KR101829545B1 - N2,N6-substituted-9H-purine-2,6-diamine derivatives having prophylatic and therapeutic effects against hepatoxicity, method for preparing the same, and pharmaceutical composition comprising the same - Google Patents

N2,N6-substituted-9H-purine-2,6-diamine derivatives having prophylatic and therapeutic effects against hepatoxicity, method for preparing the same, and pharmaceutical composition comprising the same Download PDF

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KR101829545B1
KR101829545B1 KR1020160111890A KR20160111890A KR101829545B1 KR 101829545 B1 KR101829545 B1 KR 101829545B1 KR 1020160111890 A KR1020160111890 A KR 1020160111890A KR 20160111890 A KR20160111890 A KR 20160111890A KR 101829545 B1 KR101829545 B1 KR 101829545B1
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변영주
김상겸
김세윤
한용만
이슬기
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고려대학교 세종산학협력단
한국과학기술원
충남대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/16—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/40—Heterocyclic compounds containing purine ring systems with halogen atoms or perhalogeno-alkyl radicals directly attached in position 2 or 6

Abstract

The present invention relates to a N2,N6-substituted-9H-purine-2,6-diamine derivative having prophylactic and therapeutic effects on hepatoxicity; a preparation method of the same; and a pharmaceutical composition comprising the same. More specifically, the present invention relates to a N2,N6-substituted-9H-purine-2,6-diamine derivative represented by chemical formula 1; a preparation method of the same; and a pharmaceutical composition comprising the same. In the chemical formula 1: R_1 a substituted alkyl group having 1-20 carbon atoms, a substituted cycloalkyl group having 3-20 carbon atoms, a substituted cycloheteroalkyl group having 1-20 carbon atoms, a substituted arylalkyl group having 3-20 carbon atoms, a substituted heteroalkyl group having 1-20 carbon atoms, or a substituted heteroarylalkyl group having 1-20 carbon atoms; and R_2 is a substituted alkyl group having 1-20 carbon atoms, a substituted cycloalkyl group having 3-20 carbon atoms, a substituted cycloheteroalkyl group having 1-20 carbon atoms, a substituted arylalkyl group having 3-20 carbon atoms, a substituted heteroalkyl group having 1-20 carbon atoms, or a substituted heteroarylalkyl group having 1-20 carbon atoms.

Description

간 독성 예방 및 치료 효과를 나타내는 N2,N6-치환된-9H-퓨린-2,6-다이아민 (N2,N6-substituted-9H-purine-2,6-diamine) 유도체, 그 제조방법 및 이를 포함하는 약학 조성물 {N2,N6-substituted-9H-purine-2,6-diamine derivatives having prophylatic and therapeutic effects against hepatoxicity, method for preparing the same, and pharmaceutical composition comprising the same}N2, N6-substituted-9H-purine-2,6-diamine derivatives exhibiting the effect of preventing and treating liver toxicity N6-substituted-9H-purine-2,6-diamine derivatives having prophylactic and therapeutic effects against hepatoxicity, a method for preparing same, and pharmaceutical composition comprising the same.

본 발명은 간 독성 예방 및 치료 효과를 나타내는 N2,N6-치환된-9H-퓨린-2,6-다이아민 (N2,N6-substituted-9H-purine-2,6-diamine) 유도체에 관한 것이다.The present invention relates to N2, N6-substituted-9H-purine-2,6-diamine derivatives which exhibit hepatotoxicity preventive and therapeutic effects.

TNP (N2-(m-trifluorobenzyl),N6-(p-nitrobenzyl)purine)는 상업적으로 판매되는 화학물질로서, 현재까지 알려진 바로는 이노시톨 펜타키포스페이트 키나아제 (inositol pentakisphosphate kinase)를 특이적으로 저해하는 약물이다 (비특허문헌 1). TNP는 5-이노시톨 파이로인산 (inositol pyrophosphate)의 생합성을 억제함으로써, 세포 내 5-이노시톨 다인산 (polyphosphate)의 수준을 낮출 뿐 아니라, 간세포의 인슐린 신호전달반응을 증가시킨다는 효과가 보고되어 있다 (비특허문헌 2). 또한, TNP는 골수 유래 중간엽 줄기세포에 처리할 경우, 세포의 노화진행반응을 감소시키는 효과를 갖는 것으로도 알려져 있다 (비특허문헌 3).TNP (N-2- (p-nitrobenzyl) purine) is a commercially available chemical substance, (Non-Patent Document 1). TNP has been reported to inhibit the biosynthesis of 5-inositol pyrophosphate, thereby lowering the level of intracellular 5-inositol polyphosphate and increasing the insulin signal transduction in hepatocytes Non-Patent Document 2). In addition, TNP is also known to have an effect of reducing the aging progression of cells when treated with bone marrow-derived mesenchymal stem cells (Non-Patent Document 3).

그러나, 아직까지 TNP 약물에 대한 CYP3A4 대사와의 관련성은 전혀 보고된 바가 없다. CYP450 (cytochrome P450)은 약물 대사의 75%를 담당하는 것으로 알려져 있으며, 시판되는 약물의 50% 이상이 CYP450에 의해 대사되는 것으로 보고되어있다. CYP는 다양한 동종효소 (isoform)를 가지고 있으며, CYP3A4는 간이나 장에서 약물의 대사에 관여하는 대표적인 효소이다.However, there is no report yet on the association of CYP3A4 metabolism with TNP drugs. CYP450 (cytochrome P450) is known to be responsible for 75% of drug metabolism, and more than 50% of the marketed drugs are reported to be metabolized by CYP450. CYP has a variety of isoforms, and CYP3A4 is a typical enzyme involved in drug metabolism in the liver or intestine.

비특허문헌 1: J Biol Chem. Apr 17, 2009; 284(16): 10571-10582Non-Patent Document 1: J Biol Chem. Apr 17, 2009; 284 (16): 10571-10582 비특허문헌 2: Cell. 2010; 143(6): 897-910Non-Patent Document 2: Cell. 2010; 143 (6): 897-910 비특허문헌 3: Stem Cell Res Ther. 2014 Mar 26;5(2):33Non-Patent Document 3: Stem Cell Res Ther. 2014 Mar 26; 5 (2): 33

이에, 본 발명에서는 약물의 대사에 관여하는 CYP효소의 일종인 3A4에 의해서 대사되는 반응을 억제함으로써, 간 독성 예방 및 치료 효과를 나타내는 N2,N6-치환된-9H-퓨린-2,6-다이아민 (N2,N6-substituted-9H-purine-2,6-diamine) 유도체, 그 제조방법 및 이를 포함하는 약학 조성물을 제공하고자 한다.Accordingly, in the present invention, N2, N6-substituted-9H-purine-2,6-diazabicyclo [2.2.1] quinazolin-4-yl, which exhibits hepatic toxicity prevention and therapeutic effects, is inhibited by inhibiting the metabolism by 3A4, a kind of CYP enzyme involved in drug metabolism. (N2, N6-substituted-9H-purine-2,6-diamine) derivatives, a process for preparing the same, and a pharmaceutical composition containing the same.

본 발명은 상기 과제를 해결하기 위해서, 하기 화학식 1로 표시되는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체를 제공한다:In order to solve the above-mentioned problems, the present invention provides an N2, N6-substituted-9H-purine-2,6-diamine derivative represented by the following formula

Figure 112016085009003-pat00001
Figure 112016085009003-pat00001

상기 식에서,In this formula,

R1은 탄소수 1 내지 20의 치환된 알킬, 탄소수 3 내지 20의 치환된 사이클로알킬, 탄소수 1 내지 20의 치환된 사이클로헤테로알킬, 탄소수 3 내지 20의 치환된 아릴알킬, 탄소수 1 내지 20의 치환된 헤테로알킬, 또는 탄소수 1 내지 20의 치환된 헤테로아릴알킬이고,R 1 is selected from the group consisting of substituted alkyl of 1 to 20 carbon atoms, substituted cycloalkyl of 3 to 20 carbon atoms, substituted cycloheteroalkyl of 1 to 20 carbon atoms, substituted arylalkyl of 3 to 20 carbon atoms, Heteroaryl, or substituted heteroarylalkyl of 1 to 20 carbon atoms,

R2는 탄소수 1 내지 20의 치환된 알킬, 탄소수 3 내지 20의 치환된 사이클로알킬, 탄소수 1 내지 20의 치환된 사이클로헤테로알킬, 탄소수 3 내지 20의 치환된 아릴알킬, 탄소수 1 내지 20의 치환된 헤테로알킬, 또는 탄소수 1 내지 20의 치환된 헤테로아릴알킬이다.R 2 is selected from the group consisting of substituted alkyl of 1 to 20 carbon atoms, substituted cycloalkyl of 3 to 20 carbon atoms, substituted cycloheteroalkyl of 1 to 20 carbon atoms, substituted arylalkyl of 3 to 20 carbon atoms, Heteroaryl, or substituted heteroarylalkyl of 1 to 20 carbon atoms.

본 발명의 일 구현예에 따르면, 상기 화학식 1의 화합물은 하기의 화학식 2 내지 화학식 19로 이루어진 군으로부터 선택된 어느 하나의 화합물일 수 있다:According to one embodiment of the present invention, the compound of Formula 1 may be any one selected from the following Formulas 2 to 19:

Figure 112016085009003-pat00002
Figure 112016085009003-pat00002

Figure 112016085009003-pat00003
Figure 112016085009003-pat00003

Figure 112016085009003-pat00004
Figure 112016085009003-pat00004

Figure 112016085009003-pat00005
Figure 112016085009003-pat00005

Figure 112016085009003-pat00006
Figure 112016085009003-pat00006

Figure 112016085009003-pat00007
Figure 112016085009003-pat00007

Figure 112016085009003-pat00008
Figure 112016085009003-pat00008

Figure 112016085009003-pat00009
Figure 112016085009003-pat00009

Figure 112016085009003-pat00010
Figure 112016085009003-pat00010

Figure 112016085009003-pat00011
Figure 112016085009003-pat00011

Figure 112016085009003-pat00012
Figure 112016085009003-pat00012

Figure 112016085009003-pat00013
Figure 112016085009003-pat00013

Figure 112016085009003-pat00014
Figure 112016085009003-pat00014

Figure 112016085009003-pat00015
Figure 112016085009003-pat00015

Figure 112016085009003-pat00016
Figure 112016085009003-pat00016

Figure 112016085009003-pat00017
Figure 112016085009003-pat00017

Figure 112016085009003-pat00018
Figure 112016085009003-pat00018

Figure 112016085009003-pat00019
Figure 112016085009003-pat00019

또한, 본 발명은 하기 화학식 20으로 표시되는 화합물을 아민, n-부탄올 및 NaBF4와 반응시킴으로써 하기 화학식 1로 표시되는 화합물을 제조하는 단계를 포함하는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체의 제조방법을 제공한다:In addition, the present invention relates to an amine compound represented by the formula 20, n- butanol, and NaBF 4 with reaction by the following Formula 1 N2, N6- substituted -9H- comprising the step of producing a compound represented by the purine -2 , A process for preparing a 6-diamine derivative:

Figure 112016085009003-pat00020
Figure 112016085009003-pat00020

<화학식 1>&Lt; Formula 1 >

Figure 112016085009003-pat00021
Figure 112016085009003-pat00021

상기 식에서, In this formula,

R1은 탄소수 1 내지 20의 치환된 알킬, 탄소수 3 내지 20의 치환된 사이클로알킬, 탄소수 1 내지 20의 치환된 사이클로헤테로알킬, 탄소수 3 내지 20의 치환된 아릴알킬, 탄소수 1 내지 20의 치환된 헤테로알킬, 또는 탄소수 1 내지 20의 치환된 헤테로아릴알킬이고,R 1 is selected from the group consisting of substituted alkyl of 1 to 20 carbon atoms, substituted cycloalkyl of 3 to 20 carbon atoms, substituted cycloheteroalkyl of 1 to 20 carbon atoms, substituted arylalkyl of 3 to 20 carbon atoms, Heteroaryl, or substituted heteroarylalkyl of 1 to 20 carbon atoms,

R2는 탄소수 1 내지 20의 치환된 알킬, 탄소수 3 내지 20의 치환된 사이클로알킬, 탄소수 1 내지 20의 치환된 사이클로헤테로알킬, 탄소수 3 내지 20의 치환된 아릴알킬, 탄소수 1 내지 20의 치환된 헤테로알킬, 또는 탄소수 1 내지 20의 치환된 헤테로아릴알킬이다.R 2 is selected from the group consisting of substituted alkyl of 1 to 20 carbon atoms, substituted cycloalkyl of 3 to 20 carbon atoms, substituted cycloheteroalkyl of 1 to 20 carbon atoms, substituted arylalkyl of 3 to 20 carbon atoms, Heteroaryl, or substituted heteroarylalkyl of 1 to 20 carbon atoms.

본 발명의 일 구현예에 따르면, 상기 화학식 20으로 표시되는 화합물은 하기 화학식 21로 표시되는 화합물을 DMF 중에서 아민 및 트리에틸아민과 반응시킴으로써 제조될 수 있다:According to one embodiment of the present invention, the compound represented by Formula 20 may be prepared by reacting a compound represented by Formula 21 with an amine and triethylamine in DMF:

Figure 112016085009003-pat00022
Figure 112016085009003-pat00022

더 나아가, 본 발명은 상기 화학식 1로 표시되는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체 또는 그 염을 유효성분으로 함유하는 간독성 예방 및 치료를 위한 약학 조성물을 제공한다.Furthermore, the present invention provides a pharmaceutical composition for the prevention and treatment of hepatotoxicity comprising the N2, N6-substituted-9H-purine-2,6-diamine derivative represented by the above formula (1) or its salt as an active ingredient .

본 발명의 일 구현예에 따르면, 상기 약학 조성물은 다른 간독성 예방 및 치료를 위한 약물, 담체, 희석제, 보조제 및 안정화제로 이루어진 군으로부터 선택된 하나 이상의 성분을 더 포함할 수 있다.According to an embodiment of the present invention, the pharmaceutical composition may further include one or more components selected from the group consisting of drugs, carriers, diluents, adjuvants and stabilizers for other hepatotoxicity prevention and treatment.

본 발명의 다른 구현예에 따르면, 상기 안정화제는 단백질, 당질, 완충제 및 그 혼합물로 이루어진 군으로부터 선택될 수 있다.According to another embodiment of the present invention, the stabilizing agent may be selected from the group consisting of proteins, saccharides, buffers, and mixtures thereof.

본 발명에 따르면, 약물의 대사에 관여하는 CYP효소의 일종인 3A4에 의해서 대사되는 반응을 억제함으로써, 간 독성 예방 및 치료 효과를 나타내는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체 및 이를 포함하는 약학 조성물을 제공하며, 이를 이용해서 다양한 간독성을 예방하거나 치료할 수 있다.According to the present invention, N2, N6-substituted-9H-purine-2,6-diazabicyclo [2.2.1] quinazoline, which exhibits hepatic toxicity prevention and therapeutic effects by inhibiting the metabolism by 3A4, a kind of CYP enzyme involved in drug metabolism, And derivatives thereof and pharmaceutical compositions containing the same, and can be used to prevent or treat various hepatotoxicity.

도 1은 퓨린 유도체의 6번 위치와 2번 위치에 4-설포닐벤질아민, 3-피리딜메틸아민, 이미다졸릴메틸아민, 벤즈이미다졸릴메틸아민기가 치환된 화합물들의 합성과정을 개략적으로 도시한 반응 개요도이다.
도 2a 내지 2j는 TNP 약물의 p450 CYP 효소 활성조절 평가 결과 (인간 간 마이크로좀)를 나타낸 그래프이다.
도 3은 TNP 약물의 CYP3A4 활성저해능 평가 결과 (인간 CYP3A4 마이크로좀)를 나타낸 그래프이다.
도 4는 TNP 약물의 CYP3A4 활성저해능 평가 결과 (줄기세포유래 간세포)를 나타낸 그래프이다.
도 5는 TNP 유도체들의 CYP3A4 억제효능 개선 평가 결과를 나타낸 그래프이다.
도 6은 TNP 유도체들의 CYP3A4 억제효능 상실 평가 결과를 나타낸 그래프이다.FIG. 1 schematically shows a synthesis process of compounds substituted with 4-sulfonylbenzylamine, 3-pyridylmethylamine, imidazolylmethylamine, and benzimidazolylmethylamine groups at positions 6 and 2 of a purine derivative Fig.
FIGS. 2A to 2J are graphs showing the result of evaluation of the p450 CYP enzyme activity control (human liver microsomes) of the TNP drug. FIG.
FIG. 3 is a graph showing the results of evaluation of CYP3A4 activity inhibition (human CYP3A4 microsomes) of TNP drug. FIG.
FIG. 4 is a graph showing the result of evaluation of CYP3A4 activity inhibition (stem cell-derived hepatocytes) of TNP drug. FIG.
FIG. 5 is a graph showing the results of evaluating the inhibitory effect of CYP3A4 on TNP derivatives.
FIG. 6 is a graph showing the results of the loss of CYP3A4 inhibitory activity of TNP derivatives. FIG.

이하, 본 발명을 더욱 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in more detail.

본 발명에서는 하기 화학식 1의 일반식을 갖는 퓨린 유도체들이 대사에 관여하는 CYP3A4를 효과적으로 저해함을 최초로 확인하였으며, 이를 기반으로 본 발명을 완성하게 되었다.In the present invention, it has been confirmed for the first time that purine derivatives having the general formula of the following formula 1 effectively inhibit CYP3A4 involved in metabolism. Based on this, the present invention has been completed.

따라서, 본 발명에서는 하기 화학식 1로 표시되는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체를 제공한다:Accordingly, the present invention provides an N2, N6-substituted-9H-purine-2,6-diamine derivative represented by the following Formula 1:

<화학식 1>&Lt; Formula 1 >

Figure 112016085009003-pat00023
Figure 112016085009003-pat00023

상기 식에서, In this formula,

R1은 탄소수 1 내지 20의 치환된 알킬, 탄소수 3 내지 20의 치환된 사이클로알킬, 탄소수 1 내지 20의 치환된 사이클로헤테로알킬, 탄소수 3 내지 20의 치환된 아릴알킬, 탄소수 1 내지 20의 치환된 헤테로알킬, 또는 탄소수 1 내지 20의 치환된 헤테로아릴알킬이고,R 1 is selected from the group consisting of substituted alkyl of 1 to 20 carbon atoms, substituted cycloalkyl of 3 to 20 carbon atoms, substituted cycloheteroalkyl of 1 to 20 carbon atoms, substituted arylalkyl of 3 to 20 carbon atoms, Heteroaryl, or substituted heteroarylalkyl of 1 to 20 carbon atoms,

R2는 탄소수 1 내지 20의 치환된 알킬, 탄소수 3 내지 20의 치환된 사이클로알킬, 탄소수 1 내지 20의 치환된 사이클로헤테로알킬, 탄소수 3 내지 20의 치환된 아릴알킬, 탄소수 1 내지 20의 치환된 헤테로알킬, 또는 탄소수 1 내지 20의 치환된 헤테로아릴알킬이다.R 2 is selected from the group consisting of substituted alkyl of 1 to 20 carbon atoms, substituted cycloalkyl of 3 to 20 carbon atoms, substituted cycloheteroalkyl of 1 to 20 carbon atoms, substituted arylalkyl of 3 to 20 carbon atoms, Heteroaryl, or substituted heteroarylalkyl of 1 to 20 carbon atoms.

참고로, 본 발명에 있어서, "치환된 알킬", 또는 "치환된 사이클로알킬"이라 함은, 상기 화학식 1의 퓨린 모체의 R1 또는 R2 위치에 알킬기가 부착되고, 이러한 알킬기에 사이클로알킬기가 부착된 치환기를 포함하는 의미로 사용된다.For reference, in the present invention, the term "substituted alkyl" or "substituted cycloalkyl" refers to an alkyl group attached to the R 1 or R 2 position of the purine moiety of Formula 1, and a cycloalkyl group Quot; is used herein to mean including an attached substituent.

본 발명에 따른 퓨린 유도체는 방향족 치환반응을 통해서 제조할 수 있는 바, 예를 들어 출발 물질인 2,6-다이클로로-9-퓨린에 치환된 아릴알킬아민 또는 헤테로아릴알킬아민을 사용하여 반응시킴으로써 다양한 유도체를 합성할 수 있다.The purine derivatives according to the present invention can be prepared by an aromatic substitution reaction, for example, by reacting with an arylalkylamine or a heteroarylalkylamine substituted with 2,6-dichloro-9-purine as a starting material Various derivatives can be synthesized.

따라서, 본 발명은 또한 하기 화학식 20으로 표시되는 화합물을 아민, n-부탄올 및 NaBF4와 반응시킴으로써 하기 화학식 1로 표시되는 화합물을 제조하는 단계를 포함하는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체의 제조방법을 제공한다:Accordingly, the present invention relates to also to the amine by, n- butanol, and NaBF 4 and a compound represented by the general formula N2 20, including the step of producing a compound represented by the formula 1 N6- substituted -9H- purine- A process for the preparation of 2,6-diamine derivatives is provided:

<화학식 20>(20)

Figure 112016085009003-pat00024
Figure 112016085009003-pat00024

<화학식 1>&Lt; Formula 1 >

Figure 112016085009003-pat00025
Figure 112016085009003-pat00025

상기 식에서, In this formula,

R1은 탄소수 1 내지 20의 치환된 알킬, 탄소수 3 내지 20의 치환된 사이클로알킬, 탄소수 1 내지 20의 치환된 사이클로헤테로알킬, 탄소수 3 내지 20의 치환된 아릴알킬, 탄소수 1 내지 20의 치환된 헤테로알킬, 또는 탄소수 1 내지 20의 치환된 헤테로아릴알킬이고,R 1 is selected from the group consisting of substituted alkyl of 1 to 20 carbon atoms, substituted cycloalkyl of 3 to 20 carbon atoms, substituted cycloheteroalkyl of 1 to 20 carbon atoms, substituted arylalkyl of 3 to 20 carbon atoms, Heteroaryl, or substituted heteroarylalkyl of 1 to 20 carbon atoms,

R2는 탄소수 1 내지 20의 치환된 알킬, 탄소수 3 내지 20의 치환된 사이클로알킬, 탄소수 1 내지 20의 치환된 사이클로헤테로알킬, 탄소수 3 내지 20의 치환된 아릴알킬, 탄소수 1 내지 20의 치환된 헤테로알킬, 또는 탄소수 1 내지 20의 치환된 헤테로아릴알킬이다.R 2 is selected from the group consisting of substituted alkyl of 1 to 20 carbon atoms, substituted cycloalkyl of 3 to 20 carbon atoms, substituted cycloheteroalkyl of 1 to 20 carbon atoms, substituted arylalkyl of 3 to 20 carbon atoms, Heteroaryl, or substituted heteroarylalkyl of 1 to 20 carbon atoms.

본 발명의 일 구현예에 따르면, 상기 화학식 20으로 표시되는 화합물은 하기 화학식 21로 표시되는 화합물을 DMF 중에서 아민 및 트리에틸아민과 반응시킴으로써 제조될 수 있다:According to one embodiment of the present invention, the compound represented by Formula 20 may be prepared by reacting a compound represented by Formula 21 with an amine and triethylamine in DMF:

<화학식 21>&Lt; Formula 21 >

Figure 112016085009003-pat00026
.
Figure 112016085009003-pat00026
.

하기 반응식 1에는 본 발명의 일 구현예에 따른 퓨린 유도체의 제조방법에 대한 개략적인 반응식을 도시하였다:Scheme 1 depicts a schematic reaction scheme for preparing a purine derivative according to one embodiment of the present invention.

<반응식 1><Reaction Scheme 1>

Figure 112016085009003-pat00027
Figure 112016085009003-pat00027

예를 들어, 아릴알킬아민으로서 4-설포닐벤질아민을, 헤테로아릴알킬아민으로서 3-피리딜메틸아민, 이미다졸릴메틸아민 또는 벤즈이미다졸릴메틸아민을 사용하는 경우, 하기 화학식 2 내지 19로 표시되는 다양한 퓨린 유도체들을 제조하는 것이 가능하다.For example, when using 4-sulfonylbenzylamine as the arylalkylamine and 3-pyridylmethylamine, imidazolylmethylamine or benzimidazolylmethylamine as the heteroarylalkylamine, compounds represented by the following formulas 2 to 19 It is possible to prepare various purine derivatives represented by the following formula.

<화학식 2>(2)

Figure 112016085009003-pat00028
Figure 112016085009003-pat00028

<화학식 3>(3)

Figure 112016085009003-pat00029
Figure 112016085009003-pat00029

<화학식 4>&Lt; Formula 4 >

Figure 112016085009003-pat00030
Figure 112016085009003-pat00030

<화학식 5>&Lt; Formula 5 >

Figure 112016085009003-pat00031
Figure 112016085009003-pat00031

<화학식 6>(6)

Figure 112016085009003-pat00032
Figure 112016085009003-pat00032

<화학식 7>&Lt; Formula 7 >

Figure 112016085009003-pat00033
Figure 112016085009003-pat00033

<화학식 8>(8)

Figure 112016085009003-pat00034
Figure 112016085009003-pat00034

<화학식 9>&Lt; Formula 9 >

Figure 112016085009003-pat00035
Figure 112016085009003-pat00035

<화학식 10>&Lt; Formula 10 >

Figure 112016085009003-pat00036
Figure 112016085009003-pat00036

<화학식 11>&Lt; Formula 11 >

Figure 112016085009003-pat00037
Figure 112016085009003-pat00037

<화학식 12>&Lt; Formula 12 >

Figure 112016085009003-pat00038
Figure 112016085009003-pat00038

<화학식 13>&Lt; Formula 13 >

Figure 112016085009003-pat00039
Figure 112016085009003-pat00039

<화학식 14>&Lt; Formula 14 >

Figure 112016085009003-pat00040
Figure 112016085009003-pat00040

<화학식 15>&Lt; Formula 15 >

Figure 112016085009003-pat00041
Figure 112016085009003-pat00041

<화학식 16>&Lt; Formula 16 >

Figure 112016085009003-pat00042
Figure 112016085009003-pat00042

<화학식 17>&Lt; Formula 17 >

Figure 112016085009003-pat00043
Figure 112016085009003-pat00043

<화학식 18>&Lt; Formula 18 >

Figure 112016085009003-pat00044
Figure 112016085009003-pat00044

<화학식 19>(19)

Figure 112016085009003-pat00045
.
Figure 112016085009003-pat00045
.

한편, 본 발명에서는 상기 화학식 1로 표시되는 퓨린 유도체 또는 그 염을 유효성분으로 함유하는 간 독성 예방 및 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for prevention and treatment of hepatotoxicity, which comprises the purine derivative represented by the formula (1) or a salt thereof as an active ingredient.

본 발명에서, "유효 성분으로 포함"이라는 용어는, 원하는 생물학적 효과를 실현하는데 필요하거나 또는 충분한 양으로 해당 성분이 포함되는 것을 의미한다. 실제 적용에 있어서 유효 성분으로 포함되는 양의 결정은 대상 질병을 치료하기 위한 양으로서, 다른 독성을 야기하지 않는 사항을 고려해서 결정될 수 있으며, 예를 들어 치료되는 질병 또는 병태, 투여되는 조성물의 형태, 피험체의 크기, 또는 질병 또는 병태의 심각도 등과 같은 다양한 인자에 따라서 변화될 수 있다. 본 발명이 속하는 분야에서 통상의 기술을 지닌 기술자라면 과도한 실험을 동반하지 않고 개별적 조성물의 유효량을 경험적으로 결정할 수 있다.In the present invention, the term "comprising as an active ingredient" means that the ingredient is contained in an amount necessary or sufficient to realize the desired biological effect. Determination of the amount contained as an active ingredient in an actual application is an amount for treating a subject disease and may be determined in consideration of other non-toxic factors, for example, the disease or condition to be treated, the form of the composition to be administered, The size of the subject, or the severity of the disease or condition, and the like. Those of ordinary skill in the art to which the invention pertains will be able to empirically determine the effective amount of the individual compositions without undue experimentation.

전술한 바와 같이, 상기 화학식 1의 화합물은 2 내지 19로 표시되는 화합물로부터 선택된 어느 하나의 퓨린 유도체일 수 있다.As described above, the compound of Formula 1 may be any purine derivative selected from the compounds represented by 2 to 19.

또한, 본 발명에 따른 약학 조성물은 간 독성 예방 및 치료용 다른 약물과 함께 복합 제제의 형태로 투여되거나, 또는 담체, 희석제, 보조제 및 안정화제 등과 같은 기타 성분을 포함할 수도 있다.In addition, the pharmaceutical composition according to the present invention may be administered in the form of a combined preparation together with other drugs for prevention and treatment of liver toxicity, or may contain other ingredients such as carriers, diluents, adjuvants and stabilizers.

이하, 실시예를 통해서 본 발명을 더욱 구체적으로 설명하기로 하되, 하기 실시예는 본 발명의 이해를 돕기 위한 것일 뿐, 본 발명의 범위를 제한하는 것은 아니다.EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

본 발명에 따른 퓨린 유도체의 합성Synthesis of purine derivatives according to the present invention

하기 방법에 의해서 전술한 화학식 2 내지 19에 따른 퓨린 유도체들을 합성하였다.Purine derivatives according to the above-mentioned formulas 2 to 19 were synthesized by the following method.

6-치환된 퓨린 유도체의 일반적인 합성 방법General Synthesis of 6-Substituted Purine Derivatives

DMF (7 mL)에 녹인 2,6-디클로로퓨린 (1.58 mmol) 용액에 적절한 아민 (1.74 mmol)과 트리에틸아민 (1.66 mmol)을 첨가하였다. 혼합액을 100 ℃에서 6-8 시간 동안 교반하였다. 반응액을 상온으로 식힌 후, 감압장치를 사용하여 과량의 용매를 제거하였다. 이어서, 에탄올과 물을 이용하여 재결정을 진행함으로써, 6-치환된 퓨린 유도체를 제조하였다.To a solution of 2,6-dichloropurine (1.58 mmol) in DMF (7 mL) was added the appropriate amine (1.74 mmol) and triethylamine (1.66 mmol). The mixture was stirred at 100 ° C for 6-8 hours. The reaction solution was cooled to room temperature, and then the excess solvent was removed by using a decompression apparatus. Subsequently, recrystallization was performed using ethanol and water to prepare a 6-substituted purine derivative.

N-((1H-벤조[d]이미다졸-2-일)메틸)-2-클로로-9H-퓨린-6-아민 (화학식 16)Yl) methyl) -2-chloro-9H-purin-6-amine (Formula 16)

수율: 83%, 1H NMR (300 MHz, CD3OD): δ 8.97 (s, 1H, NH), 8.28 (s, 1H, =CH-N), 7.80-7.79 (m, 2H, Ar-H), 7.46-7.755 (m, 2H, Ar-H), 5.13 (s, 2H, CH2), HRMS (ESI): [M+H] + calcd for C13H10ClN7: 300.6886, found: 300.06771H), 7.80-7.79 (m, 2H, Ar-H), 7.46 (s, 2H, Ar-H), 5.13 (s, 2H, CH2), HRMS (ESI): [M + H] + calcd for C13H10ClN7: 300.6886, found: 300.0677

2-클로로-N-(4-(메틸설포닐)벤질)-9H-퓨린-6-아민 (화학식 17)2-chloro-N- (4- (methylsulfonyl) benzyl) -9H-purin-

수율: 85%, 1H NMR (300 MHz, DMSO-d6): δ 12.96 (br, 1H, NH), 8.79 (br, 1H, NH), 8.16 (s, 1H, =CH-N), 7.87 (d, 2H, Ar-H, J = 8.1 Hz), 7.58 (d, 2H, Ar-H, J = 7.8 Hz), 5.27 (br, 1H, NH), 4.75 (s, 2H, CH2), 3.33 (s, 3H, CH3), HRMS (ESI): [M+H]+ calcd for C13H12ClN5O2S: 338.0400, found: 338.0539.(Br, IH, NH), 8.16 (s, IH, = CH-N), 7.87 (d, 2H, Ar-H, J = 8.1 Hz), 7.58 (d, 2H, Ar-H, J = 7.8 Hz), 5.27 , 3H, CH3), HRMS (ESI): [M + H] + calcd for C13H12ClN5O2S: 338.0400, found: 338.0539.

2-클로로-N-(피리딘-3-일메틸)-9H-퓨린-6-아민 (화학식 18)2-Chloro-N- (pyridin-3-ylmethyl) -9H-purin-

수율: 71%, 1H NMR (300 MHz, DMSO-d6): δ 13.05 (br, 1H, NH), 8.72 (br, 1H, NH), 8.58 (s, 1H, Ar-H), 8.44 (d, 1H, Ar-H, J = 4.5 Hz), 8.15 (s, 1H, =CH-N), 7.75 (d, 2H, Ar-H, J = 7.2 Hz), 7.40-7.30 (m, 2H, Ar-H), 5.18 (br, 1H, NH), 4.68 (s, 2H, CH2), HRMS (ESI): [M+H]+ calcd for C11H9ClN6: 261.0577, found: 261.0680.(Br, IH, NH), 8.58 (s, IH, Ar-H), 8.44 (d, 2H, Ar-H, J = 7.2 Hz), 7.40-7.30 (m, 2H, Ar- 1H), 5.18 (br, IH, NH), 4.68 (s, 2H, CH2), HRMS (ESI): [M + H] + calcd for C11H9ClN6: 261.0577, found: 261.0680.

N-((1H-이미다졸-2-일)메틸)-2-클로로-9H-퓨린-6-아민 (화학식 19)N - ((1 H-imidazol-2-yl) methyl) -2-chloro-9H- purin-

수율: 76%, 1H NMR (300 MHz, CD3OD): δ 8.13 (s, 1H, NH), 8.10 (s, 1H, =CH-N), 7.17 (s, 2H, Ar-H), 4.95 (s, 2H, CH2), HRMS (ESI): [M+H]+ calcd for C9H8ClN7: 250.0530, found: 250.0595(S, 1H, CH-N), 7.17 (s, 2H, Ar-H), 4.95 (s, , 2H, CH2), HRMS (ESI): [M + H] + calcd for C9H8ClN7: 250.0530, found: 250.0595

2,6-2,6- 이치환된Disjointed 퓨린 유도체의 일반적인 합성 방법 General Synthesis Method of Purine Derivatives

부탄올 (butanol, 5 mL)에 녹인 6-치환된 퓨린 중간체 (0.15 mmol)에 적절한 아민 (0.75 mmol)과 소듐 테트라플루오로보레이트 (0.23 mmol)를 첨가하였다. 혼합액을 180 ℃에서 마이크로웨이브 조사를 75분 동안 진행하거나, 혹은 밀봉된 튜브를 이용하여 3시간 동안 교반하였다. 반응액을 감압하여 용매를 제거하였다. 이어서, 잔류액을 역상 칼럼 크로마토그래피 (HPLC)를 이용하여 2,6-이치환된 퓨린 유도체를 제조하였다.To the 6-substituted purine intermediate (0.15 mmol) dissolved in butanol (5 mL) was added the appropriate amine (0.75 mmol) and sodium tetrafluoroborate (0.23 mmol). The mixed solution was subjected to microwave irradiation at 180 캜 for 75 minutes, or stirred for 3 hours using a sealed tube. The reaction solution was reduced in pressure to remove the solvent. The residual solution was then subjected to reverse phase column chromatography (HPLC) to prepare a 2,6-disubstituted purine derivative.

N6-(4-(메틸설포닐)벤질)-N2-(3-(트리플루오로메틸)벤질)-9H-퓨린-2,6-다이아민 (화학식 2)N6- (4- (methylsulfonyl) benzyl) -N2- (3- (trifluoromethyl) benzyl) -9H-purine-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 10% A 내지 90% A로 20분 동안; 90% 내지 10%로 30분 동안 되도록 구성하였다. Rt =11.53 min. 수율: 38%, 1H NMR (300 MHz, CD3OD): δ 8.47 (br, 1H, NH), 7.83 (d, 2H, Ar-H, J = 8.4 Hz), 7.73 (s, 1H, =CH-N), 7.61 (s, 1H, Ar-H), 7.55-7.32 (m, 5H, Ar-H), 4.79 (s, 2H, CH2), 4.59 (s, 2H, CH2), 3.01 (s, 3H, CH3), 13C NMR (75 MHz, CD3OD): 195.54, 154.35, 146.55, 142.50, 139.03, 136.14, 130.29, 128.58, 127.75, 127.07, 123.13, 122.85, 44.45 (CH2), 43.02 (CH2), HRMS (ESI): [M+H]+ calcd for C21H19F3N6O2S: 477.4748, found: 477.1087HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). The concentration gradient was 10% A to 90% A for 20 minutes at a flow rate of 2 mL / min; 90% to 10% for 30 minutes. Rt = 11.53 min. (Br, IH, NH), 7.83 (d, 2H, Ar-H, J = 8.4 Hz), 7.73 2H), 4.51 (s, 2H, CH 2), 3.01 (s, 3H, ArH), 7.61 CH3), 13C NMR (75 MHz, CD3OD): 195.54,154.35,146.55,142.50,139.03,136.14,130.29,128.58,127.75,127.07,123.13,122.85,44.45 (CH2) : [M + H] + calcd for C21H19F3N6O2S: 477.4748, found: 477.1087

N6-(4-(메틸설포닐)벤질)-N2-(4-니트로벤질)-9H-퓨린-2,6-다이아민 (화학식 3)N6- (4- (methylsulfonyl) benzyl) -N2- (4-nitrobenzyl) -9H-purine-2,6-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 10% A 내지 70% A로 35분 동안; 70% 내지 10%로 45분 동안 되도록 구성하였다. Rt =12.97 min. 수율: 55%, 1H NMR (300 MHz, DMSO-d6): δ 12.24 (br, 1H, NH), 8.19-8.07 (m, 2H, Ar-H), 7.97 (br, 1H, NH), 7.79 (d, 2H, Ar-H, J = 6.0 Hz), 7.68 (s, 1H, =CH-N), 7.59-7.43 (m, 4H, Ar-H), 7.04 (br, 1H, NH), 4.65 (br, 1H, NH), 4.50 (s, 4H, (CH2)2), 3.15 (s, 3H, CH3), HRMS (ESI): [M+H]+ calcd for C20H19N7O4S: 454.1219, found: 454.1286HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). Concentration gradients were carried out at a flow rate of 2 mL / min, 10% A to 70% A for 35 minutes; 70% to 10% for 45 minutes. Rt = 12.97 min. (Br, IH, NH), 7.79 (br, IH, NH), 8.19-8.07 (m, 2H, Ar-H) 1H), 4.65 (s, 1H, CH-N), 7.59-7.43 (m, 4H, Ar-H), 7.04 3H, CH3), HRMS (ESI): [M + H] + calcd for C20H19N7O4S: 454.1219, found: 454.1286

N2-(4-플루오로벤질)-N6-(4-(메틸설포닐)벤질)-9H-퓨린-2,6-다이아민 (화학식 4)N2- (4-fluorobenzyl) -N6- (4- (methylsulfonyl) benzyl) -9H-purine-2,6-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 10% A 내지 90% A로 30분 동안; 70% 내지 10%로 45분 동안 되도록 구성하였다. Rt =12.69 min. 수율: 43%, 1H NMR (300 MHz, CD3OD): δ 7.85 (d, 2H, Ar-H, J = 8.1 Hz), 7.75 (s, 1H, =CH-N), 7.60-7.47 (m, 2H, Ar-H), 7.32-7.17 (m, 2H, Ar-H), 7.10-6.90 (m, 2H, Ar-H), 4.84 (s, 2H, CH2), 4.50 (s, 2H, CH2), 3.10 (s, 3H, CH3), HRMS (ESI): [M+H]+ calcd for C20H19FN6O2S: 427.1274, found: 427.1279HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). The concentration gradient was 10% A to 90% A for 30 minutes at a flow rate of 2 mL / min; 70% to 10% for 45 minutes. Rt = 12.69 min. Yield: 43%, 1 H NMR (300 MHz, CD 3 OD):? 7.85 (d, 2H, Ar-H, J = 8.1 Hz), 7.75 2H, Ar-H), 7.32-7.17 (m, 2H, Ar-H), 7.10-6.90 3.10 (s, 3H, CH3), HRMS (ESI): [M + H] + calcd for C20H19FN6O2S: 427.1274, found: 427.1279

N2-(4-클로로벤질)-N6-(4-(메틸설포닐)벤질)-9H-퓨린-2,6-다이아민 (화학식 5)N2- (4-chlorobenzyl) -N6- (4- (methylsulfonyl) benzyl) -9H-purine-2,6-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 10% A 내지 70% A로 35분 동안; 70% 내지 10%로 45분 동안 되도록 구성하였다. Rt =15.32 min. 수율: 41%, 1H NMR (300 MHz, DMSO-d6): δ 8.18 (br, 1H, NH), 7.90 (br, 1H, NH), 7.80 (d, 2H, Ar-H, J = 7.8 Hz), 7.68 (s, 1H, =CH-N), 7.58-7.45 (m, 2H, Ar-H), 7.32-7.19 (s, 4H, Ar-H), 6.86 (br, 1H, NH), 4.68 (br, 1H, NH), 4.38 (s, 4H, (CH2)2), 3.15 (s, 3H, CH3), HRMS (ESI): [M+H]+ calcd for C20H19ClN6O2S: 443.0979, found: 443.0739HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). Concentration gradients were carried out at a flow rate of 2 mL / min, 10% A to 70% A for 35 minutes; 70% to 10% for 45 minutes. Rt = 15.32 min. (Br, IH, NH), 7.80 (d, 2H, Ar-H, J = 7.8 Hz) , 7.68 (s, 1H, CH-N), 7.58-7.45 (m, 2H, Ar-H), 7.32-7.19 (s, 3H, CH3), HRMS (ESI): [M + H] + calcd for C20H19ClN6O2S: 443.0979, found: 443.0739

N2-(4-니트로벤질)-N6-(피리딘-3-일메틸)-9H-퓨린-2,6-다이아민 (화학식 6)N2- (4-nitrobenzyl) -N6- (pyridin-3-ylmethyl) -9H-purine-2,6-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 1 mL/min의 유속에서, 1% A 내지 65% A로 35분 동안; 65% 내지 1% A로 45분 동안 되도록 구성하였다. Rt =6.26 min. 수율: 49%, 1H NMR (300 MHz, DMSO-d6): δ 8.45 (br, 1H, NH), 8.35 (s, 1H, Ar-H), 8.08 (d, 2H, Ar-H, J = 8.1 Hz), 7.70-7.53 (m, 2H, Ar-H), 7.48 (d, 2H, Ar-H, J = 8.4 Hz), 7.23 (s, 1H, Ar-H), 4.51 (s, 4H, (CH2)2), HRMS (ESI): [M+H]+ calcd for C18H16N8O2: 377.1396, found: 377.1434HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). The concentration gradient was 1% A to 65% A for 35 minutes at a flow rate of 1 mL / min; 65% to 1% A for 45 minutes. Rt = 6.26 min. (Br, IH, NH), 8.35 (s, IH, Ar-H), 8.08 (d, 2H, Ar-H, J = 8.1 2H, Ar-H), 7.41 (d, 2H, Ar-H, J = 8.4 Hz), 7.70-7.53 CH2) 2), HRMS (ESI): [M + H] + calcd for C18H16N8O2: 377.1396, found: 377.1434

N2-(4-클로로벤질)-N6-(피리딘-3-일메틸)-9H-퓨린-2,6-다이아민 (화학식 7)N2- (4-chlorobenzyl) -N6- (pyridin-3-ylmethyl) -9H-purine-2,6-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 1 mL/min의 유속에서, 10% A 내지 90% A로 35분 동안; 90% 내지 10% A로 45분 동안 되도록 구성하였다. Rt =6.93 min. 수율: 37%, 1H NMR (300 MHz, CD3OD): δ 8.54-8.36 (m, 2H, Ar-H), 7.78-7.68 (m, 2H, Ar-H), 7.36-7.29 (m, 1H, Ar-H), 7.28-7.18 (m, 4H, Ar-H), 4.75 (s, 2H, CH2), 4.52 (s, 2H, CH2), HRMS (ESI): [M+H]+ calcd for C18H16ClN7: 366.1156, found: 366.1208HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). Concentration gradients were carried out at a flow rate of 1 mL / min, from 10% A to 90% A for 35 minutes; 90% to 10% A for 45 minutes. Rt = 6.93 min. (M, 2H, Ar-H), 7.78-7.68 (m, 2H, Ar-H), 7.36-7.29 2H), 7.48 (s, 2H, CH2), 4.52 (s, 2H, CH2), HRMS (ESI): [M + H] + calcd for C18H16ClN7: 366.1156, found: 366.1208

N6-((1H-이미다졸-2-일)메틸)-N2-(4-플루오로페닐)-9H-퓨린-2,6-다이아민 (화학식 9)N6 - ((1H-imidazol-2-yl) methyl) -N2- (4-fluorophenyl) -9H- purine-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 1 mL/min의 유속에서, 1% A 내지 35% A로 35분 동안; 35% 내지 1% A로 45분 동안 되도록 구성하였다. Rt = 13.18 min. 수율: 33%, 1H NMR (300 MHz, CD3OD): δ 8.31 (br, 1H, NH), 7.77 (s, 1H, =CH-N), 7.32- 7.24 (m, 2H, Ar-H), 7.19 (m, 2H, Ar-H), 7.03-6.94 (m, 2H, Ar-H), 4.87 (s, 2H, CH2), 4.48 (s, 2H, CH2), 13C NMR (75 MHz, CD3OD): 165.80, 163.38, 160.16, 159.40, 154.09, 152.05, 136.56, 136.42, 128.53, 128.42, 119.63, 114.62, 114.34, 44.13 (CH2), 36.51 (CH2), HRMS (ESI): [M+H]+ calcd for C16H15FN8: 339.1404, found: 339.1501HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). The concentration gradient was 1% A to 35% A for 35 minutes at a flow rate of 1 mL / min; 35% to 1% A for 45 minutes. Rt = 13.18 min. 2H), 7.19 (br, IH, NH), 7.77 (s, IH, = CH-N), 7.32-7.24 (m, 2H, Ar-H) 2H, Ar-H), 7.03-6.94 (m, 2H, Ar-H), 4.87 (ESI): [M + H] + calcd for C16H15FN8 &lt; RTI ID = 0.0 &gt; : 339.1404, found: 339.1501

N6-((1H-이미다졸-2-일)메틸)-N2-(4-클로로벤질)-9H-퓨린-2,6-다이아민 (화학식 10)N6- ((1H-imidazol-2-yl) methyl) -N2- (4- chlorobenzyl) -9H- purine-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 0% A 내지 30% A로 40분 동안; 30% 내지 0% A로 45분 동안 되도록 구성하였다. Rt = 15.49 min. 수율: 39%, 1H NMR (300 MHz, CD3OD): δ 8.31 (br, 1H, NH), 7.77 (s, 1H, =CH-N), 7.47 (s, 1H, NH), 7.24 (s, 4H, Ar-H), 7.19 (s, 2H, Ar-H), 4.87 (s, 2H, CH2), 4.48 (s, 2H, CH2), HRMS (ESI): [M+H]+ calcd for C16H15ClN8: 355.1108, found: 355.1170HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). Concentration gradients were carried out at a flow rate of 2 mL / min, 0% A to 30% A for 40 minutes; 30% to 0% A for 45 minutes. Rt = 15.49 min. 1H), 7.47 (s, 1H, NH), 7.24 (s, 4H), 7.95 (s, 2H), 4.87 (s, 2H, CH2), 4.48 (s, 2H, CH2), HRMS (ESI): [M + H] + calcd for C16H15ClN8: 355.1108, found: 355.1170

N6-((1H-이미다졸-2-일)메틸)-N2-(3-(트리플루오로메틸)벤질)-9H-퓨린-2,6-다이아민 (화학식 11)N6 - ((1H-imidazol-2-yl) methyl) -N2- (3- (trifluoromethyl) benzyl) -9H- purine-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 0% A 내지 30% A로 45분 동안; 30% 내지 0% A로 50분 동안 되도록 구성하였다. Rt = 18.06 min. 수율: 42 %, 1H NMR (300 MHz, CD3OD): δ 8.37 (s, 1H, NH), 7.76 (s, 1H, =CH-N), 7.62-7.42 (m, 4H, Ar-H), 7.09 (s, 2H, Ar-H), 4.84 (s, 2H, CH2), 4.60 (s, 2H, CH2), HRMS (ESI): [M+H]+ calcd for C17H15F3N8: 389.1372, found: 389.1446HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). Concentration gradients were carried out at a flow rate of 2 mL / min, 0% A to 30% A for 45 minutes; 30% to 0% A for 50 minutes. Rt = 18.06 min. (M, 4H, Ar-H), 7.09 (s, 1H, NH), 7.76 2H, Ar-H), 4.84 (s, 2H, CH2), 4.60 (s, 2H, CH2), HRMS (ESI): [M + H] + calcd for C17H15F3N8: 389.1372, found: 389.1446

N6-((1H-벤조[d]이미다졸-2-일)메틸)-N2-(3-(트리플루오로메틸)벤질)-9H-퓨린-2,6-다이아민 (화학식 12)Dihydro-N6- ((1H-benzo [d] imidazol-2-yl) methyl) -N2- (3- (trifluoromethyl) benzyl)

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 10% A 내지 50% A로 30분 동안; 50% 내지 10% A로 35분 동안 되도록 구성하였다. Rt = 11.97 min. 수율: 67 %, 1H NMR (300 MHz, CD3OD): δ 8.21 (s, 1H, NH), 7.77 (s, 1H, =CH-N), 7.56-7.41 (m, 3H, Ar-H), 7.35-7.21 (m, 4H, Ar-H), 7.09-7.15 (m, 1H, Ar-H), 5.02 (s, 2H, CH2), 4.53 (s, 2H, CH2), 13C NMR (300 MHz, CD3OD): 166.45, 160.92, 156.17, 155.07, 153.70, 143.90, 138.80, 138.29, 132.24, 132.13, 131.71, 130.38, 128.08, 124.99, 124.86, 124.78, 124.73, 115.99, 114.15, 46.30 (CH2), 39.97 (CH2), HRMS (ESI): [M+H]+ calcd for C21H17F3N8: 439.1528, found: 439.1634HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). The concentration gradient was 10% A to 50% A for 30 minutes at a flow rate of 2 mL / min; 50% to 10% A for 35 minutes. Rt = 11.97 min. (S, 1H, CH-N), 7.56-7.41 (m, 3H, Ar-H), 7.35 1H NMR (300 MHz, CD3OD, s, 2H, CH2), 7.02-7.21 (m, 4H, Ar-H), 7.09-7.15 ): 166.45, 160.92, 156.17, 155.07, 153.70, 143.90, 138.80, 138.29, 132.24, 132.13, 131.71, 130.38, 128.08, 124.99, 124.86, 124.78, 124.73, 115.99, 114.15, 46.30 (CH2) HRMS (ESI): [M + H] + calcd for C21H17F3N8: 439.1528, found: 439.1634

N6-((1H-벤조[d]이미다졸-2-일)메틸)-N2-(4-니트로벤질)-9H-퓨린-2,6-다이아민 (화학식 13)N6- ((1H-benzo [d] imidazol-2-yl) methyl) -N2- (4-nitrobenzyl) -9H- purine-

화학식 13의 화합물은 아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 역상 HPLC에 의해서 정제하였다. 농도 구배는, 2 mL/min의 유속에서, 10% A 내지 40% A로 30분 동안; 40% 내지 10% A로 35분 동안 되도록 구성하였다. Rt = 6.47 min. 수율: 55 %, 1H NMR (300 MHz, CD3OD): δ 8.16 (s, 1H, NH), 7.82 (s, 1H, =CH-N), 7.80-7.68 (m, 2H, Ar-H), 7.47-7.36 (m, 2H, Ar-H), 7.28-7.15 (m, 4H, Ar-H), 4.98 (s, 2H, CH2), 4.56 (s, 2H, CH2), HRMS (ESI): [M+H]+ calcd for C20H17N9O2: 416.1505, found: 416.1585The compound of formula 13 was purified by reverse phase HPLC using 0.1% formic acid in acetonitrile (A) / water (B). The concentration gradient was 10% A to 40% A for 30 minutes at a flow rate of 2 mL / min; 40% to 10% A for 35 minutes. Rt = 6.47 min. 2H, Ar-H), 7.47 (s, 1H, NH), 7.82 (s, 2H), 7.46 (s, 2H, CH2), HRMS (ESI): [M + H] + calcd for C20H17N9O2: 416.1505, found: 416.1585

N6-((1H-벤조[d]이미다졸-2-일)메틸)-N2-(4-클로로벤질)-9H-퓨린-2,6-다이아민 (화학식 14)Yl) methyl] -N2- (4-chlorobenzyl) -9H-purine-2,6-diamine (14)

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 10% A 내지 50% A로 45분 동안; 50% 내지 10% A로 35분 동안 되도록 구성하였다. Rt = 12.85 min. 수율: 40 %, 1H NMR (300 MHz, CD3OD): δ 7.84 (s, 1H, =CH-N), 7.57-7.46 (m, 2H, Ar-H), 7.40-7.31 (m, 2H, Ar-H), 7.07-6.86 (m, 4H, Ar-H), 5.07 (s, 2H, CH2), 4.42 (s, 2H, CH2), 13C NMR (300 MHz, CD3OD): 158.26, 154.01, 152.94, 151.36, 138.87, 137.44, 135.06, 131.86, 127.97, 127.74, 123.69, 113.85, 44.02 (CH2), 37.87 (CH2), HRMS (ESI): [M+H]+ calcd for C20H17ClN8: 405.1265, found: 405.1358HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). The concentration gradient was 10% A to 50% A for 45 minutes at a flow rate of 2 mL / min; 50% to 10% A for 35 minutes. Rt = 12.85 min. (M, 2H, Ar-H), 7.57-7.46 (m, 2H, Ar-H), 7.40-7.31 1H), 7.07-6.86 (m, 4H, Ar-H), 5.07 (s, 2H, CH2), 4.42 (ESI): [M + H] + calcd for C20H17ClN8: 405.1265, found: 405.1358, found 138.87, 137.44, 135.06, 131.86, 127.97, 127.74, 123.69, 113.85, 44.02 (CH2), 37.87

N6-((1H-벤조[d]이미다졸-2-일)메틸)-N2-(4-플루오로벤질)-9H-퓨린-2,6-다이아민 (화학식 15)N6- ((lH-benzo [d] imidazol-2-yl) methyl) -N2- (4-fluorobenzyl) -9H- purine-

아세토니트릴 (A)/물 (B) 중 0.1% 포름산을 사용하는 HPLC를 수행하였다. 농도 구배는, 2 mL/min의 유속에서, 10% A 내지 50% A로 30분 동안; 50% 내지 10% A로 35분 동안 되도록 구성하였다. Rt = 7.31 min. 수율: 46 %, 1H NMR (300 MHz, CD3OD): δ 8.17 (br, 1H, NH), 7.81 (s, 1H, =CH-N), 7.56-7.46 (m, 2H, Ar-H), 7.37-7.25 (m, 2H, Ar-H), 7.13-7.01 (m, 2H, Ar-H), 6.74-6.61 (m, 1H, Ar-H), 5.05 (s, 2H, CH2), 4.42 (s, 2H, CH2), HRMS (ESI): [M+H]+ calcd for C20H17FN8: 389.1560, found: 389.1606HPLC was carried out using 0.1% formic acid in acetonitrile (A) / water (B). The concentration gradient was 10% A to 50% A for 30 minutes at a flow rate of 2 mL / min; 50% to 10% A for 35 minutes. Rt = 7.31 min. (M, 2H, Ar-H), 7.37 (s, 1H, CH-N) (M, 2H, Ar-H), 7.13-7.01 (m, 2H, Ar-H), 6.74-6.61 , 2H, CH2), HRMS (ESI): [M + H] + calcd for C20H17FN8: 389.1560, found: 389.1606

실험예Experimental Example 1:  One: CYP3A4CYP3A4 저해 효능 평가 (LC/MS 기반) Evaluation of inhibitory efficacy (based on LC / MS)

반응 용액은 최종 농도 0.2 mg/ml 마이크로좀 단백질, 0.1 M 인산 완충용액 (pH 7.4), 1 mM NADPH와 기질 혼합액 (A 세트: 페나세틴 (phenacetin, CYP1A2), 쿠마린 (coumarin, CYP2A6), 아모디아퀸 (amodiaquine, CYP2C8), S-메페니토인 (mephenytoin, CYP2C19), 덱스트로메토르판 (dextromethorphan, CYP2D6), 및 미다졸람 (midazolam, CYP3A4); B 세트: 부프로피온 (bupropion, CYP2B6), 톨부타마이드 (tolbutamide, CYP2C9), 클로르족사존 (chlorzoxazone, CYP2E1), 및 테스토스테론 (testosterone, CYP3A4))에 의해 최종 부피 200 μl로 구성되었다. 기질은 페나세틴 50 μM, 쿠마린 5 μM, 아모디아퀸 2 μM, S-메페니토인 100 μM, 덱스트로메토르판 5 μM, 미다졸람 5 μM, 부프로피온 50 μM, 툴부타마이드 100 μM, 클로르족사존 50 μM, 및 테스토스테론 50 μM 농도로 사용하였다. 모든 실험에 사용된 기질은 아세토니트릴에 녹여 사용했으며, 반응액에서 아세토니트릴의 최종농도는 0.2 % (A 세트) 및 0.25 % (B 세트)이었다. 37 ℃에서 5분 동안 사전-배양을 진행한 후에, 최종농도 1 mM NADPH를 가하여 반응을 시작하였다. 반응 용액은 10분간 37 ℃ 진탕배양기를 이용하여 배양하였다. 200 μl 부피의 얼음 냉각 반응 종결액 (내부 표준물질로 100 nM 카르바마제핀 (carbamazepine) 및 500 nM 4-메틸벨리페론 (4-methylumbelliferone)을 포함하는 아세토니트릴)을 가하여 반응을 종결하였다. 각 반응액을 4 ℃, 3,000 rpm, 20분간 원심분리한 후, A와 B 세트의 상등액을 1 : 1로 섞은 후 분석하였다. 양성대조군으로 CYP 3A4의 선택적인 저해제인 케토코나졸 (ketokonazole)을 0.5 μM 농도로 사용하였다. 시험물질인 TNP는 0, 0.62, 1.85, 5.56, 16.67과 50 μM 농도를 사용했다. The reaction solution contained a final concentration of 0.2 mg / ml microsomal protein, 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH and a substrate mixture (A set: phenacetin, CYP1A2, coumarin, CYP2A6, B: Bupropion (CYP2B6), tolbutamide (CYP2C6), aminoglycoside (CYP2C6), quinine (amodiaquine, CYP2C8) (tolbutamide, CYP2C9), chlorzoxazone (CYP2E1), and testosterone (CYP3A4)) in a final volume of 200 μl. The substrate was 50 μM phenacetin, 5 μM coumarin, 2 μM amodia queen, 100 μM S-mefenitone, 5 μM dextromethorphan, 5 μM midazolam, 50 μM bupropion, 100 μM tulobutamide, 50 μM, and testosterone 50 μM. The substrate used in all experiments was dissolved in acetonitrile and the final concentration of acetonitrile in the reaction solution was 0.2% (A set) and 0.25% (B set). After pre-incubation for 5 min at 37 ° C, the reaction was started by adding 1 mM NADPH to the final concentration. The reaction solution was incubated for 10 minutes at 37 ° C in a shaking incubator. The reaction was terminated by adding 200 μl volume of ice-cooling reaction solution (100 nM carbamazepine as an internal standard and 500 nM 4-methylumbelliferone-containing acetonitrile). Each reaction mixture was centrifuged at 4 ° C and 3,000 rpm for 20 minutes, and A and B supernatants were mixed at a ratio of 1: 1. As a positive control, ketokonazole, a selective inhibitor of CYP 3A4, was used at a concentration of 0.5 μM. TNP, the test substance, was used at concentrations of 0, 0.62, 1.85, 5.56, 16.67 and 50 μM.

시료의 분석방법은 LC-MS를 이용하여 하기와 같이 진행하였다. 샘플 10 μl를 질량분석기에 주입하여 대사체를 0.4 ml/min의 유속으로 Atlantis™ C18 컬럼 (3 μm 2.1×50 mm; Waters)과 SecuityGuard™ C18 (2.0×4.0mm i.d., Phenomenex, Torrance, CA) 가드 컬럼을 이용하여 분리하였다. 이동상으로 0.1% 포름산이 각각 포함된 탈이온수 (이동상 A)와 아세토니트릴 (이동상 B)를 이용하였다. 설정된 장비의 조건은 다음과 같다: 이온 소스 온도, 600 ℃; 분무 가스 속도, 50 l/min; 보조 가스 속도, 4.0 l/min; 커튼 가스 속도, 20 l/min; 및 충돌 가스 (질소) 압력, 3.4×105 Torr. 초순도 질소 가스가 CUR (커튼 가스), CAD (collisionally activated dissociation, 충돌 활성화 해리) 및 NEB (분무기 가스)를 위해서 사용되었다. MRM 트랜지션, 충돌 에너지와 머무름 시간은 각각 대사체와 내부 표준물질에 대해 측정되었다. 샘플의 분석은 포지티브 및 네거티브 (6-하이드록시클로르족사존, 6-hydroxychlorzoxazone) 다중 반응 모니터링 (MRM) 스캔 모드를 이용하여 각각 형성된 대사체와 내부 표준물질의 피크 넓이를 통해서 계산하였다. 분석의 머무름 시간은 각 채널 별로 0.08초로 설정하였다. 분석된 값들은 Analyst™ 소프트웨어 (version 1.5.2, Applied Biosystems, Foster City, CA)를 통해 처리하였다.The analytical method of the sample was carried out as follows using LC-MS. A 10 μl sample was injected into the mass spectrometer and the metabolites were analyzed using an Atlantis ™ C18 column (3 μm 2.1 × 50 mm; Waters) and SecuityGuard ™ C18 (2.0 × 4.0 mm id, Phenomenex, Torrance, Calif.) At a flow rate of 0.4 ml / And then separated using a guard column. Deionized water (mobile phase A) and acetonitrile (mobile phase B) each containing 0.1% formic acid were used as a mobile phase. The conditions of the set equipment are as follows: Ion source temperature, 600 ° C; Spray gas velocity, 50 l / min; Auxiliary gas velocity, 4.0 l / min; Curtain gas velocity, 20 l / min; And impact gas (nitrogen) pressure, 3.4 x 10 &lt; 5 &gt; Torr. Ultra pure nitrogen gas was used for CUR (curtain gas), CAD (collisionally activated dissociation) and NEB (atomizing gas). MRM transitions, impact energy and retention times were measured for metabolites and internal standard, respectively. Analysis of the samples was performed using the peak widths of each formed metabolite and internal standard using the positive and negative (6-hydroxychlorzoxazone) multi-reaction monitoring (MRM) scan modes. The retention time of the assay was set to 0.08 seconds for each channel. Analyzed values were processed through Analyst ™ software (version 1.5.2, Applied Biosystems, Foster City, CA).

실험예Experimental Example 2:  2: CYP3A4CYP3A4 저해 효능 평가 (형광분석기반) Evaluation of inhibition efficacy (based on fluorescence analysis)

CYP3A4 효소의 억제 효율은 Invitrogen 사에서 제공하는 Vivid®CYP4503A4Blue screening kit (CatNo.P2858)를 이용하여 측정하였다. 반응물은 다양한 농도의 TNP와 유도체 화합물들을 포함하는 시험 약물과, Vivid® CYP450 3A4 BACULOSOMES 5 nM, CYP450 3A4 기질 Vivid®BOMCC 10μM, NADP+ 100μM, 1XVivid®CYP450 반응 완충용액 I, 1XVivid® 재생 시스템에 의해 최종 부피 100 ㎕로 구성되었다. 실험에 사용된 기질 CYP450 3A4 기질 Vivid BOMCC와 3A4 선택적 저해제인 케토코나졸은 아세토니트릴에 녹여 사용하였으며, TNP와 유도체 화합물들은 DMSO에 녹여 사용하였고, 최종 반응액에서 아세토니트릴과 DMSO의 최종 농도는 각각 0.1 %이었다. 시험 약물과 Vivid®CYP450 BACULOSOMES 시약과 Vivid® 재생 시스템을 섞은 시험 약물 반응 용액 90 ㎕을 96-웰의 빈 플레이트에 분주하였고, 상온에서 20분 간 전처리하였다. 최종 농도 100 μM의 NADP+와 최종 농도 10 μM의 CYP450 3A4 기질 Vivid®BOMCC가 첨가된 Vivid®CYP450 반응 완충용액 10㎕를 첨가한 후 상온에서 10분간 반응하였다. CYP450 3A4의 억제 효과를 평가하는 반응에서 사용된 TNP와 케토코나졸의 최종 농도는 50, 100, 250, 500, 1000 nM이고, TNP와 유도체 화합물들의 억제 효과 비교 반응은 최종 농도 100 nM에서 수행되었다. 분석은 하기와 같이 진행하였다. 최종 반응물의 형광값은 Berthold 사의 Mithras LB940 플레이트 판독기의 키네틱 모드를 이용하여 여기 405 ㎚ / 발광 460 ㎚ 조건에서 1분 간격으로 10분 동안 측정하였다. 측정된 형광값을 이용해서 시험 약물의 반응 속도와 양성대조군 (케토코나졸)과 음성대조군 (0.1 % 아세토니트릴, 0.1 % DMSO)의 반응 속도를 비교함으로써 시험 약물의 CYP450 3A4 억제 효율 (%)을 계산하였다.The inhibition efficiency of CYP3A4 enzyme was measured using the Vivid® CYP4503A4Blue screening kit (Cat No. P2858) supplied by Invitrogen. The reaction was terminated by a test drug containing various concentrations of TNP and derivative compounds and a test drug containing Vivid® CYP450 3A4 BACULOSOMES 5 nM, CYP450 3A4 substrate Vivid® BOMCC 10 μM, NADP + 100 μM, 1XVivid® CYP450 Reaction Buffer I, Lt; / RTI &gt; volume. The final concentration of acetonitrile and DMSO in the final reaction solution was 0.1%, and the final concentration of CYP450 3A4 substrate Vivid BOMCC and ketoneconazole 3A4 selective inhibitor was dissolved in acetonitrile. TNP and its derivatives were dissolved in DMSO, . 90 μl of the test drug reaction solution containing the Vivid® CYP450 BACULOSOMES reagent and the Vivid® regeneration system was dispensed into a 96-well empty plate and pretreated at room temperature for 20 minutes. NADP + at a final concentration of 100 μM and 10 μl of Vivid® CYP450 reaction buffer containing a final concentration of 10 μM CYP450 3A4 substrate Vivid® BOMCC were added and reacted at room temperature for 10 min. The final concentrations of TNP and ketoconazole were 50, 100, 250, 500, and 1000 nM, respectively. The inhibitory effects of TNP and its derivatives were compared at a final concentration of 100 nM. The analysis proceeded as follows. Fluorescence values of the final reaction were measured for 10 minutes at 1 minute intervals under excitation 405 nm / emission 460 nm using the Kinetics mode of a Mithras LB940 plate reader from Berthold. The measured fluorescence values were used to calculate the CYP450 3A4 inhibition efficiency (%) of the test drug by comparing the rate of reaction of the test drug with the rate of the positive control (ketoconazole) and the negative control (0.1% acetonitrile, 0.1% DMSO) .

실험예Experimental Example 3. 인간 배아 줄기세포 ( 3. Human embryonic stem cells ( hESChESC ) 유래 간세포의 배양) Derived stem cell culture

인간 배아 줄기세포 (hESC)로부터 내배엽과 같은 다른 분화적 계통 세포로의 분화 여부를 확인하기 위하여, 인간 배아 줄기세포로부터 내배엽인 20 간세포 (hepatocytes)로의 분화를 유도하였다 (Cai, J. et. al, (2007) Hepatology 45(5): 1229-1239). 구체적으로, hESC 세포주는 CHA-hESC 세포주로 지지세포가 없는 시스템 (feeder-free system)에서 조절된 배지 (conditioned medium)에 가득 차도록 3일 동안 배양하였다. 배양 후, 상기 hESC 세포를 50 ng/㎖ 액티빈 A (Activin A; Peprotech사, 미국)를 포함하는 RPMI-1640 (Hyclone사, 미국) 배지에서 5 일간 배양하여 분화를 유도하였다. 상기 분화된 세포는 30 ng/㎖ 섬유아세포 성장인자 4 (fibroblast growth factor4; Peprotech사) 및 20 ng/㎖ 골 형성 단백질 2 (bone morphogenetic protein 2, BMP2; Peprotech사)를 포함하는 간세포 배양 배지 (hepatocyte culture medium, HCM; Lonza 사, 미국)에서 5 일간 배양한 후, 20 ng/㎖ 간세포 성장 인자 (hepatocyte growth factor, HGF; Peprotech 사)를 첨가한 간세포 배양 배지에서 5일 동안 추가로 배양하여 hESC로부터 간세포로 분화를 유도하였다. 상기 분화된 간세포는 105 ng/㎖ 온코스태틴 M (oncostatin M; R&D Systems 사, 미국) 및 0.1 μM 덱사메타손 (dexametasone; 시그마-알드리치 사, 미국)을 첨가한 간세포 배양 배지에서 5 일간 배양하면서 간세포의 성숙을 유도하여 성숙된 간세포를 수득하였다.In order to confirm the differentiation from human embryonic stem cells (hESC) into other differentiated systematic cells such as endoderm, differentiation was induced from human embryonic stem cells into endoderm 20 hepatocytes (Cai, J. et al , (2007) Hepatology 45 (5): 1229-1239). Specifically, the hESC cell line was cultured for 3 days to fill the conditioned medium in a feeder-free system with a CHA-hESC cell line. After the incubation, the hESC cells were cultured for 5 days in RPMI-1640 (Hyclone, USA) medium containing 50 ng / ml Actin A (Peprotech, USA) to induce differentiation. The differentiated cells were cultured in a hepatocyte culture medium containing 30 ng / ml fibroblast growth factor 4 (Peprotech) and 20 ng / ml bone morphogenetic protein 2 (BMP2; Peprotech) cultured for 5 days in hepatocyte growth medium supplemented with 20 ng / ml hepatocyte growth factor (HEP; Peprotech), and cultured in hESC Differentiation was induced in hepatocytes. The differentiated hepatocytes were cultured for 5 days in hepatocyte culture medium supplemented with 105 ng / ml oncostatin M (R & D Systems, USA) and 0.1 μM dexamethasone (Sigma-Aldrich, USA) Maturation was induced to obtain mature hepatocytes.

도 2a 내지 2j는 인간 간 마이크로좀에서 TNP 약물이 p450 CYP 효소 9종에 대한 활성을 억제하는 것을 평가한 그래프로서, 도 2를 참조하면, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6과 CYP2E1에 대한 TNP의 억제능은 본 실험에서 사용한 최대 농도인 50 μM에서 20% 미만으로 관찰되었다. 반면에, CYP3A4의 활성을 미다졸람 (midazolam)과 테스토스테론 (testosterone)으로 측정하였을 때, TNP는 각각에 대해 IC50 값이 65.4와 31.4 μM로 억제효과를 나타내었다. 이러한 결과는 TNP가 다른 cytochrome P450에 비하여 CYP3A4에 대한 억제효과가 선택적임을 의미한다.FIG. 2 is a graph showing that TNP drug inhibits the activity of nine p450 CYP enzymes in human liver microsomes. Referring to FIG. 2, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 The inhibitory effect of TNP on CYP2E1 was observed to be less than 20% at the maximum concentration of 50 μM used in this experiment. On the other hand, when the activity of CYP3A4 was measured by midazolam and testosterone, TNP showed an inhibitory effect on IC 50 values of 65.4 and 31.4 μM, respectively. These results suggest that the inhibitory effect of TNP on CYP3A4 is more selective than that of other cytochrome P450.

도 3은 인간 유래 cytochrome P450 동족효소 3A4로 구성된 마이크로좀에 대하여 TNP의 억제효과를 형광분석기반으로 평가한 결과를 나타낸 것이다. TNP는 50, 100, 250, 500, 1000 nM에서 음성대조군인 DMSO와 비교하였을 때, 5.2, 40.2, 71, 84, 92.4 %의 억제 효과를 가지는 것으로 관찰되었다. 반면에, cytochrome P450 동족효소 3A4의 선택적 억제제로 알려진 케토코나졸은 동일 농도 조건에서 20.8, 31.6, 88.3, 93.7, 95.2 %의 억제 효과를 보였다. 이러한 결과는 TNP의 cytochrome P450 동족효소 3A4 억제 효과가 양성대조군인 케토코나졸과 유사하게 처리 농도에 비례하여 증가함을 의미한다.Figure 3 shows the result of evaluating the inhibitory effect of TNP on microsomes composed of human-derived cytochrome P450 kinase 3A4 based on fluorescence analysis. TNP was observed to inhibit at 5.2, 40.2, 71, 84, and 92.4% when compared with the negative control DMSO at 50, 100, 250, 500 and 1000 nM. On the other hand, ketoconazole, a selective inhibitor of cytochrome P450 kinase 3A4, inhibited 20.8, 31.6, 88.3, 93.7, and 95.2% at the same concentration. These results indicate that the inhibitory effect of TNP on the cytochrome P450 kinase 3A4 is increased in proportion to the treatment concentration, similar to the positive control ketoconazole.

도 4는 인간 배아줄기세포 유래 간세포에서 TNP의 cytochrome P450 동족효소 3A4에 대한 억제 효과를 분석한 결과를 나타낸 것이다. 도 4를 참조하면, 10 uM 농도로 TNP를 처리한 그룹의 세포에서 cytochrome P450 동족효소 3A4의 활성이 전혀 관찰되지 않았다. 이는 TNP가 인간 배아줄기세포 유래 간세포의 cytochrome P450 동족효소 3A4의 활성을 효과적으로 억제함을 보여준다.Fig. 4 shows the results of analysis of the inhibitory effect of TNP on cytochrome P450 kinase 3A4 in human embryonic stem cell-derived hepatocytes. Referring to FIG. 4, no activity of cytochrome P450 kinase 3A4 was observed in the cells treated with TNP at a concentration of 10 uM. This indicates that TNP effectively inhibits the activity of cytochrome P450 kinase 3A4 in human embryonic stem cell-derived hepatocytes.

도 5는 인간 유래 cytochrome P450 동족효소 3A4로만 구성된 마이크로좀에 대한 TNP와 그 유도체 화합물 6, 7, 14, 15와 양성대조군인 케토코나졸의 억제 효과를 비교 분석한 결과이다. 도 5를 참조하면, TNP의 억제 효과를 100%으로 하였을 때, 유도체 화합물 6, 7, 14, 15은 128, 133.2, 118.9, 124.4 %의 cytochrome P450 동족효소 3A4 억제 효과를 보이는 것으로 분석되었다. 따라서, 화합물 6, 7, 14 및 15는 TNP 보다 상대적으로 높은 cytochrome P450 동족효소 3A4 억제효과를 나타내는 것으로 보인다.FIG. 5 shows the results of a comparative analysis of TNP and its derivative compounds 6, 7, 14, 15 against microsomes composed of human-derived cytochrome P450 kinase 3A4 and ketoconazole, which is a positive control. 5, when the inhibitory effect of TNP was taken as 100%, it was analyzed that derivatives 6, 7, 14 and 15 showed inhibitory effects of cytochrome P450 kinase 3A4 of 128, 133.2, 118.9 and 124.4%. Thus, compounds 6, 7, 14 and 15 appear to exhibit a relatively high cytochrome P450 kinase 3A4 inhibitory effect than TNP.

도 6은 인간 유래 cytochrome P450 동족효소 3A4로만 구성된 마이크로좀에 대하여 TNP와 그 유도체 화합물 3, 13, 16의 억제 효과를 비교 분석한 결과이다. 도 6을 참조하면, TNP의 억제 효과를 100%로 하였을 때, 화합물 3, 13, 16은 동일 농도 조건에서 26.1, 29.2, 39.3 %의 cytochrome P450 동족효소 3A4 억제 효과를 나타내었다. 따라서, 화합물 3, 13 및 16은 TNP보다 상대적으로 낮은 CYP3A4 억제 효과를 가지는 것으로 보인다.FIG. 6 shows the results of comparative analysis of the inhibitory effects of TNP and its derivative compounds 3, 13, and 16 against microsomes composed of human-derived cytochrome P450 kinase 3A4 alone. Referring to FIG. 6, when the inhibitory effect of TNP was 100%, compounds 3, 13, and 16 showed inhibitory effects of cytochrome P450 kinase 3A4 of 26.1, 29.2, and 39.3% at the same concentration. Thus, compounds 3, 13 and 16 appear to have a relatively lower CYP3A4 inhibitory effect than TNP.

종합하면, 본 발명에 따른 N2,N6-치환된-9H-퓨린-2,6-다이아민 (N2,N6-substituted-9H-purine-2,6-diamine) 유도체는 약물의 대사에 관여하는 CYP효소의 일종인 3A4에 의해서 대사되는 반응을 억제함으로써, 간 독성 예방 및 치료 효과를 나타내며, 따라서, 이를 이용하여 다양한 간독성 예방 및 치료제를 제공할 수 있다.In summary, the N2, N6-substituted-9H-purine-2,6-diamine derivatives according to the present invention are useful for the metabolism of CYP Inhibiting the metabolism by 3A4, which is one type of enzyme, exhibits hepatotoxicity preventive and therapeutic effects, and thus it is possible to provide various hepatotoxic preventive and therapeutic agents using the same.

Claims (7)

하기 <화학식 1>로 표시되고, Cytochrome P450 3A4 (CYP3A4) 효소의 활성을 억제하는 것을 특징으로 하는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체:
<화학식 1>
Figure 112017106304769-pat00046

상기 식에서,
R1은 하기 구조식 1 내지 4 중에서 선택되는 어느 하나이고, R2는 하기 구조식 5이며,
<구조식>
Figure 112017106304769-pat00084

상기 <구조식>에서, R3은 할로겐기, 니트로기 및 트리플루오로메틸기 중에서 선택되는 어느 하나이다.N2, N6-substituted-9H-purine-2,6-diamine derivative represented by the following formula (1) and inhibiting the activity of Cytochrome P450 3A4 (CYP3A4)
&Lt; Formula 1 >
Figure 112017106304769-pat00046

In this formula,
R 1 is any one selected from the following Structural Formulas 1 to 4, R 2 is the following Structural Formula 5,
<Structural Formula>
Figure 112017106304769-pat00084

In the above structural formula, R 3 is any one selected from a halogen group, a nitro group and a trifluoromethyl group. 제1항에 있어서,
상기 <화학식 1>로 표시되는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체는 하기 <화학식 2> 내지 <화학식 15>로 이루어진 군으로부터 선택된 어느 하나의 화합물인 것을 특징으로 하는 N2,N6-치환된-9H-퓨린-2,6-다이아민 유도체:
<화학식 2> <화학식 3>
Figure 112017106304769-pat00047
Figure 112017106304769-pat00085

<화학식 4> <화학식 5>
Figure 112017106304769-pat00049
Figure 112017106304769-pat00086

<화학식 6> <화학식 7>
Figure 112017106304769-pat00051
Figure 112017106304769-pat00087

<화학식 8> <화학식 9>
Figure 112017106304769-pat00053
Figure 112017106304769-pat00088

<화학식 10> <화학식 11>
Figure 112017106304769-pat00055
Figure 112017106304769-pat00089

<화학식 12> <화학식 13>
Figure 112017106304769-pat00057
Figure 112017106304769-pat00090

<화학식 14> <화학식 15>
Figure 112017106304769-pat00059
Figure 112017106304769-pat00091
The method according to claim 1,
The N2, N6-substituted-9H-purine-2,6-diamine derivative represented by Formula 1 is any one selected from the group consisting of the following Chemical Formulas 2 to 15: N2, N6-substituted-9H-purine-2,6-diamine derivatives:
&Lt; Formula 2 >< EMI ID =
Figure 112017106304769-pat00047
Figure 112017106304769-pat00085

&Lt; Formula 4 &gt;&lt; EMI ID =
Figure 112017106304769-pat00049
Figure 112017106304769-pat00086

&Lt; Formula 6 >< EMI ID =
Figure 112017106304769-pat00051
Figure 112017106304769-pat00087

&Lt; Formula 8 &gt;&lt; EMI ID =
Figure 112017106304769-pat00053
Figure 112017106304769-pat00088

&Lt; Formula 10 >< EMI ID =
Figure 112017106304769-pat00055
Figure 112017106304769-pat00089

&Lt; Formula 12 &gt;&lt; EMI ID =
Figure 112017106304769-pat00057
Figure 112017106304769-pat00090

&Lt; Formula 14 >< EMI ID =
Figure 112017106304769-pat00059
Figure 112017106304769-pat00091
 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete
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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2016087665A2 (en) * 2014-12-05 2016-06-09 Centre National De La Recherche Scientifique (Cnrs) Compounds for treating cystic fibrosis

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016087665A2 (en) * 2014-12-05 2016-06-09 Centre National De La Recherche Scientifique (Cnrs) Compounds for treating cystic fibrosis

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ChemBioChem, 2002, 제9호, 페이지 897-901*
J. Comb. Chem., 2007, 제9권, 페이지 197-199 및 추가 정보(Supporting Information)*
PubChem(http://pubchem.ncbi.nlm.nih.gov/), CID115669326, Create Date 2016.1.29.*

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