Design,Synthesis And Activity Screening Of Acid Sensing Ion Channel 1a Inhibitor

Acid sensing ion channels（ASICs）,a channel belonging to the degenerin/epithelial sodium channel（DEG/ENa C）superfamily,can be activated by proton under acid condition.To date,at least seven different ASIC subunit proteins（ASIC1a,ASIC1 b,ASIC1b2,ASIC2 a,ASIC2b,ASIC3,ASIC4）have been cloned and identified in mammalian.Because local tissue acidosis can activate the channels and then result in diseases,ASICs are likely to be key molecules in diseases related to tissue acidosis.Accumulating research suggests that ASIC1 a plays a crucial role in tissue acidification diseases such as inflammation,cancer,epilepsy and ischemic stroke.Blocking ASIC1 a has a certain degree of protection,whereas there is short of potent and selective small molecule inhibitors of ASIC1 a.Therefore,this study was to synthesize some compounds of potential ASIC1 a inhibitor firstly,subsequently to explore the effect of these compounds on ASIC1 a expression and function in rat articular chondrocytes.Objective:To synthesize ASIC1 a inhibitor（6-Styryl-naphthalene-2-amidrazone derivatives）and investigate the effect of these compounds on ASIC1 a expression and function in rat articular chondrocytes in vitro.Methods:1.6-Styryl-naphthalene-2-amidrazone derivatives were synthesized via appropriate functional group transformation and Heck reaction using 6-bromo-2-naphthoic acid as the starting material,and the structure was confirmed by hydrogen nuclear magnetic resonance（¹H NMR）and high resolution mass spectrometry（HR-MS）.2.Primary rat articular chondrocytes were separated and extracted by type II collagenase digestion method.The articular chondrocytes were divided into normal group,solvent group,five concentration compound group and positive drug Amiloride group,to observe the effect of compounds on rat articular chondrocytes viability.The chondrocytes were divided into five groups,which were incubated in different pH extracelluar solution（pH 7.4,pH 7.0,pH 6.5,pH 6.0,pH 5.5）for three hours and the chondrocytes were stimulated by extracelluar pH 6.0 solution for 0min,30min,1h,3h,6h,12h,to investigate the effect of two conditions on ASIC1 a protein expression in chondrocytes.Then selected optimum condition was used to observe the effect of compounds on ASIC1 a expression in chondrocytes.At the same time,the laser scanning confocal microscope was used to investigate the effect of compounds on [Ca²⁺]i level in chondrocytes under extracelluar pH 6.0 solution stimulation.Results:1.Eight 6-Styryl-naphthalene-2-amidrazone derivatives were synthesized as followings:5a:6-Styryl-naphthalene-2-amidrazone;5b:6-[2-（4-Fluoro-phenyl）-vinyl]-naphthalene-2-amidrazone;5c:6-[2-（4-Chloro-phenyl）-vinyl]-naphthalene-2-amidrazone;5d:6-[2-（2-Fluoro-phenyl）-vinyl]-naphthalene-2-amidrazone;5e:6-[2-（4-Methoxy-phenyl）-vinyl]-naphthalene-2-amidrazone;5f:6-[2-（3-Fluoro-phenyl）-vinyl]-naphthalene-2-amidrazone;5g:6-[2-（3-Chloro-phenyl）-vinyl]-naphthalene-2-amidrazone;5h:6-（2-p-Tolyl-vinyl）-naphthalene-2-amidrazone,whose structure were confirmed by1 H NMR and HR-MS.2.MTT assay result showed that apart from compound 5h,the rest compounds had no significant difference on chondrocytes viability compared to normal group.The western blot result of this study showed that 1)pH 6.0 group and pH 5.5 group ASIC1 a protein expression were remarkably increased compared to normal group in chondrocytes,with on significant difference between pH 6.0 and pH 5.5 group;2)the expression of ASIC1 a protein gradually increased within 3h,but gradually decreased after 3h when pH 6.0solution stimulated.Therefore,exposure to pH 6.0 medium for 3h was selected for subsequent experiments;3)all compounds 5a⁵h except compound 5b had significantly decreased the expression of ASIC1 a protein compared to pH 6.0+DMSO group in chondrocytes.The laser scanning confocal microscope result indicated that compounds5a（25 mM）、5c（50 mM）、5d（25 mM）、5e（50 mM）、5g（25 mM）and 5h（50 mM）could decrease the level of [Ca²⁺]i under extracelluar pH 6.0 solution in chondrocytes and the inhibition degree of them were 33.37%、20.83%、52.07%、81.40%、59.51%、63.23%,respectively,which might be superior to Amiloride（100 m M,inhibition degreee: 23.49%）.Among these compounds,compound 5e or 5g was the most potent.Conclusion:Eight 6-Styryl-naphthalene-2-amidrazone derivatives were synthesized,and most of them have potential inhibition effect on ASIC1 a protein expression and function.Among them,compound 5e or 5g was the most potent（inhibition degreee: 81.40%（5e,50 mM）and 59.51%（5g,25 mM）,respectively）.Together with the important role of ASIC1 a in the pathogenesis of tissue acidification diseases including rheumatoid arthritis,these results might provide a potential therapeutic choice in developing drugs targeting at tissue acidification diseases.