Synthesis Of Novel Sulfonamide-1,3,5-Triazine Derivatives And Their Protective Effects On Heart Function

ObjectsSynthesis of the novel MMP inhibitors based on sulfonamides and 1,3,5-triazine,and investigates the cardioprotective effects of the novel sulfonamide-1,3,5-triazine.BackgroundMyocardial infarction(MI)and ischemia-reperfusion injury(I/R)are the most common cardiovascular diseases,which are serious threat to human life and health.Matrix metalloproteinase(MMP)is involved in the degradation of various ECM proteins and plays an important role in regulating cardiac structure and function.A large number of studies have shown that cardiac damage can lead to a significant increase in the expression of MMP.Inhibition of MMP activity can effectively reduce the damage of cardiac structure and function induced by MI and I/R.MMP can be used as a biomarker and therapeutic target for early heart injury.A large number of studies have reported the protective effects of matrix metalloproteinase inhibitors(MMPIs)on the heart.As a hydroxamate MMP inhibitor,sulfonamides can effectively inhibit the activity of various MMPs and reduce the expression of MMP induced by heart injury,which has been shown to have a significant cardioprotective effect.In addition,1,3,5-triazine has been shown to be effective in inhibiting theexpression of MMP-9,and has great potential for use in a variety of biological enzymes and receptor inhibitors.Prompted by the excellent pharmacological activity of above scaffolds,we intended to club these moieties together in search of more potent analogues of MMP-2 and MMP-9.Therefore,the present study was intended to develop novel heterocyclic conjugates of sulphonamide-1,3,5-triazine as MMP-2 and MMP-9 inhibitor.Methods1.The sulfonamide and 1,3,5-triazine are synthesized by ring hybrid chemical bonding to synthesize a novel sulfonamide-1,3,5-triazine compound,and different kinds of aromatic agents and fatty amine functional groups are used for nucleophilic substitution.Substituting the 1,3,5-triazine R group to synthesize the target compound 8(a-j).Fourier transform infrared spectroscopy(FT-IR),hydrogen spectroscopy(1HNMR),carbon spectroscopy(C NMR),mass spectrometry(MS),elemental analysis and other methods were used to analyze the physical chemistry properties of the target compounds.The MMP-2 and MMP-9 inhibitory activities of the compounds were determined by fluorescence method using fluorescent peptides as a substrate.2.Compounds 8e and 8f were selected as the evaluation objects.The toxicity of the compounds on primary cultured myocardium and fibroblasts was detected by CCK-8 method.The antioxidant activity of the compounds was detected by anti-oxidation assay kit.The effects of compounds on the expression of MMP-2 and MMP-9 induced by PMA in primary cultured cells were analyzed by Western Blot.The solated cardiac perfusion and I/R injury model was established.Furthermore,the heart rate-pressure product(RPP)and coronary artery flow rate of I/R injury solated cardiac were evaluated.The level of Cardiac troponin I(cTnI)in coronary effluent was detected by ELISA.The degree of collagen degradation was analyzed by fluorescently labeled o-furfural.The levels of MMP-2 and MMP-9 in the effluent were determined by Quantikine immunoassay kit.3.Compound 8e was selected as the evaluation object.Rat acute MI model was established by subcutaneous injection of isoproterenol(ISO)at 100 mg/kg.And serum cTnI,Mb,CK,CK-MB,LDH,ADT,ALT and ALP levels in MI rats were analyzed.The GSH,GR,GPx and SOD expressions in tissue homogenate were also determined.The myocardial tissue damage was evaluated by H&E staining.Results1.A novel sulfonamide-1,3,5-triazine derivative compound 8(a-j)was successfully synthesized,and the target products had a high synthesis yield(59-91%).By changing the R in 1,3,5-triazine,the substituent can effectively modulate the physicochemical properties and MMP inhibitory activity of the compounds.The solubility of the compounds 8c,8e and 8f was higher than others,and the pH were in the physiological range.The compounds 8a and 8b with the allylamine substituents show lower MMP inhibitory activity(8a:MMP-2 vs MMP-9,IC50=665.56±0.73 vs 625.34±0.21 nM),and introduction of aromatic group(8d)could improve MMPase inhibitory activity to some extent(IC50:123.40±0.36 vs 105.31±0.57 nM).8e had the strongest MMP inhibitory activity(8.12±0.32 nM vs 2.34±0.56 nM).8e interfered with the reciprocal curves of MMP-2 and MMP-9,and the Km and Vmax values were gradually decreases as the concentration of the inhibitor increases.2.A certain concentration of novel sulfonamide-1,3,5-triazine derivatives 8e and 8f had no obvious toxicity to primary cultured myocardium and fibroblasts.8-10 nM of 8e significantly inhibited PMA-induced MMP-9 expression(reducing about 8.5-10.2 times),while the inhibition activity of 8f was lower(32%-57%).8e and 8f had no significant antioxidant activity.The RPP of 30 min after the heart reperfusion in vitro was significant higher in 1-10 nM of 8e treatment(increased about 10-22%)compared with the control group(P<0.05),while the reperfusion of 8f did not improve the ischemic cardiac function index(6-10%).10 nM 8e reperfusion significant increased coronary flow velocity(8e vs Control=9.7 vs 6.8 mL/min,p=0.027),reduced myocardial edema(approximately 9%,p=0.015),decreased cTnl release from ischemic heart(approximately 24%reduction,p=0.022),inhibited collagen degradation(AFU decreased by 42%,p<0.001),and significantly reduced the level of MMP-9 in coronary effluent(0.314 ± 0.18 vs 1.233 ± 0.31 ng/mL,p =0.007),and the protective effect of 8f on the isolated perused heart was significantly weaker than 8e.3.The acute MI model of rat heart was successfully established by subcutaneous injection of ISO(100 mg/kg/day,for 2days).5-10 mg/kg of 8e could effectively alleviate the increase of serum indexes,such as cTnI(22-26%),Mb(9-22%),CK(9-11%)and CK-MB(15-25%)induced by ISO subcutaneous injection.And promoted the LDH,ADT,ALT and ALP decrease to normal levels.8e treatment could also effective relief the ISO-induced increase of MDA levels in myocardial(approximately 29%-48%reduction).The expressions of heart tissue antioxidant enzymes GSH(1.32-1.44 times),GR,GPx and SOD were also elevated,H&E staining showed that 8e could effective alleviate ISO-induced cardiac tissue damage.Conclusion1.The novel sulfonamide-1,3,5-triazine derived compounds can be synthesized simply and efficiently by using benzene ring hybridization.The change of R substituent in 1,3,5-triazine can modulate the MMP inhibitory activity of the compound.Compound 8e has the highest inhibitory activity of MMP-9 and MMP-2.2.8e has high biosafety and can effectively alleviate I/R-induced cardiac structure and function damage.A certain concentration of 8e can significantly alleviate ISO-induced acute myocardial injury in rats,which may be related to its MMP-9 inhibitory activity and the activation of downstream antioxidant signals.