Novel C15 Triene Triazole, D-A Derivatives Anti-HepG2, and as HDAC2 Inhibitors: A Synergy Study

A series of novel C15 urushiol derivatives were designed by introducing a pechmann structure and F-, Cl-, and Br-nitro substituents with different electronic properties into its alkyl side chain, as well as a triazolyl functional group in its aromatic oxide. Their chemical structures were determined based on the analysis of the NMR (nuclear magnetic resonance) spectroscopic and mass spectrometric data. The results showed that compound 4 exhibited a strong inhibition of the HepG2 cell proliferation (half maximal inhibitory concentration (IC50): 2.833 μM to human hepatocellular carcinoma (HepG2), and 80.905 μM to human normal hepatocytes (LO2)). Furthermore, it had an excellent synergistic effect with levopimaric acid. The nitrogen atom of the triazole ring formed a hydrogen-bonding interaction with Gly103, Gly154, and Tyr308, which made compound 4 bind to histone deacetylase (HDAC)2 more tightly. One triazole ring and His33 formed a π–π stacking effect; the other, whose branches were deep into the pocket, further enhanced the interaction with HDAC2. Meanwhile, compound 4 involved a hydrophobic interaction with the residues Phe210 and Leu276. The hydrophobic interaction and π–π stacking provided powerful van der Waals forces for the compounds.

CI-Fa relationship and HepG2 apoptosis (CI=0.211) Statistical analysis: A p<0.05 was considered significantly. All analyses were carried out with SPSS 19.0 software (SPSS, Chicago, IL, USA). (1) Joint index determination: the index of cooperation was calculated by using CalcuSyn software (Combi•nation index, CI) The CI < 1 represented that they have synergistic effect while CI ≈ 1 and CI > 1 indicated additive and antagonism respectively. (2) Statistical method SPSS 17.0 statistical software was used for statistical analysis. The measurement data material of the experimental were described by x±s. Two samples were compared with the t test. The difference was significant statistically (p < 0.05).

MTT assay detect cell proliferation
MTT assay was used to determine the inhibitory effect on the proliferation of HepG2 cells of levopimaric acid. Compound 4 and levopimaric acid were combined with three groups below. The cells in the logarithmic growth phase were inoculated into 96-well culture plates at 3.5×10 3 cells/well, 0.2 ml per well, and incubated for 72 h with different concentrations of levopimaric acid. Then, we continually cultured it for 4 h after mixing fresh MTT working solution 20 μL (final concentration was 0.5 mg/mL), and carefully discarded the supernatant, added 1.5 mL of DMSO per well and shook up. The absorbance (A) at the wavelength of 490 nm was measured by a microplate reader. The cell proliferation inhibition rate was calculated according to the following formula=[(negative control group A value -blank group A value) -(experimental group A value -blank group A value)]/ (Negative control group A value -blank group A value) × 100%. Combination therapy group (Levopimaric acid + compound 4 (1st)) A group: (7.9, 3.95, 1.98, 0.988, 0.494, 0.247) μM of levopimaric were incubated with (29, 14.65, 7.32, 3.66, 1.83, 0.916) μM of compound 4 for 72 h, respectively.

Detection of cell proliferation by MTT
MTT assay was used to determine the inhibitory effect of the sample on the proliferation of HepG2 cells. The tumor cells of logarithmic growth stage were inoculated with 3.5×10 3 /well in 96 well culture plate. 0.2 ml/pore system was cultured for 72 h with different concentrations of samples. Then 20 μL (final concentration 0.5 mg/mL) of MTT were added to each pore for 4 h. The supernatant was carefully adsorbed, and 0.15 mL DMSO was added to each pore, and oscillatory mixing was carried out. The absorbance (A) value at 490 nm wavelength was measured by enzyme labeling instrument. The inhibition rate of cell proliferation was calculated as follows: [(A value of negative control group -A value of blank group) -(A value of experimental group A value of blank group)]/(negative pair group A-A value of blank group) × 100. The statistical analysis was carried out by SPSS 17.0 statistical software. The measured data were described by x±s, and the mean of the two samples was compared with t test (P < 0. 05).

HDAC2 expression
Materials HDAC2, with a molecular weight of 60 kD (1 KD=0.9921 Ku), was purchased from Santa Cruz Company, Santa Cruz, USA. The 60 kD HDAC2 standard was diluted with buffer solution and 1% SDS at 2 μg/ml, then stored at -20℃. ECL membrane and Polyacrylamide gel were purchased from AmershamLife Science Company, Amersham, USA. ECL membrane elution buffer was purchased from PIERCE Company, Colorado, USA. The cell culture medium and its additive were purchased from Gibco Company, New York, USA. Full-range rainbow Markers and a full molecular weight protein standard were purchased from Amersham Biosciences, USA. 10% SDS polyacrylamide gel was prepared by myself. FACS Calibur flow cytometry (BD Company, New Jersey, USA). The samples spotted or dotted with 20 μL of each sample, 10 μL of HDAC2 standard sample with 60 kD. Full molecular weight standard protein sample spotted or dotted with 10 μL, electrophoresis for 1.5 h under 100 V voltage. The white matter was transferred to ECL film (100 V, 1 h), blocked with PBST containing 5% skim milk powder (PBS solution containing 0.5% Tween-20) at room temperature (r.t) for 2 h, then diluted with compounds 4 (dilute concentration for 1: 100, 1: 200, 1: 500 by PBST containing 2% skim milk powder) at 4℃ overnight with 100 r/min. Above mixture solution was washed by PBST solution for 3 times, then reacted with horseradish peroxidase labeled goat anti-rat Ig (1: 7500) for 1.5 h at room temperature. The chemiluminescence kit's chromogenic reaction and exposure to detect the expression of HDAC2. The used ECL membrane was washed with eluent at room temperature for 15 min, then stained with Actin antibody (1: 10000) and detected.

Cell resuscitation
Firstly, removed the cryopreservation tube from the liquid nitrogen, and immediately plunged it into the prepared water that was between 37 ℃ and 40 ℃ , until the cryopreservation liquid completely dissolved. Then, transferred cell cryopreservation suspension to a centrifuge tube, added about 5 mL medium, and mix them gently. After that, centrifuged cell suspension at 800 ~ 1000 r/min for 5 min, and abandoned the above liquor. Finally, joined the cell pellet into culture medium and stir them lightly. After transferring the cell suspension to culture flask, replenished the medium.

Cell passage
Firstly, sucked out the original medium when the cell coverage in the culture bottle had reached 80% ~ 90%. Secondly, added appropriate trypsin (0.25%) to digest the cells for 1~2 min until they became round. Mixed the equal volume of serum-containing medium to terminate the digestion. Thirdly, use pipette to percuss cells and make them suspend. Then, sucked the cells into a 15 mL centrifuge tube and centrifuge them for 5 min. At last, dropped the supernatant, add 1~2 mL medium to suspend the cells, and cultivated them in the culture bottle.

Cell cryopreservation
The first step was to add suitable trypsin digestion cells and collected the cell suspension. Centrifuged the suspension at 1000 rpm for 5 min in the tube, and then abandoned the supernatant. Secondly, added cryoprotectant to the cell sediment and mixed them lightly until the cell density was 1×10 6~1 ×10 7 /ml. Later, divided them into 1~1.5 ml per tube, tightened the cap and made a mark on surface including the cell code and the freezing date. In the end, cooled the cells in following order: room temperature→ 4℃ (20 min) → the freezer (30 min) → low temperature refrigerator (-30℃ for 1 h) →gaseous nitrogen (30 min) →the liquid nitrogen.

Cell proliferation measured by MTT
First of all, digested the cells and counted them. Added 100 μL cell suspension (3×10 4 /ml) into each hole of the 96 holes cell culture plate. Secondly, placed the 96 holes cell culture plate in a 5% CO2 culture box for 24 h at 37℃. Then, used the medium to dilute the drug into the desired working fluid concentration, added 100 μL per hole to the corresponding drug medium, and established a negative control group as well as a positive control group (paclitaxel, 20 ug/ml). Next, placed 96 holes cell culture plate at 37℃ and cultivated the cells in a 5% CO2 incubator for 72 h. After treating 96 holes board with MTT staining, estimated the OD value: a. Added 20 μL MTT (5 mg/ml) per hole, and cultivated cells in the box for 4 h. b. Discarded the supernatant, added 150 μL DMSO in each hole, and mixed them gently after treatment with shaker for 10 min.
c. Under 490nm ultraviolet wavelength, read the OD value from ELISA. Finally, figured up the inhibition rate of each group *The inhibition rate (%) = (negative control group OD value -experimental group OD value) / negative control group OD value ×100%.

Detection of cell cycle by PI single staining
Firstly, digested the cells of logarithmic growth phase and inoculated them into six-well plates. The next day, joined the corresponding drug-containing medium after the cells adhered. And established the negative control group at the same time. After 72 h, use 0.25% pancreatin (not contain EDTA) to digest the cells. Thirdly, applied PBS to scrub the cells (centrifuge them at 2000 rpm for 5min), and collected 5×10 5 cells. Then, used a volume fraction of 70% ethanol to fix the single cell suspension for 2 h (or overnight), and stored it at 4℃. Before washing, employed PBS to remove the fixative solution (if needed, filter cell suspension through 200 mesh). Added 100 mL RNase A, and made it in water bath at 37℃ for 30 min. Later, placed it at 4℃ for 30 min without light. The final step was to check the machine, recorded the red fluorescence at the excitation wavelength of 488 nm.

Detection of apoptosis by Annexin-V FITC/PI double staining
Firstly, inoculated the logarithmic growth cells into the six-hole plate. The next day, after the cells adhered to the wall, joined the corresponding medicine-containing medium, and meanwhile set up the negative control group. After 72 h treatment, the cells were digested by using 0.25% trypsin (excluding EDTA) and then collected. Then, used PBS to wash the cells twice (centrifuge them at 2000 rpm for 5 min), and collected 5×10 5 cells. The next step was to add 500 μL Binding Buffered to make cells suspend. After cells were mixed with 5 μL Annexin V-FITC, stirred them again with 5 μL PI. At last, treated the mixture at room temperature for 5~15 min without light, and utilized flow cytometry to detect the apoptosis.

Detection of mitochondrial membrane potential by JC-1 staining
First of all, inoculated the logarithmic growth cell digestion into the six-hole plate. The next day, added the corresponding drug-containing medium after the cells adhered to the wall, and built up a negative control group as well. Secondly, collected the cell which had been washed by PBS (centrifuge them at 2000 rpm for 5 min), and changed the cell concentration into 1×10 6 /ml. Added 900 μL sterilized deionized water to 100 μL 10×Incubation Buffer, and diluted them into 1×Incubation buffer. Stirred well and then preheated them to 37℃. Absorbed the 500 μL 1×Incubation buffer, added 1 μL JC-1 to prepare the JC-1 working fluid. Next, incubated the cells which had been treated with the above fluid in a 5% CO2 incubator at 37℃ for 15~20 min. Then, collected the cells by centrifugation at room temperature for 5 min, and washed them twice with 1×Incubation Buffer. 500μL 1×Incubation Buffer of resuspension cells were absorbed, and did the computer test to attain the final result.
Syntheses of Compound 10. Triene uruhsiol (0.5 mmol) was dissolved in dry MeCN (3 mL), SiO2/NaHSO4 (1 mmol) and ethyl acetoacetate (100 μL, 1.25 mmol) were added. The mixture was stirred at r.t for 4 h and centrifuged with H2O (50 mL). The solution was extracted with EA and evaporated to remove EA. The organic layer was washed with saturated NaHCO3 and brine, dried over MgSO4, filtered, and finally concentrated. The residue was purified by column chromatography with PE-EA (100:10) to produce compound 10.
Syntheses of Compound 15-20. Maleic anhydride was added to the dried pressure tube and stirred evenly in an oil bath. Refined lacquer(C15 triene urushiol purity﹥95%) and maleic anhydride molar ratio was 0.5:1; Temperature kept at 160℃; refined C15 triene urushiol and maleic anhydride were mixed in the high temperature environment and stirred at 160°C for 6 h, stopped the reaction and cooled to room temperature to obtain the target organic synthesis product.