Study On Design,synthesis And Activities Of Disulfide Compounds And Design,synthesis Of Cathepsin B Inhibitors

Ischemic stroke is a kind of disease of brain tissue injure,which is caused by the occlusion of the middle cerebral artery or its branches.Stroke has the characteristics of high incidence,high mortality,high disability rate and high recurrence rate,which has become one of the main causes of human death and disability.The pathological mechanism of ischemic stroke is very complicated and is the cascade process of multi-factors,multi-mechanism and multi-link,including excessive release of the excitatory amino acid,the formation and release of free radicals,excessive inflammatory response ofthe brain,neuronal cell necrosis and apoptosis.At present,the clinical treatment drugs include thrombolysis,vasodilator,anti platelet aggregation and neuroprotective agents.Al though these drugs are targeted,there are a series of problems,such as single curative efficacy,poor safety,short treatment time window,ambigious efficacy in clinic and so on.Therefore,it is becoming more and more important to find drugs for ischemic stroke with various pathological mechanisms and new targets.Hydrogen sulfide is the third gaseous signal molecule after carbon monoxide and nitric oxide.It plays an important role in the regulation and protection of the central nervous system.However,high concentration of hydrogen sulfide can produce neurotoxicity.Therefore,slow releaseing hydrogen sulfide donors have attracted people’s attention.Allicin is one of the most active components in garlic.It has effects antiinflammatory,antimicrobial,antifungal,antiparasitic,antihypertensive,anticancer and anti cerebral ischemia effects.Therefore,the release of hydrogen sulfide donor attracts people’s attention.It is reported that diallyl diallyl sulfide（DADS）produced by degradation and rearrangement of allicin is a kind of slow release hydrogen sulfide donor.In vivo experiment,acyl disulfide ether can rapidly release the donor of hydrogen sulfide,and there are no reports about this kind of compound in ischemic stroke.On the basis of the above research,DADS analogues LYY-I_1-15,as well as acyl disulfide LYY-II_1-11 has been designed and synthesized.LYY-I₁ is a hybrid of ligustrazine,an anti ischemic drug.And We have also investigated whether these compounds exert neuroprotective effects on ischemic stroke.A total of 25 compounds were synthesized,among which compound LYY-I_1,3,LYY-II_2,4,5,6,7,10 are novel compounds.The structures of all the target compounds were confirmed by 1H-NMR,13C-NMR and HR-MS.LYY-I_1-14 was synthesized by the reaction of substituted benzyl bromide with thiourea in alkaline condition and manganese dioxide as catalyst.Compound LYY-I₁₅ is prepared by neutralization of pyritinol hydrochloride sailt with alkali.Compound LYY-II_1-11 were obtained by reaction various acyl chloride with sulfur in the presence of PEG-400 as phase transfer catalyst,sodium hydroxide as base.The neuroprotective effects of LYY-I and LYY-II compounds were evaluated in HT-22 hippocampal neurons induced by glutamate with MTT method.In the establishment of glutamate injury model,the best glutamate concentration was found to be 5mM,and the cell survival rate was 25.50%at 5 mM glutamate.The pharmacological results showed that all the fifteen LYY-I compounds possessed neuroprotection from glutamate induced injury in the HT-22 cells.Compound LYY-I₁ at the concentration of 10-100μM,compared with the glutamate group,significantly increased the cell survival rates（91.77%⁹5.00%）,showing very strong neuroprotective effect（P<0.01）.Compounds LYY-I_2,7,8,14,15 at the concentration of 10-100μM also had potent neuroprotective effects.The cell survival rates were between 55.46%⁸3.84%（P<0.01）.The cell survival rates treated by LYY-I_{3-6,9,11,12,13} at the concentration of 50-100μM were60.55%⁹9.97%（P<0.01）.The most potent LYY-I₁ was compared with that of Ligustrazine,and the results displayed that Ligustrazine had no neuroprotective effect in this model and experimental concentrations.LYY-I₁ at 1¹00μM had no effect on the growth of HT-22 cells untreated with glutamate.The neuroprotective effect of LYY-I₁ on damaged HT-22 cells induced by H₂O₂ with MTT method was further assayed.The results showed that LYY-I₁ at 50-100μM could improve the survival rate of damaged HT-22 cells,which exhibited good antioxidant activity（P<0.01）,but ligustrazine had no antioxidation at these concentrations.In the ADP induced platelet aggregation model with rats,LYY-I₁ possessed antiplatelet aggregation effect at the concentration of 0.5,0.1,1mM（P<0.05 or P<0.01）in rat blood.However,ligustrazine had no antiplatelet aggregation activity at this concentration.In the mice permanent middle cerebral artery occlusion（MCAO）model,intraperitoneal injection of0.22mmol/kg LYY-I₁ after 3h of reperfusion,ischemic infarct volumes of cortex（P<0.01）,striatum（P<0.05）and hemisphereat（P<0.05）was significantly reduced.The results indicated that compound LYY-I₁ has good effect on against cerebral ischemia.The neuroprotective effects of LYY-II compounds were evaluates on glutamate induced injury in HT-22 cells by MTT method.The results showed that LYY-II₆significantly increased the survival rates of HT-22 cells（83.19%⁸4.75%,P<0.01）at10¹00 M,which had a good neuroprotective effect.LYY-II_1-5,8-11 at 50¹00μM has neuroprotective effect（P<0.01）,and their cell survival rates were among 50.40%⁷8.59%.The survival rates of cells treated by LYY-II₇ at the all experimental concentrations,were among 29.12%³0.17%,which indicated that LYY-II₇ had not neuroprotective effect.In summary,most of LYY-I₁ and LYY-II have neuroprotective effects on HT-22 cells injured by glutamate.Among them,LYY-I₁ is the most potent compound with anti-glutamate activity and has no toxicity on HT-22 cells untreated by glutamate.LYY-I₁had neuroprotective effect on hydrogen peroxide induced HT-22 cells.LYY-I₁ inhibited platelet aggregation induced by ADP in rats.In mice MCAO model,LYY-I₁ protected cerebral ischemia in.However,ligustrazine had no activity in vitro.These results suggest that LYY-I₁ may be a lead compound for potential ischemic stroke,and the structure of tetramethylpyrazine may increase the neuroprotective effect,but the main role is produced by two disulfide bonds.Cathepsin B is a protease produced in the lysosome,which is closely related to cerebral ischemia and can regulate the apoptosis of nerve cells.Therefore,Cathepsin B provides a new target for the treatment of ischemic stroke.According to the structural characteristics of cathepsin B,LYY-III compounds were designed and synthesized.A total of 15 compounds were novel structures.Allcompounds were confirmed by ¹H-NMR,¹³C-NMR and HR-MS.The target compoundsLYY-III₄ and LYY-III₁₇ were docked with the stereostructure of cathepsin B by the computer-aided simulations technology.Various amino acids（L-,L-or L-isoleucine phenylalanine glycine）as the starting materials,respectively reacted with SOCl₂ and methanol to obtain amino acid methyl ester hydrochloride,then reacted with biphenyl 4-formyl chloride,and finally reacted with hydrazine hydrate to generate intermediate acyl hydrazide.Acyl hydrazine reacted with acyl chlorideto generated compounds LYY-III_1-16.Acyl hydrazine reacted with substituted benzoyl isothiocyanate methyl ester（reactive product of substituted benzoyl chloride and potassium thiocyanate）to get compounds LYY-III_17-20.A total of 20 novel compounds were synthesized,and their structures were confirmed by ¹H-NMR,¹³C-NMR and HR-MS.The LYY-III₄ and LYY-III₁₇ were docked with Cathepsin B by computer aided simulation.The result showed that the molecular docking fraction of LYY-III₄ was-4.56 kcal/mol and the molecular docking fraction of LYY-III₁₇was-5.88 kcal/mol,which indicated that compounds LYY-III₄ and LYY-III₁₇ have the effect of inhibiting Cathepsin B.