TY - JOUR
AU - Rahiman, Aziz, Kalilur
AB - Abstract Four new heteroleptic silver(I) complexes with the general formula [Ag(L1–4)(nap)] (1–4), where L1–4 = 2-(1-(4-substitutedphenyl)ethylidene)hydrazinecarbothioamide and nap = naproxen, have been synthesized and characterized. The geometric parameters determined from density functional theory and UV-Vis studies indicate distorted tetrahedral geometry around silver(I) ion. Fourier transform infrared (FT IR) spectra evidenced asymmetric bidentate coordination mode of carboxyl oxygen atoms of naproxen with silver(I) ion. The complexes are stable for 72 h and biocompatibility was analysed towards normal human dermal fibroblast cells, which showed non-toxic nature up to 100 ng/ml. In vitro anti-proliferative activity of the complexes by MTT assay was tested against three human cancerous cell lines and one non-tumorigenic human breast epithelial cell line (MCF-10a) in which the complex 4 exhibited enhanced activity. The morphological changes observed by acridine orange/ethidium bromide and Hoechst 33258 staining method reveal apoptosis-inducing ability of the complexes. The molecular docking studies suggest hydrogen bonding, hydrophobic and π-pair interactions with the active site of epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and lipoxygenase receptors. Graphical Abstract Open in new tabDownload slide Graphical Abstract Open in new tabDownload slide apoptosis, biocompatibility, geometry optimization, in silico studies, naproxen, silver(I) metallodrugs Introduction The ever-growing field of research in medicinal inorganic chemistry is focused on metallodrugs for past few decades because of their significant role in therapeutic and diagnostic medicine [1]. The platinum-based drugs such as cisplatin and its analogues (carboplatin, nedaplatin etc.) are effective only for a narrow spectrum of tumour cells, as it is associated with various toxicity issues like neurotoxicity and nephrotoxicity, which restricts and complicates its utility to a great extent [2–4]. The standard treatment regimens have not changed and there is still an urgent need to explore the application of more physiologically relevant, endogenous metals in cancer therapeutics and develop better molecular platforms for improved cancer-targeting efficacy. In this regard, among coinage metals, divalent copper has been exclusively investigated [5, 6], while chemistry of Cu1, Ag1 and Au1 are inadequate [7], in which silver salts are long known for their antimicrobial properties and non-toxic nature [8]. Silver compounds show prominent activity due to their interaction with the electron transport system of the cell and thiol groups of the vital enzymes [9]. These special features in addition to the non-toxic nature to human cells, silver(I) complexes have gained attractive attention as anticancer therapeutic metallodrug candidates in cancer chemotherapy [10]. Multifunctional N, S donor ligands such as thiosemicarbazones have attracted particular attention over past decades in the context of their wide biological applications due to the versatility of donor atoms, π-delocalization and configurational flexibility [11]. Thiosemicarbazones such as 3-aminopyridine-2-carbaldehydethiosemicarbazone (Triapine) are known antitumor compounds, and their metal complexes show much enhanced cytotoxicity activity [12]. It is assumed that the cytotoxicity activity of thiosemicarbazone metal complexes is due to their ability to inhibit DNA topoisomerase-II, an enzyme that regulates the topology of DNA [13]. Silver(I) complexes of 2-thiophene-N(4)-methylthiosemicarbazone and 2-formylpyridinethiosemicarbazone exhibit comparable cytotoxicity activity against liver (SMMC-7721) and colon adenocarcinoma (HCT-8) cancer cell lines, respectively, with respect to the standard drug, cisplatin [14, 15]. Ag(I) complexes of 4,6-di-tert-butyl-2,3-dihydroxybenzaldehyde thiosemicarbazone and 4,6-di-tert-butyl-2,3-dihydroxybenzaldehyde isonicotinoyl hydrazone were reported for their antibacterial and cytochrome c reduction studies [16]. The non-steroidal anti-inflammatory drugs (NSAIDs) comprise a group of drugs used in the treatment for a large number of diseases, such as inflammation, pain and fever and non-selectively inhibit cyclooxygenase (COX-1 and COX-2) and lipoxygenase (LOX) metabolism, which further inhibits cancer growth [17]. Naproxen, a member of phenylalkanoic acids, is a NSAID, used as analgesic and anti-inflammatory agent [18]. Silver(I) complexes of aspirin, naproxen and salicylic acid exhibit significant antiproliferative activity against breast cancer cell lines and were found to interact with nuclear DNA and LOX causing cell apoptosis through the mitochondrial signalling pathway [19–21]. Scheme 1 Open in new tabDownload slide Synthesis of heteroleptic silver(I) complexes Scheme 1 Open in new tabDownload slide Synthesis of heteroleptic silver(I) complexes A careful literature survey confirmed that the reports on the heteroleptic silver(I) complexes of NSAIDs with thiosemicarbazone, and their theoretical, anti-proliferative and molecular docking studies has not been reported till date. In view of these observations, and in continuation of our recent report on heteroleptic copper(II) and silver(I) complexes with terpyridines and naproxen [22, 23], the current research has been designed in developing new heteroleptic silver(I) complexes of thiosemicarbazones and naproxen. The geometry optimization, electronic structures, Mulliken charge analysis and highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) analysis were performed by density functional theory (DFT) calculations using Gaussian 03 programme. The biocompatibility of the complexes was investigated using normal human dermal fibroblast (NHDF) cells by WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) assay, and in vitro anti-proliferative activity of the complexes was tested against estrogen positive (MCF-7), estrogen negative (MDA-MB-231) and pancreatic (PANC-1) cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Interest is extended in exploring the binding affinity and the mode of interaction of silver(I) complexes with EGFR, VEGFR2 and LOX receptors by docking studies using AutoDock tools. Results and Discussion Synthesis of heteroleptic silver(I) complexes A series of heteroleptic silver(I) complexes of type [Ag(L1–4)(nap)] (1–4) were synthesized in good yield via the aerobic reaction of sodium naproxen (1 mmol) with 2-(1-(4-substituted phenyl)ethylidene)hydrazinecarbothioamide (L1–4, 1 mmol) in the presence of AgNO3 (1 mmol) in methanol (Scheme 1). The complexes were characterized by elemental analysis, Fourier transform infrared (FT IR), Electrospray ionization (ESI)-MS, 1H NMR and thermal analysis. All the complexes are soluble in acetonitrile, methanol, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), and insoluble in water and diethyl ether. Trails made to obtain crystals under various solvents and conditions were unsuccessful due to poor diffraction ability. Spectral characterization The coordination mode of the synthesized heteroleptic silver(I) complexes (1–4) were analysed by comparing the FT IR spectra of the free ligands and naproxen drug with that of the complexes (Figs S1 and S2). The stretching vibration due to υ(C=O) of the carboxylic group in naproxen appears at 1725 cm−1, which upon complexation got replaced by two characteristic bands in the range 1604–1606 and 1389–1393 cm−1 corresponding to υasym(COO) and υsym(COO) stretching, indicating the coordination of carboxylic oxygen atoms of naproxen to the silver ion. The difference between the asymmetric and symmetric vibrations, Δυ = υasym(COO)–υsym(COO), plays a major role in determining the coordination mode of the carboxylate ligands. In general, the carboxylate group (COO−) binds to the metal ion through different modes such as monodentate, bidentate or asymmetrically bidentate form, which is determined by the magnitude of Δυ values obtained in the FT IR frequencies. For a monodentate binding mode, the Δυ values are found to be in between 150 and 175 cm−1 [24–26], while for bidentate binding, it is found to be in between 180 and 200 cm−1, and the asymmetrically bidentate mode of binding is suggested if Δυ values is >200 cm−1 [27]. In the present study, the Δυ values of the complexes are observed in the range 210–218 cm−1, which authenticate the asymmetric bidentate binding mode of the carboxylate group of the naproxen with silver ion. The characteristic bands of thiosemicarbazone ligands in the range 1585–1590 and 835–855 cm−1 attributed to υ(C=N) and υ(C=S) stretching vibrations, respectively, got shifted to 1505–1550 and 814–818 cm−1 for the complexes, indicating coordination of silver ion through imine nitrogen and thione moiety. Additionally, all the silver(I) complexes exhibit bands in the low wavenumber regions of 518–524 and 471–483 cm--1 assigned to υ(Ag-O) and υ(Ag-N), respectively. The band corresponding to υ(Ag-S), in general, is obtained between 350 and 300 cm−1, which could not be predicted in the present study as FT IR is measured between 4000 and 400 cm--1. Figure 1 Open in new tabDownload slide Electrospray mass spectra of heteroleptic silver(I) complexes 1 (a) and 3 (b) Figure 1 Open in new tabDownload slide Electrospray mass spectra of heteroleptic silver(I) complexes 1 (a) and 3 (b) The UV-Vis spectral studies of the silver(I) complexes (1–4) were recorded in DMF medium (Fig. S3), which gives information about the geometry and structural properties of the complexes. All the complexes showed intense absorption bands at 264–271 nm due to an intraligand charge transfer transition (π-π*), while medium intensity bands obtained in the range 325–327 nm is due to the interligand charge-transfer with partial contribution from Ag(dπ) → L(π*) transition [28]. The observed transition and the absence of bands in visible region due to d10 configuration suggest distorted tetrahedral geometry around silver(I) ion [29]. The photoluminescence spectra of the complexes (1–4) recorded in DMF medium for the powdered sample at room temperature (Fig. S4) with excitation at 354–358 nm exhibit an intense emission bands at 732–735 nm [30]. The emission quantum yield (ϕ) of the silver(I) complexes were observed in the range between 0.045 and 0.201. The coordination of the thiosemicarbazone ligand to the silver(I) ion was further supported by 1H NMR spectra (Figs S5 and S6). The ligands L1&4 showed singlet for methyl protons at 2.36, and 2.31 and 2.27 ppm, respectively, and multiplets for aromatic protons in the region 7.17–7.86 ppm [31, 32]. The silver(I) complexes exhibited methyl and aromatic protons signals at approximately same positions. The NH2 protons of ligands L1&4 observed at 8.47 and 8.28 ppm, respectively, were shifted downfield (8.85 and 8.69 ppm) due to the coordination of thione group to metal ion. The NH protons observed at 10.30 and 10.20 ppm, respectively, for ligands L1&4 were also shifted upfield (10.02 and 10.07 ppm), indicating the involvement of amide group in coordination. The 1H NMR spectrum of sodium naproxen shows a doublet at 1.42–1.39, singlet at 3.83 and multiplets at 7.09–7.78 ppm corresponding to methyl, methoxy and aromatic protons, respectively [20], which were obtained almost at the same position for the complexes, indicating the coordination of the carboxylic group with the silver metal. The observed NMR data supported the retention of the structure of the complexes in solution state. The ESI mass spectra were recorded to authenticate the stoichiometric composition of the complexes. The mass spectra of complexes 1 and 3 are shown in Fig. 1, which exhibit molecular ion peak at m/z 529.1589 and 574.0440, respectively, due to [Ag(L1&3)(nap)]+, corresponding to their molecular weight. The base peak at m/z 299.9725 and 344.9574 corresponds to [Ag(L1&3)]+, respectively. The obtained mass spectral data were in good agreement with the proposed molecular formulae of the synthesized complexes. Thermal analysis The thermal decomposition patterns of the silver(I) complexes (2 and 3) were obtained by Thermogravimetric (TG/DTG) analysis (Fig. 2). The thermograms of the complexes were interpreted to substantiate the molecular formulas obtained from elemental analysis and mass data. The thermograms of the complexes 2 and 3 were found to be stable ~305 and 240°C, respectively. Complex 2 shows decomposition in a single step from 305 to 550°C accompanied with mass loss of 81.44% (calcd. 80.91%) attributed to thiosemicarbazone and naproxen moiety with an exothermic DTG peak at 348°C, leaving a residue of metallic silver of around 18.56% (calcd. 19.09%) above 550°C. In contrast to complex 2, the complex 3 decomposes in two stages. The first decomposition occurs from 240 to 380°C with an exothermic DTG peak at 316°C and mass loss of around 43.28% (calcd. 41.41%) can be attributed to the loss of thiosemicarbazone ligand moiety. The second weight loss occurs from 380 to 780°C with an exothermic DTG peak at 412°C and mass loss of around 39.14% (calcd. 39.86%) corresponds to the loss of naproxen moiety. Finally, >800°C, the thermogram leads to a plateau, leaving a residue of metallic silver of 17.58% (calcd. 18.73%). The TG data clearly support the higher thermal stability of the synthesized silver(I) complexes when compared to the starting ligands [33]. Figure 2 Open in new tabDownload slide TG and DTG thermograms of complexes 2 (a) and 3 (b) Figure 2 Open in new tabDownload slide TG and DTG thermograms of complexes 2 (a) and 3 (b) Molecular modelling Geometry optimization Molecular modelling is a computer based tool used in deriving and representing the structure and coordination of metal complexes in three dimensions. These representations, in the absence of crystallographic data, provide information about the structural aspects and bonding modes of the complexes along with the energy minimized conformation. The geometries of the silver(I) complexes (1–4) were optimized by theoretical calculations using DFT studies at Becke-3-Lee-Yang-Parr (B3LYP)/LANL2DZ levels in gas phase and the optimized structures are shown in Fig. 3. The calculated structural parameters (bond lengths and bond angles) of the complexes are given in Table S1, which are in reasonable agreement with the previously reported crystallographic data [19, 34, 35]. The calculated bond lengths are optimized in the gas phase because of which they are slightly longer than the experimental values, which are in a tight crystal lattice. The calculated asymmetrical Ag–O bond lengths are between 2.942–2.969 and 2.139–2.172 Å, which are 0.16–0.19 and 0.21–0.24 Å longer than the experimental values. The two different Ag–O bond length values support the unsymmetrical binding mode of carboxylic group of naproxen to silver ion and support the IR spectral data. The deviation in experimental and calculated Ag–S bond length lies in the range 0.10–0.15 Å, whereas that for Ag–N is in the range 0.16–0.24. The deviation in the calculated and experimental bond angles of N–Ag–S, S–Ag–O, O–Ag– O, O–Ag– N, N–Ag–O and O–Ag–S are 2.63, 1.21, 1.32, 4.70, 3.50 and 4.88° for complex 1, 2.30, 5.94, 1.82, 1.20, 2.13 and 3.28° for complex 2, 1.96, 3.54, 2.62, 2.84, 2.41 and 2.33° for complex 3, and 1.35, 1.48, 1.52, 4.27, 1.03 and 0.74° for complex 4, respectively. The small discrepancies observed between the calculated and experimental bond length and bond angle values are attributed to hydrogen bonding and packing interactions in the lattice [36]. Figure 3 Open in new tabDownload slide Optimized geometries of silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) Figure 3 Open in new tabDownload slide Optimized geometries of silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) By calculating τ values using the following equation, molecular geometries can be predicted [37]: $$\begin{equation} \tau =\frac{\beta -\alpha }{60} \end{equation}$$ where α and β are the equatorial and axial bond angles, respectively. τ value plays a major role in predicting the geometry of the complexes. If τ value is close to 0, the molecular geometry is square planar, while if it is close to 1 then it is tetrahedral. If τ value is between 0 and 0.5, the molecular geometry is distorted tetrahedral. The α S–Ag–N values for complexes 1–4 are 46.76, 50.26, 48.56 and 51.96, and β O–Ag–O values are 71.42, 70.79, 68.75 and 71.74, respectively. The observed τ values for the silver(I) complexes lies between 0 and 0.5, authenticating distorted tetrahedral geometry of the complexes. Mulliken charge analysis The Mulliken charge analysis of the silver(I) complexes (1–4) was calculated using DFT/B3LYP/LANL2DZ basic set, and the bar diagram is shown in Fig. S7. It may be noted that in all the complexes, silver atom has positive charge between 0.177 and 0.256, and both oxygen atoms of naproxen coordinated to silver ion has negative charge between −0.409 and −0.487. All nitrogen and oxygen atoms have negative charge and all the sulphur atoms have slight positive charge. All the hydrogen atoms have net positive charge, and in particular the hydrogen atoms attached to nitrogen atoms have significantly large net positive charge. The presence of large amount of negative charge on carbon atoms and large positive charge on hydrogen atoms facilitate the formation of a large number of intra- and inter-molecular hydrogen bonding interactions in the crystalline phase. Frontier molecular orbitals The frontier molecular orbitals, HOMO and LUMO, play an important role in structural investigation as they are closely related to the electronic excitations and transition characteristics [38]. The electronic excitation from HOMO to LUMO corresponds to the transition from the ground to the first excited state [39]. The simplest way to calculate the excitation energies is the difference between the HOMO (electron donor) and LUMO (electron acceptor) of the neutral system. Figure 4 shows the HOMO-LUMO of the synthesized silver(I) complexes (1–4). It is noticed that, in all the complexes, the LUMO is localized on thiosemicarbazone moiety and the HOMO is noticed on naproxen moiety, indicating intramolecular charge transfer of electrons within the molecule. The wide energy gap between HOMO and LUMO points towards the stability of the molecule and small energy gap indicates easy charge transfer within the molecule, which further enhances the biological activity of the molecule. Based on Koopman’s theorem, electronegativity, global hardness, global softness and electrophilicity are calculated using the following equations and tabulated in Table 1. The molecule with least HOMO-LUMO gap or global softness value indicates that it is highly reactive. $$\begin{eqnarray} \mathrm{Electronegativity}\;\Big(\chi \Big)=-\frac{1}{2}\Big({E}_{\mathrm{Homo}}+{E}_{\mathrm{Lumo}}\Big) \end{eqnarray}$$ $$\begin{equation} \mathrm{Global}\kern0.17em \mathrm{hardness}\;\left(\eta \right)=-\frac{1}{2}\left({E}_{\mathrm{Homo}}-{E}_{\mathrm{Lumo}}\right) \end{equation}$$ $$\begin{equation} \mathrm{Global}\kern0.17em \mathrm{softness}\;(S)=1/\eta \end{equation}$$ $$\begin{equation} \mathrm{Electrophilicity}\;\left(\omega \right)={\chi}^2/2\eta \end{equation}$$ Figure 4 Open in new tabDownload slide Frontier molecular orbitals of the silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) Figure 4 Open in new tabDownload slide Frontier molecular orbitals of the silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) Table 1 Electronegativity (χ), global hardness (η), global softness (S) and electrophilicity (ω) values of silver(I) complexes (1–4) in eV calculated by DFT/B3LYP/LANL2DZ basic set Complexes . Electronegativity (χ) . Hardness (η) . Softness (S) . Electrophilicity (ω) . 1 0.136 0.046 10.736 0.201 2 0.130 0.053 9.511 0.161 3 0.149 0.058 8.612 0.191 4 0.158 0.035 14.146 0.352 Complexes . Electronegativity (χ) . Hardness (η) . Softness (S) . Electrophilicity (ω) . 1 0.136 0.046 10.736 0.201 2 0.130 0.053 9.511 0.161 3 0.149 0.058 8.612 0.191 4 0.158 0.035 14.146 0.352 Open in new tab Table 1 Electronegativity (χ), global hardness (η), global softness (S) and electrophilicity (ω) values of silver(I) complexes (1–4) in eV calculated by DFT/B3LYP/LANL2DZ basic set Complexes . Electronegativity (χ) . Hardness (η) . Softness (S) . Electrophilicity (ω) . 1 0.136 0.046 10.736 0.201 2 0.130 0.053 9.511 0.161 3 0.149 0.058 8.612 0.191 4 0.158 0.035 14.146 0.352 Complexes . Electronegativity (χ) . Hardness (η) . Softness (S) . Electrophilicity (ω) . 1 0.136 0.046 10.736 0.201 2 0.130 0.053 9.511 0.161 3 0.149 0.058 8.612 0.191 4 0.158 0.035 14.146 0.352 Open in new tab NMR analysis The comparison of theoretical and experimental NMR data helps to assign and understand the relationship between chemical shift and molecular structure. Hence, the theoretical 1H NMR spectra of complexes 1 and 4 were analysed by gauge including atomic orbitals (GIAO) method using DFT/B3LYP/LANL2DZ basic set to support the experimental data (Figs S5 and S6). The impact of the solvent was considered using the polarized continuum model [40]. The relationship between theoretical and experimental values of 1H NMR chemical shifts was obtained by plotting experimental data vs computed values (Fig. 5). The chemical shifts calculated by DFT method are comparable with the experimental values. The plots showed a good linear relationship since the solid line has approximately a unit slope and zero intercept, supporting the stability of the complexes in solid and gaseous environment. Figure 5 Open in new tabDownload slide Comparision between experimental and theoretical 1H NMR chemical shifts of complexes 1 (a) and 4 (b) Figure 5 Open in new tabDownload slide Comparision between experimental and theoretical 1H NMR chemical shifts of complexes 1 (a) and 4 (b) Stability and lipophilicity of the complexes Stability of a compound is a pre-requisite criterion in pharmaceutical industries for drug approval process, which ensures the maintenance of the product quality, safety and efficacy throughout the shelf life. In this regard, the stability of the synthesized heteroleptic silver(I) complexes (1–4) in biologically relevant solutions (Tris HCl:DMF/DMSO) was measured over different time intervals during 24, 48 and 72 h using UV-Vis spectroscopy, which confirmed the stability of complexes for 24 and 48 h, with negligible differences for 72 h samples (Fig. S8). Lipophilicity is a major physicochemical property in pharmaceutical research, which affects many biological processes such as adsorption, passage through membranes, drug receptor interactions, metabolism and toxicity of molecules [41]. In this regard, the water-octanol partition coefficient (log P) value for the complexes (1–4) was determined by shake-flask method using UV–Vis spectroscopy (Table S2). All the complexes show log P values between 0.034 ± 0.021 and 1.25 ± 0.011, indicating higher concentration of complexes in n-octanol (lipophilic phase) than in water (hydrophilic phase). The substituents present subtly affect the lipophilic character of the complexes, wherein, complex 4 shows high lipophilic character due to the presence of hydrophobic substituent, which further increases the affinity of the complex for ligand-receptor interaction and biological activity. In vitro cytotoxicity studies Biocompatibility test The biocompatibility of silver(I) complexes (1–4) was evaluated by one-step WST-1 assay on NHDF cells after 1 and 3 days of incubation at different concentrations (1, 10, 50 and 100 ng/ml). The number of viable cells is directly related to the amount of formazan dye formed due to the cleavage of a tetrazolium salt, WST-1, which can be quantified by measuring the absorbance at 440 nm. All the complexes were highly biocompatible up to 100 ng/ml towards NHDF cells under physiological conditions. However, complex 4 exhibited >90% cell viability at low concentration levels on day 1 and the cell proliferation activity of the complexes increased similar to that of the control group on day 3 (Fig. 6). Live/dead cells were analysed, which exhibited that on addition of the complexes, cytotoxicity effect was not induced towards the NHDF cells as compared to that of the control group (Fig. 7), which shows high biocompatibility of the complexes under physiological conditions. Figure 6 Open in new tabDownload slide Biocompatibility analysis of the complexes 1 (a) and 4 (b) on NHDF cells Figure 6 Open in new tabDownload slide Biocompatibility analysis of the complexes 1 (a) and 4 (b) on NHDF cells Figure 7 Open in new tabDownload slide Live/dead fluorescence images of NHDF cells treated with complexes 1 (a) and 4 (b) after day 1 and day 3 of incubation Figure 7 Open in new tabDownload slide Live/dead fluorescence images of NHDF cells treated with complexes 1 (a) and 4 (b) after day 1 and day 3 of incubation Anti-proliferative activity by MTT assay The selected silver(I) complexes (3&4) were further analysed for their in vitro anti-proliferative activity by MTT assay, due to their less toxic nature. The MTT assay is based on the criterion that the live cells reduce the yellow MTT by cleaving the tetrazolium rings and form water-insoluble dark blue formazan crystals. The effect of complexes with various concentration (0.29, 0.58, 1.17, 2.34, 4.68, 9.4, 18.8, 37.5, 75.0 and 150.0 ng/ml) by dissolving in 0.1% DMSO were tested for viability of the cells for 48 h against three human cancerous cell lines [estrogen positive (MCF-7), estrogen negative (MDA-MB-231) and pancreatic (PANC-1)] in comparison with a non-tumorigenic human breast epithelial cell line (MCF–10a) and two benchmark compounds, cisplatin and carboplatin under identical conditions. A blank sample of solvent (0.1% DMSO) of same volume was taken as control to identify the activity of the solvent. The cell viability decreased with increasing concentrations of complexes, which show the concentration dependent nature, and the IC50 values are given in Table 2. Complex 3 exhibits weak cell growth inhibition activity against all three cancer cell lines and complex 4 shows moderate activity towards MCF-7 cell line compared to the standard drugs. The selectivity of complex 4 towards MCF-7 compared to MDA-MB-231 and PANC-1 cell lines may be due to the presence of the electron releasing substituent. The main advantage of the synthesized complexes was its less toxic nature on normal cell line (NHDF) than the cancer cell lines. Thus, the complexes may be useful in selectively targeting cancer cells over normal cells. Complex 4 showed higher cytotoxicity activity than the complex 3 because of the presence of hydrophobic substituent. Table 2 In vitro anti-proliferative activity (IC50 values) of heteroleptic silver(I) complexes (3 and 4) against human cancerous cell lines [estrogen positive (MCF-7), estrogen negative (MDA-MB-231) and pancreatic (PANC-1)] and a non-tumorigenic human breast epithelial cell line (MCF-10a) Compounds . IC50 values (μM)a . MCF-7 . MDA-MB-231 . PANC-1 . MCF-10a . 3 94.05 ± 0.01 107.09 ± 1.61 98.96 ± 0.76 ≥100 4 73.43 ± 0.05 83.64 ± 0.08 76.82 ± 1.01 ≥100 Cisplatin 52.80 ± 0.72 44.40 ± 0.72 52.80 ± 0.72 ≥100 Carboplatin 61.10 ± 0.19 53.60 ± 1.24 53.60 ± 1.24 ≥100 AgNO3 ≤80 ≤90 ≤90 ≥100 Compounds . IC50 values (μM)a . MCF-7 . MDA-MB-231 . PANC-1 . MCF-10a . 3 94.05 ± 0.01 107.09 ± 1.61 98.96 ± 0.76 ≥100 4 73.43 ± 0.05 83.64 ± 0.08 76.82 ± 1.01 ≥100 Cisplatin 52.80 ± 0.72 44.40 ± 0.72 52.80 ± 0.72 ≥100 Carboplatin 61.10 ± 0.19 53.60 ± 1.24 53.60 ± 1.24 ≥100 AgNO3 ≤80 ≤90 ≤90 ≥100 aAverage of three independent determinations; results are expressed as mean ± SD. Open in new tab Table 2 In vitro anti-proliferative activity (IC50 values) of heteroleptic silver(I) complexes (3 and 4) against human cancerous cell lines [estrogen positive (MCF-7), estrogen negative (MDA-MB-231) and pancreatic (PANC-1)] and a non-tumorigenic human breast epithelial cell line (MCF-10a) Compounds . IC50 values (μM)a . MCF-7 . MDA-MB-231 . PANC-1 . MCF-10a . 3 94.05 ± 0.01 107.09 ± 1.61 98.96 ± 0.76 ≥100 4 73.43 ± 0.05 83.64 ± 0.08 76.82 ± 1.01 ≥100 Cisplatin 52.80 ± 0.72 44.40 ± 0.72 52.80 ± 0.72 ≥100 Carboplatin 61.10 ± 0.19 53.60 ± 1.24 53.60 ± 1.24 ≥100 AgNO3 ≤80 ≤90 ≤90 ≥100 Compounds . IC50 values (μM)a . MCF-7 . MDA-MB-231 . PANC-1 . MCF-10a . 3 94.05 ± 0.01 107.09 ± 1.61 98.96 ± 0.76 ≥100 4 73.43 ± 0.05 83.64 ± 0.08 76.82 ± 1.01 ≥100 Cisplatin 52.80 ± 0.72 44.40 ± 0.72 52.80 ± 0.72 ≥100 Carboplatin 61.10 ± 0.19 53.60 ± 1.24 53.60 ± 1.24 ≥100 AgNO3 ≤80 ≤90 ≤90 ≥100 aAverage of three independent determinations; results are expressed as mean ± SD. Open in new tab Apoptosis by Hoechst 33258 and AO/EB staining assay Apoptosis is a unique kind of maintenance of health of multicellular organisms, which occurs due to physiological and external stimuli in a regulated and controlled fashion. Necrosis differs from apoptosis, in which uncontrolled cell death occurs leading to cell lysis. Due to the sensitivity of tumour cells in the presence of chemotherapeutic agents towards few apoptotic stimuli, apoptosis-inducing ability of drugs became a primary factor in determining their efficacy [42]. In the present work, the morphological changes and apoptosis effect of complexes 3 and 4 were tested against MCF-7 cells by acridine orange (AO)/ethidium bromide (EB) and Hoechst 33258 staining methods. The MCF-7 cells were stained with AO/EB and Hoechst 33258 dyes after treating with complexes for 48 h to determine the apoptosis-inducing ability of the complexes. The cytological changes due to chromatin condensation observed in AO/EB staining shows viable cells with green nuclei and organized structure, early apoptotic cells with green nuclei and perinuclear chromatin condensation as bright green patches, late apoptotic cells with orange to red nuclei with condensed chromatin, and necrotic cells, which are large in size with orange to red nuclei with no condensed chromatin (Fig. 8). Hoechst 33258 staining method shows the apoptotic characteristics such as nuclear swelling, cytoplasmic vacuolation, chromatin fragmentation and cytoplasmic blebbing, which showed the apoptosis-inducing ability of the complexes in MCF-7 cells (Fig. 9). These morphological changes suggest that the complexes induce cell death through apoptosis, and complex 4 shows higher apoptotic activity than complex 3 in both staining methods. Figure 8 Open in new tabDownload slide AO/EB staining of MCF-7 cells for 48 h: control (a), and complexes 3 (b) and 4 (c) Figure 8 Open in new tabDownload slide AO/EB staining of MCF-7 cells for 48 h: control (a), and complexes 3 (b) and 4 (c) Figure 9 Open in new tabDownload slide Hoechst 33 258 staining of MCF-7 cells for 48 h: control (a), and complexes 3 (b) and 4 (c) Figure 9 Open in new tabDownload slide Hoechst 33 258 staining of MCF-7 cells for 48 h: control (a), and complexes 3 (b) and 4 (c) In silico analysis Molecular docking with EGFR and VEGFR2 The epidermal growth factor receptor (EGFR) plays a crucial role in initiating signal transduction pathways, which regulate biological processes like gene expression, regulating cellular proliferation, angiogenesis and apoptosis inhibition [43, 44]. EGFR is a transmembrane glycoprotein of tyrosine kinase receptor family, which is overexpressed in majority of solid tumours like breast, head and neck, non-small-cell lung, renal, ovarian and colon cancers [45]. This over expression leads to intense signal generation resulting in aggressive growth and invasiveness of cells [46]. EGFR inhibition retards metastatic activity and enhances radiation induced apoptosis and prevents radiation damaged cells from entering the G2/M phase by inducing cell-cycle arrest in G1 phase [47]. On the other hand, vascular endothelial growth factor (VEGF) belongs to homodimeric glycoprotein, which act as a potent therapeutic target for development of new anticancer agents and currently being assessed in preclinical and clinical trials [48]. VEGF expression is induced during tumour formation, which is dependent on the formation of new capillaries from existing blood vessels called as angiogenesis [49]. VEGF binds to three different, but structurally related VEGF-receptor (VEGFR) tyrosine kinases [50] among which VEGFR2 has shown to play key role in regulation of tumour angiogenesis, acts as principal mediator of several physiological and pathological functions and mediates vascular permeability in vivo [51, 52]. These facts encouraged us to analyse the interaction of the synthesized silver(I) complexes (1–4) with these receptors (EGFR and VEGFR2). The energetically favoured docked images are shown in Figs 10 and 11, Figs S9 and S10, and the binding affinity values are given in Table S3. Figure 10 Open in new tabDownload slide Docking poses of silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) with EGFR receptor showing 3D and π-pair interactions Figure 10 Open in new tabDownload slide Docking poses of silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) with EGFR receptor showing 3D and π-pair interactions Figure 11 Open in new tabDownload slide 2D Molecular docked models of silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) with VEGFR2 receptor showing hydrogen bonding and hydrophobic interactions Figure 11 Open in new tabDownload slide 2D Molecular docked models of silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) with VEGFR2 receptor showing hydrogen bonding and hydrophobic interactions All the complexes (1–4) showed hydrogen bonding and π-pair (π–π, π–σ and π-cation) interactions with both EGFR and VEGFR2 receptors. Complexes 1, 2 and 4 binds to EGFR receptor by one hydrogen bond each between hydrogen atom of ARG 819 and oxygen atom of naproxen (NH···O = 2.84 Å, dihedral angle = 148.48° for complex 1; NH···O = 2.56 Å, dihedral angle = 130.13° for complex 2; and NH···O = 2.89 Å, dihedral angle = 155.60° for complex 4), whereas, complex 3 shows hydrogen bond between hydrogen atom of THR 830 and oxygen of nitro group of the thiosemicarbazone moiety (OH···O = 2.52 Å, dihedral angle = 101.27°). Complexes 2, 3 and 4 were further stabilized by a π-cation interaction between LYS 721 and ring of thiosemicarbazone and naproxen (bond length: 5.90, 5.54 and 5.72 Å, respectively) and complex 1 interacted via π–π interaction between ring of thiosemicarbazone and PHE 699 (bond length: 5.39 Å) of EGFR receptor. In case of VEGFR2 receptor, complexes 1, 2 and 4 are stabilized by one hydrogen bond each between oxygen atom of ASN 1033 and hydrogen atom of thiosemicarbazone ligand (O···HN = 2.92 Å, dihedral angle = 130.33° for complex 1; O···HN = 2.88 Å, dihedral angle = 118.82° for complex 2; and O···HN = 2.96 Å, dihedral angle = 120.94° for complex 4). The complex 3 is stabilized by two hydrogen bonds between hydrogen atom of CYS 919 and oxygen atom of nitro group of thiosemicarbazone (NH···O = 3.16 Å, dihedral angle = 166.38°), and hydrogen atom of CYS 1045 and oxygen atom of naproxen (NH···O = 3.19 Å, dihedral angle = 127.76°). All the complexes are further stabilized by two π-cation interactions between LYS 868 and phenyl ring of naproxen (bond length: 3.71 and 3.68 Å for complex 1; 3.73 and 3.65 Å for complex 2; 3.88 and 3.55 Å for complex 3; and 3.64 and 3.73 Å for complex 4). Complex 3 is additionally stabilized by one π–σ interaction between phenyl ring of thiosemicarbazone and LEU 1035 (bond length: 3.00 Å) of VEGFR2 receptor. Besides, the binding mode was enhanced by hydrophobic interactions formed between complexes (1–4) and residues LEU 694, PHE 699, VAL 702, ALA 719, LYS 721, GLU 738, MET 742, THR 766, GLN 767, MET 769, GLY 772, ASP 813, ARG 817, ASN 818, LEU 820, THR 830, ASP 831 and PHE 832 of EGFR receptor, whereas between complexes (1–4) and residues LEU 840, VAL 848, LYS 868, ALA 866, GLU 885, ILE 888, LEU 889, PHE 918, CYS 919, ARG 1032, ASN 1033, LEU 1035, CYS 1045, ASP 1046 and PHE 1047 of VEGFR2 receptor. Molecular docking with LOX receptor LOXs are non-heme iron containing dioxygenases belonging to oxygenases, metabolizing polyunsaturated fatty acids [53, 54]. LOXs are found in many tissues and organs, which are cytosolic and active in the sub-membrane of the cell [55]. LOX catalyses the oxidation of arachidonic acid to leukotrienes for the cell life involving in inflammation mechanism [56]. LOX inhibition induces apoptosis while lipid peroxidases derived from metabolism of fatty acids by LOX regulates cellular proliferation [57, 58]. NSAIDs are found to activate the mechanism of apoptosis through LOX inhibition, which is associated with suppression of certain cancers [59, 60]. Thus, the development of new LOX inhibitors is a potential target in new metallotherapeutic drug designs. This encouraged us to study the interaction of the synthesized silver(I) complexes (1–4) with LOX enzyme. The energetically favoured docked images are shown in Fig. 12 and Fig. S11, and the binding affinity values are given in Table S3. Figure 12 Open in new tabDownload slide Docking poses of silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) with LOX receptor showing 3D and π-pair interactions Figure 12 Open in new tabDownload slide Docking poses of silver(I) complexes 1 (a), 2 (b), 3 (c) and 4 (d) with LOX receptor showing 3D and π-pair interactions All the silver(I) complexes interacted with LOX receptor through π-cation and hydrophobic interactions. None of the complexes interacted via hydrogen bonding and other π-pair (π–π and π–σ) interactions. Complex 1 was stabilized by one π-cation interaction between LYS 552 and phenyl ring of naproxen (bond length: 6.578 Å), whereas complexes 2, 3 and 4 were stabilized by two π-cation interactions each between HIS 373/HIS 378 and nitrogen atom of thiosemicarbazone (bond length: 4.715 and 3.528 Å for complex 2; 4.992 and 4.195 Å for complex 3; and 4.898 and 4.290 Å for complex 4). Besides, all the complexes resides in the hydrophobic pocket of LOX receptor defined by TRP 158, PHE 184, VAL 249, LEU 250, HIS 373, LEU 374, HIS 376, HIS 378, LEU 379, LEU 380, PHE 384, ILE 412, ASN 413, LEU 415, ALA 416, ARG 417, LEU 419, LEU 420, ILE 421, LEU 449, LEU 453, LEU 454, ASP 459, THR 548, LYS 552, ALA 606, LEU 609, LEU 610 and ILE 676. Experimental Materials Acetophenone, 4-chloroacetophenone, 4-methylacetophenone, 4-nitroacetophenone, thiosemicarbazide and AgNO3 were purchased from Sigma-Aldrich (USA). Analytical grade solvents were purchased from E. Merck and used as received without further purification. Physical measurements Elemental analysis (CHN) of the compounds was carried out with a Carlo Erba model-1106 elemental analyser. Perkin-Elmer FT IR 8300 model spectrophotometer with attenuated total reflectance (ATR) method was used to record IR spectra in the range of 4000–400 cm−1. Electronic absorption spectra were recorded in the range of 200–900 nm using Perkin-Elmer Lambda-35 spectrophotometer. Fluorescence spectra were recorded on Horiba Jobin Yvon FluoroLog SPEX-F311 spectrofluorometer. 1H NMR spectral data were obtained in DMSO(d6) solution with tetramethylsilane as an internal standard on Varian-VNMRS-400 model. ESI mass spectra were recorded on Q-Tof mass spectrometer using methanol as a carrier solvent. TG/DTG analysis was carried out using SDT Q600 US instrument from room temperature to 1000°C under nitrogen atmosphere with a heating rate of 10°C/min. The ligands 2-(1-phenylethylidene)hydrazinecarbothioamide (L1), 2-(1-(4-chlorophenyl)ethylidene)hydrazinecarbothioamide (L2), 2-(1-(4-nitrophenyl)ethylidene)hydrazinecarbothioamide (L3) and 2-(1-(4-tolyl)ethylidene)hydrazinecarbothioamide (L4) were synthesized by the procedure reported in the literature [61]. General procedure to synthesize heteroleptic silver(I) complexes of thiosemicarbazones and naproxen (1–4) A methanolic solution of 2-(1-(4-substitutedphenyl)ethylidene)hydrazinecarbothioamide (L1–4, 1 mmol) was added slowly to the methanolic solution of AgNO3 (1 mmol) followed by sodium naproxen (1 mmol) with constant stirring. The resulting solution was stirred for 4 h, filtered and kept aside for slow evaporation. The product was washed with diethyl ether, dried in vaccum and recrystallized from methanol and acetonitrile. $$\begin{equation} \left[\mathrm{Ag}\left({\mathrm{L}}^1\right)\left(\mathrm{nap}\right)\right] \end{equation}$$(1) Yield: 0.38 g, (79.5%). Colour: colourless. Anal. Calc. for: C23H24N3O3SAg (530.3882): C, 52.08; H, 4.56; N, 7.91; Found: C, 52.64; H, 4.73; N, 8.23%. Selected IR data (υ/cm−1): 3431 υ(N–H)asym, 3325 υ(N–H)sym, 1605 υ(COO)asym, 1389 υ(COO)sym, 1550 υ(C=N), 817 υ(C=S), 518 υ(Ag-O), 471 υ(Ag-N). UV-Vis (DMF): [λmax (nm) (ε, dm3 mol−1 cm−1)]: 271 (31 400), 327 (16 500). ESI-MS (m/z): 529.1589 [Ag(L1)(nap)]+. $$\begin{equation} \left[\mathrm{Ag}\left({\mathrm{L}}^2\right)\left(\mathrm{nap}\right)\right] \end{equation}$$(2) Yield: 0.42 g, (82.4%). Colour: colourless. Anal. Calc. for: C23H23N3O3SClAg (564.8332): C, 48.91; H, 4.10; N, 7.44; Found: C, 49.44; H, 4.53; N, 7.86%. Selected IR data (υ/cm−1): 3428 υ(N–H)asym, 3349 υ(N–H)sym, 1602 υ(COO)asym, 1393 υ(COO)sym, 1524 υ(C=N), 818 υ(C=S), 523 υ(Ag-O), 483 υ(Ag-N). UV-Vis (DMF): [λmax (nm) (ε, dm3 mol−1 cm−1)]: 262 (25 710), 323 (13 410). ESI-MS (m/z): 563.0232 [Ag(L2)(nap)]+. $$\begin{equation} \left[\mathrm{Ag}\left({\mathrm{L}}^3\right)\left(\mathrm{nap}\right)\right] \end{equation}$$(3) Yield: 0.39 g, (80.2%). Colour: colourless. Anal. Calc. for: C23H23N4O5SAg (575.3857): C, 48.01; H, 4.03; N, 9.74; Found: C, 48.44; H, 4.53; N, 9.96%. Selected IR data (υ/cm−1): 3439 υ(N–H)asym, 3323 υ(N–H)sym, 1606 υ(COO)asym, 1389 υ(COO)sym, 1544 υ(C=N), 815 υ(C=S), 521 υ(Ag-O), 475 υ(Ag-N). UV-Vis (DMF): [λmax (nm) (ε, dm3 mol−1 cm−1)]: 268 (28 730), 329 (12 050). ESI-MS (m/z): 574.0440 [Ag(L3)(nap)]+. $$\begin{equation} \left[\mathrm{Ag}\left({\mathrm{L}}^4\right)\left(\mathrm{nap}\right)\right] \end{equation}$$(4) Yield: 0.40 g, (81.5%). Colour: pale brown. Anal. Calc. for: C24H26N3O3SAg (544.4147): C, 52.95; H, 4.81; N, 7.72; Found: C, 53.12; H, 4.98; N, 7.94%. Selected IR data (υ/cm−1): 3422 υ(N–H)asym, 3319 υ(N–H)sym, 1604 υ(COO)asym, 1390 υ(COO)sym, 1549 υ(C=N), 814 υ(C=S), 521 υ(Ag-O), 472 υ(Ag-N). UV-Vis (DMF): [λmax (nm) (ε, dm3 mol−1 cm−1)]: 265 (26 410), 325 (12 130). ESI-MS (m/z): 543.0746 [Ag(L4)(nap)]+. Computational studies The theoretical calculations were performed using Gaussian 03 software package by applying DFT method with B3LYP supplemented with the standard LANL2DZ basis set [62, 63] with the aid of the GaussView visualization program. The geometry optimization corresponding to minimum potential energy surface and the Mulliken charge analysis of the complexes (1–4) has been obtained by solving self-consistent field equation iteratively and symmetry constraints were not applied during optimization process [64]. Stability and lipophilicity studies The stability and lipophilicity studies of the silver(I) complexes (1–4) were executed using UV-Vis spectroscopy. The stability of the complexes in biologically relevant solution (Tris HCl:DMF/DMSO) was monitored at different time intervals of 24, 48 and 72 h. The water-octanol partition coefficient (log P) value for the complexes was determined by shake-flask method [65]. An aqueous solution of complexes (5 ml, 1 mM) with octanol (5 ml) was shaken well in 20 ml separating funnel at room temperature for 1 h, and the two phases were separated after 24 h and the concentration of the complexes in each phase was determined by UV-Vis spectroscopy. Cell growth inhibition Biocompatibility test NHDF cells cultured in Dulbecco’s Modificated Eagle’s Medium [89% (v/v), DMEN, WISENT-INC, 319–010-CL], along with penicillin-streptomycin [1% (v/v), Wisent-INC, 450–201-EL] solution and fetal bovine serum [10% (v/v), corning, 35–015-CV] were seeded in a 96-well plate and maintained at 37°C in a 5% CO2 incubator for 24 h. The media from the culture was removed and washed twice with IX Dulbecco’s phosphate buffer saline (DPBS, corning, 25-031-CV). The synthesized complexes (200 μg/ml) dissolved in 0.1% DMSO and diluted to 100, 50, 25, 10 and 1 μg/ml and final concentrations of 1, 10, 25, 50 and 100 ng/ml (100 μl) were prepared and added into the NHDF cells and the plates were incubated in a humidified atmosphere of 5% CO2 at 37°C for 1 and 3 days in a separate set. Cell viability was evaluated by water soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) assay (EZ-Cytox cell viability assay kit, DAEIL Lab Service, Korea) and live and dead (L/D) cells were analysed by fluorescence staining. WST-1 assay and fluorescence staining WST-1 assay reagent was prepared by mixing EZ-Cytox (DOGEN, EZ-3000) with culture media at a ratio of 1:10 (v/v) and the L/D fluorescence staining reagent was prepared with 2 μM of Calcein-AM (Invitrogen, C3099) and 4 μM of ethidium homodimer-1 (Invitrogen, L3224) in IX DPBS under dark conditions. The media was removed from experimental plates and the cells were washed with IX DPBS, and WST-1 assay reagent (100 μl) was added to each well and incubated for 3 h in dark at 37°C. The optical density (OD) of each well was measured at 440 nm using a well plate reader (Thermo Scientific) and % cell viability was evaluated using the following equation: $$\begin{equation} \%\mathrm{cell}\ \mathrm{viability}=\frac{\mathrm{OD}\ \mathrm{of}\ \mathrm{sample}}{\mathrm{OD}\ \mathrm{of}\ \mathrm{control}} \times 100 \end{equation}$$ The L/D cells were analysed by removing the old media and adding 100 μl of L/D fluorescent staining reagent to each well and after 30 min of incubation in the dark at 37°C, the stained cells were observed under fluorescent microscope (Nikon Ti-E). Anti-proliferative activity by MTT assay The cell viability of silver(I) complexes were assessed by MTT reduction assay as described by Mosmann [66], against human estrogen positive (MCF-7), estrogen negative (MDA-MB-231) and pancreatic (PANC-1) cancerous cell lines and one non-tumorigenic human breast epithelial cell line (MCF-10a) under identical conditions, in comparison with widely used anticancer drugs, cisplatin and carboplatin. The ability to cleave the tetrazolium rings of pale yellow MTT to a dark blue water insoluble formazan helps in assessing the metabolically active (live) cells. The complexes 3 and 4 (0.29, 0.58, 1.17, 2.34, 4.68, 9.4, 18.8, 37.5, 75.0 and 150.0 ng/ml) dissolved in 0.1% DMSO were used for the anti-proliferative studies. The cell growth inhibition analyses were carried out following the procedure reported in our earlier publication [32]. Molecular docking studies The molecular docking studies of synthesized heteroleptic silver(I) complexes (1–4) with EGFR (PDB ID: 1 M17), VEGFR2 (PDB ID: 1VR2) and LOX (PDB ID: 4NRE) were carried out by following the procedure reported in our earlier publication [32]. The rigid molecular docking studies were performed with the grid based version of AutoDock Tools (ADT) 1.5.6 [67]. Conclusions A series of heteroleptic silver(I) complexes (1–4) containing 2-(1-(4-substitutedphenyl)ethylidene)hydrazinecarbothioamide and naproxen have been synthesized and characterized. Spectral and theoretical studies suggested distorted tetrahedral geometry around silver(I) ion. Optimization of the complexes was carried out using DFT/B3LYP/LANL2DZ basic set, which showed reasonable agreement between the reported crystal data and the optimized parameters. Additionally, DFT studies revealed distribution of Mulliken charge within the molecule and the obtained smaller energy gap supports the bioefficacy of the complexes. All the complexes were stable for about 72 h in biologically relevant solutions. The viability of NHDF cells on treating with complexes increased with increasing time, indicating that the synthesized complexes accelerated cell proliferation of NHDF cells without any toxicity. All the complexes also accelerated cell proliferation of non-tumorigenic human breast cancerous cell line (MCF-10a). Complex 4 was found to exhibit moderate cytotoxicity activity against the tested cancerous (MCF-7, MDA-MB-231 and PANC-1) cell lines with respect to its IC50 values. AO/EB and Hoechst 33258 staining methods confirmed the induced apoptotic activity of the complexes against MCF-7 cells. In silico studies of the complexes showed strong binding interactions with EGFR, VEGFR2 and LOX receptors via hydrogen bonding, π-pair (π–π, π–σ and π-cation) and hydrophobic interactions. These heteroleptic complexes exhibited non-toxicity to normal cells, which helps in differentiating normal cells from cancerous cells. Conflicts of interest statement. There are no conflicts of interest to declare. Acknowledgements The authors thank Department of Chemistry, IIT Madras, Chennai—600 036, for ESI-mass data and SIF, Chemistry Division, VIT, Vellore—632 014, for 1H-NMR data. References 1. Mascini M , Bagni G, Pietro MLD et al. Electrochemical biosensor evaluation of the interaction between DNA and metallodrugs. Biometals 2006 ; 19 : 409 – 418 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Lippert B . Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug . Weinheim : Wiley-VCH , 1999 . Google Scholar Crossref Search ADS Google Scholar Google Preview WorldCat COPAC 3. Fuertes MA , Alonso C, Perez JM. Biochemical modulation of cisplatin mechanisms of action: enhancement of antitumor activity and circumvention of drug resistance. Chem Rev 2003 ; 103 : 645 – 662 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Jemal A , Bray F, Center MM et al. Global cancer statistics. Ca-Cancer J Clin 2011 ; 61 : 69 – 90 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Lobana TS , Rekha T, Butcher RJ et al. Bonding trends of thiosemicarbazones in mononuclear and dinuclear copper(I) complexes: syntheses, structures, and theoretical aspects. Inorg Chem 2006 ; 4 : 1535 – 1542 . Google Scholar Crossref Search ADS WorldCat 6. Joseph M , Kuraikose M, Kurup MRP et al. Structural, antimicrobial and spectral studies of copper(II) complexes of 2-benzoylpyridine-N-4-phenyl thiosemicarbazone. Polyhedron 2006 ; 25 : 61 – 70 . Google Scholar Crossref Search ADS WorldCat 7. Ashfield LA , Cowley AR, Dilworth JR et al. Functionalized thiosemicarbazone clusters of copper(I) and silver(I). Inorg Chem 2004 ; 43 : 4121 – 4132 . Google Scholar Crossref Search ADS PubMed WorldCat 8. Melaiye A , Simons RS, Milstead A et al. Formation of water-soluble pincer silver(I)–carbene complexes: a novel antimicrobial agent. J Med Chem 2004 ; 47 : 973 – 977 . Google Scholar Crossref Search ADS PubMed WorldCat 9. Kasuga NC , Sugie A, Nomiya K. Dalton Trans 2004 ; 21 : 3732 – 3740 . Crossref Search ADS 10. Banti CN , Hadjikakou SK. Anti-proliferative and anti-tumor activity of silver(I) compounds. Metallomics 2013 ; 5 : 569 – 596 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Casas JS , Garcia-Tasende MS, Sordo J. Main group metal complexes of semicarbazones and thiosemicarbazones: a structural review. Coord Chem Rev 2000 ; 209 : 197 – 261 . Google Scholar Crossref Search ADS WorldCat 12. West DX , Liberta AE, Padhye SB et al. Thiosemicarbazone complexes of copper(II): structural and biological studies. Coord Chem Rev 1993 ; 123 : 49 – 71 . Google Scholar Crossref Search ADS WorldCat 13. Easmon J , Purstinger G, Heinisch G et al. Synthesis, cytotoxicity and antitumor activity of copper(II) and iron(II) complexes of 4N-azabicyclo[3.2.2]nonanethiosemicarbazones derived from acyl diazines. J Med Chem 2001 ; 44 : 2164 – 2178 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Ainscough EW , Brodie AM, Denny WA et al. Nitrogen, sulfur and oxygen donor adducts with copper(II) complexes of antitumor 2-formylpyridinethiosemicarbazone analogs: physicochemical and cytotoxic studies. J Inorg Biochem 1998 ; 70 : 175 – 185 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Li MX , Zhang D, Zhang LZ et al. Synthesis, crystal structures, and biological activities of 2-thiophene N(4)-methylthiosemicarbazone and its unusual hexanuclear silver(I) cluster. Inorg Chem Commun 2010 ; 13 : 1268 – 1271 . Google Scholar Crossref Search ADS WorldCat 16. Loginova NV , Kovalchuk TV, Gres AT et al. Redox-active metal complexes of sterically hindered phenolic ligands: antibacterial activity and reduction of cytochrome c. Part IV. Silver(I) complexes with hydrazone and thiosemicarbazone derivatives of 4,6-di-tert-butyl-2,3-dihydroxybenzaldehyde. Polyhedron 2014 ; 88 : 125 – 137 . Google Scholar Crossref Search ADS WorldCat 17. Banti CN , Papatriantafyllopoulou C, Manoli M et al. Nimesulide silver metallodrugs, containing the mitochondriotropic, triaryl derivatives of pnictogen: anticancer activity against human breast cancer cells. Inorg Chem 2016 ; 55 : 8681 – 8696 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Sharma J , Singla AK, Dhawan S. Zinc -naproxen complex: synthesis, physicochemical and biological evaluation. Int J Pharm 2003 ; 260 : 217 – 227 . Google Scholar Crossref Search ADS PubMed WorldCat 19. Banti CN , Giannoulis AD, Kourkoumelis N et al. Mixed ligand -silver(I) complexes with anti-inflammatory agents which can bind to lipoxygenase and calf-thymus DNA, modulating their function and inducing apoptosis. Metallomics 2012 ; 4 : 545 – 560 . Google Scholar Crossref Search ADS PubMed WorldCat 20. Banti CN , Giannoulis AD, Kourkoumelis N et al. Novel metallo-therapeutics of the NSAID naproxen. interaction with intracellular components that leads the cells to apoptosis. Dalton Trans 2014 ; 43 : 6848 – 6863 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Banti CN , Giannoulis AD, Kourkoumelis N et al. Silver(I) compounds of the anti-inflammatory agents salicylic acid and p-hydroxylbenzoic acid which modulate cell function. J Inorg Biochem 2015 ; 142 : 132 – 144 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Mahendiran D , Gurumoorthy P, Gunasekaran K et al. Structural modeling, in vitro antiproliferative activity, and the effect of substituents on the DNA fastening and scission actions of heteroleptic copper(II) complexes with terpyridines and naproxen. New J Chem 2015 ; 39 : 7895 – 7911 . Google Scholar Crossref Search ADS WorldCat 23. Mahendiran D , Kumar RS, Rahiman AK. Heteroleptic silver(I) complexes with 2,2′: 6′,2′′-terpyridines and naproxen: DNA interaction, EGFR/VEGFR2 kinase, growth inhibition and cell cycle arrest studies. Mater Sci Eng C 2017 ; 76 : 601 – 615 . Google Scholar Crossref Search ADS WorldCat 24. Şahin ZS , Şahin O, Dağli Ö et al. Diphenic acid/nicotinamide complexes of Co(II), Cu(II) and Zn(II). Synthesis and structural investigation. Polyhedron 2016 ; 117 : 214 – 223 . Google Scholar Crossref Search ADS WorldCat 25. Dağlı Ö , Köse DA, Avcı GA et al. Novel mixed-ligand complexes of coumarilate/N, N′-diethylnicotinamide with some transition metals. J Therm Anal Calorim 2017 ; 129 : 1389 – 1400 . Google Scholar Crossref Search ADS WorldCat 26. Dağlı Ö , Köse DA, Içten O et al. The mixed ligand complexes of Co(II), Ni(II), Cu(II) and Zn(II) with coumarilic acid/1, 10-phenanthroline. J Therm Anal Calorim 2019 ; 136 : 1467 – 1480 . Google Scholar Crossref Search ADS WorldCat 27. Dimiza F , Perdih F, Tangoulis V et al. Interaction of copper(II) with the non-steroidal anti-inflammatory drugs naproxen and diclofenac: synthesis, structure, DNA-and albumin-binding . J Inorg Biochem 2011 ; 105 : 476 – 489 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Roy S , Mandal TK, Mitra P et al. Synthesis, structure, spectroscopic properties, electrochemistry, and DFT correlative studies of N-[(2-pyridyl)methyliden]-6-coumarin complexes of Cu(I) and Ag(I). Polyhedron 2011 ; 30 : 913 – 922 . Google Scholar Crossref Search ADS WorldCat 29. Tan XJ , Liu HZ, Ye CZ et al. Synthesis, characterization and in vitro cytotoxic properties of new silver(I) complexes of two novel Schiff bases derived from thiazole and pyrazine. Polyhedron 2014 ; 71 : 119 – 132 . Google Scholar Crossref Search ADS WorldCat 30. Ren CX , Ye BH, He F et al. Syntheses, structures, and photoluminescence studies of [2: 2]metallomacrocyclic silver(I) complexes with 1,3-bis(4,5-dihydro-1H-imidazol-2-yl) benzene. Cryst Eng Comm 2004 ; 6 : 200 – 206 . Google Scholar Crossref Search ADS WorldCat 31. Mahendiran D , Amuthakala S, Bhuvanesh NSP et al. Copper complexes as prospective anticancer agents: in vitro and in vivo evaluation, selective targeting of cancer cells by DNA damage and S phase arrest. RSC Adv 2018 ; 8 : 16973 . Google Scholar Crossref Search ADS WorldCat 32. Bharathi S , Mahendiran D, Kumar RS et al. Biocompatibility, in vitro antiproliferative, and in silico EGFR/VEGFR2 studies of heteroleptic metal(II) complexes of thiosemicarbazones and naproxen. Chem Res Toxicol 2019 ; 32 : 1554 – 1571 . Google Scholar Crossref Search ADS PubMed WorldCat 33. Santhakumari R , Ramamurthi K, Vasuki G et al. Synthesis and spectral characterization of acetophenone thiosemicarbazone: a nonlinear optical material. Spectrochim Acta Part A 2010 ; 76 : 369 – 375 . Google Scholar Crossref Search ADS WorldCat 34. Haribabu J , Jeyalakshmi K, Arun Y et al. Synthesis, DNA/protein binding, molecular docking, DNA cleavage and in vitro anticancer activity of nickel(II) bis (thiosemicarbazone) complexes. RSC Adv 2015 ; 5 : 46031 – 46049 . Google Scholar Crossref Search ADS WorldCat 35. Muralisankar M , Haribabu J, Bhuvanesh NSP et al. Synthesis, X-ray crystal structure, DNA/protein binding, DNA cleavage and cytotoxicity studies of N(4) substituted thiosemicarbazone based copper(II)/nickel(II) complexes. Inorg Chim Acta 2016 ; 449 : 82 – 95 . Google Scholar Crossref Search ADS WorldCat 36. Kumar R , Obrai S, Kaur A et al. Synthesis, crystal structure investigation, DFT analyses and antimicrobial studies of silver(I) complexes with N, N, N′, N′′-tetrakis (2-hydroxyethyl/propyl)ethylenediamine and tris(2-hydroxyethyl)amine. New J Chem 2014 ; 38 : 1186 – 1198 . Google Scholar Crossref Search ADS WorldCat 37. Cornman CR , Geiser-Bush KM, Rowley SP et al. Structural and electron paramagnetic resonance studies of the square pyramidal to trigonal bipyramidal distortion of vanadyl complexes containing sterically crowded Schiff base ligands. Inorg Chem 1997 ; 36 : 6401 – 6408 . Google Scholar Crossref Search ADS WorldCat 38. Zhao S-S , Shi L-L, Su Z-M et al. TD-DFT studies on electronic and spectral properties of platinum(II) complexes with phenol and pyridine groups. Chem Res Chin Univ 2013 ; 29 : 361 – 365 . Google Scholar Crossref Search ADS WorldCat 39. Demir S , Tinmaz F, Dege N et al. Vibrational spectroscopic studies, NMR, HOMO-LUMO, NLO and NBO analysis of 1-(2-nitobenzoyl)-3,5-diphenyl-4,5-dihydro-1H-pyrazole with use X-ray diffraction and DFT calculations. J Mol Struct 2016 ; 1180 : 637 – 648 . Google Scholar Crossref Search ADS WorldCat 40. Ebrahimi HP , Hadi JS, Abdulnabi ZA et al. Spectroscopic, thermal analysis and DFT computational studies of salen-type Schiff base complexes. Spectrochim Acta Part A 2014 ; 117 : 485 – 492 . Google Scholar Crossref Search ADS WorldCat 41. Platts JA , Oldfield SP, Reif MM et al. The RP-HPLC measurement and QSPR analysis of log Po/w values of several Pt(II) complexes. J Inorg Biochem 2006 ; 100 : 1199 – 1207 . Google Scholar Crossref Search ADS PubMed WorldCat 42. Komeda S , Lutz M, Spek MAL et al. A novel isomerization on interaction of antitumor-active azole-bridged dinuclear platinum(II) complexes with 9-ethylguanine. Platinum(II) atom migration from N2 to N3 on 1, 2, 3-triazole. J Am Chem Soc 2002 ; 124 : 4738 – 4746 . Google Scholar Crossref Search ADS PubMed WorldCat 43. Chan TO , Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositide-regulated kinases:kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 1999 ; 68 : 965 – 1014 . Google Scholar Crossref Search ADS PubMed WorldCat 44. Herbst RS . Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys 2004 ; 59 : 21 – 26 . Google Scholar Crossref Search ADS PubMed WorldCat 45. Herbst RS , Langer CJ. Epidermal growth factor receptors as a target for cancer treatment: the emerging role of IMC-C225 in the treatment of lung and head and neck cancers. Semin Oncol 2002 ; 29 : 27 – 36 . Google Scholar Crossref Search ADS PubMed WorldCat 46. Ethier SP . Signal transduction pathways: the molecular basis for targeted therapies. Semin Radiat Oncol 2002 ; 12 : 3 – 10 . Google Scholar Crossref Search ADS PubMed WorldCat 47. Iliakis G . Cell cycle regulation in irradiated and nonirradiated cells. Semin Oncol 1997 ; 24 : 602 – 615 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 48. Ferrara N , Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005 ; 438 : 967 – 974 . Google Scholar Crossref Search ADS PubMed WorldCat 49. Bergers G , Brekken R, McMahon G et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis . Nat. Cell Biol 2000 ; 2 : 737 – 744 . Google Scholar Crossref Search ADS PubMed WorldCat 50. Senger DR , Galli SJ, Dvorak AM et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983 ; 219 : 983 – 985 . Google Scholar Crossref Search ADS PubMed WorldCat 51. Zachary I , Gliki G. Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc Res 2001 ; 49 : 568 – 581 . Google Scholar Crossref Search ADS PubMed WorldCat 52. Bates DO , Hillman NJ, Pocock TM et al. Regulation of microvascular permeability by vascular endothelial growth factors. J Anat 2002 ; 200 : 523 – 534 . Google Scholar Crossref Search ADS WorldCat 53. Ivanov I , Heydeck D, Hofheinz K et al. Molecular enzymology of lipoxygenases. Arch Biochem Biophys 2010 ; 503 : 161 – 174 . Google Scholar Crossref Search ADS PubMed WorldCat 54. Haeggstrom JZ , Funk CD. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease. Chem Rev 2011 ; 111 : 5866 – 5898 . Google Scholar Crossref Search ADS PubMed WorldCat 55. Ponten F , Jirstrom K, Uhlen MJ. The Human Protein Atlas a tool for pathology. J Pathol 2008 ; 216 : 387 – 393 . Google Scholar Crossref Search ADS PubMed WorldCat 56. Samuelsson B , Dahlen SE, Lindgren J et al. Leukotrienes and lipoxins:structures, biosynthesis and biological effects. Science 1987 ; 118 : 1171 – 1176 . Crossref Search ADS 57. Ding XZ , Kuszynski CA, El-Metwally TH et al. Lipoxygenase inhibition induced apoptosis, morphological changes, and carbonic anhydrase expression in human pancreatic cancer cells. Biochem Biophys Res Commun 1999 ; 266 : 392 – 399 . Google Scholar Crossref Search ADS PubMed WorldCat 58. Pidgeon GP , Lysaght J, Krishnamoorthy S et al. Lipoxygenase metabolism: roles in tumor progression and survival. Cancer Metastasis Rev 2007 ; 26 : 503 – 512 . Google Scholar Crossref Search ADS PubMed WorldCat 59. Thun MJ , Henley SJ, Patrono C et al. Nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst 2002 ; 94 : 252 – 266 . Google Scholar Crossref Search ADS PubMed WorldCat 60. Dannenberg AJ , Subbaramaiah K. Targeting cyclooxygenase-2 in human neoplasia: rationale and promise. Cancer Cell 2003 ; 4 : 431 – 436 . Google Scholar Crossref Search ADS PubMed WorldCat 61. Singh VP , Singh P, Singh AK. Synthesis, structural and corrosion inhibition studies on cobalt(II), nickel(II), copper(II) and zinc(II) complexes with 2-acetylthiophene benzoylhydrazone. Inorg Chim Acta 2011 ; 379 : 56 – 63 . Google Scholar Crossref Search ADS WorldCat 62. Becke AD . Density functional theory including dispersion corrections for intermolecular interactions in a large benchmark set of biologically relevant molecules. J Chem Phys 1993 ; 98 : 5648 – 5652 . Google Scholar Crossref Search ADS WorldCat 63. Frisch MJ , Trucks GW, Schlegel HBH et al. Gaussian 03 (Revision A.9) , Pittsburgh : Gaussian, Inc. , 2003 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC 64. Mulliken RSJ . Electronic population analysis on LCAO -MO molecular wave functions . J Chem Phys 1955 ; 23 : 1833 – 1840 . Google Scholar Crossref Search ADS WorldCat 65. Danielsson LG , Zhang YH. Methods for determining n-octanol-water partition constants. Trends Anal Chem 1996 ; 15 : 188 – 196 . OpenURL Placeholder Text WorldCat 66. Mosmann T . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J Immunol Methods 1983 ; 65 : 55 – 63 . Google Scholar Crossref Search ADS PubMed WorldCat 67. Trott O , Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010 ; 31 : 455 – 461 . Google Scholar PubMed OpenURL Placeholder Text WorldCat © The Author(s) 2020. Published by Oxford University Press. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Silver(I) metallodrugs of thiosemicarbazones and naproxen: biocompatibility, in vitro anti-proliferative activity and in silico interaction studies with EGFR, VEGFR2 and LOX receptors
JO - Toxicology Research
DO - 10.1093/toxres/tfaa001
DA - 2020-04-24
UR - https://www.deepdyve.com/lp/oxford-university-press/silver-i-metallodrugs-of-thiosemicarbazones-and-naproxen-7cvMavYew3
SP - 28
EP - 44
VL - 9
IS - 1
DP - DeepDyve
ER -