98.2 (d, J = 28.4 Hz), 56.0, 31.8-31.5 (t, J = 3.0 Hz); HRMS (ESI, m/z) C 13 H 10 ClF 2 N 5 O 2 [M+H] + calculated 342.0564, found 342.0567.

실시예 1-12: 2-아미노-6-클로로-9-(2-플루오로-4-메틸벤질)-9Example 1-12: 2-Amino-6-chloro-9-(2-fluoro-4-methylbenzyl)-9 HH -퓨린-8-올 (5l)-purine-8-ol (5l)

수율: 55%, 1H NMR (400 MHz, DMSO-d 6) δ 11.36 (s, 1H), 7.07 - 7.01 (m, 2H), 6.95 (ddd, J = 7.9, 1.6, 0.8 Hz, 1H), 6.61 (s, 2H), 4.88 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 159.5 (d, J = 244.8 Hz), 158.6, 152.8, 152.4, 139.6 (d, J = 7.9 Hz), 135.8, 128.6 (d, J = 4.4 Hz), 125.1 (d, J = 3.0 Hz), 120.0 (d, J = 14.6 Hz), 115.7 (d, J = 20.7 Hz), 110.1, 36.5 (d, J = 4.6 Hz), 20.5 (d, J = 1.6 Hz); HR HRMS (ESI, m/z) C13H11ClFN5O [M+H]+ 계산치 308.0709, 실측치 308.0711.Yield: 55%, 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.36 (s, 1H), 7.07 - 7.01 (m, 2H), 6.95 (ddd, J = 7.9, 1.6, 0.8 Hz, 1H), 6.61 (s, 2H), 4.88 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 159.5 (d, J = 244.8 Hz), 158.6, 152.8, 152.4, 139.6 (d, J = 7.9 Hz), 135.8, 128.6 (d, J = 4.4 Hz) , 125.1 (d, J = 3.0 Hz), 120.0 (d, J = 14.6 Hz), 115.7 (d, J = 20.7 Hz), 110.1, 36.5 (d, J = 4.6 Hz), 20.5 (d, J = 1.6) Hz); HR HRMS (ESI, m/z) C 13 H 11 ClFN 5 O [M+H] + calculated 308.0709, found 308.0711.

실시예 1-13: 2-아미노-6-클로로-9-(2,3,6-트리플루오로벤질)-9Example 1-13: 2-Amino-6-chloro-9-(2,3,6-trifluorobenzyl)-9 HH -퓨린-8-올 (5m)-Purine-8-ol (5m)

수율: 20%; 1H NMR (400 MHz, DMSO-d 6) δ 11.30 (s, 1H), 7.48 (qd, J = 9.5, 5.0 Hz, 2H), 7.12 (tdd, J = 9.2, 3.8, 2.1 Hz, 2H), 6.57 (s, 2H), 4.97 (s, 2H); HRMS (ESI, m/z) C12H7ClF3N5O [M+H]+ 계산치 330.0364, 실측치 330.0365.Yield: 20%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 11.30 (s, 1H), 7.48 (qd, J = 9.5, 5.0 Hz, 2H), 7.12 (tdd, J = 9.2, 3.8, 2.1 Hz, 2H), 6.57 (s, 2H), 4.97 (s, 2H); HRMS (ESI, m/z) C 12 H 7 ClF 3 N 5 O [M+H] + cal 330.0364, found 330.0365.

실시예 2: 화합물 6a 내지 6g 및 6j 내지 6o의 합성Example 2: Synthesis of compounds 6a to 6g and 6j to 6o

무수 DMF (3 mL) 중 화합물 3 (0.4 mmol) 및 티오카르보닐디이미다졸 (0.48 mmol)을 질소 대기하에 실온에서 3-4 시간 동안 교반하였다. 물 (20 mL)을 첨가 한 후, 유기 물질을 EtOAc (20 mL x 3)로 추출하고, MgSO4로 건조시키고 여과하였다. 여과물을 진공하에 농축시키고, 잔류물을 실리카 겔 컬럼 크로마토그래피 (EtOAc / Hex)로 정제하여 화합물 6a-g, 6j-o를 수득하였다. Compound 3 (0.4 mmol) and thiocarbonyldiimidazole (0.48 mmol) in anhydrous DMF (3 mL) were stirred under nitrogen atmosphere at room temperature for 3-4 h. After addition of water (20 mL), the organics were extracted with EtOAc (20 mL×3), dried over MgSO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography (EtOAc / Hex) to give compounds 6a-g , 6j-o obtained.

실시예 2-1: 2-아미노-9-((6-브로모벤조[Example 2-1: 2-amino-9-((6-bromobenzo[ dd ][1,3]디옥솔-5-일)메틸)-6-클로로-9][1,3]dioxol-5-yl)methyl)-6-chloro-9 HH -퓨린-8-티올 (6a)-purine-8-thiol (6a)

수율: 18%; 1H NMR (400 MHz, DMSO-d 6) δ 13.43 (s, 1H), 7.27 (s, 1H), 6.86 (s, 1H), 6.30 (s, 1H), 6.02 (s, 1H), 5.14 (s, 1H); 13C NMR (101 MHz, DMSO-d 6) δ 169.6, 159.5, 153.6, 147.4, 147.3, 137.9, 127.3, 113.4, 112.4, 111.4, 107.0, 101.9, 45.5; HRMS (ESI, m/z) C13H9BrClN5O2S [M+H]+ 계산치 413.9422, 실측치 413.9401.Yield: 18%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.43 (s, 1H), 7.27 (s, 1H), 6.86 (s, 1H), 6.30 (s, 1H), 6.02 (s, 1H), 5.14 ( s, 1H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.6, 159.5, 153.6, 147.4, 147.3, 137.9, 127.3, 113.4, 112.4, 111.4, 107.0, 101.9, 45.5; HRMS (ESI, m/z) C 13 H 9 BrClN 5 O 2 S [M+H] + calculated 413.9422, found 413.9401.

실시예 2-2: 2-아미노-9-(4-브로모-2-플루오로벤질)-6-클로로-9Example 2-2: 2-Amino-9- (4-bromo-2-fluorobenzyl) -6-chloro-9 HH -퓨린-8-티올 (6b)-purine-8-thiol (6b)

수율: 15%; 1H NMR (400 MHz, DMSO-d 6) δ 13.45 (s, 1H), 7.58 (dd, J = 9.8, 2.0 Hz, 1H), 7.35 - 7.28 (m, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.93 (d, J = 7.9 Hz, 2H), 5.26 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 169.6, 159.7, 159.5 (d, J = 250.1 Hz), 153.6, 138.0, 130.1 (d, J = 4.6 Hz), 127.7 (d, J = 3.6 Hz), 122.3 (d, J = 14.3 Hz), 120.7 (d, J = 9.6 Hz), 118.7 (d, J = 24.5 Hz), 113.1, 39.0 (d, J = 4.6 Hz); HRMS (ESI, m/z) C12H8BrClFN5S [M+H]+ 계산치 389.9406, 실측치 389.9405.Yield: 15%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.45 (s, 1H), 7.58 (dd, J = 9.8, 2.0 Hz, 1H), 7.35 - 7.28 (m, 1H), 6.97 (d, J = 8.2) Hz, 1H), 6.93 (d, J = 7.9 Hz, 2H), 5.26 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.6, 159.7, 159.5 (d, J = 250.1 Hz), 153.6, 138.0, 130.1 (d, J = 4.6 Hz), 127.7 (d, J = 3.6 Hz) , 122.3 (d, J = 14.3 Hz), 120.7 (d, J = 9.6 Hz), 118.7 (d, J = 24.5 Hz), 113.1, 39.0 (d, J = 4.6 Hz); HRMS (ESI, m/z) C 12 H 8 BrClFN 5 S [M+H] + calculated 389.9406, found 389.9405.

실시예 2-3: 2-아미노-6-클로로-9-(2-메톡시-4-(트리플루오로메틸)벤질)-9Example 2-3: 2-Amino-6-chloro-9- (2-methoxy-4- (trifluoromethyl) benzyl) -9 HH -퓨린-8-티올 (6c)-purine-8-thiol (6c)

수율: 25%; 1H NMR (400 MHz, DMSO-d 6) δ 13.48 (s, 1H), 7.32 (d, J = 1.7 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 6.94 (s, 2H), 6.77 (d, J = 7.9 Hz, 1H), 5.25 (s, 2H), 3.98 (s, 3H); HRMS (ESI, m/z) C14H11ClF3N5OS [M+H]+ 계산치 390.0398, 실측치 390.0401.Yield: 25%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.48 (s, 1H), 7.32 (d, J = 1.7 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 6.94 (s, 2H) , 6.77 (d, J = 7.9 Hz, 1H), 5.25 (s, 2H), 3.98 (s, 3H); HRMS (ESI, m/z) C 14 H 11 ClF 3 N 5 OS [M+H] + calculated 390.0398, found 390.0401.

실시예 2-4: 2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9Example 2-4: 2-Amino-6-chloro-9- (3-chloro-4-fluorobenzyl) -9 HH -퓨린-8-티올 (6d)-purine-8-thiol (6d)

수율: 20%; 1H NMR (400 MHz, DMSO-d 6) δ 13.41 (s, 1H), 7.56 (dd, J = 7.2, 2.2 Hz, 1H), 7.40 - 7.25 (m, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 169.5, 159.6, 156.5 (d, J = 246.3 Hz), 153.5, 138.0, 133.7 (d, J = 3.9 Hz), 129.6, 128.2 (d, J = 7.7 Hz), 119.4 (d, J = 17.9 Hz), 116.9 (d, J = 21.1 Hz), 113.2, 44.0; HRMS (ESI, m/z) C12H8Cl2FN5S [M+H]+ 계산치 343.9934, 실측치 343.9936.Yield: 20%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.41 (s, 1H), 7.56 (dd, J = 7.2, 2.2 Hz, 1H), 7.40 - 7.25 (m, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.5, 159.6, 156.5 (d, J = 246.3 Hz), 153.5, 138.0, 133.7 (d, J = 3.9 Hz), 129.6, 128.2 (d, J = 7.7) Hz), 119.4 (d, J = 17.9 Hz), 116.9 (d, J = 21.1 Hz), 113.2, 44.0; HRMS (ESI, m/z) C 12 H 8 Cl 2 FN 5 S [M+H] + calculated 343.9934, found 343.9936.

실시예 2-5: 2-아미노-6-클로로-9-(3,4-디플루오로벤질)-9Example 2-5: 2-Amino-6-chloro-9- (3,4-difluorobenzyl) -9 HH -퓨린-8-티올 (6e)-purine-8-thiol (6e)

수율: 15%; 1H NMR (400 MHz, DMSO-d 6) δ 13.41 (s, 1H), 7.48 - 7.30 (m, 2H), 7.15 (s, 1H), 6.92 (s, 2H), 5.25 (s, 2H); HRMS (ESI, m/z) C12H8ClF2N5S [M+H]+ 계산치 328.0230, 실측치 328.0232.Yield: 15%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.41 (s, 1H), 7.48 - 7.30 (m, 2H), 7.15 (s, 1H), 6.92 (s, 2H), 5.25 (s, 2H); HRMS (ESI, m/z) C 12 H 8 ClF 2 N 5 S [M+H] + calculated 328.0230, found 328.0232.

실시예 2-6: 2-아미노-6-클로로-9-(4-(트리플루오로메틸)벤질)-9Example 2-6: 2-Amino-6-chloro-9-(4-(trifluoromethyl)benzyl)-9 HH -퓨린-8-티올 (6f)-purine-8-thiol (6f)

수율: 18%; 1H NMR (400 MHz, DMSO-d 6) δ 13.45 (s, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 6.96 (s, 2H), 5.37 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 169.7, 159.7, 153.6, 140.5, 138.0, 128.5 (q, J = 31.6 Hz), 126.8 (q, J = 268.7 Hz), 127.9, 125.4 (q, J = 3.5 Hz), 113.1, 44.7; HRMS (ESI, m/z) C13H9ClF3N5S [M+H]+ 계산치 360.0292, 실측치 360.0296. Yield: 18%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.45 (s, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 6.96 (s, 2H) , 5.37 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.7, 159.7, 153.6, 140.5, 138.0, 128.5 (q, J = 31.6 Hz), 126.8 (q, J = 268.7 Hz), 127.9, 125.4 (q, J) = 3.5 Hz), 113.1, 44.7; HRMS (ESI, m/z) C 13 H 9 ClF 3 N 5 S [M+H] + calculated 360.0292, found 360.0296.

실시예 2-7: 2-아미노-6-클로로-9-(2-클로로-5-(트리플루오로메틸)벤질)-9Example 2-7: 2-Amino-6-chloro-9- (2-chloro-5- (trifluoromethyl) benzyl) -9 HH -퓨린-8-티올 (6i)-purine-8-thiol (6i)

수율: 13%; 1H NMR (400 MHz, DMSO-d 6) δ 13.50 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.75 - 7.68 (m, 1H), 7.18 (d, J = 2.2 Hz, 1H), 6.96 (s, 2H), 5.36 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 169.8, 159.6, 153.8, 138.1, 135.8, 134.4, 130.7, 128.0 (q, J = 32.4 Hz), 126.0 (q, J = 3.8 Hz), 123.5 (d, J = 272.5 Hz), 124.0 (q, J = 3.7, 3.0 Hz), 113.5, 43.2; HRMS (ESI, m/z) C13H8Cl2F3N5S [M+H]+ 계산치 393.9902, 실측치 393.9907.Yield: 13%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.50 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.75 - 7.68 (m, 1H), 7.18 (d, J = 2.2 Hz, 1H), 6.96 (s, 2H), 5.36 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.8, 159.6, 153.8, 138.1, 135.8, 134.4, 130.7, 128.0 (q, J = 32.4 Hz), 126.0 (q, J = 3.8 Hz), 123.5 (d , J = 272.5 Hz), 124.0 (q, J = 3.7, 3.0 Hz), 113.5, 43.2; HRMS (ESI, m/z) C 13 H 8 C l2 F 3 N 5 S [M+H] + calculated 393.9902, found 393.9907.

실시예 2-8: 2-아미노-6-클로로-9-(3,4-디클로로벤질)-9Example 2-8: 2-Amino-6-chloro-9- (3,4-dichlorobenzyl) -9 HH -퓨린-8-티올 (6j)-purine-8-thiol (6j)

수율: 29%; 1H NMR (400 MHz, DMSO-d 6) δ 13.42 (s, 1H), 7.65 - 7.52 (m, 2H), 7.26 (dd, J = 8.3, 2.1 Hz, 1H), 6.96 (s, 2H), 5.26 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 169.5, 159.6, 153.5, 138.0, 136.9, 131.0, 130.7, 130.1, 129.4, 127.7, 113.2, 44.1; HRMS (ESI, m/z) C12H8Cl3N5S [M+H]+ 계산치 361.9610, 실측치 361.9614. Yield: 29%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.42 (s, 1H), 7.65 - 7.52 (m, 2H), 7.26 (dd, J = 8.3, 2.1 Hz, 1H), 6.96 (s, 2H), 5.26 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.5, 159.6, 153.5, 138.0, 136.9, 131.0, 130.7, 130.1, 129.4, 127.7, 113.2, 44.1; HRMS (ESI, m/z) C 12 H 8 Cl 3 N 5 S [M+H] + calculated 361.9610, found 361.9614.

실시예 2-9: 2-아미노-6-클로로-9-(2,6-디플루오로-4-메톡시벤질)-9Example 2-9: 2-Amino-6-chloro-9- (2,6-difluoro-4-methoxybenzyl) -9 HH -퓨린-8-티올 (6k)-purine-8-thiol (6k)

수율: 58%; 1H NMR (400 MHz, DMSO-d 6) δ 13.27 (s, 1H), 6.90 (s, 2H), 6.71 - 6.64 (m, 2H), 5.20 (s, 2H), 3.76 (s, 3H) ); 13C NMR (101 MHz, DMSO-d6) δ 169.4, 161.3 (dd, J = 246.3, 11.6 Hz), 160.3 (t, J = 14.4 Hz), 159.5, 153.7, 137.7, 112.8, 103.0 (t, J = 18.2 Hz), 99.3 (dd, J = 21.4, 7.6 Hz); HRMS (ESI, m/z) C13H10ClF2N5OS [M+H]+ 계산치 358.0335, 실측치 358.0335.Yield: 58%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.27 (s, 1H), 6.90 (s, 2H), 6.71 - 6.64 (m, 2H), 5.20 (s, 2H), 3.76 (s, 3H)) ; 13 C NMR (101 MHz, DMSO-d6) δ 169.4, 161.3 (dd, J = 246.3, 11.6 Hz), 160.3 (t, J = 14.4 Hz), 159.5, 153.7, 137.7, 112.8, 103.0 (t, J = 18.2 Hz), 99.3 (dd, J = 21.4, 7.6 Hz); HRMS (ESI, m/z) C 13 H 10 ClF 2 N 5 OS [M+H] + calculated 358.0335, found 358.0335.

실시예 2-10: 2-아미노-6-클로로-9-(2-플루오로-4-메틸벤질)-9Example 2-10: 2-Amino-6-chloro-9-(2-fluoro-4-methylbenzyl)-9 HH -퓨린-8-티올 (6l)-purine-8-thiol (6l)

수율: 43%; 1H NMR (400 MHz, DMSO-d 6) δ 13.42 (s, 1H), 7.05 (d, J = 11.4 Hz, 1H), 6.93 (s, 2H), 6.90 (d, J = 0.8 Hz, 1H), 6.83 (t, J = 8.0 Hz, 1H), 5.27 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 169.8, 159.7, 159.4 (d, J = 244.7 Hz), 153.7, 139.4 (d, J = 8.0 Hz), 137.9, 127.9 (d, J = 4.5 Hz), 125.0 (d, J = 3.0 Hz), 119.3 (d, J = 14.4 Hz), 115.6 (d, J = 20.7 Hz), 113.1, 20.5 (d, J = 1.6 Hz); HRMS (ESI, m/z) C13H11ClFN5S [M+H]+ 계산치 324.0480, 실측치 324.0481.Yield: 43%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.42 (s, 1H), 7.05 (d, J = 11.4 Hz, 1H), 6.93 (s, 2H), 6.90 (d, J = 0.8 Hz, 1H), 6.83 (t, J = 8.0 Hz, 1H) , 5.27 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.8, 159.7, 159.4 (d, J = 244.7 Hz), 153.7, 139.4 (d, J = 8.0 Hz), 137.9, 127.9 (d, J = 4.5 Hz) , 125.0 (d, J = 3.0 Hz), 119.3 (d, J = 14.4 Hz), 115.6 (d, J = 20.7 Hz), 113.1, 20.5 (d, J = 1.6 Hz); HRMS (ESI, m/z) C 13 H 11 ClFN 5 S [M+H] + calculated 324.0480, found 324.0481.

실시예 2-11: 2-아미노-6-클로로-9-(2,3,6-트리플루오로벤질)-9Example 2-11: 2-Amino-6-chloro-9-(2,3,6-trifluorobenzyl)-9 HH -퓨린-8-티올 (6m)-Purine-8-thiol (6m)

수율: 36%; 1H NMR (400 MHz, DMSO-d 6) δ 13.35 (s, 1H), 7.46 (qd, J = 9.4, 5.0 Hz, 1H), 7.09 (tdd, J = 9.5, 3.9, 2.2 Hz, 1H), 6.93 (s, 2H), 5.32 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 169.8, 159.61, 159.4 (d, J = 244.8 Hz), 153.6, 139.3 (d, J = 7.8 Hz), 137.9, 127.8 (d, J = 4.4 Hz), 124.9 (d, J = 3.0 Hz), 119.3 (d, J = 14.5 Hz), 115.5 (d, J = 20.6 Hz), 113.1, 39.0; HRMS (ESI, m/z) C12H7ClF3N5S [M+H]+ 계산치 346.0136, 실측치 346.0139. Yield: 36%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.35 (s, 1H), 7.46 (qd, J = 9.4, 5.0 Hz, 1H), 7.09 (tdd, J = 9.5, 3.9, 2.2 Hz, 1H), 6.93 (s, 2H), 5.32 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.8, 159.61, 159.4 (d, J = 244.8 Hz), 153.6, 139.3 (d, J = 7.8 Hz), 137.9, 127.8 (d, J = 4.4 Hz) , 124.9 (d, J = 3.0 Hz), 119.3 (d, J = 14.5 Hz), 115.5 (d, J = 20.6 Hz), 113.1, 39.0; HRMS (ESI, m/z) C 12 H 7 ClF 3 N 5 S [M+H] + calculated 346.0136, found 346.0139.

실시예 3: 화합물 7a, 7b, 7d 및 7e의 합성Example 3: Synthesis of compounds 7a, 7b, 7d and 7e

EtOH (3 mL) 중 화합물 4 (0.4 mmol) 및 시아노겐 브로마이드 (0.48 mmol)를 밀봉된 반응 용기에서 밤새 환류 가열 하였다. 용매를 진공 하에서 제거하고 EA 및 물로 희석하였다. 유기 층을 MgSO4로 건조시키고 여과하였다. 여과물을 진공하에 농축시키고, 잔류물을 실리카 겔 컬럼 크로마토그래피 (EtOAc / Hex)로 정제하여 화합물 7a-b 및 7d-e를 수득하였다. Compound 4 (0.4 mmol) and cyanogen bromide (0.48 mmol) in EtOH (3 mL) were heated to reflux in a sealed reaction vessel overnight. The solvent was removed under vacuum and diluted with EA and water. The organic layer was dried over MgSO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography (EtOAc / Hex) to give compounds 7a-b and 7d-e .

실시예 3-1: 9-((6-브로모벤조[Example 3-1: 9-((6-bromobenzo[ dd ][1,3]디옥솔-5-일)메틸)-6-클로로-9][1,3]dioxol-5-yl)methyl)-6-chloro-9 HH -퓨린-2,8-디아민 (7a)-purine-2,8-diamine (7a)

수율: 14%; 1H NMR (400 MHz, DMSO-d 6) δ 7.30 (s, 1H), 6.89 (s, 2H), 6.34 (s, 2H), 6.02 (s, 2H), 5.95 (s, 1H), 5.02 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 157.5, 155.0, 153.7, 147.5, 147.4, 141.4, 128.3, 122.8, 112.8, 111.6, 106.0, 102.1, 44.3; HRMS (ESI, m/z) C13H11BrClN6O2 [M+H]+ 계산치 396.9810, 실측치 396.9811.Yield: 14%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.30 (s, 1H), 6.89 (s, 2H), 6.34 (s, 2H), 6.02 (s, 2H), 5.95 (s, 1H), 5.02 ( s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 157.5, 155.0, 153.7, 147.5, 147.4, 141.4, 128.3, 122.8, 112.8, 111.6, 106.0, 102.1, 44.3; HRMS (ESI, m/z) C 13 H 11 BrClN 6 O 2 [M+H] + cal 396.9810, found 396.9811.

실시예 3-2: 9-(4-브로모-2-플루오로벤질)-6-클로로-9Example 3-2: 9- (4-bromo-2-fluorobenzyl) -6-chloro-9 HH -퓨린-2,8-디아민 (7b)-purine-2,8-diamine (7b)

수율: 15%; 1H NMR (400 MHz, DMSO-d 6) δ 7.59 (dd, J = 9.8, 1.9 Hz, 1H), 7.41 - 7.30 (m, 1H), 6.87 (s, 2H), 6.72 (t, J = 8.2 Hz, 1H), 6.31 (s, 2H), 5.15 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 159.4 (d, J = 250.4 Hz), 157.4, 155.0, 153.5, 141.4, 129.0 (d, J = 4.7 Hz), 127.6 (d, J = 3.1 Hz), 123.0 (d, J = 14.8 Hz), 122.7, 120.6 (d, J = 9.3 Hz), 118.6 (d, J = 24.1 Hz), 38.1 (d, J = 3.7 Hz); HRMS (ESI, m/z) C12H9BrClFN5O [M+H]+ 계산치 372.9795, 실측치 372.9798.Yield: 15%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.59 (dd, J = 9.8, 1.9 Hz, 1H), 7.41 - 7.30 (m, 1H), 6.87 (s, 2H), 6.72 (t, J = 8.2) Hz, 1H), 6.31 (s, 2H), 5.15 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 159.4 (d, J = 250.4 Hz), 157.4, 155.0, 153.5, 141.4, 129.0 (d, J = 4.7 Hz), 127.6 (d, J = 3.1 Hz) , 123.0 (d, J = 14.8 Hz), 122.7, 120.6 (d, J = 9.3 Hz), 118.6 (d, J = 24.1 Hz), 38.1 (d, J = 3.7 Hz); HRMS (ESI, m/z) C 12 H 9 BrClFN 5 O [M+H] + calculated 372.9795, found 372.9798.

실시예 3-3: 6-클로로-9-(3-클로로-4-플루오로벤질)-9Example 3-3: 6-chloro-9- (3-chloro-4-fluorobenzyl) -9 HH -퓨린-2,8-디아민 (7d)-purine-2,8-diamine (7d)

수율: 10%; 1H NMR (400 MHz, DMSO-d 6) δ 7.47 (dd, J = 7.1, 2.2 Hz, 1H), 7.38 (dd, J = 9.4, 8.6 Hz, 1H), 7.19 (ddd, J = 8.6, 4.7, 2.3 Hz, 1H), 6.90 (s, 2H), 6.35 (s, 2H), 5.11 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 13C NMR (101 MHz, DMSO-d 6) δ 157.5, 156.5 (d, J = 246.4 Hz), 155.1, 153.3, 141.3, 134.3 (d, J = 3.9 Hz), 129.2, 127.7 (d, J = 7.5 Hz), 122.7, 119.4 (d, J = 18.0 Hz), 117.1 (d, J = 21.1 Hz), 42.52; HRMS (ESI, m/z) C12H9Cl2FN6 [M+H]+ 계산치 326.0253, 실측치 327.0326.Yield: 10%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.47 (dd, J = 7.1, 2.2 Hz, 1H), 7.38 (dd, J = 9.4, 8.6 Hz, 1H), 7.19 (ddd, J = 8.6, 4.7) , 2.3 Hz, 1H), 6.90 (s, 2H), 6.35 (s, 2H), 5.11 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 13 C NMR (101 MHz, DMSO- d 6 ) δ 157.5, 156.5 (d, J = 246.4 Hz), 155.1, 153.3, 141.3, 134.3 (d, J = 3.9 Hz), 129.2, 127.7 (d, J = 7.5 Hz), 122.7, 119.4 (d, J = 18.0 Hz), 117.1 (d, J = 21.1 Hz), 42.52; HRMS (ESI, m/z) C 12 H 9 C 12 FN 6 [M+H] + calculated 326.0253, found 327.0326.

실시예 3-4: 6-클로로-9-(3,4-디플루오로벤질)-9Example 3-4: 6-chloro-9- (3,4-difluorobenzyl) -9 HH -퓨린-2,8-디아민 (7e)-purine-2,8-diamine (7e)

수율: 15%; 1H NMR (400 MHz, DMSO-d 6) δ 7.44 - 7.37 (m, 1H), 7.36 - 7.30 (m, 1H), 7.04 - 6.99 (m, 1H), 6.89 (s, 2H), 6.34 (s, 2H), 5.11 (s, 2H) ); 13C NMR (101 MHz, DMSO-d 6) δ 157.5, 155.1, 153.4, 149.3 (dd, J = 246.2, 12.7 Hz), 148.8 (dd, J = 245.4, 12.6 Hz), 141.4, 134.1 (dd, J = 5.5, 3.5 Hz), 123.8 (dd, J = 6.8, 3.6 Hz), 122.7, 117.8 (d, J = 17.1 Hz), 116.4 (d, J = 17.8 Hz), 42.7; HRMS (ESI, m/z) C12H9ClF2N6 [M+H]+ 계산치 311.0622, 실측치 311.0618. Yield: 15%; 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.44 - 7.37 (m, 1H), 7.36 - 7.30 (m, 1H), 7.04 - 6.99 (m, 1H), 6.89 (s, 2H), 6.34 (s) , 2H), 5.11 (s, 2H) ); 13 C NMR (101 MHz, DMSO- d 6 ) δ 157.5, 155.1, 153.4, 149.3 (dd, J = 246.2, 12.7 Hz), 148.8 (dd, J = 245.4, 12.6 Hz), 141.4, 134.1 (dd, J) = 5.5, 3.5 Hz), 123.8 (dd, J = 6.8, 3.6 Hz), 122.7, 117.8 (d, J = 17.1 Hz), 116.4 (d, J = 17.8 Hz), 42.7; HRMS (ESI, m/z) C 12 H 9 ClF 2 N 6 [M+H] + calculated 311.0622, found 311.0618.

[반응도식 2][Scheme 2]

Figure pat00006
Figure pat00006

a 시약 및 조건: (a) 구아니딘 히드로클로라이드, NaH, EtOH, 0 ℃ → rt, 1h; (b) 밤새 환류 (수율: 59.7%); (c) BTEA-Br, 디메틸아닐린, POCl3, 아세토니트릴, 밤새 환류 (수율: 28.5%); (d) 마이크로웨이브, 벤질아민, DIEA, 1-BuOH, 170 ℃, 1-2 시간 (수율: 16.4% -79.6%). a Reagents and conditions: (a) guanidine hydrochloride, NaH, EtOH, 0° C. → rt, 1 h; (b) reflux overnight (yield: 59.7%); (c) BTEA-Br, dimethylaniline, POCl 3 , acetonitrile, refluxed overnight (yield: 28.5%); (d) microwave, benzylamine, DIEA, 1-BuOH, 170° C., for 1-2 hours (yield: 16.4% -79.6%).

제조예 2:Preparation Example 2: 메틸 2-(2-아미노-4,6-디옥소-1,4,5,6-테트라히드로피리미딘-5-일)아세테이트 (9)의 합성Synthesis of methyl 2-(2-amino-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-5-yl)acetate (9)

소듐 하이드라이드 (1.3g, 53.3mmol)를 0 ℃에서 메탄올 (101ml)에 용해시켰다. 이어서, 구아니딘 히드로클로라이드 (2.7 g, 28.3 mmol)를 동일한 온도에서 용액에 첨가하고 혼합물을 실온에서 1 시간 동안 교반하였다. 용액을 여과하고 필터 케이크를 메탄올로 3 회 세척하였다. 트리메틸 에탄-1,1,2-트리카르복실레이트 (6.02 g, 29.4 mmol)를 여과물에 0 ℃에서 첨가하고 용액을 밤새 환류시켰다. 생성된 침전물을 여과하고 필터 케이크를 건조시켜 메틸 2-(2-아미노-4,6-디옥소-1,4,5,6-테트라히드로피리미딘-5-일)아세테이트 (3.36 g, 59.7 %)를 흰색 형태로 수득하였다. 1H NMR (400 MHz, DMSO-d 6) δ 7.4 (s, 4H), 3.5 (s, 3H), 3.2 (s, 1H), 3.1 (s, 2H); HRMS (ESI, m/z) C21H24ClN5O [M+H]+ 계산치 398.1742, 실측치 398.1746.Sodium hydride (1.3 g, 53.3 mmol) was dissolved in methanol (101 ml) at 0 °C. Then, guanidine hydrochloride (2.7 g, 28.3 mmol) was added to the solution at the same temperature and the mixture was stirred at room temperature for 1 hour. The solution was filtered and the filter cake was washed 3 times with methanol. Trimethyl ethane-1,1,2-tricarboxylate (6.02 g, 29.4 mmol) was added to the filtrate at 0° C. and the solution was refluxed overnight. The resulting precipitate was filtered and the filter cake was dried to obtain methyl 2-(2-amino-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-5-yl)acetate (3.36 g, 59.7 %). ) was obtained in white form. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.4 (s, 4H), 3.5 (s, 3H), 3.2 (s, 1H), 3.1 (s, 2H); HRMS (ESI, m/z) C 21 H 24 ClN 5 O [M+H] + cal 398.1742, found 398.1746.

제조예 3: 메틸 2-(2-아미노-4,6-디클로로피리미딘-5-일)아세테이트 (10)의 합성Preparation Example 3: Synthesis of methyl 2- (2-amino-4,6-dichloropyrimidin-5-yl) acetate (10)

옥시 염화인 (15.7mL, 169mmol)을 아세토 니트릴 (30 mL) 중 메틸 2-(2-아미노-4,6-디옥소-1,4,5,6-테트라히드로피리미딘-5-일)아세테이트 (3.36g, 16.9), 벤질트리에틸암모늄 브로마이드 (0.45 g, 1.66 mmol) 및 N,N-디메틸아닐린 (1.0 g, 8.25 mmol) 용액에 적가하였다. 밤새 환류시킨 후, 용액을 아이스 배스(ice bath)에서 냉각시키고 포화 수성 NaHCO3로 pH를 7로 조정하였다. 생성된 혼합물을 에틸 아세테이트 (50 mL)로 3 회 추출하고, 합한 유기층을 MgSO4상에서 건조시켰다. 잔류물을 EtOAc-헥산 (1:3)으로 용리하는 실리카 겔 크로마토그래피로 정제하여 메틸 2-(2-아미노-4,6-디클로로피리 미딘-5-일)아세테이트, 즉 중간체 (1.13 g, 28.3 %)를 수득하였다. 1H NMR (400 MHz, DMSO-d 6) δ 7.5 (s, 2H), 3.7 (s, 2H), 3.7 (s, 3H); 13C NMR (101 MHz, DMSO-d 6) δ 169.1, 161.5, 160.8, 111.7, 51.7, 34.0; HRMS (ESI, m/z) C21H24ClN5O [M+H]+ 계산치 398.1742, 실측치 398.1746.Phosphorus oxychloride (15.7 mL, 169 mmol) was dissolved in methyl 2-(2-amino-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-5-yl)acetate in acetonitrile (30 mL) (3.36 g, 16.9), benzyltriethylammonium bromide (0.45 g, 1.66 mmol) and N , N -dimethylaniline (1.0 g, 8.25 mmol) solution were added dropwise. After refluxing overnight, the solution was cooled in an ice bath and the pH adjusted to 7 with saturated aqueous NaHCO3. The resulting mixture was extracted three times with ethyl acetate (50 mL) and the combined organic layers were dried over MgSO4. The residue was purified by silica gel chromatography eluting with EtOAc-hexanes (1:3) to methyl 2-(2-amino-4,6-dichloropyrimidin-5-yl)acetate, i.e. the intermediate (1.13 g, 28.3) %) was obtained. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.5 (s, 2H), 3.7 (s, 2H), 3.7 (s, 3H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.1, 161.5, 160.8, 111.7, 51.7, 34.0; HRMS (ESI, m/z) C 21 H 24 ClN 5 O [M+H] + cal 398.1742, found 398.1746.

실시예 4: 화합물 11a 내지 11p의 합성Example 4: Synthesis of compounds 11a to 11p

t-BuOH (2 mL) 중 메틸 2-(2-아미노-4,6-디클로로피리미딘-5-일)아세테이트 (1 eq), 벤질 아민 (1.05 eq) 및 디이소프로필에틸아민 (2 eq)의 용액을 마이크로웨이브 조건 하에서 180 ℃에서 2 시간 동안 가열하였다. 물 (50mL) 첨가 후, 유기 물질을 에틸 아세테이트 (50mL)로 추출하고, MgSO4 상에서 건조시키고, 증발 농축시켰다. 잔류물을 실리카 겔 크로마토그래피 (EtOAc/Hex, 1:2)로 정제하여 11a-p를 수득하였다.Methyl 2-(2-amino-4,6-dichloropyrimidin-5-yl)acetate (1 eq), benzyl amine (1.05 eq) and diisopropylethylamine (2 eq) in t-BuOH (2 mL) The solution was heated at 180 °C for 2 hours under microwave conditions. After addition of water (50 mL), the organics were extracted with ethyl acetate (50 mL), dried over MgSO 4 and concentrated by evaporation. The residue was purified by silica gel chromatography (EtOAc/Hex, 1:2) to give 11a-p .

실시예 4-1: 2-아미노-7-((6-브로모벤조[Example 4-1: 2-amino-7-((6-bromobenzo[ dd ][1,3]디옥솔-5-일)메틸)-4-클로로-5,7-디히드로-6][1,3]dioxol-5-yl)methyl)-4-chloro-5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (11a)]pyrimidin-6-one (11a)

1H NMR (400 MHz, DMSO-d 6) δ 7.3 (s, 1H), 7.1 (s, 2H), 6.7 (s, 1H), 6.0 (s, 2H), 4.6 (s, 2H), 3.6 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 174.2, 165.9, 162.5, 151.6, 147.5, 147.4, 127.8, 112.5, 111.6, 107.4, 102.0, 42.6, 32.6; HRMS (ESI, m/z) C14H10BrClN4O3 [M+H]+ 계산치 398.9676, 실측치 398.9678. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.3 (s, 1H), 7.1 (s, 2H), 6.7 (s, 1H), 6.0 (s, 2H), 4.6 (s, 2H), 3.6 ( s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 174.2, 165.9, 162.5, 151.6, 147.5, 147.4, 127.8, 112.5, 111.6, 107.4, 102.0, 42.6, 32.6; HRMS (ESI, m/z) C 14 H 10 BrClN 4 O 3 [M+H] + cal 398.9676, found 398.9678.

실시예 4-2: 2-아미노-4-클로로-7-(4-(트리플루오로메틸)벤질)-5,7-디히드로-6Example 4-2: 2-Amino-4-chloro-7- (4- (trifluoromethyl) benzyl) -5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (11f)]pyrimidin-6-one (11f)

1H NMR (400 MHz, DMSO-d 6) δ 7.7 - 7.7 (m, 2H), 7.5 - 7.5 (m, 2H), 7.1 (s, 2H), 4.9 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 174.1, 165.9, 162.6, 151.8, 141.0 (d, J = 1.3 Hz), 128.0 (q, J = 32.2 Hz), 127.9, 125.4 (q, J = 3.8 Hz), 124.2 (q, J =273.0 Hz), 101.6, 41.8, 32.4; HRMS (ESI, m/z) C14H10ClF3N4O [M+H]+ 계산치 343.0568, 실측치 343.0571. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.7 - 7.7 (m, 2H), 7.5 - 7.5 (m, 2H), 7.1 (s, 2H), 4.9 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 174.1, 165.9, 162.6, 151.8, 141.0 (d, J = 1.3 Hz), 128.0 (q, J = 32.2 Hz), 127.9, 125.4 (q, J = 3.8) Hz), 124.2 (q, J =273.0 Hz), 101.6, 41.8, 32.4; HRMS (ESI, m/z) C 14 H 10 ClF 3 N 4 O [M+H] + calculated 343.0568, found 343.0571.

실시예 4-3: 2-아미노-7-(4-브로모-2-플루오로벤질)-4-클로로-5,7-디히드로-6Example 4-3: 2-Amino-7- (4-bromo-2-fluorobenzyl) -4-chloro-5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (11g)]pyrimidin-6-one (11g)

1H NMR (400 MHz, DMSO-d 6) δ 7.6 (dd, J = 9.8, 1.9 Hz, 1H), 7.4 (dd, J = 8.3, 1.7 Hz, 1H), 7.2 (t, J = 8.3 Hz, 1H), 7.1 (s, 1H), 4.8 (s, 1H), 3.6 (s, 1H); 13C NMR (101 MHz, DMSO-d 6) δ 211.6, 203.4, 200.2, 197.3 (d, J = 250.3 Hz), 189.4, 168.3 (d, J = 4.6 Hz), 165.3 (d, J = 3.6 Hz), 160.3 (d, J = 14.5 Hz), 158.4 (d, J = 9.5 Hz), 156.4 (d, J = 24.5 Hz), 139.3, 73.6 (d, J = 4.4 Hz), 70.1; HRMS (ESI, m/z) C13H9BrClFN4O [M+H]+ 계산치 372.9683, 실측치 372.9684. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.6 (dd, J = 9.8, 1.9 Hz, 1H), 7.4 (dd, J = 8.3, 1.7 Hz, 1H), 7.2 (t, J = 8.3 Hz, 1H), 7.1 (s, 1H), 4.8 (s, 1H), 3.6 (s, 1H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 211.6, 203.4, 200.2, 197.3 (d, J = 250.3 Hz), 189.4, 168.3 (d, J = 4.6 Hz), 165.3 (d, J = 3.6 Hz) , 160.3 (d, J = 14.5 Hz), 158.4 (d, J = 9.5 Hz), 156.4 (d, J = 24.5 Hz), 139.3, 73.6 (d, J = 4.4 Hz), 70.1; HRMS (ESI, m/z) C 13 H 9 BrClFN 4 O [M+H] + calculated 372.9683, found 372.9684.

실시예 4-4: 2-아미노-4-클로로-7-(2-클로로-5-(트리플루오로메틸)벤질)-5,7-디히드로-6Example 4-4: 2-Amino-4-chloro-7- (2-chloro-5- (trifluoromethyl) benzyl) -5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (11i)]pyrimidin-6-one (11i)

1H NMR (400 MHz, DMSO-d 6) δ 7.7 - 7.7 (m, 2H), 7.5 (d, J = 2.1 Hz, 1H), 7.0 (s, 2H), 4.8 (s, 2H), 3.6 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 173.9, 165.7, 162.7, 153.0, 136.5 (d, J = 1.3 Hz), 134.4, 130.2, 129.0 (q, J = 32.9 Hz), 126.5 (q, J =207.05 Hz) 125.6 (q, J = 3.8 Hz), 124.8 (q, J = 3.9 Hz), 101.6, 40.5, 32.7; HRMS (ESI, m/z) C14H9Cl2F3N4O [M+H]+ 계산치 377.0178, 실측치 377.0181. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.7 - 7.7 (m, 2H), 7.5 (d, J = 2.1 Hz, 1H), 7.0 (s, 2H), 4.8 (s, 2H), 3.6 ( s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 173.9, 165.7, 162.7, 153.0, 136.5 (d, J = 1.3 Hz), 134.4, 130.2, 129.0 (q, J = 32.9 Hz), 126.5 (q, J =207.05 Hz) 125.6 (q, J = 3.8 Hz), 124.8 (q, J = 3.9 Hz), 101.6, 40.5, 32.7; HRMS (ESI, m/z) C 14 H 9 Cl 2 F 3 N 4 O [M+H] + cald. 377.0178, found 377.0181.

실시예 4-5: 2-아미노-4-클로로-7-(3,4-디클로로벤질)-5,7-디히드로-6Example 4-5: 2-Amino-4-chloro-7- (3,4-dichlorobenzyl) -5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (11j)]pyrimidin-6-one (11j)

1H NMR (400 MHz, DMSO-d 6) δ 7.6 - 7.6 (m, 2H), 7.3 (dd, J = 8.3, 2.1 Hz, 1H), 7.1 (s, 2H), 4.8 (s, 2H), 3.6 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 174.1, 165.8, 162.5, 151.7, 137.4, 131.1, 130.6, 130.0, 129.2, 127.5, 101.7, 41.1, 32.5; HRMS (ESI, m/z) C13H9Cl3N4O [M+H]+ 계산치 342.9915, 실측치 342.9918. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.6 - 7.6 (m, 2H), 7.3 (dd, J = 8.3, 2.1 Hz, 1H), 7.1 (s, 2H), 4.8 (s, 2H), 3.6 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 174.1, 165.8, 162.5, 151.7, 137.4, 131.1, 130.6, 130.0, 129.2, 127.5, 101.7, 41.1, 32.5; HRMS (ESI, m/z) C 13 H 9 C 13 N 4 O [M+H] + calculated 342.9915, found 342.9918.

실시예 4-6: 2-아미노-4-클로로-7-(2-플루오로-4-메틸벤질)-5,7-디히드로-6H-피롤로[2,3-Example 4-6: 2-Amino-4-chloro-7- (2-fluoro-4-methylbenzyl) -5,7-dihydro-6H-pyrrolo [2,3- dd ]피리미딘-6-온 (11l)]pyrimidin-6-one (11l)

1H NMR (400 MHz, CDCl3) δ 7.2 (t, J = 7.9 Hz, 1H), 6.9 - 6.8 (m, 2H), 5.1 (s, 2H), 4.9 (s, 2H), 3.5 (s, 2H), 2.3 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 173.9, 166.6, 162.4, 160.6 (d, J = 247.5 Hz), 153.1, 140.3 (d, J = 8.0 Hz), 129.7 (d, J = 4.3 Hz), 125.0 (d, J = 3.2 Hz), 119.5 (d, J = 14.9 Hz), 116.2 (d, J = 21.1 Hz), 103.2, 36.8, 323.0, 21.2 (d, J = 1.6 Hz); HRMS (ESI, m/z) C14H12ClFN4O [M+H]+ 계산치 307.0756, 실측치 307.0759. 1 H NMR (400 MHz, CDCl 3 ) δ 7.2 (t, J = 7.9 Hz, 1H), 6.9 - 6.8 (m, 2H), 5.1 (s, 2H), 4.9 (s, 2H), 3.5 (s, 2H), 2.3 (s, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ 173.9, 166.6, 162.4, 160.6 (d, J = 247.5 Hz), 153.1, 140.3 (d, J = 8.0 Hz), 129.7 (d, J = 4.3 Hz), 125.0 (d, J = 3.2 Hz), 119.5 (d, J = 14.9 Hz), 116.2 (d, J = 21.1 Hz), 103.2, 36.8, 323.0, 21.2 (d, J = 1.6 Hz); HRMS (ESI, m/z) C 14 H 12 ClFN 4 O [M+H] + calculated 307.0756, found 307.0759.

실시예 4-7: 2-아미노-7-(벤조[d][1,3]디옥솔-5-일메틸)-4-클로로-5,7-디히드로-6Example 4-7: 2-Amino-7- (benzo [d] [1,3] dioxol-5-ylmethyl) -4-chloro-5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (11n)]pyrimidin-6-one (11n)

1H NMR (400 MHz, DMSO-d 6) δ 7.1 (s, 2H), 6.9 (d, J = 1.7 Hz, 1H), 6.8 (d, J = 8.0 Hz, 1H), 6.8 (dd, J = 8.0, 1.7 Hz, H), 6.0 (s, 2H), 4.7 (s, 2H), 3.5 (s, 2H); 13C NMR (101 MHz, CDCl3) δ 173.9, 165.9, 162.5, 151.7, 147.3, 146.5, 130.0, 120.8, 108.1, 107.8, 101.4, 100.9, 41.9, 32.3; HRMS (ESI, m/z) C14H11ClN4O3 [M+H]+ 계산치 319.0592, 실측치 319.0592. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.1 (s, 2H), 6.9 (d, J = 1.7 Hz, 1H), 6.8 (d, J = 8.0 Hz, 1H), 6.8 (dd, J = 8.0, 1.7 Hz, H), 6.0 (s, 2H), 4.7 (s, 2H), 3.5 (s, 2H); 13 C NMR (101 MHz, CDCl 3 ) δ 173.9, 165.9, 162.5, 151.7, 147.3, 146.5, 130.0, 120.8, 108.1, 107.8, 101.4, 100.9, 41.9, 32.3; HRMS (ESI, m/z) C 14 H 11 ClN 4 O 3 [M+H] + calculated 319.0592, found 319.0592.

실시예 4-8: 2-아미노-4-클로로-7-(2,3-디메톡시벤질)-5,7-디히드로-6Example 4-8: 2-Amino-4-chloro-7- (2,3-dimethoxybenzyl) -5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (11o)]pyrimidin-6-one (11o)

1H NMR (400 MHz, CDCl3) δ 7.0 (s, 2H), 7.0 - 6.9 (m, 2H), 6.6 (dd, J = 5.2, 4.1 Hz, 1H), 4.8 (s, 2H), 3.8 (s, 3H), 3.8 (s, 3H), 3.6 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 174.2, 166.2, 162.6, 152.3, 151.7, 145.9, 129.4, 123.9, 118.6, 112.0, 101.6, 60.0, 55.7, 40.1, 32.4; HRMS (ESI, m/z) C15H15ClN4O3 [M+H]+ 계산치 335.0905, 실측치 335.0907. 1 H NMR (400 MHz, CDCl 3 ) δ 7.0 (s, 2H), 7.0 - 6.9 (m, 2H), 6.6 (dd, J = 5.2, 4.1 Hz, 1H), 4.8 (s, 2H), 3.8 ( s, 3H), 3.8 (s, 3H), 3.6 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 174.2, 166.2, 162.6, 152.3, 151.7, 145.9, 129.4, 123.9, 118.6, 112.0, 101.6, 60.0, 55.7, 40.1, 32.4; HRMS (ESI, m/z) C 15 H 15 ClN 4 O 3 [M+H] + cal 335.0905, found 335.0907.

실시예 4-9: 2-아미노-4-클로로-7-(2,6-디플루오로-3-메톡시벤질)-5,7-디히드로-6Example 4-9: 2-Amino-4-chloro-7- (2,6-difluoro-3-methoxybenzyl) -5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (11p)]pyrimidin-6-one (11p)

1H NMR (400 MHz, DMSO-d 6) δ 7.1 (td, J = 9.3, 5.2 Hz, 1H), 7.0 (d, J = 2.5 Hz, 2H), 7.0 (dd, J = 9.3, 1.9 Hz, 1H), 4.8 (s, 2H), 3.8 (s, 3H), 3.5 (s, 2H); 13C NMR (101 MHz, DMSO-d 6) δ 173.5, 165.7, 162.5, 154.1 (dd, J = 241.1, 6.3 Hz), 151.7, 149.9 (dd, J = 249.1, 8.0 Hz), 143.8 (dd, J = 10.7, 3.0 Hz), 113.1 (dd, J = 9.8, 2.8 Hz), 112.2 (dd, J = 19.6, 15.0 Hz), 110.2 (dd, J = 22.9, 4.1 Hz), 101.3, 56.4, 32.1, 31.9 (t, J = 3.8 Hz).; HRMS (ESI, m/z) C14H11ClFN4O2 [M+H]+ 계산치 341.0611, 실측치 341.0614. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.1 (td, J = 9.3, 5.2 Hz, 1H), 7.0 (d, J = 2.5 Hz, 2H), 7.0 (dd, J = 9.3, 1.9 Hz, 1H), 4.8 (s, 2H), 3.8 (s, 3H), 3.5 (s, 2H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 173.5, 165.7, 162.5, 154.1 (dd, J = 241.1, 6.3 Hz), 151.7, 149.9 (dd, J = 249.1, 8.0 Hz), 143.8 (dd, J) = 10.7, 3.0 Hz), 113.1 (dd, J = 9.8, 2.8 Hz), 112.2 (dd, J = 19.6, 15.0 Hz), 110.2 (dd, J = 22.9, 4.1 Hz), 101.3, 56.4, 32.1, 31.9 (t, J = 3.8 Hz).; HRMS (ESI, m/z) C 14 H 11 ClFN 4 O 2 [M+H] + calculated 341.0611, found 341.0614.

[반응도식 3][Scheme 3]

Figure pat00007
Figure pat00007

a 시약 및 조건: (a) 구아니딘 히드로클로라이드, NaH, EtOH, 0 ℃ → rt, 1h; (b) 밤새 환류 65%; (c) BTEA-Br, 디메틸아닐린, POCl3, 아세토니트릴, 밤새 환류 45%; (d) DIEA, 1-BuOH, 120 ℃, 20 시간 (수율: 10 ~ 37%) a Reagents and conditions: (a) guanidine hydrochloride, NaH, EtOH, 0° C. → rt, 1 h; (b) 65% reflux overnight; (c) BTEA-Br, dimethylaniline, POCl 3 , acetonitrile, 45% reflux overnight; (d) DIEA, 1-BuOH, 120 ℃, 20 hours (yield: 10 ~ 37%)

제조예 4:Preparation Example 4: 에틸 2-(2-아미노-4-히드록시-6-메틸피리미딘-5-일)아세테이트 (13)의 합성Synthesis of ethyl 2- (2-amino-4-hydroxy-6-methylpyrimidin-5-yl) acetate (13)

EtOH (40 mL) 중 NaOEt (17.5 mmol)의 용액에 구아니딘 히드로클로라이드 (10.5 mmol)를 0 ℃에서 첨가하였다. 1 시간 후, EtOH (20 mL) 중 1-에틸 4-메틸 2-아세틸숙시 네이트 (10.0 mmol)의 용액을 첨가한 다음, 밤새 환류시켰다. 반응 혼합물을 냉각시킨 후, 고체를 여과로 수집하여 목적 화합물 13 (65 %)을 수득하였다. 1H NMR (400 MHz, CD3OD) δ 4.14 (q, J = 7.1 Hz, 2H), 3.45 (s, 2H), 2.14 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H); MS (ESI, m/z) C9H13N3O3 [M+H]+ 계산치 212.1, 실측치 212.0.To a solution of NaOEt (17.5 mmol) in EtOH (40 mL) was added guanidine hydrochloride (10.5 mmol) at 0 °C. After 1 h, a solution of 1-ethyl 4-methyl 2-acetylsuccinate (10.0 mmol) in EtOH (20 mL) was added and then refluxed overnight. After cooling the reaction mixture, the solid was collected by filtration to give the desired compound 13 (65%). 1 H NMR (400 MHz, CDOD) δ 4.14 (q, J = 7.1 Hz, 2H), 3.45 (s, 2H), 2.14 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H); MS (ESI, m/z) C 9 H 13 N 3 O 3 [M+H] + calculated 212.1, found 212.0.

제조예 5: 에틸 2-(2-아미노-4-클로로-6-메틸피리미딘-5-일)아세테이트 (14)Preparation 5: Ethyl 2- (2-amino-4-chloro-6-methylpyrimidin-5-yl) acetate (14)

POCl3 (0.93 mL, 10 mmol)을 아세토니트릴 (10 mL) 중 화합물 13 (1.0 mmol), 벤질트리에틸암모늄 브로마이드 (27 mg, 0.1 mmol) 및 N,N-디메틸아닐린 (60 mg, 0.5 mmol)의 용액에 적가하였다. 밤새 환류시킨 후, 용액을 아이스 배스(ice bath)에서 냉각시키고 포화 수성 NaHCO3로 pH를 7로 조정하였다. 생성된 혼합물을 에틸 아세테이트 (50 mL)로 3 회 추출하고, 합한 유기층을 MgSO4상에서 건조시켰다. 잔류물을 EtOAc-헥산으로 용리하는 실리카 겔 크로마토그래피로 정제하여 목적 화합물 (40 %)을 수득하였다. 1H NMR (400 MHz, CDCl3) δ 5.34 (bs, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.68 (s, 2H), 2.36 (s, 3H), 1.27 (t, J = 7.1 Hz, 5H); MS (ESI, m/z) C9H13ClN3O2 [M+H]+ 계산치 230.1, 실측치 230.5. POCl3 (0.93 mL, 10 mmol) was mixed with compound 13 (1.0 mmol), benzyltriethylammonium bromide (27 mg, 0.1 mmol) and N,N-dimethylaniline (60 mg, 0.5 mmol) in acetonitrile (10 mL) It was added dropwise to the solution. After refluxing overnight, the solution was cooled in an ice bath and the pH adjusted to 7 with saturated aqueous NaHCO3. The resulting mixture was extracted three times with ethyl acetate (50 mL) and the combined organic layers were dried over MgSO4. The residue was purified by silica gel chromatography eluting with EtOAc-hexane to give the title compound (40%). 1 H NMR (400 MHz, CDCl 3 ) δ 5.34 (bs, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.68 (s, 2H), 2.36 (s, 3H), 1.27 (t, J = 7.1 Hz, 5H); MS (ESI, m/z) C 9 H 13 ClN 3 O 2 [M+H] + calculated 230.1, found 230.5.

실시예 5: 화합물 15a 및 15b의 합성Example 5: Synthesis of compounds 15a and 15b

t-BuOH (2 mL) 중 화합물 14 (1 eq), 벤질 아민 (1.05 eq) 및 디이소프로필에틸아민 (2 eq)의 용액을 마이크로웨이브 조건 하에서 180 ℃에서 2 시간 동안 가열하였다. 물 (50mL) 첨가 후, 유기 물질을 에틸 아세테이트 (50mL)로 추출하고, MgSO4 상에서 건조시키고, 증발 농축시켰다. 잔류물을 실리카 겔 크로마토그래피 (EtOAc/Hex, 1:2)로 정제하여 화합물 15a 및 15b를 수득하였다. A solution of compound 14 (1 eq), benzyl amine (1.05 eq) and diisopropylethylamine (2 eq) in t-BuOH (2 mL) was heated at 180° C. for 2 h under microwave conditions. After addition of water (50 mL), the organics were extracted with ethyl acetate (50 mL), dried over MgSO 4 and concentrated by evaporation. The residue was purified by silica gel chromatography (EtOAc/Hex, 1:2) to give compounds 15a and 15b .

실시예 5-1: 2-아미노-7-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-4-메틸-5,7-디히드로-6H-피롤로[2,3-Example 5-1: 2-amino-7-((6-bromobenzo[d][1,3]dioxol-5-yl)methyl)-4-methyl-5,7-dihydro-6H- pyrrolo[2,3- dd ]피리미딘-6-온 (15a)]pyrimidin-6-one (15a)

1H NMR (400 MHz, DMSO-d 6) δ 7.25 (s, 1H), 6.60 (s, 1H), 6.48 (bs, 2H), 6.03 (s, 2H), 4.65 (s, 2H), 3.55 (s, 2H), 2.15 (s, 3H); 13C NMR (101 MHz, DMSO-d 6) δ 175.2, 164.4, 162.5, 159.1, 147.5, 147.4, 128.2, 112.5, 111.7, 107.2, 102.5, 102.0, 42.1, 32.2, 20.8; MS (ESI, m/z) C15H14BrN4O3 [M+H]+ 계산치 377.0, 실측치 377.2. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.25 (s, 1H), 6.60 (s, 1H), 6.48 (bs, 2H), 6.03 (s, 2H), 4.65 (s, 2H), 3.55 ( s, 2H), 2.15 (s, 3H); 13 C NMR (101 MHz, DMSO- d 6 ) δ 175.2, 164.4, 162.5, 159.1, 147.5, 147.4, 128.2, 112.5, 111.7, 107.2, 102.5, 102.0, 42.1, 32.2, 20.8; MS (ESI, m/z) C 15 H 14 BrN 4 O 3 [M+H] + calculated 377.0, found 377.2.

실시예 5-2: 2-아미노-7-(4-브로모-2-플루오로벤질)-4-메틸-5,7-디히드로-6Example 5-2: 2-Amino-7-(4-bromo-2-fluorobenzyl)-4-methyl-5,7-dihydro-6 HH -피롤로[2,3--pyrrolo[2,3- dd ]피리미딘-6-온 (15b)]pyrimidin-6-one (15b)

1H NMR (400 MHz, DMSO-d 6) δ 7.6 (dd, J = 9.8, 1.9 Hz, 1H), 7.6 (dd, J = 8.3, 1.8 Hz, 1H), 7.2 (t, J = 8.3 Hz, 1H), 6.5 (s, 2H), 4.8 (s, 2H), 3.5 (s, 2H), 2.1 (s, 3H); HRMS (ESI, m/z) C14H13BrFN4O [M+H]+ 계산치 351.0251, 실측치 351.0252. 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.6 (dd, J = 9.8, 1.9 Hz, 1H), 7.6 (dd, J = 8.3, 1.8 Hz, 1H), 7.2 (t, J = 8.3 Hz, 1H), 6.5 (s, 2H), 4.8 (s, 2H), 3.5 (s, 2H), 2.1 (s, 3H); HRMS (ESI, m/z) C 14 H 13 BrFN 4 O [M+H] + calculated 351.0251, found 351.0252.

실험 재료 및 방법Experimental materials and methods

1. 재조합 단백질 제조 및 형광 편광 분석1. Recombinant Protein Preparation and Fluorescence Polarization Analysis

TRAP1 (전장), Grp94 (N-말단 도메인) 및 Hsp90 (N-말단 도메인) 재조합 단백질을 이전에 보고된 방법에 따라 제조하였다(Park, H. K. et al., Paralog Specificity Determines Subcellular Distribution, Action Mechanism, and Anticancer Activity of TRAP1 Inhibitors. J. Med. Chem. 2017, 60, 7569-7578). 형광 편광 분석을 위해, 형광 프로브 PU-H71-FITC를 이전에 보고된 방법에 따라 합성 하였다(Taldone, T. et al., Experimental and structural testing module to analyze paraloguespecificity and affinity in the Hsp90 inhibitors series. J. Med. Chem. 2013, 56, 6803-6818). 정제된 재조합 단백질 (50 nM)을 PU-H71-FITC3 (10 nM)과 혼합 한 후, 형광 편광 (Fluorescence polarization, FP) 버퍼 (135 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4mM KH2PO4, 1mM DTT, 2mM MgCl2, 0.1mg/ml BSA (pH 7.3))에서 다양한 농도의 억제제(PU-H71, DN401, 또는 실시예에서 제조된 화합물)와 함께 실온에서 2 시간 동안 인큐베이션 하였다. SYNERGY NEO 마이크로플레이트 리더 (BioTek Instruments, Inc.)를 사용하여 형광 편광을 측정 하였다. IC50 값을 결정하기 위해 Prism 7.0 (GraphPad Software Inc.)을 사용하여 각 화합물에 대해 선형 억제 곡선을 수득 하였다.TRAP1 (full length), Grp94 (N-terminal domain) and Hsp90 (N-terminal domain) recombinant proteins were prepared according to previously reported methods (Park, HK et al., Paralog Specificity Determines Subcellular Distribution, Action Mechanism, and Anticancer Activity of TRAP1 Inhibitors. J. Med. Chem. 2017, 60 , 7569-7578). For fluorescence polarization analysis, a fluorescent probe PU-H71-FITC was synthesized according to a previously reported method (Taldone, T. et al., Experimental and structural testing module to analyze paraloguespecificity and affinity in the Hsp90 inhibitors series. J. Med. Chem . 2013, 56, 6803-6818). Purified recombinant protein (50 nM) was mixed with PU-H71-FITC3 (10 nM), followed by fluorescence polarization (FP) buffer (135 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM). KH 2 PO 4 , 1 mM DTT, 2 mM MgCl 2 , 0.1 mg/ml BSA (pH 7.3)) with various concentrations of inhibitors (PU-H71, DN401, or compounds prepared in the Examples) at room temperature for 2 hours. did Fluorescence polarization was measured using a SYNERGY NEO microplate reader (BioTek Instruments, Inc.). Linear inhibition curves were obtained for each compound using Prism 7.0 (GraphPad Software Inc.) to determine IC 50 values.

2.2. 결정화 및 구조 결정 Crystallization and structure determination

결정학적 연구를 위해 인간 TRAP1 NTD (잔기 60-294, hTRAP1-N) 및 C 말단 도메인이 없는 TRAP1 (잔기 60-561, hTRAP1-NM)을 이전에 보고된 방법에 따라 제조하였다(Lee, C. et al., Development of a mitochondria-targeted Hsp90 inhibitor based on the crystal structures of human TRAP1. J. Am. Chem. Soc. 2015, 137, 4358-4367). 화합물 5a와 복합체화 된 TRAP1을 결정화하기 위해, 50㎕의 hTRAP1-N 단백질 (20mg/mL) 용액을 DMSO (50mM)에 용해된 1.6㎕의 억제제와 혼합하고, 얼음에서 1시간 동안 인큐베이션 하였다. 단백질-억제제 혼합물을 20mM Tris pH 7.5, 150mM NaCl 및 5mM DTT를 함유하는 완충액으로 희석하여 최종 부피 90㎕가 되도록 하여 DMSO 농도를 감소시켰다. 1 ㎕의 단백질 용액, 및 120 mM 시트레이트 (pH 5.6), 31% PEG 4,000을 포함하는 1 ㎕의 저장 완충제를 혼합함으로써 실온에서 행잉 드롭 확산법을 사용하여 결정을 성장시켰다. 결정을 결정화 용액 + 30% 글리세롤에서 동결 보호하고, 액체 질소에서 급속 냉동시켰다. For crystallographic studies, human TRAP1 NTD (residues 60-294, hTRAP1-N) and TRAP1 without C-terminal domain (residues 60-561, hTRAP1-NM) were prepared according to previously reported methods (Lee, C. et al., Development of a mitochondria-targeted Hsp90 inhibitor based on the crystal structures of human TRAP 1. J. Am. Chem. Soc . 2015, 137, 4358-4367). To crystallize TRAP1 complexed with compound 5a , 50 μl of hTRAP1-N protein (20 mg/mL) solution was mixed with 1.6 μl of inhibitor dissolved in DMSO (50 mM), and incubated on ice for 1 hour. The DMSO concentration was reduced by diluting the protein-inhibitor mixture with a buffer containing 20 mM Tris pH 7.5, 150 mM NaCl and 5 mM DTT to a final volume of 90 μl. Crystals were grown using a hanging drop diffusion method at room temperature by mixing 1 μl of protein solution and 1 μl of storage buffer containing 120 mM citrate, pH 5.6, 31% PEG 4,000. Crystals were cryoprotected in crystallization solution + 30% glycerol and flash frozen in liquid nitrogen.

화합물 5b와의 복합체에서 hTRAP1-NM의 경우, 상기한 방법으로 단백질-억제제 혼합물을 제조하였다. TRAP1-5b 복합체의 결정을 18 ℃에서 100mM 소듐 카코딜레이트 (sodium cacodylate) pH 6.76, 100mM 칼슘 아세테이트 및 14% PEG 20K를 함유하는 웰 용액에서 성장시키고, 30% 글리세롤로 동결 보호하였다. In the case of hTRAP1-NM in complex with compound 5b , a protein-inhibitor mixture was prepared by the above method. The determination of TRAP1- 5b complex were grown in the well solution containing 100mM Sodium Kakogawa delay agent (sodium cacodylate) pH 6.76, 100mM potassium acetate, and 14% PEG 20K were from 18 ℃, freeze protection in 30% glycerol.

X-선 회절 데이터는 PAL (Pohang Accelerator Laboratory)에 수집되었고 HKL2000에 의해 처리되었다(Otwinowski, Z. et al., Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology, ed Charles W. Carter, Jr. (Academic Press), 1997, Vol 276, 307-326). X-ray diffraction data collected in PAL (Pohang Accelerator Laboratory) and processed by HKL2000 (Otwinowski, Z. et al., Processing of X-ray diffraction data collected in oscillation mode. Methods in Enzymology, ed Charles W. Carter , Jr. (Academic Press), 1997 , Vol 276, 307-326).

TRAP1-5a 및 TRAP1-5b의 구조는 apo 구조 (PDB: 4Z1I 및 5Y3N)를 모델로 사용하여 Phaser-MR 프로그램으로 분자를 대체하였다(McCoy, A. j. et al., Phaser is a Phaser crystallographic software J. Appl. Cryst. 2007, 40, 658-674). TRAP1-5a 및 TRAP1-5b의 최종 모델은 Phenix 및 COOT를 사용하여 반복적인 정제 및 재구성을 하였다(Adams, P.D. et al., PHENIX: a comprehensive Python-basedsystem for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010, 66, Pt 2, 213-221). 결정학적 통계는 표 1에 요약하였다.The structure of TRAP1- 5a and 5b are TRAP1- apo structure: using (PDB 4Z1I and 5Y3N) as a model was replaced by a molecular-MR program Phaser (McCoy, A. j et al, Phaser is a Phaser crystallographic software.. J. Appl. Cryst . 2007 , 40, 658-674). The final design of the TRAP1- 5a and 5b TRAP1- was repeated purification and reconstructed using the Phenix and COOT (Adams, PD et al, PHENIX:.. A comprehensive Python-basedsystem for macromolecular structure solution Acta Crystallogr D Biol Crystallogr. 2010 , 66 , Pt 2, 213-221). Crystallographic statistics are summarized in Table 1.

TRAP1 (잔기 60-294)TRAP1 (residues 60-294) TRAP1 (잔기 60-561)TRAP1 (residues 60-561) 리간드ligand SJT009SJT009 SJT104SJT104 X선원 (X-ray source)X-ray source Beamline 5C, PALBeamline 5C, PAL Beamline 5C, PALBeamline 5C, PAL 온도 (K)temperature (K) 100100 100100 공간군 (Space group)Space group R3R3 P41212P4 1 2 1 2 셀 디멘션 (Cell dimensions)Cell dimensions a, b, c (Å)a, b, c (Å) 109.60, 109.60, 59.90109.60, 109.60, 59.90 69.54, 69.54, 251.7869.54, 69.54, 251.78 α, β, γ(°)α, β, γ(°) 90.00, 90.00, 120.0090.00, 90.00, 120.00 90.00, 90.00, 90.0090.00, 90.00, 90.00 데이터 가공Data Processing 파장 (Å)Wavelength (Å) 0.97950.9795 0.97950.9795 해상도 (Å)Resolution (Å) 50.00-1.50 (1.53-1.50)50.00-1.50 (1.53-1.50) 50.00-3.00 (3.05-3.00)50.00-3.00 (3.05-3.00) Rmerge (%)R merge (%) 4.6 (36.9)4.6 (36.9) 10.7 (52.0)10.7 (52.0) I / σII / σI 32.7 (2.9)32.7 (2.9) 17.7 (2.1)17.7 (2.1) 완전성 (Completeness, %)Completeness (%) 99.2 (96.9)99.2 (96.9) 98.8 (98.1)98.8 (98.1) 리던던시 (Redundancy)Redundancy 3.1 (3.1)3.1 (3.1) 9.9 (6.3)9.9 (6.3) 측정된 리플렉션 (Measured reflections)Measured reflections 131146131146 128554128554 고유 리플렉션 (Unique reflections)Unique reflections 4247042470 1302313023 리파인먼트 통계학 (Refinement statistics)Refinement statistics 데이터 범위 (Å)Data range (Å) 24.10-1.5024.10-1.50 24.98-3.0024.98-3.00 리플렉션 (Reflections)Reflections 4246642466 1297112971 R-팩터 (%)R-factor (%) 17.3517.35 23.2123.21 Rfree (%)R free (%) 20.5920.59 30.3030.30 비수소 원자의 수number of non-hydrogen atoms 19841984 35003500 R. m. s. 편차R. m. s. Deviation 결합 길이 (Å)bond length (Å) 0.0080.008 0.0020.002 결합 각도 (°)bonding angle (°) 1.0841.084 0.4020.402 라마찬드란 도표ramachandran diagram Favored (%) Favored (%) 98.1998.19 97.1297.12 Allowed (%) Allowed (%) 1.361.36 2.882.88 Disallowed (%) Disallowed (%) 0.450.45 00

*괄호 안의 값은 각각 가장 높은 해상도 셸(shell)에 대한 것임.*Values in parentheses are for each highest resolution shell.

3.3. 분자 역학 (Molecular Dymanics, MD) 시뮬레이션 및 리간드 도킹Molecular Dymanics (MD) Simulation and Ligand Docking

TRAP1 또는 Hsp90 억제제-결합 NTD의 공개된 결정 구조를 슈뢰딩거 소프트웨어 패키지 (2020-1 release)에서 마에스트로 인터페이스에 적용하였다. 결합 순서를 지정하고, 수소를 첨가하고, 금속에 0 차 결합을 생성하고, 이황화 결합을 만들고, 누락된 측쇄를 수정하고, 5Å 이상의 물을 제거하고, Epik 모듈을 사용하여 het 상태를 생성하기 위해 단백질 제조 wizard 모듈을 사용하여 도킹 및 시뮬레이션을 위한 단백질을 제조하였다 (Ph: 7.0±2). OPLS3e 힘장(force field)을 사용하여 제한된 에너지 최소화를 수행하였다. 분자 역학 시뮬레이션을 위해, 용매화 시스템은 Desmond (슈뢰딩거 Release 2020-1)의 시스템 빌더 패널을 사용하여 구축되었다. SPC 용매 모델은 최소 크기의 사방 정계 박스 경계에서 선택되었고, 힘장은 OPLS2005로 설정되었다. 분자 역학 패널을 사용하여 용매화된 시스템을 작업 공간에 로딩 한 후, 시스템 에너지를 1.2로 설정하였고, 사용된 총체 클래스는 NPT였다. 시뮬레이션은 300.0K 및 1.01325 bar에서 실행되도록 설정되었으며, 각 시스템의 총 시뮬레이션 시간은 300ps 궤도 추적 간격으로 300ns로 설정되었다. 매립된 리간드의 선택을 통한 리간드 그리드 생성 후에 글라이드 도킹이 수행되었고, 0.25의 부분 전하 차단으로 반데르발스 반경 스케일링 파라미터 스케일링 팩터 1로 설정되었다.The published crystal structures of TRAP1 or Hsp90 inhibitor-binding NTDs were applied to the maestro interface in the Schrödinger software package (2020-1 release). To order bonds, add hydrogen, create zero-order bonds to metals, create disulfide bonds, correct missing side chains, remove more than 5 Å of water, and generate het states using the Epik module Proteins were prepared for docking and simulation using the protein preparation wizard module (Ph: 7.0±2). Limited energy minimization was performed using an OPLS3e force field. For molecular dynamics simulations, a solvation system was built using the System Builder panel by Desmond (Schrödinger Release 2020-1). The SPC solvent model was chosen from the orthorhombic box boundary of the smallest size, and the force field was set to OPLS2005. After loading the solvated system into the workspace using the Molecular Dynamics panel, the system energy was set to 1.2, and the aggregate class used was NPT. Simulations were set to run at 300.0 K and 1.01325 bar, and the total simulation time for each system was set to 300 ns with a 300 ps orbital tracking interval. Glide docking was performed after ligand grid creation through selection of embedded ligands, and the van der Waals radius scaling parameter scaling factor was set to 1 with partial charge cutoff of 0.25.

결과 및 분석Results and analysis

실험예 1: TRAP1, Hsp90α 및 Grp94에 대한 화합물의 결합 친화도 분석Experimental Example 1: Binding affinity analysis of compounds for TRAP1, Hsp90α and Grp94

실시예에서 제조한 화합물들의 TRAP1, Hsp90α 및 Grp94에 대한 결합 친화도는 플루오레세인 이소티오시아네이트 (FITC)-표지된 PU-H71을 사용하여 형광 편광 분석으로 분석하였다. 하기 표 2 내지 표 4는 모든 시험된 화합물의 IC50 값을 나타낸 것이다.The binding affinity of the compounds prepared in Examples to TRAP1, Hsp90α and Grp94 was analyzed by fluorescence polarization analysis using fluorescein isothiocyanate (FITC)-labeled PU-H71. Tables 2 to 4 below show the IC 50 values of all tested compounds.

화합물compound RR IC50 (nM)IC 50 (nM) 선택성 (IC50 ratio)Selectivity (IC 50 ratio) TRAP1 FPTRAP1 FP Hsp90 N FPHsp90 N FP Grp94 FPGrp94 FP Hsp90/TRAP1Hsp90/TRAP1 Grp94/TRAP1Grp94/TRAP1 PU-H71PU-H71 192.4 ± 6.7192.4 ± 6.7 72.9 ± 1.872.9 ± 1.8 131.6 ± 6.4131.6 ± 6.4 0.380.38 0.680.68 DN401DN401 143.2 ± 13.4143.2 ± 13.4 430.7 ± 8.3430.7 ± 8.3 447.8 ± 18.0447.8 ± 18.0 3.03.0 3.13.1 5a5a 3,4-메틸렌디옥시, 6-Br3,4-methylenedioxy, 6-Br 65.8 ± 1.065.8 ± 1.0 393.7 ± 33.6393.7 ± 33.6 193.9 ± 7.7193.9 ± 7.7 6.06.0 2.92.9 5b5b 2-F, 4-Br2-F, 4-Br 58.0 ± 1.658.0 ± 1.6 1,134 ± 1091,134 ± 109 493.6 ± 15.3493.6 ± 15.3 19.619.6 8.58.5 5c5c 2-MeO, 4-CF3 2-MeO, 4-CF 3 188.6 ± 8.1188.6 ± 8.1 774.0 ± 35.0774.0 ± 35.0 1,616 ± 591,616 ± 59 4.14.1 8.68.6 5d5d 3-Cl, 4-F3-Cl, 4-F 728.4 ± 48.4728.4 ± 48.4 > 5,000> 5,000 > 5,000> 5,000 > 6.9> 6.9 > 6.9> 6.9 5e5e 3-F, 4-F3-F, 4-F 1,722 ± 1151,722 ± 115 > 5,000> 5,000 > 5,000> 5,000 > 2.9> 2.9 > 2.9> 2.9 5f5f 4-CF3 4-CF 3 292.8 ± 15.7292.8 ± 15.7 > 5,000> 5,000 2,547 ± 512,547 ± 51 > 17.1> 17.1 8.78.7 5g5g 2-F, 5-Cl2-F, 5-Cl 242.8 ± 14.2242.8 ± 14.2 4,494 ± 2754,494 ± 275 3,370 ± 1773,370 ± 177 18.518.5 13.913.9 5h5h 3-CF3, 4-Cl3-CF 3 , 4-Cl 143.3 ± 4.5143.3 ± 4.5 > 5,000> 5,000 4,579 ± 1794,579 ± 179 > 34.9> 34.9 31.931.9 5i5i 2-Cl, 5-CF3 2-Cl, 5-CF 3 169.7 ± 4.2169.7 ± 4.2 3,312 ± 4443,312 ± 444 1,867 ± 721,867 ± 72 19.519.5 11.011.0 5j5j 3-Cl, 4-Cl3-Cl, 4-Cl 76.6 ± 1.076.6 ± 1.0 > 5,000> 5,000 985.6 ± 7.1985.6 ± 7.1 > 65.3> 65.3 12.912.9 5k5k 2-F, 4-MeO, 6-F2-F, 4-MeO, 6-F 1,120 ± 511,120 ± 51 > 5,000> 5,000 >5,000>5,000 > 4.5> 4.5 > 4.5> 4.5 5l5l 2-F, 4-Me2-F, 4-Me 62.4 ± 1.262.4 ± 1.2 540.8 ± 49.5540.8 ± 49.5 318.0 ± 6.0318.0 ± 6.0 8.78.7 5.15.1 5m5m 2-F, 3-F, 6-F2-F, 3-F, 6-F 101.4 ± 3.3101.4 ± 3.3 2,470 ± 1442,470 ± 144 1,674 ± 411,674 ± 41 24.424.4 16.516.5

화합물compound R1R1 IC50 (nM)IC50 (nM) 선택성 (IC50 ratio)Selectivity (IC 50 ratio) TRAP1 FPTRAP1 FP Hsp90 N FPHsp90 N FP Grp94 FPGrp94 FP Hsp90/TRAP1Hsp90/TRAP1 Grp94/TRAP1Grp94/TRAP1 PU-H71 PU - H71 192.4 ± 6.7192.4 ± 6.7 72.9 ± 1.872.9 ± 1.8 131.6 ± 6.4131.6 ± 6.4 0.380.38 0.680.68 6a6a 3,4-메틸렌디옥시, 6-Br3,4-methylenedioxy, 6-Br 738.2 ± 26.2738.2 ± 26.2 1,862 ± 861,862 ± 86 1,787 ± 631,787 ± 63 2.52.5 2.42.4 6b6b 2-F, 4-Br2-F, 4-Br 332.1 ± 18.8332.1 ± 18.8 > 5,000> 5,000 >5,000>5,000 > 15.1> 15.1 > 15.1> 15.1 6c6c 2-MeO, 4-CF3 2-MeO, 4-CF 3 638.6 ± 19.5638.6 ± 19.5 > 5,000> 5,000 2,803 ± 2122,803 ± 212 > 7.8> 7.8 4.44.4 6d6d 3-Cl,4-F3-Cl,4-F 590.1 ± 38.3590.1 ± 38.3 > 5,000> 5,000 >5,000>5,000 > 8.5> 8.5 > 8.5> 8.5 6e6e 3-F,4-F3-F, 4-F 720.6 ± 39.0720.6 ± 39.0 > 5,000> 5,000 >5,000>5,000 > 6.9> 6.9 > 6.9> 6.9 6f6f 4-CF3 4-CF 3 412.7 ± 26.4412.7 ± 26.4 > 5000> 5000 3,756 ± 773,756 ± 77 > 12.1> 12.1 9.19.1 6i6i 2-Cl,5-CF3 2-Cl,5-CF 3 1,220 ± 1261,220 ± 126 > 5,000> 5,000 >5,000>5,000 > 4.1> 4.1 > 4.1> 4.1 6j6j 3-Cl, 4-Cl3-Cl, 4-Cl 63.5 ± 1.563.5 ± 1.5 > 5,000> 5,000 1,881 ± 771,881 ± 77 > 78.7> 78.7 29.629.6 6k6k 2-F,4-MeO,6-F2-F,4-MeO,6-F 178.2 ± 4.9178.2 ± 4.9 > 5,000> 5,000 839.5 ± 38.2839.5 ± 38.2 > 28.1> 28.1 4.74.7 6l6l 2-F,4-Me2-F,4-Me 82.7 ± 2.782.7 ± 2.7 > 5,000> 5,000 1,017 ± 461,017 ± 46 > 60.5> 60.5 12.312.3 6m6m 2-F, 3-F, 6-F2-F, 3-F, 6-F 392.1 ± 33.3392.1 ± 33.3 > 5,000> 5,000 3,440 ± 1203,440 ± 120 > 12.8> 12.8 8.88.8 7a7a 3,4- 메틸렌디옥시, 6-Br3,4-Methylenedioxy, 6-Br 616.7 ± 33.0616.7 ± 33.0 778.1 ± 38.5778.1 ± 38.5 1,065 ± 531,065 ± 53 1.31.3 1.71.7 7b7b 2-F, 4-Br2-F, 4-Br 476.8 ± 26.8476.8 ± 26.8 > 5,000> 5,000 3,444 ± 1733,444 ± 173 > 10.5> 10.5 7.27.2 7d7d 3-Cl,4-F3-Cl,4-F >5,000>5,000 > 5,000> 5,000 >5,000>5,000 -- -- 7e7e 3-F,4-F3-F, 4-F >5,000>5,000 > 5,000> 5,000 >5,000>5,000 -- --

화합물compound R1R1 R2R2 IC 50 (nM)IC 50 (nM) 선택성 (IC50 ratio)Selectivity (IC 50 ratio) TRAP1 FPTRAP1 FP Hsp90 N FPHsp90 N FP Grp94 FPGrp94 FP Hsp90/TRAP1Hsp90/TRAP1 Grp94/TRAP1Grp94/TRAP1 PU-H71PU-H71 192.4 ± 6.7192.4 ± 6.7 72.9 ± 1.872.9 ± 1.8 131.6 ± 6.4131.6 ± 6.4 0.380.38 0.680.68 11a11a ClCl 3,4-메틸렌디옥시, 6-Br3,4-methylenedioxy, 6-Br 296.1 ± 23.4296.1 ± 23.4 1,097 ± 581,097 ± 58 811.6 ± 40.0811.6 ± 40.0 3.73.7 2.72.7 11f11f ClCl 4-CF3 4-CF 3 1,037 ± 1001,037 ± 100 > 5,000> 5,000 >5,000>5,000 > 4.8> 4.8 > 4.8> 4.8 11g11g ClCl 2-F, 4-Br2-F, 4-Br 99.5 ± 6.099.5 ± 6.0 > 5,000> 5,000 1,259 ± 921,259 ± 92 > 50.3> 50.3 12.712.7 11i11i ClCl 2-Cl,5-CF3 2-Cl,5-CF 3 1,790 ± 981,790 ± 98 > 5,000> 5,000 >5,000>5,000 > 2.8> 2.8 > 2.8> 2.8 11j11j ClCl 3-Cl, 4-Cl3-Cl, 4-Cl 81.5 ± 3.581.5 ± 3.5 > 5,000> 5,000 1,725 ± 1021,725 ± 102 > 61.3> 61.3 21.221.2 11l11l ClCl 2-F,4-Me2-F,4-Me 89.9 ± 2.289.9 ± 2.2 1,247 ± 761,247 ± 76 1,060 ± 681,060 ± 68 13.913.9 11.811.8 11n11n ClCl 3,4- 메틸렌디옥시,3,4-methylenedioxy, 339.1 ± 24.7339.1 ± 24.7 2,996 ± 1402,996 ± 140 3,193 ± 2553,193 ± 255 8.88.8 9.49.4 11o11o ClCl 2-MeO, 3-MeO2-MeO, 3-MeO 330.8 ± 23.7330.8 ± 23.7 1,965 ± 921,965 ± 92 2,129 ± 902,129 ± 90 5.95.9 6.46.4 11p11p ClCl 2-F,6-F, 3-MeO2-F,6-F, 3-MeO 229.5 ± 17.5229.5 ± 17.5 893.2 ± 49.8893.2 ± 49.8 648.5 ± 54.1648.5 ± 54.1 3.93.9 2.82.8 15a15a MeMe 3,4- 메틸렌디옥시 y, 6-Br3,4- methylenedioxy y, 6-Br 2,848 ± 2242,848 ± 224 > 5,000> 5,000 >5,000>5,000 > 1.8> 1.8 > 1.8> 1.8 15b15b MeMe 2-F, 4-Br2-F, 4-Br 597.3 ± 36.6597.3 ± 36.6 > 5,000> 5,000 >5,000>5,000 > 8.3> 8.3 > 8.3> 8.3

표 2 내지 표 4에 나타낸 바와 같이, 우레아 (화합물 5a), 티오우레아 (화합물6a), 구아니딘 (화합물 7a) 및 아미드 (화합물 11a) 작용기가 기존 화합물 DN401의 5-원 고리에 도입되었을 때, Hsp90 및 Grp94에 비해 TRAP1에 대한 더 우수한 선택성을 가졌다. 그 중에서, 우레아 (화합물 5a)가 TRAP1에 대해 가장 강력한 화합물이며, Hsp90 및 Grp94에 비해 더 우수한 선택성을 가졌다. 다양한 분자 중에서, 우레아 유도체 5a, 5b, 5j, 5l, 티오우레아 유도체 6j, 6l 및 아미드 유도체 11j, 11l은 90 nM 미만의 IC50 값으로 매우 강한 결합 친화도를 갖는 것으로 밝혀졌다. 5j, 6j 및 11j가 TRAP1에 대해 100 nM 이하의 IC50 값을 나타내었는데, 페닐 고리의 메타-위치 및 파라-위치 둘 모두에서의 염소 치환은 TRAP1 소수성 포켓 내로 벤질 모이어티를 수용하는 것으로 판단된다. 또한, 이들 분자의 TRAP1 선택성은 Hsp90 보다 60 배 이상이고 Grp94 보다 13 배 이상이었다. TRAP1에 대한 화합물 6j의 IC50은 63.5 nM이고 각각 Hsp90 및 Grp94에 비해 78 배 및 30 배의 선택성을 가졌다. 또한, 5l, 6l 및 11l와 같이 오르토-플루오로 및 파라-메틸 치환은 우수한 TRAP1 결합 친화도 (TRAP1의 경우 IC50 = 62~90 nM) 및 선택성을 갖도록 효능을 증가시켰다.As shown in Tables 2 to 4, when urea (compound 5a ), thiourea (compound 6a ), guanidine (compound 7a ) and amide (compound 11a ) functional groups are introduced into the 5-membered ring of the existing compound DN401, Hsp90 and better selectivity for TRAP1 compared to Grp94. Among them, urea (compound 5a ) was the most potent compound for TRAP1 and had better selectivity compared to Hsp90 and Grp94. Among the various molecules, the urea derivatives 5a , 5b , 5j , 5l , the thiourea derivatives 6j , 6l and the amide derivatives 11j , 11l were found to have very strong binding affinities with IC 50 values below 90 nM. The 5j, 6j and 11j eotneunde exhibited IC 50 values less than or equal to 100 nM for TRAP1, the phenyl ring in meta-position and para-chlorine substituted in both positions are determined to accommodate the benzyl moiety into TRAP1 hydrophobic pocket . In addition, the TRAP1 selectivity of these molecules was more than 60-fold higher than that of Hsp90 and more than 13-fold higher than that of Grp94. The IC 50 of compound 6j for TRAP1 was 63.5 nM and had 78-fold and 30-fold selectivity over Hsp90 and Grp94, respectively. In addition, ortho -fluoro and para -methyl substitutions, such as 5l , 6l and 11l , increased potency with good TRAP1 binding affinity (IC 50 = 62-90 nM for TRAP1) and selectivity.

따라서, 본 실시예에서 제조한 화합물들은 우수한 TRAP1 선택적 억제 활성을 가짐을 확인하였다.Therefore, it was confirmed that the compounds prepared in this Example have excellent TRAP1 selective inhibitory activity.

실험예 2: 구조-활성 관계 (structure-activity relationship, SAR) 분석Experimental Example 2: Structure-activity relationship (SAR) analysis

실시예에서 제조한 새로운 화합물들에 대한 구조-활성 관계 분석을 위해, 화합물 5a의 TRAP1 N-말단 도메인 (TRAP-NTD)과의 X-선 공-결정 구조 및 화합물 5b의 TRAP1 N-말단 및 중간 도메인 (TRAP1-NM)과의 X-선 공-결정 구조를 측정하고, PUH71 화합물과 비교하여 결합 방식을 평가하였다.For activity relationship analysis, X- ray of the ball and TRAP1 N- terminal domain (NTD-TRAP) of compound 5a - - structure for a new compound prepared in Example TRAP1 N- terminal and the middle of the crystal structure and the compound 5b The X-ray co-crystal structure with the domain (TRAP1-NM) was measured and the binding mode was evaluated compared to the PUH71 compound.

도 1은 (A) TRAP1 NTD에서 화합물 5a의 X-선 공-결정 구조, (B) Hsp90-NTD에서 PU-H71의 X-선 공-결정 구조, (C) TRAP1 NM 도메인에서 화합물 5b의 X-선 공-결정 구조를 나타낸 X-선 결정학 결과이다. 1 shows (A) X-ray co-crystal structure of compound 5a in TRAP1 NTD, (B) X-ray co-crystal structure of PU-H71 in Hsp90-NTD, (C) X-ray co-crystal structure of compound 5b in TRAP1 NM domain. X-ray crystallography results showing the -ray co-crystal structure.

도 1에 나타낸 바와 같이, TRAP1-5a의 NTD 구조를 이전에 보고된 Hsp90-PUH71과 비교할 때, TRAP1에서 ATP-lid의 나선형 구조의 일부가 무질서한 것으로 나타났으며, 결과적으로 Asn171 (Hsp90의 Asn106에 상응함) 및 Leu172 (Hsp90의 L107에 상응함)을 ATP 결합 포켓에 위치시켰다. 이 α-나선 무질서는 TRAP1의 ATP-결합 부위를 Hsp90보다 부분적으로 더 극성으로 만들었다. 따라서, X-선 결정학을 통해 화합물 5a 및 5b가 TRAP1에서 Ans171과 직접 상호 작용함을 알 수 있었다.1, the structure of the NTD TRAP1- 5a as compared to the Hsp90-PUH71 previously reported, was found to be a part of the spiral structure of the ATP-lid disordered in TRAP1, consequently Asn171 (on Asn106 of Hsp90 corresponding) and Leu172 (corresponding to L107 of Hsp90) were located in the ATP binding pocket. This α-helix disorder made the ATP-binding site of TRAP1 partially more polar than Hsp90. Therefore, it was found that compounds 5a and 5b directly interact with Ans171 in TRAP1 through X-ray crystallography.

도 2는 (A) DN401-TRAP1 NTD 구조, (B) 화합물 5a-TRAP1 NTD 구조를 나타낸 X-선 결정학 결과이다. 물은 붉은색 구체이다.2 is an X-ray crystallography result showing the structure of (A) DN401-TRAP1 NTD and (B) compound 5a-TRAP1 NTD. Water is a red sphere.

도 1 및 2에 나타난 바와 같이, 결정 구조는 5a의 N-1, N-3 및 2-아미노가 Asn119, Asp158 및 Thr251과 직접 또는 물-매개 수소 결합을 형성함을 보여 주었다. 이는 DN401과 유사하였다. 가장 두드러진 점은 새로 도입된 5a 및 5b의 카보닐 그룹이 설계된대로 TRAP1에서 Asn171의 아미드와 직접 상호 작용하는 반면(도 2B), DN401은 물-연결된 수소 결합을 통해 Asn171과 상호 작용한다는 것이다(도 2A). 이들 극성 상호 작용은 합성된 분자가 Hsp90에 비해 TRAP1 및 파라로그 선택성에 대한 효능이 개선된 이유를 설명 할 수 있다. 5a의 3,4-메틸렌디옥시-6-브로모페닐 및 5b의 4-브로모-6-플루오로-페닐은 Phe205 및 Trp231로 추가로 π-π 적층하여 Met163, Ile168, Phe205, Trp231 및 Ile253에 의해 형성된 TRAP1 소수성 포켓을 적절히 점유하는 것으로 보인다. 따라서, X-선 결정학을 통해 화합물 5a는 TRAP1의 Asn171과 직접 상호 작용하는 반면, DN401은 물-연결된 수소 결합을 통해 Asn171과 상호 작용하는 것을 알 수 있었다. As shown in Figures 1 and 2, the crystal structure of the N-1, N-3 and 2-amino-5a of Asn119, Asp158 and Thr251 and directly or a water-showed that parameters forming a hydrogen bond. It was similar to DN401. Most notably, the newly introduced carbonyl groups in 5a and 5b interact directly with the amide of Asn171 in TRAP1 as designed (Fig. 2B), whereas DN401 interacts with Asn171 via a water-linked hydrogen bond (Fig. 2A). These polar interactions may explain why the synthesized molecule has improved potency for TRAP1 and paralog selectivity compared to Hsp90. 5a in the 3, 4-methylenedioxy-6-bromophenyl 5b and 4- bromo-6-fluoro-phenyl by π-π stacking in addition to Phe205 Met163 and Trp231, Ile168, Phe205, Ile253, and Trp231 It appears to properly occupy the hydrophobic pocket formed by TRAP1. Therefore, X-ray crystallography revealed that compound 5a directly interacts with Asn171 of TRAP1, whereas DN401 interacts with Asn171 through water-linked hydrogen bonds.

한편, 화합물 5a와 복합체화된 Hsp90을 결정화하여 화합물 5a로 ATP-lid 장애가 가능한지 여부를 조사했지만, 공-결정은 형성되지 않았다. 이것은 또한 화합물 5a의 Hsp90에 대한 약한 결합 친화도 및 결과적으로 Hsp90에 비한 우수한 선택성을 설명 할 수 있다. On the other hand, the crystallization of the compound 5a with the complexation Hsp90 but investigate the ATP-lid disorder whether to compound 5a, co-crystal was not formed. This may also explain the weak binding affinity of compound 5a to Hsp90 and consequently good selectivity over Hsp90.

실험예 3: 분자 역학 시뮬레이션Experimental Example 3: Molecular Dynamics Simulation

TRAP1에 결합하는 화합물의 동적 과정 및 Hsp90에 비한 선택성의 기초를 추가로 연구하기 위해, 본 발명자들은 Hsp90 조절제에 대한 이전 연구에서 유용한 것으로 증명된 300ns MD 시뮬레이션을 수행하였다(Colombo, G. et al., Understanding ligand-based modulation of the Hsp90 molecular chaperone dynamics at atomic resolution Proc. Natl. Acad. Sci. 2008, 105, 7976-7981.). 단백질-리간드의 근 평균 제곱 편차 (root mean square deviation, RMSD)의 시간-의존적 변화, 국소 변화를 특성화하기 위해 사용된 근 평균 제곱 변동 (root mean square fluctuation, RMSF)의 각 잔기, 및 단백질-리간드 상호 작용 분율 계수는 슈뢰딩거 소프트웨어를 사용하여 데스몬드(Desmond) 모듈로부터 수집되었다. 시뮬레이션 궤적의 각 복합물의 결합 포즈는 30 ns 간격으로 0에서 300 ns까지 추출되어 중첩되었다. To further study the basis of the kinetic process of compounds binding to TRAP1 and selectivity relative to Hsp90, we performed 300 ns MD simulations that proved useful in previous studies of Hsp90 modulators (Colombo, G. et al. , Understanding ligand-based modulation of the Hsp90 molecular chaperone dynamics at atomic resolution Proc. Natl. Acad. Sci. 2008 , 105 , 7976-7981.). Time-dependent changes in the root mean square deviation (RMSD) of the protein-ligand, each residue in the root mean square fluctuation (RMSF) used to characterize the local changes, and the protein-ligand Interaction fraction coefficients were collected from the Desmond module using Schrödinger software. The combined poses of each complex of the simulation trajectories were extracted and superimposed from 0 to 300 ns at intervals of 30 ns.

도 3은 TRAP1-5a 복합체의 MD 시뮬레이션 (300ns)을 나타낸 것이다. (A) 30 ns 간격에서 화합물 5a의 결합 방식의 중첩. (B) TRAP1 잔기와의 화합물 5a 상호 작용의 상세한 원자의 개략도. 선택한 궤적 (0-300 ns)에서 30% 이상의 시뮬레이션 시간에 발생하는 상호 작용이 표시된다. (C) 300 ns MD 궤적에 따른 TRAP1 NTD의 RMSD 변화. (D) 5a-TRAP1 상호 작용 분획 누적 막대 차트; 수소 결합 (녹색), 소수성 결합 (회색), 이온성 결합 (빨간색) 및 물 연결 (파란색). Figure 3 shows the MD simulation (300ns) of the TRAP1-5a complex. (A) Superimposition of the binding mode of compound 5a at 30 ns intervals. (B) Schematic of detailed atoms of compound 5a interaction with TRAP1 moieties. Interactions occurring over 30% of the simulation time on the selected trajectory (0-300 ns) are shown. (C) RMSD change of TRAP1 NTD along 300 ns MD trajectory. (D) 5a-TRAP1 interaction fraction stacked bar chart; Hydrogen bonds (green), hydrophobic bonds (grey), ionic bonds (red) and water bonds (blue).

도 3에 나타낸 바와 같이, TRAP1에서 5a의 시뮬레이션 궤적은 0 내지 300 ns에서 일관된 결합 방식을 보여, 이 시뮬레이션 시스템에서 바람직한 안정성을 나타냈다. TRAP1 백본 분석의 RMSD는 또한 복합체가 1 내지 3Å의 RMSD 범위에서 상당히 안정적임을 나타냈다. TRAP1의 Asp158 (Hsp90의 Asp94)과 5a 퓨린 고리의 아민 사이의 잘 알려진 소수성 상호 작용 외에, Met163, Leu168, Leu172, Phe205, Val217 및 Trp231과 같은 소수성 포켓의 잔기를 갖는 5a의 벤질의 소수성 상호 작용 및 π-π 스태킹은 강한 결합에 기여하는 것으로 나타났다. 모든 궤적의 단백질-리간드 상호 작용 분획 계수로부터, 5a의 카르보닐기가 Asn171과 강하게 결합하는 것으로 밝혀졌다. 5a의 카르보닐과 Ans171 사이의 연속적인 직접 수소 결합은 궤적의 85% 이상에서 관찰되었고, 5a의 카르보닐과 Ans171 사이의 물 연결된 수소 결합은 궤적의 15%에서 관련되었다(도 3B 및 D).As shown in Fig. 3, the simulation trajectory of 5a in TRAP1 showed a consistent binding mode from 0 to 300 ns, indicating desirable stability in this simulation system. The RMSD of the TRAP1 backbone analysis also indicated that the complex was fairly stable in the RMSD range of 1 to 3 Å. Besides the well-known hydrophobic interactions between Asp158 of TRAP1 (Asp94 of Hsp90) and the amines of the 5a purine ring, the benzyl hydrophobic interactions of 5a with residues in the hydrophobic pocket such as Met163, Leu168, Leu172, Phe205, Val217 and Trp231 and π–π stacking has been shown to contribute to strong bonding. From the protein-ligand interaction fractional counts of all loci, it was found that the carbonyl group of 5a strongly binds to Asn171. Continuous direct hydrogen bonding between the carbonyl of 5a and Ans171 was observed in over 85% of the trajectories, and water-linked hydrogen bonding between the carbonyl of 5a and Ans171 was involved in 15% of the trajectories (Fig. 3B and D).

도 4는 화합물 5a 또는 DN401과 Hsp90의 도킹된 복합체의 MD 시뮬레이션을 나타낸 것이다. (A) 30 ns 간격에서 Hsp90-5a 복합체의 결합 방식의 중첩. (B) 30 ns 간격에서 Hsp90-DN401 복합체의 결합 방식의 중첩. (C) 300 ns MD 궤적에 따른 Hsp90 NTD의 RMSD 변화. (D) 300 ns MD 궤적에 따른 Hsp90 NTD의 RMSD 변화.Figure 4 shows the MD simulation of compound 5a or the docked complex of DN401 and Hsp90. (A) Superimposition of the binding mode of the Hsp90-5a complex at 30 ns intervals. (B) Overlapping mode of binding of Hsp90-DN401 complexes at 30 ns intervals. (C) RMSD changes of Hsp90 NTDs along the 300 ns MD trajectory. (D) RMSD changes of Hsp90 NTDs along the 300 ns MD trajectory.

도 4에 나타낸 바와 같이, 화합물 5a가 Hsp90에 도킹되고 생성된 복합체를 시뮬레이션 하였을 때, 단백질은 증가된 RMSD 값을 나타내어 70 ns에서부터 불안정성을 나타내기 시작하였다. 화합물 5a의 결합 방식은 시뮬레이션 전반에 걸쳐 변화하였다.As shown in FIG. 4 , when compound 5a was docked to Hsp90 and the resulting complex was simulated, the protein showed an increased RMSD value and started to show instability from 70 ns. The binding mode of compound 5a changed throughout the simulation.

이전에 보고된 바에 따르면, Hsp90에서 ATP-lid의 입체 형태적 유연성은 Hsp90의 약물 결합 동역학 및 열역학의 차이에도 불구하고 다양한 화학형이 특정 단백질 형태에 결합 할 수 있게 한다(Amaral, M. et al., Protein conformational flexibility modulates kinetics and thermodynamics of drug binding Nature Commun. 2017, 8, 2276.). 특히, Hsp90 NTD의 ATP-lid에서 서열 104-111이 가장 유연하고 리간드 결합의 화학형에 따라 다양한 입체구조를 채택 할 수 있다. 잔기-기반 RMSF는 국소 역학의 주요 지표이며, 여기에서, 선택된 잔기를 모니터링하여 전체 역학에 대한 형태적 변화를 이해하였다. As previously reported, the conformational flexibility of ATP-lid in Hsp90 allows different chemotypes to bind to specific protein conformations despite differences in drug binding kinetics and thermodynamics of Hsp90 (Amaral, M. et al. ., Protein conformational flexibility modulates kinetics and thermodynamics of drug binding Nature Commun . 2017 , 8 , 2276.). In particular, in the ATP-lid of Hsp90 NTD, sequences 104-111 are the most flexible and can adopt various three-dimensional structures according to the chemical type of ligand binding. Residue-based RMSF is a key indicator of local dynamics, in which selected residues were monitored to understand conformational changes to overall dynamics.

도 5는 TRAP1 NTD 및 Hsp90 NTD와 다양한 억제제들의 RMSF 값을 나타낸 것이다.5 shows the RMSF values of TRAP1 NTD and Hsp90 NTD and various inhibitors.

도 5에 나타낸 바와 같이, TRAP1의 ATP-lid의 RMSF 값은 결합된 리간드에 따라 Hsp90보다 더 가변적이었다. 그러나, 300 ns 시뮬레이션 동안 TRAP1에서 ATP-lid의 Asn171 및 Leu172 잔기의 RMSF는 매우 낮았다. Hsp90의 경우, 59-76 번째 잔기 및 ATP-lid의 일부를 포함하는 Asn106 및 Leu107의 루프 영역의 RMSF는 결합된 리간드에 따라 변하였다. PU-H71, DN401 및 화합물 5a가 리간드로서 적용될 때, Hsp90의 Asn106 및 Leu107 잔기는 TRAP1 상관 잔기인 Asn171 및 Leu172보다 더 가변적인 것으로 보인다. As shown in Fig. 5, the RMSF value of ATP-lid of TRAP1 was more variable than that of Hsp90 depending on the bound ligand. However, the RMSF of Asn171 and Leu172 residues of ATP-lid in TRAP1 during 300 ns simulation was very low. In the case of Hsp90, the RMSF of the loop regions of Asn106 and Leu107, including residues 59-76 and part of ATP-lid, varied depending on the bound ligand. When PU-H71, DN401 and compound 5a are applied as ligands, the Asn106 and Leu107 residues of Hsp90 appear to be more variable than the TRAP1 correlation residues Asn171 and Leu172.

파라로그 선택성을 설명하기 위해, 퓨린 (PU-H71, BIIB021, DN401, 5a) 또는 벤즈아미드 (SNX0723)와 복합된 Hsp90 NTD의 MD 시뮬레이션을 수행하였다.To account for paralog selectivity, MD simulations of Hsp90 NTD complexed with purines (PU-H71, BIIB021, DN401, 5a ) or benzamide (SNX0723) were performed.

도 6은 300 ns MD 시뮬레이션의 30 ns 간격에서 (A) BIIB021, (B) SNX0723 및 (C) PUH71의 Hsp90에 대한 결합 방식의 중첩을 나타낸 것이다. 이들 선택된 구조는 Hsp90의 ATP 결합 포켓의 외부에 Asn107을 갖는다.Figure 6 shows the overlap of the binding mode of (A) BIIB021, (B) SNX0723 and (C) PUH71 to Hsp90 at 30 ns interval of 300 ns MD simulation. These selected structures have Asn107 outside the ATP binding pocket of Hsp90.

도 6A 및 6B에 나타낸 바와 같이, BIIB021 또는 SNX0723의 Hsp90 복합체의 시뮬레이션 궤적은 약물 결합 부위 외부의 Asn107의 연속적인 집단을 보여주었다. 5-원 고리 상의 카르보닐기로 표시되는 합성된 TRAP1 선택적 화합물 5, 6, 7 및 11은 이러한 궤적에 적합하지 않다. As shown in Figures 6A and 6B, the simulated trajectories of the Hsp90 complex of BIIB021 or SNX0723 showed a continuous population of Asn107 outside the drug binding site. Synthesized TRAP1-selective compounds 5 , 6 , 7 and 11 represented by carbonyl groups on the 5-membered ring are not suitable for this locus.

도 6C에 나타낸 바와 같이, PU-H71과 복합된 Hsp90의 ATP-lid의 나선형 구성 요소의 일부는 초기 MD 시뮬레이션 (0-30ns) 동안 90° 주위로 회전 한 다음 나머지 시뮬레이션 시간 (30-300ns)에 걸쳐 여전히 머물렀다. 회전을 통해, 서열 104-111의 α-나선 형태가 Leu107 잔기로부터 빠르게 왜곡되었다. 결과적으로 Asn106은 Leu107의 위치를 대체하고 Leu107은 약물 결합 부위 내부로 이동했다.As shown in Figure 6C, part of the helical component of the ATP-lid of Hsp90 complexed with PU-H71 rotated around 90° during the initial MD simulation (0–30 ns) and then at the rest of the simulation time (30–300 ns). stayed still across. Through rotation, the α-helical conformation of SEQ ID NOs: 104-111 was rapidly distorted from the Leu107 residue. As a result, Asn106 displaced Leu107 and Leu107 moved inside the drug binding site.

ATP-lid 세그먼트에서의 이러한 화학형 특이적 입체형태 변화는 그의 헬리컬 함량의 현저한 감소를 초래하였고 Hsp90에서 새로운 친유성 결합 포켓을 향한 변형을 초래하였다. 이들 새로 도입된 대안적인 결합 포켓은 이전 Leu107 위치 (TRAP1에서 Asn171 위치)에 Asn106을 가졌지만, 주변 아미노산 Leu107이 5a 및 5b의 치환된 벤질 고리와 반발적으로 상호 작용하는 친유성 ATP 결합 포켓으로도 회전하기 때문에, Asn106의 아미드 측쇄와 합성된 분자의 카르보닐 사이의 수소 결합 상호 작용은 제한된다. 도 7은 Hsp90 (청록색) MD 시뮬레이션의 궤적 (90 ns)과 함께 TRAP1-5a 복합체 (TRAP1: 흰색, 5a: 녹색)를 오버레이한 것이다. 단백질-리간드 상호 작용 분획 계수에 따르면, Asn106과 Leu107이 결합 부위로 회전 될 때 Asn106과의 5a 상호 작용이 증가하지 않았고, Leu-5a 상호 작용도 50-300ns 시뮬레이션 동안 감소한 것을 보여주었다(도 7). 즉, 실시예에서 제조된 새로운 화합물들은 Leu107 방해로 인해 MD 시뮬레이션 중에 생성된 Hsp90의 다양한 궤적에 대한 결합이 제한되어 있다.This chemotype-specific conformational change in the ATP-lid segment resulted in a significant decrease in its helical content and a transformation in Hsp90 towards a new lipophilic binding pocket. These newly introduced alternative binding pockets had Asn106 at the previous Leu107 position (Asn171 position in TRAP1), but also as a lipophilic ATP binding pocket in which the peripheral amino acid Leu107 repulsively interacts with the substituted benzyl rings of 5a and 5b. Due to rotation, the hydrogen bonding interaction between the amide side chain of Asn106 and the carbonyl of the synthesized molecule is limited. 7 is an overlay of the TRAP1-5a complex (TRAP1: white, 5a: green) with the trajectories (90 ns) of Hsp90 (cyan) MD simulations. According to the protein-ligand interaction fraction coefficient, it was shown that the 5a interaction with Asn106 did not increase when Asn106 and Leu107 were rotated into the binding site, and the Leu-5a interaction also decreased during the 50-300 ns simulation (Fig. 7). . That is, the novel compounds prepared in Examples have limited binding to various loci of Hsp90 generated during MD simulations due to Leu107 interference.

결론conclusion

본 실시예에서 제조한 화합물들은 in vitro에서 파라로그성 효소 Hsp90 및 Grp94에 비해 TRAP1에 대해 매우 선택적인 것으로 확인되었다. Hsp90 패밀리 단백질에서 ATP-lid의 화학형-의존적 결합 입체형태는 이소형 선택성에 크게 영향을 미치는 것으로 보인다. ATP-lid의 Asn171 잔기의 이용을 통한 TRAP1 선택적 억제 전략을 X-선 결정학 및 MD 시뮬레이션 연구에 기초하여 조사하였다. 화합물 5a 및 화합물 5b는 Asn107 및 Asp158과의 수소 결합 및 친유성 결합 부위에서 Phe205 및 Trp231과의 π-π 적층의 조합에 의해 TRAP1에 안정적으로 결합하는 것으로 나타났다. 그러나, Leu106이 화합물 5a 및 화합물 5b의 카르보닐기를 접근하지 못하게 하거나 친유성 결합 포켓으로 벤질기가 삽입되는 것을 입체적으로 차단하기 때문에, 이들 상호 작용은 Hsp90 약물 결합 부위에서 제한된다. 특히, 화합물 5j (IC50 = 77nM)는 Hsp90에 비해 65 배 선택성 및 Grp94에 비해 13 배 선택성을 갖는다. TRAP1에 대한 화합물 6j의 IC50은 63.5 nM이었고 Hsp90 및 Grp94에 비해 각각 78 배 및 30 배 선택성을 가졌다. The compounds prepared in this Example were confirmed to be highly selective for TRAP1 compared to the paralogous enzymes Hsp90 and Grp94 in vitro. The chemotype-dependent binding conformation of ATP-lid in Hsp90 family proteins appears to significantly affect isoform selectivity. A strategy for selective inhibition of TRAP1 through the use of the Asn171 residue of ATP-lid was investigated based on X-ray crystallography and MD simulation studies. Compounds 5a and 5b were shown to bind stably to TRAP1 by a combination of hydrogen bonding with Asn107 and Asp158 and π-π stacking with Phe205 and Trp231 at the lipophilic binding site. However, these interactions are limited at the Hsp90 drug binding site, as Leu106 either disallows access to the carbonyl groups of compounds 5a and 5b or sterically blocks the insertion of the benzyl group into the lipophilic binding pocket. In particular, compound 5j (IC50 = 77nM) has a 65-fold selectivity over Hsp90 and a 13-fold selectivity over Grp94. The IC 50 of compound 6j against TRAP1 was 63.5 nM and had 78-fold and 30-fold selectivity over Hsp90 and Grp94, respectively.

따라서, 본 실시예에서 제조된 화합물들은 TRAP1 선택적 억제제로서, TRAP1 활성과 연관된 다양한 질병의 치료에 활용될 수 있다.Accordingly, the compounds prepared in this Example are TRAP1 selective inhibitors, and can be used for the treatment of various diseases associated with TRAP1 activity.

Claims (11)

하기 화학식 1로 표시되는 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염:
[화학식 1]
Figure pat00008

상기 화학식 1에서,
X1은 OH, NH2, SH, 또는 O이고;
X2는 N, NH 또는 CH2이고;
a 및 b 중 어느 하나는 단일 결합이고, 다른 하나는 이중 결합이고;
R1, R2, R3, R4 및 R5는 각각 독립적으로, H, 할로, C1-6 알킬, C2-6 알케닐, 시아노, OR7, SR7 또는 NHR7이거나,
R1, R4 및 R5는 각각 독립적으로, H, 할로, C1-6 알킬, C2-6 알케닐, 시아노, OR7, SR7 또는 NHR7이고, R2 및 R3은 함께 N, O 및 S로 이루어진 군으로부터 각각 독립적으로 선택되는 1개 또는 2개의 헤테로원자를 포함하는 4원 내지 7원의 헤테로고리를 형성하고;
R6은 H, 할로 또는 C1-6 알킬이고;
상기 R7은 H 또는 C1-6 알킬이고;
상기 C1-6 알킬 또는 C2-6 알케닐은 선택적으로 할로 또는 C1-6 알킬로 치환될 수 있다.A compound represented by the following formula (1), a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:
[Formula 1]
Figure pat00008

In Formula 1,
X1 is OH, NH 2 , SH, or O;
X2 is N, NH or CH 2 ;
one of a and b is a single bond and the other is a double bond;
R1, R2, R3, R4 and R5 are each independently H, halo, C 1-6 alkyl, C 2-6 alkenyl, cyano, OR7, SR7 or NHR7;
R1, R4 and R5 are each independently H, halo, C 1-6 alkyl, C 2-6 alkenyl, cyano, OR7, SR7 or NHR7, and R2 and R3 together are N, O and S to form a 4- to 7-membered heterocycle containing 1 or 2 heteroatoms each independently selected from;
R6 is H, halo or C 1-6 alkyl;
wherein R7 is H or C 1-6 alkyl;
The C 1-6 alkyl or C 2-6 alkenyl may be optionally substituted with halo or C 1-6 alkyl. 청구항 1에 있어서, 상기 X1은 OH, NH2 또는 SH이고, 상기 X2는 N이고, 상기 a는 단일 결합이고, 상기 b는 이중 결합인 것인, 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염.The method according to claim 1, wherein X1 is OH, NH 2 or SH, X2 is N, a is a single bond, and b is a double bond, the compound, stereoisomer, solvate, or pharmaceutical thereof acceptable salts. 청구항 1에 있어서, 상기 X1은 O이고, 상기 X2는 NH 또는 CH2이고, 상기 a는 이중 결합이고, 상기 b는 단일 결합인 것인, 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염.The compound, stereoisomer, solvate, or pharmaceutically acceptable compound of claim 1, wherein X1 is O, X2 is NH or CH 2 , a is a double bond, and b is a single bond. possible salts. 청구항 1에 있어서, 상기 R1, R2, R3, R4 및 R5는 각각 독립적으로, H, F, Cl, Br, I, C1-4 알킬, C2-4 알케닐, 시아노, OR7, SR7 또는 NHR7이거나,
R1, R4 및 R5는 각각 독립적으로, H, F, Cl, Br, I, C1-4 알킬, C2-4 알케닐, 시아노, OR7, SR7 또는 NHR7이고, R2 및 R3은 함께 N, O 및 S로 이루어진 군으로부터 각각 독립적으로 선택되는 1개 또는 2개의 헤테로원자를 포함하는 5원 내지 6원의 헤테로고리를 형성하고;
R6은 H, 할로 또는 C1-4 알킬이고;
상기 R7은 H 또는 C1-4 알킬이고;
상기 C1-4 알킬 또는 C2-4 알케닐은 선택적으로 F, Cl, Br 또는 메틸로 치환될 수 있는 것인, 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염.The method according to claim 1, wherein R1, R2, R3, R4 and R5 are each independently H, F, Cl, Br, I, C 1-4 alkyl, C 2-4 alkenyl, cyano, OR7, SR7 or or NHR7;
R1, R4 and R5 are each independently H, F, Cl, Br, I, C 1-4 alkyl, C 2-4 alkenyl, cyano, OR7, SR7 or NHR7, R2 and R3 together are N, Forming a 5- to 6-membered heterocycle containing 1 or 2 heteroatoms each independently selected from the group consisting of O and S;
R6 is H, halo or C 1-4 alkyl;
wherein R7 is H or C 1-4 alkyl;
The compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, wherein the C 1-4 alkyl or C 2-4 alkenyl may be optionally substituted with F, Cl, Br or methyl. 청구항 1에 있어서, 상기 화학식 1로 표시되는 화합물은 화학식 1a로 표시되는 것인, 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염:
[화학식 1a]
Figure pat00009

X1, X2, a, b 및 R6은 청구항 1에 정의된 바와 같고;
R은 하기로부터 선택된다:
Figure pat00010
.The method according to claim 1, wherein the compound represented by Formula 1 is represented by Formula 1a, a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:
[Formula 1a]
Figure pat00009

X1, X2, a, b and R6 are as defined in claim 1;
R is selected from:
Figure pat00010
. 청구항 1에 있어서, 상기 화학식 1의 화합물은 하기 화합물로 이루어진 군으로부터 선택되는 것인, 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염:
2-아미노-9-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-6-클로로-9H-퓨린-8-올;
2-아미노-9-(4-브로모-2-플루오로벤질)-6-클로로-9H-퓨린-8-올;
2-아미노-6-클로로-9-(2-메톡시-4-(트리플루오로메틸)벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(4-(트리플루오로메틸)벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(5-클로로-2-플루오로벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(4-클로로-3-(트리플루오로메틸)벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(2-클로로-5-(트리플루오로메틸)벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(3,4-디클로로벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(2,6-디플루오로-4-메톡시벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(2-플루오로-4-메틸벤질)-9H-퓨린-8-올;
2-아미노-6-클로로-9-(2,3,6-트리플루오로벤질)-9H-퓨린-8-올;
2-아미노-9-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-6-클로로-9H-퓨린-8-티올;
2-아미노-9-(4-브로모-2-플루오로벤질)-6-클로로-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(2-메톡시-4-(트리플루오로메틸)벤질)-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(3-클로로-4-플루오로벤질)-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(3,4-디플루오로벤질)-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(4-(트리플루오로메틸)벤질)-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(2-클로로-5-(트리플루오로메틸)벤질)-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(3,4-디클로로벤질)-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(2,6-디플루오로-4-메톡시벤질)-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(2-플루오로-4-메틸벤질)-9H-퓨린-8-티올;
2-아미노-6-클로로-9-(2,3,6-트리플루오로벤질)-9H-퓨린-8-티올;
9-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-6-클로로-9H-퓨린-2,8-디아민;
9-(4-브로모-2-플루오로벤질)-6-클로로-9H-퓨린-2,8-디아민;
6-클로로-9-(3-클로로-4-플루오로벤질)-9H-퓨린-2,8-디아민;
6-클로로-9-(3,4-디플루오로벤질)-9H-퓨린-2,8-디아민;
2-아미노-7-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-4-클로로-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-4-클로로-7-(4-(트리플루오로메틸)벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-7-(4-브로모-2-플루오로벤질)-4-클로로-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-4-클로로-7-(2-클로로-5-(트리플루오로메틸)벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-4-클로로-7-(3,4-디클로로벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-4-클로로-7-(2-플루오로-4-메틸벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-7-(벤조[d][1,3]디옥솔-5-일메틸)-4-클로로-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-4-클로로-7-(2,3-디메톡시벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-4-클로로-7-(2,6-디플루오로-3-메톡시벤질)-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온;
2-아미노-7-((6-브로모벤조[d][1,3]디옥솔-5-일)메틸)-4-메틸-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온; 및
2-아미노-7-(4-브로모-2-플루오로벤질)-4-메틸-5,7-디히드로-6H-피롤로[2,3-d]피리미딘-6-온.The method according to claim 1, wherein the compound of Formula 1 is selected from the group consisting of the following compounds, a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:
2-Amino-9 - ((6-bromo-benzo [d] [1,3] dioxol-5-yl) methyl) -6-chloro -9 H-purine-8-ol;
2-amino-9- (4-benzyl-Mo-2-fluorobenzyl) -6-chloro -9 H - purin-8-ol;
2-amino-6-chloro-9- (2-methoxy-4- (trifluoromethyl) benzyl) -9 H - purin-8-ol;
2-amino-6-chloro-9-(3-chloro-4-fluorobenzyl)-9 H -purin-8-ol;
2-amino-6-chloro-9-(3-chloro-4-fluorobenzyl)-9 H -purin-8-ol;
(4- (trifluoromethyl) benzyl) 2-Amino-6-chloro -9- -9 H - purin-8-ol;
2-amino-6-chloro-9-(5-chloro-2-fluorobenzyl)-9 H -purin-8-ol;
(4-chloro-3- (trifluoromethyl) benzyl) 2-Amino-6-chloro -9- -9 H - purin-8-ol;
(2-chloro-5- (trifluoromethyl) benzyl) 2-Amino-6-chloro -9- -9 H - purin-8-ol;
2-amino-6-chloro-9-(3,4-dichlorobenzyl)-9 H -purin-8-ol;
2-amino-6-chloro-9-(2,6-difluoro-4-methoxybenzyl)-9 H -purin-8-ol;
2-amino-6-chloro-9-(2-fluoro-4-methylbenzyl)-9 H -purin-8-ol;
2-amino-6-chloro-9-(2,3,6-trifluorobenzyl)-9 H -purin-8-ol;
2-Amino-9 - ((6-bromo-benzo [d] [1,3] dioxol-5-yl) methyl) -6-chloro -9 H-purine-8-thiol;
2-amino-9-(4-bromo-2-fluorobenzyl)-6-chloro-9 H -purine-8-thiol;
2-amino-6-chloro-9-(2-methoxy-4-(trifluoromethyl)benzyl)-9 H -purine-8-thiol;
2-amino-6-chloro-9-(3-chloro-4-fluorobenzyl)-9 H -purine-8-thiol;
2-amino-6-chloro-9-(3,4-difluorobenzyl)-9 H -purine-8-thiol;
2-amino-6-chloro-9-(4-(trifluoromethyl)benzyl)-9 H -purine-8-thiol;
2-amino-6-chloro-9-(2-chloro-5-(trifluoromethyl)benzyl)-9 H -purine-8-thiol;
2-amino-6-chloro-9-(3,4-dichlorobenzyl)-9 H -purine-8-thiol;
2-amino-6-chloro-9-(2,6-difluoro-4-methoxybenzyl)-9 H -purine-8-thiol;
2-amino-6-chloro-9-(2-fluoro-4-methylbenzyl)-9 H -purine-8-thiol;
2-amino-6-chloro-9-(2,3,6-trifluorobenzyl)-9 H -purine-8-thiol;
9 - ((6-bromo-benzo [d] [1,3] dioxol-5-yl) methyl) -6-chloro -9 H-purine-2,8-diamine;
9-(4-bromo-2-fluorobenzyl)-6-chloro-9 H -purine-2,8-diamine;
6-chloro-9-(3-chloro-4-fluorobenzyl)-9 H -purine-2,8-diamine;
6-chloro-9-(3,4-difluorobenzyl)-9 H -purine-2,8-diamine;
2-amino-7 - ((6-bromo-benzo [d] [1,3] dioxol-5-yl) methyl) -4-chloro-5,7-dihydro -6 H-pyrrolo [2, 3- d ]pyrimidin-6-one;
2-amino-4-chloro-7- (4- (trifluoromethyl) benzyl) - 5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6-one;
2-amino-7- (4-bromo-2-fluorobenzyl) -4-chloro-5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6-one;
2-Amino-4-chloro-7- (2-chloro-5- (trifluoromethyl) benzyl) -5,7-dihydro-6 H -pyrrolo [2,3- d ] pyrimidine-6- On;
2-amino-4-chloro-7- (3,4-dichlorobenzyl) -5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6-one;
2-amino-4-chloro-7-(2-fluoro-4-methylbenzyl)-5,7-dihydro-6H-pyrrolo[2,3- d ]pyrimidin-6-one;
2-amino-7- (benzo [d] [1,3] dioxol-5-ylmethyl) -4-chloro-5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidine -6-one;
2-amino-4-chloro-7- (2,3-dimethoxybenzyl) -5,7-dihydro -6 H - pyrrolo [2,3-d] pyrimidin-6-one;
2-amino-4-chloro-7- (2, 6-Difluoro-3-methoxy-benzyl) - 5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6 On;
2-amino-7-((6-bromobenzo[d][1,3]dioxol-5-yl)methyl)-4-methyl-5,7-dihydro-6H-pyrrolo[2,3 - d ]pyrimidin-6-one; and
2-amino-7- (4-benzyl-Mo-2-fluorobenzyl) -4-methyl-5,7-dihydro -6 H - pyrrolo [2,3- d] pyrimidin-6-one. 청구항 1 내지 6 중 어느 한 항에 따른 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염을 포함하는 암 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer, comprising the compound according to any one of claims 1 to 6, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof. 청구항 7에 있어서, 상기 화합물, 이의 입체이성질체, 용매화물, 또는 약학적으로 허용가능한 염은, Hsp90 (Heat shock protein 90) 또는 Grp94 (Glucose-regulated protein 94)에 비해 TRAP1 (TNF receptor-Associated Protein 1)를 선택적으로 억제하는 것인, 약학적 조성물.The method according to claim 7, wherein the compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt is, compared to Hsp90 (Heat shock protein 90) or Grp94 (Glucose-regulated protein 94) TRAP1 (TNF receptor-Associated Protein 1) ) to selectively inhibit, the pharmaceutical composition. 청구항 7에 있어서, 상기 암은 TRAP1의 활성과 연관된 암인 것인 약학적 조성물.The pharmaceutical composition according to claim 7, wherein the cancer is a cancer associated with the activity of TRAP1. 청구항 7에 있어서, 상기 암은 유방암, 췌장암, 대장암, 폐암, 전립선암, 자궁경부암, 자궁내막암, 신경교종, 간암, 난소암, 뇌암, 방광암, 신장암, 신경아세포종, 구강암, 위암, 골암, 직장암, 갑상선암, 피부암, 흑색종, 혈액암, 림프종, 및 췌장신경내분비종양으로 이루어진 군으로부터 선택되는 것인 약학적 조성물.The method according to claim 7, wherein the cancer is breast cancer, pancreatic cancer, colorectal cancer, lung cancer, prostate cancer, cervical cancer, endometrial cancer, glioma, liver cancer, ovarian cancer, brain cancer, bladder cancer, kidney cancer, neuroblastoma, oral cancer, stomach cancer, bone cancer , a pharmaceutical composition selected from the group consisting of rectal cancer, thyroid cancer, skin cancer, melanoma, hematological cancer, lymphoma, and pancreatic neuroendocrine tumor. 청구항 7에 있어서, 항암제를 더 포함하는 것인 약학적 조성물.The pharmaceutical composition of claim 7, further comprising an anticancer agent.
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