萘普生中间体2-氨基-2-(5-溴-6-甲氧基-2-萘基)丙酸的合成

采用1，3－二溴－5，5－二甲基乙丙酰脲作催化剂，在丙酮溶剂中，以盐酸作催化剂，于室温快速(8min)、高收率（96％）地对2－甲氧基萘的萘环进行溴化，再经乙酰化、Bucherer-Berg环化和水解反应，制得萘普生中间体2－氨基－2－（5－溴－6-甲氧基－2－萘基）丙酸。并对环化反应的条件进行了优化，反应总收率68％。     

溴化铜对芳基烷基酮的选择性溴化反应研究

研究溴化铜对苯丙酮、间－氯苯丙酮、6－甲氧基－2－丙酰基萘、6－甲氧基－2－乙酰基萘、对－甲氧基苯乙酮和苯乙酮等芳基烷基酮的α－溴化反应，在乙醇或乙酸乙酯－氯仿混合溶剂中，溴化铜对芳基丙酮（苯丙酮、间－氯苯丙酮和6－甲氧基－2－丙酰基萘）溴化，高收率、高选择性地得到α－溴化产物。芳基乙酮（6－甲氧基－2－乙酰基萘、对－甲氧基苯乙酮和苯乙酮）的溴化铜溴化，在乙醇中反应，选择性得到α－溴化产物。     

缩酮-α-磺酸酯重排法合成dl-萘普生

β-萘酚经甲基化、丙酰化、侧链溴化、缩酮化制成1-(6-甲氧基-2-萘基)-2-羟基丙-1-酮缩二甲醇,再经苯磺酰化重排制得 dl-萘普生,总收率50%。     

dl—萘普生重排合成的工艺研究

以2-甲氧基萘为起始原料,通过丙酰化、溴化、缩酮化、重排及水解制成2-(5-溴-6-甲氧基-2-萘基)丙酸,再经催化转移氢解脱溴即得萘普生。工艺简便,反应条件温和,总收率达74%。     

1,3—二溴—5,5—二甲基海因选择性溴化反应研究

考察了在不同极性溶剂中以1,3-二溴-5,5-二甲基海因作溴化剂对溴化6-甲氧基-2,酰基萘反应的影响,13-二-5,5-二甲基海因作为溴化剂,在二氯甲烷,三氯甲烷,二氯乙烷中,于一定温度下,搅拌反应,高选择性溴化6-甲氧基-2-酰基萘的萘环,得到高收率的溴化产物6-甲氧基-5-清算溴-2-酰基萘,该化合物是全盛非甾体消炎镇痛药萘普生(S)-( )-2-(6-甲氧基-2-萘基)丙酸的关键中间体.     

dl－萘普生重排合成的工艺研究Ⅲ

在三甲基氯硅烷的存在下，丙酰萘甲醚、乙二醇和原位形成的溴合二烷—锅反应转化成２－（１－溴乙基）－２－（６－甲氧基－２－萘基）－１，３－二氧戊环（３），再经重排、水解即得ｄｌ－萘普生。对丙酰萘甲醚计算，总收率９１．５％。探讨了反应温度和不同溶剂对３收率的影响。还考察添加不同Ｌｅｗｉｓ酸对氧化锌催化３重排时间的影响。     

萘普生重排法合成工艺研究

丙酰萘甲醚单独用溴化铜溴化，得α－溴丙酰萘甲醚，产率９４％。如反应后期加入乙二醇，可一锅反应得溴化缩酮，产率９０％。     

dl-萘普生重排合成的工艺研究Ⅱ

以2-萘酚为原料,经甲基化、丙酰化等5步反应制得dl萘普生,总收率70%。其中丙酰化反应以无水三氯化铁代替无水三氯化铝。羰基的α-单溴化以过溴型聚合物试剂代替吡啶氢溴酸盐过溴化物,并割除硝基苯,简化操作。     

dl－萘普生重排合成工艺研究IV

在溴化铜的存在下，丙酰萘甲醚（２）、乙二醇和过溴型三甲基苄基铵树脂－锅反应转化成２－（１－溴乙基）－２－（６－甲氧基－２－萘基）－１，３－二氧戊环（３），再经重排、水解制得ｄｌ－萘普生（１）。以２计算，总收率８９％。并考察了反应温度、溴化铜用量对３收率的影响。     

dl—萘普生重排合成的工艺研究Ⅵ

以２－甲氧基萘淡起始原料，经氯化、丙酰化、溴化制成２－溴－１－（５－氯－６－甲氧基－２－萘基）丙－１－酮，再经缩酮化、重排、水解、脱氯４步－锅合成ｄｌ－萘普生，总收率７６．５％。     

Synthesis of 1-deoxy-1-hydroxymethyl- and 1-deoxy-1-epi-hydroxymethyl castanospermine as new potential immunomodulating agents

Two new C-1 epimeric hydroxymethyl castanospermine congeners 2a and 2b, synthesized by stereocontrolled intramolecular double reductive amination of D-glucose derived beta-keto ester as a key step, showed impressive immuno-potentiating property. The bioactivity was mediated through up-regulation of T(H1)/T(H2) cytokine ratio. The finding suggested that immunmodulatory activity of polyhydroxylated indolizidine alkaloids can be tuned by minor structural/stereochemical alterations.     

Role of glutathione S-transferases A1-1, M1-1, and P1-1 in the detoxification of 2-phenylpropenal, a reactive felbamate metabolite

Felbamate has proven to be an effective therapy for treating refractory epilepsy. However, felbamate therapy has been limited due to the associated reports of hepatotoxicity and aplastic anemia. Previous research from our laboratory has proposed 2-phenylpropenal as the reactive metabolite in felbamate bioactivation and identified its mercapturates in the urine of rats and patients undergoing felbamate therapy. While the reaction between 2-phenylpropenal and GSH has been shown to occur spontaneously under physiological conditions, the potential catalysis by glutathione transferases (GST) has remained unknown. The work presented here demonstrates a role for GST in the detoxification of 2-phenylpropenal. The kinetic data show that 2-phenylpropenal is a substrate for all three isoforms tested, with a k(cat)/K(m) of 0.275 +/- 0.035 microM(-1) s(-1) for GSTM1-1, 0.164 +/- 0.005 microM(-1) s(-1) for GSTP1-1, and 0.042 +/- 0.005 microM(-1) s(-1) for GSTA1-1. Given that electrophilic substrates such as 2-propenal have been shown to inhibit GSTs, we also examined the inhibition of GSTM1-1, GSTP1-1 and GSTA1-1 by 2-phenylpropenal. The enzyme inhibition studies demonstrate that 2-phenylpropenal inhibits GSTP1-1 and GSTM1-1. The inhibition of GSTP1-1 was completely reversible upon filtration and reconstitution in buffer containing 10 mM GSH. However, 2-phenylpropenal inhibition of GSTM1-1 was irreversible under the same conditions. The irreversible inhibition of GSTM1-1 may be important in understanding the toxicities associated with felbamate. Given that 2-phenylpropenal is both a substrate and irreversible inhibitor for GSTM1-1, GSTM1-1 represents a potential target for 2-phenylpropenal haptenization in vivo, which may in turn mediate the observed idiosyncratic reactions.     

人细胞色素P450 1A1与谷胱甘肽S-转移酶的融合蛋白及其抗体的制备

采用融合蛋白技术原核表达CYP1A1（第241-381个氨基酸）与谷胱甘肽S-转移酶（GST）的融合蛋白作为抗原,用于制备CYP1A1多克隆抗体. 根据正反重组质粒pGEX/1A1表达的融合蛋白大小不同的原理,直接表达筛选得到正向重组质粒pGEX/1A1. 通过优化表达条件, 提高了目的蛋白的表达水平. 包涵体蛋白经制备型聚丙烯酰胺凝胶电泳（PAGE）分离, 获含纯化融合蛋白GST-1A1的PAGE凝胶. 直接用含GST-1A1的凝胶悬液免疫BALB/c小鼠,自腹水中获取CYP1A1多克隆抗体（1A1pAb）. 1A1pAb用切胶纯化的融合蛋白GST-2B6交叉吸收,蛋白A- Sepharose亲和层析柱来纯化. 用切胶纯化的融合蛋白GST-1A1及GST-2B6的免疫印迹反应初步鉴定1A1pAb的特异性. 纯化的1A1pAb对融合蛋白GST-1A1反应特异性较强,但仍对GST-2B6有弱交叉反应. 在实际应用中可根据反应强度来加以区分.     

1-羟乙基-5-巯基-1H-四唑的合成

以乙醇胺、二硫化碳和氯苄为起始原料,经缩合、取代、加成、成环及脱保护反应制得氟氧头孢关键中间1-羟乙基-5-巯基-1H-四唑,总收率约67%.     

Phenotype-genotype relationships of SULT1A1 in human liver and variations in the IC50 of the SULT1A1 inhibitor quercetin

Human sulfotransferases catalyze sulfate conjugation and 2 polymorphic genes, SULT1A1 and SULT1A2 in this family of transferases have been identified, encoding for 2 isoenzymes with very similar properties and substrate specificities. In order to test the hypothesis that variability in sulfation is due to genetic polymorphism in SULT1A1, the sulfation rate of 4-nitrophenol, a diagnostic substrate, was measured in 50 human liver samples and the genotype at the SULT1A1 locus was analyzed. The rate of 4-nitrophenol sulfation varied from 473 - 1,405 pmol/min/mg between the 5th and 95th percentiles, with a median and a mean +/- SD of 757 and 807 +/- 292 pmol/min/mg, respectively. The activities detected among the SULT1A1*2/*2 homozygotes (5 cases) were significantly lower than those of the other 2 genotypes, SULTA1*11/*1 and SULT1A1*1/*2 (5 and 40 cases, respectively), whereas there was no significant difference found between the SULT1A1*1/*1 and SULT1A1*1/*2 genotypes. To evaluate the possible influence of SULT1A2 polymorphism, genotype assays were also performed for this locus. No SULT1A2*2/*2 carrier, 26 SULT1A2*1/*1 and 24 SULT1A2*1/*2 were detected in the population sample under study. However, no correlation between the rate of 4-nitrophenol sulfation and the SULT1A2 genotype was detected. These results confirm that the variation in the rate of 4-nitrophenol sulfation in human liver is mainly due to SULT1A. Since SULT1A1*1/*2 polymorphism accounts for no more than 10% of the phenotypic variation seen in this cohort, other factors must also contribute to the variability in the rate of 4-nitrophenol sulfation in human liver. However, on the basis of the data obtained, variations in age, gender and liver function as possible causative factors can be excluded. The IC50 of quercetin, a potent inhibitor of 4-nitrophenol sulfation, was measured in the liver samples and ranged from 4.6 to 17.3 nM between the 5th and 95th percentiles. The median and the mean +/- SD were 7.7 nM and 8.3 +/- 2.5 nM, respectively. There was a weak but significant correlation between the IC50 value and age of the liver donors (r = 0.283, p = 0.046). The observed variation did not correlate with the genotypes at the SULT1A1 and SULT1A2 loci.     

人细胞色素P450 1A1与谷胱甘肽S-转移酶的融合蛋白及其抗体的制备

采用融合蛋白技术原核表达 CYP1 A1 (第 2 4 1- 381个氨基酸 )与谷胱甘肽 S-转移酶 (GST)的融合蛋白作为抗原 ,用于制备 CYP1 A1多克隆抗体 .根据正反重组质粒 p GEX/ 1 A1表达的融合蛋白大小不同的原理 ,直接表达筛选得到正向重组质粒p GEX/ 1 A1 .通过优化表达条件 ,提高了目的蛋白的表达水平 .包涵体蛋白经制备型聚丙烯酰胺凝胶电泳 (PAGE)分离 ,获含纯化融合蛋白 GST- 1 A1的 PAGE凝胶 .直接用含 GST- 1 A1的凝胶悬液免疫 BALB/ c小鼠 ,自腹水中获取 CYP1 A1多克隆抗体 (1 A1 p Ab) . 1 A1 p Ab用切胶纯化的融合蛋白GST- 2 B6交叉吸收 ,蛋白 A- Sepharose亲和层析柱来纯化 .用切胶纯化的融合蛋白 GST- 1 A1及 GST-2 B6的免疫印迹反应初步鉴定 1 A1 p Ab的特异性 .纯化的 1 A1 p Ab对融合蛋白 GST- 1 A1反应特异性较强 ,但仍对 GST- 2 B6有弱交叉反应 .在实际应用中可根据反应强度来加以区分     

Association between functional genetic polymorphisms of human sulfotransferases 1A1 and 1A2

Three human phenol sulfotransferases, provisionally named SULT1A1, 1A2 and 1A3, show 91-96% homology of their amino acid sequences and are encoded by neighbouring gene loci. Functional genetic polymorphisms are known for two of these sulfotransferases. In SULT1A1, a G to A transition leads to an Arg213 to His exchange and eliminates a Bsp143II restriction site. SULT1A1*His shows lower enzyme activity and thermostability than SULT1A1*Arg. In SULT1A2, an A to C transversion causes an Asn235 to Thr exchange and introduces a BpiI restriction site. Enzyme SULT1A2*Thr is less active than SULT1A2*Asn. These substitutions were detected by restriction fragment length polymorphism analyses of genomic sequences amplified by polymerase chain reaction. Despite the high similarity between the different human SULT1A genes, it was possible to amplify specifically the polymorphic parts of either SULT1A1 or 1A2, but not the homologous sequences of the other SULT, by setting the forward primer into intron 6. DNA from 300 adult male Caucasian subjects was analysed. Allele frequencies were 0.63 and 0.37 for SULT1A1*Arg and *His, and 0.62 and 0.38 for SULT1A2*Asn and *Thr, respectively. The frequency of the haplotype SULT1A1*Arg/SULT1A2*Asn (0.61) was nearly as high as the allele frequencies of its components. The same was observed for the haplotype SULT1A1*His/SULT1A2*Thr, whose frequency was 0.35. In contrast, haplotypes 1A1*Arg/1A2*Thr and 1A1*His/1A2*Asn were very rare. Their frequencies (0.02 each) were less than 10% of the figures expected in an independent distribution. The results demonstrate a strong association of the alleles producing the more active enzyme variants (SULT1A1*Arg and SULT1A2*Asn) and of those encoding the less active variants (SULT1A1*His and SULT1A2*Thr).     

Effects of genetic polymorphisms of CYP1A1, CYP2E1, GSTM1, and GSTT1 on the urinary levels of 1-hydroxypyrene and 2-naphthol in aircraft maintenance workers

This study was undertaken to investigate the effects of genetic polymorphisms of the cytochrome P450 1A1 (CYP1A1) and 2E1 (CYP2E1), and glutathione S-transferases mu (GSTM1) and theta (GSTT1) on urinary 1-hydroxypyrene and 2-naphthol levels, and to estimate the level of exposure to polycyclic aromatic hydrocarbons (PAHs) in aircraft maintenance workers. In 218 Korean aircraft maintenance workers, the geometric means of urinary 1-hydroxypyrene and 2-naphthol were 0.32 and 3.25 micromol/mol creatinine, respectively. These urinary concentrations were approximately at the upper limit of the general population. Mean urinary 2-naphthol concentrations were significantly different between smokers and non-smokers. CYP1A1 and GSTM1 were statistically significant in analyses on both 1-hydroxypyrene and 2-naphthol levels among smokers. The results suggest that smoking has more profound effects on urinary PAH metabolites than does genetic polymorphisms in this population, and that CYP1A1 and GSTM1 activity might be related to the metabolism of 1-hydroxypyrene and 2-naphthol.     

Metabolism of the soyabean isoflavone daidzein by CYP1A2 and the extra-hepatic CYPs 1A1 and 1B1 affects biological activity

Metabolism of the isoflavones daidzein and genistein, which may protect against some cancers, was studied using human liver microsomes and recombinant CYP isoforms. The detection of three, more polar metabolites of each isoflavone by RP-HPLC required NADPH, consistent with CYP-mediated metabolism. For different liver preparations, metabolite generation from daidzein showed a significant linear correlation with metabolite generation from genistein, indicating metabolism by the same CYP(s). The lowest rate of metabolism of both isoflavones was by the preparation with the lowest CYP1A2 activity. Metabolite peak areas were substantially and significantly reduced by the CYP1A2 inhibitor furafylline and to a lesser extent by the CYP2E1 inhibitor 4-methylpyrazole. Recombinant CYP1A2, but not CYP2E1, generated the metabolites of daidzein and genistein and recombinant CYP1A1 and CYP1B1, expressed at sites including the breast and prostate, were also active. The effects of two CYP-derived metabolites of daidzein, 6,7,4'-trihydroxyisoflavone and 7,3',4'-trihydroxyisoflavone, were studied in the MCF-7 human breast cancer cell line at a concentration (50 microM) at which daidzein induces an antiproliferative response. 7,3',4'-Trihydroxyisoflavone reduced total cell numbers to a greater extent than 6,7,4'-trihydroxyisoflavone or daidzein and increased cell death. Together, these data demonstrate proof of principle that CYP-mediated metabolism of daidzein can be an activation pathway. We conclude that CYP1A2 makes the major contribution to the hepatic metabolism of both daidzein and genistein and along with metabolism at sites of hormone-dependent tumours may enhance a cancer-protective effect of daidzein if sufficiently high concentrations are reached in target tissues.