Antiproliferative Activity of an Organometallic Sn(IV) Coordination Compound Based on 1-Methylbenzotriazole against Human Cancer Cell Lines

Abstract

A motivating class of compounds with interest in the research field of biological active metallopharmaceuticals for cancer treatment is based on organometallic complexes of Sn(IV), exhibiting advantages such as improved cellular uptake and body excretion, lower toxicity, and fewer side effects compared to platinum-based drugs. In this study, the mononuclear organotin coordination complex [(CH₃)₂SnCl₂(mebta)₂] was synthesized and characterized using vibrational spectroscopy (IR, Raman), ¹H NMR, ¹³C{¹H} NMR, and X-ray crystallography. Its antiproliferative properties were thoroughly assessed across an aggressive triple-negative human breast cancer cell line. Notably, comparative studies with precursor materials verified that the observed biological activity is intrinsic to the complex itself. This study highlights the compound’s ability to induce cell fate by disrupting essential cellular functions, such as proliferation. By exploring the antiproliferative effects of organotin(IV) derivatives, we introduce a novel class of Sn complexes with 1-methylbenzotriazole (mebta), demonstrating significant potential as promising antitumor agents in the field of organotin compounds.

1. Introduction

Cytostatic properties are critical in cancer treatment as they inhibit the proliferation of cancer cells, effectively halting tumor growth and preventing metastasis. By leveraging these properties, researchers and clinicians can develop therapies that control cancer progression and enhance patient survival rates [1,2,3].

The use of platinum-based drugs like cisplatin in cancer treatment spurred intense research into other biologically active non-platinum compounds [4]. Non-platinum metal compounds, such as organotins(IV), are noted for potentially lower toxicity, improved body excretion, and fewer side effects compared to platinum-based drugs [5,6,7,8,9,10]. A key advantage of organotin(IV) compounds is that they do not induce chemoresistance, possibly offering lower toxicity relative to cisplatin analogs [11,12,13]. Cancer research towards the anticancer activity of organotin(IV) compounds derived from Marcel Gielen in the 80s [14], being the stepping stone for the numerous studies that followed. Based on these studies, several parameters could affect the efficiency of the metallodrugs, including the coordination sphere of the compound and the type of the organic ligand [15,16,17]. In particular, based on Structure Activity Relationship (SAR) of organotin(IV) compounds, it is known nowadays that their biological activity is closely connected to the availability of coordination positions on Sn, the occurrence of relatively stable ligand–Sn bonds, and slow hydrolytic decomposition behavior [18]. In parallel, the R group of the organic ligand may play an important role on the lipophilicity of the drug. The dominant mechanism of organotin compounds is to trigger apoptosis by attaching to the external phosphate groups of DNA and disrupting the intracellular metabolism of phospholipids.

Compounds represented by the formula R_ySnX_4−y (where X = Cl or Br) contain an Sn(IV) atom and are categorized as mono-, di-, tri-, or tetraorganotins depending on the number of alkyl or aryl groups attached to the tin atom. Organotin chlorides are known to induce lipid peroxidation in cell membranes, resulting in oxidative stress [19]. These R_ySnX_4−y compounds, recognized for their antitumor activity, interact with DNA to impact cell death mechanisms through necrosis, elevate extracellular Ca²⁺ levels, and generate reactive oxygen species (ROS), thereby promoting apoptosis [19].

Developed in the late 1960s, azoles are a prominent class of heterocyclic compounds and represent a prevailing part of antifungal drugs available [20]. Benzotriazoles are important “players” in biomedical research due to a plethora of antimicrobial, antiparasitic, choleretic, and cholesterol-lowering properties; in addition, they have been reported as antiproliferative agents [21]. The popularity of benzotriazoles in organic [22] and inorganic [23] chemistry is tremendous. The rationale behind the present work was an effort to combine the antitumor properties of organotins and benzotriazoles. In addition, 1-methylbenzotriazole (mebta, Scheme 1) could be a promising ligand [24] for organotin complexes with potential antitumor properties. Its ability to form stable complexes with organotin may enhance the biological activity and efficacy of these compounds in targeting cancer cells.

In this study, the mononuclear organotin coordination complex [(CH₃)₂ SnCl₂(mebta)₂] was synthesized. This compound has been characterized by different spectroscopic methods including vibrational spectroscopy (IR, Raman), ¹H-NMR and ¹³C{¹H}-NMR, along with the determination of its crystal structure by X-ray analysis. The cytotoxic activity was accessed in detail on aggressive triple-negative breast cancer cells to evaluate the influence of this compound on cell viability. This study was also performed for the pristine starting materials in order to verify that the biological activity is attributed to the complex and not to the ligand or the initial Sn starting material. The intrinsic ability of a compound to cause cell death by disrupting essential cellular functions, such as cell proliferation, is considered a key cytotoxic property. We do believe that the present effort could initiate a new class of organotin Sn complexes with 1-methylbenzotriazole (mebta), being so far unknown in the literature, with potential interesting biological properties as alternatives of other organotin antiproliferative compounds.

2. Materials and Methods

2.1. Materials and Spectroscopic-Physical Measurements

All synthetic procedures were performed under aerobic conditions. Distilled water was received from the in-house facility. Solvents and reagents were purchased from Sigma-Aldrich (Tanfrichen, Germany) and Alfa Aesar (Karlsruhe, Germany), and used as received without further purification. Dimethyltin dichloride (CAS: 753-73-1) was purchased by Sigma-Aldrich and 1-methylbenzotriazole, abbreviated as mebta (CAS: 13351-73-0), was purchased by Alfa Aesar with purity > 99.9% and >98%, respectively. The purity of the starting materials and the product was checked by carbon, hydrogen, and nitrogen microanalyses, performed by the Instrumental Analysis Laboratory of the University of Patras. KBr pellets of the complex, the mebta organic ligand, and the organotin reagent were prepared under pressure and the FT-IR spectra were recorded using a Perkin-Elmer spectrometer (16PC, Perkin-Elmer, Waltham, MA, USA). Raman spectra were recorded by a T-64000 Jobin Yvon-Horiba micro-Raman setup operating under a single-spectrograph configuration. The excitation wavelength for mebta was 514.5 nm emitted from a DPSS laser (Cobolt Fandango TMISO Laser, Norfolk, UK). The Raman spectrum of complex 1 was recorded by a 632.8 nm excitation line, emitted from the HeNe laser source (Optronics Technologies S.A., Model HLA-20P, 20 mW, Moschato, Greece); the collection of Raman spectrum was not possible by using the 514.5 nm excitation due to overlapping fluorescence during the measurement. The laser power on the samples was 1 mW. The backscattered radiation was collected from a single configuration of the monochromator after passing through an appropriate edge filter (LP02-633RU-25, Laser2000 Ltd., Huntingdon, Cambridgeshire, UK). The calibration of the instrument was achieved via the standard peak position of Si at 520.5 cm⁻¹. The spectral resolution was 5 cm⁻¹. NMR spectra were recorded using Varian VnmrS 500 (500 MHz for ¹H NMR and 126 MHz for ¹³C{¹H} NMR). UV/Vis spectra in solution were recorded on a Hitachi U-300 spectrometer. Conductivity measurements were carried out at 25 °C with a Metrohm-Herisau E-527 bridge; the concentration of complex 1 was ca. 10⁻³ M.

2.2. Preparation of the Complex

[(CH₃)₂SnCl₂(mebta)₂] (1): In a solution of 1-methylbenzotriazole (0.027 g, 0.20 mmol) in CH₂Cl₂ (6 mL) was added an amount of solid (CH₃)₂SnCl₂ (0.022 g, 0.10 mmol). The resulting colorless solution was stirred for 20 min, filtered, and allowed to slowly evaporate at room temperature. X-ray quality, colorless crystals of the product were obtained within 7 d. The crystals were collected by filtration, washed with cold CH₂Cl₂ (2 × 0.5 mL) and Et₂O (2 × 2 mL), and dried in vacuo over anhydrous CaCl₂. Yield: 52%. Anal. Calcd. (%) for SnCl₂C₁₆N₆H₂₀: C, 39.54; H, 4.15; N, 17.3. Found (%): C, 39.21; H, 4.64; N, 17.78. IR (KBr, cm⁻¹): 3093 (w), 3026 (w), 2934 (w), 1946 (w), 1812 (w), 1720 (w), 1590 (w), 1493 (m), 1455 (s), 1309 (m), 1270 (s), 1222 (s), 1171 (m), 1136 (w), 1058 (m), 1001 (w), 935 (m), 861 (w), 764 (s), 736 (m), 659 (w), 588 (m), 529 (m), 499 (m), 437 (m). ¹H NMR (500 MHz, dmso-d₆) δ 8.03 (dt, J = 8.4, 0.9 Hz, 2H), 7.84 (dt, J = 8.3, 1.0 Hz, 2H), 7.58–7.53 (m, 2H), 7.40 (ddd, J = 8.3, 6.9, 1.0 Hz, 2H), 4.31 (s, 6H), 1.03 (s, 6H). ¹³C{¹H} NMR (126 MHz, dmso-d₆) δ 145.09, 133.33, 127.08, 123.85, 118.97, 110.56, 34.11, 22.69.

2.3. Single-Crystal X-ray Crystallography

The crystallographic data were collected with a Bruker APEX II Quasar diffractometer, equipped with a graphite monochromator centered on the path of Mo Kα radiation. Colorless single crystals were coated with CargilleTM NHV immersion oil and mounted on a fiber loop, followed by data collection at 120 K. The program SAINT was used to integrate the data, which was thereafter corrected using SADABS [25]. The structure was solved using SHELXT [26] and refined by a full-matrix least-squares method on F² using SHELXL-2019 [27]. All non-hydrogen atoms were refined with anisotropic displacement parameters, whereas hydrogen atoms were assigned to ideal positions and refined isotropically using a suitable riding model.

The CIF file has been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 2370835.

2.4. Cytostatic Analysis

• Biochemicals and reagents

Dulbecco’s modified essential medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, L-glutamine, penicillin, streptomycin, amphotericin B, and gentamycin were all obtained from Biosera LTD (France). All other chemicals used were of the best commercially available grade.

• Cell cultures and conditions

MDA-MB-231 triple-negative breast cancer cell line was obtained from the American Type Culture Collection (ATCC, Baltimore, MD, USA) and routinely cultured as monolayers at 37 °C in a humidified atmosphere of 5% (v/v) CO₂ and 95% air. Cells were grown in complete DMEM culture medium supplemented with 10% FBS, a cocktail of antimicrobial agents (100 IU/mL penicillin, 100 mg/mL streptomycin, 10 mg/mL gentamicin sulphate and 2.5 mg/mL amphotericin B), 2 mM L-glutamine and 1 mM sodium pyruvate. Cells were harvested by trypsinization with 0.05% (w/v) trypsin in PBS containing 0.02% (w/v) Na₂EDTA. All experiments were conducted in serum-free conditions (0% FBS), to remove the net effects of serum. The tested compounds were dissolved in 100% DMSO at a 1 mg/mL concentration of each stock solution and the serial dilutions for final concentrations 0.05, 0.5, 1, 5, 10 ug/mL (0.10–20.58 uM), according to each experimental design were performed in DMEM 0% FBS. Concentration range was 0.10–20.58 uM, 0.38–75.1 uM, and 0.23–45.52 uM, for complex 1 (485.93 g/mol), mebta (133.15 g/mol), and (CH₃)₂SnCl₂ (219.68 g/mol), respectively. DMSO concentration range in the working solutions of the tested compounds was 0.05–2.5% v/v, accordingly. Cells were treated with the complex and its substitutes for 24 h prior to each experimental procedure. To note, vehicle controls with the respective concentration of DMSO as in the tested compounds have been included in each experimental procedure. The highest DMSO concentration used did not significantly affect cell viability.

• Phase contrast microscopy

MDA-MB-231 cells were seeded into 96-well plates at a density of 5000 cells per well. Cells were incubated in complete medium (DMEM, 10% FBS) for 24 h and then the medium was changed to serum-free, and the cells were serum starved overnight prior to treatment with complex 1 (10 ug/mL). For phase-contrast microscopy, images of live cells growing on the culture dish were collected on an OLYMPUS CKX41 microscope equipped with a CMOS color digital camera (SC30).

• WST-1 cell viability assay

MDA-MB-231 breast cancer cells were seeded into 96-well plates at a density of 5000 cells per well. Cells were incubated in complete medium (DMEM, 10% FBS) for 24 h and then the medium was changed to serum-free, and the cells were serum starved overnight prior to treatment with the tested compounds for 24 h in the concentration range 0.05–10 ug/mL. Briefly, WST-1 premix reagent was added in each well at 1:10 ratio and the absorbance at 450 nm was measured (reference wavelength at 650 nm), according to the manufacturer’s instructions.

• Statistical analysis

For each assay, three individual experiments were conducted. Data in diagrams are expressed as mean ± standard deviation (SD). Statistical analyses and graphs were made using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Statistically significant differences are indicated by asterisks: * (p ≤ 0.05), compared with untreated (control) cells. Non-statistically significant comparisons (p > 0.05) are not displayed.

3. Results

3.1. Synthetic Comments

Different synthetic attempts were performed for the investigation of the (CH₃)₂SnCl₂/mebta system. The solvent that gave the best crystals was CH₂Cl₂. It is interesting that complex 1 was isolated by using another solvent (i.e., MeOH) apart from CH₂Cl₂ (microanalytical and IR evidence), but not with MeCN. In addition, both a 1:1 and 1:2 ratio between (CH₃)₂SnCl₂ and mebta resulted in the precipitation of the product. This is encouraging since from a synthetic point of view, it implies a rather stable product that could be easily obtained by different synthetic conditions. The colorless crystals of complex 1 suitable for single-crystal X-ray analysis were obtained from closed vials after a week; the stoichiometric equation is presented in Equation (1).

$$({\mathrm{CH}}_3{)}_2{\mathrm{SnCl}}_2+2 \mathrm{mebta} \stackrel{C{H}_{2}C{l}_2}{\to }[({\mathrm{CH}}_3{)}_2{\mathrm{SnCl}}_2{(\mathrm{mebta})}_2](\mathrm{Complex} \mathbf{1})$$

(1)

3.2. Characterization of the Products

3.2.1. Vibrational Spectroscopy

The IR spectrum of (1) is presented in Figure S1, along with the IR spectrum of the organic ligand (mebta). In the IR spectrum of mebta, the stretching vibrations attributed to the aromatic (3000–3100 cm⁻¹) and the aliphatic (2900–3000 cm⁻¹) C-H group are present. In the spectral region 1650–1500 cm⁻¹, the C=C and C=N bond stretching was observed at 1647 and 1590 cm⁻¹. The C-H deformations and the C-N stretching appear in the region 1450–1300 cm⁻¹, while the aromatic C-H in-plane and out-of-plane deformations are shown in the spectral region 1250–1000 cm⁻¹ and 1000–700 cm⁻¹, respectively. The vibration at around 650 cm⁻¹ is attributed to the C=N and N=N bonds. The majority of these peaks that are involved in the coordination, especially the vibrations of the C=N, C-N bonds, are shifted upwards in complex 1 suggesting coordination of the ring-N atom to Sn(IV) [28].

In parallel, the Raman spectra of complex 1 and mebta were recorded (Figure 1). Apparently, as in the IR spectrum, the characteristic vibrations of mebta are also present. In addition, the presence of the (CH₃)₂SnCl₂ unit is also evident in the spectrum. In particular, the stretching vibrations noticed at 342 and 553 cm⁻¹ are attributed to the Sn-Cl asymmetric and the Sn-C symmetric vibrations, respectively [29]. It is important to note that the excitation laser line used for the measurement did not promote any Resonance Raman effects, since based on the UV/Vis spectrum of the complex, there is no evidence of an absorption band in the wavelength range 500–800 nm.

3.2.2. ¹H and ¹³C{¹H} NMR Spectroscopy

The ¹H NMR and ¹³C{¹H} NMR spectra were recorded in d₆-DMSO solutions for complex 1 in order to investigate the purity of the complex and the stability of the sample in d₆-DMSO, which is the medium that was used for the subsequent cytostatic measurements. In Figure 2a the ¹H NMR spectrum of complex 1 (Figure 2a) is presented. It is evident that the ¹H NMR spectrum shows all the characteristic signals attributed to the ligand mebta in the aromatic area (i.e., δ 8.03 (dt, J = 8.4, 0.9 Hz, 2H), 7.84 (dt, J = 8.3, 1.0 Hz, 2H), 7.58–7.53 (ddd, J 8.4, 6.9 and 0.9 Hz, 2H), 7.40 (ddd, J = 8.3, 6.9, 1.0 Hz, 2H)), and the signal at δ 4.31 (s, 6H) ) in the aliphatic region, which is in accordance with previous experimental and theoretical analysis of mebta [28,30]. In addition to these signals, the signal in the aliphatic region (i.e., δ 1.03 ppm) is due to the co-existence of the two CH₃ groups of the (CH₃)₂ SnCl₂ in the product. This is also supported by the HSQC NMR spectrum of the complex (Figure S2) where the satellite peaks are clearly observed. The ²J(¹H, ¹¹⁷Sn) and ²J(¹H, ¹¹⁹Sn) coupling constants were calculated to be 109 and 113.67 Hz, respectively (Figure S3), which is in close proximity to analogous coordination complexes [31,32].

In a similar way, in the ¹³C{¹H} NMR spectrum of complex 1 (Figure 2b), all the characteristic signals of mebta attributed to the aromatic carbons are shown in the range 110–150 ppm (i.e., δ 145.09, 133.33, 127.08, 123.85, 118.97, 110.56 ppm) further supported by the previous ¹³C{¹H} NMR analysis of the ligand [28,30]. The signal at δ 34.11 ppm in the spectrum of 1 is assigned to the carbon atoms of the CH₃ groups of both mebta ligands whereas the carbon atoms of the coordinated methyl groups resonate at δ 22.69 ppm. The latter appears at δ 22.49 ppm in the spectrum of (CH₃)₂SnCl₂. The assignment of these two kinds of carbon atoms is supported by the HSQC spectrum of complex 1 (Figure S2) where they show cross-peaks with their corresponding protons resonating at δ 4.31 and 1.03 ppm, respectively. The ¹H NMR and ¹³C{¹H} NMR δ values of mebta in 1 are slightly different than the corresponding ones in the free ligand, implying that mebta remains coordinated in solution. The stability of the complex in DMSO is further proven by its molar conductivity in the same solvent, Λ_Μ (10⁻³ M, 25 °C, DMSO), which is 3 S cm² mol⁻¹; this value is indicative of the presence of neutral species in solution [33] suggesting no decomposition through Cl⁻ release. The above experimental facts strongly suggest that the solid-state structure is retained in solution. In addition, the stability of the complex after 24 h was confirmed by ¹H NMR measurements with time (t = 0, 0.5 h, 1 h, 4 h and 24 h) (Figure S4) [34]. This stability is crucial for the biological study, thus the stock solutions have been prepared in DMSO, which were then diluted in serum-free cell culture medium for each experimental procedure. In this context, future work will include stability studies of the Sn(IV) complex in aqueous media, particularly the hydrolysis rates of chloride ligands.

3.2.3. UV/Vis Spectroscopy

The UV/Vis spectrum of the complex 1 (Figure 3) was recorded in a solution of 82 uM in methanol (MeOH), showing the characteristic π-π* transitions of the organic ligand. The absence of absorbance peaks at the range 400–700 nm ensures that there is no interference of Resonance Raman effects during the Raman measurements.

3.3. Description of the Molecular Structure and Packing

Structural illustration of the molecule [(CH₃)₂SnCl₂(mebta)₂] (1) is provided in Figure 4. Complex 1 crystallizes in the triclinic space group P-1 (no 2). The Sn^IV center is located at a crystallographically imposed inversion center. The metal ion is coordinated to 2 terminal mondentate mebta ligands through one N atom of the triazole ring; this atom is the nitrogen of the position 3 of the azole ring, see Scheme 1. The octahedral coordination sphere of Sn^IV (coordination number = 6) is completed by two Cl⁻ and two CH₃⁻ ligands. Typical bond lengths for an octahedral organotin(IV) complex were observed [35,36]. Selected interatomic distances (Å) and angles (^o) are provided in the Figure 4 caption, indicating the octahedral configuration of the complex. The main crystallographic data of complex 1 (C₁₆H₂₀Cl₂N₆Sn) could be summarized as follows: triclinic, P-1 (no. 2), a = 7.1665(4), b = 7.7958(4), c = 9.5437(5) Å, α = 111.720(2)°, β = 110.205(2)°, γ = 90.404(2)°, V = 459.26(4) ų, Z = 1, T = 120 K, 11,307 reflections measured, 2479 unique (R_int = 0.0184), ϴ_max = 29.211° GoF = 1.126, R₁ = 0.0118, wR₂ (all reflections) = 0.0328.

In the crystal, the aromatic rings of the complex molecules are π-stacked to form a 2D supramolecular network by π-π interactions along the (ac) plane (Figure 5). The distance between the planes formed by the aromatic rings is ca. 3.47 Å. These 2D networks pack along the third dimension of the crystal without specific directional interactions (Figure S5).

3.4. Investigation of Antiproliferative Properties

We evaluated the potential antiproliferative effects of complex 1 and its “components” in terms of the cell viability of triple-negative breast cancer cells, MDA-MB-231. MDA-MB-231 cells were treated for 24 h in the concentration range 0.05–100 ug/mL at 10 ug/mL increment (selective concentrations are shown in Figure 6), in order to define the effective antiproliferative concentration of complex 1 and the effects, if any, of its “components”. As shown in Figure 6, no significant cytotoxicity is observed at concentrations 0.05–10 ug/mL for complex 1, while at the highest concentration, 10 ug/mL, complex 1 statistically significantly reduces MDA-MB-231 cell viability at 75% (IC₅₀ ~ 20 uM). Notably, we observed no significant effects on cell viability of MDA-MB-231 cells treated for 24 h with either mebta or (CH₃)₂SnCl₂ (Figure 6b,c). Moreover, as shown in Figure 6d, we evaluated the effects of complex 1 in the most effective concentration (10 ug/mL) in terms of cell morphology. MDA-MB-231 breast cancer cells typically appear elongated and spindle-like in the traditional 2D monolayers. As depicted in Figure 6d, no significant effects on MDA-MB-231 cell morphology were observed; however, the formation of cell aggregates and junctions may be linked to the reduced cell viability in the presence of complex 1.

Collectively, we have highlighted the promising role of the organotin(IV) complex 1 in reducing the proliferative rates of the aggressive triple-negative breast cancer cells and that this effect is due to the whole complex and not to its ingredients, i.e., mebta and/or (CH₃)₂SnCl₂.

4. Conclusions

A new organotin coordination complex, i.e., [(CH₃)₂SnCl₂(mebta)₂], was synthesized. In the crystal structure of the complex, π-π interactions result in a 2D supramolecular network. The vibration spectroscopy (IR and Raman) verified the successful synthesis and the purity of the complex. No resonance effects intervene to the Raman spectra as it was verified through UV/Vis spectroscopy. In addition, NMR study (¹H NMR and ¹³C{¹H} NMR) revealed the stability of the complex in DMSO, which was the solvent used for the cytotoxicity measurements. Complex 1 reduces the proliferative rates of the aggressive triple-negative breast cancer cells with a significantly reduction of MDA-MB-231 cell viability at 75% (IC₅₀ ~ 20 uM), while this effect was not observed for the two starting materials used for the preparation of complex 1. The present study introduces the potential of the development of a new class of mixed organometallic-coordination Sn(IV) compounds with benzotriazole ligation, with preferential profile as antiproliferative agents in aggressive breast cancer cells. In future, studies investigating the biological effects of these promising compounds as cytotoxic complexes for breast cancer cells, the use of cytostatic agents, such as cytarabine, as controls in functional assays (i.e., cell mobility studies) or incorporating cell cycle assessments, may effectively address and evaluate the compounds’ cytostatic impact. The interplay between estrogen receptor signaling and intercellular matrix components is a key factor in understanding the regulatory mechanism on breast cancer cell functional properties [37]. Work in progress in our laboratories is directed towards the study of the effect of the nature of the organometallic ligand R, the halide ligand, and the benzotriazole group in complexes [R₂SnX₂L₂], where R = Et, Ph, X = Br, I and L = various neutral benzotriazole ligands, and the development of SAR criteria. We have been also working in the preparation and study of the antiproliferative properties in compounds of the general type [R₃SnXL₂].