Synthesis And Pharmacological Study Of The2-pyrrolidone Derivative HL-5201

Objective:To synthesize the2-pyrrolidone derivative HL-5201via multicomponent reactions (MCRs), and to study the anti-inflammatory, immune and other pharmacologic activities of HL-5201.Method:1) The2-pyrrolidinone derivative HL-5201was synthesized with diethyl acetylenedicarboxylate,2-aminopyridine, cyclohexylamine and formaldehyde via the four component reactions in the solvent of absolute ethyl alcohol, and the reaction was catalyzed by acetic acid. The process of the reaction was monitored by thin layer chromatography (TLC), and the crude product was purified by silica gel column chromatography or recrystallization. The structure of HL-5201was analysed by MS,’H-NMR and13C-NMR. The optimizing of the reaction was carried out by changing the molar ratio of the different components, the reaction solvent, the feeding method and temperature.2) Mouse peritoneal macrophages were prepared and stimulated with LPS (10μg/ml) to induce the macrophages to secrete nitric oxide (NO), interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-a (TNF-a). The effects of HL-5201on secretion of NO, IL-1β, IL-6and TNF-a were measured by the Griess reagent or ELISA. The anti-inflammatory activity of HL-5201in vivo was assessed using xylene-induced ear edema model in mice. 3) The analgesic effect of HL-5201was evaluated by acetic acid-induced writhing response and hot plate test in mice.4) Mouse peritoneal macrophages and splenocytes were prepared, and the effect of HL-5201on metabolic capacity of the cells was measured by MTT assay. Signaling pathway blockers (PI3K, P38MAPK and JAK-2blockers), and inhibitors of proteasome and gene transcription were employed to explore the possible mechanisms involed in HL-5201affecting the metabolic capacity of macrophages. The effect of HL-5201on phagocytosis of mouse peritoneal macrophages was determined by neutral red phagocytosis assay. Mouse splenocytes were prepared and stimulated with LPS (10μg/ml) or Con A (4μg/ml) to proliferate. The effect of HL-5201on the proliferation was evaluated by MTT assay.5) Carbon clearance test was modeled by iv injection of mice with India ink, and the effect of HL-5201on the clearance capacity known as an indicator of non-specific immune function was examined. The model of humoral immune response was established by immunization of mice with rabbit red blood cells (RRBCs) and the level of serum hemolysin was measured to evaluate the activity of HL-5201on humoral immune function. DNFB-induced delayed-type hypersensitivity in mice was used to evaluate the effect of HL-5201on cellular immune function.6) Anti-tumor activity of HL-5201was assessed by S180tumor-bearing mice. And the effect on proliferation of S180tumor cells in vitro was evaluated by MTT assay.Results:1) Reaction was completed within3hours, and the target product (HL-5201) precipitated from the solvent as a white crystalline or solid powder. Silica gel column chromatography (mobile phase:n-hexane/ethyl acetate=6-1:1) or recrystallization was used to separate and purify the crude product. The structure of HL-5201was analysed via MS,’H-NMR and13C-NMR, its chemical name:4-cyclohexyl amino-5-oxo-1-pyridine-2-yl-2,5-dihydro-lH-pyrrole-3- carboxylic acid ethyl ester. The maximal yield was obtained when cyclohexylamine/2-aminopyridine/formaldehyde/acetic acid=1:1:1:3:2. Reaction was conducted in ethanol at room temperature. Diethyl acetylenedicarboxylate and cyclohexylamine were mixed10minutes before the reaction, and then added a mixture of2-aminopyridine, formaldehyde and acetic acid into the reaction. Under this reaction conditions, the yield of HL-5201was81.2%.2) In the experiment of LPS-stimulated mouse peritoneal macrophages that secrete inflammatory factors (NO, IL-1β, IL-6and TNF-a), LPS (10μg/ml) could significantly stimulate the mouse peritoneal macrophages to secrete NO, IL-1β, IL-6and TNF-a compared with the blank control group (P=0.000, P=0.000, P=0.000and P=0.000). HL-5201could effectively inhibit LPS-stimulated mouse peritoneal macrophages to produce NO compared with LPS group (P=0.000), the maximal inhibitory effect appeared at the concentration of60μmol/L, and the inhibition rate reached52.5%. At the concentration of60μmol/L, HL-5201significantly inhibited the LPS-stimulated peritoneal macrophages to produce IL-1β and IL-6(P=0.000and P=0.004), the inhibition rates were60.9%and33.7%. In contrast, HL-5201at concentrations of60and30μmol/L significantly promoted LPS-stimulated mouse peritoneal macrophages to produce TNF-a (P=0.000and P=0.000). In the test of xylene-induced mouse ear swelling, compared with the model group, HL-5201showed no significant inhibition on xylene-induced ear edema in mice.3) In the analgesic experiment, compared with the model group, HL-5201displayed no significant decrease in the writhes induced by acetic acid at the doses used and no impact on the pain threshold in hot plate test in mice.4) In the test of metabolic activity of mouse peritoneal macrophages and splenocytes, HL-5201could significantly enhance the metabolism of MTT by mouse peritoneal macrophages and splenocytes compared with the blank control group, in a dose-dependent manner at low concentrations, and the maximum effect appeared at the concentration of30μmol/L (P=0.000). Actinomycin D (0.1pg/ml), Wortmannin (10μmol/L), SB203580(10μmol/L), MG132(0.05μmol/L), LY294002(5μmol/L) and Genistein (10μmol/L) significantly attenuated the enhancement effect of HL-5201(30μmol/L) on the metabolic capacity of mouse macrophages or splencytes. Whereas H89(0.1μmol/L), BAY11-7082(0.1μmol/L) and AG490(100μmol/L) showed no impact on it. HL-5201showed a significantly enhancement effect on the phagocytosis of neutral red by mouse peritoneal macrophages (P=0.000). In the test of splenocytes proliferation induced by LPS and Con A, LPS (10μg/ml) and Con A (4μg/ml) could significantly stimulate the proliferation of splenocytes compared with the blank control group (P=0.000and P=0.000). Compared with the model group, HL-5201at concentrations of60and30μmol/L could significantly inhibit LPS or Con A-stimulated mouse splenocytes proliferation (P=0.000and P=0.000). When the concentrations were lower than15μmol/L, HL-5201showed an opposite effect on the proliferation of splenocytes.5) In the immunological experiment, compared with the model group, HL-5201significantly increased mouse correction phagocytic index in carbon clearance test at the dose of100mg/kg (P=0.010). However, HL-5201showed no effect on the mouse ear swelling in DNFB-induced delayed-type hypersensitivity experiment and on the serum hemolysin in humoral immune experiment.6) To observe the effects of HL-5201on tumor, HL-5201at a range of concentrations from7.5to60μmol/L showed no inhibitory effect on the proliferation of S180tumor cells compared with the blank control group, on the contrary, it significantly promoted the proliferation (P=0.000). Compared with model group, HL-5201at high doses (100mg/kg) could promote the tumor growth in S180tumor-bearing mice in vivo (P=0.016).Conclusion:1) HL-5201can be synthesized with diethyl acetylenedicarboxylate, cyclohexylamine,2-aminopyridine and formaldehyde via the four component reactions in the solvent of absolute ethyl alcohol. The reaction mainly experiences domino hydrogen amination, aza-en type reaction and amidation-cyclization. HL-5201can be purified by the silica gel column chromatography (mobile phase:n-hexane/ethyl acetate=6-1:1) or by recrystallization. This method has several advantages such as simple manipulation, mild reaction conditions, high yield of product, and so on.2) HL-5201effectively inhibited LPS-stimulated mouse peritoneal macrophages in production of NO, IL-1β, IL-6and other inflammatory cytokines, indicating it has a significant anti-inflammatory activity in vitro. HL-5201showed no obviously inhibitory effect on xylene-induced ear edema in mice, suggesting that HL-5201has no anti-inflammatory activity in vivo.3) HL-5201did not reduce the acetic acid-induced mouse writhes, and had no effect on the pain threshold in hot plate test, suggesting that HL-5201has no analgesic effect in vivo.4) HL-5201significantly promoted the metabolic capacity of mouse peritoneal macrophages and splenocytes, suggesting that HL-5201may have immune potentiating activity. This effect may be elicited through activation of several signaling pathways, such as PI3K, P38MAPK and other protein kinase pathways. HL-5201showed a significantly enhancement effect on the phagocytosis of neutral red by mouse peritoneal macrophages, indicating that this compound can promote phagocytic function. HL-5201at high concentrations (60,30μmol/L) significantly inhibited LPS or Con A-stimulated mouse splenocytes proliferative response, but it showed a promoting effects at low concentrations (3.25-15mol/L), suggesting that HL-5201has the immunosuppressive activity at high doses, and immunoenhancement activity at low doses. The mechanism underlying these dual effects needs further study.5) HL-5201significantly increased the phagocytic index in mice, but had no effect on DNFB-induced ear edema and serum hemolysin, suggesting that HL-5201can enhance non-specific immune function, but has no effect on cellular immunity or humoral immunity in vivo.6) HL-5201shows a promoting effect on tumor growth.