Design,Synthesis And Pharmacological Evaluation Of A Novel MTOR-Targeted Anti-EV71 Agent

Hand,foot and mouth disease?HFMD?is an infectious disease that seriously threatens the health of human beings,especially infants and young children.HFMD has spread widely around the world in recent decades.According to the National Health and Health Committee report,until 2015,there were more than 117,600,00cases of hand,foot and mouth disease in China,including 3,361 deaths.The annual average incidence rate was about 117/100,000,ranking first among the number of reported cases of Class C infectious diseases.With the liberalization of China's birth policy,the situation of prevention and control of HFMD has become increasingly severe.The main pathogen causing HFMD is Enterovirus 71?EV71?,which has high infectivity and variability.At present,there are no antiviral drugs that can effectively inhibit EV71 in clinical practice.Most antiviral drugs against EV71 are still in the research and development stage,so the demand for developing high-efficiency anti-EV71 drugs is particularly urgent.At present,most of the targets of antiviral drugs are concentrated on the virus itself,but the characteristics of the viral mutation make the drug highly susceptible to drug resistance.The mechanism of action of the host-based antiviral drug is mainly to inhibit the synthesis of proteins or molecules required by the virus in the host cell,affecting the life cycle of the virus,and achieving the purpose of inhibiting the virus.The development of antiviral drugs based on host cells not only solves the problem of drug resistance,but also has broad-spectrum applicability,which can effectively improve drug development efficiency and reduce research and development costs.At present,host cell-based antiviral drugs have become a new trend in the development of antiviral drugs.Mammalian target of rapamycin?mTOR?,an important functional protein in mammalian host cells,plays an important role in sensing intracellular nutrient signals and regulating protein translation.Studies have shown that when the virus infects host cells,it triggers the intracellular PI3K/Akt/mTOR signaling pathway,which in turn activates mTOR,providing a material basis for viral RNA replication and protein synthesis.By inhibiting mTOR,viral RNA replication and protein synthesis can be prevented,viral infection is inhibited.Therefore,this research choose mTOR as a target,develops mTOR inhibitors to obtain new broad-spectrum and non-resistant anti-EV71 agents.Torin2 is currently a more active mTOR inhibitor(IC₅₀=0.25 nM).In our previous work,we found that Torin2 has strong viral inhibitory activity against EV71(IC₅₀=0.01?M).However,the study found that Torin 2 is poorly water-soluble,resulting in poor drug absorption and metabolism properties,making it difficult to further manufacture.Based on the above research,this thesis uses Torin2 as the lead compound to optimize,in order to obtain a new structural mTOR inhibitor with good activity,drug metabolism and water solubility.By analyzing the interaction between Torin2 and the mTOR,it was found that the nitrogen atom on the quinoline ring forms a hydrogen bond with the VAL2240 amino acid at the ATP binding site of the mTOR,which is a key binding site to the activity of Torin2.The trifluoromethyl substituted moiety improves the drug metabolism.Therefore,it is decided to retain the two parts,and the six-membered lactam ring is changed.The flexible open-ring side chain and the five-membered ring are used to replace the six-membered lactam ring.It is expected to reduce the original three-ring rigid planar structure and reduce intermolecular forces,improve the physical properties of the drug such as solubility,and replace the pyridylamide side chain,to investigate the effect of different electrical and different ring system substituents on the activity,a total of quinoline derivatives,five-membered ring derivatives and Torin2 derivatives were screened by computer and combined with synthetic feasibility analysis.Finally,28 of 3-butenone quinolines,24 of oxazoloquinolines and 46 of Torin2 derivatives were selected.A total of 98 new structural compounds were synthesized as target compounds.According to the different compound classes,six synthetic routes were designed,and the synthesis of 98 new compounds was completed.The structure was confirmed by MS and ¹H NMR.In order to investigate the biological activity of the compounds,the in vitro anti-EV71 activity test,mTOR kinase inhibitory activity test,and mTORC1/mTORC2inhibitory activity test were carried out,and the designed compounds were evaluated biologically at the cell level,enzyme level and molecular level,respectively.The anti-EV71 activity test showed that only eight compounds?HTL-2-34,HTL-2-35,HTL-2-38,HTL-3-26,HTL-4-32,HTL-5-21,HTL-5-23,HTL-6-30?of 3-butenone quinoline derivatives and oxazoloquinoline derivatives showed moderate inhibitory activity against EV71,while most of the Torin2 derivative compounds showed strong EV71 inhibitory activity,twelve compounds?HTL-6-45,HTL-6-48,HTL-6-49,HTL-7-01,HTL-7-03,HTL-70-6,HTL-7-11,HTL-7-15,HTL-7-16,HTL-7-19,HTL-7-28,HTL-7-29?of which were close to Torin2.A total of 33 compounds with better activity were selected from the three classes of target compounds for mTOR kinase inhibitory activity assay and mTORC1/mTORC2 inhibitory activity assay.The mTOR kinase inhibitory activity results were consistent with the in vitro anti-EV71 activity test results.Torin2derivative compounds showed stronger mTOR kinase inhibitory activity than the other two classes of compounds,indicating that the anti-EV71 effect of the compound is achieved by inhibiting mTOR kinase.The results of mTORC1/mTORC2 inhibitory activity showed that among the selected 3-butenone quinolates and oxazoloquinoline compounds,only HTL-5-21 has dual inhibitory effect on mTORC1 and mTORC2,and most of Torin2 derivative compounds can inhibit both mTORC1 and mTORC2,which indirectly explains the reason why these compounds are more active than the other two classes of compounds in vitro.In order to investigate the druggability of the designed compounds,the in vivo drug metabolism tests were carried out on the five preferred compounds with better activity.The results showed that,in addition to the positive drugs,The oral bioavailability of compound HTL-6-28,HTL-7-03 and HTL-7-17 is greater than 20%,which has certain drug potential.In addition,water solubility is an important physical and chemical property affecting druggability.According to the literature,although Torin2 has good activity and drug metabolism properties,its poor water solubility?1.29?g/ml?has become a limiting factor restricting its further drug formation.Therefore,this study also tested the water solubility of the five compounds with higher activity.The results showed that the water solubility of HTL-6-48 and HTL-7-17 compounds was significantly improved compared with the positive control compounds.Compared with Torin2,it has increased by 4 times and 14 times respectively,which laid a good foundation for further medicine.