【作者】 郭兴杰；

【导师】 李发美； 今井一洋；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2002， 博士

【摘要】 本文研究了用2，1，3-苯并(口恶)二唑类衍生化试剂4-(N-氯甲酰甲基-N-甲基)氨基-7-硝基-2，1，3-苯并(口恶)二唑[4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole，NBD-COC1]和4-(N-氯甲酰甲基-N-甲基)氨基-7-N,N-二甲氨基磺酰-2，1，3-苯并(口恶)二唑[4-(N-chloroformethyl-N-methyl)amino-7-N，N-dimethylaminosulfonyl-2,1,3-benzoxadiazole，DBD-COC1]衍生化抗抑郁药物氟西汀(FLX)和去甲氟西汀(NFLX)的方法；研究了使用多糖衍生物类手性固定相拆分氟西汀的NBD-COCI衍生物(NBD-FLX)和DBD-COCI衍生物(DBD-FLX)对映体的方法；并用HPLC荧光检测法对大鼠给药后体内药代动力学及药效学进行了研究。 1．氟西汀及其代谢产物去甲氟西汀的衍生化 建立了柱前衍生化HPLC法测定氟西汀和去甲氟西汀。NBD-COC1和DBD-COC1均为酰氯类化合物，在非水溶剂中，可以和胺类化合物氟西汀和去甲氟西汀进行酰胺化反应。本文考察了衍生化试剂浓度、衍生化反应的温度和时间对反应的影响，并对两种荧光标记试剂—NBD-COC1和DBD-COC1的反应活性进行了比较。结果表明：在60℃，NBD-COC1和氟西汀的衍生化反应需2h完成，DBD-COC1和氟西汀的反应需3h完成，而NBD-COC1或DBD-COC1和去甲氟西汀的反应在30min内即可完成。NBD-COC1的氟西汀和去甲氟西汀衍生物(NBD-FLX和NBD-NFLX)的最大荧光激发和发射波长分别为478±2nm和537±2nm，DBD-COC1的氟西汀和去甲氟西汀衍生物(DBD-FLX和DBD-NFLX)的最大荧光激发和发射波长分别为454±2nm和562±2nm。用NBD-COC1作衍生化试剂，不但反应时间短，而且荧光产率高，灵敏度高。目前关于NBD-COC1用作衍生化试剂的文献还未见发表。 2．分离氟西汀衍生物对映体沈阳药科大学博士学位论文摘要 本文使用多糖类手性固定相直链淀粉一三一(3，5一二甲苯基氨基甲酸酷)(ChiralPakAo和ChiralpakAD一刃硕)和纤维素一三一(3，5一二甲苯基氨基甲酸酷)(Chiraleel on)研究了氟西汀和去甲氟西汀衍生物的对映体拆分。NBD一FLX和DBD一FLx对映体可在AD~RH，AD和OD柱上获得拆分，但去甲氟西汀的两种衍生物(NBD一FLX和DBD一FLX)在所试验的任一手性固定相上都不能获得拆分。 使用AD~RH固定相，NBD一FLx对映体的最佳拆分条件是:乙睛一甲醇一异丙醇-水(52:23:5:20)作流动相，柱温35℃。DBD一FLX对映体的最佳拆分条件是:乙睛一水(54:46)作流动相，柱温38℃。柱温和流动相组成对对映体的拆分有较大影响。在反相洗脱条件下，柱温升高，使色谱峰变窄，分离度增加。对比DBD一FLX，NBD一FLX能在AD.RH柱上获得较高的分离度(Rs)和较好的分离因子(a)。使用AD一RH固定相，两种衍生物均为S一对映体先出峰。 NBD一FLX和DBD一FLX在AD固定相上同样获得了拆分，拆分NBD一FLX的最佳条件是:异丙醇一正己烷(50:50)，柱温25℃;拆分DBD一FLX的最佳条件是:异丙醇一正己烷(20:80)，柱温25℃。在AD柱上，无论NBD一FLX还是DBD一FLX，都是R一对映体最先出峰，与使用AD一RH柱的情况正好相反。NBD一FLX比DBD一FLX在AD柱上更易保留，更易获得较高的分离度和分离因子。 使用生产商建议的流动相，即正己烷作底剂，加入甲醇、异丙醇等醇类有机改性剂作流动相，不能在OD柱上分离NBD一FLX和DBD一FLX对映体。但改用四氢吠喃、乙睛等溶剂作有机改性剂，由于减弱了溶剂和手性固定相之间的作用力，加强了被分离组分和手性固定相之间的作用力，对映体获得了较好的分离。拆分NBD一FLx的最佳条件点:正己烷一四氢吠喃(75:25)作流动相，柱温25℃(使用1 scm色谱柱)，拆分DBD一FLx的最佳条件是:正己烷一四氢吠喃一乙睛(80:20:2)作流动相，柱温25℃(使用25cm色谱柱)。AD和OD柱同为正相洗脱，但在两种柱上对映体出峰顺序恰好相反，在OD柱上，NBD一FLX和DBD一FLX均为S一对映体最先出峰。 多糖类手性固定相和溶质之间的作用力包括:氢键、偶极一偶极和二一二作用力。由于AD和OD固定相都含有3，5一二甲苯基氨基甲酸醋基团，它们的上述作用力是相同的。但淀粉一三一(3，5一二甲苯基氨基甲酸酷)(AD固定相)具有左旋四重螺旋结构，而纤维素的同类衍生物(OD固定相)具有左旋三重(3/2)螺旋构型，决定了它们有不同的对映体识别能力。本文在拆分两种衍生物对映体的过程中，研沈阳药科大学博士学位论文摘要究了流动相的组成、比例、温度等因素对对映体拆分的影响，并探讨了对映体在多糖类手性固定相上的拆分机理。3.大鼠血浆中氛西汀对映体的测定和药代动力学立体选择性研究 用NBD一COCI作荧光标记试剂，经柱前衍生化和柱切换高效液相色谱法测定了大鼠血浆中氟西汀对映体的浓度。使用ODs固定相，流动相为乙睛一水(55:45);使用ChiralPak AD固定相，流动相为乙睛一甲醇一异丙醇一水(52:23:5:加)。氟西汀的最低定量限为IOnlnol/L(100川血浆)，线性范围为10一loo0lunol几。实验结果表明:大鼠灌胃给予消旋盐酸氟西汀后，两对映体的AUC(P<0.01)、C，:(P<0.05)和T，:(P<0.01)值有显著性差异，表明氟西?更多还原

【Abstract】 Fluoxetine(FLX), N-methyl-3-phenyl-3-(2,2,2-trifiuoro-p-tolyloxy) propylamine hydrochloride, C17H18F3NO HC1, MW = 345.8, is an antidepressant which differs structurally and pharmacologically from the tricyclic agents. It has been shown to selectively inhibit the reuptake of serotonin in presynaptic neurons. Besides as a tool to increase the level of serotonin in the synaptic junction for fundamental researches on the neurosciences, FLX has been prescribed widely for the therapy of mental depression, obsessive compulsive disorders and pain syndromes. FLX is extensively metabolized by demethylation in liver, to its primary active metabolite norfluoretine (NFLX), which is also a potent serotonin (5-HT) reuptake inhibitor.In order to sensitively determine FLX, NFLX and their enantiomers in biosamples, this paper successfully explored the applicability of 2,1,3-benzoxadiazole compounds to derivatize FLX and NFLX. 4-(N-Chloroformylmethyl-N-methyl) amino-7-nitro-2,l,3-benzoxadiazole (NBD-COC1) and 4-(N-chloroformethyl-N-methyl) amino-7-N,N-dimethylamino-sulfonyl-2,l,3-benzoxadiazole(DBD-COCl) were chosen as the fluorescent derivatizing reagents. The derivatives of FLX were resolved on Chiralpak AD-RH, Chiralpak AD and Chiralcel OD columns. The derivatization and resolution techniques were applied to pharmacokinetic studies on FLX and NFLX in rats. 1. Derivatization of FLX and NFLX with NBD-COC1 and DBD-COC1.As the fluorescent labeling reagents, NBD-COC1 and DBD-COC1, both of which has an acyl halide group, were selected to derivatize the amines. The temperature, time and other conditions were examined for the reaction and the fluoresceace yield of the derivatives were compared. Between two reagents, NBD-COC1 was better reagent with regard to the sensitivity and reactivity. NBD-COC1 reacted with FLX at 60℃ for 2h ,faster than DBD-COCI , to form strong fluorescent derivative (ex=478 2nm , em=537 2nm) while DBD-COCI reacting with FLX at 60 for 3h to form weaker fluorescent derivative ( ex=454 2nm, em=562 2nm). Like FLX, NBD-NFLX derivative was more fluorescence than DBD-NFLX derivative, and both NBD-COCI and DBD-COCI reacted with NFLX at 60 within 30min. To our knowledge, no paper has been published on derirvatizing amines with NBD-COCI . 2. Resolution of FLX fluorescent derivativesThe enantiomers of NBD-FLX and DBD-FLX were separated on Chiralpak AD-RH, Chiralpak AD and Chiralcel OD columns, but no resolution was observed for NFLX derivatives. On AD-RH, the best separation was obtained with CH3CN-CH3OH-IPA-H2O (52:23:5:20) as mobile phase at 35 for NBD-FLX and with CH3CN-H2O (54:46) as mobile phase at 38 for DBD-FLX. The mobile phase components and column temperature affected the resolution greatly in the reversed phase mode. Increasing column temperature favorites the sharping the peak to gain high column efficiency and resolution. More Rs and a values were for NBD-FLX other than DBD-FLX enantiomers. As regard with the elution order, S-isomes of the derivatives eluted first on AD-RH.The separation of NBD-FLX and DBD-FLX enantiomers was also performed on the Chiralpak AD column. Baseline separation was obtained for NBD-FLX with mobile phase of IPA-hexane (50:50) and nearly baseline separation for DBD-FLX with hexane-IPA(80:20) at 25 . The elution order of the two derivatives on the AD column was reversed compared to that on the AD-RH column as expected. Just like on the AD-RH column, better separation was obtained for NBD-FLX. It may be attributed to the structural difference between two compounds which leads to a different chiral recognition ability.Alcohol modifiers such as methanol, ethanol and IPA failed to resolve NBD- and DBD-FLX on OD column. So non-alcohol modifiers such as THF, acetonitrile were tested. As a result, better enantiomeric separations of the derivatives were reached by use of THF or THF and acetonitrile as modifiers. The optimal mobile phases were hexane-THF (75:25) for NBD-FLX on 15cm OD column and hexane-THF-acetonitrile (80:20:2) for DBD-FLX on 25cm OD column. Interestin更多还原