Helical Arrangement of 2-( 4-hydroxy-3-methoxyphenyl )-Benzothiazole in Crystal Formation and Biological Evaluation on HeLa Cells

Benzothiazoles are a set of molecules with a broad spectrum of biological applications. In particular, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole is a potential breast cancer cell suppressor whose mechanism of action has been previously reported. In the present work, the title compound was synthesized, crystallized, and its biological activity on HeLa cells was evaluated. Its molecular structure was compared to that obtained by molecular modeling. Theoretical calculations suggest that the syn-rotamer is the most stable form and correlates very well with crystallographic data. The crystal structure adopts a helical arrangement formed through O13—H13···N3 intermolecular hydrogen bonding that propagates in the (14 -1 -3) plane. These results suggest that the title compound has the capacity to interleave into DNA and better explain its biological effects related to the increased CHIP expression through AhR recruitment. Finally, the biological experiments indicate that the title compound has the capacity to decrease the viability of HeLa cells with an IC50 = 2.86 μM.

synthetized, crystalized, and its molecular X-ray structure was compared with that simulated by theoretical calculations.Furthermore, its activity on cervical cancer cell line (HeLa) was evaluated.

Theoretical Molecular Modeling
The DFT theoretical calculations showed that the optimized structure is similar to the X-ray experimental structure.The geometric parameters of both the experimental and theoretical calculations are listed in Table 1.The rotational barrier around the C2-C10 bond was calculated in order to establish the structure of the most stable rotamer.Starting from the rotamer with the OMe group on the same side as the heterocyclic nitrogen (syn), the value of the rotational barrier is 7.12 kcal/mol.However, syn-and anti-rotamers are almost of the same energy with a difference of only 1.51 kcal/mol in favor of the syn structure.The title compound crystallizes in the most stable form, the syn-rotamer, whose calculated dipolar moment is smaller (2.6331 D) than the calculated value for the anti form (3.3379 D), as shown in Figure 2.

Theoretical Molecular Modeling
The DFT theoretical calculations showed that the optimized structure is similar to the X-ray experimental structure.The geometric parameters of both the experimental and theoretical calculations are listed in Table 1.The rotational barrier around the C2-C10 bond was calculated in order to establish the structure of the most stable rotamer.Starting from the rotamer with the OMe group on the same side as the heterocyclic nitrogen (syn), the value of the rotational barrier is 7.12 kcal/mol.However, synand anti-rotamers are almost of the same energy with a difference of only 1.51 kcal/mol in favor of the syn structure.The title compound crystallizes in the most stable form, the syn-rotamer, whose calculated dipolar moment is smaller (2.6331 D) than the calculated value for the anti form (3.3379 D), as shown in Figure 2.

Supramolecular Structure
The hydrogen bonding scheme contributes to the full planarity of the title compound.The graph set notation is used to describe the hydrogen bonding motifs [28].The phenol hydrogen atom is properly positioned to form an intramolecular hydrogen bond motif, S(5), with the methoxy oxygen atom, O13-H13•••O14.In addition, it is also engaged with the heterocyclic nitrogen, O13-H13•••N3, forming a three-centered hydrogen bond, O14•••H13•••N3, as the sum of angles around H13 is 360 • [29].The phenol oxygen atom also acts as the acceptor of one aryl hydrogen, C15-H15•••O13, that assembles the seven-membered ring motif R 2 2 (7), Figure 3A.The propagation of O13-H13•••N3 in the (14 -1 -3) plane gives rise to the first dimension (1D) in the form of a helix, Figure 3B.The second dimension is developed by weak interactions C16-H16A•••Ph between the helixes, through the participation of a methyl hydrogen from the methoxy group, as the donor, and the centroid of the hydroxymethoxyphenyl ring, as the acceptor.Details of the geometry of the hydrogen bonding are listed in Table 2.

Supramolecular Structure
The hydrogen bonding scheme contributes to the full planarity of the title compound.The graph set notation is used to describe the hydrogen bonding motifs [28].The phenol hydrogen atom is properly positioned to form an intramolecular hydrogen bond motif, S( 5), with the methoxy oxygen atom, O13-H13•••O14.In addition, it is also engaged with the heterocyclic nitrogen, O13-H13•••N3, forming a three-centered hydrogen bond, O14•••H13•••N3, as the sum of angles around H13 is 360° [29].The phenol oxygen atom also acts as the acceptor of one aryl hydrogen, C15-H15•••O13, that assembles the seven-membered ring motif R 2 2 (7), Figure 2A.The propagation of O13-H13•••N3 in the (14 -1 -3) plane gives rise to the first dimension (1D) in the form of a helix, Figure 2B.The second dimension is developed by weak interactions C16-H16A•••Ph between the helixes, through the participation of a methyl hydrogen from the methoxy group, as the donor, and the centroid of the hydroxymethoxyphenyl ring, as the acceptor.Details of the geometry of the hydrogen bonding are listed in Table 2.

In Vitro Citotoxicity Assay on HeLa Cells
The in vitro assay was performed in order to know the effect of the title compound on a great interest cell line such as the HeLa line.This cell line is derived from cervical cancer and is well established to explore of the effects of the novel drugs such as the title compound on cancer cells.Our results showed a clear effect on the HeLa cells by MTT assay with IC 50 = 2.86 µM, determined after 48 h of exposure, Figure 4.This value is consistent with the activity found against MDA-MB-231 cell line, a highly aggressive breast cancer cell line (IC 50 = 4.02 µM, determined in an assay after 96 h of exposure) [17].These results show a similar behavior to other cell lines because we did not observe a total death of the cells, despite increasing the concentration of the compound.However, the potency of the compound was higher in HeLa cells than in MDA-MB-231 cells, because our assay lasted half the time that was reported for breast cancer cells.Likewise, our results are consistent with the antitumor effect for other benzothiazole-containing compounds such as fluorinated 2-phenylbenzothiazole derivatives with IC 50 values between 50 to 0.0001 µM [10], as well as 2-(4-Amino-3-methylphenyl) benzothiazole derivatives showed IC 50 values between 100 nM to 100 µM [13,14].According to these data, the relative potency of this type of compound depends on the origin of the cancer cell line.For this reason, we suggest exploring the activity and mechanism action of benzothiazole-containing compounds in greater detail on HeLa cells and other cancer cell lines.

In Vitro Citotoxicity Assay on HeLa Cells
The in vitro assay was performed in order to know the effect of the title compound on a great interest cell line such as the HeLa line.This cell line is derived from cervical cancer and is well established to explore of the effects of the novel drugs such as the title compound on cancer cells.Our results showed a clear effect on the HeLa cells by MTT assay with IC50 = 2.86 μM, determined after 48 h of exposure, Figure 3.This value is consistent with the activity found against MDA-MB-231 cell line, a highly aggressive breast cancer cell line (IC50 = 4.02 μM, determined in an assay after 96 h of exposure) [17].These results show a similar behavior to other cell lines because we did not observe a total death of the cells, despite increasing the concentration of the compound.However, the potency of the compound was higher in HeLa cells than in MDA-MB-231 cells, because our assay lasted half the time that was reported for breast cancer cells.Likewise, our results are consistent with the antitumor effect for other benzothiazole-containing compounds such as fluorinated 2-phenylbenzothiazole derivatives with IC50 values between 50 to 0.0001 μM [10], as well as 2-(4-Amino-3-methylphenyl) benzothiazole derivatives showed IC50 values between 100 nM to 100 μM [13,14].According to these data, the relative potency of this type of compound depends on the origin of the cancer cell line.For this reason, we suggest exploring the activity and mechanism action of benzothiazole-containing compounds in greater detail on HeLa cells and other cancer cell lines.

Instrumental
The uncorrected melting point was measured in open-ended capillary tubes in an Electrothermal 9300 digital apparatus. 1H (300.01 MHz) and 13 C NMR (75.46 MHz) spectra were recorded on a Varian Mercury-300 spectrometer using DMSO-d6 as a solvent and TMS as an internal reference.Chemical shift values (δ) are in parts per million (ppm) and coupling constants (J values) are in Hertz (Hz), ESI-MS were recorded on a Bruker micrOTOF-Q II, infrared spectra (IR) was obtained with an ATR/FTIR PerkinElmer Spectrum v10.04.00 and UV/Vis with a Beckman Coulter spectrometer DU 650.

Chemical Synthesis and Crystallization
The compound 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole was synthesized following a reported procedure with modification [30].All chemicals and solvents were reagent grade and used as received.The reaction mixture consisted of 0.259 g (1.98 mmol) of 2-aminobenzenethiol, 0.319 g (2.1 mmol) of 4-hydroxy-3-methoxybenzaldehyde and 0.398 g (2.1 mmol) of Na2S2O5 dissolved in 5 mL of anhydrous DMSO, and was stirred at 393 K for 45 min.The reaction progress was monitored

Instrumental
The uncorrected melting point was measured in open-ended capillary tubes in an Electrothermal 9300 digital apparatus. 1H (300.01 MHz) and 13 C NMR (75.46 MHz) spectra were recorded on a Varian Mercury-300 spectrometer using DMSO-d 6 as a solvent and TMS as an internal reference.Chemical shift values (δ) are in parts per million (ppm) and coupling constants (J values) are in Hertz (Hz), ESI-MS were recorded on a Bruker micrOTOF-Q II, infrared spectra (IR) was obtained with an ATR/FTIR PerkinElmer Spectrum v10.04.00 and UV/Vis with a Beckman Coulter spectrometer DU 650.

Chemical Synthesis and Crystallization
The compound 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole was synthesized following a reported procedure with modification [30].All chemicals and solvents were reagent grade and used as received.The reaction mixture consisted of 0.259 g (1.98 mmol) of 2-aminobenzenethiol, 0.319 g (2.1 mmol) of 4-hydroxy-3-methoxybenzaldehyde and 0.398 g (2.1 mmol) of Na 2 S 2 O 5 dissolved in 5 mL of anhydrous DMSO, and was stirred at 393 K for 45 min.The reaction progress was monitored by TLC (using ethyl acetate:hexane in 1:1 proportion as eluent).The resulting mixture was cooled to room temperature, cold water was added, and the resulting precipitate was collected by filtration.The product was washed with water and left to dry at RT.The gray powder was dissolved in CH 2 Cl 2 and washed three times with brine.The product was recrystallized three times in CH 2 Cl 2 solution to obtain 0.443 g of colorless block-like crystals suitable for X-ray in 87% yield, m.p. = 162-163

X-ray Diffraction Methods
Single-crystal X-ray diffraction data was recorded on a D8 Quest CMOS (Bruker, Karlsruhe, Germany) area detector diffractometer with Mo K α radiation, λ = 0.71073 Å.The structure was solved by direct methods using SHELXS97 [31] program of WinGX package [32].The final refinement was performed by the full-matrix least-squares methods on F2 with SHELXL97 program.H atoms on C were geometrically positioned and treated as riding atoms, with C-H = 0.93-0.98Å, and with Uiso(H) = 1.2Ueq(C).The program Mercury was used for visualization, molecular graphics and analysis of crystal structures [33].The software used to prepare material for publication was PLATON [34].Crystallographic data has been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication CCDC number 1539167.Copies of the data can be obtained free of charge upon application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (Fax: +44-01223-336033 or E-Mail: deposit@ccdc.cam.ac.uk).

Molecular Modeling
Gaussian 09 software [35] with B3LYP/6-311G(d,p) basis set was used to structure the optimization and vibrational frequencies calculations.Energy calculations of the rotamers around the C2-C10 bond were performed under same basis set.

In Vitro Cytotoxicity Assay on HeLa Cells
HeLa cells (1 × 10 3 cells/well) in 100 µL of DMEM supplemented with 10% Fetal Bovine Serum were seeded in 96-well culture plates.After 24 h, the cells were treated with fresh medium containing different concentrations or not (negative control) of the title compound (1, 3, 5, 7, and 10 µM) for the following 48 h; camptothecin 1 µM was used as positive control.The plates were analyzed for cell survival using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay, as described elsewhere [36].The cytotoxic effect of each treatment was expressed as the percentage of cell survival relative to the untreated control cells, and the concentration of the compound that inhibited 50% of HeLa cell proliferation (IC 50 ) was determined by fitting the data to a typical sigmoidal dose-response curve.

Conclusions
In summary, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole, a potent inhibitor of the growth and invasiveness of breast cancer cells, was crystallized, modulated, and its cytotoxic effect on HeLa cells line was investigated by an in vitro assay.The title compound model is consistent with the obtained crystalline structure, whose conformation leads to the formation of a helix in the crystal lattice.In accordance with previous reports of this structure and other analogues in relation to its mechanism of anticancer action, it is suggested that the conformation found in its crystalline arrangement can directly interact with DNA and provoke damage and cell cycle arrest in cancer cells, in addition to CHIP expression through the recruitment of AhR, as previously demonstrated.Finally, the in vitro experiments showed that the title compound has a cytotoxic effect on HeLa cell line, therefore we suggest that the biological applications of this type of molecule, such as their anticancer effects and their interaction on nucleic acids, continue to be explored in future studies.