Design, synthesis and cytotoxic evaluation in tumor cell lines of organotin(IV) complexes with heteroditopic hydroxo-carboxylato polyaromatic ligands¶

doi:10.21203/rs.3.rs-2131319/v1

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Design, synthesis and cytotoxic evaluation in tumor cell lines of organotin(IV) complexes with heteroditopic hydroxo-carboxylato polyaromatic ligands^¶

https://doi.org/10.21203/rs.3.rs-2131319/v1

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Three polynuclear organotin(IV) derivatives of composition [n-Bu₃Sn(HL)]_n 1, [Ph₃Sn(HL)]_n 2 and [n-Bu₂Sn(HL)₂]₂ 3 were synthesized by reacting 2-((E)-(4-hydroxy-3-((E)-((4-(methoxycarbonyl)phenyl)imino)methyl)phenyl)diazenyl)benzoic acid (H′HL) with (n-Bu₃Sn)₂O, Ph₃SnOH and n-Bu₂SnO, respectively. The structures 1–3 were fully characterized by elemental analysis, IR and NMR (¹H, ¹³C, and ¹¹⁹Sn), ¹¹⁹Sn Mössbauer spectroscopy, and additionally, the molecular and crystal structures of 1–3 and its pro-ligand (H′HL) were established by single-crystal X-ray diffraction analysis. The tributyltin(IV) complex 1 is a one-dimensional coordination polymer, in which the azo ligand bridges adjacent Sn(IV) centres solely via the two carboxylate O-atoms. The hydroxy H atom forms an intramolecular O–H···N hydrogen bond with the imine N-atom, as observed in the crystal structure of H′ΗL. The triphenyltin(IV) complex 2 is also a one-dimensional coordination polymer, but in this case the azo ligand bridges adjacent Sn(IV) centres via its carboxylate group and the deprotonated phenol O-atom. Unlike in 1, the phenol H-atom has migrated to the imine N-atom, to give a zwitterionic form of the azo ligand. The tin centers in 1 and 2 are pentacoordinated and reveal a distorted trans-R₃SnO₂ trigonal-bipyramidal environment. The dibutyltin(IV) complex 3 crystallizes as discrete centrosymmetric dinuclear entities where the unique Sn(IV) center is heptacoordinated in a distorted pentagonal bipyramid coordination geometry. In vitro cytotoxicity studies of compound 1 was performed and compared with 2 across a panel of human tumor cell lines, viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR and the results were compared with the data of six clinically used anticancer drugs. Compounds 1 and 2 are potent cytotoxic agents and warrant further investigation as potential anticancer agents.

organotin carboxylates

tin(IV) compounds

Spectroscopy

Crystal structures

cytotoxicity

cell lines

Organotin(IV) compounds have attracted enormous interest in various fields, such as wood preservation to organic syntheses [1], carbon dioxide capture [2], homogenous catalysts in PVC stabilization, polyurethane formation and trans esterification [3], cytotoxic agents and various medicinal applications [4, 5]. Investigations into the multifaceted applications of organotin(IV) chemistry also involve specific spectroscopic techniques e.g., solution- and solid-state ¹¹⁹Sn NMR, ¹¹⁹Sn Mössbauer and X-ray crystallography which ultimately derive diverse structural information [6]. Furthermore, the inherent side effects, toxicity and drug resistance of antitumor drugs employing platinum based compounds sparked interest in the synthesizing and testing of new metal complexes as antitumor agents. In this respect, organotin(IV) compounds have established their status among a class of non-platinum chemotherapeutic metallo-pharmaceuticals exhibiting promising antitumor activity with fewer side effects. Organotin(IV) carboxylates containing heteroatoms, particularly nitrogen atoms, produce stable complexes, because of the coordination characteristics of the tin atom. These complexes are a focus of research for potential use in cancer chemotherapy owing to wide-ranging structural possibilities and biological activities, especially their high in vitro and in vivo antitumor activities [7–12].

The past few decades have witnessed a renaissance of organotin(IV) Schiff base carboxylates derived from amino acids bearing an imino group. These materials demonstrated potential cytotoxicity in vitro, but had low stability [13, 14]. To circumvent this, a range of new organotin(IV) carboxylates based on the diazenyl functionality has been developed, thereby increasing the stability and improved cytotoxicity in testing protocols [15–17]. Subsequently, imino and diazenyl functionalities were incorporated into a single ligand in the hope that both superior activity and extended stability would be attained in their organotin(IV) complexes [18–22]. As anticipated, tributyl and triphenyltin 2-[(E)-{3-[(E)-(adamantan-1-ylimino)methyl]-4-hydroxyphenyl}diazenyl]benzoates, which contain both diazenyl and imino functions, have demonstrated increased activity, stability and good selectivity [22]. As a furtherance of our research on the synthesis, characterization and cytotoxic potential of organotin(IV) complexes, we began our work by synthesizing and elucidating the solid-state structure elucidation of the pro-ligand 2-((E)-(4-hydroxy-3-((E)-((4-(methoxycarbonyl)phenyl)imino)methyl)phenyl)diazenyl)benzoic acid (H′HL) (Fig. 1).

The pro-ligand H′HL was prepared by condensing 2-[(E)-2-(3-formyl-4-hydroxyphenyl)-1-diazenyl]benzoic acid with methyl 4-aminobenzoate, which allow the design and construction of molecules containing diazenyl and imino functions. It is expected that the presence of diazenyl, formyl, carbonyl and ester oxygen atoms in the derived ligand moiety will facilitate hydrogen bonding interactions with the active site of amino acids of various enzymes. The presence of polyaromaticity in the ligand is expected to increase the lipophilicity of the complex. Consequently, in this study, three organotin(IV) derivatives of composition [n-Bu₃Sn(HL)]_n 1, [Ph₃Sn(HL)]_n 2 and [n-Bu₂Sn(HL)₂]₂ 3 as pre-designed target molecules loaded with pharmacological features that can act as drug. The structures of H′HL and their organotin(IV) derivatives 1–3 were deduced from spectroscopic results and X-ray diffraction studies. Cytotoxicity studies of compound 1 was carried out and compared with compound 2 across a panel of human tumor cell lines consisting of A498 (renal cancer), EVSA-T (mammary cancer), H226 (non-small-cell lung cancer), IGROV (ovarian cancer), M19 MEL (melanoma), MCF-7 (mammary cancer) and WIDR (colon cancer).

2.1. Materials

All chemicals were used, as purchased: salicylaldehyde (Merck), 4-aminobenzoic acid (Hi Media), triphenyltin hydroxide (Alfa Aesar), bis(tri-n-butyltin)oxide (Lancaster), and anthranilic acid (Sigma-Aldrich). Methyl 4-aminobenzoate (M.p. 113–115°C) was prepared by reacting 4-aminobenzoic acid with SOCl₂ in methanol at -10°C according to a suitable work-up procedure described previously for methyl 2-[(E)-(3-formyl-4-hydroxyphenyl)diazenyl]benzoate [23]. The solvents used in the reactions were of AR grade and dried using standard procedures. Toluene was distilled from sodium benzophenone ketyl.

2.2. Physical measurements

Melting points were measured using a Buchi M-560 melting point apparatus and are uncorrected. Elemental analyses were performed using a Perkin Elmer 2400 series II instrument. IR spectra in the range 4000 − 400 cm^− 1 were obtained using KBr pellets on a Bruker Alpha-II FT-IR spectrophotometer (ESI Figures S1-S4). ¹H, ¹³C NMR and ¹¹⁹Sn NMR spectra were recorded at 300.13 MHz, 75.47 MHz and 111.92 MHz, respectively, on a Bruker ACF 300 spectrometer using solutions in CDCl₃ for 1–3 and in DMSO-d₆ for the pro-ligand H′HL. ¹H, ¹³C and ¹¹⁹Sn chemical shifts were referenced to internal Me₄Si set at 0.00 ppm, CDCl₃ set at 77.0 ppm and Me₄Sn set at 0.00 ppm, respectively. ¹H and ¹³C NMR assignments of H′HL have been achieved using standard 1D ¹H and ¹³C NMR including DEPT-135 and gradient-assisted 2D ¹H-¹³C heteronuclear multiple-quantum correlation (HMQC) and heteronuclear multiple-bond correlation (HMBC) spectroscopy, on the basis of the atom-labeling shown in Fig. 1. Selected NMR spectra of pro-ligand H′HL and organotin(IV) compounds (1–3) are presented in ESI Figs. S5-S18. The Mössbauer spectra of compounds 1 and 2 in the solid state were recorded using a Model MS-900 (Ranger Scientific Co., Burleson, TX) spectrometer in the acceleration mode with moving source geometry. A 5 mCi Ca^119mSnO₃ source was used, and counts of 30,000 or more were accumulated for each spectrum. The spectra were measured at 80 K using a liquid-nitrogen cryostat (CRYO Industries of America, Inc., Salem, NH). The velocity was calibrated at ambient temperature using a composition of BaSnO₃ and tin foil (splitting 2.52 mm s^− 1). The resulting spectra were analyzed using the Web Research software package (Web Research Co., Minneapolis, MN). On the other hand, the Mössbauer spectrum of compound 3 was recorded with a conventional spectrometer operating in the transmission mode. The source was Ca¹¹⁹SnO₃ (Ritverc GmbH, St. Petersburg, Russia; 10 mCi), moving at room temperature with constant acceleration in a triangular waveform. The driving system was from Halder (Seehausen, Germany), and the NaI (Tl) detector from Harshaw (De Meern, The Netherlands). The multichannel analyzer and the related electronics were from Takes (Bergamo, Italy). The solid absorber samples, containing ca. 0.5 mg ¹¹⁹Sn cm^− 2, were held at 77.3 K in a MNC 200 liquid-nitrogen cryostat (AERE, Harwell, UK). The velocity calibration was made with a ⁵⁷Co Mössbauer source (Ritverc GmbH, St. Petersburg, Russia, 10 mCi), and with an iron foil as absorber. The isomer shifts are relative to room temperature Ca¹¹⁹SnO₃. (Mössbauer Parameters: δ, isomer shifts; Δ, quadrupole splitting; Γ: line widths; ρ = Δ/δ).

2.3. Synthesis of pro-ligand and organotin(IV) compounds 1–3

2.3.1. Synthesis of 2-((E)-(4-hydroxy-3-((E)-((4-(methoxycarbonyl)phenyl)imino)methyl)phenyl)diazenyl)benzoic acid (H′HL)

An equimolar amount of methyl 4-aminobenzoate (0.533 g, 3.53 mmol) in hot absolute ethanol solution (15 ml) was added to a hot solution of toluene (30 ml) containing 2-[(E)-2-(3-formyl-4-hydroxyphenyl)-1-diazenyl]benzoic acid [24] (0.954 g, 3.53 mmol). The reaction mixture was heated to reflux for 5 h and filtered while hot. The dark red precipitate was washed with methanol (3 x 2 ml) and dried in vacuo. The dried residue was washed with hexane and then extracted with a large amount of chloroform (approximately 150 ml). The chloroform extract after concentration afforded red crystals of H′HL. M. p.: 238–239 °C (222–225 °C [25]). Yield: 1.1 g, 77%. Anal. Found. 65.75; H, 4.20; N, 10.65. Calc. for C₂₂H₁₇N₃O₅: C, 65.50; H, 4.25; N, 10.2 %. IR (cm^− 1): 3439, 2957, 2676, 1730 ν_asym(OCO)_COOH, 1710 ν_asym(OCO)_COOMe, 1624, 1596, 1571, 1485, 1457, 1437, 1369, 1278, 1242, 1217, 1179, 1156, 1108, 1018, 876, 855, 816, 771, 689. ¹H NMR (DMSO-d₆) δ_H (ppm): 13.2 [br s, 2H, OH], 9.13 [s, 1H, H-7′], 8.30 [s, 1H, H-2′], 8.03 [d, 8.5 Hz, 2H, H-3′′, H-5′′], 7.95 [d, 8.5 Hz, 1H, H-6′], 7.77 [d, 8.5 Hz, 1H, H-3], 7.65 [d, 8.5 Hz, 1H, H-5], 7.55 [m, 4H, H-4, H-6, H-2′′, H-6′′], 7.16 [d, 8.5 Hz, 1H, H-5′], 3.85 [s, 3H, OMe]. ¹³C NMR (DMSO-d₆) δ_C (ppm): 168.3 [C = O_COOH], 165.7 [C = O_COOMe], 163.8 [C-7′], 163.7 [C-4′], 152.0 [C-1′′], 150.7 [C-1], 144.9 [C-1′], 131.6 [C-5], 130.6 [C-3′′, C-5′′], 130.1 [C-4′′], 129.8 [C-4], 129.2 [C-3], 128.5 [C-2′], 127.8 [C-2], 127.1 [C-6′], 121.7 [C-2′′, C-6′′], 119.4 [C-3′], 118.2 [C-6], 118.1 [C-5′], 52.1 [OMe].

2.2.2. Synthesis of [n-Bu₃Sn(HL)]_n 1

Compound 1 was synthesized by reacting H′HL (0.30 g, 0.74 mmol) and (n-Bu₃Sn)₂O (0.221 g, 0.37 mmol) in 40 ml of anhydrous toluene with a Dean-Stark moisture trap and a water-cooled condenser. The reaction mixture was refluxed for 6 h. The clear dark-red solution was evaporated to dryness and the gummy residue was dried in vacuo. The solid mass was placed in an ice-bath and the residue was washed with hexane (3 x 0.2 ml), then dissolved in benzene and filtered. The filtrate was kept in a refrigerator for one week, which yielded orange crystals of the desired product. Yield: 0.40 g, 77%; M. p.: 93–94 °C. Anal. Found: C, 58.75; H, 6.05; N, 5.80. Calc. for C₃₄H₄₃N₃O₅Sn: C, 58.98; H, 6.26; N, 6.07%. IR (cm^− 1): 3439, 2955, 1719 ν_asym(OCO)_COOMe, 1623 ν_asym(OCO)_COO, 1598, 1570, 1487, 1461, 1435, 1392, 1352, 1281, 1171, 1148, 1109, 878, 855, 831, 768, 694, 670, 610. ¹H NMR (CDCl₃) δ_H (ppm): 13.4 [1H, OH], 8.73 [1H, ArH], 8.13 [2H, ArH], 8.06 [1H, ArH], 8.04 [1H, ArH], 7.81 [1H, ArH], 7.51 [2H, ArH], 7.44 [1H, ArH], 7.33 [2H, ArH], 7.12 [1H, ArH], 3.94 [3H, OMe], 1.59–1.69 [6H, Sn-BuH], 1.26–1.39 [12H, Sn-BuH], 0.87 [9H, Sn-BuH]. ¹³C NMR (CDCl₃) δ_C (ppm): 166.4 [C = O_COOSn], 164.0 [C = O_COOMe], 151.8, 145.9, 131.5, 131.1, 130.8, 129.8, 129.3, 129.1, 127.9, 121.1, 118.6, 118.1, 117.6 [remaining ArC], 52.2 [OMe], 27.8, 27.0, 16.7, 13.6 [Sn-BuC]. ¹¹⁹Sn NMR (CDCl₃) δ_Sn (ppm): 114.6. Mössbauer spectrum: δ = 1.43, Δ = 3.63, Γ± = 1.12 mm s^− 1, ρ = 2.54.

2.2.3. Synthesis of [Ph₃Sn(HL)]_n 2

An analogous method to that used for the preparation of 1 was followed using Ph₃SnOH (0.090 g, 0.24 mmol) and H′HL (0.10 g, 0.24 mmol). The product was crystallized from a chloroform-toluene mixture (5:1, v/v), which upon evaporation at room temperature yielded red crystals of 2 suitable for SCXRD analysis. Yield: 0.11 g, 62%; M. p.: 153–154 °C (132–135 °C) [25]. Anal. Found: C, 64.15; H, 3.85; N, 5.50. Calc. for C₄₀H₃₁N₃O₅Sn: C, 63.85; H, 4.15; N, 5.58%. IR (cm^− 1): 3440, 1718 ν_asym(OCO)_COOMe, 1624 ν_asym(OCO)_COOH, 1598, 1523, 1495, 1432, 1369, 1281, 1250, 1190, 1170, 1141, 1111, 1017, 996, 879, 852, 768, 733, 697, 668, 612. ¹H NMR (CDCl₃) δ_H (ppm): 13.5 [1H, OH], 8.42 [1H, ArH], 8.15–6.95 [remaining ArH and Sn-PhH], 3.98 [3H, OMe]. ¹³C NMR (CDCl₃) δ_C (ppm): 166.0 [C = O_COOSn], 163.8 [C = O_COOMe], 151.8, 145.7, 138.2, 136.9, 136.6, 131.5, 131.0, 130.5, 130.1, 129.2, 128.9, 128.6, 121.2,118.5, 118.1, 113.8 [remaining ArH and Sn-PhC], 52.2 [OMe]. ¹¹⁹Sn NMR (CDCl₃) δ_Sn (ppm): −108.0. Mössbauer spectrum: δ = 1.19, Δ = 2.90, Γ± = 1.16 mm s^− 1, ρ = 2.44.

2.2.4. Synthesis of [n-Bu₂Sn(HL)₂]₂ 3

An analogous method to that used for the preparation of 1 was followed using n-Bu₂SnO (0.062 g, 0.249 mmol) and H′HL (0.20 g, 0.495 mmol). The product was crystallized from benzene-petroleum ether mixture (1:1, v/v), which upon refrigeration for several days yielded red crystals of compound 3. Yield: 0.18 g, 70%; M. p.: 168–169 °C. Anal. Found: C, 60.55; H, 4.99; N, 8.30. Calc. for C₁₀₄H₁₀₀N₁₂O₂₀Sn₂: C, 60.19; H, 4.86; N, 8.10%. IR (cm^− 1): 3649, 2956, 1723 ν_asym(OCO)_COOMe, 1622 ν_asym(OCO)_COOH, 1597, 1486, 1435, 1406, 1354, 1281, 1174, 1148, 1110, 1016, 966, 877, 855, 830, 767, 692, 672, 634, 556. ¹H NMR (CDCl₃) δ_H (ppm): 13.6 [1H, OH], 8.56 [1H, ArH], 8.05 [2H, ArH], 7.93 [2H, ArH], 7.40–7.61 [4H, ArH], 7.23 [2H, ArH], 7.11 [1H, ArH], 3.98 [3H, OMe], 1.90-1.00 [12H, Sn-BuH], 0.88 [6H, Sn-BuH]. ¹³C NMR (CDCl₃) δ_C (ppm): 166.4 [C = O_COOSn], 163.0 [C = O_COOMe], 163.6, 145.9, 152.4, 151.5, 145.9, 132.7, 131.6, 131.1, 129.6, 128.7, 127.6, 121.1, 118.7, 118.1, 117.2, 113.8 [remaining ArC], 52.2 [OMe], 26.9, 26.4, 25.5, 13.6 [Sn-BuC]. ¹¹⁹Sn NMR (CDCl₃) δ_Sn (ppm): −144.0 ppm. Mössbauer spectrum: δ = 1.37, Δ = 3.52, Γ± = 0.77 mm s^− 1, ρ = 2.57.

2.4. X-ray crystallography

Crystals of four compounds suitable for an X-ray crystal-structure determination were obtained from chloroform/acetonitrile (H′ΗL), hexane/benzene (1), chloroform/toluene (2), and benzene/petroleum ether (3) by slow evaporation of the solvent at room temperature. The measurements were made at low temperature on a Nonius KappaCCD diffractometer [26] with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) and an Oxford Cryosystems Cryostream 700 cooler. Data reduction was performed with HKL Denzo and Scalepack [27]. The intensities were corrected for Lorentz and polarization effects. An empirical absorption correction based on the multi-scan method was applied for 1–3 [28]. Equivalent reflections were merged. The data collection and refinement parameters are given in ESI Table S1, and views of the four molecular structures (segments of the polymeric chains in the cases of 1 and 2) are shown in Figs. 2–5.

The structure of 1 was solved by heavy-atom Patterson methods [29], followed by the Fourier expansion routine of DIRDIF94 [30], while all other structures were solved by direct methods by using SHELXS97 [31]. There are two symmetry-independent molecules in the asymmetric unit of H′ΗL. The atomic coordinates of the two molecules were tested carefully for a relationship from a higher symmetry space group using the program PLATON [32], but none could be found. Complex 1 exists as one-dimensional polymeric strands and the asymmetric unit contains two consecutive repeats of the principle chemical unit. Again, no higher symmetry relating these two segments could be found. The two symmetry-independent methyl ester groups are disordered through a 180° rotation of the groups. One of the butyl ligands is also disordered over two conformations. Two sets of positions were defined for the atoms of each of these disordered groups and the site occupation factors of the major conformations of these groups refined to 0.68(1), 0.71(2) and 0.57(1) for the two disordered ester groups and the disordered butyl ligand, respectively. Similarity restraints were applied to the chemically equivalent bond lengths and angles involving all disordered C-atoms, while neighboring atoms within and between each conformation of the disordered butyl ligand were restrained to have similar atomic displacement parameters. Complex 2 is also a one-dimensional polymer and the asymmetric unit is one chemical repeat of the polymeric chain. The molecule of 3 is a centrosymmetric dinuclear complex where the two halves are linked via relatively weak Sn···O(ester) interactions. In the asymmetric unit, which is one half of the molecule, one butyl ligand is disordered over two conformations. Two sets of positions were defined for the terminal propyl segment of this butyl ligand and the site occupation factor of the major conformation refined to 0.629(8). Similarity restraints were applied to the chemically equivalent bond lengths and angles involving all butyl C-atoms, including the ordered butyl ligands, while neighboring atoms within and between each conformation of the disordered butyl ligand were restrained to have similar atomic displacement parameters.

The non-hydrogen atoms in each structure were refined anisotropically. The iminium Hatom in 2 and the hydroxy and carboxylic acid H-atoms in the other structures were placed in the positions indicated by difference electron density maps and their positions were allowed to refine together with isotropic displacement parameters; the O–H distances in H′ΗL were restrained to 0.84(1) Å. All remaining H-atoms were placed in geometrically calculated positions and refined using a riding model where each H-atom was assigned a fixed isotropic displacement parameter with a value equal to 1.2U_eq of its parent C-atom (1.5U_eq for the methyl groups). The refinement of each structure was carried out on F² by using full-matrix least-squares procedures, which minimized the function Σw(F_o² – F_c²)². Corrections for secondary extinction were applied for 1 and 2. Six reflections for H′ΗL and one reflection for 3, whose intensities were considered to be extreme outliers, were omitted from the final refinement. In 1, the largest peaks of residual electron density are in the vicinity of the Sn-atoms. All calculations were performed using the SHELXL-2018 program [33]. The crystallographic diagrams were drawn using ORTEPII [34] and Mercury [35].

2.5. Experimental protocol for cytotoxicity tests

The in vitro cytotoxicity testing of tributyltin(IV) compound 1 was performed using the SRB test [36] for estimation of cell viability and the results were compared with those of its triphenyltin(IV) analogue, 2, and standard drugs in seven human tumor cell lines.

The cell lines WIDR, M19 MEL, A498, IGROV and H226 belong to the currently used anticancer screening panel of the NCI, USA [37]. The MCF7 cell line is an estrogen receptor (ER) + and progesterone receptor (PgR)+, and EVSA-T is (ER)-/(PgR)-. Prior to the experiments, a mycoplasma test was carried out on all cell lines and found to be negative. All cell lines were maintained in a continuous logarithmic culture in RPMI 1640 medium with HEPES and phenol red. The medium was supplemented with 10% FCS, 100 µg/ml of penicillin and 100 µg/ml of streptomycin. The cells were mildly trypsinized for passage and for use in the experiments. RPMI and FCS were obtained from Gibco (Paisley, Scotland). SRB, DMSO, Penicillin and streptomycin were obtained from Sigma (St. Louis MO, USA), TCA and acetic acid from Baker BV (Deventer, NL), and PBS from NPBI BV (Emmer-Compascuum, NL).

The test compound 1 and the reference compounds were dissolved to a concentration of 2.5∙10⁵ ng/ml in the complete medium, by dilution of stock solutions, which approximately contained 5 mg/ml of compound 1 and the tests compounds, respectively, in DMSO. The cytotoxicity was estimated by the microculture sulforhodamine B (SRB) test [36].

The experiment was started on day 0. On day 0, 1500–2000 cells per well were seeded into flat-bottomed micro-titer plates containing 96 wells (Cellstar, Greiner Bio-one). The plates were pre-incubated overnight at 37°C with 5% CO₂ to allow the cells to adhere to the bottom. On day 2, a three-fold dilution sequence of ten steps was realized in the complete medium, starting with the 2.5∙10⁵ ng/ml stock solution. Every dilution was used in quadruplicate by adding 50 µl to a column of four wells. This procedure results in the highest concentration of 6.25∙10⁴ ng/ml being present in column 12. Column 2 was used for the blank.

To column 1, medium was added to diminish interfering evaporation. The incubation was terminated on day 7. Subsequently, the cells were fixed with 10% trichloroacetic acid in PBS and placed at 4°C for an hour. After three washings with tap water, the cells were stained for at least 15 min with 0.4% SRB dissolved in 1% acetic acid. After staining, the cells were washed with 1% acetic acid to remove the unbound stain. The plates were air-dried and the bound stain was dissolved in 150 µl of 10 mM Tris-base. The absorbance was measured at 540 nm using an automated microplate reader (Labsystems Multiskan MS). The data were used for the construction of concentration-response curves and the determination of the ID₅₀ values by use of the Deltasoft 3 software. ID₅₀ is the dose in ng/ml that causes 50% inhibition of the tumor cells.

The variability of the in vitro cytotoxicity test depends on the cell lines used and the serum applied. With the same batch of cell lines and the same batch of serum, the inter-experimental CV (coefficient of variation) is 1–11%, depending on the cell line, and the intra-experimental CV is 2–4%. These values may be higher with other batches of cell lines and/or serum.

3.1. Synthesis and spectroscopy

The pre-ligand 2-[(E)-2-(3-formyl-4-hydroxyphenyl)-1-diazenyl]benzoic acid (Fig. 1) [24] is an excellent precursor for further functionalization of the molecule, which in subsequent reactions can be reacted with a suitable amine to obtain a Schiff base with the desired functionality or donor groups. By reacting the pre-ligand with methyl 4-aminobenzoate in absolute ethanol, the pro-ligand (free acid) 2-((E)-(4-hydroxy-3-((E)-((4-(methoxycarbonyl)phenyl)imino)methyl)phenyl)diazenyl)benzoic acid (H′HL) was obtained. Reactions of H′HL with appropriate organotin(IV) precursors, such as (n-Bu₃Sn)₂O, Ph₃SnOH and n-Bu₂SnO, yielded organotin(IV) derivatives of composition [n-Bu₃Sn(HL)]_n 1, [Ph₃Sn(HL)]_n 2 and [n-Bu₂Sn(HL)₂]₂ 3 (Fig. 1). In an another endeavor, compounds 1 and 2 were prepared by reacting methyl 4-aminobenzoate with n-Bu₃Sn- or Ph₃Sn- 2-[(E)-2-(3-formyl-4-hydroxyphenyl)-1-diazenyl]benzoates in ethanol under reflux conditions; the isolation of pure product required meticulous control of the experimental conditions. The cumbersome workup procedure was detrimental to the overall yield and the purity of the products as well and thus the one pot reaction between stoichiometric amounts of H′HL and organotin(IV) precursors is the synthesis method of choice. The reactions proceeded smoothly in anhydrous toluene and the final yields of the products were > 55%, which were dependent on the workup conditions applied for the purification and recovery of the crystals. Crystalline samples of the compounds 1–3 can be stored for several months in an inert environment and retain their color, luster and melting points. During the course of the synthesis, single crystals of the compounds 1–3 and H′HL were isolated during recrystallization, allowing the determination of their hitherto unknown crystal and molecular structures in the solid state, which was one of the prime objectives.

The microanalytical and IR spectroscopic (ESI Figs. S1-S4) data, in combination with the single-crystal X-ray diffraction (SCXRD) analysis of the compounds confirmed the formation of 1D coordination polymers (1 and 2) and centrosymmetric dinuclear entities (3). The ¹H, ¹³C, and ¹¹⁹Sn NMR data for 1–3 exhibited the expected signals for individual proton, carbon and tin signals, which indicated the presence of discrete mononuclear molecules in solution. The ¹H, ¹³C spectroscopic data of H′HL and, additionally, the ¹¹⁹Sn NMR data of compounds 1–3 (ESI Figs. S5-S18) displayed the expected resonances for the anticipated structures. The ¹¹⁹Sn NMR shift displacements of compounds 1–3 in CDCl₃ solution suggest four-coordinate coordination environments of the Sn-atom, as observed for previously reported analogues [24, 38, 39]. The coordination of the carboxylate ligand to the tin atoms in 1–3 is evidenced by the ¹³C NMR chemical shifts for the SnOC(O) carbon atoms (δ = 166 ppm) being typical for organotin carboxylates.

In the IR spectra of 1–3, a band typical for the asymmetric stretching vibration of a tin-coordinated carboxylate group, ν_asym(OCO) was detected at 1623 cm^− 1 (pro-ligand H′HL; ν_asym(OCO) 1730 cm^− 1), indicating a bonding interaction to the tin atom [24]. The assignment of the symmetric ν_sym(OCO) stretching vibration band could not be ascertained unambiguously, and hence the criterion of shift difference Δν = ν_asym(OCO) - ν_sym(OCO) could not be utilized to imply the mode of coordination of the carboxylate O atom(s). However, the mode of the carboxylate coordination in 1–3 was validated from the X-ray diffraction results (vide infra). Further, a band at 1710 cm^− 1 in H′HL was assigned to the ν_asym(C = O)_COOMe vibration, and this band showed marginal shifts to around 1720 cm^− 1 in compounds 1–3, as expected.

Mössbauer quadruple shifts (Δ) are a reliable solid-state structure indicator, at least to distinguish between the cis- and the trans-R₃SnO₂ geometry in the complexes. The tributyltin(IV) and triphenyltin (IV) compounds 1 and 2 exhibited Δ values of 3.63 [39] and 2.90 [40] mm s^− 1, respectively, which are within the specified Δ range from 3.0 to 4.1 mm s^− 1 for a trans-R₃SnO₂ coordination geometry [39]. On the other hand, the Δ value for the dibutyltin(IV) compound 3 is 3.52 mm s^− 1, which is indicative of an octahedral coordination geometry of the tin atom with trans-alkyl groups [41] and consistent with the Δ values observed for six-coordinate compounds of cognate systems whose structures have also been characterized by X-ray diffraction [38, 39]. The conclusions drawn from the Mössbauer results are in excellent agreement with the structures of 1–3 determined by X-ray crystallography (vide infra). The isomer shift (δ) values are in the range 1.43–1.19 mm s^− 1, which is typical for quadrivalent organotin derivatives, and the full width at half maximum (Γ±) of these resonance absorptions is in the range 0.77–1.13 mm s^− 1, further suggesting the presence of a single type of tin centre in each of complexes 1–3.

3.2. X-ray crystallography

The asymmetric unit in the crystal structure of the native organic acid, H′ΗL contains two symmetry-independent molecules, A (containing atom N1) and B (containing atom N4), as shown in Fig. 2. The two molecules have very similar conformations and an extended form, in which the azo and imine groups have trans configurations. The carboxylic acid and hydroxy H-atoms only form intramolecular O–H···N hydrogen bonds with the immediately adjacent azo and imine N-atoms, respectively, to form six-membered loops. There is no evidence in difference Fourier maps and the azo, imine and hydroxy bond lengths to suggest the formation of a zwitterionic species in which an acidic H-atom has migrated to the imine N-atom (Table 1). The molecules are approximately planar, but have small twists between adjacent rings, which are the largest about the imine group and overall more prominent in molecule B (Table 2). The ester group is also slightly tilted with respect to its parent ring. The molecules pack into planar sheets, which lie parallel to the (001) plane and each sheet is composed of molecules A and B. Adjacent molecules in any one column parallel to [001] through the stacked layers alternate in an ···A···B···A···B··· fashion and are slightly offset from one another, thereby precluding the presence of significant π-π interactions. The shortest centroid-centroid distance in the columns is 3.7219(12) Å, which is between the phenol rings of molecule A and B to give just a local pairwise interaction, but the slippage of the centroids is quite large at 1.68 Å. The next shortest centroid-centroid distances are all greater than 4.0 Å with slippages greater than 2.1 Å.

Table 1

Selected bond lengths (Å) and angles (º) for **H′HL**
N1–N2	1.263(2)	N4–N5	1.260(2)
N3–C14	1.284(3)	N6–C44	1.276(3)
O1–C1	1.334(3)	O6–C31	1.335(3)
O2–C1	1.210(3)	O7–C31	1.208(3)
O3–C11	1.344(2)	O8–C41	1.344(2)

Table 2

Dihedral and torsion angles (°) between various planar groups^a within the azo molecule or ligand of compounds **H′ΗL, 1, 2** and 3
Compound	A/B	B/C	C/D	D/E	C–C–N = N	C–C–C = N, C–C–N = C
H′HL mol. A	3.06(12)	3.94(10)	13.02(10)	6.9(3)	1.3(3), 1.5(3)	3.7(3), -17.0(3)
H′HL mol. B	2.69(12)	11.96(10)	21.71(10)	16.77(11)	-8.9(3), -2.7(3)	-3.6(3), 26.7(3)
1 ligand A	79.7(4)	3.4(4)	24.5(4)	2.5(5)	-1.5(11), 4.4(11)	-1.6(12), -22.0(12)
1 ligand B	84.4(4)	4.1(4)	24.3(4)	9.1(5)	-3.3(10), 7.8(11)	-0.6(12), -19.8(12)
2	68.02(17)	6.75(15)	25.85(15)	11.4(3)	-18.7(4), 22.5(4)	3.5(5), 20.9(5)
3 ligand A	28.1(2)	20.28(18)	25.78(18)	5.84(18)	36.9(5), -14.8(5)	-2.7(5), 28.5(5)
3 ligand B	27.27(19)	7.65(16)	24.42(16)	10.82(18)	29.4(4), -21.1(4)	-5.1(5), 28.9(5)
^aA = carboxylate/carboxylic acid group; B = benzoate/benzoic acid ring; C = phenol ring; D = methylbenzoate ring; E = ester group; the pairs of torsion angles are those about the C–C bonds at each end of the azo and imine groups.

The crystal structures of several related azobenzoic acids with a variety of substituents in place of the 4-methylester group of the distal phenyl ring have been reported, namely the 4-chloro (two reported structures) [42, 43], 4-bromo [44] and 4-H [45] derivatives, plus the 4-methyl derivative, which exists in a zwitterionic form [40]. The molecules in four of these five structures exhibit dihedral angles of up to 28° between at least one of the two pairs of adjacent rings: 4-chloro: 6.24(10), 27.48(10) and 21.8(2), 19.6(2)°; 4-bromo: 6.01(11), 27.78(11)°; 4-methyl: 22.84(7), 20.28(8)°. In this context, the twists seen in the molecules of H′ΗL (Table 2) are modest. However, while the ring system of the molecule in the 4-H derivative is quite planar with dihedral angles of just 1.87(12) and 2.83(12)°, the plane of the carboxylic acid group is, unusually among these azocarboxylic acid molecules, almost perpendicular to that of its parent ring at 89.09(13)°, possibly as a consequence of an intermolecular hydrogen bond between the carboxylic acid H-atom and the phenol O-atom; in all other cases, the carboxylic acid H-atom interacts intramolecularly with an azo N-atom.

In the solid state, the tributyltin(IV) and triphenyltin(IV)complexes, 1 and 2, are one-dimensional coordination polymers, but the coordinating O-atoms involved in the polymer linkages are different. For clarity, the more straightforward structure of complex 2 is described first.

In the polynuclear triphenyltin(IV) complex, 2, the azo ligand bridges adjacent Sn(IV) centres via its carboxylate group and the deprotonated phenol O-atom. The polymer spirals with 2₁ screw symmetry parallel to the [010] direction and the asymmetric unit contains one repeat of the chemically unique segment. The phenol H-atom has migrated to the imine N-atom, to give a zwitterionic form of the azo ligand. The primary coordination sphere of the Sn-atom has a slightly distorted trigonal bipyramidal geometry, being coordinated by three phenyl ligands in the equatorial plane and carboxylate and phenoxide O-atoms from two different azo ligands in axial positions to give a trans-R₃SnO₂ centre (Table 3, Fig. 3). The carboxylate group can be considered to be monodentate coordinated to the Sn-atom, as the Sn1–O2 distance is 3.213(2) Å and the C–O and C = O bond lengths within the carboxylate group are quite distinct (1.283(4) and 1.228(4) Å, respectively), although slightly less distinct than in the native azocarboxylic acid molecule (average over the two independent molecules of 1.334(3) and 1.209(3) Å, respectively). Although the Sn1–O2 distance is still within the sum of the van der Waals radii of the respective atoms (ca. 3.6 Å), the impact of this contact on the trigonal bipyramidal Sn-coordination geometry is limited, being greatest for the Ph–Sn–Ph angle that has to accommodate the approach of atom O2 (Table 3). The iminium H-atom forms an intramolecular N–H···O hydrogen bond with the adjacent phenoxide O-atom to give a six-membered loop. The azo ligand is significantly distorted from planarity at the interface between the carboxylate group and the benzoate ring, with a dihedral angle of nearly 70° (Table 2). This is probably a consequence of its coordination as a bridging ligand. Although the dihedral angle between the planes of the benzoate and phenoxide rings is small, there are significant, but opposing twists about the C–N bonds at each end of the azo group. The dihedral angles between the planes of the phenol and methylbenzoate rings and between the planes of the methylbenzoate ring and ester group are similar to those in H′ΗL. The region of the azo ligand from the benzoate ring to the phenoxide ring overlaps via weak π-π interactions in an antiparallel fashion with the same region of an adjacent ligand in another polymeric chain related by a centre of inversion. The centroid-centroid distance between the rings is 3.7284(18) Å with a slippage of the centroids of 1.46 Å and a dihedral angle between the ring planes of 6.75(15)°. In addition, there are π-π interactions between centrosymmetrically-related and thus strictly parallel methylbenzoate rings in different pairs of polymeric chains to the ones involved in the benzoate-phenol π-π interactions, with a centroid-centroid distance of 3.8927(19) Å and a slippage of 1.77 Å. A three-dimensional supramolecular framework arises as a consequence of the combination of all described interactions.

Table 3

Selected bond lengths (Å) and angles (º) for 1^a
Sn1–O1	2.220(5)	Sn2–O6	2.198(5)
Sn1–O2	3.196(5)	Sn2–O7	3.192(5)
Sn1–O7'	2.446(5)	Sn2–C63	2.119(8)
Sn1–C23	2.140(8)	Sn2–C67	1.240(8)
Sn1–C27	2.181(8)	Sn2–C71a	2.154(9)
Sn1–C31	2.146(8)	Sn2–C71b	2.155(9)
N1–N2	1.250(8)	N4–N5	1.263(9)
N3–C14	1.261(10)	N6–C54	1.282(10)
O1–C1	1.271(9)	O6–C41	1.279(9)
O2–C1	1.255(9)	O7–C41	1.233(9)
O3–C11	1.330(10)	O8–C51	1.340(10)
Sn2–O2	2.458(5)
O1–Sn1–O7'	171.58(17)	O2–Sn2–O6	170.48(17)
O1–Sn1–C23	94.7(2)	O2–Sn2–C63	87.1(2)
O1–Sn1–C27	91.2(3)	O2–Sn2–C67	90.1(2)
O1–Sn1–C31	97.0(3)	O2–Sn2–C71a	82.8(6)
O7'–Sn1–C23	86.7(2)	O2–Sn2–C71b	120.8(2)
O7'–Sn1–C27	80.8(2)	O6–Sn2–C63	94.8(3)
O7'–Sn1–C31	89.1(2)	O6–Sn2–C67	96.7(3)
C23–Sn1–C27	117.2(4)	O6–Sn2–C71a	87.8(6)
C23–Sn1–C31	123.2(3)	O6–Sn2–C71b	93.2(8)
C27–Sn1–C31	117.9(4)	C63–Sn2–C67	122.6(3)
Sn1–O1–C1	120.3(5)	C63–Sn2–C71a	112.4(4)
Sn2–O2–C1	142.1(5)	C63–Sn2–C71b	118.8(6)
Sn2–O6–C41	120.4(5)	C67–Sn2–C71a	124.0(5)
Sn1–O7'–C41'	143.6(5)	C67–Sn2–C71b	116.3(8)
^a Primed atoms refer to the molecule in the following symmetry related
positions: ' 1 + x, y, z.

The tributyltin(IV) complex, 1, is also a one-dimensional coordination polymer, but this time the azo ligand bridges adjacent Sn(IV) centres solely via the two carboxylate O-atoms and the asymmetric unit contains two symmetry-independent repeats of the chemically unique segment (segment A contains atom N1 and segment B contains atom N4). The polymeric chain extends parallel to the [100] direction with successive asymmetric units related only by translation symmetry. Unlike 2, the hydroxy H atom has not migrated to the imine N-atom, but forms an intramolecular O–H···N hydrogen bond with the imine N-atom, as in the structure of H′ΗL. The primary coordination sphere of the trans-R₃SnO₂ centre has a slightly distorted trigonal bipyramidal geometry, being coordinated by three n-butyl ligands in the equatorial plane and carboxylate O-atoms from two different azo ligands in axial positions (Table 4, Fig. 4). The distortions of the symmetry-independent coordination polyhedra are smaller than that observed in 2, despite carboxylate O-atoms O2 and O7 still being within 3.2 Å of atoms Sn1 and Sn2, respectively. The distortions from planarity of the two symmetry independent azo ligands are similar to those observed for 2 (Table 2); the planes or the carboxylate groups are even closer to being perpendicular to those of their parent benzoate rings. Despite the different modalities of azo ligand coordination, the π-π interactions between adjacent polymeric chains described for 2 are very similar to those found in the structure of 1. The benzoate-phenol ring π-π interactions are, however, weaker than in 2: the centroid-centroid distances between the benzoate and phenol rings are 3.943(5) and 3.898(5) Å for A···A and B···B ligand interactions, respectively, with slippages of the centroids of 1.95 and 1.87 Å and dihedral angles between the ring planes of 3.4(4) and 4.1(4)°, respectively. In contrast, the π-π interactions between centrosymmetrically-related methylbenzoate rings are stronger, with centroid-centroid distances of 3.781(6) and 3.723(5) Å, and slippages of 1.02 and 1.07 Å for A···A and B···B ligand interactions, respectively. Again, a three-dimensional supramolecular framework arises through the combination of all described interactions.

Table 4

Selected bond lengths (Å) and angles (º) for 2^a
Sn1–O1	2.154(2)	N1–N2	1.247(4)
Sn1–O2	3.213(2)	N3–C14	1.303(4)
Sn1–O3'	2.379(2)	O1–C1	1.283(4)
Sn1–C23	2.146(3)	O2–C1	1.228(4)
Sn1–C29	2.145(3)	O3–C11	1.300(4)
Sn1–C35	2.139(3)
O1–Sn1–O3'	169.25(8)	O3'–Sn1–C35	88.68(10)
O1–Sn1–C23	99.82(10)	C23–Sn1–C29	109.90(12)
O1–Sn1–C29	87.93(10)	C23–Sn1–C35	131.58(12)
O1–Sn1–C35	94.72(10)	C29–Sn1–C35	116.59(12)
O3'–Sn1–C23	85.37(10)	Sn1–O1–C1	120.8(2)
O3'–Sn1–C29	81.44(10)	Sn1–O3'–C11'	132.58(19)
^a Primed atoms refer to the molecule in the following symmetry related
positions: ' ½-x, ½+y, 1½-z.

The dibutyltin(IV) complex, 3, crystallises as discrete centrosymmetric dinuclear entities (Fig. 5). The unique Sn(IV) center is bidentate coordinated by the carboxylate O-atoms of each of two azo ligands, the carbonyl O-atom of the methylbenzoate group of a third azo ligand, and two n-butyl ligands. The carboxylate groups are less asymmetrically coordinated to the Sn-atom than in 1 and 2, with the longer of the Sn–O distances being in the range 2.51–2.56 Å (Table 5), and can thus be considered as truly bidentate. Taking all coordinating atoms into consideration, the coordination geometry of the Sn(IV) centre can be described as a distorted pentagonal bipyramid with the n-butyl ligands in axial positions. The angle subtended by the n-butyl ligands deviates by approximately 34° from linearity, with the ligands bent over the equatorial site occupied by the coordinating methylbenzoate O-atom. Of the two symmetry-unique azo ligands in the structure, one is terminal, while the other bridges to the second Sn(IV) centre via the carbonyl O-atom of the methylbenzoate group. The hydroxy H-atom has not migrated to the imine N-atom, but does form an intramolecular O–H···N hydrogen bond with it. The dihedral angles in Table 2 show that for the terminal and bridging azo ligands, the planes of the carboxylate groups are turned only mildly out of the planes of their parent benzoate rings by just under 30°, while the dihedral angle between the plane of the benzoate and phenol rings in the terminal azo ligand is the largest seen among the discussed structures; this dihedral angle is significantly smaller in the bridging azo ligand. The other dihedral angles listed in Table 2 for both azo ligands are similar to those in the other reported structures. The torsion angles about the C–C bonds at each end of the azo group and at the N-end of the imine group are somewhat larger than those in the other structures. One can speculate that the differences described here are a consequence of strains induced in the azo ligands by the double-bridged dinuclear molecule compared with the one-dimensional coordination polymers. The only suggestion of π-π interactions is a set of intramolecular interactions involving the three rings of each symmetry-independent azo ligand, which overlay each other head-to-tail, but are tilted slightly with respect to one another. The centroid-centroid distances between the benzoate ring of the terminal azo ligand and the methylbenzoate ring of the bridging ligand, between the phenol rings of both ligands and between the methylbenzoate ring of the terminal ligand and the benzoate ring of the bridging ligand are 3.817(2), 3.7399(19) and 3.8345(19) Å, respectively, with corresponding slippages of the centroids of 1.10, 1.33 and 1.25 Å and dihedral angles between the ring planes of 6.94(17), 8.79(16) and 11.23(17)°, respectively.

Table 5

Selected bond lengths (Å) and angles (º) for 3^a
Sn1–O1	2.130(2)	O1–C1	1.291(4)
Sn1–O2	2.563(3)	O2–C1	1.256(4)
Sn1–O6	2.129(2)	O3–C11	1.347(4)
Sn1–O7	2.509(3)	N4–N5	1.252(4)
Sn1–O10'	2.768(3)	N6–C44	1.288(5)
Sn1–C23	2.144(4)	O6–C31	1.281(4)
Sn1–C27	2.111(4)	O7–C31	1.252(4)
N1–N2	1.258(4)	O8–C41	1.344(4)
N3–C14	1.294(5)	O10–C51	1.212(4)
O1–Sn1–O2	55.21(9)	O6–Sn1–C23	110.55(3)
O1–Sn1–O6	79.49(9)	O6–Sn1–C27	97.66(15)
O1–Sn1–O7	133.98(9)	O7–Sn1–O10'	80.55(8)
O1–Sn1–O10'	145.35(9)	O7–Sn1–C23	88.77(13)
O1–Sn1–C23	99.29(13)	O7–Sn1–C27	92.78(16)
O1–Sn1–C27	103.47(16)	O10'–Sn1–C23	74.88 (11)
O2–Sn1–O6	133.84(9)	O10'–Sn1–C27	72.37(15)
O2–Sn1–O7	170.59 (8)	C23–Sn1–C27	146.47(18)
O2–Sn1–O10'	90.18(8)	Sn1–O1–C1	101.7(2)
O2–Sn1–C23	87.24(13)	Sn1–O2–C1	82.6(2)
O2–Sn1–C27	85.88(16)	Sn1–O6–C31	100.9(2)
O6–Sn1–O7	55.57(9)	Sn1–O7–C31	84.0(2)
O6–Sn1–O10'	134.89(9)	Sn1–O10'–C51'	155.1(3)
^a Primed atoms refer to the molecule in the following symmetry related
positions: ' 2-x, 1-y, 2-z.

The Cambridge Structural Database (CSD) [46] contains 15 entries for alkyltin(IV) complexes with related azobenzoate ligands possessing a variety of substituents in place of the 4-methylester group of the distal phenyl ring. Of these, six are tetranuclear bis(dicarboxylatotetrabutyldistannoxane) complexes with 4-H, 4-chloro, 4-bromo, 4-methyl or 4-ethyl substituents; none are zwitterionic [45, 47]. Two are polymeric triphenyltin(IV) chain structures in which a zwitterionic azo ligand bridges adjacent metal centres via the carboxylate and phenoxide O-atoms, as in 2 (4-bromo, 4-methyl [40]). One is a centrosymmetric dinuclear triphenyltin(IV) complex with two zwitterionic azo ligands bridging the metal centres via their carboxylate and phenoxide O-atoms, so the same linkages as in the polymeric chain just described, but with a closed dinuclear circuit (4-methoxy [40]). Three consist of one-dimensional polymeric tributyltin(IV) chains bridged through the carboxylate O-atoms of the azo ligand, as in 1 (4-chloro, 4-methyl, 4-methoxy [48–50]). Two are discrete mononuclear tribenzyltin(IV) complexes with an additional aqua ligand (4-chloro, 4-methoxy [51]). Finally, one is a discrete dinuclear triphenyltin(IV) complex with the metal centres bridged by the carboxylate groups at opposite ends of a single dicarboxylate azo ligand (4-carboxylate [25).

Reports of crystal structures with substituents at the 2- or 3-position of the distal phenyl ring of the azo ligand are rare, with only two 2-hydroxy examples being listed in the CSD [52]. One of these is a discrete dinuclear trimethyltin(IV) species with two zwitterionic azo ligands coordinating via their carboxylate and phenoxide O-atoms, while the other structure is a polymeric ladder structure in which the azo ligand coordinates to three different tributyltin(IV) centres via the carboxylate, phenoxide and deprotonated 2-hydroxy O-atoms.

From a comparison of the structures reported and discussed here, one might surmise that the presence of phenyl ligands in the triorganotin(IV) species favours the formation of azo ligand linkages that bridge metal centres solely via the carboxylate group O-atoms, as in 1, while n-butyl ligands preclude such bridging interactions and instead favour either zwitterion formation, thereby allowing bridging which involves coordination of the phenoxide O-atom, as in 2, or coordination via a more distal O-atom, as in 3, although that is a dibutyltin(IV) complex. These differences are presumably related to the steric influences of the phenyl versus n-butyl ligands.

3.4.Cytotoxicity studies

In the preceding studies, we have shown that [Ph₃Sn(Lⁿ)] compounds (Lⁿ = Schiff base carboxylates derived from amino acids) with ligands bearing an imino group demonstrated potential cytotoxicity in vitro, but had low stability [13, 14]. Recently, triphenyltin carboxylates derived from Schiff bases with increased stability and improved cytotoxicity were also developed [15]. In this quest, several triphenyltin(IV) 2-[(E)-2-(aryl)-1-diazenyl]benzoates which now contained a diazenyl group exhibited promising cytotoxity with better stability in testing protocols [16, 17]. Subsequently, imino and diazenyl functionalities were incorporated into a single ligand molecule in the hope of accomplishing both superior activity and extended stability. In this pursuit, tributyltin- and triphenyltin complexes of related ligands were prepared and their cytotoxic potentials were evaluated and found to show analogous results with enhanced stability [22, 25, 53, 54]. In accordance with this and with the intention of achieving a better cytotoxic performance, [n-Bu₃Sn(HL)]_n 1 was prepared. The cytotoxicity of 1 was determined and compared with that of its triphenyltin analogue [Ph₃Sn(HL)]_n 2 [25] whose crystal structure was not known earlier; refer to Fig. 3 for the structure of 2. The ligand scaffold of 1 and 2 now contains simultaneously a diazenyl- and an imino- group, thus allowing considerable scope for optimizing the cytotoxicity and molecular flexibility in order to probe activity across a panel of human tumor cell lines, viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. Additionally, the methyl ester moiety in 1 and 2 is incorporated in the molecule to increase the solubility.

The in vitro cytotoxic properties of 1 and 2 were compared with those of several standard drugs viz., doxorubicin (DOX), cisplatin (CDDP), 5-fluorouracil (5-FU), methotrexate (MTX), etoposide (ETO) and paclitaxel (TAX) across a panel of seven human tumor cell lines (Table 6). In general, triphenyltin derivatve 2 displayed better cytotoxic results than its tributyltin analogue 1. However, comparable cytotoxic activities were noted particularly for EVSA-T, MCF-7 and WIDR cell lines. Further, compounds 1 and 2 exhibited much superior activity than cisplatin, 5-fluorouracil and etoposide across a panel of cell lines. The encouraging cytotoxic effect may be predictive of in vivo antitumor activity. Compounds 1 and 2 both carry diazenyl- and imino-skeletons, but they differ in the constitution of Sn-R ligands. In general, Ph₃Sn compounds always demonstrated superior activity than their more soluble Bu₃Sn analog. This could be related to the stability of the molecule in the test medium. The experiment takes five days and cells in the test medium can act as pockets that may serve as a kind of storage device, from which slow release of Ph₃Sn compound takes place. However, the variations in the in vitro cytotoxicity among 1 and 2 across tumor cell lines could also be related to a different kinetic and mechanistic behavior. Recently, the cytotoxicity for nBu₃Sn(ibuprofen) and Ph₃Sn(ibuprofen) were reported; however, in practice, the results should not be compared directly, since these were investigated using DU145 (prostate cancer), HCT-15 (colon adenocarcinoma), Caco-2 (colorectal adenocarcinoma) human cancer cell lines [55]. The observed results convincingly support the view that compounds 1 and 2 are potent cytotoxic agents and deserve further investigation as potential anticancer agents.

Table 6

*In vitro* ID₅₀ values (ng/ml) of test compounds [n-Bu₃Sn(HL)]_n (1) and [Ph₃Sn(HL)]_n (2), along with standard drugs, using cell viability tests in seven human tumor cell lines.^a
Test compounds	Cell lines
Test compounds	A498	EVSA-T	H226	IGROV	M19 MEL	MCF-7	WIDR
[n-Bu₃Sn(HL)]_n (1)	185	87	180	181	165	114	103
[Ph₃Sn(HL)]_n (2)	106	69	108	112	115	103	104
DOX	90	8	199	60	16	10	11
CDDP	2253	422	3269	169	558	699	967
5-FU	143	475	340	297	442	750	225
MTX	37	5	2287	7	23	18	<3.2
ETO	1314	317	3934	580	505	2594	150
TAX	<3.2	<3.2	<3.2	<3.2	<3.2	<3.2	<3.2
^aAbbreviation: doxorubicin (DOX), cisplatin (CDDP), 5-fluorouracil (5-FU), methotrexate (MTX), etoposide (ETO) and paclitaxel (TAX). Standard drug reference values have been obtained under identical conditions and are cited immediately after the test complexes.

Three organotin(IV) compounds of formulations [n-Bu₃Sn(HL)]_n1, [Ph₃Sn(HL)]_n2 and [n-Bu₂Sn(HL)₂]₂3 were synthesized. The compounds were fully characterized by elemental analysis and well-known spectroscopic techniques; all of them, including the pro-ligand H′HL were also characterized by single-crystal XRD analysis. In the crystalline state, the tin atom in triorganotin(IV) compounds 1 and 2 is pentacoordinated within a distorted trans-R₃SnO₂ trigonal-bipyramidal coordination environment. Dibutyltin(IV) compound 3 crystallizes as discrete centrosymmetric dinuclear entities, where the unique Sn(IV) center is heptacoordinated and displays a distorted pentagonal bipyramid coordination geometry. The solid-state five-coordinate polymeric structures of 1 and 2, and the dinuclear units in 3 are not retained in solution, as revealed from ¹¹⁹Sn chemical shifts, which indicate the presence of a four-co-ordinate mononuclear structure. The cytotoxic activities of 1 and 2in vitro were assessed using the SRB test against seven human tumor cells, viz., A498, EVSA-T, H226, IGROV, M19 MEL, MCF-7 and WIDR. The data showed that triphenyltin compound 2 exhibited more pronounced in vitro cytotoxicity results than the tributyltin analogue 1. However, comparable cytotoxic activities were noted specifically with EVSA-T, MCF-7 and WIDR cells, and these activities are superior to those exhibited by cisplatin, 5-fluorouracil and etoposide. Therefore, further investigation of 1 and 2 as potential drugs is merited. Furthermore, the molecular framework of these organotin(IV) complexes offers considerable scope for optimizing the design, both in terms of the cytotoxic activity, stability and dissolution properties.

Acknowledgements

Financial support from the Department of Biotechnology, India (Grant No. BT/PR25263/NER/95/1104/2017, TSBB), is gratefully acknowledged. MRA and BH thank the University Grants Commission for the award of UGC-JRF fellowships.

Data availability statement

The data supporting the findings of this study are available in the supplementary material of this article.

Conflicts of interest

The authors declare that they have no conflicts of interest with the contents of this article.

ORCID

Tushar S. Basu Baul: 0000-0002-9587-3503

Maheswara Rao Addepalli: 0000-0002-0299-7479

Anthony Linden: 0000-0002-9343-9180

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Design, synthesis and cytotoxic evaluation in tumor cell lines of organotin(IV) complexes with heteroditopic hydroxo-carboxylato polyaromatic ligands^¶

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Materials

2.2. Physical measurements

2.3. Synthesis of pro-ligand and organotin(IV) compounds 1–3

2.3.1. Synthesis of 2-((E)-(4-hydroxy-3-((E)-((4-(methoxycarbonyl)phenyl)imino)methyl)phenyl)diazenyl)benzoic acid (H′HL)

2.2.2. Synthesis of [n-Bu₃Sn(HL)]_n 1

2.2.3. Synthesis of [Ph₃Sn(HL)]_n 2

2.2.4. Synthesis of [n-Bu₂Sn(HL)₂]₂ 3

2.4. X-ray crystallography

2.5. Experimental protocol for cytotoxicity tests

3. Results And Discussion

3.1. Synthesis and spectroscopy

3.2. X-ray crystallography

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Design, synthesis and cytotoxic evaluation in tumor cell lines of organotin(IV) complexes with heteroditopic hydroxo-carboxylato polyaromatic ligands¶

Status:

Version 1

Abstract

Figures

1. Introduction

2. Experimental

2.1. Materials

2.2. Physical measurements

2.3. Synthesis of pro-ligand and organotin(IV) compounds 1–3

2.3.1. Synthesis of 2-((E)-(4-hydroxy-3-((E)-((4-(methoxycarbonyl)phenyl)imino)methyl)phenyl)diazenyl)benzoic acid (H′HL)

2.2.2. Synthesis of [n-Bu3Sn(HL)]n 1

2.2.3. Synthesis of [Ph3Sn(HL)]n 2

2.2.4. Synthesis of [n-Bu2Sn(HL)2]2 3

2.4. X-ray crystallography

2.5. Experimental protocol for cytotoxicity tests

3. Results And Discussion

3.1. Synthesis and spectroscopy

3.2. X-ray crystallography

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Design, synthesis and cytotoxic evaluation in tumor cell lines of organotin(IV) complexes with heteroditopic hydroxo-carboxylato polyaromatic ligands^¶

2.2.2. Synthesis of [n-Bu₃Sn(HL)]_n 1

2.2.3. Synthesis of [Ph₃Sn(HL)]_n 2

2.2.4. Synthesis of [n-Bu₂Sn(HL)₂]₂ 3