| Objective:To optimize the synthesis of the compound N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide for higher yield and simpler technology. To observe the interaction between the compound N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide and BSA. To study the possible drug resistance reversal mechanism of the compound N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide. To study the anti-tumor quantitative structure-activity relationship(QSAR) of higher fatty acid derivatives to provide evidence for the further structure optimization.Methods:1. Different proportion of behenic acid and N, N’-Dicyclohexylcarbodiimide were used as starting materials to synthesize the compound N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide, and different methods were used to purify the final compound.2. By fluorospectro photometry, the interaction between the N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide and BSA at different temperatures were detected.3. Fluorospectro photometer was used to detect the accumulation effect of ADM in the cancer cells which were intervened by N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide.4.28 compounds which were synthesized earlier were calculated by MOE to develop a estimated linear model of QSAR higher fatty acids, and the ability to predict on antitumor was validated.Results:1. The synthesis scale of the compound N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide was expanded to 10 times, the rate between N,N’-Dicyclohexylcarbodiimide and behenic acid was 1 to 1.2, the reaction time was 36h, theN,N’-Dicyclohexylcarbodiimide was nearly reacted completely, the product was purified by new method, and the final yield was 64.6%.2. Fluorospectro photometer was used to detect the interaction between the N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide and BSA. The experimental results showed that bimolecular quenching constant Kq was 1.64×1011 Lmol-1s-1, quenching constant Ksv was 1.64×103Lmol-1, binding constant Kb was 5.41×102 molL-1 at the temperature of 25℃ and 1.75×104 molL-1 at 37℃, binding site was 1.The thermodynamic parameters were calculated. The enthalpy change (AH) was 2.22x 105 KJ.mol"1, entropy change (AS) was 7.99×102 J.K-1.mor-1, the gibbs free energy change (△G) was -1.56×104 KJ.mol-1 at the temperature of 25℃ and-2.52×104KJ.mol at 37℃. Compared with samples of the same concentration, the results of synchronous fluorescence showed that fluorescence intensity increased with the increase of temperature, while the intensity of quenching weakened when AX,=15, fluorescence intensity increased with the increase of temperature, while the intensity of quenching increased when △λ=60.3. RF-5301PC Fluorospectro photometer was used to tested the concentration of adriamycin, Ex was 470nm and Em was 585nm.There was a good linear relationship for ADM with the range of 50ng/ml to 5000ng/ml. The standard curve equation was F=0.0041*C+0.156 (r=0.9995). The RSD of precision was 0.73%,2.16%,2.48%,1.75%,1.12% and 3.54%, respectively. The RSD of intra-day precision was 1.21%,-2.6% and -3.09%, respectively. The RSD of inter-day precision was -048%,5.04% and 3.9%, respectively. The recovery rate was 99.52%,98.3% and 98.76%, respectively. In the experiment of intake, the ADM content in the cells of experimental group was 465.36ng at lh, 509.83ng at 2h,512.74ng at 4h, while the control group was 457.56ng,17.8ng and 515.03ng, respectively. In the experiment of exocytosis, the ADM content in the cells of experimental group was 225.56ng at 1h,26.59ng at 2h,60.88ng at 4h, while the control group was 247.92ng,113.86ng and 65.37ng, respectively.4. The estimated linear model of QSAR on antitumor of higher fatty acids was pI50=14.46369-1.48600*SlogP+3.07797*SMR+1.86576* E_sol-0.14763*vol+0.77365*E_vdw-0.20820*E_ele-0.15662*E_tor-4.20054 *dipole (R2=0.91258), the results of verification showed that predicted values and experimental ones were almost unanimously.Conclusions:l.The synthesis reaction will be more fully finished by properly adding behenic acid into the reaction system. The compound N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide can be purified quickly and efficiently by the new method.2.The interaction mechanism between N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide with BSA is static quenching, the intermolecular forces is mainly hydrophobic interaction,1:1 compounds are formed by BSA and N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide. N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide has no effect on the microenvironment of tyrosine residues and nearly no effect on the microenvironment of tryptophan residues. The combination of N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide and tryptophan residues enhances with the temperature raising, while the combination of N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide and tyrosine residues weakenes with the temperature raising.3.The drug resistance mechanism of N-cyclohexyl-N-(cyclohexylcarbamoyl)docosanamide is irrelevant to increase the accumulation effect of ADM in the cancer cells.4.The equation which was built in QSAR research has good prediction ability. |
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