| Objective:We studied the structural modification of usenamine to obtain derivatives and screened anti-cancer activity.Then evaluate the toxicological safety of anti-cancer active products,to understand the toxic target organs of the compound,and explore the toxic target organs of the compound to provide information for subsequent drug development.Methods:1.A variety of organic acids were used as acylating agents to carry out acylation reaction with usenamine,DMAP as the catalyst for the chemical reaction,and DIC as the dehydrating agent in the reaction process to prepare derivatives.2.The inhibition rate of cancer cells in vitro is used as a reference to screen anti-cancer active products.The MTT method was used to screen the anticancer activity of the structure modification product of usenamine.Finally,select the products with anti-cancer activity.3.Solvent extraction and silica gel column chromatography were used to separate and purify the product.4.Nuclear magnetic resonance hydrogen spectroscopy,carbon spectroscopy,and mass spectrometry are used to determine the structure and molecular weight of compounds.5.Four factors and three levels of orthogonal experiments were used to optimize the synthesis conditions of anti cancer active products.6.The UV spectrophotometer was used to test the solubility of pine nicotine in different solvents.And then compare with the solubility of the original drug,and analyze the change in the solubility of the modified product.7.Using in vitro anti-tumor activity test,MTT method was used to test the inhibitory effect of different concentrations of Usnea nicotinyl amine on CNE2、U-251 and HCT-116.8.We used the maximum drug resistance method for acute toxicity test.The mouse sperm deformity test and mouse bone marrow cell micronucleus test were used to observe genotoxicity.The 30-day feeding test was used to detect the toxicity of short-term repeated administration.Results:1.Usenamine can acylate with some organic acids(Benzoic acid、usenamine、cinnamic acid、O-Toluic acid、1-Naphthoic acid、Hydrocinnamic acid)and produce new compounds2.The reaction of niacin with usenamine can produce some compounds with strong anti-cancer effect in vitro.3.Anti-cancer active derivatives formed by the reaction of niacin with usenamine,The Chinese name is Usnea nicotinyl amine.The molecular formula is C24H20N2O7,The molecular weight 448,And the molecular structure is E)-N-(1-(6-acetyl-7,9-dihydroxy-8,9b-dimethyl-1,3-dioxo-3,9b-dihydrodibenzo[b,d]f uran-2(1-H)-ylidene)ethyl)isonicotinamide.4.The optimal synthesis conditions for Usnea nicotinyl amine:n(usenamine):n(niacin)=1:1.5,n(usenamine):n(DMAP)=1:0.2,the reaction temperature is 20° and the reaction time is 96 h.The yield is 28.72%.5.The saturated solubility of usnea nicotinyl amine in n-octanol,methanol,absolute ethanol,75%ethanol and 50%ethanol solutions are 2.232±0.002、1.453±0.002、1.581±0.001、1.758±0.002、0.371±0.001 mg/ml.The saturated solubility of usenamine is 2.085±0.001、2.533±0.072、3.592±0.029、1.001±0.011、0.219±0.006 mg/ml respectively.6.The inhibitory rate of Usnea nicotinyl amine to CNE2 under the conditions of 0.625,1.25,2.5,5.0,10.0 μmol/L for 72 h was-0.61±3.12%,22.91±20.84%,87.02±7.47%,91.46±4.73%,93.06±4.10%;The inhibition rates in U-251 are-1.64±3.79%,19.01±13.91%,68.09±11.65%,85.54±5.41%,91.97±2.01%at 72 h;The inhibition rates in HCT-116 were 14.97±3.35%,30.77±2.86%,87.99±2.66%,99.91±0.08%,99.86±0.10%at 72 h.IC50 values of Usnea nicotinyl amine were 1.68±0.51μmol/L,2.14 ± 0.41 μ mol/L,1.93 ± 0.13 μmol/L in CNE2,U-251 and HCT-116 cell lines.7.In the acute toxicity test,when the final dose was 10 g/kg,there was no poisoning reaction in the mice and there were no deaths.8.Mice sperm abnormality test,When the dose of sinotinamide was 1.25,2.5,and 5.0 g/kg by intragastric administration,the sperm deformity rates of the mice were 2.02±0.12%,2.14±0.24%,and 2.13±0.18%,respectively,and there was no statistical difference from the normal group academic significance(P>0.05).9.Bone marrow cell micronucleus test,When intragastric administration of Usnea nicotinyl amine was 1.25,2.5,and 5.0 g/kg,the micronucleus rates of male mice were 2.4%,2.6%,and 4.0%respectively.At the same dose concentration,the micronucleus rate of bone marrow cells in female mice was 3.0%,3.4%,4.2%.and there was no statistical difference from the normal group academic significance(P>0.05)10.In the 30-day feeding test,there was no significant difference in body weight and food utilization between the mice in the Usnea nicotinyl amine group and the normal group.Male mice:Compared with the normal group,WBC,LYMPH and NEUT decreased significantly(P<0.05),BUN and UA increased significantly(P<0.05),and liver coefficient increased(P<0.05)in the high-dose(2.6 g/kg)group.The RBC and HGB decreased significantly(P<0.05),UREA levels increased significantly(P<0.05),and liver,spleen and testicular coefficients increased significantly(P<0.05)in the middle-dose(1.3 g/kg)group.The HLD was decreased in the low-dose(0.625 g/kg)group.Female mice:Compared to the normal group,The levels of RBC,HGB,WBC,LYMPH,TC and HLD decreased significantly(P<0.05),and the levels of AST,Cr and UA increased significantly(P<0.05)in the high-dose(2.6 g/kg)group.In the middle dose(1.3 g/kg)group,HGB decreased significantly(P<0.05),and UA increased significantly(P<0.05).In the low dose(0.625 g/kg)group,LYMPH decreased significantly(P<0.05),and UA levels increased significantly(P<0.05).Conclusion:1.After the acylation of usenamine with nicotinic acid can form Usnea nicotinyl amine with anti can cer acti v ity.The molecular formula is C24H20N2O7.The optimal synthesis conditions of Usnea nicotinyl amine is:n(usenamine):n(niacin)=1:1.5,n(usenamine):n(DMAP)=1:0.2,the reaction temper-ature is 20°and the reaction time is 48 h.The yield is 28.72%.The main factor that affects the yield is the ratio of niacin to usenamine.2.The saturated solubility of Usnea nicotinyl amine in 50%ethanol,75%ethanol and n-octanol solvent is higher than usenamine.The solubility of Usnea nicotinyl amine in methanol and absolute ethanol solvent is lower than that of usenamine.3.Usnea nicotinyl amine has significant anti-tumor activity in vitro.4.The acute toxicity test in mice proves that Usnea nicotinyl amine is non-toxic.5.Existing data cannot determined whether Usnea nicotinyl amine have genotoxic.6.Oral high-dose sinotinamide may affect hematopoietic function and show nephrotoxicity in mice. |
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