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

Abstract

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.

1. Introduction

Benzothiazoles are a class of bicyclic compounds with a broad spectrum of biological applications such as neuroprotective effects [1,2], antimicrobial activity [3,4,5,6], antioxidant and radioprotective effects [7], anticonvulsant activity [8,9] and antitumor properties [10,11]. In particular, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole has been reported to be a tyrosinase inhibitor against hyperpigmentation [12]. Recent studies showed that this compound and its analogues promote signaling and nuclear translocation of Aryl hydrocarbon Receptor (AhR) and induce the carboxyl terminus of Hsp70-interacting protein (CHIP) expression through recruitment of AhR upstream of the CHIP gene. This mechanism has potential application in the suppression of tumor progression in breast cancer cells [13,14,15,16,17]. The synthesis of the title compound has been recently improved by using several catalysts such as LiBr [18], ZnO [19] and CdS [20], silica [21], nanoparticles, clays [22,23], and transition metals [24,25]. The number of synthetic methods reflects the importance of this compound. In this paper, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole was 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.

2. Results and Discussion

2.1. Molecular Structure

The title compound crystallizes in an orthorhombic system, space group P2₁2₁2₁, with four molecules in the unit cell. Its X-ray molecular structure is depicted in Figure 1. The bond distances values N3—C2 of 1.297(4) Å and N3—C9 of 1.368(4) Å are typical for an N(sp²)—C(sp²) double and single bond order character, respectively, whereas C2—C10 bond distance of 1.453(4) has a value for a typical single Csp²—Csp² bond [26]. Bond lengths values S1—C2 of 1.735(3) Å and S1—C8 of 1.724(3) Å are characteristic for a single S—Csp² bond. Phenol and methyl ether C—O distances O13—C13 of 1.337(4) Å and O14—C14 of 1.358(4) Å, respectively, are significantly smaller than the usual values (Car—O of 1.362(15) in phenols and 1.370(11) in ethers), but similar to the value found in the structure of 2-(4-hydroxyphenyl)benzothiazole [27]. Both phenyl (Ph) (C10—C15) and benzothiazole (BZT) (S1/C2/N3/C4—C9) rings are planar. The atoms C12 in the former and C2 in the latter deviate 0.004(4) Å and 0.006(3) Å, respectively, from their corresponding mean planes. The Ph ring is almost coplanar with the benzothiazole moiety; the torsion angle between the two rings is 4.69(7)°, which is more coplanar than the same angle in 2-(4-hydroxyphenyl)benzothiazole (18.49(6)°). Phenol and methyl ether moieties are coplanar with a O13—C13—C14—O14 torsion angle value of −1.0(5)°.

2.2. 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. Comparison between modeled and crystal geometric structures of 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole.

	Modeled Structure	Crystal Structure
Energy (kcal/mol)	−717 529.76
EHOMO (kcal/mol)	−136.91
ELUMO (kcal/mol)	−41.60
GAP (kcal/mol)	−178.52
Bond lengths (Å)
N3—C9	1.398	1.368(4)
N3—C2	1.299	1.297(4)
C2—S1	1.877	1.735(3)
S1—C8	1.815	1.724(3)
C2—C10	1.456	1.453(4)
C13—C14	1.409	1.400(5)
O13—C13	1.381	1.337(4)
O14—C14	1.399	1.358(4)
Bond angles (°)
N3—C2—S1	113.5	115.0(2)
C2—C10—C11	122.7	122.1(3)
C2—C10—C15	118.1	118.7(3)
H—O13—C13	109.4	109
C16—O14—C14	118.9	118.2(3)
Torsion angles (°)
N3—C2—C10—C11	−179.987	174.7(3)
N3—C2—C10—C15	0.012	−5.0(5)
C16—O14—C14—C13	−179.995	−179.2(3)
S1—C2—C10—C15	−179.985	176.2(2)
S1—C2—C10—C11	0.016	−4.1(4)
O13—C13—C14—O14	0.001	-1.0(5)

. 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.

2.3. 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 R²₂(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. Hydrogen bonding geometric features of 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole.

D—H∙∙∙A	D—H (Å)	H∙∙∙A (Å)	D∙∙∙A (Å)	D—H∙∙∙A (°)
O13—H13∙∙∙O14	0.84	2.24	2.669(4)	112
O13—H13∙∙∙N3 i	0.84	2.05	2.844(4)	157
C15—H15∙∙∙O13 ii	0.95	2.40	3.339(4)	169
C16—H16A∙∙∙Ph iii	0.95	2.77	3.571(4)	140

Symmetry codes: (i) 1 − x, 1/2 + y, 3/2 − z; (ii) 1 − x, −1/2 + y, 3/2 − z; (iii) 1 + x, y, z.

.

2.4. 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 4. 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.

3. Materials and Methods

3.1. Instrumental

The uncorrected melting point was measured in open-ended capillary tubes in an Electrothermal 9300 digital apparatus. ¹H (300.01 MHz) and ¹³C 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.

3.2. 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 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 CH₂Cl₂ and washed three times with brine. The product was recrystallized three times in CH₂Cl₂ solution to obtain 0.443 g of colorless block-like crystals suitable for X-ray in 87% yield, m.p. = 162–163 °C; UV (EtOH) λ_max(log ε) 332.6 (4.41); IR (ATR/FTIR, cm⁻¹): ν 3400–3096, 1277, 1255 (Ar-OH); 1604, 1585 (Ar-o-disubst.); 1524 (C = N); 1191, 1011 (Ar-O-CH₃); ¹H NMR δ: 10.0 (br, 1H, OH), 8.03 (d, 1H, ³J = 8.8, H-4), 7.96 (d, 1H, ³J = 7.7, H-7), 7.62 (d, 1H, ⁴J = 1.7, H-15), 7.48 (dd, 1H, ³J = 8.2, ⁴J = 1.7, H-11), 7.47 (dd, ³J = 8.2, 7.7, 1H, H-6), 7.38 (dd, 1H, ³J = 8.2, 8.8, H-5), 6.91 (d, 1H, ³J = 8.2, H-12), 3.87 (s, 3H, OCH₃); ¹³C NMR δ: 168.0 (C-2), 154.1 (C-9), 150.5 (C13), 148.5 (C-14), 134.6 (C-8), 126.9 (C-6), 125.4 (C-5), 124.8 (C-10), 122.7 (C-7), 122.5 (C-4), 121.7 (C-11), 116.2 (C-12), 110.4 (C-15), 56.1 (CH₃); m/z (ESI) 258.05 [M+].

3.3. 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 = 0.93–0.98 Å, 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: [email protected]).

Crystal Data for C₁₄H₁₁NO₂₆S (M = 257.30 g/mol): orthorhombic, space group P2₁2₁2₁ (No. 19), a = 5.4526(10) Å, b = 10.993(3) Å, c = 19.668(5) Å, α = β = γ = 90°, V = 1178.9(5) ų, Z = 4, T = 100(2) K, Dcalc = 1.450 g/cm³, 24929 reflections measured (2.1° ≤ 2Θ ≤ 26.1°), 2324 unique (Rint = 0.0733, Rsigma = 0.0287) which were used in all calculations. The final R1 was 0.045 (I > 2σ(I)) and wR2 was 0.097 (all data), GooF = 1.072 and Abs. coefficient = 0.266.

3.4. 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.

3.5. In Vitro Cytotoxicity Assay on HeLa Cells

HeLa cells (1 × 10³ 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 (IC₅₀) was determined by fitting the data to a typical sigmoidal dose-response curve.

4. 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.