We have synthesized di(3-thienyl)methanol (2) and di(3-thienyl)methane (3) and studied their anticancer effects (Scheme 1 and Figure 1). The structures of compound 2 and 3 are clearly supported by their ¹H, ¹³C-NMR spectra and microanalysis.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay has been widely accepted as a reliable way to measure the cell proliferation rate and cell death [15,16]. The data obtained by MTT assay show that compounds 2 and 3 have inhibitory effects on the growth of T98G and HEK cells in dosage-dependent manners. Compounds 2 and 3 can inhibit 50% T98G cell growth (IC₅₀) obviously in the 60–200 µg/mL range after 24, 48 and 72 h of the treatment (Figure 2). Maximum inhibitory effect was shown by compound 2 on T98G cells after 72 h of treatment, however compound 3 also effectively inhibits the growth of T98G cells at all the concentrations (0.7–2,500 µg/mL) and without any time dependent effects (Figure 2). Overall compound 2 was the most potent one, having significant a inhibitory effect on T98G cells growth. Both compounds are less toxic to the HEK cells with 75%–97% viability at 60 µg/mL concentration at all time intervals, which further increased by decreasing the concentration of the compounds (Figure 2). For further studies, we have selected compound 2 on the basis of its significant toxicity against T98G brain cancer cells.

Figure 3 shows the growth kinetics of T98G cells treated with compound 2. Cell proliferation kinetics have been studied at 24, 48, 72 h after compound treatment, following trypsinization and counting total cells per plate by using a trypan blue dye and hemocytometer. Data obtained from the growth kinetics assay shows that compound 2 has inhibitory effects on the growth of T98G cells in a concentration dependent manner.

It was also noted that the cells exposed to 2.2 and 6.6 µg/mL concentrations show less growth inhibitory effects than those treated with 20, 60 and 200 µg/mL concentration. Maximum effect was shown by 200 µg/mL concentration of compound 2, which inhibits the growth of cells up to 93% at 48 and 72 h after treatment and its viability range was 7%–8%. In the case of 20 and 60 µg/mL exposures, we found 45%–60% cells death at all time intervals. Cell morphology analysis revealed that shape and size of viable cells were also affected by treatment of compound 2 (Figure 4). From Figure 4 it is found that there was remarkable differences in shape and size of treated cells at 2.2, 20, 60 and 200 µg/mL after treatment.

The clonogenic assay shows the effect of compound 2 on the colony-forming capacity and survival of exponentially growing T98G cells. We have used the clonogenic assay for confirming the growth inhibition results of compound 2. Clonogenic assay or colony formation assay is an in vitro assay based on the ability of a single cell to grow into a colony. Only a fraction of seeded cells retains the capacity to produce colonies after cytotoxic drug treatment. As observed in Figure 5, the surviving fraction of T98G cells has been drastically decreased after its treatment by compound 2. Compound 2 treatment enhances cell death and also inhibits colony formation capability in the T98G cell population in a concentration dependent manner. After treatments with different concentrations (2.2–200 µg/mL), the surviving fraction of T98g cells declines, as evidenced by the reduction in the number of colonies formed. Even at low doses, 2.2, 6.6 and 20 µg/mL exposure of compound 2 shows a significant decline in colony survival and the surviving fractions was found to be 0.85, 0.71 and 0.58, respectively. However, a significant drastic decline in their surviving fraction could be observed after exposure to 60 and 200 µg/mL treatment and their surviving fractions were found to be 0.32 and 0 (zero), respectively. This shows that these treatments significantly inhibit the colony formation capabilities of brain cancer cells at all the dosages.

In recent years, the in vitro micronucleus (MN) assay has become an attractive tool for measuring genotoxicity by physical and chemical agents, due to its capacity to detect clastogenic and aneugenic events, simplicity of scoring, accuracy, multipotentiality and wide applicability in different cell types. For measuring micronucleus, we have treated T98G cells with compound 2 under similar conditions and concentrations to those which were used in earlier experiments on growth kinetics and the clonogenic assay. After treatment by compound 2, cell cultures were grown for a 24 and 48 h in order to allow chromosomal damage leading to the formation of micronuclei in bi- or multinucleated interphase cells.

Figure 6 shows the micronucleus frequency at 24 and 48 h for T98G brain cancer cell after treatment with 2.2–60 µg/mL concentration of compound 2. The frequency of untreated cells with micronuclei were in the range of 3.5%–3.9%; whereas the treatment with 2.2 µg/mL does not induce any significant level of micronuclei formation in T98G cells, and the range of micronucleus is 3.8–3.9. However, micronuclei frequency increased from 3.5% (control) to 5.6%, 6.21% and 7.7% for 6.6, 20 and 60 µg/mL concentration treated cells, respectively, at 24 h of culture. However, it was increased further at 48 h of culture as 6.6, 20 and 60 µg/mL concentrations of compound 2 further increased micronuclei frequency from 3.9% (control) to 5.9%, 6.43% and 7.9%, respectively. Micronuclei shown in compound 2 treated T98G cells were formed by DNA strand breaks generated during the faulty excision repair process. The remaining unsealed DNA leads to the formation of micronuclei in subsequent mitosis, and cells with micronuclei are found to be associated with loss of reproductive capacity.

For a molecule to be a probable drug, besides having a good biological activity, it must have nice pharmacokinetic accessibility in biological systems. To access the pharmacokinetic profile of the synthesized molecule, we used the well validated in silico tools: Osiris, Chemaxon and Catalyst. These tools have been validated with almost 7,000 drug molecules available in market. The analysis of theoretical toxicity risks for the thienyl derivatives using the OSIRIS program shows that all compounds were less toxic and can be used as a therapeutic molecules (Figure 7). As these compounds are considered for oral delivery, they were submitted to the analysis of Lipinski ‘rule of five’, druglikness and drug score by using the Catalyst software (Table 1).

Our results pointed that all effective compounds fulfill this rule and their druglikness property such as molecular weight (180–196), Log P (2.38–3.13), nHBA (0–1), nHBD (0–1) and number of rotatable bonds (rotb) (2) were better than commercial drugs (Table 1). Finally, we evaluated thienyl derivative 2 as a potential drug by calculating druglikeness and drug-score. Druglikeness, which is related to the similarity with trade drugs (−1.28–1.29). Drug score of all derivatives was in the range of 0.46–0.56. Among the both thienyl compounds, 2 showed the best values of drug-score (0.56) with a lower toxicity effect, which suggests it should be considered for further in vivo exploration.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), bisbenzimidazole derivative Hoechst-33342 (bis-benzimide(2-[4-ethoxyphenyl]-5-[4- methyl-1-piperazinylpiperazinyl]-2,5-bi-1H-benzimidazole)trihydrochloride), Hank’s balanced salt solution (HBSS), Dulbecco’s modified phosphate buffered saline (PBS), Dulbecco’s modified eagle’s medium (DMEM), fetal calf serum (FCS), N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] (HEPES) buffer, propidium iodide (PI), Ribonuclease-A (RNase-A), trihydro-chloride, propidium iodide and trypsin were obtained from the Sigma Chemical Co., USA. All other chemicals used in the present study were of analytical grade.

3-Bromothiophene (1, 60 g, 0.36 mol) was added into a solution of n-butyllithium (0.36 mol) in THF (120 mL) at −78 °C and a solution of the ethyl formate (13.63 g, 0.184 mol) in THF (60 mL) was added dropwise while maintaining the temperature between −78 and −70 °C [17]. The mixture was then allowed to warm slowly (over ~1 h) to RT. The resulting mixture was hydrolyzed with 10% aqHCl (76.8 g) with cooling below 0 °C. The organic layer was separated, and the aqueous layer was extracted with CHCl₃ (300 mL × 3). The combined solutions were dried with Na₂SO₄ and concentrated in vacuo. The residue was chromatographed on a short silica gel column (hexane-EA 6:1) to yield a pale-yellow viscous oil, which solidified upon standing. Yield: 88% (31.78 g); m.p. 61–62 °C. ¹H-NMR (400 MHz, CDCl₃): 7.19 (dd, 2H, J = 5.0, 3.0 Hz), 7.07 (m, 2H), 6.93 (dd, 2H, J = 5.0, 1.3 Hz), 5.76 (s, 1H), 3.10 (br s, 1H); ¹³C-NMR (100 MHz, CDCl₃): 144.71 (2C), 126.26 (2C), 125.94 (2C), 121.50(2C), 68.67. Anal Calcd for C₉H₈OS₂:C, 55.07; H, 4.11 Found: C, 54.88; H,4.17.

To a well stirred solution of chlorotrimethylsilane (55.34 g, 0.5094 mol) in anhydrous CH₃CN (240 mL) was added NaI (76.35 g, 0.5094 mol) in one portion. The resulting slurry was stirred for 20 min at 0 °C, and then a solution of corresponding di(3-thienyl) methanol (2, 20 g, 0.1018 mol) in CH₃CN (150 mL) was added dropwise over 30 min to maintain the reaction temperature below 10 °C [18]. The reaction mixture was quenched with aqueous NaOH (10.99 g in 150 mL) extracted with CH₂Cl₂ (300 mL × 3), washed with a saturated solution of Na₂S₂O₃·5H₂O (150 mL), and dried over Na₂SO₄. Organic solvents were removed in vacuo, and the brown residue was purified by column chromatography (hexane) to yield the pure di(3-thienyl)methane (3) as a white solid or pale yellow oil which solidified upon standing. Yield: 90% (16.51 g); m.p. 35–36 °C. ¹H-NMR (400 MHz, CDCl₃): 7.38 (dd, 2H), 7.09 (m, 2H), 4.14 (s, 2H); ¹³C-NMR (100 MHz, CDCl₃): 140.75 (2C), 128.21 (2C), 125.42 (2C), 121.00(2C), 30.89. Anal Calcd for C₉H₈OS₂: C, 59.96; H, 4.47 Found: C, 59.74; H, 4.19.

T98G (brain cancer) and HEK Normal (Human Embryonic Kidney) cells were used in the present studies were purchased from SNU (Seoul, Korea). We were culture these cell line in 75 cm² culture flasks (Corning, New York, NY, USA) using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% glutamine, penicillin (100 IU/mL) and streptomycin (100 mg/mL) (all from Euroclone, UK) or by distributor instructions. All cultures were maintained at 37 °C, 95% relative humidity and 5% CO₂. Prior to each cytotoxicity test, the cells were harvested using trypsin–ethylenediaminetetraacetic acid (EDTA)–PBS solution (with 0.25% trypsin–0.05mM according to the distributor’s instructions) and diluted at a density of 5 × 10⁵ cells/mL in MTT assays. Stock cultures were passaged every third day after harvesting the cells with 0.05% trypsin and seeding 8 × 10³ cells/cm² in tissue culture flasks to maintain the cells in the exponential phase. All experiments were carried out in exponentially growing cells. The cell suspension was seeded into 24-well plates (Corning, New York, NY, USA) at 100 µL well, and incubated for approximately 20–24 h before tests in order to reach confluency. Before the cells were seeded into 24-well plates, the plates were treated with 0.01% poly-D-lysine solution (Sigma-Aldrich, Germany).

Cells were seeded in 24-well plates at a concentration of 2–4 × 10³ cells/well in 200 μL of complete media and incubated for 24 h at 37 °C in 5% CO₂ atmosphere to allow for cell adhesion. All assays were performed in two independent sets of quadruplicate tests. Control group containing without treatment was run in each assay. Following after 24, 48 and 72 h of exposure of cells to compound, each well will be carefully rinsed with 200 μL PBS buffer. Cytotoxicity were assessed using MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide). MTT solutions 20 μL (5 mg·mL⁻¹dd H₂O) along with 200 μL of fresh, complete media were added to each well and plates were incubated for 3 h [19]. Following incubation, the medium were removed and the purple formazan precipitate in each well were sterilized in 200 μL DMSO. Absorbances were measured using microplate reader at 570 nm and results were expressed as % viability which is directly proportional to metabolic active cell number. Percentage (%) viability was calculated as:

Cells were seeded at 7,000–10,000 cells/cm² in 90–120 mm Petri dishes or flasks, and their proliferation kinetics will be studied at 24, 48, 72 h after treatment by compounds, following trypsinization and counting total cells per flask/disc by using a Neubauer hemocytometer.

Clonogenic assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. The colony is defined to consist of at least 50 cells. The assay essentially tests every cell in the population for its ability to undergo “unlimited” division [20]. Clonogenic assay is the method of choice to determine cell reproductive death after treatment with ionizing radiation, but we can also be used to determine the effectiveness of drug molecules. Only a fraction of seeded cells retains the capacity to produce colonies before or after treatment, cells will be seeded out in appropriate dilutions to form colonies in 1–3 weeks. After harvesting with 0.05% trypsin, 150–400 (depending on the treatment) cells will be plated 10–14 h before treatment in DMEM at 37 °C. Cultured cells will be treated with doses 20 to 100 ug/ml of compounds. After the treatment, cells will be incubated in dark under humidified, 5% CO₂ atmosphere at 37 °C for 8–10 days to allow colony formation. Colonies will be fixed with methanol and will be stained with 1% crystal violet. Colonies of more than 50 cells will be counted and the surviving fraction (SF) will calculated. Clonogenic survival curves will be constructed from three independent experiments by least-squares regression fitting averaged survival levels.

The purpose of the micronucleus assay is to detect those chemical and physical agents which modify chromosome structure and segregation in cells [21]. For measuring micronucleus we treated T98G cells by compound under similar conditions as we used in earlier experiments for growth kinetics and clonogenic assay. After treatment by compound, cell cultures are grown for a 24 and 48 h to allow chromosomal damage to lead to the formation of micronuclei in bi- or multinucleated interphase cells. Air-dried slides containing acetic acid–methanol (1:3 V/V) fixed treated cells were stained with Hoechst-33342 (10 µg/mL in PBS (0.1M), disodium phosphate (0.45 M) buffer containing 0.05% Tween-20 detergent). Slides were examined under the fluorescence microscope using an UV excitation filter. Fluorescent nuclei were visualized using a blue emission filter. Cells containing micronuclei were counted from >1,000 cells by employing the criteria of Countrymen and Heddle [21].

The fraction of cells containing micronuclei, called the M-fraction (%) or MN frequency was calculated as follows:

where Nm is the number of cells with micronuclei and Nt is the total number of cells analyzed. Since, micronuclei formation is linked to cell proliferation, the micronuclei frequencies were normalized with respect to the cell numbers.

To evaluate pharmacokinetic profile descriptors such as cLogP (octanol/water partition coefficient) and LogS (water solubility) were calculated using the Osiris Property Explorer on-line system [22]. The thienyl derivatives were submitted to in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) screening, using the Osiris program. Values of druglikeness are based on the occurrence frequency of each fragment of the molecule in commercial drugs while the drug-score evaluates the compound’s potential to qualify for a drug and is related to topological descriptors, fingerprints of molecular druglikeness, structural keys and other properties such as cLog P and molecular mass [22,23]. In silico theoretical safety analysis is also evaluated by Osiris software, whereby a score of 1 means a drug is safe and a score < 1 means a drug molecule is theoretically toxic in use. The pharmacokinetic profile, important for a good oral bioavailability of a compound, was also evaluated according to the Lipinski’s ‘rule-of-five’ by using the Catalyst and Chemaxon softwares, which analyse features that a drug should present to allow the absorption and permeation across the membranes and states molecular weight < 500 Daltons (Da), calculated octanol/water partition coefficient (cLogP) < 5, number of hydrogen-bond acceptors (nHba) < 10, and number of hydrogen-bond donors(nHbd) < 5 [22,23], as well as a fifth rule added later, which infers the number of rotatable bonds < 10.