The Synthesis Of The BSA-benzodiazepine And BSA-phencyclidine Conjugates

In this thesis, The BSA-benzodiazepine and BSA-phencyclidine(PCP) conjugates were designed and synthesized. It is divided into two chapters. In chapter one, we reviewed hapten-carrier conjugates'research and application, the biological effects and synthetic methods of benzodiazepines and PCP.Hapten can not cause the body's immune response because of its low molecular weight and simple structure. In order to produce specific antibodies in the immune, immunologist often connected the hapten to the carrier to form a hapten - carrier conjugate that stimulates the immune response. Many small molecules were connected with protein, peptide or polysaccharide to elicit the immune response. Radioactive immunoassay has often been developed. Benzodiazepine and PCP are small molecule haptens that have important biological activity, medicinal chemists and synthetic chemists have developed a variety of methods for their synthesis. On this basis, we designed the BSA-benzodiazepine and BSA-phencyclidine conjugates.In chapter two, conjugates of benzodiazepine and PCP with BSA were designed and synthesizedAt first, the hapten benzodiazepine was synthesized. The procedure started with 1-chloro-4-nitrobennzene and 2-phenylacetonitrile, through intermolecular cyclization and reduction to form (2-amino-5-chlrophenyl)(phenyl)methanone, (2-amino-5-chlrophenyl)(phenyl)methanone was coupled with tert-butyl 3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-fluoro-4-oxobutanoate, followed by removal Fmoc and intramolecular cyclization to afford tert-butyl 2-((Z)-7-chloro-2,3-dihydro-2-oxo-5-phenyl-1H-benzo[e][1,4]diazepin-3-yl)acetate. The easter was converted to the carboxyl, followed by Curtius rearrangement, alcoholysis with phenylmethanol to form the cbz protected amino compound, (Z)-3-(Aminomethyl)-7-chloro-1-methyl-5-phenyl-1H-benzo[e][1,4]diazepine-2(3H)-one was prepared by removal of cbz by BBr3, then it was coupled with glutaric anhydride to give the hapten 4-(((Z)-7-chloro-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-benzo[e][1,4]diazepin-3-yl)methylcarbamoyl)butanoic acid.The overall yield of this 11-steped sequence was 7%. After activation of the hapten with N-hydroxysuccinimide(NHS), the product reacted with BSA in the ratio of 20:1 in the solution of DMF and the phosphate buffer. The residue was dialysised, lyophilized, and tested by MALDI-TOF ,the number of benzodiazepine molecule coupled to a BSA molecule is 14.1Secondly, PCP-BSA complex was synthesized. p-Bromophenylmethyl aldehyde was protected by glycol. 1-(4-(1,3-dioxolan-2-yl)phenyl)cyclohenyl)cyclohexanol was formed by Grignard reaction between cyclohexanone and 2-(4-bromophenyl)-1,3-dioxolane.The aldehyde was given by deprotection,followed by oxidation to carboxyl by oxone, and protection by methyl ester to afford methyl 4-(1-hydroxycyclohexyl)benzoate. The hydroxyl was replaced by NaN3 and reduced with Pd/C to give methyl- 4-(1-aminocyclohexyl)benzoate. Methyl-4-(1-(piperidin-1-yl)cyclohexyl)benzoate was prepared by substitution of dibromopentane and methyl- 4-(1-aminocyclohexyl)benzoate, followed by removal of methyl ester by LiOH/H2O, coupled with methyl-4-(aminomethyl)benzoate to afford the hapten methyl- 4-((4-(1-(piperidin-1-yl)cyclohexyl)benzamido)methyl)benzoate. There were 10 steps. The overall yield was 5.1% in 10 steps. The hapten was activated by NHS, then reacted with BSA in the ratio of 20:1 and 40:1 in the solution of DMF and the phosphate buffer. The residue was dialysised, lyophilized, and tested by MALDI-TOF, The numbers of PCP molecule coupled to a BSA are 9.8 and 13.4...