| Objective:It was aimed to synthesize the tetrahydropyrimidine derivative ZL-5010 with diethyl but-2-ynedioate, cyclohexanamine and formaldehyde as starting materials and to explore anti-inflammatory and immunopharmacological activities of the compound.Methods:ZL-5010 (diethyl1,3- dicyclohexyl- 1,2,3,6- tetrahydropyrimidine-4,5-dicarboxylate) was synthesized from diethyl but-2-ynedioate, cyclohexanamine 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 ethanol. ZL-5010 was separated and purified by preparative thin-layer chromatography(PTLC). The metabolic activities of normal muose splenocytes cells were determined by methylthiazolyl tetrazolium(MTT) colorimetry assay. The amounts of tumor necrosis factorα(TNF-α) and interleukin 1 (IL-1 beta) were measured with ELISA assay. Anti-inflammatory activity was estimated with two acute inflammation models, including carrageenan-induced rat-paw edema and dimethylbenzene-induced ear edema in mice. Acetic acid-induced abdominal-writhing response in mice and hot water mouse tail-flick response were used to evaluate its analgesic activity. Results:ZL-5010 (diethyl 1,3-dicyclohexyl-1,2,3,6-tetrahydropyrimidine -4,5-dicarboxylate) appeared to be a colourless oil substance with a yield about 82.7%. The chemical structure of ZL-5010 was characterized by 1H-NMR and MS, which was in accord with the literature data. ZL-5010 (20,10,5μg/ml) inhibited the metabolic activity of normal mouse splenocytes, and their average inhibitive rates were 64.5%,52.1% and 18.9%, respectively, with statistically significant differences(P=0.000, P=0.000, P-0.036) as compared with control group. The half maximal inhibitory concentration (IC50) was about 11.95μg/ml. ZL-5010 (25,12.5, 6.25μg/ml) significantly decreased the production of TNF-a (P=0.000, P=0.000, P=0.000) in normal mouse splenocytes in vitro, compared with control group, and inhibited the secretion of IL-1 beta in mouse splenocytes induced by lipopolysaccharide (LPS) (P=0.000, P=0.000, P=0.001, vs LPS group). Effects of ZL-5010 on the production of IL-1 beta and TNF-a in mouse splenocytes were dose-dependent within a certain concentration range. Compared with control group, ZL-5010(100,200mg/kg.b.w)significantly reduced the dimethylbenzen-induced ear edema in normal mice(P=0.000, P=0.000), and their average inhibitive rates were 42.0% and 50.4%, respectively. ZL-5010 (100,200mg/kg) and aspirin group could significantly reduce the paw edema induced by carrageenan in rats (P=0.000, P=0.002, P=0.009 vs control group), and their average paw edema were 0.044ml,0.069ml,0.090ml, respectively. The average paw edema of ZL-5010 (50mg/kg) group was a little lower than that of control group, but there was not significance between them (P=0.071). ZL-5010 (200, 100mg/kg) reached the maximum inhibition at 3 hours after rats were administered ZL-5010, and aspirin group reached the maximum inhibition at 5 hours, their maximum inhibition rates were 75.0%,93.2% and 64.5%, respectively. ZL-5010(100,200/kg.b.w) and aspirin group significantly reduced the frequency of writhing induced by acetic acid in normal mice (P=0.000, P=0.000, P=0.001, vs control group), and their average rates of inhibition were 46.3%,53.5% and 33.5%, respectively.Conclusions: 1.ZL-5010(diethyl 1,3-dicyclohexyl-1,2,3,6-tetrahydropyrimidine -4,5-dicarboxylate) was successfully synthesized from diethyl but-2-ynedioate, cyclohexanamine 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. ZL-5010 was purified by preparative thin-layer chromatography with N-hexane and acetoacetate (v/v=8/1) as developing solvent. The method is simple and easy to carry out.3. ZL-5010 can significantly inhibit metabolic activity and production of TNF-a in normal mouse splenocytes, and can inhibit the secretion of IL-1 beta in mouse splenocytes induced by LPS.4. ZL-5010 can reduce the frequency of writhing induced by acetic acid in normal mice, suggesting analgesic capacity.5. ZL-5010 can decrease the paw edema induced by carrageenan in rats and the dimethylbenzene-niduced ear edema in normal mice, implying good anti-inflammatory activity.6. The preliminarily experimental results demonstrate that ZL-5010 has good anti-inflammatory and analgesic activities. |
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