Synthesis Of Anticancer Lead Compound T-OA, Quality Standards And Other Related Pharmaceutical Research

Background:the anticancer lead compound T-OA (3β-Hydroxyolea-12-en-28-oic acid-3,5,6-trimethylpyrazin-2-methyl ester; Fig.1) was previously reported by our research team. It was synthesized via conjugating the effective antitumor ingredients (oleanolic acid and ligustrazine) of a classic traditional Chinese medicine (TCM) formulation, and had been already applied for national and international patents. The present research data showed that T-OA having selective inhibition against tumors but low toxicity was worth to do further research and development. This paper got the financial support from National Natural Science Foundation of China (No.81173519) and Innovation Team Project Foundation of Beijing University of Chinese Medicine named’Lead Compounds Discovering and Developing Innovation Team Project Foundation’(No.2011-CXTD-15).Fig.1:the structure of anticancer lead compound T-OAResearch purposes:the pharmaceutical researches on T-OA were mainly divided into four parts:synthesis process, quality standards, equilibrium solubility and apparent oil-water partition coefficient, and metabolites in rat urinary; this issue aimed to improve the preclinical research materials of T-OA.Research methods:the research on synthesis process was designed to optimize laboratory preparation technology of intermediate TMP-Br and target product T-OA; firstly, applying HPLC-MS for identifying chemical compositions in reaction solution, and then investigating the effects of different factors on productivity; at last, getting the optimum preparation method by orthogonal process. The part of quality standards was aimed at listing T-OA’s quality standard projects, determination methods and limits according to’China Pharmacopoeia’2010Edition and’drug research guiding principle’. A high performance liquid chromatography method was established and used to detect the concentration of T-OA in buffer solutions and surfactant solutions; the partition coefficients for n-octanol-water/buffer solution systems of T-OA were determined by shaking flask method. In the study of metabolites in urinary, timely collecting rat urine of different groups, after freeze drying, the solid was extracted by ethyl acetate, and residues were injected into HPLC-HRMS, and then precise molecular formulas of metabolites were elucidated by comparing the differential of these samples.Results:the optimum reaction conditions of TMP-Br were NBS/TMP=0.9, CC14/TMP=5mL/g, four common energy-saving lamps (85W) being used as the initiators, and reaction time being1h (calculated by1g TMP). The best reaction conditions of T-OA were K2CO3/OA=1, TMP-Br/OA=1, THF/OA=10mL/g, and reaction time being4h (calculated by1g oleanolic acid). The part of quality standard was listing relevant investigation items. T-OA’s equilibrium solubility in buffer solutions was increased with pH (1-10) rising and the largest value was5.10μg·mL-1at37℃; the equilibrium solubility in32g-L"1lauryl sodium sulfate solution increased to706.97μg·mL-1; apparent oil/water partition coefficient of T-OA was544.71(1g Papp=2.74) and it increased with pH (1-10) rising. Study on metabolites in urinary, one metabolite of OA and two metabolites of TMP in material group were identified; besides, there’s one metabolite of T-OA rather than TMP’s and OA’s in administration group.Conclusions:this paper initially formed three technical standards, including the preparation norm of intermediate TMP-Br, the quality standard draft of anticancer lead compound T-OA, and determination norm of T-OA’s oil-water partition coefficient, which aimed at offering technical standards for future related researches of anticancer lead compound T-OA. The obtained optimum preparation process avoided using benzene (Residual Solvent Class1), and largely shortened reaction time (from13.5h to5h), increased product’s yield; using ordinary energy-saving lamps as initiator reduced cost. The yeild of the optimum process was26.02%, which provided a reference for future industrial production. Study on quality standards benefited for writing T-OA’s quality standard draft. Moreover, investigating apparent oil-water partition coefficient, equilibrium solubility and metabolites in rat urine promoted to understand T-OA’s solubilities in various media. It’s inferred that T-OA might play efficacy in forms of the whole molecular instead of material fragments; the established HPLC-HRMS method in this paper was reliable and could be used to screen metabolites of related derivatives in rat urine; in addition, the content was expected to benefit to the prodrug design based on oleanolic acid.