KR101980292B1 - Fluorescence Compounds and Preparation Method Therof - Google Patents
Fluorescence Compounds and Preparation Method Therof Download PDFInfo
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- KR101980292B1 KR101980292B1 KR1020170098313A KR20170098313A KR101980292B1 KR 101980292 B1 KR101980292 B1 KR 101980292B1 KR 1020170098313 A KR1020170098313 A KR 1020170098313A KR 20170098313 A KR20170098313 A KR 20170098313A KR 101980292 B1 KR101980292 B1 KR 101980292B1
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- compound
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- benzene ring
- ethyl acetate
- cooh
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Images
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0026—Acridine dyes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
Abstract
본 발명의 목적은 형광강도, 상대양자효율, 표지율 면에서 우수하여 조영제 조성물로도 이용될 수 있는 형광 화합물, 이를 이용한 표지방법 등을 제공하고자 한다. 본 발명에 따른 형광 화합물은 형광강도, 상대양자효율, 표지율 면에서 우수하여 타겟 물질의 표지 및 염색에 보다 효과적으로 활용될 수 있다. 또한, 광학 안정성이 우수하여 장시간의 염색에도 안정적 형광을 나타내며, 체내에 투여시 축적되지 않으면서도 형광 강도가 우수하여 종래의 염료에 비하여 소량의 사용에도 염색 및 체내 영상화가 용이하여 경제적으로 이용이 가능하다.It is an object of the present invention to provide a fluorescent compound that is excellent in fluorescence intensity, relative quantum efficiency, and labeling ratio and can also be used as a contrast agent composition, and a labeling method using the same. The fluorescent compound according to the present invention is superior in terms of fluorescence intensity, relative quantum efficiency, and labeling ratio, and can be more effectively used for labeling and dyeing a target substance. In addition, it exhibits stable fluorescence even after dyeing for a long time due to its excellent optical stability, and is excellent in fluorescence intensity without accumulating when administered to the body. Therefore, it can be used economically because dyeing and visualization in the body is easier than a conventional dye. Do.
Description
본 발명은 형광 화합물에 관한 것으로, 보다 상세하게는 알킬카르복시아미노 시아누릭 클로라이드 치환기를 포함하는 시아닌계 형광 화합물 및 이의 응용에 관한 것이다.The present invention relates to a fluorescent compound, and more particularly, to a cyanine fluorescent compound containing an alkyl carboxyaminocyanilic chloride substituent and its application.
생체 물질 자체는 가시광 및 근적외 영역의 형광이 미약하거나 없으므로 바이오 분야에서는 생체 내/외에서 세포 및 세포 이하 단계에서의 생물학적인 현상을 관찰하거나 생체 내로 투영되어 조영 및 질환부위의 광학 영상을 얻기 위하여 생체 물질에 형광 염료 또는 형광 염료가 미리 표지된 특정 생체 물질을 광학장비와 함께 활용하는 다양한 기법을 통해 영상화한 자료를 얻고 있다.Since the biomolecule itself has little or no fluorescence in the visible and near infrared regions, it is necessary to observe the biological phenomenon in the cell or subcellular phase in vivo or outside in the bio field, or to project an in vivo image, The material is imaged using a variety of techniques that utilize certain biomaterials previously labeled with fluorescent dyes or fluorescent dyes with optical equipment.
바이오 분야에서 사용되는 다양한 광학 분석(optical anylsis) 장비들은 내장된 광원 및 필터에 따라 형광을 관찰하기에 적합한 여기 파장(excitation wavelength) 및 형광 파장(emission wavelength)를 가진 형광 염료를 기본 소재나 시약으로 선택하게 된다.A variety of optical anylsis devices used in the biotechnology field include fluorescent dyes with excitation wavelengths and fluorescence wavelengths suitable for observing fluorescence according to the built-in light source and filter as basic materials or reagents .
주로 사용되는 광학 분석 장비로는 세포 관찰을 위한 형광현미경(fluorescece microscope), 공초점현미경(confocal microscope), 유세포분석기(flowcytometer), 마이크로어레이(microarray), 정량 중합효소연쇄반응 장치(qualitative PCR system), 핵산 및 단백질 분리, 분석을 위한 전기영동(electrophoresis) 장치, 실시간 생체내 영상 장비(in vivo imaging system) 등 연구 목적의 장비 외에도, 면역 분석 기법(immnuno assay)이나 PCR 분석 및 통계 기술이 접목된 핵산 및 단백질 진단 키트(또는 바이오칩) 기반 체외 진단(in vitro diagnosis) 장비와 의료 영상 수술(image-guided surgery)을 위한 수술대 및 내시경 장비 등의 진단 및 치료를 위한 것들이 알려져 있으며, 지속적으로 새로운 응용 분야 및 더 높은 수준의 해상도 및 데이터 처리 능력을 가진 장비가 개발되고 있다. The most commonly used optical analysis equipment is a fluorescence microscope, confocal microscope, flowcytometer, microarray, and qualitative PCR system for cell observation. In addition to equipment for research purposes such as nucleic acid and protein separation, electrophoresis for analysis, in vivo imaging system, etc., immunno assay, PCR analysis and statistical techniques are combined. Diagnosis and treatment of in vitro diagnosis equipment based on nucleic acid and protein diagnostic kits (or biochips) and operating table and endoscopic equipment for image-guided surgery are known, and new applications And higher resolution and data processing capabilities are being developed.
일반적으로 단백질 또는 펩타이드 등 생체 분자의 표지를 위해 사용되는 형광염료(fluorescent dye)는 대부분 안트라닐레이트(anthranilate), 1-알킬틱 이소인돌(1-alkylthic isoindoles), 피롤리논(pyrrolinones), 비메인(bimanes), 벤즈옥사졸(benzoxazole), 벤즈이미다졸(benzimidazole), 벤조퓨란(benzofurazan), 나프탈렌(naphthalenes), 쿠마린(coumarins), 시아닌(cyanine), 스틸벤(stilbenes), 카바졸(carbazoles), 페난트리딘(phenanthridine), 안트라센(anthracenes), 보디피(bodipy), 플로세인(fluoresceins), 에오신(eosins), 로다민(rhodamines), 피렌(pyrenes), 크리센(chrysenes) 및 아크리딘(acridines) 등의 구조가 포함되어 있다.In general, fluorescent dyes used for the labeling of biomolecules such as proteins or peptides are mostly anthranilates, 1-alkylthic isoindoles, pyrrolinones, But are not limited to, bimanes, benzoxazole, benzimidazole, benzofurazan, naphthalenes, coumarins, cyanine, stilbenes, carbazoles, ), Phenanthridine, anthracenes, bodipy, fluoresceins, eosins, rhodamines, pyrenes, chrysenes, and acry Acridines, and the like.
상기에서 예시한 다수의 형광 발색단 중에서 바이오 분야에서 이용 가능한 형광 염료 구조를 선별하는 경우, 일반적으로는 대부분의 생체 분자들이 존재하는 매질, 즉, 수용액 및 수용성 버퍼 내에 존재할 때 강한 형광을 내는 것과 형광 장비에 맞는 여기 및 형광 파장을 갖는 것이 중요하다.Among the many fluorescent chromophores exemplified above, when a fluorescent dye structure usable in a biotechnology field is selected, it is generally preferred to use a fluorescence dye when present in a medium in which most biomolecules exist, that is, an aqueous solution and an aqueous buffer, It is important to have an excitation and fluorescence wavelength that is suitable for the wavelength.
바이오 분야에서 주로 적용될 수 있는 염료는 가급적 수용액이나 친수성 조건에서 광표백(photobleaching) 및 소광(quenching) 현상이 적고, 다량의 빛을 흡수할 수 있도록 몰흡광계수(molecular extinction coefficient)가 커야 하며, 생체 분자 자체의 형광 범위와 멀리 떨어진 500 nm 이상의 가시광선 영역이나 근적외선 영역에 있어야 하고, 다양한 pH 조건에서 안정하여야 하나, 상기 제한 사항을 만족할 수 있는 생체 분자 표지용으로 사용 가능한 염료의 구조는 한정되어 있다.The dyes which can be applied mainly in the biotechnology field have a low molecular weight and a high molecular extinction coefficient so that they can absorb a large amount of light with little photobleaching and quenching phenomenon in aqueous solution or hydrophilic condition, It is required to be in a visible light region or a near infrared region of 500 nm or more away from its own fluorescence range and stable at various pH conditions, but the structure of dyes usable for biomolecule marking which can satisfy the above limitations is limited.
이러한 요구 조건에 부합하는 형광 색원체로는 시아닌, 로다민, 플로세인, 보디피, 쿠마린, 아크리딘, 피렌 유도체들이 있는데, 염료 단독 또는 생체 분자 구조 내의 특정 치환기와 결합이 가능하도록 반응기를 도입시키기도 하며, 그 중 잔텐(xanthane) 계열의 플로세인 및 로다민과, 폴리메틴(polymethine) 계열의 시아닌 유도체 염료 화합물들이 주로 상품화되어 있다.Fluorescent materials conforming to these requirements include cyanine, rhodamine, flocine, bodipy, coumarin, acridine, and pyrene derivatives. The dye can be introduced alone or in combination with specific substituents in biomolecular structures Among them, xanthane series floresine and rhodamine and polymethine series cyanine derivative dye compounds are mainly commercialized.
특히 시아닌 발색단을 가진 염료 화합물은 다양한 흡수/여기 파장의 화합물을 합성하기 용이하다는 장점 외에도, 일반적으로 광학 및 pH 안정성이 탁월하고, 좁은 흡수 및 발광 파장 범위를 가지며, 500 내지 800 nm의 형광 영역을 갖기 때문에 생체 분자의 자체 형광 영역과 중첩되지 않아 분석이 용이하며, 용매 및 용해도 특성에 따라 다소 차이는 있지만, 높은 몰흡광계수를 나타내는 등 많은 장점이 있어 생물학적 응용에 많이 이용된다.In particular, the dye compound having a cyanine chromophore is advantageous in that it is easy to synthesize compounds having various absorption / excitation wavelengths, and is excellent in optical and pH stability, has a narrow absorption and emission wavelength range, has a fluorescent region of 500 to 800 nm , It is easy to analyze because it does not overlap with the fluorescence region of the biomolecule itself and has many merits such as a high molar extinction coefficient although it is slightly different according to the solvent and solubility characteristics and is widely used in biological applications.
그 이외에도, 시아닌 발색단을 가진 염료 화합물은 화상표시장치용 광학필터나 레이저 용착용 수지 조성물의 용도로 유용하게 이용될 수도 있다. 특정한 광에 강도가 큰 흡수를 가지는 화합물은 액정표시장치, 플라즈마 디스플레이 패널, 전계발광디스플레이, 음극관 표시장치, 형광 표시관 등의 화상표시장치용 광학필터나 DVDㅁR 등의 광학 기록 매체의 광학 요소로서 널리 이용되고 있다. 광학 필터에는 불필요한 파장의 광들을 선택적으로 흡수하는 기능이 요구되는데, 동시에 형광등 등의 외광의 반사나 글레어를 방지하기 위해서는 480~500 nm 및 540~560 nm의 파장광 흡수가 요구되며, 화상품질을 높이기 위해서는 근적외선의 파장을 선택적으로 흡수하는 기능이 요구되고 있다.In addition, a dye compound having a cyanine chromophore may be usefully used as an optical filter for an image display device or a resin composition for a laser. A compound having a strong absorption in a specific light may be used as an optical filter for an image display device such as a liquid crystal display, a plasma display panel, an electroluminescence display, a cathode ray tube display, a fluorescent display tube or the like or an optical element for an optical recording medium such as a DVD R . In order to prevent reflection and glare of external light such as fluorescent lamps, it is necessary to absorb wavelength light of 480 to 500 nm and 540 to 560 nm, A function of selectively absorbing the wavelength of near-infrared rays is required.
상기와 같이, 산업적으로 유용하게 적용하기 위해서는 광학 및 pH 안정성이 우수하면서도 특정 파장 범위에서 좁은 흡수/발광 파장 범위를 가지면서도 높은 몰흡광계수를 나타내는 신규한 염료의 개발이 지속적으로 요구되는 바이다.As described above, there is a continuing need for the development of a novel dye having a high molar extinction coefficient while having excellent optical and pH stability and narrow absorption / emission wavelength range in a specific wavelength range.
본 발명의 목적은 형광강도, 상대양자효율, 표지율 면에서 우수하여 조영제 조성물로도 이용될 수 있는 형광 화합물, 이를 이용한 표지방법 등을 제공하고자 한다.It is an object of the present invention to provide a fluorescent compound that is excellent in fluorescence intensity, relative quantum efficiency, and labeling ratio and can also be used as a contrast agent composition, and a labeling method using the same.
본 발명의 일 측면은 하기 화학식 1로 표시되는 형광 화합물 제조용 중간체에 관한 것이다.One aspect of the present invention relates to an intermediate for the production of a fluorescent compound represented by the following general formula (1).
[화학식 1][Chemical Formula 1]
X1은 O 또는 S이고; X2는 -C6H4-SO3H, -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이며;X 1 is O or S; X 2 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -COOH;
W1은 -(CH2)4-SO3 - 또는 -(CH2)3-SO3 -이고; W2는 -(CH2)5-COOH, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 1 is - (CH 2 ) 4 -SO 3 - or - (CH 2 ) 3 -SO 3 - ; W 2 is - (CH 2 ) 5 -COOH, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
X2가 -C6H4-SO3H이면 W2는 -(CH2)5-COOH이고, X2가 -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이면 W2는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 2 is - (CH 2 ) 5 -COOH and X 2 is -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -, when X 2 is -C 6 H 4 -SO 3 H, COOH, W 2 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
A1은 H 또는 SO3H이고, A2는 H이며, 또는 A1과 A2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;A 1 is H or SO 3 H, A 2 is H, or A 1 and A 2 combine with each other to form a benzene ring, said benzene ring being substituted with two SO 3 H;
B1은 H 또는 SO3H이고, B2는 H이며, 또는 B1과 B2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있다.B 1 is H or SO 3 H, B 2 is H, or B 1 and B 2 may combine with each other to form a benzene ring, and the benzene ring may be substituted with two SO 3 H.
본 발명의 다른 측면은 하기 화학식 2로 표시되는 형광 화합물에 관한 것이다.Another aspect of the present invention relates to a fluorescent compound represented by the following general formula (2).
[화학식 2](2)
X1은 O 또는 S이고; X3는 -C6H4-SO3H, -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이며;X 1 is O or S; X 3 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -CO-Z or - (CH 2 ) 2 -CO-Z;
W1은 -(CH2)4-SO3 - 또는 -(CH2)3-SO3 -이고; W3는 -(CH2)5-CO-Z, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 1 is - (CH 2 ) 4 -SO 3 - or - (CH 2 ) 3 -SO 3 - ; W 3 is - (CH 2 ) 5 -CO-Z, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
X3가 -C6H4-SO3H이면 W3는 -(CH2)5-CO-Z이고, X3가 -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이면 W3는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 3 is - (CH 2 ) 5 -CO-Z when X 3 is -C 6 H 4 -SO 3 H, X 3 is -C 6 H 4 - (CH 2 ) 2 -CO- 2 ) 2 -CO-Z, W 3 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
-Z는 
A1은 H 또는 SO3H이고, A2는 H이며, 또는 A1과 A2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;A 1 is H or SO 3 H, A 2 is H, or A 1 and A 2 combine with each other to form a benzene ring, said benzene ring being substituted with two SO 3 H;
B1은 H 또는 SO3H이고, B2는 H이며, 또는 B1과 B2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있다.B 1 is H or SO 3 H, B 2 is H, or B 1 and B 2 may combine with each other to form a benzene ring, and the benzene ring may be substituted with two SO 3 H.
본 발명의 또 다른 측면은 본 발명의 여러 구현예에 따른 형광 화합물을 유효성분으로 포함하는 조영제 조성물에 관한 것이다.Another aspect of the present invention relates to a contrast agent composition comprising a fluorescent compound according to various embodiments of the present invention as an active ingredient.
본 발명의 또 다른 측면은 본 발명의 여러 구현예에 따른 형광 화합물을 표지대상 물질과 결합시키는 단계를 포함하는 형광 화합물 표지방법에 관한 것이다.Another aspect of the present invention relates to a fluorescent compound labeling method comprising the step of binding a fluorescent compound according to various embodiments of the present invention to a labeling substance.
본 발명의 또 다른 측면은 하기 화학식 1의 화합물로부터 하기 화학식 2의 화합물을 제조하는 형광 화합물 제조방법에 관한 것이다.Another aspect of the present invention relates to a method of preparing a fluorescent compound for producing a compound of the following formula (2) from a compound of the formula (1).
[화학식 1][Chemical Formula 1]
[화학식 2](2)
상기 화학식 1 및 2에서, X1은 O 또는 S이고; X2는 -C6H4-SO3H, -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이며;In the above formulas (1) and (2), X 1 is O or S; X 2 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -COOH;
W1은 -(CH2)4-SO3 - 또는 -(CH2)3-SO3 -이고; W2는 -(CH2)5-COOH, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 1 is - (CH 2 ) 4 -SO 3 - or - (CH 2 ) 3 -SO 3 - ; W 2 is - (CH 2 ) 5 -COOH, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
X2가 -C6H4-SO3H이면 W2는 -(CH2)5-COOH이고, X2가 -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이면 W2는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 2 is - (CH 2 ) 5 -COOH and X 2 is -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -, when X 2 is -C 6 H 4 -SO 3 H, COOH, W 2 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
A1은 H 또는 SO3H이고, A2는 H이며, 또는 A1과 A2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;A 1 is H or SO 3 H, A 2 is H, or A 1 and A 2 combine with each other to form a benzene ring, said benzene ring being substituted with two SO 3 H;
B1은 H 또는 SO3H이고, B2는 H이며, 또는 B1과 B2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;B 1 is H or SO 3 H, B 2 is H, or B 1 and B 2 may combine with each other to form a benzene ring, said benzene ring may be substituted with two SO 3 H;
X3는 -C6H4-SO3H, -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이며;X 3 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -CO-Z or - (CH 2 ) 2 -CO-Z;
W3는 -(CH2)5-CO-Z, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 3 is - (CH 2 ) 5 -CO-Z, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
X3가 -C6H4-SO3H이면 W3는 -(CH2)5-CO-Z이고, X3가 -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이면 W3는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 3 is - (CH 2 ) 5 -CO-Z when X 3 is -C 6 H 4 -SO 3 H, X 3 is -C 6 H 4 - (CH 2 ) 2 -CO- 2 ) 2 -CO-Z, W 3 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
-Z는 
본 발명에 따른 형광 화합물은 형광강도, 상대양자효율, 표지율 면에서 우수하여 타겟 물질의 표지 및 염색에 보다 효과적으로 활용될 수 있다. 또한, 광학 안정성이 우수하여 장시간의 염색에도 안정적 형광을 나타내며, 체내에 투여시 축적되지 않으면서도 형광 강도가 우수하여 종래의 염료에 비하여 소량의 사용에도 염색 및 체내 영상화가 용이하여 경제적으로 이용이 가능하다.The fluorescent compound according to the present invention is superior in terms of fluorescence intensity, relative quantum efficiency, and labeling ratio, and can be more effectively used for labeling and dyeing a target substance. In addition, it exhibits stable fluorescence even after dyeing for a long time due to its excellent optical stability, and is excellent in fluorescence intensity without accumulating when administered to the body. Therefore, it can be used economically because dyeing and visualization in the body is easier than a conventional dye. Do.
도 1은 화합물 1과 대조형광염료의 형광 스펙트럼을 보여준다.
도 2는 Rhodamine 6G (TCI)를 기준으로, 화합물 1과 대조형광염료(IRDyeㄾ 800CW NHS ester, DyLight™ 800 NHS ester, ICG NHS ester)의 상대양자효율을 측정한 결과이다.
도 3a 내지 3d는 화합물 1과 대조형광염료의 세포독성 평가 결과이다.
도 4는 화합물 1과 2종의 대조형광염료에 대한 FOBI Image이다.
도 5a 및 5b는 화합물 1과 2종의 대조형광염료를 이용하여 Plate reader 측정한 결과를 보여주는 그래프이다.
도 6은 화합물 1과 대조형광염료의 In vivo 이미징 결과를 보여준다.
도 7은 화합물 1과 대조형광염료를 이용하여 각 장기별 형광 강도를 측정한 후 Organ to liver ratio를 구한 결과를 보여준다.
도 8은 화합물 1을 도입한 나노파티클(화합물1-HGC)의 암 타겟능을 보여주는 in vivo 이미징 분석 결과이다.
도 9는 화합물 1을 도입한 나노파티클(화합물1-HGC)의 암 타겟능을 보여주는 FL 강도 그래프이다. 좌측은 in vivo 분석 결과이고, 우측은 ex-vivo 분석 결과이다.Figure 1 shows the fluorescence spectra of
FIG. 2 shows the result of measurement of the relative quantum efficiency of
Figs. 3a to 3d show the cytotoxicity evaluation results of the
4 is an FOBI image of
FIGS. 5A and 5B are graphs showing the results of a plate reader
Figure 6 shows the in vivo imaging results of
FIG. 7 shows the result of measuring the fluorescence intensity of each
8 is an in vivo imaging analysis showing the cancer targeting ability of the nanoparticle (compound 1-HGC) into which the
FIG. 9 is a graph of FL intensity showing the cancer targeting ability of the nanoparticle (compound 1-HGC) into which the
이하에서, 본 발명의 여러 측면 및 다양한 구현예에 대해 더욱 구체적으로 살펴보도록 한다.Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.
본 발명의 일 측면은 하기 화학식 1로 표시되는 형광 화합물 제조용 중간체에 관한 것이다.One aspect of the present invention relates to an intermediate for the production of a fluorescent compound represented by the following general formula (1).
[화학식 1][Chemical Formula 1]
X1은 O 또는 S이고; X2는 -C6H4-SO3H, -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이며;X 1 is O or S; X 2 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -COOH;
W1은 -(CH2)4-SO3 - 또는 -(CH2)3-SO3 -이고; W2는 -(CH2)5-COOH, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 1 is - (CH 2 ) 4 -SO 3 - or - (CH 2 ) 3 -SO 3 - ; W 2 is - (CH 2 ) 5 -COOH, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
X2가 -C6H4-SO3H이면 W2는 -(CH2)5-COOH이고, X2가 -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이면 W2는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 2 is - (CH 2 ) 5 -COOH and X 2 is -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -, when X 2 is -C 6 H 4 -SO 3 H, COOH, W 2 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
A1은 H 또는 SO3H이고, A2는 H이며, 또는 A1과 A2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;A 1 is H or SO 3 H, A 2 is H, or A 1 and A 2 combine with each other to form a benzene ring, said benzene ring being substituted with two SO 3 H;
B1은 H 또는 SO3H이고, B2는 H이며, 또는 B1과 B2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있다.B 1 is H or SO 3 H, B 2 is H, or B 1 and B 2 may combine with each other to form a benzene ring, and the benzene ring may be substituted with two SO 3 H.
일 구현예에 있어서, 상기 중간체는 하기 화화합물 중에서 선택되는 것을 특징으로 하는 형광 화합물 제조용 중간체가 개시된다.In one embodiment, an intermediate for the preparation of a fluorescent compound is provided, wherein the intermediate is selected from the following compounds:
본 발명의 다른 측면은 하기 화학식 2로 표시되는 형광 화합물에 관한 것이다.Another aspect of the present invention relates to a fluorescent compound represented by the following general formula (2).
[화학식 2](2)
X1은 O 또는 S이고; X3는 -C6H4-SO3H, -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이며;X 1 is O or S; X 3 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -CO-Z or - (CH 2 ) 2 -CO-Z;
W1은 -(CH2)4-SO3 - 또는 -(CH2)3-SO3 -이고; W3는 -(CH2)5-CO-Z, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 1 is - (CH 2 ) 4 -SO 3 - or - (CH 2 ) 3 -SO 3 - ; W 3 is - (CH 2 ) 5 -CO-Z, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
X3가 -C6H4-SO3H이면 W3는 -(CH2)5-CO-Z이고, X3가 -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이면 W3는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 3 is - (CH 2 ) 5 -CO-Z when X 3 is -C 6 H 4 -SO 3 H, X 3 is -C 6 H 4 - (CH 2 ) 2 -CO- 2 ) 2 -CO-Z, W 3 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
-Z는 
A1은 H 또는 SO3H이고, A2는 H이며, 또는 A1과 A2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;A 1 is H or SO 3 H, A 2 is H, or A 1 and A 2 combine with each other to form a benzene ring, said benzene ring being substituted with two SO 3 H;
B1은 H 또는 SO3H이고, B2는 H이며, 또는 B1과 B2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있다.B 1 is H or SO 3 H, B 2 is H, or B 1 and B 2 may combine with each other to form a benzene ring, and the benzene ring may be substituted with two SO 3 H.
일 구현예에 있어서, 상기 형광 화합물은 섬유, 생체분자, 나노입자 및 유기화합물 중에서 선택되는 표지대상 물질에 표지되는 것을 특징으로 하는 형광 화합물이 개시된다.In one embodiment, the fluorescent compound is labeled with a labeling substance selected from a fiber, a biomolecule, a nanoparticle, and an organic compound.
다른 구현예에 있어서, 상기 생체분자는 단백질, 펩타이드, 탄수화물, 당, 지방, 항체, 프로테오글라이칸, 글라이코프로틴 및 siRNA 중에서 선택되는 것을 특징으로 하는 형광 화합물이 개시된다.In another embodiment, the fluorescent molecule is characterized in that the biomolecule is selected from a protein, a peptide, a carbohydrate, a sugar, a fat, an antibody, a proteoglycan, a glycoprotein and an siRNA.
본 발명의 또 다른 측면은 본 발명의 여러 구현예에 따른 형광 화합물을 유효성분으로 포함하는 조영제 조성물에 관한 것이다.Another aspect of the present invention relates to a contrast agent composition comprising a fluorescent compound according to various embodiments of the present invention as an active ingredient.
본 발명의 또 다른 측면은 본 발명의 여러 구현예에 따른 형광 화합물을 표지대상 물질과 결합시키는 단계를 포함하는 형광 화합물 표지방법에 관한 것이다.Another aspect of the present invention relates to a fluorescent compound labeling method comprising the step of binding a fluorescent compound according to various embodiments of the present invention to a labeling substance.
이때, 상기 표지대상 물질은 섬유, 생체분자, 나노입자 및 유기화합물 중에서 선택된 1종 이상이며,At this time, the labeling substance is at least one selected from fibers, biomolecules, nanoparticles and organic compounds,
상기 표지대상 물질은 아민기, 수산화기 및 티올기 중에서 선택된 적어도 1개의 기능기를 포함하며,Wherein the labeling substance comprises at least one functional group selected from an amine group, a hydroxyl group and a thiol group,
상기 기능기에 상기 제1항의 형광 화합물이 결합한다.And the functional group is bonded to the fluorescent compound of the first aspect.
일 구현예에 있어서, 상기 생체분자는 단백질, 펩타이드, 탄수화물, 당, 지방, 항체, 프로테오글라이칸, 글라이코프로틴 및 siRNA으로 이루어진 군 중에서 선택된다.In one embodiment, the biomolecule is selected from the group consisting of proteins, peptides, carbohydrates, sugars, fats, antibodies, proteoglycans, glycoproteins and siRNA.
본 발명의 또 다른 측면은 하기 화학식 1의 화합물로부터 하기 화학식 2의 화합물을 제조하는 형광 화합물 제조방법에 관한 것이다.Another aspect of the present invention relates to a method of preparing a fluorescent compound for producing a compound of the following formula (2) from a compound of the formula (1).
[화학식 1][Chemical Formula 1]
[화학식 2](2)
상기 화학식 1 및 2에서, X1은 O 또는 S이고; X2는 -C6H4-SO3H, -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이며;In the above formulas (1) and (2), X 1 is O or S; X 2 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -COOH;
W1은 -(CH2)4-SO3 - 또는 -(CH2)3-SO3 -이고; W2는 -(CH2)5-COOH, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 1 is - (CH 2 ) 4 -SO 3 - or - (CH 2 ) 3 -SO 3 - ; W 2 is - (CH 2 ) 5 -COOH, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
X2가 -C6H4-SO3H이면 W2는 -(CH2)5-COOH이고, X2가 -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이면 W2는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 2 is - (CH 2 ) 5 -COOH and X 2 is -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -, when X 2 is -C 6 H 4 -SO 3 H, COOH, W 2 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
A1은 H 또는 SO3H이고, A2는 H이며, 또는 A1과 A2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;A 1 is H or SO 3 H, A 2 is H, or A 1 and A 2 combine with each other to form a benzene ring, said benzene ring being substituted with two SO 3 H;
B1은 H 또는 SO3H이고, B2는 H이며, 또는 B1과 B2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;B 1 is H or SO 3 H, B 2 is H, or B 1 and B 2 may combine with each other to form a benzene ring, said benzene ring may be substituted with two SO 3 H;
X3는 -C6H4-SO3H, -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이며;X 3 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -CO-Z or - (CH 2 ) 2 -CO-Z;
W3는 -(CH2)5-CO-Z, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 3 is - (CH 2 ) 5 -CO-Z, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
X3가 -C6H4-SO3H이면 W3는 -(CH2)5-CO-Z이고, X3가 -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이면 W3는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;W 3 is - (CH 2 ) 5 -CO-Z when X 3 is -C 6 H 4 -SO 3 H, X 3 is -C 6 H 4 - (CH 2 ) 2 -CO- 2 ) 2 -CO-Z, W 3 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
-Z는 
-Z is
이하에서 실시예 등을 통해 본 발명을 더욱 상세히 설명하고자 하며, 다만 이하에 실시예 등에 의해 본 발명의 범위와 내용이 축소되거나 제한되어 해석될 수 없다. 또한, 이하의 실시예를 포함한 본 발명의 개시 내용에 기초한다면, 구체적으로 실험 결과가 제시되지 않은 본 발명을 통상의 기술자가 용이하게 실시할 수 있음은 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연하다.Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.
또한 이하에서 제시되는 실험 결과는 상기 실시예 및 비교예의 대표적인 실험 결과만을 기재한 것이며, 아래에서 명시적으로 제시하지 않은 본 발명의 여러 구현예의 각각의 효과는 해당 부분에서 구체적으로 기재하도록 한다.In addition, the experimental results presented below only show representative experimental results of the embodiments and the comparative examples, and the respective effects of various embodiments of the present invention which are not explicitly described below will be specifically described in the corresponding part.
실시예Example
실시예 1: 화합물 a의 제조Example 1: Preparation of compound a
(1) 화합물 1-1의 합성(1) Synthesis of Compound 1-1
p-히드라지노벤젠설폰산 (p-Hydrazinobenzenesulfonic acid) (10 g, 53 mmol, 1 eq, Aldrich)과 3-메틸-2-부탄온 (3-Methyl-2-butanone) (17.18 mL, 160 mmol, 3.02 eq, TCI)을 아세트산 30 mL에 가한 후, 4 시간 동안 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 생성된 고체 입자를 여과하였다. 에틸아세테이트(Ethyl acetate)로 2, 3회 세정한 후 감압 건조시켰다. (11.34 g, 89%) Hydrazinobenzenesulfonic acid (10 g, 53 mmol, 1 eq., Aldrich) and 3-Methyl-2-butanone (17.18 mL, 160 mmol, 3.02 eq, TCI) was added to 30 mL of acetic acid, and the mixture was heated and refluxed for 4 hours. The mixture was cooled to room temperature, and the resulting solid particles were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. (11.34 g, 89%).
Rf = 0.68 (역상, C18, 아세토니트릴/물 1:4 v/v)R f = 0.68 (reversed phase, C18, acetonitrile / water 1: 4 v / v)
수산화칼륨 (1.427 g, 25.4 mmol, 1.2 eq)을 프로판올(n-Propanol) 35 mL에 용해시키고, 앞에서 얻은 고체 물질 (5.073 g, 21.2 mmol, 1 eq)을 메탄올(Methanol) 35 mL에 용해시키고 적가한 후, 상온에서 24 시간 교반하고, 여과하여 노란색의 고체 입자를 얻었다. (5.35 g, 90%)Potassium hydroxide (1.427 g, 25.4 mmol, 1.2 eq) was dissolved in 35 mL of n-propanol and the solid material (5.073 g, 21.2 mmol, 1 eq) obtained above was dissolved in methanol (35 mL) The mixture was stirred at room temperature for 24 hours and filtered to obtain yellow solid particles. (5.35 g, 90%).
Rf = 0.68 (역상, C18, 아세토니트릴/물 1:4 v/v)R f = 0.68 (reversed phase, C18, acetonitrile / water 1: 4 v / v)
(2) 화합물 1-2의 합성(2) Synthesis of Compound 1-2
1-1 (6 g, 21.6 mmol, 1 eq)과 1,4-부탄설톤(1,4-Butanesultone) (6.4 mL, 68.9 mmol, 3.01 eq, TCI), 아세트산 나트륨(Sodium acetate) (2.1 g, 25.9 mmol, 1,2 eq)을 아세토나이트릴(Acetonitrile) 10 ml에 가한 후, 12 시간 동안 100℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. (5 g, 61.6 %) (1. 6 g, 21.6 mmol, 1 eq), 1,4-butanesultone (6.4 mL, 68.9 mmol, 3.01 eq, TCI), sodium acetate (2.1 g, 25.9 mmol, 1.2 eq) was added to 10 ml of acetonitrile, and the mixture was reacted by heating at 100 ° C for 12 hours under reflux. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. (5 g, 61.6%).
Rf = 0.31 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.31 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(3) 화합물 1-3의 합성(3) Synthesis of Compound 1-3
1-1 (5 g, 20.9 mmol, 1 eq)과 6-브로모헥사노익 산(6-Bromohexanoic acid) (8.1 g, 41.8 mmol, 2 eq, TCI)을 1,2-다이클로로벤젠(1,2-Dichlorobenzene) 20 ml에 가한 후, 12 시간 동안 120℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. (5.2 g, 70.3 %) (8.1 g, 41.8 mmol, 2 eq, TCI) was added to a solution of l, 2-dichlorobenzene (1, 2-Dichlorobenzene), and the mixture was reacted for 12 hours at 120 ° C under reflux. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. (5.2 g, 70.3%).
Rf = 0.23 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.23 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(4) 화합물 1-4의 합성(4) Synthesis of Compound 1-4
N,N-다이메틸포름아마이드(DMF, N,N-Dimethylformamide) (60.8 ml, 2 mol, 5 eq, 덕산)와 디클로로메탄(Dichloromethane) 40 ml을 혼합하고 온도를 0 ℃로 냉각시켰다. 디클로로메탄 40 ml에 포스포러스 옥시클로라이드(Phosphorus oxychloride) (45.2 ml, 1.5 mol, 3.7 eq, Aldrich)를 용해시킨 후 상기 냉각시킨 용액에 10분 동안 적가하였다. 상기 홍합액에 디클로로메탄 60 ml에 싸이클로헥사논(Cyclohexanone) (11.7 ml, 0.4 mol, 1 eq, Aldrich)을 용해시킨 용액을 10분 동안 적가하였다. 3시간 동안 가열 환류시킨뒤 상온으로 냉각시키고, 아닐린(Aniline) (31.0 ml, 1.0 mol, 2.7 eq, Aldrich)을 넣고 추가로 1시간 동안 상온에서 교반한다. 반응 종료 후, 50 ml의 증류수를 혼합 용액에 넣고 냉장 보관 한 후 생성된 어두운 보라색 침전을 여과하여 감압 건조하였다. (38 g, 26 %)40 ml of N, N-Dimethylformamide (60.8 ml, 2 mol, 5 eq, Duksan) and 40 ml of dichloromethane were mixed and the temperature was cooled to 0 ° C. Phosphorus oxychloride (45.2 ml, 1.5 mol, 3.7 eq, Aldrich) was dissolved in 40 ml of dichloromethane, and the solution was added dropwise to the cooled solution for 10 minutes. A solution of cyclohexanone (11.7 ml, 0.4 mol, 1 eq, Aldrich) dissolved in 60 ml of dichloromethane was added dropwise to the above mussel solution for 10 minutes. The mixture was heated to reflux for 3 hours and then cooled to room temperature. Aniline (31.0 ml, 1.0 mol, 2.7 eq, Aldrich) was added thereto and stirred at room temperature for further 1 hour. After completion of the reaction, 50 ml of distilled water was added to the mixed solution, and the mixture was stored in the refrigerator. The resulting dark purple precipitate was filtered and dried under reduced pressure. (38 g, 26%).
Rf = 0.99 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.99 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(5) 화합물 1-5의 합성(5) Synthesis of Compound 1-5
1-2 (941 mg, 2.67 mmol, 1 eq)과 1-3 (1 g, 2.67 mmol, 1 eq), 1-4 (862 mg, 2.67 mmol, 1 eq), 아세트산 나트륨(Sodium Acetate) (438 mg, 5.34 mmol, 2 eq, 덕산)을 에탄올(Ethanol) 20 ml에 가한 후, 2 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 1-5를 얻었다. (180 mg, 7.8 %) 1-2 (941 mg, 2.67 mmol, 1 eq) and 1-3 (1 g, 2.67 mmol, 1 eq), 1-4 (862 mg, 2.67 mmol, 1 eq), sodium acetate mg, 5.34 mmol, 2 eq, Duksan) was added to 20 ml of ethanol and the mixture was reacted by heating at 50 ° C for 2 hours. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the developing solution to obtain pure compound 1-5. (180 mg, 7.8%).
Rf = 0.45 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.45 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C40H49ClN2O11S3 864.22, 측정치 863.0LC / MS, Calcd. C 40 H 49 ClN 2 O 11 S 3 864.22, found 863.0
(6) 화합물 a의 합성(6) Synthesis of compound a
1-5 (160 mg, 0.186 mmol, 1 eq)과 소듐 4-하이드로시벤젠-설포네이트(Sodium 4-Hydroxybenzene-sulfonate dihydrate, TCI) (146 mg, 0.74 mmol, 4 eq), 포타슘 카보네이트(Potassium carbonate) (51 mg, 0.372 mmol, 2 eq, 덕산)를 다이메틸포름아마이드 5 ml에 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. (5 g, %) 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 a를 얻었다. (83 mg, 44.6 %) (146 mg, 0.74 mmol, 4 eq), potassium carbonate (160 mg, 0.186 mmol, 1 eq) and sodium 4-hydroxybenzene-sulfonate dihydrate ) (51 mg, 0.372 mmol, 2 eq, Duksan) was added to 5 ml of dimethylformamide, followed by heating at 50 ° C for 12 hours. After cooling to room temperature, the particles were collected with diethylether, washed 2 or 3 times, and dried under reduced pressure. (5 g,%) acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using a developing solution to obtain pure compound a. (83 mg, 44.6%).
Rf = 0.21 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.21 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C40H54N2O15S4 1002.24, 측정치 1001.6LC / MS, Calcd. C 40 H 54 N 2 O 15 S 4 1002.24, Measured 1001.6
1H NMR (500 MHz, D2O) : δ 8.110-8.079 (t, 4H, J = 8Hz), δ 7.969-7.918 (t, 1H), δ 7.890-7.859 (t, 1H), δ 7.833-7.734 (m, 3H), δ 7.656-7.629 (t, 1H), δ 7.610-7.578 (t, 1H, J = 8Hz), δ 7.377-7.318 (m, 2H), δ 6.292-6.204 (q, 1H, J = 14Hz), δ 4.144 (s, 3H), δ 3.790-3.765 (t, 4H), δ 3.376 (s, 2H), δ 3.002-2.971 (t, 1H), δ 2.940 (s, 1H), δ 2.884 (s, 1H), δ 2.753 (s, 3H), δ 2.090 (s, 1H), δ 1.965-1.832 (m, 8H), δ 1.656-1.626 (t, 2H, J = 7Hz), δ 1.382-1.271 (m, 8H), δ 1.182 (s, 1H), δ 1.144-1.119 (t, 1H, J = 6.5Hz)
1 H NMR (500 MHz, D 2 O): δ 8.110-8.079 (t, 4H, J = 8Hz), δ 7.969-7.918 (t, 1H), δ 7.890-7.859 (t, 1H), δ 7.833-7.734 (m, 3H), δ 7.656-7.629 (t, 1H), δ 7.610-7.578 (t, 1H, J = 8Hz), δ 7.377-7.318 (m, 2H), δ 6.292-6.204 (q, 1H, J = 14 Hz),? 4.144 (s, 3H),? 3.790-3.765 (t, 4H),? 3.376 (s, 2H),? 3.002-2.971 (t, 2H, J = 7 Hz),? 1.382-1.271 (s, 1H),? (m, 8H),? 1.182 (s, 1H),? 1.144-1.119 (t, 1H, J = 6.5Hz)
실시예 2: 화합물 b의 제조Example 2: Preparation of compound b
(1) 화합물 2-1의 합성(1) Synthesis of Compound 2-1
1-1 (23 g, 82.9 mmol, 1 eq)과 1,3-프로판설톤(1,3-Propanesultone) (32.5 mL, 331.6 mmol, 4 eq, TCI), 아세트산 나트륨(Sodium acetate) (8.2 g, 99.4 mmol, 1,2 eq)을 아세토나이트릴(Acetonitrile) 70 ml에 가한 후, 12 시간 동안 120℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. (30 g, 100 %) (32 g, 331.6 mmol, 4 eq., TCI), sodium acetate (8.2 g, 1 eq) and 1-propanol 99.4 mmol, 1,2 eq) was added to 70 ml of acetonitrile, and the mixture was reacted for 12 hours at 120 ° C under reflux. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. (30 g, 100%).
Rf = 0.26 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.26 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(2) 화합물 2-2의 (2) Compound 2-2 합성synthesis
2-1 (22.4 g, 60.1 mmol, 2 eq)와 1-4 (10 g, 30 mmol, 1 eq), 아세트산 나트륨(Sodium Acetate) (6.3 g, 75 mmol, 2.5 eq, 덕산)을 에탄올(Ethanol) 50 ml에 가한 후, 2 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 2-2를 얻었다. (3 g, 11.5 %) 2-one (22.4 g, 60.1 mmol, 2 eq), 1-4 (10 g, 30 mmol, 1 eq) and sodium acetate (6.3 g, 75 mmol, 2.5 eq, Duksan) ), And the mixture was reacted by heating at 50 DEG C for 2 hours. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography with developing solution to give pure 2-2. (3 g, 11.5%).
Rf = 0.50 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.50 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C36H43ClN2O12S4 858.14, 측정치 856.9LC / MS, Calc'd C 36 H 43 ClN 2 O 12 S 4 858.14, found 856.9
(3) 화합물 b의 합성(3) Synthesis of compound b
2-1 (3 g, 3.5 mmol, 1 eq)과 소듐 3-(4-하이드록시페닐)프로피오닉 산(3-(4-Hydroxyphenyl)propionic acid, TCI) (2.3 g, 12 mmol, 4 eq), 포타슘 카보네이트(Potassium carbonate) (1 g, 7.0 mmol, 2 eq, 덕산)를 다이메틸포름아마이드 50 ml에 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 b를 얻었다. (1.5 g, 41.8 %) (2.3 g, 12 mmol, 4 eq) of 2-1 (3 g, 3.5 mmol, 1 eq) and sodium 3- (4- hydroxyphenyl) propionic acid , Potassium carbonate (1 g, 7.0 mmol, 2 eq, Duksan) were added to 50 ml of dimethylformamide, and the mixture was reacted by heating at 50 ° C for 12 hours. After cooling to room temperature, the particles were collected with diethylether, washed 2 or 3 times, and dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain pure compound b. (1.5 g, 41.8%).
Rf = 0.45 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.45 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C45H52N2O15S4 988.23, 측정치 987.7
LC / MS, calculated C 45 H 52 N 2 O 15 S 4 988.23, found 987.7
실시예 3: 화합물 c의 제조Example 3: Preparation of compound c
(1) 화합물 3-1의 합성(1) Synthesis of Compound 3-1
2,3,3-트리메틸인돌레닌(2,3,3-Trimethylindolenine) (6 g, 21.6 mmol, 1 eq)과 1,3-프로판설톤(1,3-Propanesultone) (6.4 mL, 68.9 mmol, 3.01 eq, TCI), 아세트산 나트륨(Sodium acetate) (2.1 g, 25.9 mmol, 1,2 eq)을 아세토나이트릴(Acetonitrile) 10 ml에 가한 후, 12 시간 동안 100℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. (3.5 g, 58.3 %) 2,3,3-Trimethylindolenine (6 g, 21.6 mmol, 1 eq) and 1,3-propanesultone (6.4 mL, 68.9 mmol, 3.01 eq, TCI) and sodium acetate (2.1 g, 25.9 mmol, 1,2 eq) were added to 10 ml of acetonitrile and the mixture was reacted for 12 hours at 100 ° C under reflux. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. (3.5 g, 58.3%).
Rf = 0.31 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.31 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(2) 화합물 3-2의 합성(2) Synthesis of Compound 3-2
3-1 (3.5 g, 12.3 mmol, 2 eq)과 1-4 (2 g, 6.16 mmol, 1 eq), 아세트산 나트륨(Sodium Acetate) (0.5 g, 6.16 mmol, 1 eq, 덕산)을 에탄올(Ethanol) 20 ml에 가한 후, 2 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 3-2를 얻었다. (4 g, 93 %) (2 g, 6.16 mmol, 1 eq) and sodium acetate (0.5 g, 6.16 mmol, 1 eq, Duksan) were dissolved in ethanol ), And the mixture was reacted by heating at 50 DEG C for 2 hours. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain pure compound 3-2. (4 g, 93%).
Rf = 0.62 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.62 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C36H43ClN2O6S2 698.23, 측정치 698.1LC / MS, Calcd. C 36 H 43 ClN 2 O 6 S 2 698.23, found 698.1
(3) 화합물 c의 합성(3) Synthesis of Compound c
3-2 (2 g, 2.85 mmol, 1 eq)과 소듐 3-(4-하이드록시페닐)프로피오닉 산(3-(4-Hydroxyphenyl)propionic acid, TCI) (1.42 g, 8.55 mmol, 3 eq), 포타슘 카보네이트(Potassium carbonate) (0.79 g, 5.7 mmol, 2 eq, 덕산)를 다이메틸포름아마이드 5 ml에 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 a를 얻었다. (700 mg, 30.4 %) 3-2 (2 g, 2.85 mmol, 1 eq) and sodium 3- (4-hydroxyphenyl) propionic acid (TCI) (1.42 g, 8.55 mmol, 3 eq) , Potassium carbonate (0.79 g, 5.7 mmol, 2 eq, Duksan) was added to 5 ml of dimethylformamide, and the mixture was reacted by heating at 50 ° C for 12 hours. After cooling to room temperature, the particles were collected with diethylether, washed 2 or 3 times, and dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain pure compound a. (700 mg, 30.4%).
Rf = 0.57 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.57 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C45H52N2O9S2 828.31, 측정치 827.9
LC / MS, Calcd. C 45 H 52 N 2 O 9 S 2 828.31, found 827.9
실시예 4: 화합물 d의 제조Example 4: Preparation of compound d
(1) 화합물 4-1의 합성(1) Synthesis of Compound 4-1
2,3,3-트리메틸인돌레닌(2,3,3-Trimethylindolenine) (20 g, 125.6 mmol, 1 eq)과 1,4-부탄설톤(1,4-Butanesultone) (38.3 mL, 376.8 mmol, 3 eq, TCI), 아세트산 나트륨(Sodium acetate) (12.4 g, 15.7 mmol, 1,2 eq)을 아세토나이트릴(Acetonitrile) 100 ml에 가한 후, 12 시간 동안 100℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. (16 g, 43 %) 2,3,3-Trimethylindolenine (20 g, 125.6 mmol, 1 eq) and 1,4-butanesultone (38.3 mL, 376.8 mmol, 3 eq, TCI) and sodium acetate (12.4 g, 15.7 mmol, 1,2 eq) were added to 100 ml of acetonitrile and the mixture was reacted for 12 hours at 100 ° C under reflux. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. (16 g, 43%).
Rf = 0.38 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.38 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(2) 화합물 4-2의 합성(2) Synthesis of Compound 4-2
4-1 (16 g, 54.2 mmol, 2.5 eq)과 1-4 (8.4 g, 21.7 mmol, 1 eq), 아세트산 나트륨(Sodium Acetate) (3.6 g, 43.4 mmol, 2 eq, 덕산)을 에탄올(Ethanol) 160 ml에 가한 후, 2 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 4-2를 얻었다. (3 g, 19 %) 4-one (16 g, 54.2 mmol, 2.5 eq), 1-4 (8.4 g, 21.7 mmol, 1 eq) and sodium acetate (3.6 g, 43.4 mmol, 2 eq, Duksan) ), And the mixture was reacted by heating at 50 DEG C for 2 hours. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain pure compound 4-2. (3 g, 19%).
Rf = 0.49 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.49 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C38H47ClN2O6S2 726.26, 측정치 726.3LC / MS, calculated C 38 H 47 ClN 2 O 6 S 2 726.26, found 726.3
(3) 화합물 d의 합성(3) Synthesis of compound d
4-2 (3 g, 4.12 mmol, 1 eq)과 4-머캡토하이드로시나믹 산(4-Mercaptohydrocinnami acid, TCI) (1.5 g, 8.24 mmol, 2 eq), 포타슘 카보네이트(Potassium carbonate) (1.14 g, 8.24 mmol, 2 eq, 덕산)를 다이메틸포름아마이드 30 ml에 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 d를 얻었다. (1.5 g, 41.8 %) 4-mercaptohydrocinnamic acid (TCI) (1.5 g, 8.24 mmol, 2 eq), potassium carbonate (1.14 g, , 8.24 mmol, 2 eq, Duksan) was added to 30 ml of dimethylformamide, followed by heating at 50 ° C for 12 hours. After cooling to room temperature, the particles were collected with diethylether, washed 2 or 3 times, and dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain pure compound (d). (1.5 g, 41.8%).
Rf = 0.5 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.5 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C47H56N2O8S3 872.32, 측정치 873.1
LC / MS, calculated C 47 H 56 N 2 O 8 S 3 872.32, found 873.1
실시예 5: 화합물 e의 제조Example 5: Preparation of Compound e
4-2 (90 mg, 0.124 mmol, 1 eq)과 3-하이드록시프로파녹 산(3-Hydroxypropanoic acid, TCI) (0.139 ml, 0.5 mmol, 4 eq), 포타슘 카보네이트(Potassium carbonate) (34 mg, 0.25 mmol, 2 eq, 덕산)를 다이메틸포름아마이드 3 ml에 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 e를 얻었다. (61 mg, 63.0 %) 3-hydroxypropanoic acid (TCI) (0.139 ml, 0.5 mmol, 4 eq), potassium carbonate (34 mg, 0.1 mmol) 0.25 mmol, 2 eq, Duksan) was added to 3 ml of dimethylformamide, followed by heating at 50 ° C for 12 hours. After cooling to room temperature, the particles were collected with diethylether, washed 2 or 3 times, and dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the developing solution to obtain a pure compound e. (61 mg, 63.0%).
Rf = 0.47 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.47 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C41H52N2O9S2 780.31, 측정치 781.3
LC / MS, Calcd. C 41 H 52 N 2 O 9 S 2 780.31, found 781.3
실시예 6: 화합물 f의 제조Example 6: Preparation of compound f
(1) 화합물 5-1의 합성(1) Synthesis of Compound 5-1
6-아미노-1,3-나프탈렌디설포닉 산 모노소듐 염 수화물) (6-Amino-1,3-naphthalene disulfonic acid monosodium salt hydrate, TCI) (15g)을 증류수 200mL에 가한 후 12 시간 동안 이온교환수지를 이용하여 이온교환 후 감압 건조 시킨다. (15g, 100%)Amino-1,3-naphthalene disulfonic acid monosodium salt hydrate (TCI) (15 g) was added to 200 mL of distilled water, and the mixture was stirred for 12 hours in an ion exchange resin Followed by ion exchange and drying under reduced pressure. (15 g, 100%).
염화제일주석 (Tin(II) chloride)에 증류수 40ml, 염산(HCl) 8ml을 혼합하고 냉각시켰다. 앞에서 얻은 고체 물질(15 g, 49.5mmol, 1eq)에 증류수 120ml, 염산(HCl) 25ml을 가한 후 5분 동안 0℃에서 반응시킨다. 아질산나트륨 (Sodium nitrite) (3.4 g, 49.5mmol, 1 eq)에 증류수 50ml 가한 것을 상기 용매에 적가 후 30분 동안 반응시킨다. 상기 냉각시킨 용액을 혼합액에 적가 후 12 시간 동안 상온에서 반응시킨다. 회전농축기(evaporator)로 용매를 건조 후 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. (18g, 114%)40 ml of distilled water and 8 ml of hydrochloric acid (HCl) were mixed with tin chloride (Tin (II) chloride) and cooled. 120 ml of distilled water and 25 ml of hydrochloric acid (HCl) were added to the solid material (15 g, 49.5 mmol, 1 eq) obtained above, and the mixture was reacted at 0 ° C for 5 minutes. 50 ml of distilled water was added to sodium nitrite (3.4 g, 49.5 mmol, 1 eq), and the mixture was added dropwise to the solvent, followed by 30 minutes of reaction. The cooled solution is added dropwise to the mixed solution and reacted at room temperature for 12 hours. After drying the solvent with an evaporator, the particles were grabbed with diethylether, washed 2 or 3 times, and dried under reduced pressure. (18 g, 114%).
(2) 화합물 5-2의 합성(2) Synthesis of Compound 5-2
5-1 (6 g, 18.8 mmol, 1 eq,)과 3-메틸-2-부탄온 (3-Methyl-2-butanone) (6.05 mL, 56.4 mmol, 3.02 eq, TCI), 포타슘 아세테이트(Potassium acetate) (3.7 g, 37.6 mmol, 2 eq, 덕산)을 아세트산 50 mL에 가한 후, 12 시간 동안 140℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고 용매를 감압 건조 후 에틸아세테이트(Ethyl acetate)로 2, 3회 세정한 후 감압 건조시켰다. (7.4 g, 107%) 3-Methyl-2-butanone (6.05 mL, 56.4 mmol, 3.02 eq, TCI), 5-1 (6 g, 18.8 mmol, 1 eq.) And potassium acetate ) (3.7 g, 37.6 mmol, 2 eq, Duksan) was added to 50 mL of acetic acid, and the mixture was reacted by heating at 140 ° C under reflux for 12 hours. The reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, and the residue was washed with ethyl acetate two or three times, followed by drying under reduced pressure. (7.4 g, 107%).
Rf = 0.57 정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.57 normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(3) 화합물 5-3의 합성(3) Synthesis of Compound 5-3
5-2 (30 g, 81.2 mmol, 1 eq)과 1,4-부탄설톤(1,4-Butanesultone) (33.2 mL, 328.8 mmol, 4 eq, TCI), 아세트산 나트륨(Sodium acetate) (8.05 g, 39.8 mmol, 1.2 eq)을 아세토나이트릴(Acetonitrile) 70 ml에 가한 후, 12 시간 동안 120℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 감압 건조하였다. (35g, 85%)4-butanesultone (33.2 mL, 328.8 mmol, 4 eq., TCI), sodium acetate (8.05 g, 39.8 mmol, 1.2 eq) was added to 70 ml of acetonitrile, and the mixture was reacted by heating at 120 ° C under reflux for 12 hours. Cooled to room temperature, and dried under reduced pressure. (35 g, 85%).
Rf = 0.25 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.25 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(4) 화합물 5-4의 합성(4) Synthesis of Compound 5-4
5-3 (2.2 g, 4.33 mmol, 2 eq), 1-4 (862 mg, 2.17 mmol, 1 eq), 아세트산 나트륨(Sodium Acetate) (450 mg, 5.42 mmol, 2.5 eq, 덕산)을 에탄올(Ethanol) 20 ml, 증류수 10ml에 가한 후, 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 5-4를 얻었다. (248.7 mg, 23.3 %) (862 mg, 2.17 mmol, 1 eq) and sodium acetate (450 mg, 5.42 mmol, 2.5 eq, Duksan) were dissolved in ethanol ) And 10 ml of distilled water, and the mixture was reacted by heating at 50 캜 for 12 hours. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain pure compound 5-4. (248.7 mg, 23.3%).
Rf = 0.15 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.15 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C46H51ClN2O18S6 1146.11, 측정치 1147.4LC / MS, Calcd. C 46 H 51 ClN 2 O 18 S 6 1146.11, Meas. 1147.4
(5) 화합물 f의 합성(5) Synthesis of compound f
5-4 (220 mg, 0.19 mmol, 1 eq)과 3-(4-하이드로시페닐) 프로피오닉 산(3-(4-hydroxyphenyl)propionic acid, TCI) (127.4 mg, 0.76 mmol, 4 eq), 포타슘 카보네이트(Potassium carbonate) (53 g, 0.38 mmol, 2 eq, 덕산)를 다이메틸포름아마이드 30 ml에 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 f를 얻었다. (1.5 g, 41.8 %) 4- (4-hydroxyphenyl) propionic acid (TCI) (127.4 mg, 0.76 mmol, 4 eq), 5-4 (220 mg, 0.19 mmol, Potassium carbonate (53 g, 0.38 mmol, 2 eq, Duksan) was added to 30 ml of dimethylformamide and reacted by heating at 50 캜 for 12 hours. After cooling to room temperature, the particles were collected with diethylether, washed 2 or 3 times, and dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain a pure compound (f). (1.5 g, 41.8%).
Rf = 0.31 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.31 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C53H56N2O21S6 1248.17, 측정치 1247.86
LC / MS, Calcd. C 53 H 56 N 2 O 21 S 6 1248.17, Measurement 1247.86
실시예 7: 화합물 g의 제조Example 7: Preparation of compound g
(1) 화합물 6-1의 합성(1) Synthesis of Compound 6-1
1,1,2-트리메틸벤즈 인돌(1,1,2-Trimethylbenz[e] indole) (2 g, 9.56 mmol, 1 eq)과 1,4-부탄설톤(1,4-Butanesultone) (2.9 mL, 28.7 mmol, 3.01 eq, TCI), 아세트산 나트륨(Sodium acetate) (941 mg, 11.5 mmol, 1,2 eq)을 아세토나이트릴(Acetonitrile) 10 ml에 가한 후, 12 시간 동안 100℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. (1 g, 31.3 %) 1,1,2-Trimethylbenz [e] indole (2 g, 9.56 mmol, 1 eq) and 1,4-butanesultone (2.9 mL, Was added to 10 ml of acetonitrile and the mixture was heated at 100 ° C for 12 hours under reflux to obtain a reaction mixture. The reaction mixture was stirred at room temperature for 2 hours, . The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. (1 g, 31.3%).
Rf = 0.37 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.37 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(2) 화합물 6-2의 합성(2) Synthesis of Compound 6-2
6-1 (1 g, 2.90 mmol, 2 eq)과 1-4 (465 mg, 1.45 mmol, 1 eq), 아세트산 나트륨(Sodium Acetate) (238 mg, 2.90 mmol, 2 eq, 덕산)을 에탄올(Ethanol) 20 ml에 가한 후, 2 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 용매 제거 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 6-2를 얻었다. (517.5 mg, 43 %) (468 mg, 1.45 mmol, 1 eq) and sodium acetate (238 mg, 2.90 mmol, 2 eq, Duksan) were dissolved in ethanol ), And the mixture was reacted by heating at 50 DEG C for 2 hours. The reaction mixture was cooled to room temperature, and solid particles produced after solvent removal were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the developing solution to obtain pure Compound 6-2. (517.5 mg, 43%).
Rf = 0.53 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.53 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C38H47ClN2O6S2 826.29, 측정치 827.1LC / MS, calculated C 38 H 47 ClN 2 O 6 S 2 826.29, found 827.1
(3) 화합물 g의 합성(3) Synthesis of Compound g
6-2(200 mg, 0.242 mmol, 1 eq)과 3-(4-하이드록시페닐)프로피오닉 산(3-(4-Hydroxyphenyl)propionic acid, TCI) (161 mg, 0.968 mmol, 4 eq), 포타슘 카보네이트(Potassium carbonate) (67 mg, 0.48 mmol, 2 eq, 덕산)를 다이메틸포름아마이드 5 ml에 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 g를 얻었다. (14.1 g, 6.1 %) (161 mg, 0.968 mmol, 4 eq) and 3- (4-hydroxyphenyl) propionic acid (TCI) (200 mg, 0.242 mmol, Potassium carbonate (67 mg, 0.48 mmol, 2 eq, Duksan) was added to 5 ml of dimethylformamide and reacted by heating at 50 ° C for 12 hours. After cooling to room temperature, the particles were collected with diethylether, washed 2 or 3 times, and dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain a pure compound g. (14.1 g, 6.1%).
Rf = 0.60 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.60 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C55H60N2O9S2 956.37, 측정치 9567.6
LC / MS, calculated C 55 H 60 N 2 O 9 S 2 956.37, found 9567.6
실시예 8: 화합물 h의 제조Example 8: Preparation of compound h
6-2(300 mg, 0.36 mmol, 1 eq)과 4-머캡토하이드로시나믹 산(4-Mercaptohydrocinnami acid, TCI) (264 mg, 1.45 mmol, 4 eq) (161 mg, 0.968 mmol, 4 eq), 포타슘 카보네이트(Potassium carbonate) (97 mg, 0.7 mmol, 2 eq, 덕산)를 다이메틸포름아마이드 5 ml에 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터로 입자를 잡은 뒤 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 h를 얻었다. (94 mg, 26.9 %) (161 mg, 0.968 mmol, 4 eq) and 4-mercaptohydrocinnamic acid (TCI) (264 mg, 1.45 mmol, 4 eq) , Potassium carbonate (97 mg, 0.7 mmol, 2 eq, Duksan) were added to 5 ml of dimethylformamide, and the mixture was reacted by heating at 50 ° C for 12 hours. After cooling to room temperature, the particles were collected with diethylether, washed 2 or 3 times, and dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain a pure compound h. (94 mg, 26.9%).
Rf = 0.47 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.47 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C55H60N2O8S3 972.35, 측정치 9723.2
LC / MS, Calcd. C 55 H 60 N 2 O 8 S 3 972.35, Measurement 9723.2
실시예 9: 화합물 i의 제조Example 9: Preparation of compound i
(1) 화합물 7-1의 합성(1) Synthesis of Compound 7-1
7-2 (7.4 g, 20.3 mmol, 1 eq)과 1,3-프로판설톤(1,3-Propanesultone) (7.9 mL, 90.5 mmol, 4.46 eq, TCI), 아세트산 나트륨(Sodium acetate) (2 g, 40.6 mmol, 2 eq)을 아세토나이트릴(Acetonitrile) 20 ml에 가한 후, 12 시간 동안 120℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 감압 건조하였다. (15g, 153%)7-2 (7.4 g, 20.3 mmol, 1 eq), 1,3-propanesultone (7.9 mL, 90.5 mmol, 4.46 eq. TCI), sodium acetate (2 g, 40.6 mmol, 2 eq) was added to 20 ml of acetonitrile, and the mixture was reacted at 120 ° C under reflux for 12 hours. Cooled to room temperature, and dried under reduced pressure. (15 g, 153%).
(2) 화합물 7-2의 합성(2) Synthesis of Compound 7-2
7-1 (15 g, 30.4 mmol, 1 eq)과 말론알데하이드 다이아닐리드 하이드로클로라이드(Malonaldehyde Dianilide Hydrochloride) (7.8 g, 30.4 mmol, 1 eq, TCI), 트리에틸아민(Triethylamine) (4.2 mL, 30.4 mmol, 1 eq, TCI)를 아세트산(Acetic acid) 100ml에 가한 후 4 시간 동안 140℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 그 후 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v의 전개액으로 Silica gel 60 정상 크로마토그래피로 정제하였다. (12 g, 60 %) (7.8 g, 30.4 mmol, 1 eq., TCI) and triethylamine (4.2 mL, 30.4 mmol) were added to a solution of 7-1 (15 g, 30.4 mmol, 1 eq) and malonaldehyde Dianilide Hydrochloride , 1 eq, TCI) was added to 100 ml of acetic acid, and the mixture was reacted for 4 hours at 140 ° C under reflux. The mixture was cooled to room temperature, washed with ethyl acetate two or three times, and dried under reduced pressure. Thereafter, the supernatant was purified by
Rf = 0.5 정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.5 Normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(3) 화합물 7-3의 합성(3) Synthesis of Compound 7-3
에틸 2-메틸아세토아세테이트(Ethyl 2-methylacetoacetate) (24.2 mL, 0.16 mol, 1 eq, TCI)과 에틸-6-브로모헥사노에이트(Ethyl 6-bromohexanoate) ( 34 mL, 0.17 mol, 1.1 eq, TCI), 소듐 에톡사이드(Sodium ethoxide) (64 mL, 0.77 mol, 4.8 eq, TCI)에 에탄올(Ethanol) 200ml을 가한 후 12 시간 동안 120℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 1M 염산을 소량 적가 후 pH를 중성(7)로 만든다. 클로로포름(Chloroform)과 증류수를 이용하여 추출 후 용매를 감압 건조한다. 그 후 핵산과 에틸아세테이트를 전개액으로 Silica gel 60 정상 크로마토그래피로 정제하였다. (48g, 106.6%)Ethyl 6-bromohexanoate (34 mL, 0.17 mol, 1.1 eq.) Was added to a solution of ethyl 2-methylacetoacetate (24.2 mL, 0.16 mol, 1 eq, TCI) TCI), sodium ethoxide (64 mL, 0.77 mol, 4.8 eq, TCI), and the mixture was reacted at 120 ° C for 12 hours under reflux. Cool to room temperature, add a small amount of 1 M hydrochloric acid, and adjust the pH to neutral (7). After extraction with chloroform (chloroform) and distilled water, the solvent is dried under reduced pressure. Then, the nucleic acid and ethyl acetate were purified by using
Rf = 0.4 (정상, 실리카겔, 핵산:에틸아세테이트=9:1 v/v)R f = 0.4 (normal, silica gel, nucleic acid: ethyl acetate = 9: 1 v / v)
(4) 화합물 7-4의 합성(4) Synthesis of Compound 7-4
7-3 (48 g, 0.16 mol, 1 eq)과 수산화나트륨(Sodium hydroxide) (21.4 g, 0.51 mol, 3.2 eq, 덕산)에 메탄올(Methanol) (165 mL, 3.9 mol, 24.4 eq)과 증류수 (54.6 mL, 2.89 mol, 18.1 eq)를 가한 후 12 시간 동안 50℃에서 가열하며 반응시켰다. 감압 건조 후 1M 염산을 200ml 적가하여 pH를 산성(1)로 만들었다. 에틸아세테이트와 증류수로 추출 후 용매를 감압 건조한다. (30g, 95.2%)Methanol (165 mL, 3.9 mol, 24.4 eq) and distilled water (1 mL) were added to 7-3 (48 g, 0.16 mol, 1 eq) and sodium hydroxide (21.4 g, 0.51 mol, 3.2 eq, 54.6 mL, 2.89 mol, 18.1 eq) was added, and the mixture was reacted by heating at 50 ° C for 12 hours. After drying under reduced pressure, 200 ml of 1M hydrochloric acid was added dropwise to make the pH acidic (1). After extraction with ethyl acetate and distilled water, the solvent is evaporated to dryness under reduced pressure. (30 g, 95.2%).
Rf = 0.0 (정상, 실리카겔, 핵산:에틸아세테이트=9:1 v/v)R f = 0.0 (normal, silica gel, nucleic acid: ethyl acetate = 9: 1 v / v)
(5) 화합물 7-5의 합성(5) Synthesis of Compound 7-5
7-4 (15.8 g, 84.8 mmol, 1.5 eq)과 5-1 (18 g, 56.5 mmol, 1 eq)에 아세트산(Acetic acid) 87ml을 가한 후 5시간 동안 120℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고 생성된 고체를 제거하기 위해 여과하였으며 여액을 감압 건조시켰다. 그 후 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v의 전개액으로 Silica gel 60 정상 크로마토그래피로 정제하였다. (30g, 115.3%)87 ml of acetic acid was added to 7-4 (15.8 g, 84.8 mmol, 1.5 eq) and 5-1 (18 g, 56.5 mmol, 1 eq) and the mixture was heated at 120 ° C under reflux for 5 hours. The solution was cooled to room temperature and filtered to remove the resulting solid. The filtrate was dried under reduced pressure. Thereafter, the supernatant was purified by
Rf = 0.45 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.45 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
(6) 화합물 7-6의 합성(6) Synthesis of Compound 7-6
7-5 (30 g, 63.9 mmol, 1 eq) 1,3-프로판설톤(1,3-Propanesultone) (37.5 mL, 428.1 mmol, 6.7 eq, TCI), 아세트산 나트륨(Sodium acetate) (7.2 g, 105.4 mmol, 1,65 eq)을 아세토나이트릴(Acetonitrile) 45 ml에 가한 후, 5 시간 동안 120℃에서 가열 환류하며 반응시켰다. 상온으로 냉각시키고, 감압 건조하였다. 그 후 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하였다. (52g, 140%) 7-5 (30 g, 63.9 mmol, 1 eq) 1,3-Propanesultone (37.5 mL, 428.1 mmol, 6.7 eq, TCI), sodium acetate (7.2 g, 105.4 mmol, 1.65 eq) was added to 45 ml of acetonitrile, and the mixture was reacted for 5 hours at 120 ° C under reflux. Cooled to room temperature, and dried under reduced pressure. The aqueous acetonitrile solution was then purified by RP-C18 reverse phase chromatography using the eluent. (52 g, 140%).
(7) 화합물 i의 합성(7) Synthesis of compound i
7-6 (15 g, 25.9 mmol, 1.5 eq)과 7-2 (11.5 g, 17.3 mmol, 1 eq)를 디메틸포름아마이드(Dimethylformamide)에 완용한다. 상기 용액에 트리에틸아민(Triethylamine) (20.5 mL, 147 mmol, 8.5 eq, TCI)를 아세트산무수물(Acetic anhydride) (9 mL, 95.1 mmol, 5.5 eq, 덕산)를 가한 후 1 시간 동안 상온에서 반응시켰다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 그 후 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v의 전개액으로 Silica gel 60 정상 크로마토그래피로 정제하였다. (12 g, 60 %) 7-6 (15 g, 25.9 mmol, 1.5 eq) and 7-2 (11.5 g, 17.3 mmol, 1 eq) are dissolved in dimethylformamide. Acetic anhydride (9 mL, 95.1 mmol, 5.5 eq, Duksan) was added to the solution, followed by reaction at room temperature for 1 hour . After washing with ethyl acetate two or three times, it was dried under reduced pressure. Thereafter, the supernatant was purified by
Rf = 0.5 정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)
R f = 0.5 Normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
실시예 10: 화합물 j의 제조Example 10: Preparation of compound j
(1) 화합물 8-1의 합성(1) Synthesis of Compound 8-1
화합물 7-6 (8.165 g, 0.0438 mol, 1 eq)에 p-하이드라지노벤젠설포닉 산(p-Hydrazinobenzensulfonic Acid Hemihydrate) (8.25 g, 0.0438 mol, 1 eq), 아세트산를 첨가한 뒤 120℃에서 5시간동안 환류시켰다. 이를 감압건조 시킨 뒤 정상 크로마토그래피법을 이용하여 정제하였다. (12.6g, 84.8%)Hydrazinobenzenesulfonic acid hemihydrate (8.25 g, 0.0438 mol, 1 eq) and acetic acid were added to compound 7-6 (8.165 g, 0.0438 mol, 1 eq) Lt; / RTI > After drying it under reduced pressure, it was purified using a normal chromatographic method. (12.6 g, 84.8%)
Rf = 0.51 (Silicagel, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3)R f = 0.51 (Silicagel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3)
(2) 화합물 8-2의 합성(2) Synthesis of Compound 8-2
화합물 8-1 (12.57 g, 0.037 mol, 1 eq)에 아세트산 나트륨 (4.16 g, 0.061 mol, 1.65 eq), 1,3-프로판 설톤(1,3-Propane Sultone) (21.3ml, 0.243 mol, 6.57 eq), 아세토니트릴 (24.8 ml)을 첨가한 뒤 110℃에서 5시간동안 환류시켰다. 그 뒤 감압건조 한 뒤 역상 크로마토그래피법을 이용하여 정제하였다. (12g, 70.6%)Sodium acetate (4.16 g, 0.061 mol, 1.65 eq) and 1,3-propane sultone (21.3 ml, 0.243 mol, 6.57 eq) were added to compound 8-1 (12.57 g, 0.037 mol, eq) and acetonitrile (24.8 ml) were added, and the mixture was refluxed at 110 ° C for 5 hours. After that, it was dried under reduced pressure and then purified by reversed phase chromatography. (12 g, 70.6%)
Rf = 0.3 (Silicagel, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3)R f = 0.3 (Silicagel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3)
(3) 화합물 j의 합성(3) Synthesis of compound j
8-2 (15 g, 25.9 mmol, 1.5 eq)과 7-4 (11.5 g, 17.3 mmol, 1 eq)를 디메틸포름아마이드(DMF, Dimethylformamide)에 완용한다. 상기 용액에 트리에틸아민(Triethylamine) (20.5 mL, 147 mmol, 8.5 eq, TCI)를 아세트산무수물(Acetic anhydride) (9 mL, 95.1 mmol, 5.5 eq, 덕산)를 가한 후 1 시간 동안 상온에서 반응시켰다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 그 후 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 j를 얻었다. (1.5 g, 41.8 %) 8-2 (15 g, 25.9 mmol, 1.5 eq) and 7-4 (11.5 g, 17.3 mmol, 1 eq) are dissolved in dimethylformamide (DMF). Acetic anhydride (9 mL, 95.1 mmol, 5.5 eq, Duksan) was added to the solution, followed by reaction at room temperature for 1 hour . After washing with ethyl acetate two or three times, it was dried under reduced pressure. Thereafter, an aqueous acetonitrile solution was purified by RP-C18 reverse phase chromatography using the developing solution to obtain a pure compound j. (1.5 g, 41.8%).
Rf = 0.02 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.02 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C44H50N2O20S6 1118.23 측정치 1117.0
LC / MS, Calcd. C 44 H 50 N 2 O 20 S 6 1118.23 Measured 1117.0
실시예Example
11: 화합물 1의 제조 11: Preparation of
(1) 화합물 a-1의 합성(1) Synthesis of compound a-1
a (500 mg, 0.5 mmol, 1 eq)와 N,N-다이석시니미딜 카보네이트(DSC, N,N-Disuccinmidyl carbonate) (384 mg, 1.5 mmol, 3 eq, TCI), N,N-다이아이소프로필에틸아민(DIPEA, N,N-Diisopropylethylamine) (870 μl, 5 mmol, 10 eq, TCI)을 다이메틸포름아마이드 50 ml에 가한 후, 1 시간 동안 40℃에서 가열하며 반응시켰다. 상온으로 냉각시키고, 다이에틸 이터를 이용하여 입자를 잡은 뒤 생성된 고체 입자를 감압 건조시켰다 (500 mg, 91%) N, N-disuccinimidyl carbonate (384 mg, 1.5 mmol, 3 eq, TCI), N, N-disuccinimidyl carbonate (DSC, 500 mg, 0.5 mmol, (870 μl, 5 mmol, 10 eq., TCI) was added to 50 ml of dimethylformamide, and the mixture was reacted by heating at 40 ° C. for 1 hour. After cooling to room temperature, the particles were collected by using a diethylether, and the resulting solid particles were dried under reduced pressure (500 mg, 91%).
Rf = 0.31 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.31 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C50H57N3O17S4 1099.26, 측정치 1100.5LC / MS, calculated C 50 H 57 N 3 O 17 S 4 1099.26, measured 1100.5
(2) 화합물 a-2의 합성(2) Synthesis of compound a-2
화합물 a-1 (500 mg, 0.035 mmol, 1 eq)에 1,3-다이아미노프로판 (1,3-Diaminopropane) (9.72 ul, 0.1165 mmol, 1 eq)을 디엠에프(DMF) 10 mL에 가한 후, 30분 동안 상온에서 반응시켰다. 반응 후 생성된 고체 입자를 여과하였다. 에틸아세테이트로 2, 3회 세정한 후 감압 건조시켰다. 아세토니트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 a-2를 얻었다. (400mg, 75.6%) To DMF (10 mL) was added 1,3-diaminopropane (9.72 ul, 0.1165 mmol, 1 eq) to compound a-1 (500 mg, 0.035 mmol, 1 eq) , And reacted at room temperature for 30 minutes. The solid particles produced after the reaction were filtered. After washing with ethyl acetate two or three times, it was dried under reduced pressure. The acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the eluent to obtain pure compound a-2. (400 mg, 75.6%).
Rf = 0.33 (정상, 실리카겔, 아세토니트릴:증류수=4:1 v/v)R f = 0.33 (normal, silica gel, acetonitrile: distilled water = 4: 1 v / v)
LC/MS, 계산치 C49H62N4O14S4 1058.31, 측정치 LC / MS, Calcd. C 49 H 62 N 4 O 14 S 4 1058.31, measured
(3) 화합물 a-3의 합성(3) Synthesis of compound a-3
시아누릭 클로라이드 (Cyanuric chloride) (383 mg, 2.08 mmol, 5.5 eq)를 아세토나이드릴 35 ml와 얼음 35 ml에 넣은 후 온도를 0~5℃로 0.5 시간 동안 섞어주었다. 화합물 a-2 (400 mg, 0.378 mmol, 1eq), 중탄산나트륨 (Sodium bicarbonate) 191 mg를 첨가하여 온도를 0~5℃로 2시간 동안 반응 하였다. 감압 건조 뒤, 아세토나이트릴 수용액을 전개액으로 RP-C18 역상 크로마토그래피로 정제하여 순수한 화합물 a-3을 얻었다. (373 mg, 81.9 %) Cyanuric chloride (383 mg, 2.08 mmol, 5.5 eq) was added to 35 ml of acetonimate and 35 ml of ice, and the mixture was stirred at 0-5 ° C for 0.5 hour. Compound a-2 (400 mg, 0.378 mmol, 1 eq) and sodium bicarbonate (191 mg) were added and reacted at 0-5 ° C for 2 hours. After drying under reduced pressure, the acetonitrile aqueous solution was purified by RP-C18 reverse phase chromatography using the developing solution to obtain pure compound a-3. (373 mg, 81.9%).
Rf = 0.47 (이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3)R f = 0.47 (isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3)
LC/MS, 계산치 C52H61Cl2N7O14S4 1205.25 측정치 LC / MS, Calcd. C 52 H 61 Cl 2 N 7 O 14 S 4 1205.25 Measure
(4) 화합물 a-4의 합성(4) Synthesis of compound a-4
a-3 (373 mg, 0.309 mmol, 1eq), 6-아미노헥사노익산 (6-Aminohexanoic acid) (284 mg, 2.16 mmol, 7 eq), N,N-다이아이소프로필에틸아민(DIPEA, N,N-Diisopropylethylamine) (538 μl, 3.09 mmol, 10 eq, TCI)을을 증류수 30 ml에 가한 후, 1시간 동안 상온에서 반응시켰다. 그 후 아세토나이트릴 수용액을 전개액으로 이용하는 역상 크로마토그래피로 정제하여 순수한 화합물 a-4를 얻었다. (235 mg, 58.5 %) N-diisopropylethylamine (DIPEA, N, N-dimethylformamide) was added to a-3 (373 mg, 0.309 mmol, 1 eq), 6-aminohexanoic acid (284 mg, 2.16 mmol, N-Diisopropylethylamine (538 μl, 3.09 mmol, 10 eq, TCI) was added to 30 ml of distilled water and reacted at room temperature for 1 hour. Thereafter, the solution was purified by reverse phase chromatography using an aqueous solution of acetonitrile as a developing solution to obtain pure compound a-4. (235 mg, 58.5%).
Rf = 0.33 (이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3)R f = 0.33 (isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3)
LC/MS, 계산치 C58H73ClN8O16S4 1300.37, 측정치 1300.9LC / MS, Calcd. C 58 H 73 ClN 8 O 16 S 4 1300.37, measurement 1300.9
(5) 화합물 1의 합성(5) Synthesis of
a-4 (235 mg, 0.18 mmol, 1 eq)와 N,N-다이석시니미딜 카보네이트(DSC, N,N-Disuccinmidyl carbonate) (139 mg, 0.542 mmol, 3 eq, TCI), N,N-다이아이소프로필에틸아민(DIPEA, N,N-Diisopropylethylamine) (314 μl, 1.8 mmol, 10 eq, TCI)을 다이메틸포름아마이드 20 ml에 가한 후, 1시간 동안 상온에서 반응시켰다. 다이에틸 이터를 이용하여 입자를 잡은 뒤 생성된 고체 입자를 감압 건조시켰다. 그 후 아세토나이트릴 수용액을 전개액으로 이용하는 역상 크로마토그래피로 정제하여 순수한 화합물 1를 얻었다. (39 mg, 15.5 %) a-4 N, N-disuccinimidyl carbonate (139 mg, 0.542 mmol, 3 eq, TCI), N, N-diisobutylaluminum hydride (235 mg, 0.18 mmol, 1 eq) (314 μl, 1.8 mmol, 10 eq., TCI) was added to 20 ml of dimethylformamide, and the mixture was allowed to react at room temperature for 1 hour. The particles were grabbed with diethylether and the resulting solid particles were dried under reduced pressure. Thereafter, purified by reversed phase chromatography using an aqueous solution of acetonitrile as a developing solution to obtain
Rf = 0.65 (정상, 실리카겔, 이소부탄올:프로판올:에틸아세테이트:증류수=2:4:1:3 v/v)R f = 0.65 (normal, silica gel, isobutanol: propanol: ethyl acetate: distilled water = 2: 4: 1: 3 v / v)
LC/MS, 계산치 C62H76ClN9O18S4 1397.39, 측정치 1397.3LC / MS, Calcd C 62 H 76 ClN 9 O 18 S 4 1397.39, Measurement 1397.3
1H NMR (600 MHz, (CD3)2SO) : δ 7.729-7.648 (m, 3H), δ 7.424-7.411 (d, 1H), δ 7.342-7.328 (d, 1H), δ 7.223-7.195 (d, 1H), δ 7.134-7.120 (d, 1H), δ 7.078-6.987 (d, 3H), δ 6.380-6.356 (d, 1H), δ 6.242-6.219 (d, 1H), δ 6.117-6.093 (d, 1H), δ 6.023-5.999 (d, 1H), δ 4.196-4.112 (t, 1H), δ 4.112-4.061 (m, 7H), δ 3.262-3.251 (q, 2H, J = 2.75Hz), δ 3.251-3.201 (t, 3H), δ 3.201-3.123 (m, 5H), δ 3.004-2.981 (d, 3H), δ 2.791-2.708 (m, 10H), δ 2.645-2.618 (t, 5H, J = 12Hz), δ 2.591-2.536 (q, 7H, J = 21Hz), δ 2.010-1.951 (m, 1H), δ 1.637-1.588 (m, 6H), δ 1.543-1.433 (m, 6H)
1 H NMR (600 MHz, (CD 3 ) 2 SO):? 7.729-7.648 (m, 3H),? 7.424-7.411 (d, 1H),? 7.342-7.328 (d, 1H),? 7.223-7.195 (d, 1H),? 7.134-7.120 (d, 1H),? 7.078-6.987 (d, 3H),? 6.380-6.356 1H, d, 1H),? 6.023-5.999 (d, 1H),? 4.196-4.112 (t, 1H),? 4.112-4.061 (m, 7H),? 3.262-3.251 (q, 2H, J = 3H),? 2.791-2.708 (m, 10H),? 2.645-2.618 (t, 5H, J = 12 Hz),? 2.591-2.536 (q, 7H, J = 21 Hz),? 2.010-1.951 (m, 1H),? 1.637-1.588 (m, 6H),? 1.543-1.433
시험예 1: 화합물의 광학특성 평가Test Example 1: Evaluation of Optical Properties of Compound
(1) 형광강도 비교 - 800 파장 염료(1) Fluorescence intensity comparison - 800 wavelength dye
화합물 1과 대조형광염료(IRDyeㄾ 800CW NHS ester, DyLight™ 800 NHS ester, ICG NHS ester)의 형광강도를 비교하였다. 4 종의 형광염료에 DMF를 넣어 Stock solution을 제조한다. 농도는 10 mg/mL로 동일하게 만들었다. 이 후 pH 7.4 10 mM Phosphate buffered saline(이하 1X PBS)을 이용하여 78.12 nM의 농도로 희석한 후 Excitation 774 nm 설정 하에 형광을 측정하였다. 측정은 PerkinElmer의 LS 55 Fluorescence spectrometer를 활용하였다. 도 1은 두 화합물 1의 형광 스펙트럼을 나타낸 것이며, 본 분석으로 화합물 1의 형광강도가 모든 대조형광염료(IRDyeㄾ 800CW NHS ester, DyLight™ 800 NHS ester, ICG NHS ester)보다 형광강도가 상대적으로 강함을 확인하였다.The fluorescence intensities of
(2) 800 파장 화합물의 상대양자효율(Relative quantum yield) 측정(2) Measurement of relative quantum yield of 800 wavelength compound
Rhodamine 6G (TCI)를 기준으로, 화합물 1과 대조형광염료(IRDyeㄾ 800CW NHS ester, DyLight™ 800 NHS ester, ICG NHS ester)의 상대양자효율을 측정하였다. 4 개의 형광염료와 Rhodamine 6G에 DMF를 넣어 10 mg/mL Stock solution을 제조한다. 이 후 pH 7.4 1X PBS을 이용하여 10 uM의 농도로 희석한 후 흡광 및 형광을 측정하였다. 10 uM 농도로부터 1/2 dilution을 시행하여 1/128x 농도 샘플까지 총 8회씩 흡ㅇ형광을 측정하였다. 수학식 1에 측정값을 대입하여 상대양자효율을 분석하였고, 표 1 및 도 2와 같은 결과를 얻었다. 화합물 1의 상대 양자효율은 모든 대조형광염료보다 더 높았다.The relative quantum efficiencies of
6GRhodamine
6G
(3) 800 파장 염료의 세포독성(3) Cytotoxicity of 800 wavelength dyes
화합물 1과 대조형광염료(IRDyeㄾ 800CW NHS ester, DyLight™ 800 NHS ester, ICG NHS ester)를 HeLa Cell에 처리 후 MTT assay를 통해 염료의 세포독성을 평가하였다. 동일한 양의 Cell(2*105개)에 화합물 1과 대조형광염료들을 농도(0, 5, 10, 15, 20, 25 μg/mL)별로 2시간 동안 처리 후 5mg/mL의 MTT assay 용액을 4시간 동안 추가 처리하였다. Plate reader기를 통해 540nm에서 흡광을 측정하여 Cell viability를 산출하였다. 화합물 1은 도 3과 같이 Cell viability에 영향을 주지 않았으며, 염료 독성 또한 없었다. 모든 대조형광염료 또한 Cell viability에 영향을 주지 않았다.
시험예 2 : 단백질에 표지(labeling)한 후의 성능 비교Test Example 2: Performance comparison after labeling with protein
(1) 800 파장 염료의 표지율(1) labeling rate of 800 wavelength dyes
화합물 1과 대조형광염료(IRDyeㄾ 800CW NHS ester, DyLight™ 800 NHS ester) 총 3 종에 대하여 항체(Thermo, Goat anti-Mouse IgG H+L Secondary Ab, 140 kDa)에 표지를 진행하고, 표지율 (Dye/Protein ratio, 이하 D/P ratio)을 비교하였다. 표지 전 화합물 1과 대조형광염료는 모두 DMF에 10 mg/mL로 녹여 Stock solution을 만들어 사용하였고, 반응 조건은 항체 0.5 mg에 형광염료 0.1 mg으로 각 Dye 분자량에 관계없이 동일한 무게량을 반응시켰다. 반응 버퍼는 최종 pH 8.5 가 되도록 10 mM pH 7.4 Phosphate buffered saline (이하 1X PBS) 및 1M pH 9.4 Sodium carbonate-Bicarbonate buffer (이하 1 M CBC)를 섞어 제조하였으며, 항체 최종 반응 농도는 2 mg/mL이 되게 하였다. 화합물 1 및 대조형광염료들을 앞선 실험 조건 하에 상온, 암실 환경에서 1 시간 자석 교반하며 반응시켰고, 이후 1X PBS로 미리 Buffer equilibrium 해 둔 PD-10 Column (GE Healthcare)을 이용하여 정제하여 반응물을 획득하였다. 획득한 반응물을 각 1/4 dilution (원액으로 분석할 경우 기기 상 Saturation으로 표시 됨)하여 280, 771, 774 nm 파장에서 흡광도를 분석(Agilent, Cary 8454 UV-Vis spectrophotometer)하였고, 보편적으로 알려진 수식에 따라 표지율을 산출하였다. 표 2에 그 결과를 나타내었다.Labeled antibody (Thermo, Goat anti-Mouse IgG H + L Secondary Ab, 140 kDa) was labeled with
표 2에서 D/P 1은 각 형광염료 고유의 기준 (Ex 파장에서의 흡광 값, Extinction coefficient, Correction factor)을 적용한 표지율이며, D/P 2는 IRDyeㄾ 800CW NHS ester 제품 기준 (Ex 파장에서의 흡광 값, Extinction coefficient, Correction factor)을 적용 해 산출한 결과이다. 표시한 Ex/Em, Ext.co., CF 값, 분자량은 각 회사의 Datasheet 참고 및 자사에서 진행한 광학분석 결과를 기반으로 하였다. 표지율을 비교 분석 해 보면, IRDyeㄾ 800CW NHS ester와는 비슷한 정도, DyLightTM 800 NHS ester와 비교하면 화합물 1이 더 높은 표지율을 나타내었다. 주시할 점은, 화합물 1의 분자량이 1397.39 g/mol로 대조형광염료 IRDyeㄾ 800CW NHS ester 보다는 약 20 %, DyLightTM 800 NHS ester에 비해서는 약 33 %가 큰데, 그렇기 때문에 동일 중량으로 반응한 본 실험의 경우 몰비 환산 시 화합물 1이 상대적으로 적은 몰수로 투입된 것이다. 따라서 화합물 1의 표지율은 대조형광염료들에 비해 최소 동등 혹은 그 이상의 수준을 갖는다고 할 수 있다.In Table 2, D /
(2) 800 파장 염료의 단백질 반응물(Conjugates) 간 형광강도 비교(2) Comparison of fluorescent intensities between protein reactants (Conjugates) of 800 wavelength dyes
시험예 2-(1)에서 수득한 각 반응물을 활용하여, 화합물 1 및 대조형광염료의 단백질(항체) 표지 후 형광강도를 비교하였다. 육안으로 비교할 수 있는 FOBI (NeoScience, Fluorescence- labeled Organism Bioimaging Instrument, FOBI mini BR)와 측정치로 비교가 가능한 Multi-label plate reader (PerkinElmer, Enspire 2300)로 이중 분석을 진행하였다. 각 반응물의 원액과 1/2, 1/4, 1/10 희석 샘플(1X PBS 이용), 그리고 Blank로 1X PBS를 96-well Black plate에 순서대로 well 당 100 uL 씩 주입하고 분석하였다. FOBI Imaging은 기기 상 Light source NIR (735 nm) channel 및 NIR Emission filter 적용, Exposure time 1600 ms, Gain 7x 설정 하에 수행하였고, Plate reader 분석의 경우 화합물 1 및 IRDyeㄾ 800CW NHS ester 제품의 Ex/EmMax 파장 774/789 nm를 적용하여 1 차(DyLight™ 800 NHS ester의 최대 흡ㅇ형광 파장도 크게 차이 나지 않음), 응용 연구에 보편적으로 활용하는 Indocyanine green(ICG)의 파장 785/812 nm를 적용하여 2 차 분석하였다. 1, 2 차 분석에서 Wavelength 이외의 기기설정은 동일하게 하였다 (Measure -ment height 9.5 mm, Number of flashes 200 등). 도 4에 FOBI Image를 제시하였고, 도 5에는 Plate reader 측정한 결과의 비교 그래프를 나타내었다. 도 5의 형광강도는 Blank 보정 (각 Blank well의 형광강도 측정 평균치를 각 well 측정값에서 뺀 것으로, 보정 전과 경향에 있어서의 차이는 없다)한 자료이다. 예상되는 바와 같이, ICG 최대 흡형광 파장 설정에서의 측정에 비해 화합물 1 및 대조형광염료의 실제 최대 흡형광 파장 설정 하의 측정 결과가 전반적으로 높은 강도로 나타나기는 하였으나, 이미지 및 측정치 분석 모두에서 화합물 1 > IRDyeㄾ 800CW NHS ester > DyLight™ 800 NHS ester 반응물의 순서로 형광세기가 강한 것으로 확인된다. 이러한 경향은 원액을 포함한 1/2, 1/4, 1/10 희석 샘플에서 모두 동일하였다.
Using the reactants obtained in Test Example 2- (1), the fluorescence intensities after the labeling of the protein (antibody) of the
시험예 3: 화합물 1을 도입한 나노파티클의 제조(화합물1-HGC)Test Example 3: Preparation of nanoparticles incorporating Compound 1 (Compound 1-HGC)
(1) 나노파티클의 제조(HGC)(1) Production of nanoparticles (HGC)
글라이콜 키토산(Glycol Chitosan, GC)을 1.0g을 80mL의 초순수수에 녹인 후 3일 동안 투석을 진행하며, 투석 완료 후 동결건조 진행한다. (587 mg, 58%)Dissolve 1.0 g of Glycol Chitosan (GC) in 80 mL of ultrapure water and dialyse for 3 days. After dialysis, freeze-drying proceeds. (587 mg, 58%).
정제된 글라이콜 키토산(GC) 200mg를 초순수수 24mL에 완용한 후 24mL의 메탄올을 첨가하여 교반한다. 60mg의 담즙산(5β-Chloanic acid)을 48mL의 메탄올에 넣고 완용한다. 48mg의 N-(3-디메틸아미노프로필)-N'-에틸카보디이미드 염산염(EDC)을 1mL의 메탄올에 넣고 교반한 후 글라이콜 키토산(GC) 용액에 넣어 교반한다. 28.8mg의 N-하이드록시석신이미드(NHS)에 메탄올 1mL을 넣고 교반한 후 담즙산 용액에 투입하여 교반하고 상기 혼합물을 글라이콜 키토산(GC)혼합용액에 투입하여 12 시간 동안 상온에서 교반을 진행한다. 상기 반응액을 여과한 후, 4일 동안 투석 진행하며 투석 완료 후 동결건조하여 담즙산-키토산(HGC) 나노입자를 얻었다. (208.8 mg, 104%)200 mg of purified glycol chitosan (GC) is added to 24 mL of ultrapure water, and 24 mL of methanol is added and stirred. 60 mg of 5β-Chloanic acid is added to 48 mL of methanol. 48 mg of N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC) is added to 1 mL of methanol and stirred, and the mixture is stirred in a glycol chitosan (GC) solution. After adding 1 mL of methanol to 28.8 mg of N-hydroxysuccinimide (NHS) and stirring, the mixture was added to a bile acid solution and stirred. The mixture was added to a glycol chitosan (GC) mixed solution and stirred at room temperature for 12 hours Go ahead. The reaction solution was filtered, dialyzed for 4 days, dialyzed and lyophilized to obtain cholanic acid-chitosan (HGC) nanoparticles. (208.8 mg, 104%).
(2) 화합물1-HGC의 제조(2) Production of Compound 1-HGC
담즙산-키토산(HGC) 나노입자 (50 mg, 0.53 μmol, 1 eq)을 0.1M pH9.0 Carbonate buffer 20ml에 가한 후 12 시간 동안 상온에서 용해시켰다. 화합물 1 (6 mg, 4.25 μmol, 8 eq)을 다이메틸 설폭사이드(Dimethyl sulfoxide, DMSO) 0.6ml에 용해 후 상기 용액에 적가 후 12 시간 동안 상온에서 교반한다, 5일 동안 투석 진행하며 투석 완료 후 동결건조하여 화합물 1을 도입한 나노파티클(화합물1-HGC)을 얻었다. (45 mg, 90%)
Cholanic acid-chitosan (HGC) nanoparticles (50 mg, 0.53 μmol, 1 eq) were added to 20 ml of 0.1 M pH 9.0 Carbonate buffer and dissolved at room temperature for 12 hours. Compound 1 (6 mg, 4.25 μmol, 8 eq) was dissolved in 0.6 ml of dimethyl sulfoxide (DMSO), added dropwise to the solution, and stirred at room temperature for 12 hours. The mixture was dialyzed for 5 days, And lyophilized to obtain a nanoparticle (Compound 1-HGC) into which
시험예 4 : 화합물 1의 동물실험 분석 Test Example 4: Animal experiment analysis of
(1) 800 파장 염료의 체내분포양상 측정(1) Measurement of distribution pattern of 800 wavelength dye
화합물 1과 대조형광염료(IRDyeㄾ 800CW Carboxylic acid)를 DMSO에 10mg/ml로 녹여 Stock solution으로 만들어 사용하였고, 1X PBS를 이용하여 희석하여 0.25g/kg의 농도로 마우스의 꼬리정맥을 통해 주사 하였다. 마우스는 6주령의 수컷 Balb/c-nu (20g)의 마우스를 사용하였다. 이미징은 FOBI (NeoScience, Fluorescence- labeled Organism Bioimaging Instrument, FOBI mini BR)를 이용하여 염료를 주사한 후 1시간, 3시간, 24시간이 지난 뒤 In vivo 이미징 하였고, 24시간 이미징을 한 후 Liver, Lung, Spleen, Kidney, Heart를 적출하여 ex vivo 이미징을 수행하였다. 이 때 FOBI Imaging은 기기 상 Light source NIR (735 nm) channel 및 NIR Emission filter 적용, Exposure time 1000 ms, Gain 5x 설정 하에 수행하였다. 분석을 위해 각 장기에 Liver 대비 얼마나 축적 되는지 확인하기 위해 FOBI 기기의 소프트웨어인 NEOimaging for FOBI를 이용하여 각 장기의 형광 강도를 측정한 뒤 Organ to liver ratio를 구하였고, 도 7에 그 결과를 나타내었다. 도 6은 In vivo 이미징 결과로 화합물 1과 대조형광염료인 IRDyeㄾ 800CW는 유사한 결과를 보였다. 염료를 주사한 후 1시간이 지났을 때 마우스의 몸 전체에 염료가 퍼져있었고, 3시간이 지나 면서 점차 소변을 통해 빠져나가면서 24시간이 지났을 때는 거의 모든 염료가 빠져 나간 것을 확인 하였다. ex vivo 이미징을 비교 하였을 때 화합물 1이 IRDyeㄾ 800CW에 비해 조금 형광강도가 강한 것으로 보이나, 두 가지의 염료 모두 Liver 와 Kidney에 축적되어 있는 것을 확인하였고, 도 7의 Organ to liver Ratio를 통해 유사한 경향의 체내분포양상을 보이는 것을 확인 하였다.
(2) 화합물1을 도입한 나노파티클(화합물1-HGC)의 Xenograft mouse model을 이용한 암 타겟능 확인(2) Confirmation of cancer targeting ability using Xenograft mouse model of nanoparticle (Compound 1-HGC) into which
화합물 1을 도입한 나노파티클(화합물1-HGC)의 암 타겟능을 확인하기 위해 6주령의 수컷 Balb/c-nu (20g)에 Squamous cell carcinoma 세포주인 SCC7 세포(1x106/0.1ml)를 마우스의 왼쪽 허벅지 위에 Subcutaneous injection하여 Xenograft mouse model을 제작하였다. 화합물1-HGC는 1mg/ml의 농도로 Distilled water (이하 DW)에 녹여 Stock solution을 제조하고, 마우스 종양의 부피가 60~80 mm3가 되었을 때 화합물1-HGC를 120 μg 꼬리정맥을 통해 주사하였다. In vivo 이미징은 IVIS spectrum (PerkinElmer)을 이용하여 745nm/800nm Filter에서 화합물1-HGC 주사 후 5분, 12시간, 1일, 2일, 3일, 4일, 5일 수행하였고, ex vivo 이미징은 5일 이미징을 한 후 Liver, Lung, Spleen, Kidney, Heart, Tumor를 적출하여 수행하였다. 분석은 IVIS spectrum의 software를 이용하여 수행하였다. 도 8의 In vivo 이미징의 분석 결과로 12시간 때부터 종양에 축적되기 시작하며 2일째에 가장 강한 형광를 보이고, 이 후 점차 형광의 강도가 감소하기 시작하는 것을 확인 하였다. 도 9에서 화합물1-HGC가 실제로 종양에 축적되는지 확인하기 위해 장기를 적출하고, ex vivo 이미징을 하여 장기의 형광 강도를 비교한 결과로 화합물1-HGC가 종양에 축적된 것을 확인하였다. 종양이외에 Kidney에 많이 축적되는 것을 확인되었는데 이 것은 화합물1-HGC가 소변으로 배출되기 때문인 것으로 보인다, In vivo, ex vivo 이미징으로 화합물1-HGC의 암 타겟능을 확인하였고, 화합물 1이 다양한 동물실험에서 사용이 가능한 것을 확인하였다.SCC7 cells (1x106 / 0.1ml), a squamous cell carcinoma cell line, were injected into 6-week-old male Balb / c-nu (20g) to examine the cancer targeting ability of the nanoparticle (compound 1-HGC) Xenograft mouse model was constructed by subcutaneous injection on the left thigh. Compound 1-HGC was dissolved in distilled water (DW) at a concentration of 1 mg / ml to prepare a stock solution. Compound 1-HGC was injected through a 120 μg tail vein when the tumor volume reached 60-80 mm 3 Respectively. In vivo imaging was performed 5 minutes, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days after compound 1-HGC injection at 745 nm / 800 nm filter using IVIS spectrum (PerkinElmer) Liver, Lung, Spleen, Kidney, Heart, and Tumor were extracted after imaging for 5 days. The analysis was performed using the IVIS spectrum software. As a result of the in vivo imaging analysis of FIG. 8, it was confirmed that accumulation in the tumor began at 12 hours, the strongest fluorescence was observed at the second day, and then the intensity of fluorescence gradually began to decrease. In FIG. 9, organs were excised to confirm that compound 1-HGC actually accumulated in the tumor, and ex vivo imaging was performed to compare the fluorescence intensities of the organs. As a result, it was confirmed that the compound 1-HGC was accumulated in the tumor. In addition to the tumor, accumulation of Kidney was confirmed, which is probably due to the excretion of Compound 1-HGC into the urine. In vivo and ex vivo imaging confirmed the cancer targeting ability of Compound 1-HGC, And that it is possible to use it.
Claims (9)
[화학식 2]
X1은 O 또는 S이고; X3는 -C6H4-SO3H, -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이며;
W1은 -(CH2)4-SO3 - 또는 -(CH2)3-SO3 -이고; W3는 -(CH2)5-CO-Z, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;
X3가 -C6H4-SO3H이면 W3는 -(CH2)5-CO-Z이고, X3가 -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이면 W3는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;
-Z는
A1은 H 또는 SO3H이고, A2는 H이며, 또는 A1과 A2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;
B1은 H 또는 SO3H이고, B2는 H이며, 또는 B1과 B2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있다.A fluorescent compound represented by the following formula (2):
(2)
X 1 is O or S; X 3 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -CO-Z or - (CH 2 ) 2 -CO-Z;
W 1 is - (CH 2 ) 4 -SO 3 - or - (CH 2 ) 3 -SO 3 - ; W 3 is - (CH 2 ) 5 -CO-Z, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
W 3 is - (CH 2 ) 5 -CO-Z when X 3 is -C 6 H 4 -SO 3 H, X 3 is -C 6 H 4 - (CH 2 ) 2 -CO- 2 ) 2 -CO-Z, W 3 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
-Z is
A 1 is H or SO 3 H, A 2 is H, or A 1 and A 2 combine with each other to form a benzene ring, said benzene ring being substituted with two SO 3 H;
B 1 is H or SO 3 H, B 2 is H, or B 1 and B 2 may combine with each other to form a benzene ring, and the benzene ring may be substituted with two SO 3 H.
상기 표지대상 물질은 섬유, 생체분자, 나노입자 및 유기화합물 중에서 선택된 1종 이상이며,
상기 표지대상 물질은 아민기, 수산화기 및 티올기 중에서 선택된 적어도 1개의 기능기를 포함하며,
상기 기능기에 제3항 내지 제5항 중 어느 한 항에 따른 형광 화합물이 결합하는 것을 특징으로 하는 형광 화합물 표지방법.A fluorescent compound labeling method comprising the step of binding a fluorescent compound according to any one of claims 3 to 5 with a labeling substance,
The labeling substance is at least one selected from a fiber, a biomolecule, a nanoparticle and an organic compound,
Wherein the labeling substance comprises at least one functional group selected from an amine group, a hydroxyl group and a thiol group,
The fluorescent compound labeling method according to any one of claims 3 to 5, wherein the functional group is bonded to the fluorescent compound according to any one of claims 3 to 5.
[화학식 1]
[화학식 2]
상기 화학식 1 및 2에서, X1은 O 또는 S이고; X2는 -C6H4-SO3H, -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이며;
W1은 -(CH2)4-SO3 - 또는 -(CH2)3-SO3 -이고; W2는 -(CH2)5-COOH, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;
X2가 -C6H4-SO3H이면 W2는 -(CH2)5-COOH이고, X2가 -C6H4-(CH2)2-COOH 또는 -(CH2)2-COOH이면 W2는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;
A1은 H 또는 SO3H이고, A2는 H이며, 또는 A1과 A2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;
B1은 H 또는 SO3H이고, B2는 H이며, 또는 B1과 B2는 서로 결합하여 벤젠고리를 형성하고, 상기 벤젠 고리는 2개의 SO3H로 치환될 수 있으며;
X3는 -C6H4-SO3H, -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이며;
W3는 -(CH2)5-CO-Z, -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;
X3가 -C6H4-SO3H이면 W3는 -(CH2)5-CO-Z이고, X3가 -C6H4-(CH2)2-CO-Z 또는 -(CH2)2-CO-Z이면 W3는 -(CH2)3-SO3H 또는 -(CH2)4-SO3H이며;
-Z는
[Chemical Formula 1]
(2)
In the above formulas (1) and (2), X 1 is O or S; X 2 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -COOH;
W 1 is - (CH 2 ) 4 -SO 3 - or - (CH 2 ) 3 -SO 3 - ; W 2 is - (CH 2 ) 5 -COOH, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
W 2 is - (CH 2 ) 5 -COOH and X 2 is -C 6 H 4 - (CH 2 ) 2 -COOH or - (CH 2 ) 2 -, when X 2 is -C 6 H 4 -SO 3 H, COOH, W 2 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
A 1 is H or SO 3 H, A 2 is H, or A 1 and A 2 combine with each other to form a benzene ring, said benzene ring being substituted with two SO 3 H;
B 1 is H or SO 3 H, B 2 is H, or B 1 and B 2 may combine with each other to form a benzene ring, said benzene ring may be substituted with two SO 3 H;
X 3 is -C 6 H 4 -SO 3 H, -C 6 H 4 - (CH 2 ) 2 -CO-Z or - (CH 2 ) 2 -CO-Z;
W 3 is - (CH 2 ) 5 -CO-Z, - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
W 3 is - (CH 2 ) 5 -CO-Z when X 3 is -C 6 H 4 -SO 3 H, X 3 is -C 6 H 4 - (CH 2 ) 2 -CO- 2 ) 2 -CO-Z, W 3 is - (CH 2 ) 3 -SO 3 H or - (CH 2 ) 4 -SO 3 H;
-Z is
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