Design, Synthesis And Activity Evaluation Of N-substituted Amino-4-benzensulfonyl Guanidine And5-nitro-1H-indazole-3-carboxylic Acid Derivatives As Novel Acrosin Inhibitors

The world population has exceeded7billion now, which brings about a heavy burden for social and economic development. China is a country with a large population and family planning is our basic national policy. With the rising status of women today, more and more women ask men to bear the corresponding responsibility for contraception. But so far, there is no clinical use records of male anti-fertility agent. Therefore, there is an urgent need for designing and developing a kind of efficient, reversible, safe and convenient male contraception agents.The epididymis is an important part of the male reproductive system and plays a decisive role in sperm maturation, storage and protection. Acrosome is the core structure of the sperm and as a specialized lysosome, it contains a variety of enzymes associated with fertilization. Acrosin not only affects sperm motility, promotes sperm motility but also disperses acrosome matrix in the process of fertilization and and hydrolyzes the zona pellucida, which helps sperm enter and fusion to ovum. Therefore acrosin becomes the research hotspot target in recent years.Acrosin inhibitors are few and mostly obtained by random screening. Most of them have the drawbacks of toxicity, poor specificity and unstable and so on. In addition, due to the information lack of binding mode of the inhibitor and target enzyme, the progress of acrosin inhibitor research and development based on the structure of the target enzyme is relatively slow.In our previous studies, we have built the homologous3D model of human acrosin and analyzed the properties of the activity site of human acrosin. It was found that active cavity is an irregular concaveslot and can be divided into three parts:PI, P2and G, where P1is the polar region of the ligand binding and there is a certain degree of hydrophobicity in the bottom of P2and G. In addition, there are some critical amino acid residues in every cavity, such as T216, Q218, S221, R199, E120and so on.In this study, we designed and synthesized two types of acrosin inhibitors. They are N-substituted amino-4-benzensulfonyl guanidine and5-nitro-1H-indazole-3-carboxylic acid derivatives, respectively.The design of the first class compounds are based on the binding mode of most existing inhibitors and KF950with the best inhibitory activity. From their docking results, we found that the guanidine groups are all located in P1and have several hydrogen bonding ineractions with P1, which shows that the guanidine group is an important pharmacophore for the high acrosin inhibitory activity. Therefore, we keep the group retained. As KF950has the drawbacks of unstability and strong basicity, we introduced the stable group amide-bond and liposolubility group sullfonyl on the basis of retained guanidine group and a new type of acrosin inhibitors N-substituted amino-4-benzensulfonyl guanidine are designed. In accordance with the design route, the key intermediate N-substituted amino-4-benzensulfonyl guanidine was obtained by two steps from the starting substance p-nitro-benzensulfonyl chloride, the reaction of the intermediate with different substitute acyl chlorides yield the target compounds. All35target compounds are firstly reported and all the structure of intermediates and target compounds were confirmed by1HNMR and MS.The second class compounds are designed according to the literature and the nature of the active cavity. From the literatures we found that1H-indazole-3-carboxylic acid derivatives has the action of anti-sperm production. The docking results of the lead compound and acrosin shows that the compound nucleus is located in the edge of P1and there is no polar group forming hydrogen binding interactions with P1, so we introdued nitro group in the hydrophobic aromatic ring. After docking, we got the intended consequences, we found that the nitro group can form strong hydrogen bonding interactions with P1. The molecules could be extended to P2/G cavity by modifying the carboxyl groups. So a novel of acrosin inhibitor5-nitro-1H-indazole-3-carboxamide were designed and synthesized. The intermediate1H-indazole-3-carboxylic acid was obtained by a reported procedure from the starting material β-acetyl phenylhydrazine. Then the intermediate5-nitro-1H-indazole-3-carboxylic acid was obtained by nitrification reaction, and finally the carboxylic acid group was replaced with a variety of amines to afford the target compounds. All21target compounds were firstly reported and all the structure of intermediates and target compounds were confirmed by1HNMR and MS.The acrosin inhibitory activity of all title compounds were assessed accroding to the method of Kenndy et al.. The experimental results showed that all compounds had better activity than the control compound TLCK. IC50of the compoud with best inhibitory activity reached0.01μmol/ml, which is deserved for further study. Next we disscussed the structure-activity relationship, providing a reference for further study.This study improved the rational design platform of the human acrosin further. Acording to the nature of acrosin activity site, phsicochemical property and structural features of KF950, we designed and synthesized a series of N-substituted amino-4-benzensulfonyl guanidine and5-nitro-lH-indazole-3-carboxamide derivatives and tested their in vitro acrosin inhibitory activity. We found that4p and6d are worthy of further research.