A preliminary investigation of anticancer activity of novel benzothiazole derivatives against A549 lung carcinoma cell line Yeni benzotiyazol türevlerinin A549 akciğer karsinomu hücre hattına karşı antikanser etkinliğinin ilk basamak araştırması

Objective(s): In this study, it was aimed to synthesize new chemotherapeutic agents based on known antiproliferative properties of benzothiazol-2-amine moiety. The antitumor activity of the newly synthesized compounds was determined against A549 lung cancer cell lines. Methods: Eighteen compounds were obtained by two steps synthetic route. The anticancer potency of the compounds were detected using MTT assay and flow cytometric analysis on A549 cell line. Some physicochemical properties of the compounds were calculated, virtually. Results: Compounds 15 and 18 showed the highest cytotoxic activity even than cisplatin. Also, it was determined that compound 18 caused 19.1% (early and late) apoptosis whereas cisplatin caused 21.6% (early and late). Conclusion: Considering the calculated virtual data, eighteen new compounds were found within the boundaries of Lipinski rule of five to be an oral drug. Besides, the most potent compounds 15 and 18 were detected that both have 1-methylbenzimidazole structure, and also methoxy and ethoxy substituents on benzothiazole ring.


Introduction
Lung cancer is the most common cause of cancer-related death which represents 29% of all cancer deaths worldwide. Among its types, non-small cell lung cancer (NSCLC) is higher than eighty percent of all lung cancer diseases [1,2]. Chemotherapy provides symptomatic treatment and modest improvement in survival in advanced non-small cell lung cancer [3,4]. However, recent literature indicate that newer chemotherapeutic agents extend life span and relieve symptoms in all stages of lung cancer, such as NSCLC [5].
Benzothiazoles have been known to have a large scale of biological activities including antimalarial, antitubercular, antihelmintic, anticonvulsant, analgesic, anti-inflammatory, antidiabetic and antitumor activities [6][7][8]. The diversity of biological activity varies according to the alkyl, aryl group or any other functional groups at the second position of benzothiazole ring. It has been reported that the benzothiazole structure modified with an aryl group and imidazole ring can affect inhibition of the growth of certain cancer cell lines [9][10][11]. In various studies, new benzothiazole compounds were focused on substituting 2-aminobenzothiazoles, 2-arylbenzothiazoles and benzothiazole-2-thiol as functional groups [12][13][14][15][16][17]. Among them, a large number of 2-aminobenzothiazoles are generated one of the privileged part of medicinal chemistry which are also extensively studied [18,19]. Related compounds were reported to possess cytotoxic properties on different cancer cells which were comparable to cisplatin [20]. Besides, combination of 2-aminobenzothiazoles with other heterocycles is a well-known approach to design new drug like molecules, which allows achieving new pharmacological profile, action, toxicity lowering [21].
Considering these literature, we have designed novel benzothiazole derivatives including different heterocyclic rings, imidazole, triazole and benzimidazole. Compounds have been studied to determine their anticancer potency against A549 tumor cell line (human lung carcinoma cell) compared with cisplatin.

Materials and methods Chemistry
All chemicals were purchased from Sigma-Aldrich Chemical Co (Sigma-Aldrich Corp., St. Louis, MO, USA) and Merck Chemicals (Merck KGaA, Darmstadt, Germany). Melting points were determined by MP90 digital melting point apparatus (Mettler Toledo, OH) and were uncorrected. Spectroscopic data were recorded on the following instruments: Bruker Tensor 27 IR spectrophotometer; 1 H NMR (nuclear magnetic resonance) Bruker DPX-300 FT-NMR spectrometer, 13 C NMR, Bruker DPX 75 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA); M + 1 peaks were determined by Shimadzu LC/MS ITTOF system (Shimadzu, Tokyo, Japan). Elemental analyses were performed in a Perkin Elmer EAL 240 elemental analyser for C, H, N, O and S.

Synthesis of the compounds General method for the synthesis of 2-chloro-N-(6substituted benzothiazol-2-yl)acetamides (1-6)
A solution of 2-amino-6-substituted benzothiazol (1g, 6 mmol) in 250 mL of tetrahydrofuran was prepared and triethylamine (1.1 mL, 7.2 mmol) was added. Chloroacetyl chloride (0.64 mL, 7.2 mmol) in 5 mL of tetrahydrofuran was added dropwise in ice bath. After completion of dropping, the resulting mixture was stirred for 1 h at room temperature and evaporated to dryness. The residue was washed with water, dried and recrystallized from ethanol to afford the pure compounds 1-6.

MTT assay
MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was carried out to determine cytotoxicity of the compounds against A549 (human lung adenocarcinoma cells) cell line according to the reported data [22,23]. A549 cells were cultured in 96-well flat-bottom plates at 37 o C for 24 h (2 x 10 4 cells per well). After 24 h drug incubation, 20 μL MTT solution (5 mg/mL MTT powder in PBS) was added to each well and incubated for 2 h in the same conditions. Formazan crystals were dissolved in 200 μL DMSO and the absorbance was read by ELISA reader (OD570 nm). The percentage of viable cells was calculated based on the medium control. All experiments were performed at least three times.  Structure clarifications of the final compounds (13-30) were carried out through FT-IR, 1 H-NMR, 13 C-NMR and HRMS spectroscopic data and elemental analysis. Characteristic stretching absorptions of C=O and N-H bonds were observed in the region of 1699-1668 cm −1 and 3404-3307 cm −1 , respectively. The stretching bands for C=N and C=C were recorded at about 1614-1440 cm −1 .

Annexin V/ PI staining
In the 1 H-NMR spectra, the signal due to methylene and NH protons of acetamide (-NHCOCH 2 ) moiety appeared as singlet peaks at 4.00-5.08 ppm and 12.50-13.36 ppm, respectively. N-CH 3 protons of 1H-imidazole, 1H-triazole and 1H-benzimidazole rings were observed as singlet peaks about 3.60-4.33 ppm. All the other aromatic and aliphatic protons were observed at the expected regions.

Biochemistry
The synthesized compounds were screened for their in vitro antiproliferative activity against A549 non-small cell lung cancer cell line. The results were represented in Table 1 as in inhibition concentration (IC 50 ). The compounds were displayed cytotoxic profile within ranges 9.0 ± 1.0 μg/mL-500 μg/mL whereas standard drug showed cytotoxicity at 21.0 ± 2.0 μg/mL concentration. showed higher cytotoxicity, were studied further in the flow cytometry to detect apoptotic cell death ratios that they caused. Compounds and cisplatin were performed to the system at IC 50 concentrations. The results of the Annexin V/PI staining protocol were summarized in Table 2 and the diagrams were represented in Figure 2. The survival of A549 cell line was confirmed and viable cells were found 96.7% for control group. Early, late apoptotic and viable cell ratios were determined as 17.0, 4.6, 37.0% for cisplatin treated tumor cells survived with a ratio of 37.0%. None of the compounds did not reach cisplatin's potency. Only compound 18 caused 19.1% (early and late) apoptosis totally whereas standard drug had 21.6% (early and late). Also, compound 26 exhibited half apoptotic potency of cisplatin.
Some physicochemical characteristics were calculated virtually and demonstrated in Table 3. For all compounds, log octanol/water partition coefficient, molecular weight, total polar surface area, number of hydrogen acceptors, number of hydrogen donors, number of rotatable bonds were calculated using Molinspiration Calculation of Molecular Properties toolkit. According to Lipinski rule of five [25] and its variations; an oral drug has log P lower than five, hydrogen bond donors lower than five, hydrogen bond acceptors lower than 10, molecular mass less than 500 daltons, polar surface area equal to or less than 140 Å 2 and number of rotable bonds less than 10. Eighteen new compounds were found within the boundaries of this rule and they may be oral drugs considering the data.

Conclusion
Compounds ( .0 μg/mL against A549 lung cancer cell line. Among these two compounds, 18 caused 19.1% (early and late) apoptosis which was very close to cisplatin caused. Compounds were all detected to be an oral drug and some of them were found to be a potential antitumor agent. In further studies, new and similar compounds are planned to be sythesized for developing this issue. Besides, promising results are expected to be obtained with increased number of cell lines.