| Objective:It was aimed to synthesize the tetrahydropyrimidine derivative LH-2703with diethyl but-2-ynedioate, phenylamine and formaldehyde as starting materials and to explore its anti-inflammatory and immunopharmacological activities.Methods:1. LH-2703(1,3-Diphenyl-olyl-1,2,3,6-tetrahydro-pyrimidine-4,5-dicarboxylic acid diethyl ester) was synthesized from diethyl but-2-ynedioate, phenylamine and formaldehyde through a domino process of one-pot multicomponent reaction,followed by hydroamination, Mannich-type reaction, nucleophilic addition, and dehydration-cyclization. This reaction was catalyzed by acetic acid in the solvent of methanol. LH-2703was separated and purified by column chromatography.2. Fifty mice were randomly divided into five groups, including DMSO group, aspirin group (200mg/kg.b.w) and LH-2703compound group (200,100,50mg/kg.b.w).Each group was dissolved with0.5%CMC-Na solution.One hour after the last administration,40μl xylene was spread evenly on right ear, after thirty mimutes, mice were to put to death, the ears were cut, draw the materials from the same place of two ears by hole puncher,then weighing in electronic balance and calculate it, the weight of double ears were weighed to evaluate the influence of LH-2703on the swelling of the ear tissues.3. Forty SD rats were randomly divided into five groups, including DMSO group, aspirin group (200mg/kg.b.w) and LH-2703compound group (200,100,50mg/kg.b.w).Each group was dissolved with0.5%CMC-Na solution.One hour after the last administration,0.1ml1%carrageenan saline solution by high pressure sterilization with hypodermic injection from rat right toe each rat, and marked at the ankle well. Rat paw volume meter respectively measured after the injected1%carrageenan1,2,3,4and5hour. Record and calculate the degree of vola swelling.4. Fifty mice were randomly divided into five groups, including DMSO group, aspirin group (200mg/kg.b.w) and LH-2703compound group(200,100,50mg/kg.b.w).Each group was dissolved with0.5%CMC-Na water solution.One hour after single intragastric administration, each mouse is intraperitoneal injection by0.7%acetic acid,and the number of writhing was recorded in twenty minutes.5. Fifty female mice tails were put on hot water during(55±0.5)□,and recorded the time when the mice drifting as pain threshold. Screening the mice that pain threshold was during5and30secends, were randomly divided into five groups, including DMSO group, tramadol hydrochloride group (10mg/kg.b.w) and LH-2703compound group (200,100,50mg/kg.b.w). Thrity mimutes after single intragastric administration,and recorded the pain threshold.6. Fifty female mice were put on hot plate during(55±0.5)℃,and recorded the time when the mice licking the foot as pain threshold.Screening the mice that pain threshold was during5and30secends,were randomly divided into five groups, including DMSO group, tramadol hydrochloride group (10mg/kg.b.w) and LH-2703compound group (200,100,50mg/kg.b.w). Thrity mimutes after single intragastric administration,and recorded the pain threshold. 7. Griess reagent assay kit was used to assess nitric oxide (NO) produced by mouse peritoneal marophages. Interleukin-1β(IL-1β) was measured with ELISA assay.8. The metabolic activities of normal mouse splenocytes cells were determined by methylthiazolyl tetrazolium (MTT) colorimetry assay.9. Non-specific immunity,humoral immunity and cellular immunity were estimat ed with carbon particle clearance model, hemolysin model and DTH model of mice respectively.Results:LH-2703(1,3-Diphenyl-olyl-1,2,3,6-tetrahydro-pyrimidine-4,5-dicarboxylic acid diethyl ester) was separated by column chromatography. The chemical structure of LH-2703was characterized by MS,1H-NMR and13C-NMR. LH-2703(200,100and50mg/kg.b.w) significantly reduced the dimethylbenzene-induced ear edema in normal mice(P<0.001, P<0.001, P=0.013,vs control group), and their average inhibitive rates were53.2%,46.3%and26.6%, respectively. LH-2703(200,100mg/kg.b.w) could significantly reduce the paw edema induced by carrageenan in rats at3hours after rats were administered (P<0.001, P=0.007, vs control group), and their average inhibitive rate were46.4%,33.9%. LH-2703(200,100and50mg/kg.b.w) significantly reduced the frequency of writhing induced by acetic acid in normal mice (P<0.001, P=0.002, P=0.047, vs control group), and their average rates of inhibition were51.3%,40.8%and25.1%, respectively. LH-2703(200mg/kg.b.w) could significantly prolong the incubation period of tail-flick respose induced by hot water in mice. The pain threshold value was increased by36.4%(P=0.044, vs control group). LH-2703(200,100mg/kg.b.w) could significantly prolong the HPPT by Hot plate Test in mice. HPPT were increased by48.0%and23.7%, respectively (P=0.006, P<0.001,vs control group). LH-2703(20,10and5μg/ml) significantly decreased the production of NO and IL-1β by normal mouse peritoneal macrophages induced by lipopolysaccharide (LPS) in vitro, with inhibibition by22.7%and29.9%, respectively (P=0.036, P=0.009,vs LPS group).LH-2703(200mg/kg.b.w) did not show any significant effect on non-specific, humoral and cellular immunity in normal mice. Conclusions:1. LH-2703(1,3-Diphenyl-olyl-1,2,3,6-tetrahydro-pyrimidine-4,5-dicarboxylic acid diethyl ester) was successfully synthesized from diethyl but-2-ynedioate, phenylamine, orthotoluidine, paratoluidine, and formaldehyde through a domino process of one-pot multicomponent reaction,followed by hydroamination, Mannich-type reaction, nucleophilic addition, and dehydration-cyclization, which is a simple and feasible method with excellent yields.2. LH-2703was purified by column chromatography with petroleum and acetoacetate (v:v=7:1～5:1) as mobile phase. The method is simple and easy to carry out.3. LH-2703can decrease the paw edema induced by carrageenan in rats and the dimethylbenzene-induced ear edema in normal mice, implying good anti-inflammatory activity.4. LH-2703can reduce the frequency of writhing induced by acetic acid in normal mice, suggesting peripheral analgesic capacity.5. LH-2703can significantly increase hot water mouse tail-flick time and HPPT, suggesting central analgesic capacity.6. LH-2703can significantly inhibit production of NO and IL-1βin normal mouse peritoneal macrophages induced by lipopolysaccharides (LPS) in vitro.7. LH-2703did not show any significant effect on non-specific, humoral and cellular immunity in normal mice.8. LH-2703inhibits metabolic activity of normal mouse splenocytes. |
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