KR102589266B1 - peptidomimetic compound having an ability to inhibit the formation of dementia-inducing beta-amyloid aggregates and a composition for preventing or treating dementia containing the same - Google Patents
peptidomimetic compound having an ability to inhibit the formation of dementia-inducing beta-amyloid aggregates and a composition for preventing or treating dementia containing the same Download PDFInfo
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- KR102589266B1 KR102589266B1 KR1020230039967A KR20230039967A KR102589266B1 KR 102589266 B1 KR102589266 B1 KR 102589266B1 KR 1020230039967 A KR1020230039967 A KR 1020230039967A KR 20230039967 A KR20230039967 A KR 20230039967A KR 102589266 B1 KR102589266 B1 KR 102589266B1
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- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
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- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
본 발명은 치매 예방 또는 치료능을 갖는 펩티도미메틱 화합물 및 이를 포함하는 조성물에 관한 것으로서, 더욱 상세하게는 치매유발성 베타아밀로이드의 응집체 형성 저해능을 갖는 펩티도미메틱 화합물 및 이를 포함하는 치매 예방 또는 치료용 조성물에 관한 것이다.
본 발명의 히스티딘 유도체를 포함하는 펩티도미메틱 화합물은 치매유발 베타아밀로이드의 피브릴 형성 저해 효과가 우수하며, 인체 적용시 체내 안정성이 우수하므로, 의약품 등의 산업 분야에 매우 유용하다. The present invention relates to a peptidomimetic compound having the ability to prevent or treat dementia and a composition containing the same, and more specifically, to a peptidomimetic compound having the ability to inhibit the formation of dementia-causing beta-amyloid aggregates and the prevention or treatment of dementia containing the same. It relates to a composition for use.
The peptidomimetic compound containing the histidine derivative of the present invention has an excellent effect of inhibiting fibril formation of beta-amyloid that causes dementia, and has excellent stability in the body when applied to the human body, so it is very useful in industrial fields such as pharmaceuticals.
Description
본 발명은 치매 예방 또는 치료능을 갖는 펩티도미메틱 화합물 및 이를 포함하는 조성물에 관한 것으로서, 더욱 상세하게는 치매유발성 베타아밀로이드의 응집체 형성 저해능을 갖는 펩티도미메틱 화합물 및 이를 포함하는 치매 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a peptidomimetic compound having the ability to prevent or treat dementia and a composition containing the same, and more specifically, to a peptidomimetic compound having the ability to inhibit the formation of dementia-causing beta-amyloid aggregates and the prevention or treatment of dementia containing the same. It relates to a composition for use.
펩타이드는 바이오의 핵심소재로 단백질의 기능적 최소 단위이며, 아미노산이 2개로부터 50개 이하로 구성된다. 소량으로 우수한 효능을 보일 뿐만 아니라 독성이 없어 의약품, 식품 및 화장품의 주요 원료로 많이 활용되고 있다. 특히 인체에 안전하고 효과가 좋은 펩타이드 화장품 소재는 날로 사용량이 늘어날 뿐만 아니라 새로운 소재의 개발은 수입대체 효과 등 당 산업에서 매우 중요하다고 할 수 있다.Peptides are core biomaterials and the minimum functional unit of proteins, and consist of 2 to 50 amino acids. Not only does it show excellent efficacy in small amounts, but it is also non-toxic, so it is widely used as a major raw material for medicines, foods, and cosmetics. In particular, not only is the use of peptide cosmetic materials that are safe and effective for the human body increasing day by day, but the development of new materials is very important in the industry, including the import substitution effect.
또한 펩티도미메틱 (Peptidomimetic)은 펩타이드의 유사체로서, 펩타이드의 장점인 높은 효능은 그대로 유지시키면서 단점인 체내 안정성을 획기적으로 극복시킬수 있는 핵심 차세대 바이오 소재이다.In addition, peptidomimetic is an analogue of peptide and is a key next-generation bio material that can dramatically overcome the disadvantage of stability in the body while maintaining the high efficacy of peptides.
2000년 이후 합성 기술의 발달로 그동안 상업화가 이루어지지 않았던 펩타이드의 상업화 및 연구가 본격적으로 이루어지기 시작하였으며, 이를 바탕으로 한 신약 연구 또한 매년 꾸준히 증가하고 있는 추세이다.Since 2000, with the development of synthetic technology, commercialization and research on peptides, which had not been commercialized until now, began to take place in earnest, and research on new drugs based on these has also been steadily increasing every year.
한편, 치매(dementia)는 뇌의 인지 기능 장애로 인해 일상생활을 스스로 유지하지 못하는 상태, 혹은 그러한 질병을 말한다. 치매관리법 제2조 제1호에서는 치매를 '퇴행성 뇌질환 또는 뇌혈관계 질환 등으로 인하여 기억력, 언어능력, 지남력(指南力), 판단력 및 수행능력 등의 기능이 저하됨으로써 일상 생활에서 지장을 초래하는 후천적인 다발성 장애'로 정의한다.Meanwhile, dementia refers to a condition or disease in which a person is unable to maintain daily life on his or her own due to cognitive dysfunction in the brain. Article 2, Paragraph 1 of the Dementia Management Act defines dementia as 'a condition that causes disruption in daily life due to a decline in functions such as memory, language ability, orientation, judgment, and performance due to degenerative brain disease or cerebrovascular disease. It is defined as ‘acquired multiple disorder’.
치매의 주요 원인은 베타아밀로이드에 의한 것으로 알려져 있으며, 베타아밀로이드가 잘못 접히면서 서로 뭉쳐 플라크(plaque)를 형성하면 독성을 띠면서 신경세포의 신호전달 시스템인 시냅스(synapse)를 파괴하면, 치매 증상이 나타나는 것으로 알려져 있다. It is known that the main cause of dementia is beta-amyloid. When beta-amyloid folds incorrectly and clumps together to form plaques, it becomes toxic and destroys synapses, the signaling system of nerve cells, causing dementia symptoms. It is known to appear.
대한민국 등록특허 제10-1173677호는 EPPS를 유효성분으로 함유하는 베타아밀로이드 집적 관련 질환의 예방 또는 치료용 약학적 조성물을 개시하였으나, EPPS(N-(2-하이드록시에틸)피페라진-N'-(3-프로판술폰산))는 일반 chemical의 치료제 물질이며, 합성과정이 복잡하여 사업화에 용이하지 않은 점과 베타아밀로이드 응집 저해 효과가 그렇게 우수하지 않은 단점이 있었다.Republic of Korea Patent No. 10-1173677 discloses a pharmaceutical composition for preventing or treating diseases related to beta-amyloid accumulation containing EPPS as an active ingredient, but EPPS (N-(2-hydroxyethyl)piperazine-N'- (3-Propanesulfonic acid)) is a general chemical treatment material, but its synthesis process is complicated, so it is not easy to commercialize, and its effect in inhibiting beta-amyloid aggregation is not very good.
또한 대한민국 등록특허 제10-0730988호는 베타아밀로이드집적 방해 및 베타아밀로이드 화이브릴 용해 기능이 있는 화합물, 이의 제조방법, 용도 및 베타아밀로이드 집적 방해 및 베타아밀로이드 화이브릴 용해 기능이 있는 화합물을 검색하는 방법을 개시하였다. 하지만, 상기 특허의 물질 역시 일반 chemical이며, 상업화하기에는 어려운 공정을 포함하며, 효과 또한 우수하지 않은 단점이 있었다.In addition, Republic of Korea Patent No. 10-0730988 discloses compounds with the function of interfering with beta-amyloid accumulation and dissolving beta-amyloid fibrils, their manufacturing methods, uses, and methods for searching for compounds with the function of interfering with beta-amyloid accumulation and dissolving beta-amyloid fibrils. started. However, the material in the patent was also a general chemical, involved a process that was difficult to commercialize, and had the disadvantage of not being effective.
이에, 본 발명자들은 위에서 언급한 바와 같이 치매의 주요 원인인 베타아밀로이드의 주요 구성 서열을 기반으로 구조를 디자인하여 새로운 물질을 합성하고 다양한 펩티도미메틱을 연구, 개발하는 과정에서 특정 서열을 갖는 펩티도미메틱이 치매의 주요 원인인 베타아밀로이드 피브릴(Fibril)과 같은 응집체 형성을 억제한다는 사실을 확인하였으며, 다양한 효능평가를 통해 치매 치료제로써의 가능성을 확인하여 본 발명을 완성하게 되었다.Accordingly, as mentioned above, the present inventors designed a structure based on the main structural sequence of beta-amyloid, the main cause of dementia, synthesized a new material, and in the process of researching and developing various peptidomimetics, peptidomimetic with a specific sequence. It was confirmed that Metic inhibits the formation of aggregates such as beta-amyloid fibrils, which are the main cause of dementia, and its potential as a dementia treatment was confirmed through various efficacy evaluations, leading to the completion of the present invention.
본 발명의 목적은 치매유발성 베타아밀로이드의 응집체 형성 저해능을 갖는 펩티도미메틱 화합물 및 이를 포함하는 치매 예방 또는 치료용 조성물을 제공하는데 있다.The purpose of the present invention is to provide a peptidomimetic compound that has the ability to inhibit the formation of dementia-causing beta-amyloid aggregates and a composition for preventing or treating dementia containing the same.
본 발명의 다른 목적은 치매유발성 베타아밀로이드의 응집체 형성 저해능을 갖는 펩티도미메틱 화합물의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing a peptidomimetic compound that has the ability to inhibit the formation of dementia-inducing beta-amyloid aggregates.
상기 목적을 달성하기 위하여, 본 발명은 하기 화학식 I 또는 II로 표시되는 펩티도미메틱 화합물을 제공한다.In order to achieve the above object, the present invention provides a peptidomimetic compound represented by the following formula (I) or (II).
[화학식 I][Formula I]
[화학식 II] [Formula II]
본 발명은 또한, 상기 화학식 I 또는 II로 표시되는 펩티도미메틱 화합물을 유효성분으로 포함하는 치매 예방 또는 치료용 조성물을 제공한다.The present invention also provides a composition for preventing or treating dementia comprising the peptidomimetic compound represented by Formula (I) or (II) as an active ingredient.
본 발명에 있어서, 상기 화학식 I 또는 II로 표시되는 펩티도미메틱 화합물은 치매유발성 베타아밀로이드의 응집체 형성 저해능을 갖는 것을 특징으로 한다.In the present invention, the peptidomimetic compound represented by Formula (I) or (II) is characterized by its ability to inhibit the formation of dementia-causing beta-amyloid aggregates.
본 발명은 또한, (a) 고체상 (Solid-phase) 합성방법으로 화학식 1로 표시되는 Fmoc-Ala-NH-레진을 수득하는 단계; (b) 상기 단계 (a)에서 수득한 레진에 Fmoc-His(Bis-cyclohexylpropyl)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH 및 Fmoc-γ-Glu-OtBu를 순차적으로 결합시켜 화학식 2로 표시되는 보호화된 펩타이드가 결합된 레진을 수득하는 단계; (c) 상기 단계 (b)에서 수득한 보호화된 펩타이드가 결합된 레진으로부터 레진 및 아미노산의 보호기를 동시에 제거하여 화학식 3으로 표시되는 비정제 펩티도미메틱 화합물을 수득하는 단계; 및 (d) 상기 단계 (c)에서 수득한 비정제 펩티도미메틱 화합물을 정제하여 화학식 I로 표시되는 펩티도미메틱 화합물을 수득하는 단계를 포함하는 화학식 I로 표시되는 펩티도미메틱 화합물의 제조방법을 제공한다.The present invention also includes the steps of (a) obtaining Fmoc-Ala-NH-resin represented by Formula 1 by a solid-phase synthesis method; (b) Fmoc-His(Bis-cyclohexylpropyl)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH in the resin obtained in step (a). , sequentially combining Fmoc-Cys(Trt)-OH and Fmoc-γ-Glu-OtBu to obtain a resin to which the protected peptide represented by Formula 2 is bound; (c) simultaneously removing the protective groups of the resin and the amino acid from the resin to which the protected peptide obtained in step (b) is bound, thereby obtaining a purified peptidomimetic compound represented by Formula 3; and (d) purifying the crude peptidomimetic compound obtained in step (c) to obtain a peptidomimetic compound represented by Formula I. to provide.
[화학식 I][Formula I]
[화학식 1][Formula 1]
[화학식 2][Formula 2]
상기 화학식 2에서 R1은 수소 또는 수산기 보호기이고, R2는 티올 보호기이며, R3는 아민 보호기이다.In Formula 2, R 1 is a hydrogen or hydroxyl protecting group, R 2 is a thiol protecting group, and R 3 is an amine protecting group.
[화학식 3][Formula 3]
본 발명은 또한, 화학식 I로 표시되는 펩티도미메틱 화합물에 -SH기 산화 시약을 혼합하고, pH를 6~9로 조절하여 디설파이드 결합(S-S)을 수행하는 것을 특징으로 하는 화학식 II로 표시되는 펩티도미메틱 화합물의 제조방법을 제공한다.The present invention also provides a peptidomimetic compound represented by Formula II, which is characterized in that the peptidomimetic compound represented by Formula I is mixed with an -SH group oxidation reagent, and the pH is adjusted to 6 to 9 to perform disulfide bonding (S-S). A method for producing a dometic compound is provided.
[화학식 II][Formula II]
본 발명에 있어서, 상기 레진은 링크아마이드 레진 (Rink Amide Resin), 링크아마이드 엠비에이치에이 레진 (Rink Amide MBHA Resin), 링크아마이드 에이엠 레진 (Rink Amide AM Resin) 및 링크아마이드 렘 레진 (Rink Amide RAM Resin)으로 구성된 군에서 선택되는 것을 특징으로 한다.In the present invention, the resin is Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, and Rink Amide RAM Resin. ) is characterized in that it is selected from the group consisting of.
본 발명에 있어서, 레진 및 아미노산의 보호기를 동시에 제거하는 산성 용액은 부피% 기준으로 TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA/phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/water (95/5), TFA/DCM/indole (70/28/2) 및 TFA/TIPS/water (95/2.5/2.5)로 구성된 군에서 선택되는 것을 특징으로 한다.In the present invention, the acidic solution that simultaneously removes the protecting groups of resin and amino acid is TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/ 5/5/5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA /phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/ It is characterized in that it is selected from the group consisting of water (95/5), TFA/DCM/indole (70/28/2), and TFA/TIPS/water (95/2.5/2.5).
본 발명의 히스티딘 유도체를 포함하는 펩티도미메틱 화합물은 치매의 주요원인인 베타아밀로이드의 피브릴 형성을 저해하여 치매 치료제로써 활용이 가능하므로, 의약품 등의 관련 산업 분야에 매우 유용하다.The peptidomimetic compound containing the histidine derivative of the present invention can be used as a treatment for dementia by inhibiting the fibril formation of beta-amyloid, the main cause of dementia, and is therefore very useful in related industries such as pharmaceuticals.
도 1은 본 발명에 따른 히스티딘 유도체를 포함하는 펩티도미메틱 화합물의 합성도식이다.
도 2는 본 발명에 따른 펩티도미메틱 화합물 I 및 II에 대한 ThT 분석(Thioflavin T assay)결과이다.
도 3은 ThT 분석이 끝난 펩티도미메틱 화합물 I 및 II에 대한 TEM (Transmission Electron Microscopy) 사진이다.Figure 1 is a schematic diagram of the synthesis of a peptidomimetic compound containing a histidine derivative according to the present invention.
Figure 2 shows the results of ThT analysis (Thioflavin T assay) for peptidomimetic compounds I and II according to the present invention.
Figure 3 is a TEM (Transmission Electron Microscopy) photograph of peptidomimetic compounds I and II after ThT analysis.
본 명세서에서 특별한 표시가 없는 한, 아미노산 및 보호기의 지정에 사용되는 약어는 IUPAC-IUB의 생화학 용어 위원회 (Commission of Biochemical Nomenclature)에서 권장하는 용어에 기초한다 (Biochemistry, 11:1726-1732(1972); Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984).Unless specifically indicated herein, the abbreviations used to designate amino acids and protecting groups are based on the terminology recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB ( Biochemistry, 11:1726-1732 (1972) ; Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984).
본 명세서에서 용어 "펩타이드"는 펩타이드 결합에 의해 아미노산 잔기들이 서로 결합되어 형성된 선형의 분자를 의미한다.As used herein, the term “peptide” refers to a linear molecule formed by linking amino acid residues together through peptide bonds.
본 명세서에서 용어 "펩티도미메틱"은 펩타이드 유사체로 펩타이드의 단점인 체내 안정성이 좋아 지도록 아미노산에 치환체를 도입하는 등 구조가 변형된 펩타이드의 일종이다.As used herein, the term "peptidomimetic" is a peptide analog and is a type of peptide whose structure has been modified, such as by introducing substituents to amino acids, to improve stability in the body, which is a disadvantage of peptides.
본 명세서에서 사용한 용매, 시약, 보호기 및 아미노산의 약어는 다음과 같다:The abbreviations for solvents, reagents, protecting groups and amino acids used herein are as follows:
DCM : 디클로로메탄 (Dichloromethane)DCM: Dichloromethane
DIEA : 엔,엔-디이소프로필에틸아민 (N,N-Diisopropylethylamine)DIEA: N,N-Diisopropylethylamine
DMF : 엔,엔-디메틸포름아미드 (N,N-Dimethylformamide)DMF: N,N-Dimethylformamide
DMSO : 디메틸설폭시드 (Dimethylsulfoxide)DMSO: Dimethylsulfoxide
DTT : 디티올쓰레이톨 (Dithiolthreitol)DTT: Dithiolthreitol
EDT : 1,2-에탄디티올 (1,2-Ethanedithiol)EDT: 1,2-Ethanedithiol
MC : 메틸렌클로라이드 (Methylene chloride)MC: Methylene chloride
ACN : 아세토니트릴 (Acetonitrile)ACN: Acetonitrile
Ala : 알라닌 (Alanine)Ala: Alanine
Arg : 아르기닌 (Arginine)Arg: Arginine
Boc : 복 (tert-Butyloxycarbonyl)Boc: Bok (tert-Butyloxycarbonyl)
Cys : 시스테인 (Cysteine)Cys: Cysteine
γ-Glu : 감마-글루타믹산 (γ-Glutamic acid)γ-Glu: Gamma-Glutamic acid
Gly : 글라이신 (Glycine)Gly: Glycine
His : 히스티딘 (Histidine)His: Histidine
HPLC : 고성능액체크로마토그래피 (High Performance Liquid Chromatography)HPLC: High Performance Liquid Chromatography
Fmoc : 9-플루오레닐옥시카보닐 (9-Fluorenyloxycarbonyl)Fmoc: 9-Fluorenyloxycarbonyl
Trt : 트리페닐메틸 (또는 트리틸) (Triphenylmethyl or Trityl)Trt: Triphenylmethyl (or Trityl)
Pbf : 2,2,4,6,7-펜타메틸-디히드로벤조퓨란-5-설포닐 (2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl)Pbf: 2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl (2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl)
Pmc : 2,2,5,7,8-펜타메틸크로만-6-설포닐 (2,2,5,7,8-Pentamethylchroman-6-sulfonyl)Pmc: 2,2,5,7,8-Pentamethylchroman-6-sulfonyl (2,2,5,7,8-Pentamethylchroman-6-sulfonyl)
Leu : 류신 ( Leucine)Leu: Leucine
Lys : 라이신 (Lysine)Lys: Lysine
Mtr : 4-메톡시-2,3,6-트리메틸페닐-설포닐 (4-Methoxy-2,3,6-trimethylphenyl-sulfonyl)Mtr: 4-Methoxy-2,3,6-trimethylphenyl-sulfonyl
Tos : 파라-톨루엔설포닐 (para-Toluenesulfonyl)Tos: Para-Toluenesulfonyl
TES : 트라이에틸실란 (Triethylsilane)TES: Triethylsilane
TFA : 트라이플루오로아세틱 엑시드 (Trifluoroacetic acid)TFA: Trifluoroacetic acid
TIPS : 트리이소프로필실란 (Triisopropylsilane)TIPS: Triisopropylsilane
Trt : 트리틸 (Triphenylmethyl)Trt: Triphenylmethyl
Val : 발린 (Valine)Val: Valine
본 발명에서는 치매의 주요 원인인 베타아밀로이드의 피브릴 형성을 효과적으로 제어할 수 있는 히스티딘 유도체를 포함하는 다양한 펩티도미메틱을 연구, 개발하는 과정에서 특정 서열을 갖는 히스티딘 유도체를 포함하는 펩티도미메틱을 제조하였고, 이는 피브릴 형성을 저해하는 효과가 우수함을 확인하였다.In the present invention, in the process of researching and developing various peptidomimetics containing histidine derivatives that can effectively control the fibril formation of beta-amyloid, the main cause of dementia, a peptidomimetic containing a histidine derivative having a specific sequence is manufactured. It was confirmed that the effect of inhibiting fibril formation was excellent.
따라서, 본 발명은 일 관점에서 하기 화학식 I 또는 II로 표시되는 펩티도미메틱 화합물에 관한 것이다. Accordingly, in one aspect, the present invention relates to a peptidomimetic compound represented by the following formula (I) or (II).
[화학식 I][Formula I]
[화학식 II][Formula II]
본 발명은 또한, 상기 화학식 I 또는 II로 표시되는 펩티도미메틱 화합물을 유효성분으로 포함하는 치매 예방 또는 치료용 조성물에 관한 것이다.The present invention also relates to a composition for preventing or treating dementia comprising the peptidomimetic compound represented by Formula (I) or (II) as an active ingredient.
상기 화학식 I 또는 II로 표시되는 펩티도미메틱 화합물은 치매유발성 베타아밀로이드의 응집체 형성 저해능을 갖는 것을 특징으로 한다.The peptidomimetic compound represented by Formula I or II is characterized by its ability to inhibit the formation of dementia-causing beta-amyloid aggregates.
본 발명에 따른 치매 예방 또는 치료용 조성물은 약학적 조성물로 이용될 수 있는데, 본 발명의 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 본 발명에 따른 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 이에 제한되지 않는다.The composition for preventing or treating dementia according to the present invention can be used as a pharmaceutical composition. The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The pharmaceutical composition according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. may, but is not limited to this.
본 발명의 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조 에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있으며, 이에 제한되지 않는다.Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Examples include, but are not limited to, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
본 발명의 약학적 조성물을 제제화 할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 유효성분에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글라이콜 (propylene glycol), 폴리에틸렌 글라이콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있으며, 이에 제한되지 않는다.When formulating the pharmaceutical composition of the present invention, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain the active ingredients plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. may be used, but are not limited thereto.
본 발명의 약학적 조성물은 피부, 주사, 피하주사, 복용제제 등에 사용할 수 있다.The pharmaceutical composition of the present invention can be used on the skin, for injection, subcutaneous injection, or as a dosage form.
본 발명의 약학적 조성물에 포함된 화학식 I 또는 II로 표시되는 펩티도미메틱 화합물의 함량은 용도, 적용 형태, 사용 목적 및 소망하는 효과에 따라서 적절히 조절 가능하며, 함량 대비 효과를 고려하여, 예컨대 전체 조성물 중량에 대하여 0.0001 내지 99.9 중량% 이내에서 사용할 수 있다.The content of the peptidomimetic compound represented by Formula I or II contained in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the use, application form, purpose of use, and desired effect, and considering the effect compared to the content, for example, the total It can be used within 0.0001 to 99.9% by weight based on the weight of the composition.
본 발명의 약학적 조성물은 경구 및 비경구로 투여할 수 있으며, 경구, 경피, 피하, 정맥투여가 가능하며, 또한 경피 부여와 도포에 의한 국부 투여 (Topical application) 방식으로 적용될 수 있다.The pharmaceutical composition of the present invention can be administered orally and parenterally, and can be administered orally, transdermally, subcutaneously, and intravenously. It can also be applied by topical application by transdermal administration and application.
본 발명은 또한, (a) 고체상 (Solid-phase) 합성방법으로 화학식 1로 표시되는 Fmoc-Ala-NH-레진을 수득하는 단계; (b) 상기 단계 (a)에서 수득한 레진에 Fmoc-His(Bis-cyclohexylpropyl)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH 및 Fmoc-γ-Glu-OtBu를 순차적으로 결합시켜 화학식 2로 표시되는 보호화된 펩타이드가 결합된 레진을 수득하는 단계; (c) 상기 단계 (b)에서 수득한 보호화된 펩타이드가 결합된 레진으로부터 레진 및 아미노산의 보호기를 동시에 제거하여 화학식 3으로 표시되는 비정제 펩티도미메틱 화합물을 수득하는 단계; 및 (d) 상기 단계 (c)에서 수득한 비정제 펩티도미메틱 화합물을 정제하여 화학식 I로 표시되는 펩티도미메틱 화합물을 수득하는 단계를 포함하는 화학식 I로 표시되는 펩티도미메틱 화합물의 제조방법에 관한 것이다.The present invention also includes the steps of (a) obtaining Fmoc-Ala-NH-resin represented by Formula 1 by a solid-phase synthesis method; (b) Fmoc-His(Bis-cyclohexylpropyl)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH in the resin obtained in step (a). , sequentially combining Fmoc-Cys(Trt)-OH and Fmoc-γ-Glu-OtBu to obtain a resin to which the protected peptide represented by Formula 2 is bound; (c) simultaneously removing the protective groups of the resin and the amino acid from the resin to which the protected peptide obtained in step (b) is bound, thereby obtaining a purified peptidomimetic compound represented by Formula 3; and (d) purifying the crude peptidomimetic compound obtained in step (c) to obtain a peptidomimetic compound represented by Formula I. It's about.
[화학식 I][Formula I]
[화학식 1][Formula 1]
[화학식 2][Formula 2]
상기 화학식 2에서 R1은 수소 또는 수산기 보호기이고, R2는 티올 보호기이며, R3는 아민 보호기이다.In Formula 2, R 1 is a hydrogen or hydroxyl protecting group, R 2 is a thiol protecting group, and R 3 is an amine protecting group.
[화학식 3][Formula 3]
이하 각 단계별로 본 발명의 화학식 I로 표시되는 펩티도미메틱 화합물의 제조방법을 설명한다. Hereinafter, the method for producing the peptidomimetic compound represented by Formula I of the present invention will be described step by step.
(a) 단계: 고체상 (Solid-phase) 합성방법으로 화학식 1로 표시되는 Fmoc-Ala-NH-레진을 수득Step (a): Obtain Fmoc-Ala-NH-resin represented by Formula 1 by solid-phase synthesis method.
상기 레진으로는 링크아마이드 레진 (Rink Amide Resin), 링크아마이드엠비에이치에이 레진 (Rink Amide MBHA Resin), 링크아마이드에이엠 레진 (Rink Amide AM Resin), 링크아마이드렘 레진 (Rink Amide RAM Resin) 등을 이용할 수 있다.The above resins include Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, and Rink Amide RAM Resin. You can.
본 발명에 있어서 사용되는 용매로는 디클로로메탄 (Dichloromethane), N,N-디메틸포름아마이드 (N,N-Dimethylformamide), N,N-디메틸아세트아마이드 (N,N-Dimethylacetamide), N-메틸피롤리돈 (N-Methylpyrrolidone), 클로로포름 (Chloroform), 1,2-디클로로에탄 (1,2-Dichloroethane), 테트라히드로퓨란 (Tetrahydrofurane), 1,4-디옥산 (1,4-Dioxane), 메탄올 (Methanol), 에탄올 (Ethanol), 이소프로판올 (Isopropanol), 에틸렌 글리콜 (Ethylene glycol), 메틸 아세테이트 (Methyl acetate), 에틸 아세테이트 (Ethyl acetate) 등을 예시할 수 있으나 이에 한정되는 것은 아니다. Solvents used in the present invention include dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrroli. N-Methylpyrrolidone, Chloroform, 1,2-Dichloroethane, Tetrahydrofurane, 1,4-Dioxane, Methanol ), ethanol, isopropanol, ethylene glycol, methyl acetate, ethyl acetate, etc., but are not limited thereto.
(b) 단계: (a)에서 수득한 레진에 Fmoc-His(Bis-cyclohexylpropyl)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH 및 Fmoc-γ-Glu-OtBu를 순차적으로 결합시켜 화학식 2로 표시되는 보호화된 펩타이드가 결합된 레진을 수득Step (b): Add Fmoc-His(Bis-cyclohexylpropyl)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH to the resin obtained in (a). , Fmoc-Cys(Trt)-OH and Fmoc-γ-Glu-OtBu were sequentially combined to obtain a resin to which the protected peptide represented by Formula 2 was bound.
상기 (b) 단계는 다시 (ⅰ) 레진에 결합되어 있는 아미노산 보호기 (N-말단의 아미노기)를 염기로 처리하여 제거하고 레진을 세척하는 단계; 및 (ⅱ) 새롭게 결합시킬 보호화된 아미노산을 세척된 레진에 반응 및 결합시키는 단계를 포함한다.Step (b) further includes (i) removing the amino acid protecting group (N-terminal amino group) bound to the resin by treating it with a base and washing the resin; and (ii) reacting and binding the protected amino acid to be newly combined to the washed resin.
본 발명의 화학식 2에서 카르복실산 보호기 R1은 당 업계에서 통상적으로 이용하는 수소 또는 수산기 보호기를 이용할 수 있다. 상기 수산기 보호기는 터트-부틸 (tert-butyl)기, 트리페닐메틸 (triphenylmethyl 또는 trityl)기, 아세트아미노메틸 (acetaminomethyl)기, 벤조일 (benzoyl)기, 디페닐메틸 (diphenylmethyl)기, 벤질 (benzyl)기, 파라-메톡시벤질 (para-methoxybenzyl)기, 벤질옥시카보닐 (benzyloxycarbonyl)기, 파라-니트로벤질 (para-nitrobenzyl)기, 알릴 (allyl)기, 디메틸실릴 (dimethylsilyl)기, 터트-부틸디메틸실릴 (tert-butyldimethylsilyl)기, 트리이소프로필실릴 (triisopropylsilyl)기, 알킬 (alkyl, 탄소수 10개 이하)기 등을 예시할 수 있으며, 터트-부틸 (tert-butyl)기, 트리페닐메틸 (triphenylmethyl)기 또는 아세트아미노메틸 (acetaminomethyl)기가 바람직하며, 터트-부틸 (tert-butyl)기를 이용하는 것이 보다 바람직하다.In Formula 2 of the present invention, the carboxylic acid protecting group R 1 may be a hydrogen or hydroxyl protecting group commonly used in the art. The hydroxyl protecting group is tert -butyl group, triphenylmethyl or trityl group, acetaminomethyl group, benzoyl group, diphenylmethyl group, and benzyl group. group, para -methoxybenzyl group, benzyloxycarbonyl group, para -nitrobenzyl group, allyl group, dimethylsilyl group, tert-butyl Examples include dimethylsilyl ( tert -butyldimethylsilyl) group, triisopropylsilyl group, alkyl (10 or less carbon atoms) group, tert-butyl ( tert -butyl) group, triphenylmethyl ) group or acetaminomethyl group is preferable, and it is more preferable to use a tert -butyl group.
상기 화학식 2에서 R2는 당 업계에서 통상적으로 이용하는 티올 보호기를 이용할 수 있다. 상기 티올 보호기는 메톡시메틸 (Methoxymethyl)기, 벤질옥시메틸 (Benzyloxymethyl)기, 트리페닐메틸 (Triphenylmethyl)기, 터트-부틸디메틸실릴 (tert-Butyldimethylsilyl)기, 트리페닐실릴 (Triphenylsily)기, 트리이소프로필실릴 (Triispropylsilyl)기, 파라-메톡시벤질 (para-Methylxybenzyl)기, 테트라히드로피란 (Tetrahydropyran)기, 테트라히드로퓨란 (Tetrahydrofuran)기, 터트-부틸 (tert-Butyl)기, 디페닐메틸 (Diphenylmethyl)기, 2-클로로트리틸 (2-Chlorotrityl)기, 벤질 (Benzyl)기, 4-메톡시벤질 (4-Methoxybenzyl)기, 알릴 (Allyl)기, 터트-부틸옥시카르보닐 (tert-Butyloxycarbonyl)기, 아세틸 (Acetyl)기, 벤조일 (Benzoyl)기 등을 예시할 수 있으며, 메톡시메틸 (Methoxymethyl)기, 벤질옥시메틸 (Benzyloxymethyl)기, 트리페닐메틸 (Triphenylmethyl)기, 터트-부틸디메틸실릴 (tert-Butyldimethylsilyl)기, 트리페닐실릴 (Triphenylsily)기 또는 트리이소프로필실릴 (Triisopropylsilyl)기를 이용하는 것이 바람직하며, 2-클로로트리틸 (2-Chlorotrityl)기 또는 트리페닐메틸 (Triphenylmethyl)기를 이용하는 것이 보다 바람직하며, 티올 보호기가 트리페닐메틸 (Triphenylmethyl)기를 이용하는 것이 가장 바람직하지만 이에 국한되는 것은 아니다.In Formula 2, R 2 may use a thiol protecting group commonly used in the industry. The thiol protecting group includes a methoxymethyl group, a benzyloxymethyl group, a triphenylmethyl group, a tert -Butyldimethylsilyl group, a triphenylsilyl group, and a triiso Triispropylsilyl group, para -Methylxybenzyl group, Tetrahydropyran group, Tetrahydrofuran group, tert -Butyl group, Diphenylmethyl ) group, 2-Chlorotrityl group, Benzyl group, 4-Methoxybenzyl group, Allyl group, tert -Butyloxycarbonyl Group, acetyl (Acetyl) group, benzoyl (Benzoyl) group, etc. can be exemplified, methoxymethyl group, benzyloxymethyl group, triphenylmethyl group, tert-butyldimethylsilyl ( It is preferable to use a tert -Butyldimethylsilyl group, a Triphenylsilyl group, or a Triisopropylsilyl group, and it is more preferable to use a 2-Chlorotrityl group or a Triphenylmethyl group. It is most preferable that the thiol protecting group uses a triphenylmethyl group, but is not limited to this.
상기 화학식에서 R3은 당 업계에서 통상적으로 이용하는 아민 보호기를 이용할 수 있다. 아세틸 (Acetyl)기, 벤조일(Benzoyl), 벤질옥시메틸 (Benzyloxymethyl)기, 트리틸 (Trityl)기, Ddz (α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz))기, Bpoc (2-(4-Biphenyl)isopropoxycarbonyl (Bpoc))기, Nps (2-Nitrophenylsulfenyl (Nps))기, Fmoc (9-Fluorenylmethoxycarbonyl (Fmoc))기, Nsc (2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc))기, Bsmoc (1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc))기, α-Nsmoc ( (1,1-Dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc))기, Dde & ivDde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-mehtylbutyl (ivDde))기, Fmoc* (2,7-Di-tert-butyl-Fmoc (Fmoc*))기, Fmoc(2F) (2-Fluoro-Fmoc (Fmoc(2F)))기, 알릴 (allyl)기, mio-Fmoc & dio-Fmoc (2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7-Diisooctyl-Fmoc (dio-Fmoc))기, TCP (Tetrachlorophthaloyl (TCP))기, Pms (2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms))기, Esc (Ethanesulfonylethoxycarbonyl (Esc))기, Sps (2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps))기, Z (Benzyloxycarbonyl (Z))기, Alloc (Allyloxycarbonyl (Alloc))기, oNBS & pNBS (o-Nitrobenzenesulfonyl (oNBS) and p-nitrobenzenesulfonyl (pNBS)기, dNBS (2,4-Dinitrobenzenesulfonyl (dNBS))기, Bts (Benzothiazole-2-sulfonyl (Bts))기, Troc (2,2,2-Trichloroethyloxycarbonyl (Troc))기, Dts (Dithiasuccinoyl (Dts))기, pNZ (p-Nitrobenzyloxycarbonyl (pNZ))기, Poc (Propargyloxycarbonyl (Poc))기, oNZ & NVOC (o-Nitrobenzyloxycarbonyl (oNZ) and 6-Nitroveratryloxycarbonyl (NVOC))기, NPPOC (2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC))기, MNPPOC ( 2-(3,4-Methylenedioxy-6-nitrophenyl)propyloxycarbonyl (MNPPOC))기, BrPhF (9-(4-Bromophenyl)-9-fluorenyl (BrPhF))기, Azoc (Azidomethyloxycarbonyl (Azoc))기, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-Z))기, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기 등을 예시할 수 있으며, Azoc (Azidomethyloxycarbonyl (Azoc))기, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-Z))기, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기를 이용하는 것이 바람직하며, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기를 이용하는 것이 보다 바람직하며, 아민 보호기가 Boc (tert-Butyloxycarbonyl (Boc))기를 이용하는 것이 가장 바람직하다.In the above formula, R 3 may use an amine protecting group commonly used in the art. Acetyl group, Benzoyl, Benzyloxymethyl group, Trityl group, Ddz (α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz)) group, Bpoc (2-( 4-Biphenyl)isopropoxycarbonyl (Bpoc)) group, Nps (2-Nitrophenylsulfenyl (Nps)) group, Fmoc (9-Fluorenylmethoxycarbonyl (Fmoc)) group, Nsc (2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc)) group, Bsmoc (1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc)) group, α-Nsmoc ( (1,1-Dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc)) Group, Dde & ivDde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-2,6-dioxocyclohex- 1-ylidene)-3-mehtylbutyl (ivDde)) group, Fmoc* (2,7-Di-tert-butyl-Fmoc (Fmoc*)) group, Fmoc(2F) (2-Fluoro-Fmoc (Fmoc(2F) )) group, allyl group, mio-Fmoc & dio-Fmoc (2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7-Diisooctyl-Fmoc (dio-Fmoc)) group, TCP (Tetrachlorophthaloyl (TCP) ) group, Pms (2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms)) group, Esc (Ethanesulfonylethoxycarbonyl (Esc)) group, Sps (2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps)) group, Z (Benzyloxycarbonyl (Z)) group, Alloc (Allyloxycarbonyl (Alloc)) group, oNBS & pNBS (o-Nitrobenzenesulfonyl (oNBS) and p-nitrobenzenesulfonyl (pNBS) group, dNBS (2,4-Dinitrobenzenesulfonyl (dNBS)) group, Bts (Benzothiazole -2-sulfonyl (Bts)) group, Troc (2,2,2-Trichloroethyloxycarbonyl (Troc)) group, Dts (Dithiasuccinoyl (Dts)) group, pNZ (p-Nitrobenzyloxycarbonyl (pNZ)) group, Poc (Propargyloxycarbonyl (Poc) )) group, oNZ & NVOC (o-Nitrobenzyloxycarbonyl (oNZ) and 6-Nitroveratryloxycarbonyl (NVOC)) group, NPPOC (2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC)) group, MNPPOC ( 2-(3,4-Methylenedioxy -6-nitrophenyl)propyloxycarbonyl (MNPPOC)) group, BrPhF (9-(4-Bromophenyl)-9-fluorenyl (BrPhF)) group, Azoc (Azidomethyloxycarbonyl (Azoc)) group, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-) Z)) group, Boc (tert-Butyloxycarbonyl (Boc)) group, Mtt (4-Methyltrityl (Mtt)) group, etc., Azoc (Azidomethyloxycarbonyl (Azoc)) group, Cl-Z (2-Chlorobenzyloxycarbonyl ( Cl-Z)) group, Boc (tert-Butyloxycarbonyl (Boc)) group, and Mtt (4-Methyltrityl (Mtt)) group are preferably used. Boc (tert-Butyloxycarbonyl (Boc)) group, Mtt (4-Methyltrityl ( It is more preferable to use the Mtt)) group, and it is most preferable to use the Boc (tert-Butyloxycarbonyl (Boc)) group as the amine protecting group.
상기 아미노산 보호기 (N-말단의 아미노기)를 제거하는 염기로는 피페리딘 (Piperidine), 4-메틸피페리딘 (4-Methylpiperidine), 피롤리딘 (Pyrrolidine), 피페라진 (Piperazine), 하이드라진 하이드레이트 (Hydrazine hydrate), DBU (1,8-DiazaBisyclo[5.4.0]undec-7-ene), 4-메틸피페리딘 (4-Methylpiperidine), 1-메틸-3-부틸이미다졸리윰 테트라플로오로보란 (1-methyl-3-butyl imidazolium BF4), 에탄올아민 (Ethanolamine), 시클로헥실아민 (Cyclohexylamine), 디시클로헥실아민 (Dicyclohexylamine), 트리스(2-아미노에틸)아민 (Tris(2-aminoethyl)amine), 1,3-디시클로헥산비스-(메틸아민) (1,3-Dicyclohexanebis-(methylamine)), 1,4-비스-(3-아미노프로필)피페라진 (1,4-Bis-(3-aminopropyl)piperazine), 디에틸아민 (Diethylamine), 4-디메틸아미노피리딘 (4-Dimethylaminopyridine) 등의 유기 염기 또는 수산화 리튬 (Lithium hydroxide), 수산화 나트륨 (Sodium Hydroxide), 수산화 칼슘 (Calcium Hydroxide), 수산화 칼륨 (Potassium Hydroxide) 등의 무기 염기를 이용할 수 있다.Bases that remove the amino acid protecting group (N-terminal amino group) include piperidine, 4-methylpiperidine, pyrrolidine, piperazine, and hydrazine hydrate. (Hydrazine hydrate), DBU (1,8-DiazaBisyclo[5.4.0]undec-7-ene), 4-Methylpiperidine, 1-methyl-3-butylimidazolium tetrafluoro Borane (1-methyl-3-butyl imidazolium BF4), Ethanolamine, Cyclohexylamine, Dicyclohexylamine, Tris(2-aminoethyl)amine ), 1,3-Dicyclohexanebis-(methylamine) (1,3-Dicyclohexanebis-(methylamine)), 1,4-bis-(3-aminopropyl)piperazine (1,4-Bis-(3 -aminopropyl)piperazine), diethylamine, 4-dimethylaminopyridine, or organic bases such as lithium hydroxide, sodium hydroxide, calcium hydroxide, hydroxide Inorganic bases such as potassium hydroxide can be used.
본 발명에 있어서, 보호화된 아미노산을 세척된 레진에 반응 및 결합시킬 때 사용하는 시약으로는 DCC (N,N-Dicyclohexylcarbodiimide), DIC (N,N-Diisopropylcarbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), PyBrOP (Bromo-tripyrrolidino-phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate), PyOxim (Ethyl cyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), HCTU (2-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N',N',-tetramethylaminium hexafluorophosphate), HDMC (N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uronium hexafluorophosphate N-oxide), TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminium), COMU (1-[1-(Cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate), TOTT (2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethyl-isothiouronium tetrafluoroborate), EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), TFFH (Tetramethylfluoroformamidinium hexafluorophosphate), EEDQ (N-Ethoxycarbony-2-ethoxy-1,2-dihydro-quinoline), T3P (2-Propanephosphonic acid anhydride), DEPBT (3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate), HOBt (1-Hydroxybenzotriazole), HOOBt(HODhbt, Hydroxy-3,4-dihydro-4-ox-1,2,3-benzo-triazine), BTC (bis-Trichloromethylcarbonate 또는 Triphosgene), CDI (1,1'-Carbonyldiimidazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole), HOSu (N-Hydroxysuccinimide) 등을 예시할 수 있다. In the present invention, the reagents used to react and bind the protected amino acid to the washed resin include DCC (N,N-Dicyclohexylcarbodiimide), DIC (N,N-Diisopropylcarbodiimide), and BOP (Benzotriazole-1-yl- oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), PyBrOP (Bromo-tripyrrolidino-phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate) , PyOxim (Ethyl cyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), HCTU (2-(6 -Chloro-1H-benzotriazol-1-yl)-N,N,N',N',-tetramethylaminium hexafluorophosphate), HDMC (N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino] -uronium hexafluorophosphate N-oxide), TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1 ,1,3,3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminium), COMU (1-[1-(Cyano-2-ethoxy -2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate), TOTT (2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethyl-isothiouronium tetrafluoroborate), EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), TFFH (Tetramethylfluoroformamidinium hexafluorophosphate), EEDQ (N-Ethoxycarbony-2-ethoxy-1,2-dihydro-quinoline), T3P (2-Propanephosphonic acid anhydride), DEPBT (3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate), HOBt (1-Hydroxybenzotriazole), HOOBt(HODHbt, Hydroxy-3,4-dihydro-4 -ox-1,2,3-benzo-triazine), BTC (bis-Trichloromethylcarbonate or Triphosgene), CDI (1,1'-Carbonyldiimidazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt ( Examples include 1-Hydroxyazabenzotriazole), HOSu (N-Hydroxysuccinimide), etc.
(c) 단계: (b)에서 수득한 보호화된 펩타이드가 결합된 레진으로부터 레진 및 아미노산의 보호기를 동시에 제거하여 화학식 3으로 표시되는 비정제 펩티도미메틱 화합물을 수득Step (c): Simultaneously remove the protecting groups of the resin and the amino acid from the resin to which the protected peptide obtained in (b) is bound, to obtain a purified peptidomimetic compound represented by Formula 3.
상기 레진 및 아미노산의 보호기를 동시에 제거하는 과정은 산성 용액의 존재 하에서 수행되는데, 상기 산성 용액은 TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA/phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/water (95/5), TFA/DCM/indole (70/28/2) 및 TFA/TIPS/water (95/2.5/2.5) 등의 용액을 예시할 수 있다. 상기 산성 용액의 혼합비는 부피%를 기준으로 한다.The process of simultaneously removing the protecting groups of the resin and the amino acid is performed in the presence of an acidic solution, which includes TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2 ), TFA/phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5) , TFA/water (95/5), TFA/DCM/indole (70/28/2), and TFA/TIPS/water (95/2.5/2.5). The mixing ratio of the acidic solution is based on volume %.
(d) 단계: (c) 단계에서 수득한 비정제 펩티도미메틱 화합물을 정제하여 화학식 I로 표시되는 펩티도미메틱을 수득Step (d): Purifying the crude peptidomimetic compound obtained in step (c) to obtain the peptidomimetic represented by formula (I).
본 발명에 있어서, 정제는 펩타이드 정제에 통상적으로 이용되는 방법으로 정제 가능하며, 순상 또는 역상 고성능액체크로마토그래피 (HPLC)를 이용할 수 있다. 정제시 사용되는 용매로는 메탄올 (Methanol), 에탄올 (Ethanol), 이소프로판올 (Isopropanol), 아세토니트릴 (Acetonitrile), 정제수 및 이들의 혼합용액으로 구성된 군에서 선택될 수 있으며, 삼불화아세트산 (Trifluoroacetic acid), 아세트산 (Acetic acid), 포름산 (Formic acid) 등의 산을 함께 이용할 수 있다.In the present invention, purification can be done by a method commonly used for peptide purification, and normal phase or reverse phase high performance liquid chromatography (HPLC) can be used. Solvents used during purification may be selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, purified water, and mixed solutions thereof, and trifluoroacetic acid. , Acetic acid, formic acid, etc. can be used together.
본 발명은 또한, 화학식 I로 표시되는 펩티도미메틱 화합물에 -SH기 산화 시약을 혼합하고, pH를 6-9로 조절하여 디설파이드 결합(S-S)을 수행하는 것을 특징으로 하는 화학식 II로 표시되는 펩티도미메틱 화합물의 제조방법에 관한 것이다.The present invention also provides a peptidomimetic compound represented by Formula II, which is characterized in that the peptidomimetic compound represented by Formula I is mixed with an -SH group oxidation reagent, and the pH is adjusted to 6-9 to perform disulfide bonding (S-S). It relates to a method for producing dometic compounds.
[화학식 II] [Formula II]
본 발명에 있어서, -SH기 산화 시약은 Dithiothreitol (DTT), 2-Mercaptoethanol (2-ME), Glutathione (GSH), Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), Dithiobutylamine (DTBA), Cysteine hydrochloride (Cys-HCl), 2-Aminoethanethiol (2-MEA-HCl), 탄산수소나트륨(NaHCO3) 수용액, 탄산칼륨(K2CO3) 수용액, NH4OH(암모니아수), 수산화나트륨(NaOH) 수용액, Oxido-Shuffling Systems(GSH/GSSG, 2-ME/HED, DTT/oxidized DTT, GroEL/GroES, Hsp70, Hsp100) 등을 이용할 수 있으며, 반응 농도는 10-1~ 10-5 M 농도의 범위에서 가능하다.In the present invention, the -SH group oxidation reagent includes Dithiothreitol (DTT), 2-Mercaptoethanol (2-ME), Glutathione (GSH), Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), Dithiobutylamine (DTBA), Cysteine hydrochloride ( Cys-HCl), 2-Aminoethanethiol (2-MEA-HCl), sodium bicarbonate (NaHCO 3 ) aqueous solution, potassium carbonate (K 2 CO 3 ) aqueous solution, NH 4 OH (ammonia water), sodium hydroxide (NaOH) aqueous solution, Oxido -Shuffling Systems (GSH/GSSG, 2-ME/HED, DTT/oxidized DTT, GroEL/GroES, Hsp70, Hsp100) can be used, and the reaction concentration is possible in the range of 10 -1 to 10 -5 M concentration. .
본 발명에 있어서, pH를 6~9로 조절하기 위한 시약으로서, 상기 범위의 pH로 조절 가능한 것이라면 특별한 제한없이 사용이 가능하지만, NH4OH(암모니아수)를 사용하는 것이 바람직하다.In the present invention, as a reagent for adjusting pH to 6 to 9, any reagent that can be adjusted to pH in the above range can be used without particular limitation, but NH 4 OH (ammonia water) is preferably used.
제조된 화학식 II로 표시되는 펩티도미메틱 화합물의 정제는 펩타이드 정제에 통상적으로 이용되는 방법으로 정제 가능하며, 순상 또는 역상 고성능액체크로마토그래피 (HPLC)를 이용할 수 있다. 정제시 사용되는 용매로는 메탄올 (Methanol), 에탄올 (Ethanol), 이소프로판올 (Isopropanol), 아세토니트릴 (Acetonitrile), 정제수 및 이들의 혼합용액으로 구성된 군에서 선택될 수 있으며, 삼불화아세트산 (Trifluoroacetic acid), 아세트산 (Acetic acid), 포름산 (Formic acid) 등의 산을 함께 이용할 수 있다.The prepared peptidomimetic compound represented by Formula II can be purified by a method commonly used for peptide purification, and normal phase or reverse phase high performance liquid chromatography (HPLC) can be used. Solvents used during purification may be selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, purified water, and mixed solutions thereof, and trifluoroacetic acid. , Acetic acid, formic acid, etc. can be used together.
이하, 본 발명을 실시 예에 의해 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시 예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
[실시예][Example]
본 명세서 전체에 거쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 한 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and liquid (weight/volume) %, unless otherwise specified. /Liquid is (volume/volume) %.
실시 예 1 : H-Ala-NH-레진 (Rink Amide)의 제조Example 1: Preparation of H-Ala-NH-Resin (Rink Amide)
여과막이 장착된 고체상 (solid-phase) 합성 반응기에 링크아마이드 에이엠 레진 (치환율 = 0.70 mmol/g, 100 mmole) 및 N,N-디메틸포름아마이드 (1000 ml, 이하 DMF)를 넣고, 10분간 레진을 팽창시킨 후, 감압 하에서 여과막을 통하여 용매를 제거하였다. Fmoc-Ala-OH (분자량 = 311.3 g/mol) (93.39 g, 300 mmol, 3.0 당량), Oxyma (분자량 = 142.11 g/mol, 46.90 g, 330 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 41.7 g, 330 mmole)을 적가한 다음 상온에서 4시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반한 다음, 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하였다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-Ala-NH-레진(Rink Amide)을 수득하였다 (치환율: 0.7 mmol/g, 수율 >99%).Linkamide AM resin (substitution rate = 0.70 mmol/g, 100 mmole) and N,N-dimethylformamide (1000 ml, hereinafter referred to as DMF) were added to a solid-phase synthesis reactor equipped with a filtration membrane, and the resin was mixed for 10 minutes. After swelling, the solvent was removed through a filtration membrane under reduced pressure. Fmoc-Ala-OH (molecular weight = 311.3 g/mol) (93.39 g, 300 mmol, 3.0 equivalent) and Oxyma (molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution and then gently stirred at room temperature for 4 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine/DMF solution was added and stirred for 15 minutes, then the reaction solution was removed by filtration under reduced pressure, and the resin was treated one more time in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-Ala-NH-resin (Rink Amide) was obtained (substitution rate: 0.7 mmol/g, yield >99%).
실시 예 2 : H-His(Bis-cyclohexylpropyl)-Ala-NH-레진 (Rink Amide)의 제조Example 2: Preparation of H-His(Bis-cyclohexylpropyl)-Ala-NH-Resin (Rink Amide)
H-Ala-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-His(Bis-cyclohexylpropyl)-OH (분자량 = 626.85 g/mol) (188.06 g, 300 mmol, 3.0 당량) 과 Oxyma (분자량 = 142.11 g/mol, 46.90 g, 330 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 41.7 g, 330 mmole)을 적가한 다음 상온에서 8시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반하였다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하고, DMF 1000 ml으로 총 6회 레진을 세척한 후 H-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-His(Bis-cyclohexylpropyl)-OH (molecular weight = 626.85 g/mol) (188.06 g, 300 mmol, 3.0 equivalent) and Oxyma in a reactor containing H-Ala-NH-resin (Rink Amide) (100 mmole) (Molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) was dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution and then gently stirred at room temperature for 8 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, the resin was treated one more time in the same manner, the resin was washed a total of 6 times with 1000 ml of DMF, and H-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) was added. Obtained (yield >99%).
실시 예 3 : H-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진 (Rink Amide) 제조Example 3: Preparation of H-Val-His(Bis-cyclohexylpropyl)-Ala-NH-Resin (Rink Amide)
H-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-Val-OH (분자량 = 339.4 g/mol, 101.82 g, 300 mmole)과 Oxyma (분자량 = 142.11 g/mol, 46.90 g, 330 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 41.7 g, 330 mmole)을 적가한 다음 상온에서 4시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반하였다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하였다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-Val-OH (molecular weight = 339.4 g/mol, 101.82 g, 300 mmole) and Oxyma (molecular weight) were added to a reactor containing H-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) (100 mmole). = 142.11 g/mol, 46.90 g, 330 mmole) was dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution and then gently stirred at room temperature for 4 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated one more time in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) was obtained (yield >99%).
실시 예 4 : H-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진 (Rink Amide) 제조Example 4: Preparation of H-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-Resin (Rink Amide)
H-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-Leu-OH (분자량 = 353.4 g/mol, 101.82 g, 300 mmole)과 Oxyma (분자량 = 142.11 g/mol, 46.90 g, 330 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 41.7 g, 330 mmole)을 적가한 다음 상온에서 4시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반하였다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리였다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-Leu-OH (molecular weight = 353.4 g/mol, 101.82 g, 300 mmole) and Oxyma were added to a reactor containing H-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) (100 mmole). (Molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) was dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution and then gently stirred at room temperature for 4 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated one more time in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) was obtained (yield >99%).
실시 예 5 : H-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진 (Rink Amide) 제조Example 5: Preparation of H-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-Resin (Rink Amide)
H-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-Lys(Boc)-OH (분자량 = 468.5 g/mol, 140.55 g, 300 mmole)과 Oxyma (분자량 = 142.11 g/mol, 46.90 g, 330 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 41.7 g, 330 mmole)을 적가한 다음 상온에서 4시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반하였다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하였다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-Lys(Boc)-OH (molecular weight = 468.5 g/mol, 140.55 g, 300 mmole) and Oxyma (molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution and then gently stirred at room temperature for 4 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated one more time in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) was obtained (yield >99%).
실시 예 6 : H-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진 (Rink Amide) 제조Example 6: Preparation of H-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-Resin (Rink Amide)
H-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-Gly-OH (분자량 = 297.3 g/mol, 148.65 g, 300 mmole)과 Oxyma (분자량 = 142.11 g/mol, 78.16 g, 550 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 69.41 g, 550 mmole)을 적가한 다음 상온에서 4시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반하였다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하였다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-Gly-OH (molecular weight = 297.3 g/mol, 148.65 g, 300 mmole) and Oxyma (molecular weight = 142.11 g/mol, 78.16 g, 550 mmole) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 69.41 g, 550 mmole) was added dropwise to the mixed reaction solution and then gently stirred at room temperature for 4 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated one more time in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) was obtained (yield >99%). .
실시 예 7 : H-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진 (Rink Amide) 제조Example 7: Preparation of H-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-Resin (Rink Amide)
H-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-Cys(Trt)-OH (분자량 = 585.7 g/mol, 292.85 g, 300 mmole)과 Oxyma (분자량 = 142.11 g/mol, 78.16 g, 550 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 69.41 g, 550 mmole)을 적가한 다음 상온에서 4시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반하였다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하였다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-Cys(Trt)-OH (molecular weight = 585.7) in a reactor containing H-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) (100 mmole). g/mol, 292.85 g, 300 mmole) and Oxyma (molecular weight = 142.11 g/mol, 78.16 g, 550 mmole) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 69.41 g, 550 mmole) was added dropwise to the mixed reaction solution and then gently stirred at room temperature for 4 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated one more time in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) was obtained ( Yield >99%).
실시 예 8 : H-γ-Glu-(OtBu)-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진 (Rink Amide) 제조Example 8: Preparation of H-γ-Glu-(OtBu)-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-Resin (Rink Amide)
H-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)(100 mmole)이 들어있는 반응기에 Fmoc-γ-Glu-OtBu (분자량 = 425.5 g/mol, 212.75 g, 500 mmole)과 Oxyma (분자량 = 142.11 g/mol, 78.16 g, 550 mmole)을 DMF 1000 ml에 용해한 다음 투입하였다. 혼합된 반응액에 DIC (분자량 = 126.2 g/mol, 69.41 g, 550 mmole)을 적가한 다음 상온에서 4시간 동안 서서히 교반하였다. 레진을 DMF 1000 ml으로 2회 세척한 후, 20% 피페리딘/DMF 용액 1000 ml를 더하고 15분 동안 교반하였다. 감압 여과하여 반응액을 제거하고, 레진을 동일하게 1회 더 처리하였다. DMF 1000 ml으로 총 6회 레진을 세척한 후 H-γ-Glu-(OtBu)-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)을 수득하였다 (수율 >99%).Fmoc-γ-Glu-OtBu in a reactor containing H-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) (100 mmole). (molecular weight = 425.5 g/mol, 212.75 g, 500 mmole) and Oxyma (molecular weight = 142.11 g/mol, 78.16 g, 550 mmole) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 69.41 g, 550 mmole) was added dropwise to the mixed reaction solution and then gently stirred at room temperature for 4 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated one more time in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-γ-Glu-(OtBu)-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-resin (Rink Amide) was obtained (yield >99%).
실시 예 9 : Crude H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl) -Ala-NH₂ 제조Example 9: Preparation of Crude H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH₂
H-γ-Glu-(OtBu)-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-레진(Rink Amide)을 반응기에 투입하고 냉각된 TFA/TIPS/water (95/2.5/2.5) 용액 3 L를 서서히 부어 넣은 다음 상온에서 3시간 동안 교반하였다. 냉각된 디에틸에테르 12 L에 반응액을 서서히 적가하여 펩타이드를 석출시켰다. 실온에서 30분 동안 교반한 다음 여과하여 펩타이드를 회수한 후, 용매를 감압 제거하였다. 진공 건조기에서 7시간 동안 건조 시켜, 백색의 Crude H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH2 105.9 g을 수득하였다. (분자량 1105.46 g/mol, Crude peptide 순도 72.5%, 수율 95.8%) H-γ-Glu-(OtBu)-Cys(Trt)-Gly-Lys(Boc)-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH-Resin (Rink Amide) was added to the reactor and cooled TFA. 3 L of /TIPS/water (95/2.5/2.5) solution was slowly poured into the solution and stirred at room temperature for 3 hours. The reaction solution was slowly added dropwise to 12 L of cooled diethyl ether to precipitate the peptide. After stirring at room temperature for 30 minutes, the peptide was recovered by filtration, and the solvent was removed under reduced pressure. Dry in a vacuum dryer for 7 hours to produce white Crude H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH 2 105.9 g was obtained. (Molecular weight 1105.46 g/mol, crude peptide purity 72.5%, yield 95.8%)
실시 예 10 : H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl) -Ala-NH₂제조Example 10: Preparation of H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH₂
실시예 9에서 수득한 Crude H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH2 105.9 g을 정제수에 용해한 다음 0.46μm membrane으로 여과하였다. 여과액을 산업용 HPLC (230 nm, 500 ml/분, 10 미크론 C18 컬럼에서 20분 내에 0.1% TFA 내 아세토니트릴 초기농도 15%에서 45%로 증가)로 반복 주입한 후 main fraction 부분을 분획하여 회전 증발기로 용매를 농축한 다음 동결건조하여 화학식 I로 표시되는 펩티도미메틱 화합물 I(WP-30104GTT, 서열번호 1) 46.7 g (수율: 44.1%, 순도: 97.3%)을 수득하였다.Crude H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH 2 obtained in Example 9 105.9 g was dissolved in purified water and then filtered through a 0.46μm membrane. The filtrate was repeatedly injected into industrial HPLC (230 nm, 500 ml/min, 10 micron C18 column, initial concentration of acetonitrile in 0.1% TFA increased from 15% to 45% within 20 minutes), then the main fraction was separated and spun. The solvent was concentrated using an evaporator and then lyophilized to obtain 46.7 g (yield: 44.1%, purity: 97.3%) of peptidomimetic compound I (WP-30104GTT, SEQ ID NO: 1) represented by Formula I.
실시 예 11 : H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl) -Ala-NH₂ S-S Dimer 제조Example 11: Preparation of H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH₂ S-S Dimer
실시예 10에서 수득한 H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH₂ 20 g을 0.1M 암모니아수 2L에 용해한 다음 정제수에 용해한 다음 상온에서 12시간 동안 교반하여 디설파이드 결합(S-S)을 수행하였다. 이후 결합의 안정화를 위하여 반응액에 TFA를 첨가하여 pH 3.0 이하로 산성화시킨 다음 농축하고 0.46μm membrane으로 여과하였다.20 g of H-γ-Glu-Cys-Gly-Lys-Leu-Val-His(Bis-cyclohexylpropyl)-Ala-NH₂ obtained in Example 10 was dissolved in 2L of 0.1M aqueous ammonia and then dissolved in purified water and incubated at room temperature for 12 hours. Disulfide bonding (S-S) was performed by stirring for a while. Afterwards, to stabilize the binding, TFA was added to the reaction solution to acidify it to pH 3.0 or lower, and then concentrated and filtered through a 0.46μm membrane.
여과액을 산업용 HPLC (230 nm, 500 ml/분, 10 미크론 C18 컬럼에서 20분 내에 0.1% TFA 내 아세토니트릴 초기농도 15%에서 45%로 증가)로 주입한 후 main fraction 부분을 분획하여 동결건조 후 화학식 II로 표시되는 펩티도미메틱 화합물 II(WP-30104GTT S-S Dimer, 서열번호 2) 16.8 g (Dimer 분자량 : 2208.9 g/mol, 수율: 84.0%, 순도: 98.4%)을 수득하였다.The filtrate was injected into industrial HPLC (230 nm, 500 ml/min, 10 micron C18 column, initial concentration of acetonitrile in 0.1% TFA increased from 15% to 45% within 20 minutes), then the main fraction was fractionated and lyophilized. Then, 16.8 g of peptidomimetic compound II (WP-30104GTT S-S Dimer, SEQ ID NO: 2) represented by Formula II (Dimer molecular weight: 2208.9 g/mol, yield: 84.0%, purity: 98.4%) was obtained.
실험 예 1 : 티오플라빈 T(ThT) 형광세기를 이용한 베타아밀로이드 응집체 형성 저해능 평가Experimental Example 1: Evaluation of the ability to inhibit beta-amyloid aggregate formation using thioflavin T (ThT) fluorescence intensity
본 발명에 따른 펩티도미메틱 화합물의 베타아밀로이드 응집체 형성 저해능을 측정하기 위해 티오플라빈 T(ThT) 형광세기를 이용하여 하기의 실험을 수행하였다. 티오플라빈 T(ThT)는 용액 중에 있는 베타아밀로이드 피브릴에 잘 결합하여 베타아밀로이드 피브릴의 농도가 증가함에 따라 형광 세기가 증가하는 경향을 나타낸다.To measure the ability of the peptidomimetic compound according to the present invention to inhibit beta-amyloid aggregate formation, the following experiment was performed using thioflavin T (ThT) fluorescence intensity. Thioflavin T (ThT) binds well to beta-amyloid fibrils in solution, and its fluorescence intensity tends to increase as the concentration of beta-amyloid fibrils increases.
실시예 10 및 실시예 11에서 제조된 펩티도미메틱 화합물(I 및 II)과 ThT의 결합 활성은 검은색 96 well 평평한 바닥 플레이트에서 측정하였다. The binding activity of the peptidomimetic compounds (I and II) prepared in Examples 10 and 11 and ThT was measured in a black 96 well flat bottom plate.
응집에 대한 펩티도미메틱 화합물의 영향을 조사하는 아밀로이드 베타42(이하 "Aβ42") ThT 분석의 경우, Aβ42(10μM)를 CuCl2 (10μM) 및 펩티도미메틱 화합물(30μM)의 부재 또는 존재하에 HEPES 용액(20mM, 150mM NaCl, ThT(20μM)를 포함하는 pH 7.4) 플레이트를 흔들지 않고 37℃에서 인큐베이션 하였다. For the amyloid beta42 (hereafter “Aβ42”) ThT assay investigating the effect of peptidomimetic compounds on aggregation, Aβ42 (10 μM) was incubated with HEPES in the absence or presence of CuCl 2 (10 μM) and peptidomimetic compounds (30 μM). The plate of solution (pH 7.4 containing 20mM, 150mM NaCl, ThT (20μM)) was incubated at 37°C without shaking.
특정 시점에서 Aβ42 응집체에 대한 ThT의 결합에 의해 생성된 형광은 각각 430 및 492 nm의 여기 및 방출 파장에서 플레이트 판독기 (FLUOstarOmega, BMG labtech, Ortenberg, Germany)를 사용하여 측정하였다. 반응 조건에서 주로 IFE(1차 내부 필터 효과)를 고려해야 할 경우 Beer-Lambert의 법칙에 따라 아래 식을 실험 결과에 적용하였다.The fluorescence produced by the binding of ThT to Aβ42 aggregates at specific time points was measured using a plate reader (FLUOstarOmega, BMG labtech, Ortenberg, Germany) at excitation and emission wavelengths of 430 and 492 nm, respectively. When IFE (first-order internal filter effect) had to be mainly considered in the reaction conditions, the equation below was applied to the experimental results according to Beer-Lambert's law.
즉, Aβ42 10 μM 농도, 37℃에서 double orbital agitation을 통해 Aβ42의 fibrillation을 유도하였으며, excitation 파장과 emission 파장은 각각 445nm, 490nm로 설정하여 형광(fluorescence)를 측정하였다. 총 17시간 동안 측정하여 Aβ42의 fibrillation이 saturation 되는 것을 확인하였다. That is, fibrillation of Aβ42 was induced through double orbital agitation at a concentration of 10 μM Aβ42 at 37°C, and fluorescence was measured by setting the excitation and emission wavelengths to 445 nm and 490 nm, respectively. Measurements were made for a total of 17 hours to confirm that saturation of Aβ42 fibrillation was observed.
도 2로부터, 펩티도미메틱 화합물을 처리하지 않고, Aβ42만 사용한 경우에는 반응 시작 직후 형광강도(fluorescence intensity)가 증가하기 시작하였으며, 약 3시간 후에 saturation되는 것을 확인하였다. From Figure 2, when only Aβ42 was used without treatment with the peptidomimetic compound, the fluorescence intensity began to increase immediately after the start of the reaction, and saturation was confirmed after about 3 hours.
반면, Aβ42에 펩티도미메틱 후보물질을 1:2 (펩티도미메틱 후보물질 농도 20 μM)와 1:10 (펩티도미메틱 후보물질 농도 100 μM)의 비율로 추가하여 ThT assay를 진행한 경우에는 펩티도미메틱 화합물이 Aβ42의 아밀로이드 피브릴 형성 lag-time을 지연시켰으며 saturation 후의 형광강도도 낮춘다는 것을 알 수 있었다. On the other hand, when the ThT assay was performed by adding the peptidomimetic candidate to Aβ42 at a ratio of 1:2 (peptidomimetic candidate concentration 20 μM) and 1:10 (peptidomimetic candidate concentration 100 μM), the peptidomimetic candidate was It was found that the domimetic compound delayed the lag-time of amyloid fibril formation of Aβ42 and also lowered the fluorescence intensity after saturation.
Aβ42와 펩티도미메틱 화합물의 몰비율이 1:2인 경우에도 화합물 I 및 화합물 II는 Aβ42 아밀로이드 피브릴 형성을 40% 이상 저해하는 효능을 나타내었다.(도 2)Even when the molar ratio of Aβ42 and peptidomimetic compound was 1:2, Compound I and Compound II showed the efficacy of inhibiting Aβ42 amyloid fibril formation by more than 40% (Figure 2).
실험 예 2 : Transmission electron microscopy (TEM) 평가Experimental Example 2: Transmission electron microscopy (TEM) evaluation
단량체화된 Aβ42를 HEPES 완충액(20mM, 150mM NaCl, pH 7.4)의 최종 단백질 농도가 10μM이 되도록 하고 펩티도미메틱 화합물(30μM)의 부재 또는 존재 하에서 교반없이 37℃에서 24시간 동안 인큐베이션하였다. 37℃에서 흔들지 않고 200 메쉬 탄소 코팅된 Copper 그리드(SPI 공급, West Chester, PA)에 응집 분석의 부분 표본(5 μL)을 스포팅하여 TEM 연구를 위해 샘플을 준비하였다. 그런 다음 샘플을 여과지로 닦아내고 건조한 다음 샘플을 2% uranyl acetate in water(5 μL)로 30초 동안 음성 염색하고 물로 세척하고 여과지로 닦아내고 다시 건조하였다. 그런 다음 샘플을 200kV에서 작동하는 TecnaiG2 F20 TEM(FEI Tecnai™ G2, Hillsboro, Oregon)으로 분석하였다.Monomerized Aβ42 was incubated in HEPES buffer (20mM, 150mM NaCl, pH 7.4) to a final protein concentration of 10μM and incubated at 37°C for 24h without agitation in the absence or presence of peptidomimetic compounds (30μM). Samples were prepared for TEM studies by spotting aliquots (5 μL) of the aggregation assay onto 200 mesh carbon-coated copper grids (SPI Supply, West Chester, PA) without shaking at 37°C. Then, the sample was wiped with filter paper and dried, and the sample was negatively stained with 2% uranyl acetate in water (5 μL) for 30 seconds, washed with water, wiped with filter paper, and dried again. The samples were then analyzed on a TecnaiG2 F20 TEM (FEI Tecnai™ G2, Hillsboro, Oregon) operating at 200 kV.
ThT assay가 끝난 샘플을 회수하여, 2% Potassium phosphotungstate 용액으로 샘플을 negative stain 시킨 뒤 carbon coated copper grid에 로딩시켜 Tecnai G2 F30 S-Twin 기기를 이용하여 분석을 수행하였다. Samples after the ThT assay were collected, negatively stained with a 2% potassium phosphotungstate solution, loaded onto a carbon coated copper grid, and analyzed using a Tecnai G2 F30 S-Twin device.
도 3에 나타난 바와 같이, 펩티도미메틱 화합물(I 및 II)을 첨가하지 않은 Aβ42 피브릴에 비해 펩티도미메틱 후보물질이 첨가되어 함께 fibrillation이 진행된 경우 Aβ42의 아밀로이드 피브릴 형성이 저해됨을 확인하였다.As shown in Figure 3, it was confirmed that the formation of amyloid fibrils of Aβ42 was inhibited when fibrillation was carried out with the addition of peptidomimetic candidates compared to Aβ42 fibrils without the addition of peptidomimetic compounds (I and II).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당 업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
Claims (7)
[화학식 I]
[화학식 II]
A peptidomimetic compound having the ability to inhibit the formation of dementia-inducing beta-amyloid aggregates, represented by the following formula (I) or (II).
[Formula I]
[Formula II]
[화학식 I]
[화학식 II]
A composition for preventing or treating dementia comprising as an active ingredient a peptidomimetic compound having the ability to inhibit the formation of dementia-causing beta-amyloid aggregates represented by the following formula (I) or (II).
[Formula I]
[Formula II]
(a) 고체상 (Solid-phase) 합성방법으로 화학식 1로 표시되는 Fmoc-Ala-NH-레진을 수득하는 단계;
(b) 상기 단계 (a)에서 수득한 레진에 Fmoc-His(Bis-cyclohexylpropyl)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH 및 Fmoc-γ-Glu-OtBu를 순차적으로 결합시켜 화학식 2로 표시되는 보호화된 펩타이드가 결합된 레진을 수득하는 단계;
(c) 상기 단계 (b)에서 수득한 보호화된 펩타이드가 결합된 레진으로부터 레진 및 아미노산의 보호기를 동시에 제거하여 화학식 3으로 표시되는 비정제 펩티도미메틱 화합물을 수득하는 단계; 및
(d) 상기 단계 (c)에서 수득한 비정제 펩티도미메틱 화합물을 정제하여 화학식 I로 표시되는 펩티도미메틱 화합물을 수득하는 단계;
[화학식 I]
[화학식 1]
[화학식 2]
상기 화학식 2에서 R1은 수소 또는 수산기 보호기이고, R2는 티올 보호기이며, R3는 아민 보호기이다.
[화학식 3]
A method for preparing a peptidomimetic compound represented by formula (I) comprising the following steps:
(a) Obtaining Fmoc-Ala-NH-resin represented by Formula 1 by a solid-phase synthesis method;
(b) Fmoc-His(Bis-cyclohexylpropyl)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH in the resin obtained in step (a). , sequentially combining Fmoc-Cys(Trt)-OH and Fmoc-γ-Glu-OtBu to obtain a resin to which the protected peptide represented by Formula 2 is bound;
(c) simultaneously removing the protective groups of the resin and the amino acid from the resin to which the protected peptide obtained in step (b) is bound, thereby obtaining a purified peptidomimetic compound represented by Formula 3; and
(d) purifying the crude peptidomimetic compound obtained in step (c) to obtain a peptidomimetic compound represented by Formula I;
[Formula I]
[Formula 1]
[Formula 2]
In Formula 2, R 1 is a hydrogen or hydroxyl protecting group, R 2 is a thiol protecting group, and R 3 is an amine protecting group.
[Formula 3]
[화학식 I]
[화학식 II]
Preparation of a peptidomimetic compound represented by Formula II, characterized in that the peptidomimetic compound represented by Formula I is mixed with an -SH group oxidation reagent, and the pH is adjusted to 6 to 9 to perform disulfide bond (SS). method.
[Formula I]
[Formula II]
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