Synthesis And Metabolism Study Of Anti-Echinococcus Bemzimidazole Prodrug

Echinococcosis(hydatid disease), caused by the Echinococcosis larva parasiting on the body of human and animals, is one of the most important zoonotic diseases. China is one of the countries where hydatid disease is most seriously epidemic, the latest survey estimated that there are approximately400,000people suffering from hydatid disease,50million people being threatened. Echinococcosis mainly distribute in Sichuan, Qinghai, Xinjiang, Tibet and Gansu provinces(autonomous regions). There are two main kinds Echinococcus in China:one is Echinococcus granulosus (cystic hydatid disease) which is caused by the larva infection of Echinococcus granulosus, the other is Echinococcus multilocularis (alveolar hydatid disease) which is caused by the larva infection of Echinococcus multilocularis.The use of benzimidazoles(Alb and Meb) since1980s opened up a new approach to the chemotherapy of echinococcosis, and the Alb and Meb are recommended by the WHO for the treatment of human echinococcosis. Though Alb is more frequently used in the clinical treatment, mebendazloe showed better efficacy in animal experiments, especially the damage to the germinal layer of cells. This is because gastrointestinal absorption of Meb in humans is worse, the absorption rate only5%～10%of the total intake, the bioavailiability, even lower, only about2.3%, long duration and high dose of medication is required for the disease. If the bioavailability is improved, the efficacy may increase significantly. Besides, experiments show that Fen and oxfendazole showed better efficacy in the treatment of animal echinococcosis. Many efforts have been devoted to the improvement of their efficacy, mainly by the formulations, which can’t shorten the treatment course and reduce side effects, and plasma drug concentration is difficult to increase exponentially. The status quo of clinic treatment cannot be changed radically.Prodrug can change the drug action time and ester water distribution coefficient, improve the solubility and membrane permeability, etc."Prodrug" was first coined by Albert in1958, about5%～7%of drugs approved worldwide can be classified as prodrugs. As a drug optimization strategy, prodrugs are widely used against oncology, cardiovascular, nervous system and infectious diseases, mainly to solve drug permeability, poor solubility and low bioavailability.1Synthesis of anti-echinococcus bemzimidazole prodrugsIn light of the commonly used functional group modification strategies, the amide prodrugs of Meb (compound1and2) and acyclic thiourea prodrug of Fen (compound3) were designed. The synthesis routs were found through SCiFinder. Those compounds were synthesized, separated and purified, with the reaction conditions being explored. Their structures and purity were certified by HPLC/MS and Nuclear magnetic resonance for the further biological experiments.2In vitro metabolism of anti-echinococcus bemzimidazole prodrugsThe HPLC methods of compound1,2and3were established for the in vitro metabolism tests. Two acidic solutions with pH1.5and pH3.5separately and the other three biological medium which are artificial gastric juice, intestinal juice and mice liver homogenate have been prepared. Compound1,2and3were mixed with those solutions and incubated at37℃in oscillator, samples were collected at different time points and analyzed quantitatively by HPLC instrument, drawing the metabolic curves. Compound1is instable and decomposed at room temperature. Compound2can be metabolized in five solutions above, but the parent drug Meb can’t be detected. In three biological medium, the added compound3can be metabolized, but only in the liver homogenate, the parent drug Fen was generated, with the highest7.92%metabolic rate. 3In vivo metabolism of anti-echinococcus bemzimidazole prodrugsHPLC methods for the detection of prodrug plasma concentration were established. Mice plasma were collected at different time points after oral administration of suspension of compound2dispersing in1%tragacanth gum solution and measured by HPLC. Compound2and Meb can’t be observed. Suspension of compound3dispersing in1%tragacanth gum solution was administrated orally, compound3and Fen can’t be observed in serum but can be detected in gastric contents as well as Oxf which is Fen’s metabolite.4In vitro activity test against Echinococcus granulosus protoscolex of anti-echinococcus bemzimidazole prodrugsCyst fluid containing Echinococcus granulosus protoscolex were collected for the in vitro activity test. The prodrugs were added respectively to the3wells of the96-well plate, cultured, observing the protoscolex activity and the surface damage. Three days later, the mortability were calculated using methylene blue staining method. The test showed that at20μg/mL, the mortality of compound2was100%, equal to Meb, and at10μg/mL100%, better than70.5%of Meb. The mortality of protoscolex treated by compound3was45.9%at10μg/mL, lower than100%by Fen.