KR101715127B1 - Pyrimidine-carboxylate derivative for preventing or treating neuromyelitis optica - Google Patents

Pyrimidine-carboxylate derivative for preventing or treating neuromyelitis optica Download PDF

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KR101715127B1
KR101715127B1 KR1020150152289A KR20150152289A KR101715127B1 KR 101715127 B1 KR101715127 B1 KR 101715127B1 KR 1020150152289 A KR1020150152289 A KR 1020150152289A KR 20150152289 A KR20150152289 A KR 20150152289A KR 101715127 B1 KR101715127 B1 KR 101715127B1
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carboxylate
phenyl
methyl
methylthio
ethyl
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박기덕
배애님
김동진
조남철
박종현
최지원
연슬기
하태환
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주은지
김성민
천소영
김희정
장재완
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한국과학기술연구원
서울대학교병원
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    • 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
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • 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

Abstract

The present invention relates to a pharmaceutical composition capable of preventing or treating diseases caused by the combination of NMO-IgG and aquaporin 4 protein, and preferably neuromyelitis optica or degenerative nervous system diseases. The composition comprises a pyrimidine-carboxylate derivative or pharmaceutically acceptable salt thereof, or a solvate thereof, as an active ingredient, thereby inhibiting the combination of the NMO-IgG and the aquaporin 4 protein.

Description

시신경 척수염 예방 또는 치료용 피리미딘 카복실레이트 유도체{Pyrimidine-carboxylate derivative for preventing or treating neuromyelitis optica}[0001] The present invention relates to a pyrimidine-carboxylate derivative for preventing or treating optic neuritis,

본 발명은 NMO-IgG와 아쿠아포린 4의 결합을 저해할 수 있어 NMO-IgG와 아쿠아포린 4의 결합에 의해 유발되는 질병, 바람직하게는 시신경 척수염 또는 퇴행성 신경계 질환을 예방 또는 치료할 수 있는 피리미딘 카복실레이트 유도체 화합물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by binding of NMO-IgG and aquaporin 4, preferably optic neuritis or degenerative nervous system diseases, which can inhibit the binding of NMO-IgG and aquaporin 4, Lt; / RTI > derivatives.

시신경 척수염(neuromyelits optica, NMO)는 대표적인 불치 뇌질환 중 하나로, 중추신경과 시신경에 영향을 주는 염증성 탈수초성 자가면역질환이다. 시신경 척수염은 성상 세포에서 주로 발현되는 아쿠아포린 4라는 수분 전달 통로 단백질에 대한 시신경 척수염 특이항체인 NMO-IgG의 유무로 다른 질병과 구별될 수 있다. 상기 시신경 척수염 특이항체는 환자의 약 75%에서 발견된다. 아쿠아포린 4 단백질은 NMO-IgG의 주 항원으로 뇌와 중추신경을 염증세포로부터 보호하는 혈 뇌 장벽을 구성하고 있는 성상세포에 주로 분포되어 있다. NMO-IgG와 아쿠아포린 4의 결합은 시신경척수염 발병 과정에서 핵심이 되는 주요 기전이다. 시신경 척수염 특이항체인 NMO-IgG 가 항원인 아쿠아포린 4에 결합을 하면서 도움 체 매게 세포독성 혹은 항체의존성 세포 매게 성 세포독성을 유발하여 성상세포에 손상을 주게 되어 시신경 척수염을 발병시킨다.Neuromyelits optica (NMO) is one of the most common invasive brain diseases. It is an inflammatory dehydrative autoimmune disease affecting the central nervous system and optic nerve. Optic neuritis is distinguished from other diseases by the presence of NMO-IgG, a optic neuritis specific antibody to the water transport pathway protein, aquaporin 4, which is mainly expressed in astrocytes. The optic nerve spindle-specific antibody is found in about 75% of patients. The aquaporin 4 protein is a major antigen of NMO-IgG and is mainly distributed in astrocytes that constitute a blood-brain barrier that protects the brain and central nervous system from inflammatory cells. Binding of NMO-IgG to aquaporin 4 is a key mechanism in the pathogenesis of optic neuritis. NMO-IgG, an optic nerve spindle-specific antibody, binds to an antigen, aquaporin 4, to induce cognitive cytotoxicity or antibody-dependent cell-mediated cytotoxicity, damaging astrocytes and causing optic neuritis.

현재 시신경 척수염의 치료제로는 대부분 항암제나 면역 억제제, 면역 조절제, 혈액 정화 요법이 쓰이고 있고, 이큘리주맙이라는 항체에 대한 안정성 임상시험이 진행단계에 있다. 하지만 면역 억제제들은 강한 독성을 띄고 중추신경계 감염, 백혈병, 심장독성, 또는 사망과 같은 심한 부작용을 동반하기 때문에, 이런 부작용들을 최소화하고 효능을 극대화시킬 수 있는 치료제를 개발하는 것이 아주 시급하고 중요하다. 그럼에도 불구하고 심한 뇌 질환의 병리기전은 아직까지도 정확히 알려진 바가 없기 때문에 기본적인 치료방법이 없고, 시신경 척수염과 같은 희귀 질병에는 치료에 활성을 띄는 화합물의 개발도 미비한 상태이다. Currently, anticancer drugs, immunosuppressants, immunomodulators, and blood purification therapies are mostly used as therapeutic agents for optic neuritis, and clinical trials for the safety of this antibody are in progress. However, it is very urgent and important to develop therapeutic agents that minimize these side effects and maximize efficacy, as immunosuppressants are highly toxic and have severe side effects such as central nervous system infection, leukemia, cardiac toxicity, or death. Nevertheless, the pathology of severe brain diseases has not yet been well known, so there is no basic treatment, and rare diseases such as optic neuropathy are under development.

미국공개특허 2014-0170140호에서는 아쿠아포린 4에 결합하는 항체를 이용하여 단일요법 또는 면역억제제 또는 혈장분리 반출술과 같은 기본 NMO 요법과 함께 시신경 척수염을 치료하는 방법에 관한 특징을 기재하고 있으며, 미국등록특허 7872032호에서는 과산화소체 증식체 활성화 수용체 델타 조절물질로서 1,3,4-옥사디아졸-2-온 유도체 또는 이의 입체 이성질체 호변이성체 또는 이의 용매화물 또는 약제학적으로 허용되는 염을 이용하여 다발성 경화증, 시신경척수염 등을 포함하는 질병을 치료하는 방법에 관한 특징을 개시하고 있다. U.S. Patent Publication No. 2014-0170140 discloses a method for treating optic neuropathy with basic NMO therapy such as monotherapy or immunosuppressant or plasma separation using an antibody binding to aquaporin 4, In patent No. 7872032, a 1,3,4-oxadiazol-2-one derivative or a stereoisomer isomer thereof or a solvate thereof or a pharmaceutically acceptable salt thereof as a peroxide activator receptor agonist delta modulating substance is used to treat multiple Inflammatory bowel disease, inflammatory bowel disease, scleroderma, optic neuritis, and the like.

시신경 척수염과 증상이 비슷한 다발성 경화증의 상황과 비교해 보면, 다발성 경화증은 서양에서 비교적 높을 비율을 차지하는 반면, 시신경 척수염은 한국과 중국을 비롯한 아시아 지역에서 환자의 비율이 높게 나타났다고 보고되었다. 다발성 경화증은 치료제가 개발되어 이미 서양에서 큰 시장을 형성하고 있으므로 시신경 척수염 또한 치료제 개발이 되면 빠른 시일 내에 한국과 중국을 포함한 아시아에서 큰 시장을 형성할 것이라고 예상한다.Compared with multiple sclerosis with symptoms similar to those with optic neuritis, multiple sclerosis was reported to be relatively high in the West, while optic nerve sheath inflammation was reported to be high in Asia, including Korea and China. As multiple sclerosis has already been developed in a large market in Western countries, optic nerve spondylitis is expected to form a big market in Asia, including Korea and China, soon after the development of therapeutic drugs.

본 발명이 해결하고자 하는 과제는 피리미딘 카복실레이트 유도체 또는 이의 약제학적으로 허용 가능한 염 또는 이의 용매화물을 유효성분으로 포함하여 시신경 척수염 또는 퇴행성 신경계 질환을 치료 또는 예방할 수 있는 약학 조성물을 제공하는 것이다. The object of the present invention is to provide a pharmaceutical composition comprising a pyrimidine carboxylate derivative or a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient to treat or prevent optic neuritis or degenerative nervous system diseases.

상술한 과제를 달성하기 위하여 본 발명의 일 측면은 NMO-IgG(Neuro myelitis Optica-IgG)와 아쿠아포린 4 단백질의 결합을 저해할 수 있는 하기 [화학식 1]로 표시되는 피리미딘 카복실레이트 유도체 또는 이의 약제학적으로 허용 가능한 염 또는 이의 용매화물을 유효성분으로 포함하는 조성물을 제공하고자 한다. In order to achieve the above object, an aspect of the present invention is to provide a pyrimidine carboxylate derivative represented by the following formula (1) capable of inhibiting the binding of NMO-IgG (Neuro myelitis Optica-IgG) and aquaporin- A pharmaceutically acceptable salt or a solvate thereof as an active ingredient.

[화학식 1][Chemical Formula 1]

Figure 112015105890340-pat00001
Figure 112015105890340-pat00001

상기 [화학식 1]에서, In the above formula (1)

R1 및 R2는 서로 동일하거나 상이하고 C1 -8알킬이며,R 1 and R 2 are the same or different and each is a C 1 -8 alkyl each other,

R3, R4, R5, R6 및 R7은 서로 동일하거나 상이하고, 각각 독립적으로, 수소, 니트로, 카르복시, 히드록시, 할로겐, C1 -8알킬, 할로겐화 C1 - 6알킬 및 C1 - 6알킬옥시 중에서 선택된다. R 3, R 4, R 5 , R 6 and R 7 are the same or different from each other, each independently, hydrogen, nitro, carboxy, hydroxy, halogen, C 1 -8 alkyl, halogenated C 1 - 6 alkyl, and C 1 - 6 is selected from alkyloxy.

본 발명에 있어서, 상기 할로겐은 플루오로, 클로로, 브로모 및 요오도 중에서 선택될 수 있고, In the present invention, the halogen may be selected from among fluoro, chloro, bromo and iodo,

상기 C1 - 8알킬은 직쇄상 또는 분쇄상 알킬일 수 있으며, 구체적으로 메틸, 에틸, 프로필, 이소프로필, 부틸, 이소부틸, t-부틸, 펜틸, 헥실, 헵틸 및 옥틸 중에서 선택될 수 있고, The C 1 - 8 alkyl may be a straight-chain or branched alkyl, a, can be specifically selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t- butyl, pentyl, hexyl, heptyl, and octyl, the

상기 할로겐화 C1 - 6알킬은 트리플루오로메틸 또는 트리플루오로에틸일 수 있으며,The halogenated C 1 - 6 alkyl may be a methyl or ethyl trifluoroacetate in trifluoroacetic acid,

상기 C1 -7의 알킬옥시기는 메톡시, 에톡시, 프로폭시, 부톡시, 펜톡시, 헥실옥시, 헵틸옥시 및 옥틸옥시 중에서 선택될 수 있다. The alkyloxy C 1 -7 group may be selected from methoxy, ethoxy, propoxy, butoxy, pentoxy, hexyloxy, heptyloxy and octyloxy.

상기 [화학식 1]은 하기 [화학식 1a] 또는 [화학식 1b]로 표시되는 화합물 일 수 있다. The above formula (1) may be a compound represented by the following formula (1a) or (1b).

[화학식 1a][Formula 1a]

Figure 112015105890340-pat00002
Figure 112015105890340-pat00002

[화학식 1b][Chemical Formula 1b]

Figure 112015105890340-pat00003
Figure 112015105890340-pat00003

상기 [화학식 1a] 또는 [화학식 1b]에서, R1, R2, R3, R4, R5, R6 및 R7은 상히 [화학식 1]에서 정의한 바와 동일하다.R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are as defined in formula (I).

상기 [화학식 1]로 표시되는 화합물은 구체적으로 The compound represented by the above formula (1)

메틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Methyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

프로필 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Propyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

부틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Butyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

펜틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Pentyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-(4-(메틸티오)페닐)-2-옥소-6-(페닐설포닐메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6- (phenylsulfonylmethyl) hexahydropyrimidine-5-carboxylate;

부틸 4-(4-(메틸티오)페닐)-2-옥소-6-(페닐설포닐메틸)헥사히드로피리미딘-5-카복실레이트;Butyl 4- (4- (methylthio) phenyl) -2-oxo-6- (phenylsulfonylmethyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(4-(메틸티오)페닐)-2-옥소-6-(m-톨일설포닐메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6- (m-tolylsulfonylmethyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(4-(메틸티오)페닐)-2-옥소-6-(토실메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6- (tosylmethyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(4-(메틸티오)페닐)-2-옥소-6-((3,4,5-트리메틸페닐설포닐)메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6 - ((3,4,5-trimethylphenylsulfonyl) methyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(4-(메틸티오)페닐)-2-옥소-6-((4-트리플루오로메틸)페닐설포닐)메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6 - ((4-trifluoromethyl) phenylsulfonyl) methyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(4-(메틸티오)페닐)-2-옥소-6-((3-트리플루오로메틸)페닐설포닐)메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6 - ((3-trifluoromethyl) phenylsulfonyl) methyl) hexahydropyrimidine-5-carboxylate;

에틸 4-((3-메톡시페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3-methoxyphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((4-메톡시페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((4-methoxyphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((2,4-디메톡시페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((2,4-dimethoxyphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3-히드록시페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3-hydroxyphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-(4-(메틸티오)페닐)-6-((3-니트로페닐설포닐)메틸)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (4- (methylthio) phenyl) -6 - ((3-nitrophenylsulfonyl) methyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((4-클로로페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((4-chlorophenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3-클로로페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3-chlorophenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3-브로모페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3-bromophenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((4-브로모페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((4-bromophenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3,4-디메틸페닐설포닐)메틸)-2-옥소-6-(4-프로필티오)페닐)헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -2-oxo-6- (4-propylthio) phenyl) hexahydropyrimidine-5-carboxylate;

메틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Methyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

프로필 4-((3,4-디메틸페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Propyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

부틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Butyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

펜틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Pentyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-(3-(메틸티오)페닐)-2-옥소-6-(페닐설포닐메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (3- (methylthio) phenyl) -2-oxo-6- (phenylsulfonylmethyl) hexahydropyrimidine-5-carboxylate;

부틸 4-(3-(메틸티오)페닐)-2-옥소-6-(페닐설포닐메틸)헥사히드로피리미딘-5-카복실레이트;Butyl 4- (3- (methylthio) phenyl) -2-oxo-6- (phenylsulfonylmethyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(3-(메틸티오)페닐)-2-옥소-6-(m-톨일설포닐메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (3- (methylthio) phenyl) -2-oxo-6- (m-tolylsulfonylmethyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(3-(메틸티오)페닐)-2-옥소-6-(토실메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (3- (methylthio) phenyl) -2-oxo-6- (tosylmethyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(3-(메틸티오)페닐)-2-옥소-6-((3,4,5-트리메틸페닐설포닐)메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (3- (methylthio) phenyl) -2-oxo-6 - ((3,4,5-trimethylphenylsulfonyl) methyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(3-(메틸티오)페닐)-2-옥소-6-((4-트리플루오로메틸)페닐설포닐)메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (3- (methylthio) phenyl) -2-oxo-6 - ((4-trifluoromethyl) phenylsulfonyl) methyl) hexahydropyrimidine-5-carboxylate;

에틸 4-(3-(메틸티오)페닐)-2-옥소-6-((3-트리플루오로메틸)페닐설포닐)메틸)헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (3- (methylthio) phenyl) -2-oxo-6 - ((3-trifluoromethyl) phenylsulfonyl) methyl) hexahydropyrimidine-5-carboxylate;

에틸 4-((3-메톡시페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3-methoxyphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((4-메톡시페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((4-methoxyphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((2,4-디메톡시페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((2,4-dimethoxyphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3-히드록시페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3-hydroxyphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-(3-(메틸티오)페닐)-6-((3-니트로페닐설포닐)메틸)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4- (3- (methylthio) phenyl) -6 - ((3-nitrophenylsulfonyl) methyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((4-클로로페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((4-chlorophenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3-클로로페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3-chlorophenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3-브로모페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((3-bromophenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((4-브로모페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;Ethyl 4 - ((4-bromophenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;

에틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(3-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트; 및Ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (3- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate; And

에틸 4-((3,4-디메틸페닐설포닐)메틸)-2-옥소-6-(3-프로필티오)페닐)헥사히드로피리미딘-5-카복실레이트 중에서 선택되는 것일 수 있다. Ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -2-oxo-6- (3-propylthio) phenyl) hexahydropyrimidine-5-carboxylate.

본 발명에 있어서, 상기 약제학적으로 허용 가능한 염은 당해 기술 분야에서 통상적으로 사용되는 것이면 특별히 제한은 없으며, 구체적인 예로는 염산, 브롬산, 술폰산, 아미도황산, 인산 또는 질산과 같은 무기산이나 카르복시산 또는 술폰산과 같은 유기산을 이용하여 염을 형성할 수 있으나 이에 한정되는 것은 아니다. In the present invention, the pharmaceutically acceptable salt is not particularly limited as long as it is commonly used in the art. Specific examples thereof include inorganic acids such as hydrochloric acid, bromic acid, sulfonic acid, amidosulfuric acid, phosphoric acid or nitric acid, The salt may be formed using an organic acid such as sulfonic acid, but is not limited thereto.

또한, 상기 카르복시산의 종류로는 아세트산, 말레산, 푸마르산, 말산, 시트르산, 타르타르산, 락트산, 벤조산, 숙신산, 프로피온산, 글리콜산, 스테아르산 및 젖산 등이 포함될 수 있으며, 술폰산의 종류로는 메탄술폰산, 에탄술폰산, 벤젠술폰산, 톨루엔 술폰산 및 나프탈렌디술폰산 등이 포함될 수 있으나, 상기 카르복시산 및 술폰산의 종류가 상기 화합물들로 한정되는 것은 아니다. Examples of the carboxylic acid include acetic acid, maleic acid, fumaric acid, malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, succinic acid, propionic acid, glycolic acid, stearic acid and lactic acid, Ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, and naphthalenedisulfonic acid, but the types of the carboxylic acid and sulfonic acid are not limited to the above-mentioned compounds.

아쿠아포린 4는 혈액 뇌 관문을 둘러싸고 있는 별아교세포 말단에 존재하며, 세포 막을 통해 수분을 수송하는 역할을 하는 통로로 작용한다. 시신경 척수염의 특이 항체인 NMO-IgG는 수분 통로 단백질인 아쿠아포린 4에 결합하여 면역 반응을 유발한다. Aquaporin 4 is located at the end of the astrocyte surrounding the blood-brain barrier, and serves as a pathway for transporting moisture through the cell membrane. NMO-IgG, a specific antibody to optic neuritis, binds to the aquaporin 4, a water channel protein, to trigger an immune response.

본 발명에 따른 상기 [화학식 1]로 표시되는 피리미딘 카복실레이트 유도체는 NMO IgG와 아쿠아포린 4의 단백질의 결합을 저해하는데 핵심 역할을 한다. 또한 농도의존적으로 NMO IgG와 보체의 결합에 의한 세포독성을 저해한다. The pyrimidine carboxylate derivatives represented by the above formula (1) according to the present invention play a key role in inhibiting the binding of proteins of NMO IgG and aquaporin 4. In addition, it inhibits cytotoxicity by binding of NMO IgG and complement in a concentration-dependent manner.

따라서 상기 [화학식 1]로 표시되는 화합물, 이의 약제학적으로 허용가능한 염 또는 이의 용매화물을 유효성분으로 포함하는 조성물은 NMO IgG와 아쿠아포린 4의 단백질의 결합저해제로 유용하게 이용될 수 있다. Therefore, a composition comprising the compound represented by the formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient can be usefully used as a binding inhibitor of the protein of NMO IgG and aquaporin 4.

본 발명에 의하면, 상기 "시신경 척수염"이란, 단안 또는 양안의 심안 시신경염(optic neuritis)과 척추 3분절 이상을 침범하는 긴 척수염(longitudinally extensive myelitis)이 특징적으로 발현하는 질환을 의미한다.According to the present invention, the term "optic nerve spondylitis" means a disease characterized by optic neuritis of the monocular or bilateral and longitudinally extensive myelitis involving more than three segments of the vertebra.

상기 시신경 척수염은 성상교세포 등에 존재하는 아쿠아포린 4에 항-아쿠아 자가항체가 결합하여 보체-의존성 세포독성을 유도함으로써, BBB(blood brain barrier)의 파괴 등이 유발되는 것을 주요 발병 및 진행 기전으로 한다. The optic neuropathy is a major pathogenesis and progression mechanism in which the anti-aqua autoantibody binds to aquaporin 4 present in astrocytes and induces complement-dependent cytotoxicity, thereby causing destruction of the blood brain barrier (BBB) .

본 발명에 의하면, 상기 [화학식 1]로 표시되는 피리미딘 카복실레이트 유도체는 NMO IgG와 아쿠아포린 4의 단백질의 결합을 저해하는 능력이 우수하여 NMO IgG와 아쿠아포린 4의 단백질의 결합에 의해 발생하는 세포의 사멸을 방지할 수 있다. 따라서 상기 [화학식 1]로 표시되는 피리미딘 카복실레이트 유도체, 이의 약제학적으로 허용가능한 염 또는 이의 용매화물을 유효성분으로 포함하는 조성물은 NMO IgG와 아쿠아포린 4의 단백질의 결합에 의한 세포 사멸에 의해 유발되는 질병, 구체적으로 시신경 척수염 또는 퇴행성 신경계 질환의 치료 또는 예방 효과를 나타낼 수 있으므로 시신경 척수염의 치료 또는 예방용 약학조성물 또는 퇴행성 신경계 질환의 치료 또는 예방용 약학조성물로 유용하게 이용될 수 있다. According to the present invention, the pyrimidine carboxylate derivatives represented by the above formula (1) are excellent in the ability to inhibit the binding of the proteins of NMO IgG and aquaporin 4 and are produced by the binding of proteins of NMO IgG and aquaporin 4 It is possible to prevent cell death. Therefore, a composition comprising the pyrimidine carboxylate derivative represented by the above formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient can be obtained by the cell death by the binding of the protein of NMO IgG and aquaporin 4 Can be useful as a pharmaceutical composition for the treatment or prevention of optic neuritis or for the treatment or prevention of degenerative nervous system diseases because it can show a therapeutic or preventive effect of induced diseases, specifically optic neuritis or degenerative nervous system diseases.

본 발명에 의하면 상기 퇴행성 신경계 질환은 데빅병, 다발성경화증 및 루게릭병 중에서 선택되는 어느 하나 일 수 있다. According to the present invention, the degenerative neurological disease may be any one selected from DeVic disease, multiple sclerosis and Lou Gehrig's disease.

본 발명에 의하면 상기 피리미딘 카복실레이트 유도체, 이의 약제학적으로 허용 가능한 염 또는 이의 용매화물은 통상적인 담체, 보조제 또는 희석제와 혼합하여 통상의 제제화 방법으로 제형화하여 경구투여 또는 비경구투여에 적합한 형태로 제조될 수 있다. According to the present invention, the pyrimidine carboxylate derivative, its pharmaceutically acceptable salt or a solvate thereof may be formulated by a conventional formulation method by mixing with a conventional carrier, adjuvant or diluent, and then formulated into a form suitable for oral administration or parenteral administration . ≪ / RTI >

상기 담체, 보조제 또는 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. Examples of the carrier, adjuvant or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, poly Vinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

또한, 제제화할 경우에는 통상의 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등을 더 포함할 수도 있으며, 마그네슘 스티레이트, 탈크 같은 윤활제를 더 첨가할 수도 있다. When formulating the composition, the composition may further contain conventional fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like, and lubricants such as magnesium stearate and talc may be further added.

상기 경구투여의 경우에는 정제, 캡슐제, 용액, 시럽제, 현탁제 등의 형태로 제조될 수 있고, 비경구투여의 경우에는 복강, 피하, 근육, 경피에 대한 주사제의 형태로 제조될 수 있다.The oral administration may be in the form of tablets, capsules, solutions, syrups, suspensions, etc. In the case of parenteral administration, it may be prepared in the form of injections for peritoneal, subcutaneous, muscular and transdermal administration.

본 발명의 일 실시예에 따르면, 상기 약학조성물은 NMO-IgG와 아쿠아포린 4 단백질의 결합을 방지하는 것으로서, 상기 약학조성물 내의 [화학식 1]의 피리미딘 카복실레이트 유도체 또는 이의 약제학적으로 허용 가능한 염의 1일 유효투여량은 성인을 기준으로 0.01 내지 1000 mg/day이나, 투여용량은 환자의 나이, 몸무게, 성별, 투여형태, 건강상태 및 질환 정도에 따라 달라질 수 있으며, 의사 또는 약사의 판단에 따라 일정 시간간격으로 1일 1회 내지 수회로 분할 투여할 수도 있다. According to one embodiment of the present invention, the pharmaceutical composition is a composition for preventing the binding of NMO-IgG and aquaporin 4 protein, comprising a pyrimidine carboxylate derivative of the formula 1 or a pharmaceutically acceptable salt thereof The effective dose per day is 0.01 to 1000 mg / day on an adult basis, but the dose may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity, And may be administered once or several times a day at a predetermined time interval.

본 발명에 따른 피리미딘 카복실레이트 유도체 또는 이의 약제학적으로 허용가능한 염 또는 이의 용매화물은 NMO-IgG와 아쿠아포린 4 단백질의 결합으로 발생하는 세포의 사멸을 방지할 수 있어, NMO-IgG와 아쿠아포린 4 단백질의 결합에 의한 세포 사멸에 의해 유발되는 질병, 구체적으로 시신경 척수염, 퇴행성 신경계 질환 등의 치료 및 예방 효과를 나타낸다. The pyrimidine carboxylate derivative or its pharmaceutically acceptable salt or solvate thereof according to the present invention can prevent the death of cells caused by the binding of NMO-IgG and aquaporin 4 protein, and can prevent NMO-IgG and aquaporin 4 protein, specifically, optic neuritis, degenerative nervous system diseases, and the like.

도 1은 본 발명의 일 실시예에 따른 젖산탈수효소 분비 검사법을 도식화하여 나타낸 도이다.
도 2는 본 발명의 일 실시예에 따라 일반인의 IgG와 시신경척수염(NMO) 환자의 IgG를 각각 U87MG와 U87MG-AQP4로 처리하여 얻은 LDH-releasing 결과 그래프이다.
도 3은 본 발명의 일 실시예에 따른 화합물이 농도의존적으로 NMO-IgG와 보체의 결합에 의한 세포독성을 저해하는 결과를 그래프로 나타낸 도이다.
도 4는 본 발명의 일 실시예에 따른 화합물이 AQP4와 NMO-IgG 결합을 저해하는지 확인하기 위한 간접면역형광항체 검사법을 도식화하여 나타낸 도이다.
도 5는 본 발명의 일 실시예에 따른 화합물의 AQP4와 NMO-IgG 결합 저해능을 나타낸 간접면역형광항체 검사 결과이다.
도 6은 본 발명의 일 실시예에 따른 임피던스 측정을 기반으로 한 실시간세포지수측정법을 도식화하여 나타낸 도이다.
도 7은 NMO-IgG와 보체의 결합에 의한 세포독성에 의해 감소된 세포지수가 수 시간 후 본 발명의 일 실시예에 따른 화합물에 의해 농도 의존적으로 증가된 결과를 나타낸 도이다. 1 is a diagram illustrating a lactate dehydratase secretion test according to an embodiment of the present invention.
FIG. 2 is a graph of LDH-releasing results obtained by treating IgG of patients with generalized IgG and optic neuropathy (NMO) with U87MG and U87MG-AQP4, respectively, according to an embodiment of the present invention.
FIG. 3 is a graph showing a result of inhibiting cytotoxicity of a compound according to an embodiment of the present invention by concentration-dependent binding of NMO-IgG and complement.
FIG. 4 is a diagram illustrating an indirect immunofluorescent antibody test method for confirming whether a compound according to an embodiment of the present invention inhibits AQP4 and NMO-IgG binding.
FIG. 5 shows the result of indirect immunofluorescent antibody test showing the inhibition of AQP4 and NMO-IgG binding of a compound according to an embodiment of the present invention.
6 is a diagram illustrating a real-time cell index measurement method based on impedance measurement according to an embodiment of the present invention.
FIG. 7 is a graph showing the results of a concentration-dependent increase of the cell index reduced by cytotoxicity by binding of NMO-IgG and complement to the compound according to one embodiment of the present invention after several hours.

이하, 바람직한 실시예를 들어 본 발명을 더욱 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 보다 구체적으로 성명하기 위한 것으로, 본 발명의 범위가 이에 의하여 제한되지 않는다는 것은 당업계의 통상의 지식을 가진 자에게 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent to those skilled in the art, however, that these examples are for the purpose of describing the present invention more specifically and that the scope of the present invention is not limited thereby.

시험예Test Example 1.  One. 젖산탈수소효소Lactate dehydrogenase 분비( secretion( actateactate dehydrogenasedehydrogenase releaserelease assayassay ; ; LDHLDH assay) 검사 assay

하기 도 1에 나타낸 방법으로 젖산탈수소효소 분비검사(LDH assay)를 수행하였다. The lactate dehydrogenase secretion test (LDH assay) was performed by the method shown in Fig.

Complement dependent cytotoxicity 저해 정도는 CytoTox-ONE Homogeneous Membrane Integrity Assay Kit(Promega, Madison, USA)를 사용하여 NMO환자의 IgG와 인간 보체 human complement를 처리하여 보체에 의한 cell death(compelent dependent cytotoxicity)를 LDH-releasing 수치의 감소정도에 의하여 측정하였다. U87MG-AQP4 over-expressed stable cell은 완전 배지[10% 우태아혈청(FBS, Thermo Scientific), 1% 페니실린/스트렙토마이신(Penstripe, Gibco), 400ng/mL G418-disulfate(Duchefa Biochemie)]이 첨가된 DMEM 배지에 섞어주고 96 well plate에 2.5×104세포/well로 분주한 후 37 ℃ 배양기에서 이틀 동안 배양하였다. 그 후, 배지를 제거하고 HBSS(Sigma)로 1회 세척한 후 50 μL의 0.2% DMSO가 포함된 후보 물질들을 농도별로 처리한 후 50 μL의 NMO-IgG 100 μg/mL 와 5% human complement를 혼합하여 96 well에 반응시켰다.The complement dependent cytotoxicity inhibition was assessed by LDH-releasing (compelent dependent cytotoxicity) of complement-dependent cytotoxicity by treating IgG and human complement of NMO patients using the CytoTox-ONE Homogeneous Membrane Integrity Assay Kit (Promega, Madison, Were measured by the degree of decrease. U87MG-AQP4 over-expressed stable cells were stained with complete medium [10% fetal bovine serum (FBS, Thermo Scientific), 1% penicillin / streptomycin (Penstripe, Gibco), 400 ng / mL G418-disulfate (Duchefa Biochemie) DMEM medium, and the cells were seeded at 2.5 × 10 4 cells / well in a 96-well plate and incubated at 37 ° C. for 2 days. Thereafter, the medium was removed, washed once with HBSS (Sigma), and then treated with 50 μL of 0.2% DMSO-containing candidate substances at a concentration of 50 μL of NMO-IgG 100 μg / mL and 5% human complement Mixed and reacted in 96 wells.

1시간 후, 100 μL의 LDH detecting reagent를 넣어주고 10분 후 microplate reader에서 형광(fluorescence)을 excitation 560 nm, emission 590 nm에서 측정하였으며, 이를 하기 도 2에 나타내었다.
After 1 hour, 100 μL of LDH-detecting reagent was added. After 10 minutes, fluorescence was measured in a microplate reader at excitation 560 nm and emission 590 nm, which is shown in FIG.

시험예Test Example 2.1  2.1 ComplementComplement dependentdependent cytotoxicitycytotoxicity (( CDCCDC ) ) assayassay 법 검증 Legal verification

CDC assay는 NMO-IgG의 AQP-4 결합과 함께 complement의 IgG 결합으로 인해 cell membrane이 손상됨으로 인한 cytotoxicity를 측정하는 방법으로 시험예 1번의 방법에 따라 손상된 세포 밖으로 나오는 lactate dehydrogenase (LDH)를 이용해 cytotoxicity 정도를 측정하는 LDH assay를 진행 하였다. 이 방법을 검증하기 위해 AQP-4가 trasfection 된 U87-MG 세포주와 대조군 세포주 모두에서 CDC assay를 진행하였다. 또한 NMO 환자의 IgG와 건강한 사람의 IgG를 함께 비교 시험하여 효능 평가시스템을 검증하였다. The CDC assay is a method for measuring the cytotoxicity due to the damage of the cell membrane due to IgG binding of complement with the AQP-4 binding of NMO-IgG. Using the lactate dehydrogenase (LDH) exiting the damaged cell according to the method of Test Example 1, cytotoxicity LDH assay was performed. To validate this method, CDC assays were performed in both U87-MG cell lines and control cell lines transfected with AQP-4. In addition, the efficacy evaluation system was verified by comparing the IgG of NMO patients with the IgG of healthy persons.

하기 도 2e에 나타낸 바와 같이, AQP-4가 발현되는 세포에 NMO 환자의 IgG를 처리했을 때 농도 의존적으로 CDC가 일어남을 확인할 수 있었다. 이를 통해 본 발명에서 사용한 CDC assay 법은 NMO-IgG가 AQP-4와의 결합에 의해 일어남을 검증하였다.
As shown in FIG. 2E, it was confirmed that CDC was induced in a concentration-dependent manner when the cells expressing AQP-4 were treated with IgG of NMO patients. Thus, the CDC assay method used in the present invention proved that NMO-IgG is caused by binding with AQP-4.

시험예Test Example 2.2  2.2 ComplementComplement dependentdependent cytotoxicitycytotoxicity (( CDCCDC ) ) assayassay

본 발명에 따른 화합물이 NMO-IgG에 일어나는 세포사멸을 저해하는지 확인하기 위하여, 본 발명에 따른 화합물 및 LDH assay(CDC assay)를 이용하여 NMO-IgG와 human complement에 의해 cytotoxicity가 일어난 군 보다 LDH release 수치가 적은 화합물을 도출하였다. In order to confirm whether the compounds according to the present invention inhibited the cell death caused by NMO-IgG, the compounds according to the present invention and the LDH assay (CDC assay) were used to compare LDH release Compounds with low numbers were derived.

대표 화합물로 에틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트(이하, "실시예 1"라 한다)의 CDC assay에 대한 LDH release 수치를 대조군(100%) 대비 퍼센트 환율로 계산하여 하기 표 1 및 도 3에 나타내었다. 표 1 및 도 3에 나타낸 바와 같이 본 발명에 따른 실시예1 화합물은 기존에 보고된 활성 화합물인 arbidol 보다 더 낮은 LDH release 수치를 나타내는 것을 확인하였다. 이에, 본 발명에 따른 화합물이 NMO-IgG에 일어나는 세포사멸을 저해하는 효과가 있음을 확인하였다. As a representative compound, ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine- 1 ") was calculated in terms of percent exchange rate relative to the control (100%) for the CDC assay, and is shown in Table 1 and FIG. As shown in Table 1 and Fig. 3, it was confirmed that the compound of Example 1 according to the present invention exhibited a lower LDH release value than the arbidol, which is the previously reported active compound. Thus, it was confirmed that the compound of the present invention has an effect of inhibiting the cell death that occurs in NMO-IgG.

구분division CDC assay(대조군 대비 LDH relese %)CDC assay (LDH relese% compared to the control) vehiclevehicle 100100 실시예 1Example 1 6161 arbidolarbidol 7575

시험예Test Example 4.  4. AQP4AQP4 와 Wow NMONMO -- IgGIgG 결합 저해  Inhibition of binding 간접면역형광항체Indirect immunofluorescent antibody 검사법( Test method IndirectIndirect Immunofluorescence  Immunofluorescence antibodyantibody assayassay ))

본 발명에 따른 [화학식 1]로 표시되는 화합물인 실시예 1이 AQP4 와 NMO-IgG의 결합을 저해하는지 확인하기 위하여 간접면역형광항체 검사법을 실시하였으며, 이를 하기 도 4에 도식화하여 나타내었다. AQP4가 과발현되어있는 stable cell line에 NMO-IgG를 처리한 후 FITC 형광물질이 결합되어 있는 secondary antibody를 반응시켜 결합정도를 측정함으로써 본 발명에 따른 화합물이 결합수치를 감소시키는지를 확인하였다. In order to confirm whether or not Example 1, which is a compound represented by Formula 1 according to the present invention, inhibits the binding of AQP4 and NMO-IgG, an indirect immunofluorescent antibody test was performed, which is illustrated in FIG. A stable cell line in which AQP4 is overexpressed was treated with NMO-IgG and secondary antibody with FITC fluorescent material was reacted to measure the degree of binding, thereby confirming whether the compound according to the present invention decreases the binding level.

U87MG-AQP4 over-expressed stable cell은 완전 배지[10% 우태아혈청(FBS, Thermo Scientific), 1% 페니실린/스트렙토마이신(Penstripe, Gibco), 400 ng/mL G418-disulfate(Duchefa Biochemie)]이 첨가된 DMEM 배지에 섞어주고 96well plate에 2.5×104세포/1well로 분주한 후 37 ℃ 배양기에서 이틀 동안 배양하였다. 세척과정을 거쳐 4% paraformaldehyde 용액을 처리하여 15분 동안 고정시켰다. 4% PFA를 빨아들인 후, Blocking buffer [1% Bovine Serum Albumin, 6 mM Glucose in PBS]에 20분 동안 배양하였다. Blocking buffer를 빨아들인 후, 50 uL의 0.2% DMSO가 포함된 후보 물질들을 농도별로 처리한 후 50 uL의 NMO-IgG 100 ug/uL를 혼합하여 96 well에 반응시켰다. 1시간 반응시킨 후, Blocking buffer로 세척과정 후 500배 희석된 FITC-conjugated Goat Anti-human IgG antibodies(Sigma)를 각 웰에 적하하였다. 다시 37 ℃ 배양기에서 30분 동안 반응시킨 후 세척과정을 거치고 형광현미경 하에서 관찰하였다. 자동화시스템을 탑재한 multiplate reader(Operetta, Perkin Elmer)에서 secondary antibody의 형광정도를 측정한 후 본 발명에 따른 ㅅ실시예 1이 NMO-IgG만 결합한 웰보다 수치가 적은 것을 확인하였다. U87MG-AQP4 over-expressed stable cells were stained with complete medium [10% fetal bovine serum (FBS, Thermo Scientific), 1% Penicillin / Streptomycin (Gibco), 400 ng / mL G418-disulfate (Duchefa Biochemie) The cells were mixed with DMEM medium at a density of 2.5 × 10 4 cells / well in a 96-well plate, and cultured in a 37 ° C. incubator for 2 days. After washing, 4% paraformaldehyde solution was applied and fixed for 15 minutes. After 4% PFA was aspirated, it was incubated in blocking buffer [1% Bovine Serum Albumin, 6 mM Glucose in PBS] for 20 min. Blocking buffer was aspirated, and 50 μL of 0.2% DMSO-containing candidate substances were treated at different concentrations. Then, 50 μL of NMO-IgG 100 μg / μL was mixed and reacted in 96 wells. After 1 hour of reaction, the cells were washed with blocking buffer and then FITC-conjugated goat anti-human IgG antibodies (Sigma) diluted 500-fold were added to each well. After incubating for 30 minutes in a 37 ° C incubator, the cells were washed and observed under a fluorescence microscope. The degree of fluorescence of the secondary antibody was measured in a multiplate reader (Operetta, Perkin Elmer) equipped with an automation system, and it was confirmed that Example 1 according to the present invention had fewer numbers than NMO-IgG alone wells.

본 발명에 따른 실시예 1의 화합물은 CDC assay에서의 결과와 마찬가지로 간접면역형광항체 검사법을 통해서 AQP4와 NMO-IgG의 결합을 저해하는 것으로 나타났다. 특히, 하기 도 5에 나타낸 바와 같이, 실시예 1의 화합물은 종래의 알려진 화합물인 arbidol 보다 더 우수한 AQP4와 NMO-IgG의 결합 저해 효능을 보여주었다. The compounds of Example 1 according to the present invention were found to inhibit the binding of AQP4 and NMO-IgG by indirect immunofluorescent antibody assay as in the CDC assay. In particular, as shown in FIG. 5 below, the compound of Example 1 showed the binding inhibition effect of AQP4 and NMO-IgG, which is superior to the known compound arbidol.

상기와 같은 결과를 바탕으로 본 발명에 따른 화합물은 AQP4와 NMO-IgG의 결합을 억제함으로써 이로 인해 발생되는 세포독성을 현저히 줄일 수 있으므로, AQA4에 항-아쿠아포린 4 자가항체가 결합하여 보체-의존성 세포독성을 유도하여 유발되는 질환, 구체적으로 시신경 척수염의 치료제로 유용하게 적용할 수 있다.
Based on the above results, the compounds according to the present invention inhibit the binding of AQP4 and NMO-IgG, thereby significantly reducing the cytotoxicity caused thereby. Therefore, the anti-aquaporin 4 autoantibody binds to AQA4, And can be usefully used as a therapeutic agent for a disease caused by inducing cytotoxicity, specifically, optic neuritis.

시험예Test Example 5. 임피던스 측정을 기반으로 한 실시간세포지수측정법( 5. Real-time cell index measurement based on impedance measurement ( ImpedanceImpedance based  based realreal timetime cellcell analyzeranalyzer assayassay ))

NMO-IgG와 complement의 결합으로 인해 cell membrane이 손상됨으로 인한 세포의 변화 및 독성을 실시간으로 오랜 시간 측정하기 위해 임피던스 저항값 측정을 기반으로 한 실시간 세포지수 측정법을 도입하였으며, 이를 하기 도 6에 도식화하여 나타내었다. iCELLigence (ACEA Biosciences, San Diego, USA) 는 세포분석장비로 E-plate well 바닥에 심어진 미세전극 집합체를 통해 세포가 자라는 동안 발생하는 임피던스 (impedance)라는 저항값을 측정하여 세포의 반응을 실시간으로 모니터링하는 장치이다. 세포에 의해 감지된 저항값은 세포지수(cell index)라는 지표로 나타나게 되고, 세포지수의 변화는 세포 모양변화와 밀접한 관련성을 나타낸다. 별도의 형광 표지가 없이도 세포의 변화를 실시간으로, 또한 지속적으로 분석할 수 있는 스크리닝 시스템을 통해 화합물을 검색하였다. CHO-K1-AQP4 over-expressed stable cell은 완전 배지[10% 우태아혈청(FBS, Thermo Scientific), 1% 페니실린/스트렙토마이신(Penstripe, Gibco), 400ng/mL G418-disulfate(Duchefa Biochemie)]이 첨가된 DMEM 배지에 섞어주고 E-plate 에 5.0×104세포/well로 분주한 후 37 ℃ 배양기에서 여섯 시간 동안 배양하였다. 그 후, 배지에 0.4% DMSO가 포함된 후보 물질들을 농도별로 포함하여 NMO-IgG 10 μg/mL 와 0.5% human complement를 혼합한 100 μL의 용액을 E-plate well에 반응시켰다.In order to measure the change and toxicity of cells due to cell membrane damage due to binding of NMO-IgG and complement for a long time in real time, a real-time cell index measurement method based on measurement of impedance resistance value was introduced. Respectively. The iCELLigence (ACEA Biosciences, San Diego, USA) is a cell analyzer that measures the resistance of an electrode during cell growth through a collection of microelectrodes embedded in the bottom of an E-plate well, . The resistance value detected by the cell appears as an index of the cell index, and the change of the cell index is closely related to the change of the cell shape. The compounds were screened through a screening system capable of continuously and continuously analyzing changes in cells in the absence of a separate fluorescent label. CHO-K1-AQP4 over-expressed stable cells were cultured in complete medium (FBS, Thermo Scientific, 1% Penicillin / Streptomycin, Gibco, 400 ng / mL G418-disulfate (Duchefa Biochemie) The cells were mixed with the DMEM medium, and the cells were seeded on the E-plate at 5.0 × 10 4 cells / well and incubated at 37 ° C. for 6 hours. Subsequently, 100 μL of a mixture of NMO-IgG 10 μg / mL and 0.5% human complement containing 0.4% DMSO-containing candidate substances was added to the E-plate well.

배양기 안에 있는 분석기기에 무선으로 연결되어있는 아이패드 출력기기에 그래프로 결과가 저장된 것을 확인하였다. 24시간 후, 세포지수의 변화가 실시간으로 24시간 동안 입력된 결과에서 화합물을 처리하기 전인 세포배양 5시간 후의 세포지수와 10시간 후의 세포지수를 비교분석하였으며, 이를 하기 도 7에 나타내었다. 도 7을 참고로 하면, 본 발명에 따른 실시예 1의 화합물이 첨가되지 않은 대조군(DMSO)은 세포의존성 독성에 의해 세포지수가 감소된 반면, 본 발명에 따른 실시예 1의 화합물이 첨가된 군은 농도 의존적으로 세포지수가 증가하는 경향을 나타내었다. 상기 결과를 통해 본 발명에 따른 화합물이 보체의존성 세포독성 저해능이 있음을 확인하였다. It was confirmed that the graphical results were stored in the iPad output device wirelessly connected to the analyzer in the incubator. After 24 hours, the change of cell index was input for 24 hours in real time, and the cell index after 5 hours and the cell index after 10 hours before the compound treatment were compared and analyzed, and it is shown in FIG. 7, the control group (DMSO) to which the compound of Example 1 according to the present invention was not added had a decreased cell index due to cell-dependent toxicity, whereas the group to which the compound of Example 1 according to the present invention was added Showed a tendency that the cell index increased in a concentration dependent manner. From the above results, it was confirmed that the compound according to the present invention has the ability to inhibit complement dependent cytotoxicity.

Claims (7)

삭제delete 삭제delete 삭제delete 하기 [화학식 1]로 표시되는 피리미딘 카복실레이트 유도체 또는 이의 약제학적으로 허용 가능한 염 또는 이의 용매화물을 유효성분으로 포함하는 시신경 척수염 예방 또는 치료용 약학조성물:
[화학식 1]
Figure 112016090744296-pat00016

상기 [화학식 1]에서,
R1 및 R2는 서로 동일하거나 상이하고 C1-6알킬이며,
R3, R4, R5, R6 및 R7은 서로 동일하거나 상이하고, 각각 독립적으로, 수소 또는 메틸이다. A pharmaceutical composition for preventing or treating optic neuritis, comprising as an active ingredient, a pyrimidine carboxylate derivative represented by the following formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof:
[Chemical Formula 1]
Figure 112016090744296-pat00016

In the above formula (1)
R 1 and R 2 are the same or different and are C 1-6 alkyl,
R 3 , R 4 , R 5 , R 6 and R 7 are the same as or different from each other, and each independently hydrogen or methyl. 제4항에 있어서, 상기 [화학식 1]로 표시되는 화합물은
메틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;
에틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;
프로필 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;
부틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;
펜틸 4-((3,4-디메틸페닐설포닐)메틸)-6-(4-(메틸티오)페닐)-2-옥소헥사히드로피리미딘-5-카복실레이트;
에틸 4-(4-(메틸티오)페닐)-2-옥소-6-(페닐설포닐메틸)헥사히드로피리미딘-5-카복실레이트;
부틸 4-(4-(메틸티오)페닐)-2-옥소-6-(페닐설포닐메틸)헥사히드로피리미딘-5-카복실레이트;에틸 4-(4-(메틸티오)페닐)-2-옥소-6-(m-톨일설포닐메틸)헥사히드로피리미딘-5-카복실레이트;
에틸 4-(4-(메틸티오)페닐)-2-옥소-6-(토실메틸)헥사히드로피리미딘-5-카복실레이트;
에틸 4-(4-(메틸티오)페닐)-2-옥소-6-((3,4,5-트리메틸페닐설포닐)메틸)헥사히드로피리미딘-5-카복실레이트; 및
에틸 4-((3,4-디메틸페닐설포닐)메틸)-2-옥소-6-(4-프로필티오)페닐)헥사히드로피리미딘-5-카복실레이트;중에서 선택되는 것을 특징으로 하는 시신경 척수염 예방 또는 치료용 약학조성물.The compound according to claim 4, wherein the compound represented by the formula (1)
Methyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;
Ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;
Propyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;
Butyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;
Pentyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -6- (4- (methylthio) phenyl) -2-oxohexahydropyrimidine-5-carboxylate;
Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6- (phenylsulfonylmethyl) hexahydropyrimidine-5-carboxylate;
Butyl 4- (4- (methylthio) phenyl) -2-oxo-6- (phenylsulfonylmethyl) hexahydropyrimidine-5-carboxylate ethyl 4- Oxo-6- (m-tolylsulfonylmethyl) hexahydropyrimidine-5-carboxylate;
Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6- (tosylmethyl) hexahydropyrimidine-5-carboxylate;
Ethyl 4- (4- (methylthio) phenyl) -2-oxo-6 - ((3,4,5-trimethylphenylsulfonyl) methyl) hexahydropyrimidine-5-carboxylate; And
Wherein the optic nerve is selected from the group consisting of ethyl 4 - ((3,4-dimethylphenylsulfonyl) methyl) -2-oxo-6- (4-propylthio) phenyl) hexahydropyrimidine- ≪ / RTI > 하기 [화학식 1]로 표시되는 피리미딘 카복실레이트 유도체 또는 이의 약제학적으로 허용 가능한 염 또는 이의 용매화물을 유효성분으로 포함하는 퇴행성 신경계 질환 치료 또는 예방용 약학조성물:
[화학식 1]
Figure 112016090744296-pat00008

상기 [화학식 1]에서,
R1 및 R2는 서로 동일하거나 상이하고 C1-6알킬이며,
R3, R4, R5, R6 및 R7은 서로 동일하거나 상이하고, 각각 독립적으로, 수소 또는 메틸이다. A pharmaceutical composition for treating or preventing degenerative neurological diseases comprising a pyrimidine carboxylate derivative represented by the following formula (1), a pharmaceutically acceptable salt thereof or a solvate thereof as an active ingredient:
[Chemical Formula 1]
Figure 112016090744296-pat00008

In the above formula (1)
R 1 and R 2 are the same or different and are C 1-6 alkyl,
R 3 , R 4 , R 5 , R 6 and R 7 are the same as or different from each other, and each independently hydrogen or methyl. 제6항에 있어서, 상기 퇴행성 신경계 질환은 데빅병, 다발성경화증 및 루게릭병 중에서 선택되는 것을 특징으로 하는 약학조성물.7. The pharmaceutical composition according to claim 6, wherein the degenerative neurological disease is selected from DeVic disease, multiple sclerosis and Lou Gehrig's disease.
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KR20140059168A (en) * 2011-04-21 2014-05-15 더 리젠츠 오브 더 유니버시티 오브 콜로라도, 어 바디 코포레이트 Compositions and methods for the treatment of neuromyelitis optica

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