Synthesis of 4-thio-5-(2-thienyl)uridine and cytotoxicity activity against colon cancer cells in vitro

A novel anti-tumor agent 4-thio-5-(2′′-thienyl)uridine (6) was synthesized and the in vitro cytotoxicity activity against mice colon cancer cells (MC-38) and human colon cancer cells (HT-29) was evaluated by MTT assay. The results showed that the novel compound had antiproliferative activity toward MC-38 and HT-29 cells in a dose-dependent manner. The cell cycle analysis by flow cytometry indicated that compound 6 exerted in tumor cell proliferation inhibition by arresting HT-29 cells in the G2/M phase. In addition, cell death detected by propidium iodide staining showed that compound 6 efficiently induced cell apoptosis in a concentration-dependent manner. Moreover, the sensitivity of human fibroblast cells to compound 6 was far lower than that of tumor cells, suggesting the specific anti-tumor effect of 4-thio-5-(2′′-thienyl)uridine. Taken together, novel compound 6 effectively inhibits colon cancer cell proliferation, and hence would have potential value in clinical application as an antitumor agent.


Introduction
The ow of genetic information is directed by nucleic acids (DNA and RNA). The base analogues structurally similar to the four canonical bases can be incorporated into DNA, which alter their properties and facilitate basic studies of DNA-related processes such as its interaction with proteins, replication and transcription. 1,2 Structural modication of nucleosides is biologically meaningful, and has led to the discovery of a variety of new therapeutic agents, which includes antiviral and anticancer agents. Pyrimidines and fused pyrimidines, being an integral part of DNA and RNA have considerable chemical and pharmacological importance. Thio-pyrimidine compounds in which an oxygen atom in the base is replaced by sulfur have been reported to show improved anti-cancer activity and immune enhancement. [3][4][5][6][7] Thiophene and its derivatives as important sulfurcontaining ve-member heterocyclic compounds have various biological and pharmacological properties in terms of antimicrobial and antitumor activities. [8][9][10] As a rich electronic aromatic ring compound, the charge density of thiophene is greater than that of benzene ring, and thus, it is easy for thiophene to occur the p-p interactions. Moreover, the sulfur atom in thiophene is certain nucleophilic and easy to interact with electrophilic receptors or form metal or metal ion. From the viewpoint of reducing the drug side effects, enhancing the drug targeting and improving the pharmacokinetic properties of drugs and the corresponding therapeutic index, new compound with the characteristics of both thiophene and thio-pyrimidine are certainly with explore as attractive antitumor agent. 11,12 Colon cancer is a kind of digestive tract malignant tumor, commonly occurring at the junction of the rectum and sigmoid colon. The incidence of colon cancer begins to rise at age 40 and peaks at age 60 to 75, and the main life-style related cause is high-fat diet and inadequate cellulose intake. Colon cancer ranks third among the leading causes of cancer-associated death aer lung and prostate cancer for men and aer lung and breast cancer for women. 13 In recent years, there is an upward trend in China as the improvement of people's living standard and changes in diet. Accordingly, the development of novel chemotherapeutic agents is an urgent issue.
In this study, a novel 4-thio-5-(2 00 -thienyl)uridine (6) compound was synthesized by introducing a thiophene to uridine by a series of reaction (Scheme 1). The cytotoxicity of compound was evaluated by in vitro administering to mice colon cancer cells line (MC-38) and human colon cancer cells line (HT-29) by MTT assay, respectively. Compound 6 was found to have antiproliferative activity against colon cancer cells. Flow cytometry examination revealed that compound 6 inhibited cell growth through cell cycle arrest at G2/M phase. Combined with no cytotoxic and apoptotic effect on human broblast cells, 4-thio-5-(2 00 -thienyl)uridine (6) have potential clinical applications.

Apparatus and materials
Reagents were obtained from commercial suppliers and were used without further purication. Melting point was determined on a XR-4-type micro-melting point detector, and was uncorrected. The compounds synthesized were puried by column chromatography using silica gel (200-300 mesh) except for recrystallization and thin-layer chromatography (TLC) using silica gel 60 F254 plates (250 mm; Qingdao Ocean Chemical Company, China). IR spectra were recorded using a Nicolet 550 Spectrophotometer (4000-400 cm À1 ) with a crystalline sample spread on KBr pellets. UV spectra were recorded using UV-vis spectrophotometer (JASCO, Japan); 1 H NMR and 13 C NMR spectra were obtained by a 500 MHz Bruker AV-400 spectrometer with TMS as an internal standard. The mass spectrum was obtained on Hewlett-Packard 1100 LC/MSD spectrometer.

Crystal structure
The crystallographic data for 4-thio-5-(2 00 -thienyl)uridine were summarized in Table 1. X-ray diffraction data were collected at 120 K using a Bruker Nonius Kappa CCD diffractometer mounted at the window of a Bruker FR591 rotating anode (Mo Ka, l ¼ 0.71073Å) and equipped with Bruker APEX II detector and Oxford Cryosystems Cryostream device. ORTEP views of 4thio-5-(2 00 -thienyl)uridine were given in Fig. S4. † The structure was solved by direct methods using SHELXS97. 22 The crystal structure has been deposited in Cambridge Structural Database: CCDC depository number 1479957.

Toxicity test on colon cancer cells
The MTT colorimetric method and ow cytometry were employed to analyze the anti-tumor activity of 4-thio-5-(2 00thienyl)uridine on mice colon cancer cell line MC-38 and human colon cancer cell line HT-29, which were purchased from the American Type Culture Collection.
2.4.1. Cell antiproliferative activity assay. The proliferation inhibitory activities of the synthesized target compounds was evaluated using mouse and human colon tumor cell lines (MC-38 and HT-29) by the MTT method in vitro. MC-38 and HT-29 cells were cultured in DMEM and RPMI1640 medium, 10% fetal calf serum, 100 mg mL À1 penicillin, and 100 mg mL À1 streptomycin at 37 C under 5% CO 2 , respectively. Then the cells were seed into 96-well plate at 1 Â 10 4 cells per well and coincubated with 4-thio-5-(2 00 -thienyl)uridine under different concentrations (nal concentration: 100 mM, 200 mM, 300 mM, 400 mM and 500 mM) for 24 h, respectively. At the end of this time interval, 20 mL (5 mg mL À1 ) MTT (3-(4,5 dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) was added to each well, and aer incubation at 37 C for 4 h the MTT solution was removed and 100 mL of dimethylsulfoxide (DMSO) was added to dissolve the crystals. The absorbance of each well was measured at 570 nm.
2.4.2. Cell cycle distribution and apoptosis analysis by ow cytometry detection. Cell cycle distribution and apoptosis analysis was conducted according to the reported procedure. 24 Briey, the HT-29 cells were inoculated into a 6-well cell culture plate at 5 Â 105 cells per well and cultured overnight. Then, 2 mL of the culture medium containing different concentrations of 4-thio-5-(2 00 -thienyl)uridine were added to each well respectively and co-incubated for another 48 hours. Aerwards, the cells oating in the supernatant were combined with the adherent fraction, washed with phosphate buffered saline (PBS) thrice, xed with cool ethanol, treated with RNase, and then stained with propidium iodide (50 mg mL À1 ) at 37 C in a dark environment. The samples were immediately analyzed by ow cytometry.

Statistical analysis
Data were presented as mean AE SD of three independent experiments. One-way analysis of variance (ANOVA) was performed on the data to assess the impact of the variables on the results. SPSS 13.0 statistical soware (SPSS Inc., Chicago, USA) was used for the statistical analysis of all data. A p value of #0.05 was considered to be statistically signicant.

Results and discussion
3.1. Chemistry synthesis 3.1.1. Synthetic route to 4-thio-5-(2 00 -thienyl)uridine. The synthetic route to the target 4-thio-5-(2 00 -thienyl)uridine was depicted in Scheme 1. The starting uridine 1 was rst acetylated at its 2 0 -, 3 0 -and 5 0 -positions to produce the acetylated compound 2. The compound 3, an important intermediate was easily synthesized via iodine and CAN (ceric ammonium nitrate) in MeCN. Then, the synthesis of compound 4 was carried out through palladium catalyzed stille cross coupling reaction to produce the thien-conjugated uridine 4 with a yield of over 90%. [14][15][16][17] Next, compound 5 was prepared according to the reported procedure with some modication. 18,19 Finally the protecting groups were removed with ammonia in methanol to produce 4-thio-5-(2 00 -thienyl)uridine 6 with good yields. Aer the target compound was successfully prepared, structural characteristics of the compound were examined on UV, MS and NMR spectroscopies, the results were shown in ESI (Fig. S1-S3 †).

Cytotoxicity activities
3.2.1. Anti-proliferative activity of compound 6 against mouse colon cancer cell MC-38 in vitro. The cytotoxic effect of the prepared compound was evaluated against mouse colon cancer cell MC-38. The MC-38 cell line was treated with compound 6 (nal concentrations: 100, 200, 300, 400, 500 mM) for 48 h, and the viable cells were measured by a MTT assay. As shown in Fig. 1, the cell growth inhibition was observed under different concentration of 4-thio-5-(2 00 -thienyl)uridine. At a low concentration of 100 mM, the cell survival rate dropped to around 65%, then gradually declined at a slower rate along with the increase of compound concentration, nally reached the minimal value of 44% at 500 mM, suggesting that 4-thio-5-(2 00 -thienyl)uridine induced a dose-dependent proliferation inhibition of MC-38 cells. Additional increase in dosage did not further decrease the cell survival rate, and no signicant cell death was detected and maintained at a relatively stable state (data not shown). The variance analysis data suggested that compound 6 had signicant inhibitory inuence on MC-38 cell growth (p < 0.03). In comparison of the adjacent low-dose groups, only the 200 mM group showed signicant difference (p < 0.05) from the adjacent groups in the inhibition of MC-38 cell growth, while no signicant differences were found between the remaining groups, exhibiting a slow antiproliferative trend of the target compound along with the increase of concentrations.
3.2.2. Anti-proliferative activity against human colon cancer cells HT-29. The in vitro proliferation inhibitory activity of the prepared compounds was evaluated against the human colon cancer cell HT-29. Aer 48 h treatment under different concentrations (nal concentrations: 100, 200, 300, 400, 500 mM), MTT assay was adopted to examine the cell survival rate. As shown in Fig. 2, compound 6 exhibited low antiproliferative activities at 100 mM level, with cell viability of 92%. However, the numbers of living cells considerably decreased accompanied with the concentration increase of compound, and reached the minimal value of approximately 43% at 500 mM level, suggesting that compound 6 induced dose-dependent proliferation inhibition of HT-29 cells. Subsequent increases in dosage did not further reduce the HT-29 cell survival rate, but rather maintained at a relatively stable state (data not shown). The variance analysis of the data suggested that the title compound exerted a signicant antiproliferative activity against HT-29 cell from concentration of 200 mM (p < 0.001). In addition, the signicant difference between the test and adjacent low-dose groups indicated that the inhibitory effect considerably enhanced when the compound 6 concentrations was elevated (p < 0.01).  for 24 h, then subjected to ow cytometry to examine the cell cycle distribution. As showed in Fig. 3 and 4, low concentration (100 mM) of compound 6 almost no inuence on cell cycle progression, while higher concentration resulted in G2 phase and mitosis arrest in a dose-dependent manner, which reected by the population of cells in the G2/M phase increasing from 10.85% at 200 mM to 36.76% at 500 mM. On the other hand, there was no signicant impact of compound 6 on the numbers of cells at G1 phase and S phase. Since the cells in G2/M phase had completed DNA replication (containing 4N DNA) and entered the mitosis process, the accumulation of cells at this stage implied that compound 6 plays an important role in mitotic arrest. In addition, a fraction of cell population with DNA content lower than that of G1 (sub-G1 phase) gradually increased aer exposure to compound 6, from 10.3% (control group) to 20.54% (100 mM), 21.197% (200 mM), 31.47% (300 mM), 40.46% (400 mM), and 44.63% (500 mM), respectively. As the DNA replication had not yet occurred at G1 phase, the total DNA content lower than the value in G1 phase indicated the occurrence of DNA degradation in the cell, which commonly was considered as an important feature of cell apoptosis. 20,21 Therefore, the increase of cell numbers at the sub-G1 phase revealed that compound 6 could induce apoptosis of HT-29 cell at such low concentration of 100 mM. Meanwhile, the population of apoptotic cells elevated under higher concentration, suggesting the apoptosis induction occurred in a dose-dependent manner. These results indicated that cell cycle arrest in the G2/M stage and apoptosis induction was important factors by which compound 6 exerted its inhibitory effects on HT-29 cells.
3.2.4. Anti-proliferative activity against normal human broblasts. The ideal anti-cancer drug should target tumors specically while causing minimal side effects. The impact of the drug on normal cell proliferation was one of the key checkpoints in determining its specic anti-tumor activity and side effect prole. Therefore, we extended our study on examining the effects of different concentrations of compound 6 on the proliferation of broblast cells. Aer 48 hours of drug treatment under different concentrations, MTT was added to the broblast cells. The OD values were measured and the cell proliferation rates were calculated. The results obtained revealed that the sensitivity of normal human broblast cells to compound 6 were lower than that of tumor cells, and higher concentration gave no inuence on cell proliferation (Fig. 5), indicating that 4-thio-5-(2 00 -thienyl)uridine is a promising agent with specic anti-tumor effect.

Conclusions
In this study, we successfully synthesized a novel compound: 4thio-5-(2 00 -thienyl)uridine (6) by connecting thiophene to 5position of thiopyrimidine. In vitro pharmacological analysis demonstrated the compound 6 exerted proliferation inhibitory activity against MC-38 and HT-29 cells by arresting cell cycle at the G2/M phase and inducing apoptosis in a dose-dependent manner. The sensitivity of the normal human broblasts to compound 6 was found to be substantially lower than that of the tumor cells and different concentrations showed no signicant inhibition of normal broblast proliferation, suggesting that 4-thio-5-(2 00 -thienyl)uridine (6) and its analogues would be promising candidates for anti-tumor drug development.