Effects Of A New Series Of Pyridazinone Compounds On Myocardial Mechanical Properties

Objective: To investigate the influences of NO.1, NO.2, NO.3, NO.4 and NO.5 compounds on the functional changes and mechanism invoided for preserving heart in vitro.Methods:1 Synthesis of the new pyridazinone compounds (NO.1, NO.2, NO.3, NO.4 and NO.5 compounds) A new series of 4,5-Dihydro-6-[(1-R)-1,2,5,6-tetrahydropyrid-4-yl]pyridazin-3(2H)-one were synthesized by simple, high yielding routes. The key step was the combination of pyridazinone which is as a mother nucleus. In this experiment, firstly, we synthesized 4-O-4(4-pyridine)butadiene-acrylonitrile, and then we synthesized 4,5-Dihydro-6-(4-pyridinyl)-3(2H)-pyridazinone (NO.1 compound).1.1 NO.1 compound was used as a nucleus, reacted with some compounds which contain halogen in the reflux. Two quaternary amine salts (NO.2 compound) was obtained, its yield can reach more than 70%.1.2 NO.2 compound was reduced through sodium borohydride into a compound containing the tetrahydropyridine (NO.3 and NO.5 compounds). Finally, NO.3 and NO.5 compounds were hydrolyzed in order to improve their water-soluble. We examined two methods as the acid hydrolysis and alkaline hydrolysis and founded that the reaction rate was so fast that it was not easy to control to make the product suffer a greater loss when using alkaline, hence, we chose acid to hydrolyze NO.3 and NO.5 compounds. In order to increase the yield, the action was carried out under a separator. The target compounds were organic acids, their water solubility were more suitable for oral administration.1.3 The general procedure of 1-ethoxyethanoyl-4-(4,5-dihydro pyridazin-3(2H)-one-6-yl) pyridinium bromide and 1-ethoxypropionyl-4-(4,5-dihydropyridazin-3(2H)-one-6-yl) pyridinium bromide (NO.2 compound): NO.1 compound (0.7g, 4mmol) and ethyl bromoacetate (1.5g, 7mmol) or Ethylβ-bromopropionate (1.4g, 7mmol) were dissolved in absolute ethanol (25 ml), refluxed for 12 hours and then stirred at room temperature overnight. The solvent was removed under reduced pressure. The resulting precipitate was collected by filtration after adding 50ml acetone to obtain the titled compound as white columns.1.4 The general procedure of 4,5-Dihydro-6-[(1-ethoxy ethanoyl)-1,2,5,6-tetrahydropyrid-4-yl]pyridazin-3(2H)-one And 4,5-Dihydro-6-[(1-ethoxypropionyl)-1,2,5,6-tetrahydropyrid-4-yl]pyridazin-3(2H)-one (NO.3 and NO.5 compounds): NO.2 compound (0.96g, 2.8mmol) was dissolved in methanol (15 ml), gradually added with sodium borohydride (1.1g, 30 mmol) under ice cooling and then stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue obtained was added with dichloromethane, washed with water and dried over anhydrous magnesium sulfate. The solvent was removed and then the formed crude solid was filtered, washed thoroughly with anhydrous ethanol to obtain the titled compound as white crystals.1.5 The general procedure of 1-[4-(4,5-Dihydro3(2H)pyri dazinone-6-yl)-1,2,5,6-tetrahydropyridyl] acetic acid hydrochloride (NO.4 compound): NO.3 compound (330mg, 1.25mmol) was dissolved in 1 M HCl (20ml) and refluxed for 5 h under a separator. The solution was decolored by using active carbon (0.5 g), then filtered and evaporated under reduced pressure. The solid residue was finally recrystallized from EtOH.2 Preparation of the isolated atrium and papillary muscles and administration of drug2.1 Preparation of isolated atrium of rats and guinea-pigs Healthy rats and guinea-pigs were anaesthetized in 25% urethane. Then we removed the heart by thoracotomy rapidly and immediately, placed the heart in pre-cooling oxygen-saturated by 95%O2+5%CO2 Krebs solution, cut ventricle along the atrioventricular groove, intercepted left and right atrium carefully (including sinus), paid attention not to damage sinus node, then put the isolated left and right atrium of rats or guinea-pigs into the continuously oxygenated wheat's bottom, gave a pre-load and adjusted until the muscle tension of the specimen achieved maximum value, then balanced 45 minutes, and then the experiment was began.2.2 Preparation of isolated papillary muscles of rabbitsHealthy rabbits were anaesthetized in 25% urethane, then we removed the heart by thoracotomy rapidly and immediately placed it in pre-cooling oxygen-saturated by 95%O2+5%CO2 Krebs solution, cut ventricle along the atrioventricular groove, and then selected a complete papillary muscle rapidly, then put it into the bottom of Mak's slot which contained 20 ml continuously oxygenated Krebs solution. The specimen end was connected with the tension transducer. Papillary muscle was driven by electrical stimulus. Gave it a pre-load and adjusted it until the muscle tension of the specimen achieved maximum value, balanced 45 minutes, then the experiments were began.3 Measurement of influences of new pyridazinone compounds on myocardial mechanical properties.3.1 Measurement of inotropic effects of new pyridazinone compounds After giving drugs (10-7-3x10-4 M), the MS4000U-1C biological signal collecting system was used to record and analyzed the changes of spontaneous beating and contractility.3.2 Influences of different blocking agents on positive inotropic effects of NO.3We usedα-recepter blocking agent,β-recepter blocking agent and blocking agent of Ca2+ channel to investigate the mechanisms for the positive inotropic effects of NO.3.3.3 Measurement of chronotropic effects of new pyridazinone compounds NO.1, NO.2, NO.3 and NO.5 compounds were given to study their myocardial chronotropic effects.3.4 Measurement of lusitropic effects of new pyridazinone compounds To study the relaxation phase, the following parameters were measured: time to peak tension (TTP), time to half relaxation (T50%), relaxation time (Rt), total twitch duration (Tt).Results:1 Inotropic effects of NO.1, NO.2, NO.3, NO.4 and NO.5 compounds on contractile force of isolated muscle strips1.1 Inotropic effects of NO.1, NO.2, NO.3 and NO.5 compounds on contractile force of isolated atrials in rats and guinea pigs NO.1 and NO.2 compounds increased no significantly on isometric tension of isolated atrials in rats. NO.3 and NO.5 compounds at 3×10-4 mol/L increased average tension significantly. ISO (10-10-10-5 mol/L) concentration-dependently increased the contractile force of isolated atrium in rats. NO.1 and NO.2 compounds increased no significantly on isometric tension of isolated atrials in guinea pigs. NO.3 and NO.5 compounds at 3×10-4 mol/L increased average tension significantly. ISO (10-10-10-5 mol/L) concentration-dependently increased the contractile force of isolated atrium in guinea pigs.1.2 Influence of different blocking agents on the inotropic effects of NO.3 compoundIt proved that different blocking agents had no Influence on the inotropic effects of NO.3 compound.1.3 Inotropic effects of NO.1, NO.2, NO.3, NO.4 and NO.5 compounds on contractile force of isolated papillary muscles in rabbits. NO.1 and NO.2 compounds increased no significantly on isometric tension of isolated papillary muscles in rabbits. NO.3, NO.4 and NO.5 compounds at 3×10-4 mol/L increased average tension significantly. ISO (10-10-10-5 mol/L) concentration-dependently increased the contractile force of isolated atrium in rabbits.2 Chronotropic effects of NO.1, NO.2, NO.3, NO.4 and NO.5 compounds on frequency of isolated atrials in rats and guinea pigs. NO.1, NO.2, NO.3, NO.4 and NO.5 compounds at 3x10-4 mol/L had no significant effects on the heart rate of muscle strips of isolated atrials in rats and guinea pigs. ISO dose-dependently increased the rate of isolated atrials in rats and guinea pigs.3 Lusitropic effects of NO.3, NO.4 and NO.5 compounds for isolated muscle stripsNO.3 and NO.5 compounds had no significant effects on TPT, Tt, Rt, T50% of isolated left atrials in rats and guinea pigs. NO.3, NO.4 and NO.5 compounds had no significant effects on TPT, Tt, Rt, T50% of isolated papillary muscles in rabbits.Conclusion: NO.1 and NO.2 compounds increased no significantly on isometric tension of isolated atrials in rats. NO.3 ,NO.4 and NO.5 compounds at 3×10-4 mol/L increased average tension of isolated atrials and papillary muscles significantly. NO.1, NO.2, NO.3, NO.4 and NO.5 compounds at 3x10-4 mol/L had no significant effects on the heart rate of isolated atrials in rats and guinea pigs. NO.3 and NO.5 compounds had no significant effects on TPT, Tt, Rt, T50% of isolated left atrials in rats and guinea pigs.