| ObjectiveTo investigate the anti-inflammatory and analgesic effects of the tetrahydro-pyrimidine derivative ZL-5015(1,3-dicyclopentyl-1,2,3,6-tetrahydro-pyrimidine-4,5-dicarboxylic acid diethyl ester) and to explore the mechanisms of action.Methods1. ZL-5015was synthesized from diethyl but-2-ynedioate, cyclopentylamine 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. ZL-5015was separated and purified by preparative column chromatography.2. Xylene-induced murine ear swelling was used to evaluate the anti-inflammatory activity in vivo.50BALB/c mice were randomly divided into five groups, including control group, aspirin group (100mg/kg.b.w) and ZL-5015compound group (100,50and25mg/kg.b.w). The compound was dissolved with 0.5%CMC-Na solution. One hour after the last administration,20μl xylene was applied evenly to the right ear of the animal. After another40min, all mice were euthanised by cervical dislocation and both ears were removed. Round pieces of ear samples were made at the same size (7mm in diameter) from both ears and weighed. Swelling degree was calculated by subtracting the left ear sample weight from the right.3. Carrageenan-induced rat paw edema was used to evaluate the anti-inflammatory activity in vivo. Forty SD rats were randomly divided into five groups, including control group, aspirin group (100mg/kg.b.w) and ZL-5015compound group (100,50and25mg/kg.b.w). The compound was dissolved with0.5%CMC-Na solution. One hour after the last administration, the right hind paw of each animal was measured in volume (mL)(designated hour0time point) prior to administration with100μl carrageenan (1%, w/v) by intraplantar injection. After that, the volume (mL) of the injected paw was taken again at hours1,2,3,4and5time points after the administration of carrageenan. Edema was calculated by subtracting the right hind paw volume (mL) at hour0time point from those after the administration of carrageenan.4. Acetic acid-induced mouse writhing was used to evaluate the anti-inflammatory and analgesic activities in vivo. Fifty BALB/c mice were randomly divided into five groups, including control group, aspirin group (100mg/kg.b.w) and compound ZL-5015group(100,50and25mg/kg.b.w). The compound was dissolved with0.5%CMC-Na water. On the third day,1h after the last treatment, each mouse is intraperitoneal injection by0.7%acetic acid, and the number of writhes that occurred within20min of the challenge was totaled for each mouse.5. Hot plate test was used to evaluate the analgesic activity in vivo. Female mice were screened in advance by placing them on a hot plate with temperature maintained at50℃, and the time between the mice touching the plate and beginning to lick the hind paw was recorded as latency of response or defined as pain threshold. Fifty suitable female mice with body weight at20±2g, whose pain threshold was between10and30secends, were included and randomly divided into five groups, including control group, tramadol hydrochloride group (10mg/kg.b.w) and ZL-5015compound group (100,50and25mg/kg.b.w). On the third day,1h after the last treatment, the animals were placed on the plate and pain threshold was recorded.6. The metabolic activities of murine peritoneal macrophages and RAW264.7cells were determined by methylthiazolyl tetrazolium (MTT) colorimetry assay.7. Murine peritoneal macrophages and RAW264.7cells activated by lipopolysaccharides (LPS,10(μg/ml) were used as in vitro inflammatory models to investigate the effect of ZL-5015on the production of inflammatory mediators. Griess reagent assay kit was used to assess nitric oxide (NO) and Interleukin-1∞(IL-1β), Interleukin-10(IL-10), TNF-α and PGE2were measured with ELISA assay.8. Murine peritoneal macrophages and RAW264.7cells activated by LPS (10μg/ml) were used as in vitro inflammatory models to investigate the effect of ZL-5015on gene expression of inflammatory factors at mRNA level.6well plates were used for cell culture. Real time fluorescence quantitative PCR was exploited to assess the mRNA of interest. The content of mRNA was expressed as2-ΔCt using GAPDH as internal reference and normalized to control group.ResultsZL-5015C1,3-dicyclopentyl-1,2,3,6-tetrahydro-pyrimidine-4,5-dicarboxylic acid diethyl ester) was successfully synthesized and separated by column chromatography. The chemical structure of ZL-5015was characterized by MS,1H-NMR and 13C-NMR.ZL-5015(100and50mg/kg.b.w) significantly reduced the xylene-induced ear edema in normal mice(P=0.011and P=0.031vs control group), and the average inhibitive rates were49.2%and41.8%, respectively.ZL-5015(100,50and25mg/kg.b.w) could significantly reduce the paw edema induced by carrageenan in rats at3h after rats were administered (P=0.000, P=0.003and P=0.031vs control group), and the average inhibitive rate were53.2%,41.3%and28.7%. ZL-5015(100,50and25mg/kg.b.w) significantly reduced the frequency of writhing induced by acetic acid in normal mice (P=0.000, P=0.001and P=0.005vs control group), and the average rates of inhibition were51.8%,39.6%and32.7%, respectively. ZL-5015(100、50、25mg/kg.b.w) could significantly prolong the pain threshold by hot plate test in mice. The pain threshold was increased by23.4%(P=0.006,vs control group),13.0%and5.5%, respectively.After LPS stimulation of murine peritoneal macrophages for6h, the amounts of proinflammatory cytokines IL-1β and TNF-a in the supernatants were increased dramatically, and24h stimulation was required for inflammatory factors or mediators PGE2and NO to rise remarkably in the supernatants at the same experimental setting. ZL-5015(40μM and20μM) partially inhibited the macrophages production of the proinflammatory cytokines and inflammatory factors or mediators induced by LPS, and their average inhibitive rates were41.1%and29.1%for IL-1β;42.9%and33.8%for TNF-a;57%and45.1%for PGE2; and67.1%and49.4%for NO, respectively. The differences of means for the examined parameters between ZL-5015groups and LPS group were statistically significant (IL-1β:P=0.000and P=0.000; TNF-α: P=0.005and P=0.019; PGE2:P=0.001and P=0.005; NO:P=0.000and P=0.000) ZL-5015at80μM could partially inhibit the metabolic activities of murine peritoneal macrophages, the inhibitive rate was25.2%, whereas no significant inhibition effect was observed at the concentration of40μM or below80uM of ZL-5015partially inhibited the macrophages production of IL-10, the inhibitive rate was66.3%, whereas20μM and lOμM of ZL-5015could increase the macrophages production of IL-10, the increase rate were7.1%and6.1%, respectively. After LPS stimulation of RAW264.7cells for6h, the amounts of pro inflammatory cytokines IL-1β and TNF-α in the supernatants were increased dramatically, and after LPS stimulation of RAW264.7cells for24h, the amounts of inflammatory factors or mediators PGE2and NO were noticed to rise significantly. ZL-5015(40μM and20μM) partially inhibited the RAW264.7cells production of the proinflammatory cytokines and inflammatory factors or mediators induced by LPS, and their average inhibitive rates were43.4%and28%for IL-iβ;34.8%and26.3%for TNF-α;66.5%and51.6%for PGE2; as well as60.9%and36.2%for NO, respectively. The differences of means for the examined parameters between ZL-5015groups and LPS group were statistically significant (IL-1β:P=0.000and P=0.017; TNF-α:P=0.004and?=0.025; PGE2: P=0.000and P=0.002; as well as NO:P=0.000and P=0.000).80μM of ZL-5015could partially inhibit the metabolic activity of RAW264.7cells with an inhibitive rate for29.7%and reduce the RAW264.7cells production of IL-10with a reduction rate for62.9%. Whereas ZL-5015at concentrations of40μM、20μM and10μM exhibited potentiating effect on RAW264.7cells production of IL-10, the potentiating rates were4%、12%and7%, respectively.After LPS stimulation of murine peritoneal macrophages for24h, the mRNA expressions of COX-2and iNOS were markedly increased. ZL-5015(40μM and20μM) partially inhibited the expressions of the genes of interest induced by LPS, the inhibitive rates were37.9%and29.1%for COX-2; and41.5%and36.5%for iNOS, respectively. The differences of means for the examined parameters between ZL-5015groups and LPS group were statistically significant (COX-2:P=0.001and P=0.008; and iNOS:P=0.000and P=0.01). RAW264.7cells were treated with the same protocol as that for murine peritoneal macrophages, ZL-5015(40μM and20μM) partially inhibited the expressions of the genes of interest induced by LPS, the inhibitive rate were39.5%and31.6%for COX-2; and38.5%and31.6%for iNOS, respectively. The differences of means for the examined parameters between ZL-5015groups and LPS group were statistically significant (COX-2:P=0.001and P=0.007; and iNOS:P=0.001and P=0.01).Conclusions1. ZL-5015(1,3-dicyclopentyl-1,2,3,6-tetrahydro-pyrimidine-4,5-dicarboxylic acid diethyl ester) can be obtained by synthesis from diethyl but-2-ynedioate, cyclopentylamine, 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 yield. Column chromatography with N-hexane and acetoacetate (v/v=7/1-4/1) as developing solvent is suitable for purification of ZL-5015. The method is simple and easy to carry out.2. ZL-5015can decrease the paw edema induced by carrageenan in rats and xylene-induced ear edema in normal mice, implying that the compound has anti-inflammatory activity.3. ZL-5015can reduce the frequency of writhing induced by acetic acid in normal mice, suggesting peripheral anti-inflammatory or analgesic capacity of the compound.4. ZL-5015can increase pain threshold in hot plate test at doses larger than those for anti-inflammatory action, suggesting a minor central analgesic activity.5. ZL-5015can partially inhibit endotoxin-induced production of IL-1β and TNF-a by murine peritoneal macrophages and RAW264.7cells in vitro, which may be explained as a part of the anti-inflammatory mechanisms.6. ZL-5015can slightly promote endotoxin-induced production of IL-10by murine peritoneal macrophages and RAW264.7cells in vitro, which may contribute to the anti-inflammatory effect through inhibiting the secretion of pro-inflammatory cytokines.7. ZL-5015can partially inhibit endotoxin-induced production of PGE2and NO by murine peritoneal macrophages and RAW264.7cells in vitro, suggesting that the anti-inflammatory effect of ZL-5015is resulted from inhibiting the production of PGE2and NO.8. ZL-5015can partially inhibit endotoxin-induced increases in mRNA expressions of COX-2and iNOS in murine peritoneal macrophages and RAW264.7cells in vitro, suggesting that the inhibitory effects of ZL-5015on PGE2and NO production are mediated by inhibiting the mRNA expressions of the related enzymes COX-2and iNOS, respectively. However, the potency of the inhibition of mRNA expression is lower than that for inhibition of PGE2and NO production, indicating other mechanisms may be also involved in the anti-inflammatory effect, which remains to be further studied. |
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