Studies On Synthesis,Biological Activity And Pharmacokinetics Of Novel Pyxinol Derivatives

In order to improve the physicochemical properties of the lead compound pyxinol and enhance its pharmacological activity,based on the review of the research progress of pyxinol and its derivatives and the anti-myocardial ischemia drugs,combined the drug synthesis,biological activity screening and pharmacokinetic evaluation,we effectively prepared a series of new pyxinol fatty acid ester derivatives,deeply screened the anti-myocardial ischemia and anti-heart failure activities,early evaluated the pharmacokinetic parameters of the active derivative,and successfully obtained an innovative drug candidate with good cardioprotective activity.The main innovative achievements are as follows:1.Synthesis of new pyxinol fatty acid ester derivativesIn order to avoid the synthesized derivatives being too fat-soluble,saturated fatty acids(2⁸ C)were used for esterification with C3/C12/C25-OH of pyxinol,and a series of derivatives were designed and synthesized.The structures of 32 derivatives(including 7 monoesterification products at C-3 position,7 monoesterification products at C-12 position,5 double esterification products at C-3 and C-12 positions,6 double esterification products at C-12 and C-25 positions,7 triesterification products at C-3,C-12 and C-25 positions)were identified by physical and chemical properties analysis,NMR and HR-MS.Except derivatives 1 and 7,the other 30 derivatives are new compounds synthesized for the first time.2.Cardioprotective effects and mechanism of pyxinol fatty acid ester derivativesBased on literature reports that the lead compound pyxinol had a good cardioprotective effect,the paper continued to evaluate the cardioprotective effects(including anti-myocardial ischemia and anti-heart failure effect)of the synthesized pyxinol fatty acid ester derivatives and to explore the mechanism of action.2.1 Effects on H9c2 cardiomyocyte injury induced by H₂O₂Rat cardiomyoblast cell line H9c2 cells were stimulated by H₂O₂ to establish cardiomyocyte injury model in vitro.The cell viability of H9c2 cells of 32 fatty acid ester derivatives was determined by the CCK-8 method.The results indicated that pyxinol and its derivatives showed various degrees of protective effect against the injured H9c2 cells.The order of the activity was C-3 monoesterification products > C-3,12,25 triesterification products > C-3,12 double esterification products > pyxinol > C-12 monoesterification products > C-12,25 double esterification products.It was divulged that the ester bond at the C-3 position contributing the most effect to the activity was the main active group.Among all derivatives,compound 5(3-hexanoyl-pyxinol)exhibited the strongest dose-dependent protective effect against myocardial injury induced by H₂O₂ in H9c2 cells.Compound 5 might play a protective role in cardiomyocytes by increasing the activity of SOD in cells,reducing the production of MDA,inhibiting lipid peroxidation and reducing the degree of cell injury.2.2 Intervention effect of compound 5 on myocardial ischemia model ratThe acute myocardial ischemia model in rat was established by using left anterior descending coronary artery ligation method.The rats were intragastrically administrated with compound 5(5,10,20 mg/kg).The cardiac contractility,myocardial infarction area,LDH,AST,CK,c Tn T,MDA,and SOD were used as evaluation indicators to investigate the cardioprotective effect of compound 5.The results showed that compound 5 could dose-dependently reduce the left ventricular volume(LVVs)and myocardial infarction area,increase the ejection fraction(EF)and cardiac contractility,reduce the levels of LDH,CK,AST,c Tn T,and MDA,and increase the content of SOD compared with model group.Compound 5 had a similar effect with metoprolol,the positive control drug.It was indicated that compound 5 had a good protective effect on the heart of myocardial ischemia rats.2.3 Inhibition effects of pyxinol fatty acid ester derivatives on ACEACE plays an important role in heart failure and is the main cause of congestive heart failure.With lisinopril being as positive control drug,the ACE inhibition effect of 32 fatty acid ester derivatives was evaluated by using colorimetric method in vitro.Among the derivatives,both compound 5 and compound 9(3,12,25-tripropionyl-pyxinol)both displayed similar activities to lisinopril(89.17%)with the inhibition rates of 90.31% and 87.02%,respectively.And the effects were better than pyxinol(57.23%).The IC50 values of compound 5,compound 9 and lisinopril were 105 n M,114 n M and 81 n M,respectively.It had been proved that compound 5 and compound 9 exhibited good ACE inhibitory activity in vitro.Moreover,the molecular docking results showed that the inhibitory selectivity of compounds 5 and 9 was the C-domain of ACE enzyme.2.4 Intervention effect of compound 5 and compound 9 on zebrafish heart failure modelZebrafish is one of the ideal model animals for studying heart failure.Zebrafish at 48 hours after fertilization were selected,and then pretreated with compounds 5 and 9(0.5,1,and 10 ?g/m L,respectively)for 4 h.Then,the zebrafish were treated with verapamil to develop the heart failure model.The heart beats,cardiac output,ejection fraction,fractional shortening,enlarged heart,and venous congestion were used as the evaluation indicators to evaluate the cardiac functions.The results showed that compounds 5 and 9 at 1 ?g/m L could both significantly reduce heart dilatation and venous congestion,and increase cardiac output as well as heart rate.The bioactivity of compound 5 was stronger than that of compound 9,and showed similar activity to enalapril.2.5 Metabolomics study of compound 5 against heart failureUPLC-Q/TOF-MS-based metabolomics technology was used to analyze the heart failure zebrafish pretreated with compound 5.The results showed that the contents of many endogenous metabolites in heart failure zebrafish had changed significantly compared with normal zebrafish.After pretreated with compound 5,the levels of 25 endogenous metabolites,such as choline,pyruvate,and arachidonic acid,had been significantly re-regulated.It was then inferred that compound 5 exerted anti-heart failure activity by intervening 8 metabolic pathways such as arachidonic acid metabolism,phenylalanine metabolism,linoleic acid metabolism,pyruvate metabolism,folate biosynthesis,sphingolipid metabolism,glycerophospholipid metabolism,purine metabolism.Among them,the pathway of folate biosynthesis was found to be related to heart failure for the first time.3.Study on the pharmacokinetics of orally administered compound 5 in rats 3.1 Study on plasma concentration-time profileThe HPLC-ELSD quantitative method for the determination of compound 5 in plasma was established for the first time,and the plasma concentration-time profiles of compound 5 in rat after intragastric administration(20.0,40.0,80.0 mg/kg)were determined.Then,the pharmacokinetic parameters were obtained.The results showed that the kinetic process of compound 5 in vivo was a linear process,and there was no gender difference in the pharmacokinetic behavior in rats.Compared with the parameters of pyxinol reported in the literature,the elimination time of compound 5 was significantly prolonged,and compound 5 had a longer circulation time in rats.Therefore,it was deduced that compound 5 would have sufficient pharmacological action time in vivo.The plasma concentration of the main metabolite(pyxinol)was also quantitatively analyzed for the first time.The results showed that the prototype drug(compound 5)was the main component before Tmax.While both prototype drug and its metabolite(pyxinol)were detected during the elimination period(Tmax 20 h).During the period of 20 h⁴0 h,pyxinol was mainly detected in plasma.3.2 Study on bioavailability of orally administered compound 5The plasma concentration-time profile of compound 5 in rat after intravenous injection(10.0 mg/kg)was determined for the first time,and some basic parameters such as T1/2?AUC?CL and Vd were obtained.The absolute bioavailability of compound 5 calculated by AUC(0-t)was 41.36%,which was similar to the absolute bioavailability(43%)of the lead compound pyxinol reported in the literature.3.3 Determination of the oil/water partition coefficient of compound 5The lipophilicity of the compound has an important impact on the entire pharmacokinetic process,especially the absorption of oral drugs in the body.The paper simulated different parts of the gastrointestinal tract,and used the shake flask method to make compound 5 reach equilibrium in the water-n-octanol buffer solution under the conditions of p H 2.0,p H 5.8,and p H 7.4.The concentration of compound 5 in two phases was determined by using HPLC-ELSD technology,respectively.The oil/water partition coefficient of compound 5 was acquired for the first time.The Log P of compound 5 is 4.18,which is less than the Log P value of the lead compound pyxinol(3.76),indicating that the introduction of fatty acyl groups in the structure could increase the fat solubility.3.4 Study on biotransformation of orally administered compound 5UPLC-Q/TOF-MS technology combined with the UNIFI platform of metabolite analysis was used to quickly analyze and identify the main metabolites of orally administered compound 5(80.0 mg/kg)in bile,urine and feces for the first time.21 metabolites were identified,including 6 phase I metabolites and 15 phase II metabolites.Phase I metabolic reactions included dehydration,dehydrogenation,hydration,oxidation,dehexanoyl,dehexanoyloxy,and phase II metabolic reactions included methylation,acetylation,phosphorylation,sulfation,cysteine conjugation,glycine conjugation,glutathione conjugation and glucuronidation.In summary,this paper enriched the structural modification of pyxinol,and also screened an innovative candidate drug(compound 5,3-hexanoyl-pyxinol)with better activity and higher bioavailability.Compound 5 exhibited good anti-myocardial ischemia and anti-heart failure effects,slow elimination,and high bioavailability.The study could provide the theoretical basis and scientific data for further research and development.