CN104311504A - 一种环烷基单取代氨基萤光素化合物及其制备方法与应用 - Google Patents
一种环烷基单取代氨基萤光素化合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种环烷基单取代氨基萤光素化合物及其制备方法与应用。具有结构通式如下或者其游离形式:其中,n为0至9,m为1至9;n=m,R为氢基,甲基,乙基,丙基,卤素等单取代集团;当n≠m,R为氢基。该类化合物作为生物发光底物的应用,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;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;能够在萤光素酶、ATP、Mg2+的存在下作为报告信号检测药物在酶水平、细胞水平、动物水平的药理作用。
Description
技术领域
本发明属于药物技术领域,特别涉及一种环烷基单取代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%。
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.但是生物发光是一种爆发型的发光现象,半衰期较短,这些原因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 enables true real-timebioluminescence imaging of luciferase activity in vivo.Mol Imaging,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 allowsfor long-term imaging of firefly luciferase 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.,RationalDesign and Development of Near-Infrared-Emitting Firefly Luciferins Available In Vivo.Angew.Chem.Int.Ed.,2013.52:p.1175-1179)。
在某种程度上,以上研究解决了限制生物发光应用的问题,但是以上前期材料合成繁琐,花费较高,发光强度较弱,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
针对以上问题,我们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.该底物合成方法简单,经济适用,对于生物发光在体内的应用有着较大的推进作用。
为实现上述目的,本发明采用下述技术方案:
一种环烷基单取代氨基萤光素化合物,具有通式(Ⅰ)的结构:
其中,n为0至9的自然数,m为1至9的自然数;n=m,R为氢基,甲基,乙基,正丙基,正丁基,氟,氯,溴,碘;R为氢基。
当n≠m,R为氢基时,m,n之和为2,3,4,5,6,7,8,9,10,10,11,12,13,14,15,16,17,18。
上述的具有通式(Ⅰ)的结构的环烷基单取代氨基萤光素化合物,包括表1所示的化合物,其名称如下:
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环己烷基)氨基(L1),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环戊烷基)氨基(L2),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环丁烷基)氨基(L3),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(4-甲基-环己烷基)氨基(L4),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(3-甲基-环丁烷基)氨基(L5),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(4-乙基-环已烷基)氨基(L6),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(3-乙基-环丁烷基)氨基(L7),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(4-丙基-环己烷基)氨基(L8),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(3-丙基-环丁烷基)氨基(L9),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(4-溴-环已烷基)氨基(L10),
化合物名称后的括号中为其对应的代号。
所述的环烷基单取代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2-氰基6-氨基苯并噻唑溶于醋酸中,搅拌5分钟,再加入环烷酮或环戊酮或环丁酮,搅拌5分钟,搅拌结束后,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5分钟,再分次加入饱和碳酸钠溶液,直至反应液中无气泡生成;反应液用饱和碳酸钠溶液洗涤后,用乙酸乙酯萃取,得到的乙酸乙酯层用无水硫酸钠干燥、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体积比为1:1的甲醇和二氯甲烷的混合溶液中,将无水碳酸钾与一水合半胱氨酸盐酸盐溶于质量浓度为40%的甲醇水溶液中,将上述两液混合,反应15分钟后,将反应液中的有机溶剂蒸除,调节反应液的pH值为5.5~6.5,待大量固体析出后,过滤,即得目标化合物。
步骤1)中所述2-氰基6-氨基苯并噻唑的加入量为所述醋酸重量的1%~5%,所述环烷酮或环戊酮或环丁酮的加入量为所述醋酸重量的8~10%;所述氰基硼氢化钠的加入量为所述醋酸重量的3%~8%;
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1%~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%。
本发明的化合物作为生物发光底物的应用。
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,Mg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.本发明的化合物与氨基荧光素或者是D-荧光素相比,化合物溶液在低浓度时对荧光素酶有着较高的响应,在作为探针应用时有着较高的灵敏性;
2.本发明的化合物有一个环烷基,脂溶性较好,所以有着更好的组织相容性,更易跨膜,作为探针,对于细胞水平的检测将会更及时迅速;
3.本发明的化合物体内吸收较好,发光较为稳定,在体内有较长发光时间,所以可用于体内药物代谢实时检测。
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)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环己基)氨基(L1)的制备】
中间体2-氰基6-环己烷基化氨基苯并噻唑(1-1)的制备
将2-氰基6-氨基苯并噻唑(50mg,0.28mmol)溶于2mL醋酸中,搅拌五分钟,未溶解,再将环戊酮(1mL,mmol)加入其中搅拌5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,在反应液中加入乙酸乙酯,搅拌五分钟,再分次加入饱和碳酸钠溶液,直至反应液中无气泡生成.反应液用饱和碳酸钠溶液洗涤,用乙酸乙酯萃取,乙酸乙酯层用无水硫酸钠干燥,过滤,之后过硅胶柱纯化,得50mg2-氰基6-环己烷基化氨基苯并噻唑(1-1)黄色晶体,产率为68%。
目标化合物(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环己基)氨基(L1)的制备
将2-氰基6-环戊烷基化氨基苯并噻唑(30mg,0.12mmol)溶于甲醇和二氯甲烷的混合溶液(2mL,1:1)中,将无水碳酸钾(33mg,0.24mmol)与一水合半胱氨酸盐酸盐(42mg,0.24mmol)溶于水与甲醇的混合液(2.5mL,1.5:1),两液混合,十五分钟后反应完毕,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18mg橙红色(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环己基)氨基(L1),产率为43%。
1H-NMR(300MHz,DMSO-d6)δ13.101(s,1H)7.766(d,J=9Hz,1H),7.097(d,J=2.1Hz,1H),6.849(dd,J=2.4Hz,J=9Hz,1H),6.241(d,J=6.9Hz,1H),5.353(dd,J=8.4Hz,J=9.6Hz,1H),3.755~3.586(m,2H),1.977~1.942(m,2H),1.753~1.710(m,2H),1.634~1.594(m,1H),1..431~1.309(m,2H),1.242~1.128(m,4H);
13C-NMR(75MHz,DMSO-d6)δ171.31,164.28,152.75,148.01,138.37,124.33,115.86,100.01,77.96,50.62,34.46,32.31,25.45,24.50;
HRMS m/z calcd.for C17H18N3O2S2[M-H]-360.0840,found 360.0846.
【实例二:(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环戊基)氨基(L2)的制备】
中间体2-氰基6-环丁烷基化氨基苯并噻唑(2-1)的制备
将2-氰基6-氨基苯并噻唑(50mg,0.28mmol)溶于2mL醋酸中,搅拌五分钟,未溶解,再将环戊酮(50mg,0.59mmol)加入其中搅拌5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,在反应液中加入乙酸乙酯,搅拌五分钟,再分次加入饱和碳酸钠溶液,直至反应液中无气泡生成。反应液用饱和碳酸钠溶液洗涤,用乙酸乙酯萃取,乙酸乙酯层用无水硫酸钠干燥,过滤,之后过硅胶柱纯化,得40mg 2-氰基6-环戊烷基化氨基苯并噻唑(2-1)黄色晶体,产率为58%。
目标化合物(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环戊基)氨基(L2)的制备
将2-氰基6-环戊烷基化氨基苯并噻唑(30mg,0.12mmol)溶于甲醇和二氯甲烷的混合溶液(2mL,1:1)中,将无水碳酸钾(33mg,0.24mmol)与一水合半胱氨酸盐酸盐(42mg,0.24mmol)溶于水与甲醇的混合液(2.5mL,1.5;1),两液混合,十五分钟后反应完毕,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20mg橙红色(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环戊基)氨基(L2),产率为49%。
1H-NMR(300MHz,DMSO-d6)δ13.084(s,1H)7.774(d,J=9Hz,1H),7.069(d,J=2.1Hz,1H),6.896(dd,J=2.4Hz,J=9Hz,1H),6.393(d,J=6.3Hz,1H),5.339(t,J=8.4Hz,1H),3.820~3.589(m,3H),2.010~1.928(m,2H),1.743~1.462(m,6H);
13C-NMR(75MHz,DMSO-d6)δ171.33,164.20,152.85,148.57,143.82,138.27,124.22,115.92,100.22,78.04,53.63,34.49,32.34(2C),24.67(2C);
HRMS m/z calcd.for C16H16N3O2S2[M-H]-346.0684,found 346.0689.
【实例三(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环丁基)氨基(L3)的制备】
中间体2-氰基6-环丁烷基化氨基苯并噻唑(3-1)的制备
将2-氰基6-氨基苯并噻唑(50mg,0.28mmol)溶于2mL醋酸中,搅拌五分钟,未溶解,再将环丁酮(200μL,)加入其中搅拌5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,在反应液中加入乙酸乙酯,搅拌五分钟,再分次加入饱和碳酸钠溶液,直至反应液中无气泡生成.反应液用饱和碳酸钠溶液洗涤,用乙酸乙酯萃取,乙酸乙酯层用无水硫酸钠干燥,过滤,之后过硅胶柱纯化,得40mg 2-氰基6-环丁烷基化氨基苯并噻唑(3-1)黄色晶体,产率为62.5%。
目标化合物(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2-氰基6-环丁烷基化氨基苯并噻唑(30mg,0.13mmol)溶于甲醇和二氯甲烷的混合溶液(2mL,1:1)中,将无水碳酸钾(35.9mg,0.26mmol)与一水合半胱氨酸盐酸盐(46mg,0.26mmol)溶于水与甲醇的混合液(2.5mL,1.5;1),两液混合,十五分钟后反应完毕,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24mg黄白色(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),产率为56%。
1H-NMR(300MHz,DMSO-d6)δ13.109(s,1H)7.785(d,J=9Hz,1H),7.990(d,J=2.1Hz,1H),6.855(dd,J=2.1Hz,J=9Hz,1H),6.679(d,J=6.3Hz,1H),5.358(dd,J=8.4Hz,J=7.8Hz,1H),3.968~3.847(m,1H),3.758~3.590(m,2H),2.418~2.354(m,2H),1.938~1.673(m,4H);
13C-NMR(75MHz,DMSO-d6)δ171.29,164.27,153.15,147.70,144.11,138.22,124.36,115.60,100.34,77.97,77.15,47.76,34.48,30.06,30.01,14.91;
HRMS m/z calcd.for C15H14N3O2S2[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(0.01,0.05,0.1,025,0.5,1μM)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
结果显示,在低浓度时,目标化合物较D-萤光素和氨基萤光素的发光强度强,而且目标化合物的发光强度有明显的浓度依赖性,可以用于定量检测。
【实例五:萤火虫萤光素酶底物脂溶性评价】
挑选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。目标化合物,D-萤光素,氨基萤光素用Chembiodraw ultra 12.0软件对其脂溶性进行了计算,得到CLogP,结果如下表:
结果显示,目标化合物的CLogP较D-萤光素和氨基萤光素大,所以有较好的脂溶性,这可能使其在细胞跨膜过程占有一定的优势。【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
结果显示目标化合物测试得到的生物发光波长与天然底物D-萤光素相比,波长红移了40-50nm,较氨基萤光素相比,波长红移了10~20nm,这说明受试化合物的生物发光更适合应用生物体内测试。
【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200μL ES-2-LUC细胞悬液(4×106/mL),于37℃,5%CO2细胞培养箱中孵育24h。倒掉培养基,加入50μL不同浓度目标化合物,D-萤光素,氨基萤光素(0.01,0.05,0.1,0.25,0.5,1μM),用活体成像仪测试,得结果如下表:
表3
结果显示,在低浓度下目标化合物的发光强度较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4mM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
结果显示,目标化合物L3体内活性较D-萤光素,氨基萤光素有明显优势,所以检测灵敏度较高,适合应用于体内检测。
【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,氨基萤光素配制成浓度为4mM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,得到消失时间,如下表:
表5
由表中数据可得,目标化合物L3的消失时间是D-萤光素的2.5倍多,是氨基萤光素的4倍。因此我们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-萤光素配制成浓度为4mM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
结果显示,在滤光片cy 5.5条件下,目标化合物L2是D-萤光素光强的4倍多,而L3是其6倍,因此,目标化合物较原始底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,但并非对本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以内。
Claims (8)
1.一种环烷基单取代氨基萤光素化合物,具有通式(Ⅰ)的结构:
其中,n为0至9自然数,m为1至9自然数;n=m,R为氢基,甲基,乙基,正丙基,正丁基,氟,氯,溴,碘;当n≠m,R为氢基。
2.根据权利要求1所述的环烷基单取代氨基萤光素化合物,其特征在于,所述n≠m,R为氢基时,m,n之和为2,3,4,5,6,7,8,9,10,10,11,12,13,14,15,16,17,18。
3.如权利要求2所述的化合物,其特征在于,包括表1所示的化合物,其名称如下:
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环己烷基)氨基(L1),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环戊烷基)氨基(L2),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(环丁烷基)氨基(L3),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(4-甲基-环己烷基)氨基(L4),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(3-甲基-环丁烷基)氨基(L5),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(4-乙基-环已烷基)氨基(L6),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(3-乙基-环丁烷基)氨基(L7),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(4-丙基-环己烷基)氨基(L8),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(3-丙基-环丁烷基)氨基(L9),
(S)-4-((2-(4-羧基-4,5-二氢噻唑-2-基)苯并噻唑-6-(4-溴-环已烷基)氨基(L10),
化合物名称后的括号中为其对应的代号。
4.权利要求1所述的环烷基单取代氨基萤光素化合物作为生物发光底物的应用。
5.权利要求1所述的环烷基单取代氨基萤光素化合物在检测萤光素酶在体外,细胞和体内分布成像方面的应用。
6.权利要求1所述的环烷基单取代氨基萤光素化合物在检测ATP的存在和数量以及检测ATP在体外,细胞和体内分布成像方面的应用。
7.权利要求1所述的环烷基单取代氨基萤光素化合物在检测药物在酶水平、细胞水平、动物水平的药理作用方面的应用。
8.根权利要求1~5中任一项所述的环烷基单取代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2-氰基6-氨基苯并噻唑溶于醋酸中,搅拌5分钟,再加入环烷酮或环戊酮或环丁酮,搅拌,搅拌结束后,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;反应液用饱和碳酸钠溶液洗涤后,用乙酸乙酯萃取,得到的乙酸乙酯层用无水硫酸钠干燥、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体积比为1:1的甲醇和二氯甲烷的混合溶液中,将无水碳酸钾与一水合半胱氨酸盐酸盐溶于质量浓度为40%的甲醇水溶液中,将上述两液混合,反应15分钟后,将反应液中的有机溶剂蒸除,调节反应液的pH值为5.5~6.5,待大量固体析出后,过滤,即得目标化合物;
步骤1)中所述2-氰基6-氨基苯并噻唑的加入量为所述醋酸重量的1%~5%,所述环烷酮或环戊酮或环丁酮的加入量为所述醋酸重量的8~10%;所述氰基硼氢化钠的加入量为所述醋酸重量的3%~8%;
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1%~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%。
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