KR20210055172A - Novel sesquiterpene derivatives and use thereof - Google Patents

Novel sesquiterpene derivatives and use thereof Download PDF

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KR20210055172A
KR20210055172A KR1020190141336A KR20190141336A KR20210055172A KR 20210055172 A KR20210055172 A KR 20210055172A KR 1020190141336 A KR1020190141336 A KR 1020190141336A KR 20190141336 A KR20190141336 A KR 20190141336A KR 20210055172 A KR20210055172 A KR 20210055172A
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황성관
이홍우
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엠에프씨 주식회사
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Abstract

A novel sesquiterpene derivative or an optical isomer thereof of the present invention has excellent neuron protective effects and apoptosis inhibition effects, thereby being usefully used for the prevention, alleviation and treatment of Parkinson's disease.

Description

신규 세스퀴테르펜 유도체 (3) 및 이의 용도 {Novel sesquiterpene derivatives and use thereof}Novel sesquiterpene derivatives and use thereof {Novel sesquiterpene derivatives and use thereof}

본 발명은 신규 세스퀴테르펜 유도체 및 이의 용도에 관한 것으로, 보다 구체적으로 신규 세스퀴테르펜 유도체 또는 이의 광학 이성질체; 이를 포함하는 약학적 조성물, 의약외품 조성물 및 식품 조성물; 및 이의 파킨슨병(Parkinson's disease) 예방, 개선 또는 치료 용도에 관한 것이다.The present invention relates to novel sesquiterpene derivatives and uses thereof, and more particularly to novel sesquiterpene derivatives or optical isomers thereof; Pharmaceutical compositions, quasi-drug compositions, and food compositions comprising them; And its use in the prevention, amelioration or treatment of Parkinson's disease.

파킨슨병은 인간 뇌의 흑질 치밀부(substantia nigra pars compacta, SNc)에서 도파민 작동성 뉴런(dopamine acting neuron)의 신경세포 퇴화가 특징으로 나타나는 것으로서, 이는 선조체(striatum)에서 점진적으로 도파민의 감소 또는 고갈을 유발시킨다. 파킨슨병의 특징은 기저핵 순환(basal ganglia circulation)의 균형을 망가뜨리고 운동뉴런(motor neuron)이 본래의 기능을 못하게 하여, 신체운동의 경직(rigidity), 진전(tremor), 운동불능(akinesia)를 일으킨다. 대략 50대 이상 인구의 3~5%가 이 질환으로 고통 받는 것으로 추정되는 뇌신경 퇴화 질병이다. Parkinson's disease is characterized by neuronal degeneration of dopamine acting neurons in the substantia nigra pars compacta (SNc) of the human brain, which is a progressive decrease or depletion of dopamine in the striatum. Causes. The characteristic of Parkinson's disease is that it disrupts the balance of the basal ganglia circulation and prevents the motor neurons from functioning, thereby reducing the rigidity, tremor, and akinesia of physical movement. Raises. It is a neurodegenerative disease that is estimated to suffer from the disease in approximately 3 to 5% of the population over 50.

파킨슨병의 근본적인 원인이 아직 정확하게 규명되지 않았으나, 최근 일련의 연구결과, 정상인 뇌(brain)의 중뇌 흑질부(mesencephalon substantia nigra)부위의 신경세포에서 만들어지는 뇌신경전달 neuro transmitted substances)물질 중 하나인 도파민(dopamine)의 결핍으로 인해 여러 운동기능 실조가 나타나는 것으로 알려져 있다. Although the underlying cause of Parkinson's disease has not yet been accurately identified, a recent series of studies have shown that dopamine, one of the substances produced by neurons in the mesencephalon substantia nigra region of the normal brain. (Dopamine) is known to cause several motor ataxia due to deficiency.

최근, 파킨슨병 치료를 위해 신경전달물질 수용체(receptors)에 대한 길항제(agonists), GABA(gamma aminobutyric acid) 효능제, 세포 내 칼슘 감소제(calcium reducer), 유리 라디칼 제거제(free radical scavenger), 글루타메이트 유리기 억제제 등 다양한 약물이 시도 되고 있으나 대부분 위험요소(toxicity)와 부작용(adverse effects)을 가지고 있어 보다 안전한 약물(safety candiadtes) 개발이 절실히 요구되고 있는 현실이다.Recently, for the treatment of Parkinson's disease, agonists against neurotransmitter receptors, gamma aminobutyric acid (GABA) agonists, calcium reducers in cells, free radical scavengers, glutamate. Various drugs such as free radical inhibitors have been tried, but most of them have toxicity and adverse effects, so the development of safer drugs (safety candiadtes) is urgently required.

상기의 문제점들로 인해 부작용이 거의 없거나 무시할 수 있는 정도의 보다 더 안전한 저독성(lower toxicity)을 갖는 파킨슨병 치료제를 개발하고자 하는 연구가 진행되고 있다. 그 예로는, 천연의 백목 또는 향나무의 주성분인 (+)-세드롤(cedrol), 위드롤(widdrol)을 추출하여 추출물을 유효성분으로 하는 파킨슨 질환 예방 및 치료용 조성물이 대한민국 공개특허공보 제10-2018-0114267호에 개시되어 있으며, 편백나무, 측백나무, 구상나무 혼합 시료로부터 추출한 오일 성분물질(oleoresins)을 유효성분으로 하는 뇌의 집중효과 개선용 조성제가 대한민국 공개특허공보 10-2017-0128716호에 개시되어 있다.Due to the above problems, research is being conducted to develop a Parkinson's disease treatment that has little or negligible side effects and has a safer, lower toxicity. As an example, a composition for preventing and treating Parkinson's disease using the extract as an active ingredient by extracting (+)-cedrol, which is the main component of natural cedar or juniper, is disclosed in Korean Patent Laid-Open Publication No. 10 -It is disclosed in 2018-0114267, and a composition for improving the concentration effect of the brain using as an active ingredient an oil component (oleoresins) extracted from a mixture of cypress, cypress, and bulbus trees is the Republic of Korea Patent Laid-Open Patent Publication 10-2017-0128716 It is disclosed in the issue.

하지만 상기 발명과 같이 천연 추출물을 그대로 사용하는 경우, 천연물질(natural products) 자체가 가지고 있는 본질적 독성(intact toxicity)에 노출될 위험이 존재하며, 이는 천연 추출물이 함유하는 본질적 문제(intrinsic major risk)로 개선 또는 감소 등을 인위적으로 조절(control) 할 수 없다. 또한 이들 정유성분(oleoresins) 물질은 물에 대한 용해도가 매우 낮아(예: 세드롤 경우 22mg/l) 약물을 효과적으로 체내(host body)에 투입할 수 없는 중대한 단점이 있다. However, if the natural extract is used as it is, as described above, there is a risk of exposure to the intrinsic toxicity of the natural products themselves, which is an intrinsic major risk. The improvement or reduction cannot be controlled artificially. In addition, these oleoresins have a very low solubility in water (eg, 22 mg/l in the case of sedrol), so that drugs cannot be effectively injected into the host body.

이를 개선하기 위해서는 다양한 약물 가용화 기술 등 고난도 약제학적 기술이 요구되나 이 역시 주성분(active pharmaceutical ingredients)이 갖는 물에 대한 낮은 용해도 범위에서 크게 벗어나지 않기 때문에 본질적으로 물에 대한 용해도가 매우 낮아 약물로 개발이 어려운 부분이 있다. 뿐만 아니라 투여된 약물이 물에 대한 낮은 용해도 때문에(lower solubility for water) 쉽게 결정화(crystallization)되어 체내 신장(kidney in body)에 축적되어 신장기능을 망가뜨려(kidney dysfuction)를 유발하여 신부전(renal failure) 등 중대한 부작용(severe adverse effects)을 야기시키는 문제가 있어 이에 대한 심도 깊은 연구가 요구되고 있다.In order to improve this, high-level pharmaceutical technologies such as various drug solubilization technologies are required, but this also does not deviate significantly from the low solubility range in water of the active pharmaceutical ingredients, so the solubility in water is inherently very low, making it difficult to develop as a drug. There is a difficult part. In addition, due to the low solubility of the administered drug in water (lower solubility for water), it is easily crystallized and accumulates in the kidney in the body, causing kidney dysfuction to cause kidney failure. There is a problem that causes severe adverse effects, and in-depth research on this is required.

대한민국 공개특허공보 10-2017-0128716호Korean Patent Application Publication No. 10-2017-0128716 대한민국 공개특허공보 10-2018-0114267호Korean Patent Application Publication No. 10-2018-0114267

본 발명은 신규 세스퀴테르펜 유도체 또는 이의 광학 이성질체를 제공한다.The present invention provides novel sesquiterpene derivatives or optical isomers thereof.

본 발명은 상기 신규 세스퀴테르펜 유도체 또는 이의 광학 이성질체를 유효성분으로 포함하는 파킨슨병 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease comprising the novel sesquiterpene derivative or an optical isomer thereof as an active ingredient.

본 발명은 상기 신규 세스퀴테르펜 유도체 또는 이의 광학 이성질체를 유효성분으로 포함하는 파킨슨병 예방 또는 개선용 의약외품 조성물을 제공한다.The present invention provides a quasi-drug composition for preventing or improving Parkinson's disease, comprising the novel sesquiterpene derivative or an optical isomer thereof as an active ingredient.

본 발명은 상기 신규 세스퀴테르펜 유도체 또는 이의 광학 이성질체를 유효성분으로 포함하는 파킨슨병 예방 또는 개선용 식품 조성물을 제공한다.The present invention provides a food composition for preventing or improving Parkinson's disease comprising the novel sesquiterpene derivative or an optical isomer thereof as an active ingredient.

본 발명자들은 백목(학명 cupressus fenebris) 또는 향나무(juniprus chinensis) 등의 목질부에서 추출되는 주요 활성성분 중 하나인 (+)-세드롤(cedrol, 분자량 222.36, C15H26O)과 (+)-위드롤(widdrol, 분자량 222.36, C15H26O)을 이용하여 이들이 가지는 내재성(intrinsic) 독성성분(toxicity)을 감소시키고 물에 대한 용해도를 크게 증가시켜 보다 우수한 신경보호기능(neuron protective function) 및 세포사멸사(apoptosis)를 감소시키는 약물 효과를 극대화하기 위해 다양한 약리활성단(active pharmacopeia site)을 갖는 신약 후보물질을 개발하고자 노력하였다. 이를 위해, 분자 화학적 지식에 기반한(medicinal chemistry based on drug design concept) 다년간의 노력을 기울인 결과 새로운 세스퀴테르펜 유도체 계열의 신규 화합물을 합성하여 파킨슨병 예방 및 치료에 탁월한 효과를 갖는 것을 확인함으로써 본 발명을 완성하였다.The inventors of the present invention are one of the main active ingredients extracted from woody parts such as white tree (scientific name cupressus fenebris) or juniper tree (juniprus chinensis), which is (+)-cedrol (molecular weight 222.36, C 15 H 26 O) and (+)- Widrol (molecular weight 222.36, C 15 H 26 O) is used to reduce their intrinsic toxicity and greatly increase the solubility in water, resulting in a better neuroprotective function. And in order to maximize the effect of drugs that reduce apoptosis, efforts have been made to develop new drug candidates having various active pharmacopeia sites. To this end, as a result of years of efforts based on molecular chemistry knowledge (medicinal chemistry based on drug design concept), the present invention was confirmed to have excellent effects in the prevention and treatment of Parkinson's disease by synthesizing a novel compound of a new sesquiterpene derivative series. Was completed.

이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific description described below.

신규 세스퀴테르펜 유도체Novel sesquiterpene derivatives

본 발명은 신규 세스퀴테르펜(sesquiterpene) 유도체, 즉 하기 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체를 제공한다.The present invention provides a novel sesquiterpene derivative, that is, a compound represented by the following Chemical Formulas 1 to 12, or an optical isomer thereof.

Figure pat00001
Figure pat00001

1) (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-히드록시-3-메톡시벤조네이트;1) (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-4-hydroxy-3-methoxy Benzoate;

2) (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-메톡시-4-피발로일옥시벤조네이트;2) (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulene-6-yl-3-methoxy-4-pivalo Monooxybenzoate;

3) (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-(피발로일옥시)벤조네이트;3) (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-4-(pivaloyloxy)benzo Nate;

4) (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-히드록시페닐)아크릴레이트;4) (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-(4- Hydroxyphenyl)acrylate;

5) (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-히드록시-3-메톡시페닐)아크릴레이트;5) (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-(4- Hydroxy-3-methoxyphenyl)acrylate;

6) (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-(2-(디메틸아미노)에톡시)페닐)아클릴레이트;6) (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-(4- (2-(dimethylamino)ethoxy)phenyl)acrylate;

7) (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-히드록시-3-메톡시벤조네이트;7) (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulen-7-yl 4-hydroxy-3-methoxybenzoate;

8) (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-메톡시-4-피발로일옥시벤조네이트;8) (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulen-7-yl 3-methoxy-4-pivaloyloxybenzoate;

9) (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-피발로일옥시벤조네이트;9) (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulen-7-yl 4-pivaloyloxybenzoate;

10) (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-히드록시페닐)아크릴레이트;10) (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene -7-yl 3-(4-hydroxyphenyl)acrylate;

11) (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-히드록시-3-메톡시페닐)아크릴레이트;11) (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene -7-yl 3-(4-hydroxy-3-methoxyphenyl)acrylate;

12) (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-(2-(디메틸아미노)에톡시)페닐)아클릴레이트.12) (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene -7-yl 3-(4-(2-(dimethylamino)ethoxy)phenyl)acrylate.

또한, 본 발명은 하기 화학식 1, 화학식 2, 화학식 5, 화학식 7, 화학식 8, 화학식 11로 표시되는 화합물 또는 이의 광학 이성질체를 제공한다.In addition, the present invention provides a compound represented by Formula 1, Formula 2, Formula 5, Formula 7, Formula 8, and Formula 11, or an optical isomer thereof.

Figure pat00002
Figure pat00002

본 발명의 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체는 신경세포 보호효과(neuron protective effects) 및 신경세포자살사 억제효과(apoptosis inhibition effects)가 있으므로 파킨슨병(Parkinson disease) 예방 및 치료용 약학적 조성물로 유용하게 사용 될 수 있다.Since the compounds represented by Chemical Formulas 1 to 12 of the present invention or optical isomers thereof have neuroprotective effects and apoptosis inhibition effects, pharmaceuticals for the prevention and treatment of Parkinson disease It can be usefully used as an appropriate composition.

본 발명의 구체적인 일 실시예에서는, 세포독성 물질인 L-글루탐산 또는 6-히드록시도파민을 SH-SY5Y 세포에 처리하여 신경 독성을 유발시킨 다음 본 발명의 화합물을 처리한 결과 본 발명의 화합물이 신경세포 보호효과가 있음을 확인하였고(실험예 3-2 참조), 다른 일 실시예에서는 본 발명의 화합물이 신경세포 세포자살사 저해효과도 있음을 확인하였다(실험예 3-3 참조).In a specific embodiment of the present invention, a cytotoxic substance, L-glutamic acid or 6-hydroxydopamine, is treated on SH-SY5Y cells to induce neurotoxicity, and then the compound of the present invention is treated with the compound of the present invention. It was confirmed that there was a cell protective effect (see Experimental Example 3-2), and in another example, it was confirmed that the compound of the present invention also had an inhibitory effect on neuronal cell apoptosis (see Experimental Example 3-3).

신규 세스퀴테르펜 유도체 또는 이의 광학 이성질체를 포함하는 조성물, 이의 용도 및 이를 이용한 치료 방법Composition comprising novel sesquiterpene derivative or optical isomer thereof, use thereof, and treatment method using the same

본 발명은 하기 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상을 유효성분으로 포함하는 파킨슨병 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease, comprising as an active ingredient any one or more of a compound represented by the following Chemical Formulas 1 to 12 or optical isomers thereof.

Figure pat00003
Figure pat00003

본 발명에 있어서, 상기 약학적 조성물은 하기 화학식 1, 화학식 2, 화학식 5, 화학식 7, 화학식 8, 화학식 11로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상을 유효성분으로 포함하는 것일 수 있다.In the present invention, the pharmaceutical composition may include a compound represented by the following Formula 1, Formula 2, Formula 5, Formula 7, Formula 8, Formula 11, or any one or more of optical isomers thereof as an active ingredient.

Figure pat00004
Figure pat00004

본 발명에서 사용하는 용어 “파킨슨병(Parkinson disease)”은 뇌의 흑질(substantia nigra)에 분포하는 도파민의 신경세포가 점차 소실되어 발생하며 안정떨림, 경직, 운동완만 및 자세 불안정성이 특징적으로 나타나는 신경계의 만성 진행성 퇴행성 질환을 의미한다.The term “Parkinson disease” used in the present invention is caused by the gradual loss of dopamine neurons distributed in the substantia nigra of the brain, and is characterized by stable tremor, stiffness, locomotion, and postural instability. Refers to a chronic progressive degenerative disease.

본 발명에서 사용하는 용어, "예방"은 화합물 또는 조성물의 투여에 의해 질환의 발병을 억제시키거나 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any action that suppresses or delays the onset of a disease by administration of a compound or composition.

본 발명에서 사용하는 용어, "치료"는 화합물 또는 조성물의 투여에 의해 질환의 의심 및 발병 개체의 증상이 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" refers to any action in which symptoms of an individual suspected of and onset of a disease are improved or beneficially altered by administration of a compound or composition.

본 발명의 구체적인 일 실시예에서는, 세포독성 물질인 L-글루탐산 또는 6-히드록시도파민을 SH-SY5Y 세포에 처리하여 신경 독성을 유발시킨 다음 본 발명의 화합물을 처리한 결과 본 발명의 화합물이 신경세포 보호효과가 있음을 확인하였고(실험예 3-2), 다른 일 실시예에서는 본 발명의 화합물이 신경세포 세포자살사 저해효과도 있음을 확인하였다(실험예 3-3).In a specific embodiment of the present invention, a cytotoxic substance, L-glutamic acid or 6-hydroxydopamine, is treated on SH-SY5Y cells to induce neurotoxicity, and then the compound of the present invention is treated with the compound of the present invention. It was confirmed that there was a cell protective effect (Experimental Example 3-2), and in another example, it was confirmed that the compound of the present invention also had an effect of inhibiting neuronal cell suicide (Experimental Example 3-3).

본 발명의 약학적 조성물은 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상 외에 추가로 약학적으로 허용 가능한 담체를 1종 이상 포함할 수 있다. 약학적으로 허용 가능한 담체는 당업계에서 통상적으로 이용되는 것으로, 구체적으로 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알지네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리딘, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸 히드록시벤조네이트, 프로필 히드록시벤조네이트, 활석, 스테아르산 마그네슘, 미네랄, 또는 오일일 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 약학적 조성물들은 상기 성분들 외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제, 분산제, 안정화제 등을 추가로 포함할 수 있다. 또한, 본 발명의 약학적 조성물은 약학적으로 허용 가능한 담체 및 부형제를 이용하여 정제, 산제, 과립제, 환제, 캡슐제, 현탁액, 에멀젼, 내용액제, 유제, 시럽 등의 경구용 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제제화하여 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 제제는 당업계에서 제제화에 사용되는 통상의 방법 또는 Remington's Pharmaceutical Science(19th ed., 1995)에 개시되어 있는 방법으로 제조될 수 있으며, 각 질환 또는 성분에 따라 다양한 제제로 제제화될 수 있다.The pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to any one or more of the compounds represented by Chemical Formulas 1 to 12 or optical isomers thereof. Pharmaceutically acceptable carriers are commonly used in the art, and specifically lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidine, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral, or oil may be, but is not limited thereto. The pharmaceutical compositions of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, dispersing agents, stabilizers, and the like in addition to the above components. In addition, the pharmaceutical composition of the present invention uses pharmaceutically acceptable carriers and excipients to form tablets, powders, granules, pills, capsules, suspensions, emulsions, solutions, emulsions, syrups, and other oral formulations, external preparations, and suppositories. Alternatively, it may be formulated in the form of a sterile injectable solution and prepared in a unit dose form, or may be prepared by being placed in a multi-dose container. The formulation may be prepared by a conventional method used for formulation in the art or a method disclosed in Remington's Pharmaceutical Science (19th ed., 1995), and may be formulated into various formulations according to each disease or ingredient.

본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여(oral administration) 또는 비경구 투여(transdermal administration)할 수 있으며, 바람직하게는 경구 투여할 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method, and preferably may be administered orally, but is not limited thereto.

본 발명의 약학적 조성물의 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성별, 병적 상태, 식이섭취 종류, 투여 경로, 배설 속도, 및 반응 감응성과 같은 요인들에 따라 그 범위가 다양할 수 있다. 본 발명의 약학적 조성물의 바람직한 투여량은 성인 기준으로 0.01mg/kg 내지 1000mg/kg이며, 하루 일회 내지 수회에 나누어 투여할 수 있다.The dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, dietary intake type, administration route, excretion rate, and response sensitivity. can do. A preferred dosage of the pharmaceutical composition of the present invention is 0.01mg/kg to 1000mg/kg on an adult basis, and may be administered once to several times a day.

본 발명은 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는 파킨슨병 예방 또는 치료 방법을 제공한다.The present invention provides a method for preventing or treating Parkinson's disease comprising administering the pharmaceutical composition to an individual.

본 발명의 용어, "투여"는 적절한 방법으로 개체에게 소정의 물질을 도입하는 것을 의미한다.The term "administration" of the present invention means introducing a given substance to an individual in an appropriate manner.

본 발명의 용어, "개체"는 질환이 발병하였거나 발병할 수 있는 인간을 포함한 쥐, 생쥐, 가축 등의 모든 동물을 의미하며, 구체적으로 인간을 포함하는 포유동물일 수 있으나, 이에 제한되는 것은 아니다.The term "individual" of the present invention refers to all animals such as mice, mice, livestock, etc., including humans who have or may develop disease, and may specifically be mammals including humans, but is not limited thereto. .

본 발명의 파킨슨병 예방 또는 치료 방법은 상기 약학적 조성물을 약학적으로 유효한 양으로 투여하는 것일 수 있다.The method of preventing or treating Parkinson's disease of the present invention may be administering the pharmaceutical composition in a pharmaceutically effective amount.

본 발명의 용어, "약학적으로 유효한 양"이란 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 이는 환자의 성별, 연령, 체중, 건강상태, 질병의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로, 및 배출 비율, 치료 기간, 배합 또는 동시에 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 당업자에 의해 결정될 수 있다.As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, which is the sex, age, and weight of the patient. , Health status, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or simultaneously, and other medical fields. It can be determined by a person skilled in the art according to known factors.

본 발명은 하기 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상을 유효성분으로 포함하는 파킨슨병 예방 또는 개선용 의약외품 조성물을 제공한다.The present invention provides a quasi-drug composition for preventing or improving Parkinson's disease, comprising as an active ingredient any one or more of a compound represented by the following Chemical Formulas 1 to 12 or optical isomers thereof.

Figure pat00005
Figure pat00005

상기 “파킨슨병”, “예방” 등의 의미는 전술한 바와 같다.The meanings of “Parkinson's disease” and “prevention” are as described above.

본 발명의 용어, "개선"은 상기 조성물의 투여로 질환이 호전되거나 이롭게 변경되는 모든 행위를 의미한다.The term "improvement" of the present invention refers to any action in which a disease is improved or beneficially altered by administration of the composition.

본 발명에서 사용하는 용어 본 발명의 용어, "의약외품"은 사람이나 동물의 질병을 진단, 치료, 개선, 경감, 처치 또는 예방할 목적으로 사용되는 물품들 중 의약품보다 작용이 경미한 물품들을 의미한다. 예를 들어, 약사법에 따른 의약외품은 의약품의 용도로 사용되는 물품을 제외한 것으로, 사람/동물의 질병 치료나 예방에 쓰이는 제품, 인체에 대한 작용이 경미하거나 직접 작용하지 않는 제품 등이 포함된다.The term used in the present invention The term "quasi-drug" of the present invention refers to items that are less effective than medicines among items used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals. For example, quasi-drugs according to the Pharmaceutical Affairs Act exclude items used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases of humans/animals, and products that have mild or no direct action on the human body.

본 발명의 화합물 또는 조성물을 의약외품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 의약외품 또는 의약외품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합량은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.When the compound or composition of the present invention is used as a quasi-drug additive, the composition may be added as it is or used with other quasi-drug or quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

상기 의약외품 조성물은 특별히 이에 제한되지 않으나, 구체적으로 연고제, 로션제, 스프레이제, 패취제, 크림제, 산제, 현탁제, 겔제 또는 젤의 형태로 제조되어 사용될 수 있다.The quasi-drug composition is not particularly limited thereto, but may be specifically prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel.

본 발명은 하기 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상을 유효성분으로 포함하는, 파킨슨병(Parkinson disease) 예방 또는 개선용 식품 조성물을 제공한다.The present invention provides a food composition for preventing or improving Parkinson disease, comprising as an active ingredient any one or more of a compound represented by the following Chemical Formulas 1 to 12 or optical isomers thereof.

Figure pat00006
Figure pat00006

상기 “파킨슨병”, “예방”, “개선” 등의 의미는 전술한 바와 같다.The meanings of “Parkinson's disease”, “prevention”, and “improvement” are as described above.

본 발명의 용어, "식품"은 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료, 비타민 복합제, 건강 기능 식품, 건강 식품 및 건강 보조 식품 등이 있으며, 통상적인 의미에서의 식품을 모두 포함한다.As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , Vitamin complexes, health functional foods, health foods, and health supplements, and all foods in the usual sense are included.

상기 "건강기능(성)식품(functional food)"은 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미한다. 여기서 '기능(성)'이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다.The term "functional food" is the same term as food for special health use (FoSHU). In addition to supplying nutrients, the processed medicine and medical effects are effective so that the bioregulatory function is effectively displayed. Means high food. Here, the term'function (sex)' means to control nutrients for the structure and function of the human body or to obtain useful effects for health purposes such as physiological actions.

상기 "건강식품(health food)"은 일반식품에 비해 적극적인 건강유지나 증진 효과를 가지는 식품을 의미하고, "건강보조식품(health supplement food)"은 건강보조 목적의 식품을 의미한다. 경우에 따라, 건강기능식품, 건강식품, 건강보조식품의 용어는 혼용된다.The "health food" refers to a food having an active health maintenance or promotion effect compared to general food, and "health supplement food" refers to a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement food are used interchangeably.

본 발명의 식품은 당 업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당 업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 구체적으로, 단백질, 탄수화물, 지방, 영양소, 조미제, 및 향미제를 포함할 수 있으며, 상기 탄수화물의 예는 포도당, 과당, 말토스, 수크로스, 올리고당, 덱스트린, 사이클로덱스트린, 자일리톨, 소르비톨, 에리트롤, 사카린, 또는 합성 향미제가 있으나, 이에 제한되는 것은 아니다. 본 발명의 식품 조성물은 식품으로 인정되는 제형이면 제한 없이 다양한 형태의 제형으로 제조될 수 있다.The food product of the present invention can be prepared by a method commonly used in the art, and during the production, raw materials and ingredients commonly added in the art may be added to prepare the food product. Specifically, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents may be included, and examples of the carbohydrates are glucose, fructose, maltose, sucrose, oligosaccharide, dextrin, cyclodextrin, xylitol, sorbitol, ery Troll, saccharin, or synthetic flavoring agents, but are not limited thereto. The food composition of the present invention may be prepared in a variety of forms without limitation as long as it is a formulation recognized as a food.

본 발명의 신규 세스퀴테르펜 유도체 또는 이의 광학 이성질체는 신경세포 보호효과(neuron protective effects) 및 신경세포자살사 억제효과(apoptosis inhibition effects)가 뛰어나므로 파킨슨병의 예방, 개선 및 치료에 유용하게 사용 될 수 있다.The novel sesquiterpene derivatives or optical isomers thereof of the present invention are excellent in neuroprotective effects and apoptosis inhibition effects, so they can be usefully used in the prevention, improvement and treatment of Parkinson's disease. have.

도 1 내지 도 4는 화학식 1 내지 12로 표시되는 화합물을 다양한 농도로 SH-SY5Y 세포에 처리하여 세포생존율을 측정한 결과이다.
도 5 내지 도 10은 다양한 농도의 본 발명 화합물이 전 처리된 SH-SY5Y 세포에 L-글루탐산을 처리한 경우 세포생존율 증가를 확인한 것이다.
도 11 내지 도 16은 다양한 농도의 본 발명 화합물이 전 처리된 SH-SY5Y 세포에 6-히드록시도파민을 처리한 경우 세포생존율 증가를 확인한 것이다.
도 17은 다양한 농도의 본 발명의 화합물이 전 처리된 SH-SY5Y 세포에 L-글루탐산을 처리한 경우 세포자살사 억제효과를 나타낸 것이다.
도 18은 다양한 농도의 본 발명의 화합물이 전 처리된 SH-SY5Y 세포에 6-히드록시도파민을 처리한 경우 세포자살사 억제효과를 나타낸 것이다.1 to 4 are results of measuring cell viability by treating SH-SY5Y cells with various concentrations of compounds represented by Chemical Formulas 1 to 12.
5 to 10 show the increase in cell viability when the SH-SY5Y cells pre-treated with various concentrations of the compound of the present invention were treated with L-glutamic acid.
11 to 16 show the increase in cell viability when 6-hydroxydopamine was treated in SH-SY5Y cells pre-treated with various concentrations of the compound of the present invention.
17 shows the inhibitory effect of apoptosis when the compound of the present invention of various concentrations was treated with L-glutamic acid to pre-treated SH-SY5Y cells.
18 shows the inhibitory effect of apoptosis when 6-hydroxydopamine was treated in SH-SY5Y cells pre-treated with various concentrations of the compound of the present invention.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

한편, 하기 본 발명의 화합물 제조를 위한 출발물질은 특별한 언급이 없는 한 중국 J&H Chem 에서 구입하여 사용하였다.On the other hand, the starting materials for the preparation of the compounds of the present invention were purchased and used from J&H Chem in China unless otherwise noted.

실시예 1: (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-히드록시-3-메톡시벤조네이트의 제조: [화합물 1]Example 1: (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-4-hydroxy-3- Preparation of methoxybenzoate: [Compound 1]

[화학식 1][Formula 1]

Figure pat00007
Figure pat00007

(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-올 13.3g (1.2equiv.) 과 4-히드록시-3-메톡시벤조산 8.4g (0.05mol, 1equiv.)을 디메틸아세트아미드 250mL에 넣고 교반하여 용해하였다. 상기 용액에 탄산칼륨 103.6g (1.5equiv.), 4-톨루엔설포닐 클로라이드 9.5g (1.2equiv.) 과 4-디메틸아미노피리딘 0.9g (15mol%)을 순차적으로 넣고 실온에서 2시간 교반하였다. 그 다음 에틸아세테이트 550mL, 염화암모늄 53.6g과 정제수 600mL의 혼합액을 가하고 30분간 교반하였다. 그 후 유기층을 모으고 무수황산마그네슘으로 건조 후 여과하고 감압 농축하여 표지의 화합물 (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-히드록시-3-메톡시벤조네이트 13.4g (72%)을 얻었다.(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulene-6-ol 13.3g (1.2equiv.) and 4-hydroxy 8.4 g (0.05 mol, 1equiv.) of -3-methoxybenzoic acid was added to 250 mL of dimethylacetamide and stirred to dissolve. Potassium carbonate 103.6g (1.5equiv.), 4-toluenesulfonyl chloride 9.5g (1.2equiv.) and 4-dimethylaminopyridine 0.9g (15mol%) were sequentially added to the solution and stirred at room temperature for 2 hours. Then, a mixture of 550 mL of ethyl acetate, 53.6 g of ammonium chloride and 600 mL of purified water was added, followed by stirring for 30 minutes. Thereafter, the organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure, and the labeled compound (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7- Methanoazulen-6-yl-4-hydroxy-3-methoxybenzoate 13.4g (72%) was obtained.

1H-NMR (400 MHz, CDCl3) δ 0.99(m, 9H), 1.2-1.3(m, 4H), 1.46(m, 2H), 1.48(s, 3H), 1.56(m, 1H), 1.66(m, 3H), 1.72(m, 1H), 1.95(m, 2H), 3.83(s, 3H), 5.35(s, 1H), 7.15(d, 1H, J=7.5), 7.45(s, 1H), 7.46(d, 1H, J=7.5). IR(KBr) 3440, 2995,　1710,　1645,　1420,　1391,　1250,　1165,　760cm-1．MS(ESI) m/z (M+1), 372.23 (100.0%), 373.23 (25.0%), 374.24 (3.1%). 1 H-NMR (400 MHz, CDCl 3 ) δ 0.99 (m, 9H), 1.2-1.3 (m, 4H), 1.46 (m, 2H), 1.48 (s, 3H), 1.56 (m, 1H), 1.66 (m, 3H), 1.72(m, 1H), 1.95(m, 2H), 3.83(s, 3H), 5.35(s, 1H), 7.15(d, 1H, J=7.5), 7.45(s, 1H) ), 7.46 (d, 1H, J=7.5). IR(KBr) 3440, 2995, 1710, 1645, 1420, 1391, 1250, 1165, 760cm -1 ．MS(ESI) m/z (M+1), 372.23 (100.0%), 373.23 (25.0%), 374.24 (3.1%).

실시예 2: (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-메톡시-4-피발로일옥시벤조네이트의 제조: [화합물 2]Example 2: (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-methoxy-4- Preparation of pivaloyloxybenzoate: [Compound 2]

[화학식 2][Formula 2]

Figure pat00008
Figure pat00008

디클로로메탄 250mL에 실시예 1에서 제조한 (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-히드록시-3-메톡시벤조네이트 18.6g (0.05mol, 1equiv.)을 넣고 교반하였다. 상기 용액에 4-디메틸아미노피리딘 0.9g (15mol%), 피발로일 클라라이드 7.2g (1.2equiv.), 트리에틸아민 10.5mL (1.5equiv.)를 순차적으로 넣고 실온에서 4시간 교반하였다. 반응이 끝난 후 차가운 정제수 600mL을 넣고 30분간 교반하였다. 그 다음 교반을 정지하여 층 분리후 유기층을 모으고 수층을 디클로로메탄 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine), 포화수소탄산나트륨 용액을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압 농축하여 조결정 화합물을 얻고 핵산을 가하여 재결정 하여 표지의 화합물 (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-메톡시-4-피발로일옥시벤조네이트 16.4g (72%)을 얻었다.(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-4 prepared in Example 1 in 250 mL of dichloromethane -Hydroxy-3-methoxybenzoate 18.6g (0.05mol, 1equiv.) was added and stirred. To the solution, 0.9g (15mol%) of 4-dimethylaminopyridine, 7.2g (1.2equiv.) of pivaloyl claride, and 10.5mL (1.5equiv.) of triethylamine were sequentially added and stirred at room temperature for 4 hours. After the reaction was over, 600 mL of cold purified water was added and stirred for 30 minutes. Then, the stirring was stopped, the layers were separated, and the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of dichloromethane. The organic layer was washed with water, brine, and saturated sodium hydrogencarbonate solution, respectively, and then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude compound, and then recrystallized by adding nucleic acids to the labeled compounds (3R, 3aS, 6R, 7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-methoxy-4-pivaloyloxybenzoate 16.4 g (72% ).

1H-NMR (400 MHz, CDCl3) δ 0.99(m, 9H), 1.2-1.3(m, 4H), 1.23 (s, 9H), 1.46(m, 2H), 1.48(s, 3H), 1.56(m, 1H), 1.66(m, 3H), 1.72(m, 1H), 1.95(m, 2H), 3.83(s, 3H), 7.29(d, 1H, J=7.5), 7.59 (s, 1H), 7.62(d, 1H, J=7.5). IR(KBr) 2928, 2875, 1695, 1640, 1466，1379,　1251,　1165,　726cm-1．MS(ESI) m/z (M+1) 456.29 (100.0%), 457.29 (30.9%), 458.29 (5.4%). 1 H-NMR (400 MHz, CDCl 3 ) δ 0.99 (m, 9H), 1.2-1.3 (m, 4H), 1.23 (s, 9H), 1.46 (m, 2H), 1.48 (s, 3H), 1.56 (m, 1H), 1.66 (m, 3H), 1.72 (m, 1H), 1.95 (m, 2H), 3.83 (s, 3H), 7.29 (d, 1H, J=7.5), 7.59 (s, 1H ), 7.62 (d, 1H, J=7.5). IR(KBr) 2928, 2875, 1695, 1640, 1466, 1379, 1251, 1165, 726cm -1 ．MS(ESI) m/z (M+1) 456.29 (100.0%), 457.29 (30.9%), 458.29 ( 5.4%).

실시예 3: (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-(피발로일옥시)벤조네이트의 제조: [화합물 3]Example 3: (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulene-6-yl-4-(pivaloyloxy ) Preparation of benzoate: [Compound 3]

실시예 3-1: (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-히드록시벤조네이트의 제조Example 3-1: (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-4-hydroxybenzo Preparation of Nate

Figure pat00009
Figure pat00009

(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-올 13.3g (1.2equiv.)과 4-히드록시벤조산 6.9g (0.05mol, 1equiv.)을 디메틸아세트아미드 250mL에 넣고 교반하여 용해하였다. 상기 용액에 탄산칼륨 103.6g (1.5equiv.), 4-톨루엔설포닐 클로라이드 9.5g (1.2equiv.)과 4-디메틸아미노피리딘 0.9g (15mol%)을 순차적으로 넣고 실온에서 2시간 교반하였다. 에틸아세테이트 550mL, 염화암모늄 53.6g과 정제수 600mL의 혼합액을 가하고 30분간 교반하였다. 유기층을 모으고 무수황산마그네슘으로 건조 후 여과하고 감압 농축하여 표지의 화합물 (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-히드록시벤조네이트 10.8g (63%)을 얻었다.(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulene-6-ol 13.3g (1.2equiv.) and 4-hydroxyl 6.9 g (0.05 mol, 1equiv.) of benzoic acid was added to 250 mL of dimethylacetamide and stirred to dissolve. Potassium carbonate 103.6g (1.5equiv.), 4-toluenesulfonyl chloride 9.5g (1.2equiv.) and 4-dimethylaminopyridine 0.9g (15mol%) were sequentially added to the solution and stirred at room temperature for 2 hours. A mixture of 550 mL of ethyl acetate, 53.6 g of ammonium chloride and 600 mL of purified water was added, followed by stirring for 30 minutes. The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the labeled compound (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoa 10.8 g (63%) of julen-6-yl-4-hydroxybenzoate were obtained.

1H-NMR(400 MHz, CDCl3) δ 0.99(m, 9H), 1.2-1.3(m, 4H), 1.23 (s, 9H), 1.46(m, 2H), 1.48(s, 3H), 1.56(m, 1H), 1.66(m, 3H), 1.72(m, 1H), 1.95(m, 2H), 5.35(s, 1H), 6.81(d, 2H, J=7.5), 7.90(d, 2H, J=7.5). IR(KBr) 3560, 2983, 1647, 1519, 1468, 1221, 767cm-1. MS(ESI) m/z(M+1) 342.22(100.0%), 343.22(23.9%), 344.23(2.8%). 1 H-NMR (400 MHz, CDCl 3 ) δ 0.99 (m, 9H), 1.2-1.3 (m, 4H), 1.23 (s, 9H), 1.46 (m, 2H), 1.48 (s, 3H), 1.56 (m, 1H), 1.66 (m, 3H), 1.72 (m, 1H), 1.95 (m, 2H), 5.35 (s, 1H), 6.81 (d, 2H, J=7.5), 7.90 (d, 2H) , J=7.5). IR(KBr) 3560, 2983, 1647, 1519, 1468, 1221, 767cm -1 . MS (ESI) m/z (M+1) 342.22 (100.0%), 343.22 (23.9%), 344.23 (2.8%).

실시예 3-2: (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-(피발로일옥시)벤조네이트의 제조: [화합물 3]Example 3-2: (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulene-6-yl-4-(pivalo Preparation of monooxy)benzoate: [Compound 3]

[화학식 3][Formula 3]

Figure pat00010
Figure pat00010

디클로로메탄 250mL에 실시예 3-1에서 제조한 (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-히드록시-벤조네이트 17.1g(0.05mol, 1equiv.)을 넣고 교반하였다. 상기 용액에 4-디메틸아미노피리딘 0.9g(15mol%), 피발로일 클라라이드 7.2g(1.2equiv.), 트리에틸아민 10.5mL(1.5equiv.) 를 순차적으로 넣고 실온에서 4시간 교반하였다. 반응이 끝나면 차가운 정제수 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층 분리후 유기층을 모으고 수층을 디클로로메탄 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine), 포화수소탄산나트륨 용액을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압 농축하여 조결정 화합물을 얻고 핵산을 가하여 재결정 하여 표지의 화합물 (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-4-(피발로일옥시)벤조네이트 16.2g (76%)을 얻었다.(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl prepared in Example 3-1 in 250 mL of dichloromethane 17.1 g (0.05 mol, 1equiv.) of -4-hydroxy-benzoate was added and stirred. To the solution, 0.9g (15mol%) of 4-dimethylaminopyridine, 7.2g (1.2equiv.) of pivaloyl claride, and 10.5mL (1.5equiv.) of triethylamine were sequentially added and stirred at room temperature for 4 hours. Upon completion of the reaction, 600 mL of cold purified water was added and stirred for 30 minutes. Stirring was stopped, the layers were separated, the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of dichloromethane. The organic layer was washed with water, brine, and saturated sodium hydrogencarbonate solution, respectively, and then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude compound, and then recrystallized by adding nucleic acids to the labeled compounds (3R, 3aS, 6R, 7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-4-(pivaloyloxy)benzoate 16.2g (76%) was obtained. .

1H-NMR (400 MHz, CDCl3) δ 0.99(m, 9H), 1.2-1.3(m, 4H), 1.23 (s, 9H), 1.46(m, 2H), 1.48(s, 3H), 1.56(m, 1H), 1.66(m, 3H), 1.72(m, 1H), 1.95(m, 2H), 7.40(d, 2H, J=7.5), 8.04(d, 2H, J=7.5). IR(KBr) 2959, 2928, 1692, 1640, 1463，1378,　1225,　1048,　760cm-1．MS(ESI) m/z (M+1) 426.28 (100.0%), 427.28 (29.8%), 428.28 (5.0%). 1 H-NMR (400 MHz, CDCl 3 ) δ 0.99 (m, 9H), 1.2-1.3 (m, 4H), 1.23 (s, 9H), 1.46 (m, 2H), 1.48 (s, 3H), 1.56 (m, 1H), 1.66 (m, 3H), 1.72 (m, 1H), 1.95 (m, 2H), 7.40 (d, 2H, J=7.5), 8.04 (d, 2H, J=7.5). IR(KBr) 2959, 2928, 1692, 1640, 1463, 1378, 1225, 1048, 760cm -1 ．MS(ESI) m/z (M+1) 426.28 (100.0%), 427.28 (29.8%), 428.28 ( 5.0%).

실시예 4: (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-히드록시페닐)아크릴레이트의 제조: [화합물 4]Example 4: (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-( Preparation of 4-hydroxyphenyl)acrylate: [Compound 4]

[화학식 4][Formula 4]

Figure pat00011
Figure pat00011

(E)-3-(4-히드록시페닐)아크릴산 16.4g(0.1mol, 1.2equiv.)과 (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-올 26.7g(1.2equiv.) 을 테트라히드로푸란 500ml에 넣고 교반하여 용해하였다. 상기 용액에 4mL 황산을 넣고 30분간 교반 한 후 30g 황산마그네슘을 넣고 반응액을 가열하여 4시간 환류하였다. 반응이 종료되면 감압 농축하여 테트라히드로푸란을 제거하고 에틸아세테이트 550mL와 포화수소탄산나트륨 용액 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층 분리 후 유기층을 모으고 수층을 에틸아세테이트 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine)을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압 농축하여 조결정(Crude) 화합물을 얻었다. 조결정 화학물은 컬럼 크로마토그래피로(에틸아세테이트: 핵산=1:5) 정제하여 표지의 화합물 (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-히드록시페닐)아크릴레이트 23.6g (64%)을 얻었다.(E)-3-(4-hydroxyphenyl)acrylic acid 16.4 g (0.1 mol, 1.2equiv.) and (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro- 26.7 g (1.2equiv.) of 1H-3a,7-methanoazulene-6-ol was added to 500 ml of tetrahydrofuran and stirred to dissolve. 4 mL sulfuric acid was added to the solution, stirred for 30 minutes, 30 g magnesium sulfate was added, and the reaction solution was heated to reflux for 4 hours. When the reaction was completed, the mixture was concentrated under reduced pressure to remove tetrahydrofuran, and 550 mL of ethyl acetate and 600 mL of saturated sodium hydrogencarbonate solution were added, followed by stirring for 30 minutes. After the stirring was stopped, the layers were separated, the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of ethyl acetate. The organic layer was washed with water and brine, respectively, then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude (Crude) compound. The crude crystal was purified by column chromatography (ethyl acetate: nucleic acid = 1:5) and labeled compound (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyl Octahydro-1H-3a,7-methanoazulen-6-yl-3-(4-hydroxyphenyl)acrylate 23.6g (64%) was obtained.

1H-NMR (400 MHz, CDCl3) δ 0.99(m, 9H), 1.2-1.3(m, 4H), 1.46(m, 2H), 1.48(s, 3H), 1.56(m, 2H), 1.66(m, 3H), 1.72(m, 1H), 1.99(m, 1H), 5.35(s, 1H), 6.30(d, 1H, J=15.1), 6.65(d, 2H, J=7.5), 7.48(d, 1H, J=15.1), 7.56(d, 2H, J=7.5). IR(KBr) 3560, 2959, 1692, 1640, 1520, 1463,　1225,　760cm-1．MS(ESI) m/z (M+1) 368.24 (100.0%), 369.24 (26.4%), 370.24 (4.0%). 1 H-NMR (400 MHz, CDCl 3 ) δ 0.99 (m, 9H), 1.2-1.3 (m, 4H), 1.46 (m, 2H), 1.48 (s, 3H), 1.56 (m, 2H), 1.66 (m, 3H), 1.72(m, 1H), 1.99(m, 1H), 5.35(s, 1H), 6.30(d, 1H, J=15.1), 6.65(d, 2H, J=7.5), 7.48 (d, 1H, J=15.1), 7.56 (d, 2H, J=7.5). IR(KBr) 3560, 2959, 1692, 1640, 1520, 1463, 1225, 760cm -1 ．MS(ESI) m/z (M+1) 368.24 (100.0%), 369.24 (26.4%), 370.24 (4.0%) ).

실시예 5: (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-히드록시-3-메톡시페닐)아크릴레이트의 제조: [화합물 5]Example 5: (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-( Preparation of 4-hydroxy-3-methoxyphenyl)acrylate: [Compound 5]

[화학식 5][Formula 5]

Figure pat00012
Figure pat00012

(E)-3-(4-히드록시-3-메톡시페닐)아크릴산 9.7g(0.05mol, 1equiv.)과 (3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-올 13.3g(1.2equiv.)을 테트라히드로푸란 250ml에 넣고 교반하여 용해하였다. 상기 용액에 2mL 황산을 넣고 30분간 교반 한 후 15g 황산마그네슘을 넣고 반응액을 가열하여 6시간 환류하였다. 반응이 종료되면 감압 농축하여 테트라히드로푸란을 제거하고 에틸아세테이트 550mL와 포화수소탄산나트륨 용액 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층 분리후 유기층을 모으고 수층을 에틸아세테이트 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine)을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압 농축하여 조결정(Crude) 화합물을 얻었다. 조결정 화학물은 컬럼 크로마토그래피로(에틸아세테이트: 핵산=1:10) 정제하여 표지의 화합물 (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-히드록시-3-메톡시페닐)아크릴레이트 13.4g (67%)을 얻었다.(E)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid 9.7 g (0.05 mol, 1equiv.) and (3R,3aS,6R,7R,8aS)-3,6,8,8-tetra Methyloctahydro-1H-3a,7-methanoazulene-6-ol 13.3g (1.2equiv.) was added to 250ml of tetrahydrofuran and stirred to dissolve. 2 mL sulfuric acid was added to the solution, stirred for 30 minutes, 15 g magnesium sulfate was added, and the reaction solution was heated to reflux for 6 hours. When the reaction was completed, the mixture was concentrated under reduced pressure to remove tetrahydrofuran, and 550 mL of ethyl acetate and 600 mL of saturated sodium hydrogencarbonate solution were added, followed by stirring for 30 minutes. Stirring was stopped, the layers were separated, the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of ethyl acetate. The organic layer was washed with water and brine, respectively, then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude (Crude) compound. The crude crystal was purified by column chromatography (ethyl acetate: nucleic acid = 1:10), and the labeled compound (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyl Octahydro-1H-3a,7-methanoazulen-6-yl-3-(4-hydroxy-3-methoxyphenyl)acrylate 13.4g (67%) was obtained.

1H-NMR (400 MHz, CDCl3) δ 0.99(m, 9H), 1.2-1.3(m, 4H), 1.46(m, 2H), 1.48(s, 3H), 1.56(m, 2H), 1.66(m, 3H), 1.72(m, 1H), 1.99(m, 1H), 3.82(s, 3H), 5.35(s, 1H), 6.30(d, 1H, J=15.1), 6.79(d, 1H, J=7.5), 6.99 (d, 1H, J=7.5), 7.16(s, 1H), 7.48(d, 1H, J=15.1). IR(KBr) 3550, 2979, 1640, 1522, 1466,　1223,　760cm-1．MS(ESI) m/z (M+1) 398.25 (100.0%), 399.25 (27.6%), 400.25 (4.4%). 1 H-NMR (400 MHz, CDCl 3 ) δ 0.99 (m, 9H), 1.2-1.3 (m, 4H), 1.46 (m, 2H), 1.48 (s, 3H), 1.56 (m, 2H), 1.66 (m, 3H), 1.72(m, 1H), 1.99(m, 1H), 3.82(s, 3H), 5.35(s, 1H), 6.30(d, 1H, J=15.1), 6.79(d, 1H) , J=7.5), 6.99 (d, 1H, J=7.5), 7.16 (s, 1H), 7.48 (d, 1H, J=15.1). IR(KBr) 3550, 2979, 1640, 1522, 1466, 1223, 760cm -1 ．MS(ESI) m/z (M+1) 398.25 (100.0%), 399.25 (27.6%), 400.25 (4.4%).

실시예 6: (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-(2-(디메틸아미노)에톡시)페닐)아클릴레이트의 제조: [화합물 6]Example 6: (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-( Preparation of 4-(2-(dimethylamino)ethoxy)phenyl)acrylate: [Compound 6]

[화학식 6][Formula 6]

Figure pat00013
Figure pat00013

디클로로메탄 250mL에 실시예 4에서 제조한 (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-히드록시페닐)아크릴레이트 18.4 g(0.05mol, 1equiv.)을 넣고 교반하였다. 상기 용액에 4-디메틸아미노피리딘 0.9g(15mol%), 2-클로로-N,N-디메틸에텐아민 6.5g(1.2equiv.), 트리에틸아민 10.5mL(1.5equiv.) 를 순차적으로 넣고 실온에서 3시간 교반하였다. 반응이 끝나면 차가운 정제수 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층 분리후 유기층을 모으고 수층을 디클로로메탄 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine), 포화수소탄산나트륨 용액을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압 농축하여 조결정 화합물을 얻고 핵산을 가하여 재결정 하여 표지의 화합물 (E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-테트라메틸옥타히드로-1H-3a,7-메타노아줄렌-6-일-3-(4-(2-(디메틸아미노)에톡시)페닐)아클릴레이트 16.7g (76%)을 얻었다.(E)-(3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulene-6 prepared in Example 4 in 250 mL of dichloromethane -Yl-3-(4-hydroxyphenyl)acrylate 18.4 g (0.05 mol, 1equiv.) was added and stirred. To the above solution, 0.9 g (15 mol%) of 4-dimethylaminopyridine, 6.5 g (1.2equiv.) of 2-chloro-N,N-dimethylethenamine, and 10.5 mL (1.5equiv.) of triethylamine were sequentially added to the solution at room temperature. The mixture was stirred for 3 hours. Upon completion of the reaction, 600 mL of cold purified water was added and stirred for 30 minutes. Stirring was stopped, the layers were separated, the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of dichloromethane. The organic layer was washed with water, brine, and saturated sodium hydrogencarbonate solution, respectively, and then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude compound, and then recrystallized by adding nucleic acid to the labeled compound (E)-(3R, 3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl-3-(4-(2-(dimethylamino)ethoxy )Phenyl)acrylate 16.7g (76%) was obtained.

1H-NMR (400 MHz, CDCl3) δ 0.99(m, 9H), 1.2-1.3(m, 4H), 1.46(m, 2H), 1.48(s, 3H), 1.56(m, 2H), 1.66(m, 3H), 1.72(m, 1H), 1.99(m, 1H), 2.76(t, 2H, J=7.1), 2.82(s, 6H) 4.11(t, 2H, J=7.1), 6.31(d, 1H, J=15.1), 6.94(d, 2H,J=7.5), 7.48 (d, 1H, J=15.1) 7.62(d, 2H, J=7.5). IR(KBr) 2993, 1647, 1521, 1468, 1221, 767cm-1．MS(ESI) m/z (M+1) 439.31 (100.0%), 440.31 (31.2%), 441.32 (4.6%). 1 H-NMR (400 MHz, CDCl 3 ) δ 0.99 (m, 9H), 1.2-1.3 (m, 4H), 1.46 (m, 2H), 1.48 (s, 3H), 1.56 (m, 2H), 1.66 (m, 3H), 1.72(m, 1H), 1.99(m, 1H), 2.76(t, 2H, J=7.1), 2.82(s, 6H) 4.11(t, 2H, J=7.1), 6.31( d, 1H, J=15.1), 6.94 (d, 2H, J=7.5), 7.48 (d, 1H, J=15.1) 7.62 (d, 2H, J=7.5). IR(KBr) 2993, 1647, 1521, 1468, 1221, 767cm -1 ．MS(ESI) m/z (M+1) 439.31 (100.0%), 440.31 (31.2%), 441.32 (4.6%).

실시예 7: (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-히드록시-3-메톡시벤조네이트의 제조: [화합물 7]Example 7: (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene-7 Preparation of -yl 4-hydroxy-3-methoxybenzoate: [Compound 7]

[화학식 7][Formula 7]

Figure pat00014
Figure pat00014

(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-올 13.3g(1.2equiv.) 과 4-히드록시-3-메톡시벤조산 8.4g(0.05mol, 1equiv.)을 디메틸아세트아미드 250mL에 넣고 교반하여 용해하였다. 상기 용액에 탄산칼륨 103.6g(1.5equiv.), 4-톨루엔설포닐 클로라이드 9.5g(1.2equiv.)과 4-디메틸아미노피리딘 0.9g (15mol%)을 순차적으로 넣고 실온에서 4시간 교반하였다. 에틸아세테이트 550mL, 염화암모늄 53.6g과 정제수 600mL의 혼합액을 가하고 30분간 교반하였다. 유기층을 모으고 무수황산마그네슘으로 건조 후 여과하고 감압농축하여 표지의 화합물 (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-히드록시-3-메톡시벤조네이트 14.1g (76%)을 얻었다.(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene-7-ol 13.3 g (1.2equiv.) and 8.4 g (0.05 mol, 1equiv.) of 4-hydroxy-3-methoxybenzoic acid were added to 250 mL of dimethylacetamide and stirred to dissolve. Potassium carbonate 103.6g (1.5equiv.), 4-toluenesulfonyl chloride 9.5g (1.2equiv.) and 4-dimethylaminopyridine 0.9g (15mol%) were sequentially added to the solution and stirred at room temperature for 4 hours. A mixture of 550 mL of ethyl acetate, 53.6 g of ammonium chloride and 600 mL of purified water was added, followed by stirring for 30 minutes. The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and labeled compound (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8- Octahydro-1H-benzo[7]annulen-7-yl 4-hydroxy-3-methoxybenzoate 14.1 g (76%) was obtained.

1H-NMR(400 MHz, CDCl3) δ 1.13~1.38(m, 8H), 1.25(s, 6H), 1.30(s, 3H), 1.48(s, 3H), 1.54(m, 1H), 1.79(m, 1H), 2.21(m, 1H), 2.46(m, 1H), 3.83(m, 3H), 5.20(t, 1H), 5.35(s, 1H), 7.15(d, 1H, J=7.3), 7.45(m, 2H). IR(KBr) 3640, 2905,　1710,　1645,　1465, 1391, 1015,　917cm-1. MS(ESI) m/z(M+1), 372.23 (100.0%), 373.23 (25.0%), 374.24 (3.1%). 1 H-NMR (400 MHz, CDCl 3 ) δ 1.13-1.38 (m, 8H), 1.25 (s, 6H), 1.30 (s, 3H), 1.48 (s, 3H), 1.54 (m, 1H), 1.79 (m, 1H), 2.21(m, 1H), 2.46(m, 1H), 3.83(m, 3H), 5.20(t, 1H), 5.35(s, 1H), 7.15(d, 1H, J=7.3 ), 7.45 (m, 2H). IR(KBr) 3640, 2905, 1710, 1645, 1465, 1391, 1015, 917cm -1 . MS (ESI) m/z (M+1), 372.23 (100.0%), 373.23 (25.0%), 374.24 (3.1%).

실시예 8: (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-메톡시-4-피발로일옥시벤조네이트의 제조: [화합물 8]Example 8: (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene-7 Preparation of -yl 3-methoxy-4-pivaloyloxybenzoate: [Compound 8]

[화학식 8][Formula 8]

Figure pat00015
Figure pat00015

디클로로메탄 250mL에 실시예 7에서 제조한 (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-히드록시-3-메톡시벤조네이트 18.6g(0.05mol, 1equiv.)을 넣고 교반하였다. 상기 용액에 4-디메틸아미노피리딘 0.9g(15mol%), 피발로일 클로라이드 7.2g(1.2equiv.), 트리에틸아민 10.5mL(1.5equiv.)를 순차적으로 넣고 실온에서 6시간 교반하였다. 반응이 끝난 후 차가운 정제수 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층 분리 후 유기층을 모으고 수층을 디클로로메탄 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine), 포화수소탄산나트륨 용액을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압농축하여 조결정 화합물을 얻었다. 조결정 화합물은 핵산을 가하여 재결정하여 표지의 화합물 (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-메톡시-4-피발로일옥시벤조네이트 16.0g (70%)을 얻었다.(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[ prepared in Example 7 in 250 mL of dichloromethane. 7] Anulen-7-yl 4-hydroxy-3-methoxybenzoate 18.6g (0.05mol, 1equiv.) was added and stirred. To the solution, 0.9g (15mol%) of 4-dimethylaminopyridine, 7.2g (1.2equiv.) of pivaloyl chloride, and 10.5mL (1.5equiv.) of triethylamine were sequentially added and stirred at room temperature for 6 hours. After the reaction was over, 600 mL of cold purified water was added and stirred for 30 minutes. After the stirring was stopped, the layers were separated, and the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of dichloromethane. The organic layer was washed with water, brine, and saturated sodium hydrogencarbonate solution, respectively, and then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude compound. The crude crystal compound is recrystallized by adding nucleic acid to the labeled compound (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H- Benzo[7]annulen-7-yl 3-methoxy-4-pivaloyloxybenzoate 16.0 g (70%) were obtained.

1H-NMR(400 MHz, CDCl3) δ 1.13~1.38(m, 8H), 1.23(s, 9H), 1.25(s, 6H), 1.30(s, 3H), 1.48(s, 3H), 1.54(m, 1H), 1.79(m, 1H), 2.21(m, 1H), 2.46(m, 1H), 3.83(m, 3H), 5.20(t, 1H), 7.29 (d, 1H, J=7.3), 7.59(m, 2H). IR(KBr) 3444, 2910,　1712,　1654,　1450, 1394, 1003,　917cm-1. MS(ESI) m/z(M+1) 454.27 (100.0%), 455.28 (30.9%), 456.28 (5.6%). 1 H-NMR (400 MHz, CDCl 3 ) δ 1.13-1.38 (m, 8H), 1.23 (s, 9H), 1.25 (s, 6H), 1.30 (s, 3H), 1.48 (s, 3H), 1.54 (m, 1H), 1.79 (m, 1H), 2.21 (m, 1H), 2.46 (m, 1H), 3.83 (m, 3H), 5.20 (t, 1H), 7.29 (d, 1H, J=7.3 ), 7.59 (m, 2H). IR(KBr) 3444, 2910, 1712, 1654, 1450, 1394, 1003, 917cm -1 . MS (ESI) m/z (M+1) 454.27 (100.0%), 455.28 (30.9%), 456.28 (5.6%).

실시예 9: (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-피발로일옥시벤조네이트의 제조: [화합물 9]Example 9: (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene-7 Preparation of -yl 4-pivaloyloxybenzoate: [Compound 9]

실시예 9-1: (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-히드록시벤조네이트의 제조Example 9-1: (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene Preparation of -7-yl 4-hydroxybenzoate

Figure pat00016
Figure pat00016

(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-올 13.3g(1.2euiqv.)과 4-히드록시벤조산 6.9g(0.05mol, 1equiv.)을 디메틸아세트아미드 250mL에 넣고 교반하여 용해하였다. 상기 용액에 탄산칼륨 103.6g (1.5equiv.), 4-톨루엔설포닐 클로라이드 9.5g(1.2equiv.)과 4-디메틸아미노피리딘 0.9g(15mol%)을 순차적으로 넣고 실온에서 2.5시간 교반하였다. 에틸아세테이트 550mL, 염화암모늄 53.6g과 정제수 600mL의 혼합액을 가하고 30분간 교반하였다. 유기층을 모으고 무수황산마그네슘으로 건조 후 여과하고 감압농축하여 표지의 화합물 (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-히드록시벤조네이트 11.3g (66%)을 얻었다.(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene-7-ol 13.3 g (1.2euiqv.) and 6.9 g (0.05 mol, 1equiv.) of 4-hydroxybenzoic acid were added to 250 mL of dimethylacetamide and stirred to dissolve. Potassium carbonate 103.6g (1.5equiv.), 4-toluenesulfonyl chloride 9.5g (1.2equiv.) and 4-dimethylaminopyridine 0.9g (15mol%) were sequentially added to the solution and stirred at room temperature for 2.5 hours. A mixture of 550 mL of ethyl acetate, 53.6 g of ammonium chloride and 600 mL of purified water was added, followed by stirring for 30 minutes. The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and labeled compound (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8- Octahydro-1H-benzo[7]annulen-7-yl 4-hydroxybenzoate 11.3 g (66%) was obtained.

1H-NMR(400 MHz, CDCl3) δ 1.13~1.38(m, 8H), 1.25(s, 6H), 1.30(s, 3H), 1.48(s, 3H), 1.54(m, 1H), 1.79(m, 1H), 2.21(m, 1H), 2.46(m, 1H), 5.20(t, 1H), 5.35(s, 1H), 6.80(d, 2H, J=7.3), 7.90(d, 2H, J=7.3). IR(KBr) 3560, 2983, 1647, 1519, 1468, 1221, 767cm-1. MS(ESI) 342.22 (100.0%), 343.22 (23.9%), 344.23 (2.8%). 1 H-NMR (400 MHz, CDCl 3 ) δ 1.13-1.38 (m, 8H), 1.25 (s, 6H), 1.30 (s, 3H), 1.48 (s, 3H), 1.54 (m, 1H), 1.79 (m, 1H), 2.21(m, 1H), 2.46(m, 1H), 5.20(t, 1H), 5.35(s, 1H), 6.80(d, 2H, J=7.3), 7.90(d, 2H) , J=7.3). IR(KBr) 3560, 2983, 1647, 1519, 1468, 1221, 767cm -1 . MS (ESI) 342.22 (100.0%), 343.22 (23.9%), 344.23 (2.8%).

실시예 9-2: (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-피발로일옥시벤조네이트의 제조: [화합물 9]Example 9-2: (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene Preparation of -7-yl 4-pivaloyloxybenzoate: [Compound 9]

[화학식 9][Formula 9]

Figure pat00017
Figure pat00017

디클로로메탄 250mL에 실시예 9-1에서 제조한 (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-히드록시벤조네이트 17.1g(0.05mol, 1equiv.)을 넣고 교반하였다. 상기 용액에 4-디메틸아미노피리딘 0.9g(15mol%), 피발로일 클로라이드 7.2g(1.2equiv.), 트리에틸아민 10.5mL(1.5equiv.)를 순차적으로 넣고 실온에서 4시간 교반하였다. 반응이 끝나면 차가운 정제수 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층분리 후 유기층을 모으고 수층을 디클로로메탄 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine), 포화수소탄산나트륨 용액을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압농축하여 조결정 화합물을 얻었다. 조결정 화합물은 핵산을 가하여 재결정하여 표지의 화합물 (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 4-피발로일옥시벤조네이트 13.7g (64%)을 얻었다.(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H- prepared in Example 9-1 in 250 mL of dichloromethane Benzo[7]annulen-7-yl 4-hydroxybenzoate 17.1g (0.05mol, 1equiv.) was added and stirred. To the solution, 0.9g (15mol%) of 4-dimethylaminopyridine, 7.2g (1.2equiv.) of pivaloyl chloride, and 10.5mL (1.5equiv.) of triethylamine were sequentially added and stirred at room temperature for 4 hours. Upon completion of the reaction, 600 mL of cold purified water was added and stirred for 30 minutes. After the stirring was stopped, the layers were separated, and the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of dichloromethane. The organic layer was washed with water, brine, and saturated sodium hydrogencarbonate solution, respectively, and then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude compound. The crude crystal compound is recrystallized by adding nucleic acid to the labeled compound (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H- Benzo[7]annulen-7-yl 4-pivaloyloxybenzoate 13.7 g (64%) was obtained.

1H-NMR(400 MHz, CDCl3) δ 1.13~1.38(m, 8H), 1.23(s, 9H), 1.25(s, 6H), 1.30(s, 3H), 1.48(s, 3H), 1.54(m, 1H), 1.79(m, 1H), 2.21(m, 1H), 2.46(m, 1H), 5.20(t, 1H), 7.40 (d, 2H, J=7.3), 8.04(d, 2H, J=7.3). IR(KBr) 2959, 2928, 1692, 1640, 1463，1378,　1225,　1048,　760cm-1. MS(ESI) m/z(M+1) 426.28 (100.0%), 427.28 (29.8%), 428.28 (5.0%). 1 H-NMR (400 MHz, CDCl 3 ) δ 1.13-1.38 (m, 8H), 1.23 (s, 9H), 1.25 (s, 6H), 1.30 (s, 3H), 1.48 (s, 3H), 1.54 (m, 1H), 1.79 (m, 1H), 2.21 (m, 1H), 2.46 (m, 1H), 5.20 (t, 1H), 7.40 (d, 2H, J=7.3), 8.04 (d, 2H) , J=7.3). IR(KBr) 2959, 2928, 1692, 1640, 1463，1378, 1225, 1048, 760cm -1 . MS (ESI) m/z (M+1) 426.28 (100.0%), 427.28 (29.8%), 428.28 (5.0%).

실시예 10: (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-히드록시페닐)아크릴레이트의 제조: [화합물 10]Example 10: (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7] Preparation of annulene-7-yl 3-(4-hydroxyphenyl)acrylate: [Compound 10]

[화학식 10][Formula 10]

Figure pat00018
Figure pat00018

(E)-3-(4-히드록시페닐)아크릴산 16.4g(0.1mol, 1.2equiv.)과 (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-올 22.2g(1.0euiqv.) 을 테트라히드로푸란 500ml에 넣고 교반하여 용해하였다. 상기 용액에 황산 4mL를 넣고 30분간 교반한 후 황산마그네슘 30g을 넣고 반응액을 가열하여 3시간 환류하였다. 반응이 종료되면 감압농축하여 테트라히드로푸란을 제거하고 에틸아세테이트 550mL와 포화수소탄산나트륨 용액 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층분리 후 유기층을 모으고 수층을 에틸아세테이트 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine)을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압농축하여 조결정(Crude) 화합물을 얻었다. 조결정 화합물은 컬럼 크로마토그래피(에틸아세테이트: 핵산=1:6)로 정제하여 표지의 화합물 (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-히드록시페닐)아크릴레이트 26.9g (73%)을 얻었다.(E)-3-(4-hydroxyphenyl)acrylic acid 16.4 g (0.1 mol, 1.2equiv.) and (4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a, 22.2 g (1.0euiqv.) of 5,6,7,8-octahydro-1H-benzo[7]annulene-7-ol was added to 500 ml of tetrahydrofuran and stirred to dissolve. 4 mL of sulfuric acid was added to the solution, stirred for 30 minutes, 30 g of magnesium sulfate was added, and the reaction solution was heated to reflux for 3 hours. When the reaction was completed, the mixture was concentrated under reduced pressure to remove tetrahydrofuran, and 550 mL of ethyl acetate and 600 mL of saturated sodium hydrogencarbonate solution were added, followed by stirring for 30 minutes. After the stirring was stopped, the layers were separated, and the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of ethyl acetate. The organic layer was washed with water and brine, respectively, then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude (Crude) compound. The crude compound was purified by column chromatography (ethyl acetate: nucleic acid = 1:6) and labeled compound (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4, 26.9 g (73%) of 4a,5,6,7,8-octahydro-1H-benzo[7]annulen-7-yl 3-(4-hydroxyphenyl)acrylate were obtained.

1H-NMR(400 MHz, CDCl3) δ 1.13~1.38(m, 9H), 1.25(s, 6H), 1.30(s, 3H), 1.48(s, 3H), 1.62(m, 1H), 2.04(m, 1H), 2.29(m, 1H), 5.20(t, 1H), 5.35(s, 1H), 6.31(d, 1H, J=5.2), 6.65(d, 2H, J=7.3), 7.48(d, 1H, J=5.3), 7.56(d, 2H, J=7.3). IR(KBr) 3560, 2959, 1692, 1640, 1520, 1463,　1225,　760cm-1. MS(ESI) m/z(M+1) 368.24 (100.0%), 369.24 (26.4%), 370.24 (4.0%). 1 H-NMR (400 MHz, CDCl 3 ) δ 1.13-1.38 (m, 9H), 1.25 (s, 6H), 1.30 (s, 3H), 1.48 (s, 3H), 1.62 (m, 1H), 2.04 (m, 1H), 2.29(m, 1H), 5.20(t, 1H), 5.35(s, 1H), 6.31(d, 1H, J=5.2), 6.65(d, 2H, J=7.3), 7.48 (d, 1H, J=5.3), 7.56 (d, 2H, J=7.3). IR(KBr) 3560, 2959, 1692, 1640, 1520, 1463, 1225, 760cm -1 . MS (ESI) m/z (M+1) 368.24 (100.0%), 369.24 (26.4%), 370.24 (4.0%).

실시예 11: (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-히드록시-3-메톡시페닐)아크릴레이트의 제조: [화합물 11]Example 11: (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7] Preparation of annulen-7-yl 3-(4-hydroxy-3-methoxyphenyl)acrylate: [Compound 11]

[화학식 11][Formula 11]

Figure pat00019
Figure pat00019

(E)-3-(4-히드록시-3-메톡시페닐)아크릴산 9.7g(0.05mol, 1equiv.)과 (4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-올 13.3(1.2euiqv.)을 테트라히드로푸란 250ml에 넣고 교반하여 용해하였다. 상기 용액에 황산 2mL를 넣고 30분간 교반 한 후 황산마그네슘 15g을 넣고 반응액을 가열하여 4시간 환류하였다. 반응이 종료되면 감압농축하여 테트라히드로푸란을 제거하고 에틸아세테이트 550mL와 포화수소탄산나트륨 용액 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층 분리 후 유기층을 모으고 수층을 에틸아세테이트 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine)을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압 농축하여 조결정(Crude) 화합물을 얻었다. 조결정 화합물은 컬럼 크로마토그래피(에틸아세테이트: 핵산=1:6)로 정제하여 표지의 화합물 (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-히드록시-3-메톡시페닐)아크릴레이트 13.7g (69%)을 얻었다.(E)-3-(4-hydroxy-3-methoxyphenyl)acrylic acid 9.7 g (0.05 mol, 1equiv.) and (4aR,7R)-1,1,4a,7-tetramethyl-2,3, 4,4a,5,6,7,8-octahydro-1H-benzo[7]annulene-7-ol 13.3 (1.2euiqv.) was added to 250 ml of tetrahydrofuran and stirred to dissolve. 2 mL of sulfuric acid was added to the solution, stirred for 30 minutes, 15 g of magnesium sulfate was added, and the reaction solution was heated to reflux for 4 hours. When the reaction was completed, the mixture was concentrated under reduced pressure to remove tetrahydrofuran, and 550 mL of ethyl acetate and 600 mL of saturated sodium hydrogencarbonate solution were added, followed by stirring for 30 minutes. After the stirring was stopped, the layers were separated, the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of ethyl acetate. The organic layer was washed with water and brine, respectively, then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude (Crude) compound. The crude compound was purified by column chromatography (ethyl acetate: nucleic acid = 1:6), and the labeled compound (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4, 13.7 g (69%) of 4a,5,6,7,8-octahydro-1H-benzo[7]annulen-7-yl 3-(4-hydroxy-3-methoxyphenyl)acrylate were obtained.

1H-NMR(400 MHz, CDCl3) δ 1.13~1.38(m 9H), 1.25(s, 6H), 1.30(s, 3H), 1.48(s, 3H), 1.62(m, 1H), 2.04(m, 1H), 2.29(m, 1H), 3.83(s, 3H), 5.20(t, 1H), 5.35(s, 1H), 6.31(d, 1H, J=5.3), 6.79(d, 1H, J=7.3), 6.99 (d, 1H, J=7.3), 7.16(s, 1H), 7.48(d, 1H, J=5.3). IR(KBr) 3550, 2979, 1640, 1522, 1466,　1223,　760cm-1. MS(ESI) m/z(M+1) 398.25 (100.0%), 399.25 (27.6%), 400.25 (4.4%). 1 H-NMR (400 MHz, CDCl 3 ) δ 1.13-1.38 (m 9H), 1.25 (s, 6H), 1.30 (s, 3H), 1.48 (s, 3H), 1.62 (m, 1H), 2.04 ( m, 1H), 2.29(m, 1H), 3.83(s, 3H), 5.20(t, 1H), 5.35(s, 1H), 6.31(d, 1H, J=5.3), 6.79(d, 1H, J=7.3), 6.99 (d, 1H, J=7.3), 7.16 (s, 1H), 7.48 (d, 1H, J=5.3). IR(KBr) 3550, 2979, 1640, 1522, 1466, 1223, 760cm -1 . MS (ESI) m/z (M+1) 398.25 (100.0%), 399.25 (27.6%), 400.25 (4.4%).

실시예 12: (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-(2-(디메틸아미노)에톡시)페닐)아클릴레이트의 제조: [화합물 12]Example 12: (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro-1H-benzo[7] Preparation of annulen-7-yl 3-(4-(2-(dimethylamino)ethoxy)phenyl)acrylate: [Compound 12]

[화학식 12][Formula 12]

Figure pat00020
Figure pat00020

디클로로메탄 250mL에 실시예 10에서 제조한 (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-히드록시페닐)아크릴레이트 18.4g(0.05mol, 1equiv.)을 넣고 교반하였다. 상기 용액에 4-디메틸아미노피리딘 0.9g(15mol%), 2-클로로-N,N-디메틸에텐아민 6.5g(1.2equiv.), 트리에틸아민 10.5mL(1.5equiv.)를 순차적으로 넣고 실온에서 6시간 교반하였다. 반응이 끝나면 차가운 정제수 600mL을 넣고 30분간 교반하였다. 교반을 정지하여 층분리 후 유기층을 모으고 수층을 디클로로메탄 500ml로 2회 추출하였다. 유기층을 물과 브라인(Brine), 포화수소탄산나트륨 용액을 사용하여 각각 세척 후 황산나트륨을 사용하여 건조하고 용매를 감압농축하여 조결정 화합물을 얻었다. 조결정 화합물은 핵산을 가하여 재결정하여 표지의 화합물 (E)-(4aR,7R)-1,1,4a,7-테트라메틸-2,3,4,4a,5,6,7,8-옥타히드로-1H-벤조[7]아눌렌-7-일 3-(4-(2-(디메틸아미노)에톡시)페닐)아클릴레이트 14.5g (66%)을 얻었다.(E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octahydro- prepared in Example 10 in 250 mL of dichloromethane 1H-benzo[7]annulen-7-yl 3-(4-hydroxyphenyl)acrylate 18.4g (0.05mol, 1equiv.) was added and stirred. To the above solution, 0.9 g (15 mol%) of 4-dimethylaminopyridine, 6.5 g (1.2equiv.) of 2-chloro-N,N-dimethylethenamine, and 10.5 mL (1.5equiv.) of triethylamine were sequentially added to the solution at room temperature. The mixture was stirred for 6 hours. Upon completion of the reaction, 600 mL of cold purified water was added and stirred for 30 minutes. After the stirring was stopped, the layers were separated, and the organic layer was collected, and the aqueous layer was extracted twice with 500 ml of dichloromethane. The organic layer was washed with water, brine, and saturated sodium hydrogencarbonate solution, respectively, and then dried over sodium sulfate, and the solvent was concentrated under reduced pressure to obtain a crude compound. The crude crystal compound is recrystallized by adding nucleic acid to the labeled compound (E)-(4aR,7R)-1,1,4a,7-tetramethyl-2,3,4,4a,5,6,7,8-octa Hydro-1H-benzo[7]annulen-7-yl 3-(4-(2-(dimethylamino)ethoxy)phenyl)acrylate 14.5g (66%) was obtained.

1H-NMR (400 MHz, CDCl3) δ 1.13~1.38(m 9H), 1.25(s, 6H), 1.30(s, 3H), 1.48(s, 3H), 1.62(m, 1H), 2.04(m, 1H), 2.29(m, 1H), 2.76(t, 2H), 2.82(s, 6H), 4.11(t, 2H), 5.20(t, 1H),, 6.31(d, 1H, J=5.3), 6.94(d, 2H, J=7.5), 7.48 (d, 1H, J=5.3) 7.62(d, 2H, J=7.5). IR(KBr) 2993, 1647, 1521, 1468, 1221, 767cm-1. MS(ESI) m/z(M+1) 439.31 (100.0%), 440.31 (31.2%), 441.32 (4.6%). 1 H-NMR (400 MHz, CDCl 3 ) δ 1.13~1.38(m 9H), 1.25(s, 6H), 1.30(s, 3H), 1.48(s, 3H), 1.62(m, 1H), 2.04( m, 1H), 2.29(m, 1H), 2.76(t, 2H), 2.82(s, 6H), 4.11(t, 2H), 5.20(t, 1H),, 6.31(d, 1H, J=5.3 ), 6.94 (d, 2H, J=7.5), 7.48 (d, 1H, J=5.3) 7.62 (d, 2H, J=7.5). IR(KBr) 2993, 1647, 1521, 1468, 1221, 767cm -1 . MS (ESI) m/z (M+1) 439.31 (100.0%), 440.31 (31.2%), 441.32 (4.6%).

<실험예1> 신규 세스퀴테르펜 유도체 화합물의 물리화학적 특성 분석<Experimental Example 1> Analysis of physicochemical properties of novel sesquiterpene derivative compounds

상기 실시예 1 내지 12와 같이 합성한 신규 세스퀴테르펜 유도체 화합물 (화학식 1 내지 12로 표시되는 화합물)의 물리화학적 특성(chemico-physical properties)을 조사하였고 대조물질로 (+)-세드롤(cedrol)과 (+)-위드롤(widdrol)을 사용하여 상기 신규 합성한 화합물과 물리화학적 특성을 비교하였다. 실험항목으로 성상(appearance), 용해도(solubility for water), 인습성(hygroscopicity for humidity), 조해성(deliquesce for new material), 가속 6개월 안정성(accelerated chemical stabilities for 6 months) 및 열적안정성(behavior of thermal stability, 50±2℃ 24시간 처리) 등을 비교 평가하였다. The physico-physical properties of the novel sesquiterpene derivative compounds (compounds represented by Formulas 1 to 12) synthesized as in Examples 1 to 12 were investigated, and as a control material (+)-cedrol ) And (+)-widdrol were used to compare the physicochemical properties with the newly synthesized compound. Experimental items include appearance, solubility for water, hygroscopicity for humidity, deliquesce for new material, accelerated chemical stabilities for 6 months, and behavior of thermal. stability, 50±2°C, 24 hours treatment), etc. were compared and evaluated.

화합물 No.Compound No. 성상Appearance
(appearance)(appearance) 용해도Solubility
(mg/L)(mg/L) 인습성Conventionality
(hygroscopic)(hygroscopic) 조해성Deliquescent
(deliqueusce)(deliqueusce) 가속조건Acceleration condition
화학안정성(6개월)Chemical stability (6 months) 열안정성Thermal stability
(thermal behavior)(thermal behavior) 세드롤
(cedrol)Sedrol
(cedrol) 흰색분말White powder 2222 인습성 있음There is a custom RH 15~25%RH 15~25% 6~10% 불순물 발생6~10% impurity generation 약간 불안정Slightly unstable 위드롤(widdrol)Widdrol 흰색분말White powder 2020 인습성 있음There is a custom RH 17~22%RH 17~22% 8~10% 불순물 발생8~10% impurity generation 약간 불안정Slightly unstable 화학식1Formula 1 흰색결정White crystal 450450 인습성 없음No custom RH 5~12%RH 5~12% 0.1~0.2%0.1~0.2% 안정함Stable 화학식 2Formula 2 희색결정White crystal 480480 인습성 없음No custom RH 9~12%RH 9~12% 0.1~0.2%0.1~0.2% 안정함Stable 화학식 3Formula 3 흰색결정White crystal 380380 인습성 없음No custom RH 6~15%RH 6~15% 0.3~0.5%0.3~0.5% 안정함Stable 화학식 4Formula 4 연미색결정Light off-white crystal 350350 인습성 없음No custom RH 8~18%RH 8~18% <0.8%<0.8% 안정함Stable 화학식 5Formula 5 흰색분말White powder 510510 인습성 없음No custom RH 12~53%RH 12~53% 0.1~0.2%0.1~0.2% 안정함Stable 화학식 6Formula 6 회백색결정Grayish white crystal 466466 인습성 없음No custom RH 18~28%RH 18~28% 0.1~0.8%0.1~0.8% 안정함Stable 화학식 7Formula 7 회백색결정Grayish white crystal 440440 인습성 없음No custom RH 17~19%RH 17~19% 0.1~0.3%0.1~0.3% 안정함Stable 화학식 8Formula 8 연미색결정Light off-white crystal 450450 인습성 없음No custom RH 10-15%RH 10-15% 0.1~0.2%0.1~0.2% 안정함Stable 화학식 9Formula 9 흰색분말White powder 180180 인습성 없음No custom RH 20-26%RH 20-26% 0.9~1.2%0.9~1.2% 안정함Stable 화학식10Formula 10 흰색분말White powder 270270 인습성 없음No custom RH 24-27%RH 24-27% 0.8~1.5%0.8~1.5% 안정함Stable 화학식11Formula 11 흰색결정White crystal 470470 인습성 없음No custom RH 20-22%RH 20-22% 0.1~0.5%0.1~0.5% 안정함Stable 화학식12Formula 12 흰색결정White crystal 355355 인습성 없음No custom RH 28-30%RH 28-30% 0.8~1.5%0.8~1.5% 안정함Stable

상기 표 1에 나타난 바와 같이 신규 합성된 화합물 모두 대조물질로 사용된 기 공지된 천연추출물인 (+)-세드롤(cedrol)과 (+)-위드롤(widdrol)에 비해 우수한 물리화학적 특성을 보여 주었다. 상기 신규 화합물 모두 약제학적 중요한 인자(significant factors for new drug profile)인 물에 대한 우수한 용해도(water solubility) (450, 480, 450mg/L 대비 대조물질; 세드롤 22mg/L, 위드롤 20mg/L)를 보인다. 또한 실시예 1, 2, 5, 7, 8 및 11의 신규 화합물들은 신약개발시 또 다른 중요 인자(critical factors)인 유효기간과 보존성을 결정하는 물리화학적 안정성(physico-chemical stability for new drug)이 6개월 가속조건(상대습도(RH) 75%, 온도(temp.) 40℃)에서 종래 공지의 화합물보다 우수한 결과를 보여 주었다. As shown in Table 1, all of the newly synthesized compounds show superior physicochemical properties compared to the known natural extracts (+)-cedrol and (+)-widdrol, which are used as a control material. gave. All of the new compounds have excellent water solubility in water, which is a significant factor for new drug profile (450, 480, 450mg/L compared to the control material; Sedrol 22mg/L, withrol 20mg/L) Looks. In addition, the new compounds of Examples 1, 2, 5, 7, 8 and 11 have physico-chemical stability for new drugs that determine shelf life and preservability, which are other critical factors when developing new drugs. The results were superior to those of conventionally known compounds under 6 months acceleration conditions (relative humidity (RH) 75%, temperature (temp.) 40°C).

<실험예 2> 신규 세스퀴테르펜 유도체 화합물의 신경세포 독성 유발효과 측정<Experimental Example 2> Measurement of neurocytotoxic inducing effect of novel sesquiterpene derivative compounds

2-1: 신경세포주의 배양2-1: cultivation of neuronal cell lines

사람 유래 신경모세포종 SH-SY5Y 세포는 American Type culture collecteion, ATCC, USA로 부터 구입 분양 받아 사용하였으며 10% fetal bovine serum(FBS)및 페니실린 및 스트렙토마이신이 포함된 돌베코수정배지(DMEM)를 사용하여 37oC, 5% CO2 조건하에서 배양하여 사용하였다.Human-derived neuroblastoma SH-SY5Y cells were purchased from American Type culture collecteion, ATCC, USA, and were used. 10% fetal bovine serum (FBS) and dolbeco fertilization medium (DMEM) containing penicillin and streptomycin were used. It was used after culturing under conditions of 37 o C and 5% CO 2.

2-2: 신경세포 독성 유발여부 확인2-2: Confirmation of neural cell toxicity

상기 신규 세스퀴테르펜 유도체인 화학식 1 내지 12로 표시되는 화합물이 신경세포에 직접적인 독성을 유발 하는지 확인하기 위하여 Western assay method 이용하여 SH-SY5Y 세포의 생존력(viability)을 측정하였다. 이를 위해, SH-SY5Y 세포(5x104 cell/well)를 96-well plate에 분주하고 부착시킨 후, 0 내지 50μg/ml의 화학식 1 내지 12로 표시되는 화합물을 48시간 처리하였다. 배양이 끝난 후 SH-SY5Y 세포 배양액을 제거하고 Western assay method reagent(DeailLab, Korea)이 포함된 배지를 분주한 후 37oC에서 1시간 배양하였다. 이후 microplate reader(molecular Devices, USA)를 사용하여 450nm에서 흡광도를 측정하였다. 측정값은 3회 반복하여 평균값으로 나타내었다.The viability of SH-SY5Y cells was measured using a Western assay method to determine whether the compounds represented by Formulas 1 to 12, which are novel sesquiterpene derivatives, induce direct toxicity to neurons. To this end, SH-SY5Y cells (5x10 4 cells/well) were dispensed and attached to a 96-well plate, and then 0 to 50 μg/ml of compounds represented by Formulas 1 to 12 were treated for 48 hours. After the culture was completed, the SH-SY5Y cell culture medium was removed, and a medium containing a Western assay method reagent (DeailLab, Korea) was dispensed, followed by incubation at 37 o C for 1 hour. Then, the absorbance was measured at 450 nm using a microplate reader (molecular Devices, USA). The measured value was repeated three times and expressed as an average value.

그 결과 도 1 내지 도 4에 나타난 바와 같이 본 발명의 화합물은 세포독성이 낮게 나타났으며, 특히 [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 및 [화학식 11]로 표시되는 화합물은 타 화합물에 비해 동일 용량에서 세포독성이 더 적은 것으로 나타났다.As a result, as shown in Figs. 1 to 4, the compound of the present invention has low cytotoxicity, and in particular [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8] and The compound represented by [Formula 11] was found to have less cytotoxicity at the same dose than other compounds.

<실험예 3> 신규 세스퀴테르펜 유도체 화합물에 의한 파킨슨 질환 모델에서 치료효과 확인 <Experimental Example 3> Confirmation of therapeutic effect in Parkinson's disease model by novel sesquiterpene derivative compounds

3-1: 신경세포 독성 유발3-1: Induce neurocytotoxicity

SH-SY5Y 세포 5x106 cell/well 를 96-well plate에 분주하여 부착시켰으며 본 발명의 신규 세스퀴테르펜 유도체 화합물은 다양한 농도(0~5μg/ml)로 제조하였다. 상기 부착시킨 SH-SY5Y 세포에 독성을 유발시키기 위하여 L-글루탐산(monosodium salt hydrate, 75mM) 또는 6-히드록시도파민(6-hydroxydopamin, 6-HODP) 100μM을 37oC에서 48시간 처리하여 독성을 유발시켰다.SH-SY5Y cells 5×10 6 cells/well were dispensed and attached to a 96-well plate, and the novel sesquiterpene derivative compounds of the present invention were prepared in various concentrations (0-5 μg/ml). To induce toxicity to the adhered SH-SY5Y cells, 100 μM of L-glutamic acid (monosodium salt hydrate, 75 mM) or 6-hydroxydopamin (6-HODP) was treated at 37 o C for 48 hours to prevent toxicity. Triggered.

3-2: 신규 세스퀴테르펜 유도체 화합물에 의한 파킨슨 질환 모델에서 신경세포 보호효과 측정3-2: Measurement of neuronal protective effect in Parkinson's disease model by novel sesquiterpene derivative compounds

본 발명의 세스퀴테르펜 유도체 화합물이 세포 독성 물질인 L-글루탐산 또는 6-히드록시도파민에 의해 유발되는 독성에 대하여 SH-SY5Y 세포 보호효과를 가지는지 확인하기 위해 Western assay method 이용하여 SH-SY5Y 세포의 세포 생존력(cell viability)을 측정하였다.SH-SY5Y cells using a Western assay method to determine whether the sesquiterpene derivative compound of the present invention has a protective effect on SH-SY5Y cells against toxicity caused by cytotoxic substances such as L-glutamic acid or 6-hydroxydopamine. Cell viability (cell viability) was measured.

[화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 및 [화학식 11]로 표시되는 화합물을 여러 농도(0~3μg/ml)로 제조하고 상기 화합물 및 양성 대조군(positive control compound)인 아스코빈산(ascorbic acid, 75mM)을 각각 24시간 SH-SY5Y 세포에 전 처리하였다. 그 다음 L-글루탐산 75mM을 48시간 처리하여 신경독성을 유발시켰다. 이후 대조군(control group), 양성대조군(아스코빈산 처리군) 또는 본 발명의 화합물 처리에 의해 신경세포 생존율이 증가되었는지 측정하였다.Compounds represented by [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8] and [Chemical Formula 11] were prepared in various concentrations (0-3 μg/ml), and the compound and positive Ascorbic acid (75 mM), a positive control compound, was pre-treated on SH-SY5Y cells for 24 hours, respectively. Then, 75mM L-glutamic acid was treated for 48 hours to induce neurotoxicity. Then, it was measured whether the neuron survival rate was increased by the control group, the positive control group (ascorbic acid treatment group), or the compound treatment of the present invention.

그 결과 대조군(DMSO 단독 처리군, 본 발명의 화합물 처리 전 시료)에 비해 [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 또는 [화학식 11]로 표시되는 화합물을 처리한 경우 세포생존율이 유의미하게 증가(도 5 내지 10 참조)하였으며 이 중 본 발명의 화합물 0.5μg/ml 처리시 값을 하기 표 2에 기재하였다. As a result, compared to the control group (DMSO alone treatment group, sample before compound treatment of the present invention), it is represented by [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8] or [Chemical Formula 11] When the compound was treated, the cell viability was significantly increased (see FIGS. 5 to 10), and the values of 0.5 μg/ml of the compound of the present invention were shown in Table 2 below.

신규 화합물New compound 세포생존증가율(%)Cell viability increase rate (%) 신규 화합물New compound 세포생존증가율(%)Cell viability increase rate (%) 화학식 1Formula 1 14.3414.34 화학식 7Formula 7 13.1213.12 화학식 2Formula 2 16.7116.71 화학식 8Formula 8 15.8815.88 화학식 5Formula 5 14.2014.20 화학식 11Formula 11 13.3413.34

세포 생존증가율(%) = (신규 화합물 처리 후 - 처리 전 시료)/처리 후 시료 Cell viability increase rate (%) = (after new compound treatment-sample before treatment)/sample after treatment

또한, 상기와 같이 [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 및 [화학식 11] 로 표시되는 화합물을 여러 농도(0~3μg/ml)로 24시간 SH-SY5Y 세포에 전 처리한 다음, 6-히드록시도파민 100mM을 48시간 처리하여 신경세포 보호효과를 측정하였다.In addition, compounds represented by [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8], and [Chemical Formula 11] as described above are used in various concentrations (0-3 μg/ml). Time SH-SY5Y cells were pretreated and then treated with 100 mM 6-hydroxydopamine for 48 hours to measure the neuronal protective effect.

그 결과 대조군(DMSO 단독 처리군, 본 발명의 화합물 처리 전 시료)에 비해 [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 또는 [화학식 11]로 표시되는 화합물을 처리한 경우 세포생존율이 유의미하게 증가(도 11 내지 16 참조)하였고 이 중 본 발명의 화합물 0.5μg/ml 처리시 값을 하기 표 3에 나타내었다.As a result, compared to the control group (DMSO alone treatment group, sample before compound treatment of the present invention), it is represented by [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8] or [Chemical Formula 11] When the compound of the present invention was treated, the cell viability was significantly increased (see FIGS. 11 to 16), and values of 0.5 μg/ml of the compound of the present invention were shown in Table 3 below.

신규 화합물New compound 세포생존증가율(%)Cell viability increase rate (%) 신규 화합물New compound 세포생존증가율(%)Cell viability increase rate (%) 화학식 1Formula 1 13.2413.24 화학식7Formula 7 12.5212.52 화학식 2Formula 2 18.0918.09 화학식 8Formula 8 19.6019.60 화학식 5Formula 5 19.7919.79 화학식 11Formula 11 20.1320.13

세포 생존증가율(%) = (신규 화합물 처리 후 - 처리 전 시료)/처리 후 시료상기 결과를 통해 본 발명의 [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 및 [화학식 11]로 표시되는 화합물은 L-글루탐산 또는 6-히드록시도파민에 의해 유발되는 신경세포 독성으로부터 신경세포의 세포생존율을 증가시키는 효과가 있고 나아가, 양성대조군으로 사용한 아스코르브산(Ascrobic acid) 보다 더 높은 증가율을 보임을 확인하였다. 이는 상기 화합물은 L-글루탐산 또는 6-히드록시도파민에 의해 유발되는 신경세포 독성으로부터 신경세포를 보호함으로써 파킨슨병에 대한 우수한 치료 효과를 가진다는 것을 의미한다.Cell viability increase rate (%) = (after new compound treatment-sample before treatment) / sample after treatment [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Formula 7] 8] and [Formula 11] have the effect of increasing the cell viability of neurons from neurocytotoxicity induced by L-glutamic acid or 6-hydroxydopamine, and further, ascorbic acid used as a positive control (Ascrobic acid). acid) showed a higher rate of increase. This means that the compound has an excellent therapeutic effect against Parkinson's disease by protecting neurons from neurocytotoxicity caused by L-glutamic acid or 6-hydroxydopamine.

3-3: 신규 세스퀴테르펜 유도체 화합물에 의한 파킨슨 질환 모델에서 신경세포 세포자살사(apoptosis)의 저해효과 측정3-3: Measurement of inhibitory effect on neuronal apoptosis in Parkinson's disease model by novel sesquiterpene derivative compounds

신규 세스퀴테르펜 유도체 화합물인 [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 및 [화학식 11]로 표시되는 화합물에 의한 신경세포 보호의 기전을 확인하기 위하여 상기 화합물이 L-글루탐산 또는 6-히드록시도파민에 의해 유발되는 신경세포의 세포자살사(apoptosis)를 저해하는지를 확인하였다.To confirm the mechanism of neuronal protection by compounds represented by the novel sesquiterpene derivative compounds [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8] and [Chemical Formula 11] For this, it was confirmed whether the compound inhibited apoptosis of neurons caused by L-glutamic acid or 6-hydroxydopamine.

SH-SY5Y 세포의 세포자살사를 측정하기 위하여 MuseTM Annexin V & Dead Cell Kit를 사용하였다. 우선 SH-SY5Y 세포를 2x105 cells/well로 24-well plate에 분주한 다음 상기 [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 또는 [화학식 11]로 표시되는 화합물을 24시간 전 처리하고 L-글루탐산 또는 6-히드록시도파민을 48시간 처리하여 세포독성을 유발시켰다. 이후 배양이 끝난 세포를 회수하고 MuseTM Annexin V & Dead Cell reagent 100μl를 첨가하고 실온에서 빛을 차단하고 20분간 염색(dying)한 다음, 염색된 세포들은 MuseTM Annexin V & Dead Cell Analyzer (Merck Millipore, Germany)를 사용하여 분석하였다.To measure the apoptosis of SH-SY5Y cells, Muse TM Annexin V & Dead Cell Kit was used. First, the SH-SY5Y cells are dispensed into a 24-well plate at 2x10 5 cells/well, and then [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8] or [Chemical Formula 11] Cytotoxicity was induced by treatment with the compound represented by 24 hours before and treatment with L-glutamic acid or 6-hydroxydopamine for 48 hours. After the cultured cells were recovered, 100 μl of Muse TM Annexin V & Dead Cell reagent was added, light was blocked at room temperature, dyed for 20 minutes, and then the stained cells were treated with Muse TM Annexin V & Dead Cell Analyzer (Merck Millipore). , Germany).

그 결과, SH-SY5Y 세포에 대조군(DMSO)를 전 처리한 다음, L-글루탐산을 처리한 경우, 간세포(Liver cell, annexin V-/7-AAD cell)의 비율이 감소하는 한편, 세포자살사(apoptosis) 세포(Annexin V+) 비율이 크게 증가하였다. 이로부터 L-글루탐산에 의해 SH-SY5Y 세포에 독성이 유발되어 신경 세포자살사(neuron apoptosis)가 일어난 것을 알 수 있다(도 17 참조).As a result, the SH-SY5Y treated before the control group (DMSO) in the cell when the then treated with L- glutamic acid, Hepatocellular (Liver cell, annexin V - / 7-AAD cell) which the other hand, the reduction ratio of the cell jasalsa ( Apoptosis) cells (Annexin V + ) ratio increased significantly. From this, it can be seen that toxicity is induced in SH-SY5Y cells by L-glutamic acid, resulting in neuronal apoptosis (see FIG. 17).

반면, 상기 [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 또는 [화학식 11]로 표시되는 화합물을 전 처리한 경우, L-글루탐산에 의한 유발되는 신경세포의 세포자살사(apoptotic)가 하기 표 4와 같이 크게 억제되었다(도 17 참조).On the other hand, when the compound represented by [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8] or [Chemical Formula 11] is pretreated, nerves caused by L-glutamic acid Cell apoptosis (apoptotic) was greatly suppressed as shown in Table 4 below (see FIG. 17).

신규 화합물New compound Apoptosis(%)Apoptosis(%) 신규 화합물New compound Apoptosis(%)Apoptosis(%) DMSO단독(%)DMSO alone (%) DMSO+L-Glu(%)DMSO+L-Glu(%) 화학식 1Formula 1 농도 0.1: 19.34Concentration 0.1: 19.34 화학식 7Formula 7 농도 0.1: 19.27Concentration 0.1: 19.27 10.0510.05 27.3727.37 화학식 2Formula 2 농도 0.5: 19.47Concentration 0.5: 19.47 화학식 8Formula 8 농도 0.5: 22.50Concentration 0.5: 22.50 -- -- 화학식 5Formula 5 농도 0.1: 21.14Concentration 0.1: 21.14 화학식 11Formula 11 농도 0.1: 20.75Concentration 0.1: 20.75 -- --

농도 단위: (μg/ml), apoptosis 값: % 표시 함Concentration unit: (μg/ml), apoptosis value:% displayed

또한, 상기와 같이 SH-SY5Y 세포에 대조군(DMSO)를 전 처리한 다음, 6-히드록시도파민 처리한 경우, 간세포(liver cell, annexin V-/7-AAD cell)의 비율이 감소하는 한편 세포자살사(apoptosis) 세포(Annexin V+) 비율이 크게 증가하였다. 이로부터 6-히드록시도파민에 의해 SH-SY5Y 세포의 독성이 유발되어 세포자살사(apoptosis)가 일어난 것을 알 수 있다(도 18 참조).In addition, in the case where a pre-treatment to the control group (DMSO) in SH-SY5Y cells as described above, and then 6-hydroxy dopamine treatment, stem cells - which reduces the ratio of (liver cell, annexin V / 7 -AAD cell) The cell The proportion of suicide cells (Annexin V + ) was significantly increased. From this, it can be seen that the toxicity of SH-SY5Y cells was induced by 6-hydroxydopamine, resulting in apoptosis (see FIG. 18).

반면, [화학식 1], [화학식 2], [화학식 5], [화학식 7], [화학식 8] 또는 [화학식 11]로 표시되는 화합물을 전 처리한 결과, 6-히드록시도파민에 의해 유발된 신경세포 세포자살사(apoptotic)가 하기 표 5에 기재한 바와 같이 크게 억제되었다(도 18 참조). On the other hand, as a result of pretreatment of a compound represented by [Chemical Formula 1], [Chemical Formula 2], [Chemical Formula 5], [Chemical Formula 7], [Chemical Formula 8] or [Chemical Formula 11], 6-hydroxydopamine induced Neuronal cell apoptosis (apoptotic) was greatly suppressed as shown in Table 5 below (see FIG. 18).

신규 화합물New compound Apoptosis(%)Apoptosis(%) 신규 화합물New compound Apoptosis(%)Apoptosis(%) DMSO단독(%)DMSO alone (%) DMSO+6-OHDA(%)DMSO+6-OHDA (%) 화학식 1Formula 1 농도 0.1: 14.80Concentration 0.1: 14.80 화학식 7Formula 7 농도 0.1: 15.45Concentration 0.1: 15.45 12.5012.50 25.9025.90 화학식 2Formula 2 농도 0.5: 10.76Concentration 0.5: 10.76 화학식 8Formula 8 농도 0.5: 11.66Concentration 0.5: 11.66 -- -- 화학식 5Formula 5 농도 0.1: 11.51Concentration 0.1: 11.51 화학식 11Formula 11 농도 0.1: 12.30Concentration 0.1: 12.30 -- --

농도 단위: (μg/ml), apoptosis 값: % 표시 함Concentration unit: (μg/ml), apoptosis value:% displayed

상기 실시예 3-2 및 3-3 실험결과는 본 발명의 화합물이 뛰어난 신경세포 보호효과(cell viability) 및 신경세포 세포사멸사(apoptosis) 억제효과가 있어 탁월한 파킨슨병 치료효과를 나타낸다는 것을 의미한다.The experimental results of Examples 3-2 and 3-3 indicate that the compound of the present invention has excellent neuronal cell viability and neuronal cell apoptosis inhibitory effect, thus exhibiting excellent Parkinson's disease treatment effect. do.

Claims (6)

하기 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체.
Figure pat00021
A compound represented by the following Chemical Formulas 1 to 12 or an optical isomer thereof.
Figure pat00021
 하기 화학식 1, 화학식 2, 화학식 5, 화학식 7, 화학식 8, 화학식 11로 표시되는 화합물 또는 이의 광학 이성질체.
Figure pat00022
A compound represented by the following Formula 1, Formula 2, Formula 5, Formula 7, Formula 8, and Formula 11, or an optical isomer thereof.
Figure pat00022
 제1항의 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상을 유효성분으로 포함하는, 파킨슨병(Parkinson disease) 예방 또는 치료용 약학적 조성물.
A pharmaceutical composition for preventing or treating Parkinson disease, comprising as an active ingredient any one or more of the compound represented by Chemical Formulas 1 to 12 of claim 1 or optical isomers thereof.
 제3항에 있어서, 상기 약학적 조성물은 화학식 1, 화학식 2, 화학식 5, 화학식 7, 화학식 8, 화학식 11로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상을 유효성분으로 포함하는 것인, 약학적 조성물.
Figure pat00023
The pharmaceutical composition of claim 3, wherein the pharmaceutical composition comprises any one or more of a compound represented by Formula 1, Formula 2, Formula 5, Formula 7, Formula 8, Formula 11 or optical isomers thereof as an active ingredient. Ever composition.
Figure pat00023
 제1항의 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상을 유효성분으로 포함하는, 파킨슨병(Parkinson disease) 예방 또는 개선용 의약외품 조성물.
A quasi-drug composition for preventing or improving Parkinson disease, comprising as an active ingredient any one or more of the compound represented by Chemical Formulas 1 to 12 of claim 1 or optical isomers thereof.
 제1항의 화학식 1 내지 화학식 12로 표시되는 화합물 또는 이의 광학 이성질체 중 어느 하나 이상을 유효성분으로 포함하는, 파킨슨병(Parkinson disease) 예방 또는 개선용 식품 조성물.A food composition for preventing or improving Parkinson's disease, comprising as an active ingredient any one or more of the compound represented by Chemical Formulas 1 to 12 of claim 1 or optical isomers thereof.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170128716A (en) 2016-05-13 2017-11-23 이민용 Essential Oil Compound from Chamaecyparis obtusa and Cupressaceae and Abies koreana WILS to increase RSMT (ratio of (SMR~Mid beta) to theta), RB (relative beta power spectrum), RMT (ratio of mid beta to theta), AFA(absolute fast alpha power spectrum)
KR20180114267A (en) 2017-04-07 2018-10-18 동의대학교 산학협력단 Pharmaceutical composition for preventing or treating Parkinson's disease comprising Juniperus chinensis extract or Juniperus chinensis-derived compound

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170128716A (en) 2016-05-13 2017-11-23 이민용 Essential Oil Compound from Chamaecyparis obtusa and Cupressaceae and Abies koreana WILS to increase RSMT (ratio of (SMR~Mid beta) to theta), RB (relative beta power spectrum), RMT (ratio of mid beta to theta), AFA(absolute fast alpha power spectrum)
KR20180114267A (en) 2017-04-07 2018-10-18 동의대학교 산학협력단 Pharmaceutical composition for preventing or treating Parkinson's disease comprising Juniperus chinensis extract or Juniperus chinensis-derived compound

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