Synthesis, Theoretical Calculation, and Biological Studies of Mono- and Diphenyltin(IV) Complexes of N-Methyl-N-hydroxyethyldithiocarbamate

In this study, chlorophenyltin(IV) [(C6H5)(Cl)Sn(L)2] and diphenyltin(IV) [(C6H5)2Sn(L)2] of N-methyl-N-hydroxyethyldithiocarbamate were prepared and characterized using various spectroscopic methods (FTIR, 1H, 13C, and 119Sn NMR) and elemental analysis. The FTIR and NMR spectral data, used to establish the structure of the compounds, showed the formation of the complexes via coordination to the two sulfur atoms from the dithiocarbamate ligand and the respective phenyltin(IV) derivatives. This coordination mode was further explored by DFT calculations, which showed that the bonding around the Sn center in [(C6H5)2Sn(L)2] was more asymmetric compared to the bonding around [(C6H5)(Cl)Sn(L)2]. However, the Sn–S bonds in [(C6H5)(Cl)Sn(L)2] were found to be more covalent than those in [(C6H5)2Sn(L)2]. Furthermore, the charge density of the frontier orbitals showed that the Sn atom in the complexes is relatively electrophilic and the Sn atom in [(C6H5)2Sn(L)2] has a lower atomic dipole moment than that of [(C6H5)(Cl)Sn(L)2]. The cytotoxicity and anti-inflammatory study revealed that [(C6H5)2Sn(L)2], with the higher number of phenyl substituents, has a higher potency than [(C6H5)(Cl)Sn(L)2]. The bio-efficacy study of these complexes as cytotoxic and anti-inflammatory agents showed that the complexes possessed moderate to high activity in comparison to the camptothecin and diclofenac in each case. Nevertheless, the diphenyltin(IV) derivative [(C6H5)2Sn(L)2] was found to possess a better activity than its counterpart due to the number of phenyl rings attached to the Sn center.


Introduction
Metal dithiocarbamate and its derivatives have continued to attract growing research attention due to their diverse structural variations, unique coordination chemistry, and wide biological properties [1][2][3][4][5][6]. Their organometallic derivatives such as the organotin(IV) dithiocarbamate complexes have been reported to possess interesting properties which include the possibility for ligand exchange, structural diversity, catalytic and redox capacity, and a variety of useful medicinal properties [7]. One characteristic feature of the organometallic compounds, which also makes them generally useful, is the presence of at least one metal-carbon bond within their organometallic moiety [8]. This has been The FTIR spectroscopy of the complexes showed three major regions often associated with the dithiocarbamate compounds: the stretching band frequencies attributed to ν(C=N), ν(C−S), and ν(M−S) in the regions 1580-1450, 1060-940, and 455-440 cm −1 , respectively, as already established in the literature [22]. In the spectra of these complexes, the v(C=N) stretching vibration bands were found in the frequency range 1453-1456 cm −1 [23], which is between the stretching vibration of ν(C=N) and ν(C-N), that is 1690-1640 and 1350-1250 cm -l , hence suggestive of a partial double bond character [24]. Furthermore, the stretching ν(C−S) in this present study was found in both complexes as a single band between 990 and 988 cm −1 , indicative of bidentate coordination between the metal and the dithiocarbamate ligand [23]. Additionally, the stretching bands attributed to ν(M−S), supporting the existence of coordination between the metal center and the ligand moiety, were found between 450 and 440 cm −1 , and this confirmed the presence of Sn-S bonds in the complexes [25] The proton ( 1 H) NMR spectra of the different metal complexes and their derivatives with the ligand have already been reported in the literature [26], and the spectra obtained are presented in the Supplementary Data. The influence of the phenyl group on the metal center could be observed in the three main regions of the chemical shifts. Multiple signals in both complexes were found between 7.50 and 7.25 ppm, attributed to the phenyl groups of the organotin(IV) moiety, similarly to other previous reports [13]. Other notable protons, which conform with previous reports, the methylene proton signals found between 4.06 and 3.45 ppm, due to the deshielding effect of the attached electronegative O and N atoms [27,28]. Furthermore, the proton signals of the hydroxyl group for both complexes were found in the region between 7.90 and 7.80 ppm [19].
Similarly, the carbon signals associated with the carbon atom of the thioureide πsystem δ(N 13 CS2) in each of the complexes were found around 200 pm in the 13 C spectra (see Supplementary Data). The existence of this system has been found to contribute to the stability of complexes and which, in turn, results in the reduction in the electron density [29]. Other carbon atom signals found within the range of 150 and 124 ppm are associated with the carbons of the phenyltin(IV) derivatives similar to other reports [26,30]. The other three observed carbon atom signals found around 60, 59, and 45 ppm have all been attributed to the methylene and methyl carbons, respectively. The Sn signals were found at −467 ppm from the 119 Sn NMR spectra obtained, which is indicative of a hexacoordinated geometry around the tin center [31].

Molecular and Electronic Structures
The ground state equilibrium molecular structures of the studied Sn complexes are shown in Figure 1, which presents some salient bond lengths and angles. The possibility of rotation of ethanoyl and methyl groups on the dithiocarbamate ligands was considered such that two different conformations (syn-syn or A and anti-anti or B) are presented for each of the complexes. The conformer with the ethanoyl group on each ligand pointing in the same direction was found to have relatively lower energy than its counterpart. Accordingly, conformer A of [(C6H5)(Cl)Sn(L)2] is about 0.06 kJ/mol more stable than B, Scheme 1. Synthetic scheme of the phenyltin(IV) N-methyl-N-hydroxyethyldithiocarbamate complexes.
The FTIR spectroscopy of the complexes showed three major regions often associated with the dithiocarbamate compounds: the stretching band frequencies attributed to ν(C=N), ν(C−S), and ν(M−S) in the regions 1580-1450, 1060-940, and 455-440 cm −1 , respectively, as already established in the literature [22]. In the spectra of these complexes, the v(C=N) stretching vibration bands were found in the frequency range 1453-1456 cm −1 [23], which is between the stretching vibration of ν(C=N) and ν(C-N), that is 1690-1640 and 1350-1250 cm -l , hence suggestive of a partial double bond character [24]. Furthermore, the stretching ν(C−S) in this present study was found in both complexes as a single band between 990 and 988 cm −1 , indicative of bidentate coordination between the metal and the dithiocarbamate ligand [23]. Additionally, the stretching bands attributed to ν(M−S), supporting the existence of coordination between the metal center and the ligand moiety, were found between 450 and 440 cm −1 , and this confirmed the presence of Sn-S bonds in the complexes [25] The proton ( 1 H) NMR spectra of the different metal complexes and their derivatives with the ligand have already been reported in the literature [26], and the spectra obtained are presented in the Supplementary Data. The influence of the phenyl group on the metal center could be observed in the three main regions of the chemical shifts. Multiple signals in both complexes were found between 7.50 and 7.25 ppm, attributed to the phenyl groups of the organotin(IV) moiety, similarly to other previous reports [13]. Other notable protons, which conform with previous reports, the methylene proton signals found between 4.06 and 3.45 ppm, due to the deshielding effect of the attached electronegative O and N atoms [27,28]. Furthermore, the proton signals of the hydroxyl group for both complexes were found in the region between 7.90 and 7.80 ppm [19].
Similarly, the carbon signals associated with the carbon atom of the thioureide πsystem δ(N 13 CS 2 ) in each of the complexes were found around 200 pm in the 13 C spectra (see Supplementary Data). The existence of this system has been found to contribute to the stability of complexes and which, in turn, results in the reduction in the electron density [29]. Other carbon atom signals found within the range of 150 and 124 ppm are associated with the carbons of the phenyltin(IV) derivatives similar to other reports [26,30]. The other three observed carbon atom signals found around 60, 59, and 45 ppm have all been attributed to the methylene and methyl carbons, respectively. The Sn signals were found at −467 ppm from the 119 Sn NMR spectra obtained, which is indicative of a hexacoordinated geometry around the tin center [31].

Molecular and Electronic Structures
The ground state equilibrium molecular structures of the studied Sn complexes are shown in Figure 1, which presents some salient bond lengths and angles. The possibility of rotation of ethanoyl and methyl groups on the dithiocarbamate ligands was considered such that two different conformations (syn-syn or A and anti-anti or B) are presented for each of the complexes. The conformer with the ethanoyl group on each ligand pointing in the same direction was found to have relatively lower energy than its counterpart. Accordingly, conformer A of [(C 6 H 5 )(Cl)Sn(L) 2 ] is about 0.06 kJ/mol more stable than B, while conformer A of [(C6H5)2Sn(L)2] is about 0.56 kJ/mol more stable than B. The geometry parameters listed in Figure 1 fall within the range of experimental bond lengths and angles for tin dithiocarbamate complexes reported in the literature [32,33]. Similar to what was observed in the experimental IR data (vide supra Section 2.1), the predicted stretching vibration bands of the C-N bonds in [(C 6 H 5 )(Cl)Sn(L) 2 ] range between 1410 cm −1 and 1513 cm −1 (unscaled), while similar bonds in [(C 6 H 5 )(Cl)Sn(L) 2 ] showed predicted stretching vibration bands between 1404 cm −1 and 1507 cm −1 . Furthermore, the C-N bond lengths for the two complexes are in the ranges of 1.3346(2) Å-1.3359(9) Å and 1.3385(3) Å-1.34118(1) Å, respectively, which are in-between the values for C-N single (ca. 1.482 Å, e.g., as in azetidine) and double (ca. 1.280 Å, e.g., as in diazirine) bonds [34]. These observations suggest that the C-N bonds in the complexes exhibit partial double bonds, which also correlates with the experimentally observed and inferred nature of the C-N bonds based on the experimental IR data. The four Sn-S bonds in [(C 6 H 5 )(Cl)Sn(L) 2 ] are approximately 2.56 Å, indicating that the Sn center is covalently bonded to the S atoms of the dithiocarbamate chelates. However, the bond lengths for the four Sn-S bonds in [(C6H5)2Sn(L)2] range from 2.53 to 2.97 Å, exhibiting both covalent and coordinate bonding features in pairs. The observed features of the Sn-S bonds in [(C6H5)2Sn(L)2] are characteristic of tin dithiocarbamates with monodentate co-ligands [19,33]. The presence of organic groups as co-ligands (the phenyl groups in the case of [(C6H5)2Sn(L)2]) makes the dithiocarbamate ligand behave as a bidentate unsymmetric or anisobidentate ligand [33]. Unlike in the case of alkyl co-ligands, in which the geometry parameters of Sn-dithiocarbamates are not largely affected by the alkyl chain length [19], The values of selected second-order perturbation/interaction energies (E (2) ) between atomic orbitals resulting from natural bond orbital (NBO) calculations are listed in Table 1. The values of E (2) recorded for ligand-Sn interactions revealed a strong overlap between the delocalized electronic orbitals of the S-C bonds of the dithiocarbamate ligands and the central Sn atom. An obvious trend in the values of E (2) listed in the table is the higher values for the corresponding donor-acceptor interactions in [(C6H5)2Sn(L)2] compared to [(C6H5)2Sn(L)2]. This further confirms a stronger ligand-metal bond in the former than in the latter. Furthermore, the strong overlap was also recorded for S/Sn interactions based on lone pair donation from S orbitals to the unoccupied orbitals of Sn, as exemplified by the E (2) values of the LP(3)S2 → LP*(2)Sn1 donor-acceptor interaction. Many of these LP/LP* interactions were observed in the complexes, though it was tricky to use them to explain the comparative bond strengths of the two complexes. The values of selected second-order perturbation/interaction energies (E (2) ) between atomic orbitals resulting from natural bond orbital (NBO) calculations are listed in Table  1. The values of E (2)    Orbital composition analyses of the frontier molecular orbitals (FMOs) of both complexes were carried out with the aid of the Multiwfn software module [35,36]   Numerical compositions (%) of the atomic orbitals for the FMOs are listed in Table 2 The HOMO of

Cytotoxicity Study
The cytotoxicity assay is generally considered a useful tool for the initial determination of the toxicity of test samples [37]. The cytotoxicity of the phenyltin(IV) complexes was studied against human immortalized cancer (Caco-2 and PC-3) and non-cancer (KMST-6) cells using MTT assay. Under the same conditions, the activity of the complexes was compared to that of camptothecin, which is a standard anticancer drug. The concentration of the complexes that inhibited 50% cell growth (IC 50 ) was estimated after an incubation period of 24 h, and the IC 50 of the complexes are summarized in Table 3. The activity of the complexes at various concentrations and the effect of the test complexes on the selected cell lines are presented in Figures 3 and 4, respectively (using some representative images). The complex [(C6H5)2Sn(L)2] showed a superior activity and a largely better activity than the [(C6H5)2Sn(L)2]. This activity, in comparison to the standard drug (camptothecin) in all of the tested cell lines, showed that the diphenytin(IV) N-methyl-Nhydroxyethyldithiocarbamate is 5.5, >40, and 15.3 times more active in KMST, Caco-2; and PC-3 cell lines, respectively (Figure 3). This observed very good activity is in agreement with the previous report on the cytotoxicity study of organotin(IV)-based compounds [33]. The current study supports previous reports which suggest that, as the alkyl chain or aryl groups increases in an organotin compound, increased biological activity is often observed [38]. This has been attributed to Tweedy's chelation theory, which affirms that complexation reduces the polarity of a metal ion, and in turn enhances the lipophilic properties of the complex, which consequently allows for the easy permeation of the complex through the cellular membranes [39]. Hence, as the aryl substituent increases from one in [(C6H5)2Sn(L)2] to two in [(C6H5)2Sn(L)2], the polarity becomes lower, thereby resulting in increased lipophilicity of the complex. The increase in lipophilicity of the complex increases their cytotoxicity. Although the diphenyltin complex showed better activity than the mono-phenyl derivative and the standard drug, its nonspecific action toward both normal and cancer cell lines may be improved using drug carriers. This complex could be a potential lead anticancer drug, further studies and clinical screenings are warranted to investigate its anticancer mechanism and improvise strategies to increase the selectivity of the complex. images). The complex [(C6H5)2Sn(L)2] showed a superior activity and a largely better activity than the [(C6H5)2Sn(L)2]. This activity, in comparison to the standard drug (camptothecin) in all of the tested cell lines, showed that the diphenytin(IV) N-methyl-Nhydroxyethyldithiocarbamate is 5.5, >40, and 15.3 times more active in KMST, Caco-2; and PC-3 cell lines, respectively (Figure 3). This observed very good activity is in agreement with the previous report on the cytotoxicity study of organotin(IV)-based compounds [33]. The current study supports previous reports which suggest that, as the alkyl chain or aryl groups increases in an organotin compound, increased biological activity is often observed [38]. This has been attributed to Tweedy's chelation theory, which affirms that complexation reduces the polarity of a metal ion, and in turn enhances the lipophilic properties of the complex, which consequently allows for the easy permeation of the complex through the cellular membranes [39]. Hence, as the aryl substituent increases from one in [(C6H5)2Sn(L)2] to two in [(C6H5)2Sn(L)2], the polarity becomes lower, thereby resulting in increased lipophilicity of the complex. The increase in lipophilicity of the complex increases their cytotoxicity. Although the diphenyltin complex showed better activity than the mono-phenyl derivative and the standard drug, its nonspecific action toward both normal and cancer cell lines may be improved using drug carriers. This complex could be a potential lead anticancer drug, further studies and clinical screenings are warranted to investigate its anticancer mechanism and improvise strategies to increase the selectivity of the complex.

In Vitro Anti-Inflammatory Assay
Different studies have reported that most compounds that show cytotoxic properties possess some measure of anti-inflammatory activities [40][41][42]. Thus, the anti-inflammatory properties of the complexes were studied, and the results were compared to that of diclofenac as a standard drug. The IC 50 values showed that the complexes [(C6H5)2Sn(L)2] (2.52 ± 0.02 µM) and [(C 6 H 5 )(Cl)Sn(L) 2 ] (2.67 ± 0.03 µM) possess good anti-inflammatory properties, that is comparable and slightly better than the standard drug used (2.94 ± 0.01 µM). However, the diphenyltin(IV) derivative showed better activity compared to the mono-phenyltin(IV) derivative, which is in line with the generally observed trends from organotin(IV) compounds bearing aryl groups, due to increased lipophilicity emanating from reduced polarity around the tin metal center. Furthermore, the good activity found for these complexes has been attributed to the transportation of [(C 6 H 5 ) 2 Sn(IV)] + moiety through the cell membrane, due to the increased lipophilicity observed [43,44].

Materials and Methods
The alcoholic amine and all other reagents used in this study were acquired from Merck chemical Co. (Darmstadt, Germany), while the organotin(IV) salts were purchased from Sigma-Aldrich (Darmstadt, Germany). All of the reagents were used without further purification. Gallenkamp melting point apparatus was used to determine the melting point of the respective complexes. The percentage elemental composition (C, H, N, and S) was determined using Elementar, Vario EL Cube (Langenselbold, Germany). Furthermore, the proton, carbon, and tin ( 1 H, 13 C, 119 Sn) spectra of the complexes were obtained from 600 MHz on a Bruker Avance III NMR spectrometer (Billerica, MA, USA), using tetramethylsilane as the internal standard at 25 • C. Additionally, an Alpha Bruker FTIR spectrophotometer (Billerica, MA, USA) was used to measure the infrared properties of the complexes.

Computational Details
The molecular structures of the two Sn complexes were modeled with the GaussView 5.0 and were used as the starting geometries for full geometry optimization using the density functional theory (DFT) model. Based on the free rotation of the ethanoyl and methyl groups on the sp 3 N on the dithiocarbamate ligand, two possible tagged conformers, syn-syn and anti-anti, were considered, and the geometry optimization was conducted without symmetry constraints. The M06 hybrid functional of Truhlar and Zhao [45] together with the Dunning's correlation consistent (triple-zeta) basis set (cc-pVTZ) for non-metallic atoms [46][47][48] and LANL2DZ for Sn atom were used. The adopted theoretical model can be coded M06/cc-pVTZ//LANL2DZ. This model has been successfully used for similar systems in a previous study [19]. The molecular structures were fully optimized in the ground state and the optimized geometries were characterized as the true ground state minimum by the absence of a negative vibrational frequency in the force constant calculations. All the calculations were carried out in gas phase at 298 K with the aid of Gaussian 16 [49]. Natural bond orbitals (NBO) calculations were also conducted on the optimized geometry by using the link 607 NBO 7.0 code [50] implemented in Gaussian 16. The atomic dipole moment of the Sn central atom was calculated using the Hirshfeld atomic charge (ADCH) analysis proposed by Lu and Chen [51] and implemented in Multiwfn (freeware). The ADCH calculation was carried out for the possible correlation of the biological activity of the complexes with lipophilicity around the active central metal.

Cytotoxicity Study
An already established procedure was used for the cytotoxicity assay using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma), and the cytotoxicity potentials of the as-prepared complexes were evaluated in the following cell lines: immortalized colorectal adenocarcinoma (Caco-2) and human non-tumorigenic immortalized fibroblast (KMST-6). They were acquired from American Type Cell Culture (Manassas, VA, USA). These cells were seeded at a density of 1 × 10 5 cells/mL in Dulbecco's modified Eagle medium (DMEM) on a 96 well plate. To supplement the media, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) were used. These cells were then treated with different concentrations of the complexes (0-100 µM) for 48 h, followed by the addition of the MTT dye (which contained 10 µL of 5 mg/mL per 100 µL of media) for 3 h. Subsequently, 100 µL of DMSO was added to the media to dissolve the insoluble formazan. Camptothecin and 1% DMSO were both used as positive and vehicular control, respectively. The absorbance of each of the wells in 96-well plates was determined at 570 and 700 nm using the POLARstar Omega microplate reader (BMG Labtech, Offenburg, Germany). This study was carried out in triplicate and the percentage of the viability of these cell lines was measured using the formula in Equation (1). Furthermore, changes in the cell morphology because of the complexes added to the assay were monitored and captured using EVOS XL Core Imaging System (Invitrogen, Waltham, MA, USA). % cell viability = mean value of test compounds mean value of untreated × 100 (1)

In Vitro Anti-Inflammatory Assay
Using the same procedure reported in the literature [52], 2 mL of the complexes and the standard drug (Diclofenac) were mixed with 0.2 mL of egg albumin and 2.8 mL of phosphate-buffered saline (pH 6.4). These mixtures were incubated for 20 min at 37 • C and then heated up to 70 • C in a water bath for 5 min. Thereafter, the obtained mixtures were cooled and dispensed into a 96-well plate. The absorbance was then measured at 655 nm using a microplate reader (model 680-Bio-Rad, made in the USA). The used concentration for all the samples and the standard drugs used were 50, 25, 12.5, 6.25, 3.12, and 1.56 µM.
All concentrations were carried out in triplicate. The percentage inhibition of the protein denaturation was estimated in terms of percentage inhibition using Equation (2). % Inhibition = absorbance of the test sample absorbance of the control − 1 × 100 The compounds' concentration for 50% inhibition (IC 50 ) was determined by the dose-response curve.

Conclusions
Diphenyltin(IV) N-methyl-N-hydroxyethyldithiocarbamate [(C6H5)2Sn(L)2] and chlorophenyltin(IV) N-methyl-N-hydroxyethyldithiocarbamate [(C 6 H 5 )(Cl)Sn(L) 2 ] were reported, and spectroscopic and computational studies were used to confirm the mode of coordination. The complexes possess a distorted octahedral geometry around the tin center. The cytotoxicity studies, carried out using some human cell lines, indicated that the complex [(C6H5)2Sn(L)2] showed a useful activity and a far better activity than [(C 6 H 5 )(Cl)Sn(L) 2 ]. Similarly, the anti-inflammatory study revealed the same trend. The observed trends and high activity found for the diphenyltin(IV) derivative [(C6H5)2Sn(L)2], were due to the reduced polarity, which in turn favors permeation through the cell wall, due to the number of phenyl substituents on the tin metal. This was supported by the comparative values of the atomic dipole moment of Sn in the complexes. The cytotoxicity and the anti-inflammatory studies revealed that the diphenyltin complex had a better cytotoxicity and anti-inflammatory properties than the mono-phenyl derivative and both camptothecin and diclofenac, respectively. Thus, the study suggests that [(C6H5)2Sn(L)2] could be a promising anticancer agent.