bState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;
cSchool of Pharmacy, Nantong University, Nantong 226001, China;
d. Department of Hepatobiliary Surgery, Affiliated Hospital, Nantong University, Nantong 226001, China
Malignant neoplasm is severe life threatening. Ras proteins encoding by ras genes serve as molecular switches tightly regulating intracellular signal transduction pathways controlling cell proliferation,differentiation,and cell apoptosis in normal cells [1, 2, 3]. However,oncogenic mutations in ras genes result in development of the biological process disorders,especially the occurrence of tumors in humans [4, 5, 6]. Therefore,Ras proteins and Ras related signaling are considered as promising targets in anticancer drug discovery. Farnesylthiosalicylic acid (FTS),a potent competitive Ras inhibitor,has been reported to display chemopreventive activities in clinical trials [7, 8, 9, 10, 11]; however,it displays a limited therapeutic effect [12, 13]. Our previous studies have developed a number of promising FTS derivatives that displayed significant cytotoxicity against cancer cells [14, 15, 16, 17]. Among these FTS derivatives,FTS-diamines evidently improved the antitumor activities of FTS,but failed to be selective to tumor cells . It would be great significant to searching for more potent and safer inhibitors targeting Ras proteins and Ras-related signaling pathway.
Acetylsalicylic acid (aspirin),a well known nonsteroidal antiinflammatory agent,has been revealed to inhibit cyclooxygenase (COX) activity and exhibit the extraordinary potent for the treatment of cancer [18, 19, 20, 21]. Epidemiological studies suggested that the regular intake of aspirin was associated with a reduction in the incidence of malignancies,including colorectal, gastrointestinal,and lung cancer [21, 22]. What’s more,reports demonstrated that acetylsalicylic acid and its metabolite salicylic acid (SA) could selectively induce apoptosis in several colorectal carcinoma cell lines [23, 24, 25]. Thus,acetylsalicylic acid or SA would be an excellent antitumor active fragment for the development of novel anticancer agents.
Given these,novel series of FTS/SA hybrids were designed by introducing salicylic acid fragment into parent molecule FTS with linkers of different length of diamines. We hypothesized that these new hybrids would exert inhibitory activity to tumor cells in a synergistic effect,leading to tumor cell apoptosis. Herein,we reported ten novel FTS/SA hybrids and the in vitro biological evaluation of their antitumor activity,selective cytotoxicity and apoptosis-inducing effects. 2. Experimental 2.1. Chemistry
General: Infrared (IR) spectra were recorded on a Nicolet Impact 410 instrument (KBr pellet). 1H NMR spectra were recorded with a Bruker Avance 300 MHz spectrometer at 300 K with TMS as an internal standard. MS spectra were recorded on a Mariner Mass Spectrum (ESI). Element analysis was performed on an Eager 300 instrument. All compounds were routinely checked by TLC and 1H NMR. TLCs and preparative thin-layer chromatography were performed on silica gel GF254,and the chromatograms were conducted on silica gel (200-300 mesh,Merck) and visualized under UV light at 254 and 365 nm. All solvents were reagent grade and,when necessary,were purified and dried by standards methods. Solutions after reactions and extractions were concentrated using a rotary evaporator operating at a reduced pressure of ca.20 Torr. Organic solutions were dried over anhydrous sodium sulfate. Compounds 2 and 3 were commercially available.
The synthetic route of 9a-e and 10a-e was outlined in Scheme 1.
(E,E)-Bromofarnesyl 3 was reacted with methyl thiosalicylate 2 in the presence of K2CO3 and KI to obtain methyl (E,E)-farnesylthiosalicylicate 4,which was followed hydrolyzed with NaOH solution to gain parent compound1(FTS). In addition, different diamines 5a-e was treated with (Boc)2O to generate one sideN-protection of6a-e. Then FTS was respectively reacted with 6a-e to form 7a-e in the presence of ethyl chloroformate andN-methylmorpholine. Compounds 9a-e were deprotected by treating with trifluoroacetic acid (TFA),then treated with O-acetylsalicylryl chloride prepared from the acid with SOCl2 to yield target compound 9a-e. Finally,compounds 9a-e was hydrolyzed with NaOH to gain 10a-e. The detailed experimental procedures and data of selected compounds were shown in Ref. . 2.2. Biological evaluation
MTT assay: Human hepatocellular carcinoma cells (SMMC-7721 and HepG2),human bladder carcinoma cells (EJ),human gastric cancer cells (SGC7901),human lung cancer cells (H460), human breast cancer cells (MCF-7) and human hepatocellular normal cells (LO2) at 104 cells per well were cultured in 10% FBS DMEM in 96-well flat-bottom microplates overnight. The cells were incubated in triplicate with,or without,different concentrations of each tested compound for 48 h. During the last 4 h incubation,30mL of tetrazolium dye (MTT) solution (5 mg/mL) was added to each well. The resulting MTT-formazan crystals were dissolved in 150mL DMSO,and absorbance was measured spectrophotometrically at 570 nm using an ELISA plate reader. The inhibition induced by each tested compound at the indicated concentrations was expressed as a percentage. The concentration required for 50% inhibition (IC50) was calculated using the software (GraphPadPrism Version 4.03).
Flow cytometry assay of cell apoptosis: SMMC-7721 cells were cultured overnight and incubated in triplicate with the tested compound 10a(3.0,6.0 and 12mmol/L) or vehicle for 48 h. The cells were harvested and stained with FITC-Annexin V and PI (BioVision) at room temperature for 15 min. The percentage of apoptotic cells was determined by flow cytometry (Beckman Coulter) analysis.
Western blot assay: The mechanisms of the cell apoptosis by western blot assay. SMMC-7721 cells at 1.5×105/mL were treated with 3.0,6.0 or 12mmol/L 10a or vehicle control for 8 h. After harvested and lysed,the cell lysates (50mg/lane) were separated by SDS-PAGE (12% gel) and transferred onto nitrocellulose membranes. After blocked with 5% fat-free milk,the target proteins were probed with anti-Bcl-2,anti-Bax,anticaspase-3 and anti-β-actin antibodies (Cell Signaling,Boston), respectively. The bound antibodies were detected by HRPconjugated second antibodies and visualized using the enhanced chemiluminescent reagent. The relative levels of each signaling event to control β-actin were determined by densimetric scanning. 3. Results and discussion
3.1. The anti-proliferation activities of compounds 9a-e and 10a-e
The growth inhibitory activity of target compounds 9a-e and 10a-e against human hepatocellular carcinoma cells (SMMC-7721 and HepG2),human bladder carcinoma cells (EJ),human gastric cancer cells (SGC7901),human lung cancer cells (H460) and human breast cancer cells (MCF-7) were evaluated in vitro by MTT assay,and the positive controls were FTS and sorafenib. Their IC50values were shown in Table 1. It was found that most of compounds displayed moderate to good anti-proliferation activities against each tested cancer cell,and their antitumor activities were clearly better than that of FTS (IC50= 41.3-103.2mmol/L). Several compounds,such as 9a-c and 10a-c,exhibited comparable to or even better antitumor activities than sorafenib (IC50= 7.87-22.9mmol/L). Especially,antitumor activities of10awere strongest with IC50 values of 5.72-9.76mmol/L,which were 5-16 fold less than those of FTS.
Selectivity experiment was also carried out to evaluate the selective cytotoxicity of 10a against SMMC-7721 and human hepatocellular normal cells (LO2). As presented in Fig. 1,compound10adiaplayed weak growth inhibitory activity against human normal cells,while strong anti-proliferation activity against SMMC-7721 was observed in a dose-dependent manner, which demonstrated 10a possessed selectively cytotoxicity against tumor cells.
|Fig. 1. Inhibitory effects of 10a on the proliferation of SMMC-7721 and LO2 cells. SMMC-7721 and LO2 cells were incubated with the indicated concentrations of 10a for 48 h. Cell proliferation was assessed using the MTT assay. Data are means±SD of the inhibition (%) from three independent experiments.|
To determine whether the inhibitory activities on tumor cells of 10a was due to induce cell apoptosis,apoptosis assay was further investigated employing SMMC-7721 cells which were incubated with vehicle alone,10a at 3.0,6.0,and 12.0mmol/L final concentrations,and the percentages of apoptotic cells were determined by stained with FITC-Annexin V/PI staining and flow cytometry. Fig. 2 showed that the frequency of SMMC-7721 cell apoptosis was unobvious in the untreated group. However,the percentages of the cell apoptosis showed significantly increased in SMMC-7721 cells by treated with 10a at gradually increased dose. Treatment with 12.0mmol/L of 10a induced over 80% of SMMC-7721 cell apoptosis. The results showed that10acould induce tumor cell apoptosis in a dose-dependent manner.
|Fig. 2. Compound 10a induced SMMC-7721 cell apoptosisin vitro. SMMC-7721 cells were incubated with the indicated concentrations of 10afor 48 h,and the cells were stained with FITC-Annexin V and PI,followed by flow cytometry analysis. (A) Flow cytometry analysis. (B) Quantitative analysis of apoptotic cells. Data are expressed as means±SD of the percentages of apoptotic cells from three independent experiments. *P<0.01vs.control.|
To further investigate the preliminarily molecular mechanisms underlying the cell apoptosis profiles in 10a-treated cells,the expression of Bcl-2,Bax and caspase-3 proteins was detected using western blot assay,andb-actin as the control. It is well known that Bcl-2 and Bax are anti-apoptotic and pro-apoptotic factor,respectively,and caspase-3 is the execution factor of apoptosis. As shown in Fig. 3A,the protein levels of Bcl-2 were dramatically reduced and the expression of Bax proteins was significantly increased in 10a-treated cells in a concentration-dependent manner. Moreover,activation levels of caspase-3 were also dosedependently improved.
|Fig. 2. Effect of 10a on the expression of apoptosis-related proteins in SMMC-7721 cells. (A) The expression of Bax,Bcl-2,caspase 3 andb-actin was examined by Western blot analysis. SMMC-7721 cells were incubated with,or without10aat the indicated concentrations for 48 h and the levels of protein expression were detected using specific antibodies. Data shown are representative images of each protein for three separate experiments. (B) Quantitative analysis: The relative levels ofeach protein compared to controlb-actin were determined by densimetric scanning. Data are expressed as means±SD from three separate experiments. *P<0.01vs.control.|
The SAR analysis revealed that most of the FTS/SA hybrids exhibited stronger antitumor activities than FTS and SA. The explosion of phenolic hydroxyl group of 10a-e by removing the acetyl group of 9a-e resulted in more potent against cancer cells. In addition,hybrids with relatively shorter diamine linker displayed more potent,for instance, both9aand10aexhibited optimal antitumor activities in their respective type of compounds. However,the precise mechanisms underlying the SAR of these hybrids remain further investigation. 4. Conclusion
In summary,a series of FTS/SA hybrids was designed and synthesized and theirin vitroantitumor activities were evaluated. Most of them displayed stronger anti-proliferation activities than FTS and SA against six cancer cellsin vitro,and the most potent compound10ashowed even stronger antitumor activities than sorafenib. Furthermore,10a could selectively inhibit tumor cell proliferation. Moreover,10a could significantly induce cancer cell apoptosis in a dose-dependent manner and up-regulated the expression of Bax and caspase-3 and down-regulated Bcl-2. Therefore,our novel findings may provide a new framework for the design of new hybrids for the intervention of human cancers.Acknowledgments
We gratefully acknowledge the Natural Science Foundation of China (No. 81302628) and Jiangsu Province (No. BK2011389), China Pharmaceutical University for the Open Project Program of State Key Laboratory of Natural Medicines (No. SKLNMKF201415) for the financial support,and also thank a project funded by the Priority Academic Programs Development (PAPD) of Jiangsu Higher Education Institutions.
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|||The experimental procedures and data of selected compounds: 2-(2-(2-((2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trienylthio)benzamido)ethylcarbamoyl)-phenyl acetate (9a): To the CH2Cl2 (5 mL) solution of compound 7a (0.5 g, 1 mmol) was added TFA (5 mL), and the mixture was stirred at room temperature overnight. The solvent was removed in vacuo, and the product was dissolved in dry CH2Cl2 (10 mmol) and triethylamine (0.21 mL, 1.5 mmol) was added, then p-acetoxyphenylpropenoic acid chloride (0.2 g, 1 mmol) dissolved in dry CH2Cl2 (10 mmol) was dropwise added at 0 ℃, and the reaction mixture was stirred at room temperature. After the reaction completed (1 h later), the resulting mixture was allowed to pour into ice-water (30 mL), and extracted with ethyl acetate (30 mL × 3). The organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated to afford the crude product. Then it was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 2/1, v/v) to give compound 9a, pale yellow oil, and yield 81%. IR (KBr, cm-1): ν 3262, 3074, 2925, 1748, 1565, 1245, 1198; 1H NMR (300 MHz, CDCl3): δ 7.62-7.67 (m, 2H, Ar-H), 7.34-7.39 (m, 2H, Ar-H), 7.25-7.31 (m, 4H, Ar-H), 5.25 (m, 1H, SCH2CH), 5.07 (m, 2H, 2 × CH2CH=CCH3), 3.44-3.55 (m, 6H, SCH2, 2 × NCH2), 2.28 (s, 3H, Ac), 1.97-2.02 (m, 8H, 2 × CCH2CH2CH), 1.50-1.75 (m, 12H, 4 × CH=CCH3); MS (ESI): m/z 563 [M+H]+; Anal. Calcd. for C33H42N2O4S: C, 70.43; H, 7.52; N, 4.98; Found: C, 70.28; H, 7.65; N, 5.84. 2-Hydroxy-N-(2-(2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienylthio)benzamido)ethyl)benzamide (10a): Compound 9a (0.28 g, 0.5 mmol) was dissolved in methanol (5 mL) and 1 mol/L NaOH (1 mL) was added, and the mixture was stirred at room temperature for 2 h, then was added water (20 mL), neutralized with 2 mol/L HCl to pH 5 and extracted with ethyl acetate (20 mL × 3). The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give 10a, pale yellow oil, and yield 81%. IR (KBr, cm-1): ν 3285, 3048, 2927, 1643, 1552, 1232, 1185; 1H NMR (300 MHz, CDCl3): δ 7.57-7.62 (m, 2H, Ar-H), 7.32-7.38 (m, 2H, Ar-H), 7.24-7.31 (m, 4H, Ar-H), 5.26 (m, 1H, SCH2CH), 5.08 (m, 2H, 2 × CH2CH=CCH3), 3.48-3.60 (m, 6H, SCH2, 2 × NCH2), 1.98-2.01 (m, 8H, 2 × CCH2CH2CH), 1.50-1.79 (m, 12H, 4 × CH=CCH3); MS (ESI): m/z 521 [M+H]+; Anal. Calcd. for C31H40N2O3S: C, 71.50; H, 7.74; N, 5.38; Found: C, 71.37; H, 7.82; N, 5.31. 2-Hydroxy-N-(3-(2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienylthio)benzamido)-propyl)benzamide (10b): Refer to the synthesis of 10a, compound 10b was obtained from 9b, pale yellow oil, and yield 94%. IR (KBr, cm-1): ν 3275, 3056, 2928, 1636, 1544, 1228, 1191; 1H NMR (300 MHz, CDCl3): δ 7.61-7.65 (m, 2H, Ar-H), 7.36-7.42 (m, 2H, Ar-H), 7.26-7.34 (m, 4H, Ar-H), 5.26 (m, 1H, SCH2CH), 5.07 (m, 2H, 2 × CH2CH=CCH3), 3.48-3.55 (m, 6H, SCH2, 2× NCH2), 3.32-3.36 (m, 2H, NCH2CH22), 1.98-2.02 (m, 8H, 2 × CCH2CH2CH), 1.54-1.71 (m, 12H, 4 × CH=CCH3); CCH3); MS (ESI): m/z 635 [M+H]+; Anal. Calcd. for C32H42N2O3S: C, 71.87; H, 7.92; N, 5.24; Found: C, 71.73; H, 8.01; N, 5.37.|