Discovery of Novel Cyclin-dependent Kinase (CDK) and Histone Deacetylase (HDAC) Dual Inhibitors with Potent In Vitro and In Vivo Anticancer Activity
Chunhui Cheng†, Fan Yun†, Sadeeq Ullah, Qipeng Yuan*
A B S T R A C T
In the current study, we reported a series of novel 1-H-pyrazole-3-carboxamide-based inhibitors targeting histone deacetylase (HDAC) and cyclin-dependent kinase (CDK). The representative compounds N-(4-((2-aminophenyl)carbamoyl)benzyl)-4-(2,6-dichlorobenzamido)-1H-pyrazole-3carboxamide (7c) and N-(4-(2-((2-aminophenyl)amino)-2-oxoethyl)phenyl)-4-(2,6-dichlorobenzamido)-1Hpyrazole-3-carboxamide (14a) with potent antiproliferative activities towards five solid cancer cell lines, showed excellent inhibitory activities against HDAC2 (IC50 =0.25 and 0.24nM respectively) and CDK2 (IC50 =0.30 and 0.56nM respectively). In addition, compounds 7c and 14a significantly inhibited the migration of A375 and H460 cells. Further studies revealed that compounds 7c and 14a could arrest cell cycle in G2/M phase and promote apoptosis in A375, HCT116, H460 and Hela cells, which was associated with increasing the intracellular reactive oxygen species (ROS) levels. More importantly, compound 7c possessed favorable pharmacokinetic properties with the intraperitoneal bioavailability of 63.6% in ICR mice, and potent in vivo antitumor efficacy in the HCT116 xenograft model. Our study demonstrated that compound 7c provides a promising strategy for the treatment of malignant tumors.
Key words: Histone deacetylase, Cyclin-dependent kinase, Antiproliferative activity, Pharmacokinetic properties, In vivo antitumor activity
1 Introduction
Tumorigenesis is closely associated with gene mutations and abnormal chromosome modifications which are the category of epigenetic aberrations[1]. Histone deacetylases (HDACs) and histone acetyltransferases (HATs) are epigenetic enzymes, which jointly governed the acetylation of histone [2, 3]. Overexpressed HDACs could remove the acetyl group from the terminal lysine residues of histones, thus silencing the expression of tumor suppressor genes and resulting in tumor carcinoma[4-9]. HDAC inhibitors have received considerable success for cutaneous cell lymphoma therapy. Recently, four HDAC inhibitors, vorinostat (SAHA), romidepsin (FK228), belinostat (PXD-101), and panobinostat (LBH589) have been approved by U.S. Food and Drug Administration (FDA) for the therapy of hematological malignancies[10-13]. Chidamide (CS055) has been approved by China Food and Drug Administration for the treatment of peripheral T-cell lymphoma[14] (Figure 1A). Single HDAC inhibitors have been proved to be effective for the treatment of hematological malignancies, but they show low inhibitory activity against solid tumors[15, 16]. Moreover, many clinical researches have demonstrated that HDAC inhibitors exploit synergistic effects in combination with other anticancer agents[17-19]. However, multicomponent drugs raised the risks involved in complex pharmacokinetic properties, unpredictable drug-drug interactions and different drug solubilities[20, 21]. On the contrary, developing HDAC-based multitargeting drugs could provide an effective and practical strategy to overcome the limitations of single and multicomponent agents.
The dysfunction of cell cycle regulation is a remarkable future of the occurrence and development of malignant tumors[22]. Cyclin-dependent kinases (CDKs), belonging to a family of serine/threonine protein kinases, regulate cell cycle and promote cell growth, proliferation and apoptosis[23, 24]. To date, the CDK family have consisted of 20 members (CDK1-20), which form a CDK/ cyclin complex with associated cyclin to exert their function[25]. CDK1, 2, 4 and 6 play a crucial role in cell cycle, while CDK7, 8 and 9 are involved in transcriptional regulation[26, 27]. Abnormal expression of CDK2/cyclin A or CDK2/cyclin E leads to cell carcinoma, and overexpression of CDK2 induced by gene mutation has been identified in various human tumors, containing breast cancer, ovarian carcinoma, bladder cancer, endometrial carcinoma and gastric carcinoma[28-30]. As a consequence, CDK inhibitors have been considered as promising antineoplastic agents. So far, two CDK inhibitors have been approved by FDA: palbociclib[31] and ribociclib[32] for the treatment of breast cancer and HER2-negative advanced breast cancer respectively (Figure 1B). Several CDK inhibitors have been at different stages of clinical trials, for instance, (R)-roscovitine[33, 34], AT-7519[35] and flavopiridol[36, 37].
It’s reported that vorinostat and flavopiridol, a pan-CDK inhibitor, have synergized in leukemia, breast, lung and esophageal cancer[38, 39]. In addition, Fan et al. reported a series of novel CDK4/9 and HDAC1 dual inhibitors against malignant cancer[40]. These evidence indicated that designing rationally a multitargeting molecule inhibiting CDK and HDAC could be a promising strategy for cancer therapy.
4-(2,6-difluorobenzamido)-N-(4-fluorophenyl)-1H-pyrazole-3-carboxamide, was proven to reveal high inhibitory activity against CDK1 and CDK2 enzymes, and good pharmacokinetic (PK) properties, indicating it to be a lead compound (Figure 2) [33, 35]. Additionally, pharmacophore models of most HDAC inhibitors comprise a cap group, a linker and a zinc-binding group (ZBG). The cap structure containing a hydrophobic ring interacts with amino acid residues at the protein surface; the linker occupies the tubular channel to connect the cap group and the zinc site; the zinc-binding group, such as hydroxamic acid and 2-aminobenzamide, chelates with zinc ion and forms hydrogen bonds in the catalytic site (Figure 1)[41, 42]. Considering that there is a large hydrophobic area on the surface of HDAC, we envisaged that the essential pharmacophore of compound 1 was merged with hydroxamic acid or o-aminobenzamide moiety to acquire a single molecule that could inhibit both HDAC as well as CDK enzymes. Furthermore, on basis of the strategy as shown in Figure 2 , we synthesized a series of dual CDK/ HDAC compounds, and evaluated their antitumor activity in vitro and in vivo.
2. Results and discussion
2.1 Chemistry
The synthetic routes of all target compounds were listed in Scheme 1-3. The preparation procedures of compounds 7a-7g and 8a-8g were illustrated in Scheme 1. Commercially available 4-nitro-1H-pyrazole-3-carboxylic acid (2) reacted with methyl 4-(aminomethyl)benzoate hydrochloride (3) to synthesize 4, which was reduced by palladium on activated carbon to yield compound 5. Then compound 5 was treated with different substituted benzoyl chlorides to afford the key intermediate 6. The intermediate 6 was hydrolyzed, and then treated with benzene-1,2-diamine to give the target compounds 7a-7g. In addition, the hydroxamic acids 8a-8g were directly generated by compound 6 with fresh hydroxylamine solution.
As shown in Scheme 2, firstly, compound 10 was prepared by the reduction of methyl 4-nitro-1H-pyrazole-3-carboxylate (9) in the presence of palladium on activated carbon. 10 reacted with various benzoyl chlorides to afford compound 11, which was subjected to hydrolysis in 1,4-dioxane to give the corresponding acid 12. After that, the key intermediate 13 was obtained by 12 with ethyl 2-(4-aminophenyl)acetate. The synthesis of hydroxamic acids 15a-15g was similar to that of 8a-8g. Then target compounds 14a-14g were prepared via the hydrolysis and condensation reaction from 13. In Scheme 3, compound 12 was treated with available aminoalkylester hydrochlorides to provide the intermediate 16, which was consequently converted to final products 17a-17g.
2.2 Biological evaluation
2.2.1 Assays of antiproliferative activity and enzyme inhibition
The results of antiproliferative activity and enzyme inhibition assays were displayed in Table 1 with CS055, SAHA and compound 1 as the reference drugs. The growth inhibition of all compounds was examined at the concentrations of 8µM and 2µM towards human colorectal cancer cell line (HCT116) and human lung cancer cell line (H460). In addition, we also evaluated the enzyme inhibition for HDAC1 and CDK2 at 10µM. Compounds 17a-17g with the long alkyl linker at the R3 position significantly decreased the antiproliferative activity relative to those with the aromatic ring. The introduction of electron-donating methoxyl group (7g, 8g, 14g and 15g) gave the weaker activity than that of electron-withdrawing groups, such as 7e, 8e 14e and 15e. The o-aminobenzamide-based compounds 7b-7d caused a remarkable increase of activity compared to 8b-8d with hydroxamic acid as ZBG. Compounds 14a-14c, simultaneously substituted with fluorine or chlorine moieties at the R1 and R2 positions exhibited better activity than those with R2=H, such as 14d-14g. Moreover, the antiproliferative activities were substantially consistent with enzyme inhibitory activities. For example, compounds 7a-8a with higher cell growth inhibitory effects were also more effective against HDAC1 and CDK2 than compounds 15b-15f.
HDACs or CDKs were overexpressed in many malignant tumors[43-48]. Particularly, it was reported that Class I HDACs showed aberrant upregulation in more than 75% tumor tissues[49, 50]. Studies have indicated that several novel CDK or HDAC inhibitors showed potent antiproliferative activity against human colorectal, lung, malignant, cervical and hepatoma cancer cell lines[51-60]. Consequently, IC50 values of compounds that revealed superior or similar antiproliferative activity to the positive controls were further investigated towards human cancer cell lines: HCT116, H460, A375, Hela and SMMC7721. As depicted in Table 2, compounds 7c and 14a, in which two chlorine atoms were linked at the R1 and R2 positions, exhibited excellent antiproliferative activities with IC50 values ranging from 0.71 to 7.76µM against five cell lines. Moreover, these two compounds were more potent for HCT116 cells (IC50 = 0.71 and 1.45µM, respectively) than CS055 (IC50 =2.77µM) and 1 (IC50 =1.96µM). The activity was weakened when R1 was a fluorine atom and the R2 position included fluorine or chlorine (7b and 7c, IC50 from 0.70 to 16.26µM; 14b and 14c, IC50 from 2.57 to 16.46µM). These data clearly proved that the chlorine atoms at the R1 and R2 positions were the most optimal replacement, which illustrated that 2,6-dichlorobenzamide group was crucial to enhance the potency. However, most compounds showed poor cell inhibition towards SMMC7721 cells. In addition, compound 7c was tested for the cell morphology at concentrations of 4, 1 and 0.25µM respectively against all cancer cells (Figure 3). The image analysis suggested that compounds could great change cell morphology while inhibiting cell growth.
Next, compounds 7c and 14a were tested in the mouse embryonic fibroblast cells NIH 3T3 to determine their effects on normal cells. As shown in Table 3, compounds 7c and 14a exhibited low cytotoxicity with IC50 values of 4.47 and 4.64µM respectively compared with CS055 (IC50= 2.46µM). Considering the antiproliferative activity and cytotoxicity, compounds 7c and 14a were evaluated for further biological activity.
2.2.2 In vitro HDAC and CDK inhibitory activity assay
Compounds 7c and 14a were selected for in vitro inhibitory assays against HDACs and CDKs since these compounds showed high potency. It’s evident from Table 3 that compounds 7c and 14a revealed significant isoform selectivity for HDAC2 (IC50= 0.25 and 0.24nM respectively) over other HDACs. Compound 7c was above 40-fold and 4-fold more potent for HDAC1 and HDAC3 compared with CS055. Compound 14a exhibited comparable HDAC1 inhibition to CS055, whereas for HDAC3, 14a was less active with IC50 >1000nM. These two compounds have little inhibition effects on HDAC6 and HDAC8. In addition, among CDKs, compound 7c and 14a displayed higher inhibitory activity against CDK2 with IC50 values of 0.30 and 0.56nM respectively. It was noteworthy that these two compounds also had inhibitory activity against CDK1 (7c, IC50 = 8.63nM; 14a, IC50 =12.58nM). The IC50 values of 7c and 14a towards other CDKs were >1000nM. From these results, we can confirm that compounds 7c and 14a possessed dual HDAC and CDK inhibitory activity.
2.2.3 Molecular docking study
We successfully performed molecular docking studies to disclose the binding interactions of the representative compounds (7c and 14a) with HDAC2 and CDK2. As we speculated, compounds 7c and 14a were approached to the narrow binding-site channel, and the 2- aminobenzamide moiety chelated with zinc ion at the bottom catalytic site of HDAC2 (Figure S1Aand S1C in Supporting Information). It’s observed that both compounds 7c and 14a could form four important hydrogen bonds with HIS145, HIS146, GLY154 and TYR308 in the HDAC2 active site (Figure 4A and 4C). In addition, the compound 7c formed two other hydrogen bonding interactions with ASP181 and PHE210, and the cap group of 14a also formed one additional hydrogen bond with LEU276. It might explain the reason that the HDAC1 inhibitory activity of compound 7c was superior to that of 14a. Furthermore, the pyrazole-3-carboxamide group of the compounds 7c and 14a could form hydrogen bonds to the backbone residues GLU81 and LEU83 in the hinge area of CDK2, which was essential for CDK2 inhibitory effects of compounds. Besides, 7c formed extra hydrogen bonding interaction with HIS84 (Figure 4B), while 14a was able to form similar hydrogen bond with LYS89 (Figure 4D). The molecular docking results provided a valid explanation for the interactions of compounds (7c and 14a) with HDAC2 and CDK2, and also unveiled the appropriate dual inhibitor design.
2.2.4 Cell scratch assay
Cell scratch assay is considered as an effective method to measure the cell migration and repair ability. To evaluate the inhibitory effect of the compounds on cells migration, A375 cells and H460 cells were cultured, and then treated with 7c, 14a, 1, and CS055 for 48h. The images were mentioned in Figure 5. It’s observed that A375 and H460 cells migration were apparently suppressed by 7c and 14a as compared to the positive controls 1 and CS055. The inhibitory ability of 14a toward A375 cells migration was stronger than that of 7c.
2.2.5 Cell cycle analysis
To further explore the intracellular mechanisms of the representative compounds, 7c and 14a were carefully conducted for their cell cycle effects on A375 cell line, HCT116 cell line, H460 cell line and Hela cell line. Cells were treated with 7c and 14a at concentrations of 2, 1 and 0.5µM, as well as control (DMSO) for 24h, which were measured by the flow cytometry with propidium iodide (PI) staining. As presented in Figure 6, after treatment with 7c and 14a, cancer cells could show a loss in the proportion of cells in G0/G1 phase and an obvious increase in G2/M phase compared to control (DMSO). It’s evidently observed that these compounds significantly arrested A375 and HCT116 cells in G2/M phase. For instance, the percentages of A375 cells for 7c and 14a at dose of 2µM were increased from 1.13% to 57.74% and 66.48% respectively (the data from Figure S2 in Supporting Information). Taken together, the patterns of 7c and 14a blocking cell cycle were consistent with that of compound 1, which could be relevant to CDK2 inhibitory activity (Figure S2 in Supporting Information).
2.2.6 Cell apoptosis assay
We next wanted to determine that the capacity of compounds 7c and 14a to induce cell death via apoptosis in A375 cell line, HCT116 cell line, H460 cell line and Hela cell line. Cells were treated with 7c and 14a, CS055 and compound 1 as the reference drugs for 48h, which were stained with FITC-Annexin V and propidium iodide (PI), and then analyzed by flow cytometry. Figure 7 indicated that the compounds 7c and 14a could expedite cell apoptosis in a concentration-dependent manner. Compounds 7c and 14a could more effectively induce apoptosis than control (DMSO) towards all four cell lines. The apoptosis rates of 7c and 14a towards A375 cells were 97.22% and 73.08% respectively, which were higher than that of compound 1 (66.71%) and CS055 (62.60%) at the concentration of 2µM. The apoptosis rates of HCT116 cells induced by 7c and 14a were 60.6% and 77.1% respectively, however, compound 1 and CS055 induced 48.00% and 44.30% respectively. They showed stronger effects on cell apoptosis against A375 and HCT116 cells. For H460 and Hela cells, the apoptosis induced by 7c and 14a was better than that of CS055. Hence, it can be concluded that 7c and 14a could cause cell death through apoptosis.
2.2.7. Determination of immunofluorescence and ROS generation
Since the docking results revealed a potent affinity between CDK2 and HDAC1 with the representative compounds, we assessed the effects of 7c and 14a on the inhibition of CDK2 and the deacetylation of histone H3. Compounds showed favorable antiproliferative activity for A375 cells which possessed a good cell adhesion property, so A375 cell line was selected as the model. The immunofluorometric analysis was illustrated in Figure 8A and 8B. It’s demonstrated that 7c and 14a could lead to an apparent inhibition of CDK2 compared to control (DMSO) (Fig. 8A). They also induced an increase of acetylation of H3K9(Fig. 8B), which suggested that 7c and 14a might alter the acetylation of H3, inhibiting HDAC activity. These findings elucidated that compounds 7c and 14a acted as efficient CDK2 and HDAC inhibitors.
Reactive oxygen species (ROS) play a critical role in cellular processes involving cell growth, proliferation, development, differentiation, senescence and apoptosis[61, 62]. High levels of intracellular ROS might cause DNA damage, and ultimately resulted in cell death. The 2,7-dichlorofluorescein diacetate (DCFH-DA) is frequently applied for intracellular ROS detection. As mentioned in Figure 8C, the green fluorescence signal was hardly observed in the control group. In contrast, after treatment with the compounds 7c and 14a, cells displayed strong green fluorescence, indicating a significant increase in intracellular ROS levels. Accordingly, compounds 7c and 14a might accelerate intracellular ROS accumulation, leading to cancer cell death.
2.2.8 Pharmacokinetic parameters
Considering that compounds 7c and 14a exhibited excellent in vitro antitumor activity, the pharmacokinetic properties (PK) were investigated in ICR male mice. Compounds 7c and 14a were administered intraperitoneally (IP) at a dose of 20mg/kg respectively. The PK parameters were presented Table 4. The half-life (t1/2) of compound 7c was 2.61h, the maximum plasma concentration (Cmax) was 7570 ng/mL, and the area under concentration-time curve (AUC0-∞) was 30700 ng h/mL. However, compound 14a showed a short half-life of 1.63h, a Cmax of 2170 ng/mL and an AUC0-∞ of 7200 ng h/mL. In addition, compared with 14a (F=27.8%), compound 7c showed higher bioavailability with F= 63.6%. The data indicated that compound 7c possessed better pharmacokinetic properties than 14a.
2.2.9 In vivo antitumor activity of compound 7c
As compound 7c exhibited excellent antiproliferative activity towards HCT116 cells (IC50= 0.71µM) over other cell lines, we decided to evaluate the in vivo antitumor efficacy of compound 7c in the HCT116 xenograft nude mice models. When tumors grew to a volume of 100-300 mm3, the BALB/c female mice were randomly divided into treatment and control groups (6 mice per group). Compound 7c was intraperitoneally administered at 25 and 12.5 mg/kg once daily (QD) for 21 days. No significant body weight changes and toxicity signs were observed in the treatment groups (Fig. 9A). Compound 7c effectively inhibited the growth of HCT116 xenograft tumors compared to control. The tumor growth inhibitions (TGI) of 7c at 12.5 and 25 mg/kg were 37.0% and 51.0% respectively (Fig.9B-D). These results demonstrated that compound 7c possessed remarkable in vivo antitumor efficacy.
3. Conclusions
In summary, we have successfully designed and synthesized novel dual HDAC/CDK inhibitors. The representative compounds (7c and 14a) displayed potent antiproliferative activities against five selected human cancer cell lines with IC50 values ranging from 0.71 to 7.76µM. The compounds 7c and 14a also showed excellent HDAC and CDK inhibition with IC50 values at the nanomolar level. Moreover, compounds 7c and 14a significantly arrested cell cycle at G2/M phase and promoted apoptosis towards A375 cell line, HCT116 cell line, H460 cell line and Hela cell line. In addition, the immunofluorometric analysis indicated that 7c and 14a displayed significant inhibition of CDK2 and an increase of the acetylation of H3K9. They could also enhance ROS levels, and then cause cell death. Furthermore, the in vivo studies indicated that compound 7c showed good pharmacokinetic profile, and also exhibited potent antitumor efficacy in the HCT116 xenograft model (TGI= 51.0%).
4. Experimental section 4.1 Chemistry
H NMR and C NMR spectra were recorded on a Bruker AV III 400 spectrometer (400MHz) using DMSO-d6 as solvent. The chemical shifts were measured in parts per million (ppm) relative to Me4Si as internal standard. High-resolution mass spectra (HRMS) were obtained on an Agilent Q-TOF 6540 mass spectrometer. All reagents and solvents were purchased from commercial sources and were used without further purification. The progress of all reactions was monitored by thin layer chromatography (TLC), and precoated plates with silica gel F254 were purchased from Qingdao Haiyang Chemical Co. Ltd. All the final compounds were obtained ≥ 90% purity, as tested by high-performance liquid chromatography (HPLC) on a Thermo Scientific TM UltiMate TM 3000 systerm (column, C18, 5mm, 4.6mm × 250mm; mobile phase, gradient elution of methanol/ H2O (0.1% H3PO4); flow rate, 1.0mL/min; temperature, 35°C). Analyses indicated by the symbols of the elements or functions were within 0.4% of the theoretical values.
4.2 Biological evaluation
4.2.1 Cell antiproliferative activity assay
Human colorectal cancer cell line (HCT116), human lung cancer cell line (H460), human malignant melanoma cancer cell line (A375), human cervical cancer cell line (Hela) and human hepatoma cancer cell line (SMMC7721) were used in the current researches. We also selected the mouse embryonic fibroblast cells as the normal cells for cytotoxicity assays. The cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (ThermoFisher, USA) and 1% penicillin/streptomycin at 37 , 5%CO2 atmosphere. The cells were seeded into 96-well plates at a density of 4 × 103 cells / well. After 12h, the cells were treated with 10µL of various concentrations of compounds for 72h. Then 10µL per well of CCK-8 reagent were added, followed incubation for another 1h. The absorbance was measured at 450nm using an EnSpire multimode plate reader (PerkinElmer, USA).
4.2.2 In vitro HDAC inhibition assay
HDAC1 (BML-AK511), HDAC2 (BML-AK512), HDAC3 (BML-AK531), HDAC6 (BML-AK516) and HDAC8 (BML-AK518) were purchased by Enzo Life Sciences Inc. According to the instructions, various concentrations of the tested compounds (10µL) and HDAC enzyme solution (15µL) were mixed into a 96-well microplate. The mixture was cultured at 37 for 10min. Then 25µL of diluted HDAC substrate was added to start the deacetylation reaction at 37 for 30min.Then 50µL of 1x developer was added, and the mixture was incubated at 37 for 20min, which stopped the reaction. Fluorescence was analyzed using an EnSpire multimode plate reader (PerkinElmer, USA).
4.2.3 In vitro CDK inhibition assay
The CDK1, 2, 4, 6 and 7 inhibitory activity were measured by CDK1/ Cyclin A2 Kinase Enzyme System (Promega Catalog # V2961), CDK2/ Cyclin A2 Kinase Enzyme System (Promega Catalog # V2971), CDK4/ Cyclin D1 Kinase Enzyme System (CDK4/Cyclin D1, Active Catalog No. C31-10G signalchem), CDK6/ Cyclin D3 Kinase Enzyme System (Promega Catalog # V4510), and CDK7/ Cyclin H1 Kinase Enzyme System (CDK7/ Cyclin H1 , Active Catalog No. C36-102H signalchem), respectively. The kinase reaction mixture contained 1µL of the tested compound, 2µL of enzyme, and 2µL of 2.5 x ATP/substrate mix. After incubation at room temperature for 10min, the reaction was stopped by adding 5µL of ADP Glo™ Reagent. Forty minutes later, 10µL of Kinase Detection Reagent was added, and the plate was incubated at room temperature for 30min. Luminescence was recorded by an EnSpire multimode plate reader (PerkinElmer, USA).
4.2.4 Molecular docking
The three-dimensional structures of HDAC2 (PDB code: 4LXZ) and CDK2 (PDB code: 1PYE) were performed from the Protein Data Bank. The molecular docking was carried out via Surflexdock in Sybyl-X 2.0 (Tripos Inc.). The water and ligands were removed and the polar hydrogens were added to the protein structure. The molecular structures of tested compounds 7c and 14a were optimized with Tripos force field. Then compounds 7c and 14a were docked into HDAC2 and CDK2 using Surflex-Dock module.
4.2.5 Cell migration assay
A375 or H460 cells were plated in a 12-well plate at a density of 1.0 × 104 cells / well. Twelve hours later, a straight line was scratched at the bottom of each well using a 10µL pipette tip. After washing with phosphate-buffered saline (PBS), A375 cells (H460 cells) were treated with compounds 7c and 14a at the concentration of 0.25µM (0.5µM). Then cells were cultured for 48h. The cell images were captured by an OPTEC BDS200 microscope.
4.2.6 Cell cycle analysis
The different cells were plate in 6-well plates at a density of 1.0 × 106 cells / well. Twelve hours later, 20µL of compounds 7c, 14a, 1 and CS055 were added into 6 wells at the concentrations of 2, 1 and 0.5µM respectively for 24h. Then cells were harvested, washed with phosphate-buffered saline (PBS), and fixed in cold 75% ethanol at -20 for 24h. After adding 200µL of RNase A, cells were incubated at 37°C for 30min, and then stained with 200µL of propidium iodide (PI) at 4°C for 30min. Cells were screened by the flow cytometry.
4.2.7 Cell apoptosis assay
The different cells were plated in 6-well plates at a density of 1.0 × 106 cells / well. Twelve hours later, 20µL of compounds 7c, 14a, 1 and CS055 were added into 6 wells at the concentrations of 2, 1 and 0.5µM respectively for 48h. Then cells were harvested, washed with phosphate-buffered saline (PBS), and resuspended in 500µL of 1 X annexin-binding buffer. The resuspended cells were stained with 5µL of Annexin V-FITC (10 mg/ml) and 2µL of propidium iodide in the dark for 15min. Cells were detected by the flow cytometry.
4.2.8 Immunofluorometric assay
A375 cells were plated into confocal dishes with 8.0 × 103 cells / well. Twelve hours later, 20µL of compounds 7c and 14a were added into dishes at the concentration of 1µM for 12h respectively. Then cells were washed with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde. Thirty minutes later, cells were permeabilized with 0.3% Triton X-100, and incubated in 3% bovine serum albumin (BSA) for 1h. Then cells were treated with the diluted primary antibody (anti-Histone H3 (acetyl K9)) overnight at 4 , and incubated with the secondary antibody (IgG H&L (Alexa Fluor® 488)) for 2h in the dark. After adding DAPI, the cells were cultured at room temperature for 3min in the dark. The samples were analyzed by Leica TCS SP8 confocal fluorescence microscope (Leica Microsystems, Germany). For the immunofluorometric assay of CDK2 in A375 cells treated with compounds 7c and 14a, cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and incubated in 3% bovine serum albumin (BSA). One hour later, the cells were incubated with anti-CDK2 antibody (Alexa Fluor® 488) overnight at 4 , and then were stained with DAPI for 3min. The samples were analyzed by Leica TCS SP8 confocal fluorescence microscope (Leica Microsystems, Germany).
4.2.9 ROS accumulation assay
A375 cells were plated into confocal dishes with 1.0 × 106 cells / well. Twelve hours later, 20µL of compounds 7c and 14a were added into dishes at the concentration of 1µM for 12h respectively. The cells were washed with phosphate-buffered saline (PBS), and incubated with 1µL of DCFH-DA at 37°C for 0.5h. Then the cells were washed with PBS three times, and analyzed by Leica TCS-SP8 confocal fluorescence microscope (Leica Microsystems, Germany).
4.2.10 Pharmacokinetic parameters
The pharmacokinetic properties (PK) were carried out in ICR male mice, by administering 7c and 14a intraperitoneally (IP) at a dose of 20mg/kg respectively. Compounds 7c and 14a were dissolved in a solution of DMSO: CrEL: Saline=10:10:80 (v/v/v). The blood samples were collected at 0.25h, 0.5h, 1h, 2h, 4h, 8h, 12h and 24h after intraperitoneal injection, and then analyzed by LC-MS/MS system. The PK parameters were estimated by noncompartmental model using WinNonlin 8.0.
4.2.11 In vivo antitumor activity assay
All animal experiments were approved by the local ethics committee. BALB/c nude female mice (5-6 weeks old) were afforded by Vital River Laboratory Animal Technology Co. Ltd. To establish HCT116 xenograft models, 3×106 human colorectal cancer cells were subcutaneously injected into the front-right axilla region of nude mice. When tumors grew to a volume of 100-300 mm3, the BALB/c female mice were randomly divided into treatment and control groups (6 mice per group). Mice in the treatment groups were intraperitoneally injected with compound 7c at a dose of
12.5mg/kg and 25mg/kg once daily (QD) for 21 days respectively, and mice in the control group were injected with equal volume of 0.5% MC aqueous solution. During treatment, tumor volumes and body weighs were measured every 3 days. The tumor growth inhibition (TGI) was calculated according to the following formula: TGI =100%×[1－(TVt(T)－TVinitial(T))/(TVt(C)－TVinitial(C))], where TVt(T) and TVinitial(T) are the tumor volume measured at initial time and final time of treatment in the treatment group respectively, and TVt(C) and TVinitial(C) are the tumor volume for the control group.
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