CN104860947B - 一种氮杂环烷氨基萤光素化合物及其制备方法与应用 - Google Patents
一种氮杂环烷氨基萤光素化合物及其制备方法与应用 Download PDFInfo
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- -1 amino luciferin compound Chemical class 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 150000001875 compounds Chemical class 0.000 claims abstract description 40
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 69
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 37
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 26
- 239000011259 mixed solution Substances 0.000 claims description 18
- 239000000376 reactant Substances 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 229960000583 acetic acid Drugs 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical class [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 12
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- FAYAYUOZWYJNBD-UHFFFAOYSA-N 1,3-benzothiazol-6-amine Chemical compound NC1=CC=C2N=CSC2=C1 FAYAYUOZWYJNBD-UHFFFAOYSA-N 0.000 claims description 7
- 230000036571 hydration Effects 0.000 claims description 7
- 238000006703 hydration reaction Methods 0.000 claims description 7
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims description 7
- UMHJEEQLYBKSAN-UHFFFAOYSA-N Adipaldehyde Chemical compound O=CCCCCC=O UMHJEEQLYBKSAN-UHFFFAOYSA-N 0.000 claims description 6
- PCSMJKASWLYICJ-UHFFFAOYSA-N Succinic aldehyde Chemical compound O=CCCC=O PCSMJKASWLYICJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 6
- 230000004044 response Effects 0.000 claims description 6
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 claims description 5
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- 238000005352 clarification Methods 0.000 claims description 5
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- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 abstract description 2
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- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 abstract description 2
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- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 24
- UYHQUNLVWOAJQW-UHFFFAOYSA-N 1,3-benzothiazole-2-carbonitrile Chemical compound C1=CC=C2SC(C#N)=NC2=C1 UYHQUNLVWOAJQW-UHFFFAOYSA-N 0.000 description 17
- HKSJKXOOBAVPKR-SSDOTTSWSA-N (4s)-2-(6-amino-1,3-benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound S1C2=CC(N)=CC=C2N=C1C1=N[C@@H](C(O)=O)CS1 HKSJKXOOBAVPKR-SSDOTTSWSA-N 0.000 description 15
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical compound C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 12
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical group OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 10
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
- C07D471/14—Ortho-condensed systems
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- 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
-
- 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
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- 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
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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Abstract
本发明涉及一种氮杂环烷氨基萤光素化合物及其制备方法与应用,其结构通式为:
Description
技术领域
本发明属于药物技术领域,特别涉及一种氮杂环烷氨基萤光素化合物及其制备方法与应用。
背景技术
生物发光(Bioluminescence)是生物体内的化学物质在酶的催化下将化学能转化为光能的一种过程,这是一种特殊类型的化学发光,不依赖于机体对光的吸收,化学能转化为光能的效率很高,接近100%。生物发光现象广泛存在于自然界生物有机体中,包括细菌、昆虫和海洋生物等。不同的生物发光体系的萤光素酶和萤光素结构也各有差别,目前只完成为数不多的几个物种的活性物质分离和分子结构确定,并被应用于哺乳动物的成像研究。虽然每种发光体系的发光底物不尽相同,但其生物发光的机制确大致相同:底物被萤光素酶催化氧化,产生含有激发态的电子中间体,当该电子由激发态返回基态时将化学能转化为光能,释放出光子。
生物发光成像(bioluminescence imaging)是通过灵敏的光学检测仪器监控萤光素酶标记的细胞或基因在活体生物内的活动和行为过程的一种新兴的成像技术.生物发光成像技术具有操作简便快速、灵敏度高、能够实现实时动态观测以及非侵袭性等优点。随着理论研究的深入,该技术已经逐步地应用到微生物学、生物化学、环境科学、分子生物学、医学等多个学科和领域,成为了一种重要的成像手段.生物发光成像技术在肿瘤生长监测和转移示踪、目标基因表达的检测、蛋白-蛋白相互作用、药物高通量筛选、细胞内ATP水平探测和研究细菌病毒感染宿主过程等领域有着不可替代的技术优势。生物发光信号的实时检测与萤光素的半衰期和组织的渗入量紧密相关。然而生物发光是一种爆发型的发光现象,半衰期较短,这些因素在很大程度上限制了萤光素在体内实验的应用。为了解决这些问题,Piwnica-Worms等人用微渗透泵来保证组织中萤光素的浓度(Gross,S.,et al.,Continuous delivery of D-luciferin by implanted micro-osmotic pumps enablestrue real-time bioluminescence imaging of luciferase activity in vivo.MolImaging,2007.6(2):p.121-30.),而Denmeade等人则利用聚乙二醇与D-萤光素的类似物氨基萤光素连接来延长萤光素的半衰期(Chandran,S.S.,S.A.Williams,and S.R.Denmeade,Extended-release PEG-luciferin allows for long-term imaging of fireflyluciferase activity in vivo.Luminescence,2009.24(1):p.35-8.)。除此之外,生物发光的发光波长只在可见光范围,与部分生物分子(例如,血红素)吸收光谱的重叠 限制了其在深组织的应用,将生物发光的波长范围扩展到近红外区域是一个较为理想的目标。Yasuteru Urano等人利用生物发光能量共振转移(BRET)的方法,将一系列Cy5荧光团链接在氨基萤光素上,得到近红外的发光范围(Kojima,R.,et al.,Rational Design andDevelopment of Near-Infrared-Emitting Firefly Luciferins Available InVivo.Angew.Chem.Int.Ed.,2013.52:p.1175-1179)。虽然上述研究在某种程度上解决了限制生物发光应用的问题,但是以上方法合成繁琐,发光强度较弱和花费较高,这也使得这些方法不能大范围推广。萤火虫萤光素酶和萤光素酶底物有着较高的特异性。对萤光素结构的改造可能会使其发光性质受到影响。氨基萤光素的生物发光发射光波长红移(557nm红移至593nm),且与萤光素酶具有更强的亲和力。D-萤光素的类似物以及具有新骨架的萤光素酶底物的发现能够拓展生物发光成像在生命科学领域的应用,对于整个成像领域的进步有着较大的推动作用。
发明内容
本发明的目的是提供一种氮杂环烷氨基萤光素化合物及其制备方法与应用,在氨基萤光素的基础上,对其氨基进行简单的环烷基取代,得到了能延长体内半衰期,提高组织渗透性和增强近红外区域发光强度的生物发光底物。
为实现上述目的,本发明采用下述技术方案:
一种氮杂环烷氨基萤光素化合物,具有通式(Ⅰ)的结构:
其中,n为1至9的自然数,m为1至9的自然数;n=m时,R为氢基、甲基、乙基、正丙基、正丁基、氟、氯、溴或碘;当n≠m时,R为氢基。
所述R为氢基时,m,n之和为2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17或18。
上述的具有通式(Ⅰ)的结构的氮杂环烷氨基萤光素化合物,包括表1所示的化合物,其名称如下:
(S)-2-(6-(氮杂环庚烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L1),
(S)-2-(6-(氮杂环己烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L2),
(S)-2-(6-(氮杂环戊烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L3),
(S)-2-(6-(氮杂环丁烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L4),
(S)-2-(6-(4-甲基氮杂环己烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L5),
(S)-2-(6-(3-甲基氮杂环丁烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L6),
(S)-2-(6-(4-乙基氮杂环己烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L7),
(S)-2-(6-(3-乙基氮杂环丁烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L8),
(S)-2-(6-(4-丙基氮杂环己烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L9),
(S)-2-(6-(3-丙基氮杂环丁烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L10),
(S)-2-(6-(4-溴氮杂环己烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L11),
(S)-2-(6-(3-溴氮杂环丁烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L12),化合物名称后的括号中为其对应的代号。
所述的氮杂环烷氨基萤光素化合物的制备方法,具体制备步骤如下:
1)将丙二醛或丁二醛或戊二醛或己二醛溶于冰醋酸中,随后加入2-氰基6-氨基苯并噻唑,搅拌,再加入氰基硼氢化钠,待反应液呈现澄清的橙红色溶液时,加入乙酸乙酯,搅拌,再分次加入饱和碳酸钠溶液,直至反应液中无气泡生成;用乙酸乙酯萃取三次(3×50mL),乙酸乙酯相依次用饱和碳酸钠溶液和饱和氯化钠溶液洗涤,乙酸乙酯相用无水硫酸钠干燥、过滤、过硅胶柱纯化,即得中间体化合物;
2)将中间体化合物溶于甲醇和二氯甲烷的混合溶液中,得混合溶液1,将无水碳酸钾与一水合半胱氨酸盐酸盐溶于甲醇水溶液中,得混合溶液2,在氮气保护条件下将上述两混合溶液混合,反应后,将反应液中的有机溶剂蒸除,调节反应液的pH值为弱酸性,将所得固体过滤,即得目标化合物。
步骤1)中所述丙二醛或丁二醛或戊二醛或己二醛的加入量为所述醋酸重量的2~10%;所述2-氰基6-氨基苯并噻唑的加入量为所述醋酸重量的1~5%;所述氰基硼氢化钠的加入量为所述醋酸重量的3~8%,所述每次加入2.5-3.5mL饱和碳酸钠溶液,总量为醋酸重量的4-6倍,每次加入的乙酸乙酯的量为醋酸重量的15-30倍;
步骤1)中,加入2-氰基6-氨基苯并噻唑后,搅拌的时间为10-15分钟。
步骤1)中,加入乙酸乙酯后,搅拌的时间为5分钟。
步骤2)中,甲醇和二氯甲烷的混合溶液中,甲醇和二氯甲烷的体积比为1:1。
步骤2)中所述中间体化合物的加入量为所述甲醇和二氯甲烷的混合溶液重量的0.5~3%,所述无水碳酸钾和一水合半胱氨酸盐酸盐的加入量分别为所述甲醇水溶液重量的1~2%和1.5~2.5%。以上百分数为质量百分数。
步骤2)中,所述甲醇水溶液中甲醇的质量分数为40-55%。
步骤2)中,两混合溶液混合后,反应15-30分钟。
步骤2)中,所述混合溶液1和混合溶液2的体积比为1-2:1-2。
步骤2)中,调节pH为5-6。
本发明的化合物作为生物发光的底物应用。
本发明的化合物能利用生物发光检测萤光素酶的存在和数量(包括酶水平、细胞水平和动物水平),检测萤光素酶在体外,细胞和体内分布成像的应用。
本发明的化合物能利用生物发光检测ATP的存在和数量(包括酶水平、细胞水平和动物水平),检测ATP在体外,细胞和体内分布成像的应用。
本发明的化合物能够在萤光素酶,ATP,Mg2+的存在下作为报告信号检测药物的吸收,分布,代谢,排泄,毒性作用。
本发明的有益效果:
1.本发明的化合物与D-荧光素或者氨基荧光素相比,化合物溶液对荧光素酶具有较低的检测限,在作为探针应用时拥有较高的灵敏性;
2.本发明的化合物结构中的一个氮杂环烷基团增加了脂溶性,化合物较于D-荧光素或者氨基荧光素具有更好的组织相容性,更容易跨膜进入细胞,因此在细胞水平检测的探针应用时响应更迅速;
3.本发明的化合物在体内生物发光强度较为稳定,,稳定发光时间大于7小时,并且具有较长的体内代谢时长,所以可用于体内药物代谢实时检测。
4.合成制备过程中所需的己二醛、戊二醛和丁二醛等二醛类原料,价格便宜,容易获得,所以得到的氨基荧光素衍生物合成成本比较低廉,经济适用。
5.本发明制得的化合物具有较强的细胞活性以及具有很高的近红外光区发光强度。
具体实施方式
下面的实施例可以使本专业技术人员更全面地理解本发明,但不以任何方式限制本发明。
【实例一:(S)-2-(6-(氮杂环庚烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L1)的制备】
中间体6-(氮杂环庚烷-1-基)苯并[d]噻唑-2-腈(1-1)的制备
将己二醛(65mg,0.57mmol)溶于2mL冰醋酸中,随后加入2-氰基6-氨基苯并噻唑(50mg,0.285mmol),未溶解,搅拌10分钟,再将氰基硼氢化钠(62mg,1mmol)加入,有大量气泡产生,待反应液呈现澄清的橙红色溶液时,在反应液中加入20mL乙酸乙酯,搅拌5分钟,再分次加入饱和碳酸钠溶液(3×3mL),直至反应液中无气泡生成,用乙酸乙酯萃取三次(3×50mL),乙酸乙酯层依次用饱和碳酸钠溶液和氯化钠溶液洗涤,乙酸乙酯层用无 水硫酸钠干燥,过滤,之后将目标物过硅胶柱纯化,得26mg 6-(氮杂环庚烷-1-基)苯并[d]噻唑-2-腈(1-1)黄色固体,产率为36%。
(S)-2-(6-(氮杂环庚烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L1)的制备
将6-(氮杂环庚烷-1-基)苯并[d]噻唑-2-腈(16mg,0.062mmol)溶于甲醇和二氯甲烷的混合溶液(2mL,1:1)中,将无水碳酸钾(21mg,0.124mmol)与一水合半胱氨酸盐酸盐(28.5mg,0.124mmol)溶于水与甲醇的混合液(2mL,1:1),在氮气保护条件下将上述两液混合,反应20分钟后反应完毕,将反应液中的有机溶剂蒸除,用0.1M盐酸将反应液的pH值调节至6,有大量固体析出,过滤,得14mg(S)-2-(6-(氮杂环庚烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L1)橙红色固体,产率为63%。
1H-NMR(400MHz,DMSO-d6)δ13.07(s,1H),δ7.87(d,J=9.2Hz,1H),7.30(d,J=2.4Hz,1H),7.04(dd,J=9.3,2.5Hz,1H),5.53–5.19(m,1H),3.76–3.59(m,2H),3.55(t,J=5.9Hz,4H),1.76(m,4H),1.48(m,4H).
13C NMR(101MHz,DMSO)δ174.39,157.53,152.69,152.45,149.03,138.65,124.69,117.95,107.78,82.80,51.01(2C),40.37,27.58(2C),26.84(2C).
HRMS m/z calcd.for C17H19N3O2S2[M-H]-360.0919,found 360.0846.
【实例二:(S)-2-(6-(氮杂环己烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L2)的制备】
中间体6-(氮杂环己烷-1-基)苯并[d]噻唑-2-腈(2-1)的制备
将戊二醛(57mg,0.57mmol)溶于2mL冰醋酸中,随后加入2-氰基6-氨基苯并噻唑(50mg,0.285mmol),未溶解,搅拌10分钟,再将氰基硼氢化钠(62mg,1mmol)加入,有大量气泡产生,待反应液呈现澄清的橙红色溶液时,在反应液中加入20mL乙酸乙酯,搅拌6分钟,再分次加入饱和碳酸钠溶液(3×3mL),直至反应液中无气泡生成,用乙酸乙酯萃取三次(3×50mL),乙酸乙酯层依次用饱和碳酸钠溶液和氯化钠溶液洗涤,乙酸乙酯层用无水硫酸钠干燥,过滤,之后过硅胶柱纯化,得33mg 6-(氮杂环庚烷-1-基)苯并[d]噻唑-2-腈(1-1)黄色固体,产率为48%。
(S)-2-(6-(氮杂环己烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L2)的制备
将6-(氮杂环己烷-1-基)苯并[d]噻唑-2-腈(2-1)(21mg,0.088mmol)溶于甲醇和二氯甲烷的混合溶液(2mL,1:1)中,将无水碳酸钾(25mg,0.177mmol)与一水合半胱氨酸盐酸盐(32mg,0.177mmol)溶于水与甲醇的混合液(2mL,1:1),在氮气保护条件下将上述两液混合,反应20分钟后反应完毕,将反应液中的有机溶剂蒸除,用0.1M盐酸将反应液的pH值调节至5,有大量固体析出,过滤,得21mg(S)-2-(6-(氮杂环己烷-1-基)苯并[d]噻唑-2- 基)-4,5-二氢噻唑-4-羧酸(L2)黄白色固体,产率为68%。
1H NMR(400MHz,DMSO)δ13.21(s,1H),7.92(d,J=9.1Hz,1H),7.58(s,1H),7.30(d,J=8.4Hz,1H),5.40(t,J=9.0Hz,1H),3.91–3.52(m,2H),3.31(t,J=5.9,4H),1.62(m,6H);
13CNMR(101MHz,DMSO)δ171.70,164.85,156.11,155.50,152.45,138.16,124.60,117.64,106.22,78.53,50.76(2C),35.08,25.49(2C),24.32;
HRMS m/z calcd.for C16H17N3O2S2[M-H]-346.0762,found 346.0687.
【实例三(S)-2-(6-(氮杂环戊烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L3)的制备】
中间体6-(氮杂环戊烷-1-基)苯并[d]噻唑-2-腈(3-1)的制备
将丁二醛(49mg,0.57mmol)溶于2mL冰醋酸中,随后加入2-氰基6-氨基苯并噻唑(50mg,0.285mmol),未溶解,搅拌10分钟,再将氰基硼氢化钠(62mg,1mmol)加入,有大量气泡产生,待反应液呈现澄清的橙红色溶液时,在反应液中加入20mL乙酸乙酯,搅拌5分钟,再分次加入饱和碳酸钠溶液(3×50mL),直至反应液中无气泡生成,用乙酸乙酯萃取三次(3×50mL),乙酸乙酯层分别用饱和碳酸钠溶液和氯化钠溶液洗涤,乙酸乙酯层用无水硫酸钠干燥,过滤,之后过硅胶柱纯化,得39mg 6-(氮杂环戊烷-1-基)苯并[d]噻唑-2-腈(3-1)亮黄色固体,产率为60%。
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环戊基)氨基(L3)的制备
将6-(氮杂环戊烷-1-基)苯并[d]噻唑-2-腈(3-1)(23mg,0.10mmol)溶于甲醇和二氯甲烷的混合溶液(2mL,1:1)中,将无水碳酸钾(28mg,0.20mmol)与一水合半胱氨酸盐酸盐(36mg,0.20mmol)溶于水与甲醇的混合液(2mL,1:1),在氮气保护条件下将上述两液混合,反应20分钟后反应完毕,将反应液中的有机溶剂蒸除,用0.1M盐酸将反应液的pH值调节至6,有大量固体析出,过滤,得28mg(S)-2-(6-(氮杂环戊烷-1-基)苯并[d]噻唑-2-基)-4,5-二氢噻唑-4-羧酸(L3)黄色固体,产率为84%。
1H NMR(400MHz,CDCl3)δ13.108(s,1H)δ7.97(d,J=9.1Hz,1H),6.91(d,J=2.3Hz,1H),6.86(dd,J=9.1,2.4Hz,1H),5.43(t,J=9.7Hz,1H),3.86–3.64(m,2H),3.41(t,J=6.5Hz,4H),2.19–1.90(m,4H);
13CNMR(101MHz,DMSO)δ171.79,164.79,153.75,147.70,144.21,138.63,124.87,114.25,102.04,78.48,48.15(2C),35.03,25.51(2C).
HRMS m/z calcd.for C15H15N3O2S2[M-H]-332.0527,found 332.0533.
【实例四:萤火虫萤光素酶底物的体外酶活性研究】
将50μL不同浓度目标化合物,D-萤光素,氨基萤光素(0.01,0.05,0.1,0.5,1μM)中分别加入50μL luciferase(10μg/ml,含2mM ATP)到黑色的96孔板中,用活体成像仪进行生物发光强度的测定,得结果如下表:
表1
结果表明,相较于D-萤光素和氨基萤光素,目标化合物在低浓度时生物发光强度更强,而且发光强度有明显的浓度依赖性,因此非常适合用于定量检测。
【实例五:萤火虫萤光素酶底物脂溶性评价】
根据萤火虫萤光素酶底物的体外酶活性研究结果筛选出活性较好的三个化合物L1,L2,L3为后续深入研究的的目标化合物。用Chembiodraw ultra 12.0软件对目标化合物,D-萤光素,氨基萤光素的脂溶性进行了计算,得到CLogP,结果如下表:
结果表明,目标化合物的CLogP较D-萤光素和氨基萤光素大,具有较好的脂溶性,因此受试化合物在细胞跨膜过程中占有一定的优势。
【实例六:检测探针化合物的生物发光波长性质】
向0.5mL目标化合物,D-萤光素,氨基萤光素(125μM)分别加入0.5mL萤光素酶(20μg/mL,含2mM ATP),用荧光分光光度计测试其最大波长。结果如下表:
表2
结果表明,目标化合物与天然底物D-萤光素相比,最大生物发光波长红移了40-50nm,较于氨基萤光素,最大生物发光波长红移了10~20nm,这说明目标化合物的生物发光更适合生物体内测试。
【实例七:萤火虫萤光素酶底物的细胞活性研究(细胞水平)】
在黑色的96孔板中每孔加入100μL ES-2-LUC细胞悬液(4×105/mL),于37℃,5%CO2细胞培养箱中孵育24h。吸出培养基,加入50μL不同浓度目标化合物,D-萤光素,氨基萤光素(0.01,0.025,0.05,0.1,0.25,0.5,1,2.5,5,10μM),用活体成像仪测试,得结果如下表:
表3
结果表明,目标化合物不仅较较D-萤光素和氨基萤光素高,而且有较低的检测限。。所以目标化合物可以用于细胞的生物发光检测。
【实例八:萤火虫萤光素酶底物的动物活性研究(全波长测试)】
将底物目标化合物,D-萤光素,氨基萤光素配制成浓度为1mM的氯化钠溶液,取荷瘤2周的裸鼠,腹腔注射底物100μL,注射20min后,用活体成像仪在滤光片为open条件下进行测试,结果如下表。
表4
结果表明,目标化合物的动物发光强度明显高于D-萤光素和氨基萤光素,所以受试化 合物在体内检测方面较于较D-萤光素和氨基萤光素具有明显优势。
【实例九:萤火虫萤光素酶底物的动物活性研究(体内代谢时长)】
将底物目标化合物,D-萤光素和氨基萤光素配制成浓度为1mM的氯化钠溶液,取荷瘤2周的裸鼠,腹腔注射底物100μL,注射20min后,用活体成像仪在滤光片为open条件下进行测试.对老鼠进行不间断的成像,直至信号消失,测得消失时间,如下表:
表5
结果表明,目标化合物L1的体内代谢时长是D-萤光素和氨基萤光素的3倍多。因此L1可以用于体内的长期监测。
【实例十:萤火虫萤光素酶底物的动物活性研究(近红外发光强度测试)】
将底物目标化合物,D-萤光素和氨基萤光素配制成浓度为1mM的氯化钠溶液,取荷瘤2周的裸鼠,腹腔注射底物100μL,注射20min后,用活体成像仪在滤光片cy 5.5条件下进行测试。结果如下表:
表6
结果表明,在滤光片cy 5.5条件下,目标化合物L1生物发光强度是D-萤光素和氨基萤光的17倍多,L2和L3是其6倍。因此,目标化合物更适合于体内深组织的监测。
上述虽然对本发明的具体实施方式进行了描述,但并非对发明保护范围的限制,所属领域技术人员应该明白,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围内。
Claims (3)
1.一种氮杂环烷氨基萤光素化合物的制备方法,其特征在于:具体制备步骤如下:
1)将丙二醛或丁二醛或戊二醛或己二醛溶于冰醋酸中,随后加入2-氰基6-氨基苯并噻唑,搅拌,再加入氰基硼氢化钠,待反应液呈现澄清的橙红色溶液时,加入乙酸乙酯,搅拌,再分次加入饱和碳酸钠溶液,直至反应液中无气泡生成;用乙酸乙酯萃取,乙酸乙酯相依次用饱和碳酸钠溶液和饱和氯化钠溶液洗涤,然后,用无水硫酸钠干燥、过滤、过硅胶柱纯化,即得中间体化合物;
2)将中间体化合物溶于甲醇和二氯甲烷的混合溶液中,得混合溶液1,将无水碳酸钾与一水合半胱氨酸盐酸盐溶于甲醇水溶液中,得混合溶液2,在氮气保护条件下将上述两混合溶液混合,反应后,将反应液中的有机溶剂蒸除,调节反应液的pH值为弱酸性,将所得固体过滤,即得目标化合物。
2.根据权利要求1所述的制备方法,其特征在于:步骤1)中所述丙二醛或丁二醛或戊二醛或己二醛的加入量为所述醋酸重量的2~10%;所述2-氰基6-氨基苯并噻唑的加入量为所述醋酸重量的1~5%;所述氰基硼氢化钠的加入量为所述醋酸重量的3~8%,所述每次加入2.5-3.5mL饱和碳酸钠溶液,总量为醋酸重量的4-6倍,每次加入的乙酸乙酯的量为醋酸重量的15-30倍;加入2-氰基6-氨基苯并噻唑后,搅拌的时间为10-15分钟;加入乙酸乙酯后,搅拌的时间为4-7分钟。
3.根据权利要求1所述的制备方法,其特征在于:步骤2)中,甲醇和二氯甲烷的混合溶液中,甲醇和二氯甲烷的体积比为1:1,所述中间体化合物的加入量为所述甲醇和二氯甲烷的混合溶液重量的1%~3%,所述无水碳酸钾和一水合半胱氨酸盐的加入量分别为所述甲醇水溶液重量的1%~2%和1.5~2.5%,所述混合溶液1和混合溶液2的体积比为1-2:1-2。
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