Keywords

Organotin(IV) complexes, IR, NMR, DNA interaction, Cytotoxicity, Human ovarian cell line A2780

Introduction

Owing to intriguing variety of crystal structures and topologies, organotin(IV) carboxylates are thought to provide a better perceptive of biological systems, as there is a significant influence of the structure of the molecule and the coordination number of the tin atoms on the activities of organotin(IV) compounds [1,2].

It is known that the formation of metal carboxylates is strongly dependent on various factors including reaction conditions, such as pH values, temperature, solvent systems, stoichiometry, and choice of appropriate precursors as a source of carboxylate ligands [3]. Organotin(IV) complexes with carboxylates show immense structural variety. However, such a structural diversity makes it difficult in some cases to characterize them fully just by analytical techniques. Thus, X-ray crystallographic study makes full identification possible [4].

Organotin carboxylates find applications both in industry as PVC stabilizers, homogeneous catalysts, used as antifouling paints, flame retardant and smoke suppressants, antiwar agents, homogeneous catalysts and recycling agents as well as in agriculture as biocides due to their antibacterial and antifungal activities, as wood preservatives, biodiesel catalysts, antioxidants for polypropylene and anti-herpes agents [5-7].

Antitumor activities of some organotin(IV) carboxylates are reported in literature [8-12] and these are found to show promising cytotoxic activities against various cancer cells like, sarcoma cancer cells, lungs, liver, breast and colon carcinoma etc. Antitumor activities of some of the complexes are also found to be competing with cis-platin [9,12].

Extensive investigations regarding antitumor activity have been performed with organotin(IV) compounds, however the mechanism of their toxicity remains obscure. One of the assumptions may be that these compounds are capable of reacting with cell membranes leading to leakage, which accelerates ion exchange processes, and inhibits oxidative or photochemical phosphorylation [13]. We have synthesized organotin(IV) complexes with sodium salt of 3-(¹H-indol- 3-yl) propanoic acid and characterized by IR, multinuclear (¹H, ¹³C and ¹¹⁹Sn) NMR. DNA binding studies and anticancer activity were also performed.

Materials and Methods

Trimethyltin chloride (97%), tributyltin chloride (96%), triphenyltin chloride (95%), dimethyltin chloride (97%), dibutyltin chloride (96%) and diphenyltin dichloride (96%) used were purchased from Sigma Aldrich. Analytical grade solvents were purchased from E. Merck and Fluka and were dried by standard procedures [14]. Melting points were determined in capillary tube using electrothermal melting point apparatus model MP-D Mitamura Riken Kogyo (Japan) and were uncorrected. Elemental analysis was done using a thermoscientific Flash 2000 CHN/S organic elemental analyser. Infrared spectra were recorded as KBr/CsBr discs on Perkin Elmer spectrum 1000 (USA) in range of 4000–250 cm^-1. ¹H and ¹³C NMR were recorded on Bruker AC 300 MHFT-NMR. ¹¹⁹Sn NMR spectra were taken on Avance 400 MHz TXO 10 mm XWINNMR. Me₄Sn was used as standard reference. Viscosity was measured by an Ubbelohde viscometer at room temperature. The absorption spectra were recorded on a Shimadzu 1800 UV–Vis spectrophotometer.

Synthesis

Synthesis of sodium 3-(¹H-indol-3-yl) propanoate NaL

Sodium salt of 3-(¹H-indol-3-yl) propanic acid was prepared by 1:1 molar reaction of 3-(¹H-indol-3-yl) propanoic acid with aqueous solution of sodium hydroxide in methanol with continuous stirring at room temperature for 1 hr. The product obtained was soluble in methanol and was isolated by evaporating the solvent under vacuum. Ligand salt thus obtained was washed with diethyl ether and was air dried.

(Yield: 96.05 %). M.p: 277-279 °C. FT-IR (cm^-1): ν_as (COO) 1618, ν_s (COO) 1387, Δν (231). ¹H-NMR (ppm): 1.94 (t, H₂, ³J=7 Hz), 2.14 (t, H₃, ³J=7Hz), 10.65 (N-H), 6.87 (s, H₁₂), 7.02-7.36 (m, Ar-H_6-9). ¹³C-NMR (ppm):203.1 (C-1), 42.1 (C-2), 39.8 (C-3), 108.3 (C-4), 121.5 (C-12), 137.1, 125.4, 127.8, 128.8, 130.8 134.2 (Ar-C).

General procedure for synthesis of complexes 1-6

Tri and diorganotin(IV) complexes were synthesized by refluxing ligand salt with tri and diorganotin chlorides in 1:1 and 2:1 molar ratios, respectively in dry toluene for 6-7 hrs. The NaCl formed was removed by filtration and solvent was evaporated under vacuum. All synthesized compounds were recrystallized in chloroform: n-hexane mixture (4:1) (Scheme 1).

Scheme 1: Synthesis of complexes 1-6.

Trimethylstannyl 3-(¹H-indol-3-yl)propanoate 1: (Yield: 71.4 %). M.p:194°C, Elemental analysis, % Calculated (Found), for C₁₄H₁₉NO₂Sn: C, 47.77 (47.82); H, 5.44 (5.49); N, 3.98 (4.02). FT-IR (cm^-1): ν_as (COO) 1575, ν_s (COO) 1395, Δν (180). ¹H-NMR (ppm): 2.13(t, H₂, ³J=7Hz), 2.45 (t, H₃, ³J=7Hz), 10.63 (N-H), 6.87 (s, H₁₂), 7.04-7.35 (m, Ar-H_6-9), 0.89(s, CH₃-Sn, [56]).¹³C-NMR (ppm): 198.6 (C-1), 42.3 (C-2), 39.8 (C-3), 108.5 (C-4), 123.2 (C-12) 135.1, 125.8, 126.0, 128.8, 127.8 134.2 (Ar-C), 2.6 {(CH₃-Sn), ¹J[¹¹⁹Sn-¹³C=352 Hz]}.¹¹⁹Sn NMR (CDCl₃, ppm): -10

Tributylstannyl 3-(¹H-indol-3-yl)propanoate 2: (Yield: 75.22 %). Elemental analysis, % Calculated (Found), for C₂₃H₃₇NO₂Sn: C, 57.76 (57.80); H, 7.80 (7.93); N, 2.93 (2.84). FT-IR (cm^-1): ν_as (COO) 1583, ν_s (COO) 1396, Δν (187). ¹H-NMR (ppm): 2.13 (t, H₂, ³J=7Hz), 2.46 (t, H₃, ³J=7Hz), 10.54 (N-H), 6.86 (s, H₁₂), 7.05-7.38 (m, Ar-H_6-9), 1.21-1.63 (m, H- α, H- β,H- γ ), 0.87 (t, H_δ, ³J=7.4Hz ).¹³C-NMR (ppm): 197.9 (C-1), 41.8 (C-2), 39.5 (C-3), 108.3 (C-4), 122.2 (C-12) 135.4, 125.7, 126.1, 128.8, 127.6 134.4 (Ar-C), 13.4 {(C- α,¹J[¹¹⁹Sn-¹³C=356 Hz]}, 23.2 (C- β ), 25.5 (C- γ ), 28.3 (C- δ ). ¹¹⁹Sn NMR (CDCl₃, ppm): -29

Triphenylstannyl 3-(¹H-indol-3-yl)propanoate 3: (Yield: 73.53 %). M.p: 102-104°C, Elemental analysis, % Calculated (Found), for C₂₉H₂₅NO₂Sn: C, 64.71 (64.75); H, 4.68 (4.64); N, 2.60 (2.69). FT-IR ν_as (COO) 1594, ν_s (COO) 1406, Δν (188).¹H-NMR (ppm): 2.14 (t, H₂, ³J=7Hz), 2.45 (t, H₃, ³J=7Hz), 10.59 (N-H), 6.85 (s, H₁₂), 7.06-7.38 (m, Ar-H_6-9), 7.43-7.52 (m, Sn-C₆H₅),7.49-7.85 (m, Sn-C₆H₅).¹³C-NMR (ppm):197.1 (C-1), 42.2 (C-2), 39.5 (C-3), 108.1 (C-4), 122.5 (C-12) 134.5, 125.4, 126.5, 128.8, 127.1 134.0 (Ar-C),139.5 (C- α ), 136.9(C- β ), 129.1 (C- γ ), 131.2 (C- δ ). ¹¹⁹Sn NMR (CDCl₃, ppm): -221

Dimethylstannanediyl bis[3-(¹H-indol-3-yl)propanoate 4: (Yield: 64.11 %). M.p: 150-156°C, Elemental analysis, % Calculated (Found), for C₂₄H₂₆N₂O₄Sn: C, 54.89(54.65); H, 4.99 (4.94); N, 5.33 (5.27). FT-IR (cm^-1): ν_as (COO) 1589, ν_s (COO) 1408, Δν (181). ¹H-NMR (ppm): 2.15 (t, H_2,2’, ³J=7Hz), 2.45 (t, H_3,3’, ³J=7Hz), 10.62 (N-H), 6.82(s, H₁₂,₁₂,), 7.09-7.42 (m, Ar-H_6-9), 0.95 (s, CH₃-Sn [60]). ¹³C-NMR (ppm): 197.0 (C-1), 42.3 (C-2), 39.8 (C-3), 108.7 (C-4), 122.5 (C-12) 135.5, 125.2 126.9, 128.8, 127.5 134.1 (Ar-C), 1.4 {(CH₃-Sn), ¹J[¹¹⁹Sn-¹³C=352 Hz]}. ¹¹⁹Sn NMR (CDCl₃, ppm): -45

Dibutylstannanediyl bis[3-(¹H-indol-3-yl)propanoate 5: (Yield: 71.74 %). M.p: 94-98 °C, Elemental analysis, % Calculated (Found), for C₃₀H₃₈N₂O₄Sn: C, 59.13 (59.17); H, 6.29 (6.24); N, 4.60 (4.65). FT-IR (cm^-1): ν_as (COO) 1582, ν_s (COO) 1403, Δν (179). ¹H-NMR (ppm):2.13 (t, H_2,2’, ³J=7Hz), 2.44 (t, H_3,3’, ³J=7HZ), 10.62 (N-H), 6.86 (s, H_12,12’), 7.05-7.36 (m, Ar-H_6-9),1.25-1.59 (m, H- α, H- β, H- γ, H- δ ).¹³C-NMR (ppm): 197.3 (C-1), 42.2 (C-2), 39.9 (C-3), 108.4 (C-4), 122.5 (C-12) 135.8, 124.9, 126.7, 129.0, 127.5 134.7 (Ar-C),12.9, 17.3, 26.5, 29.3 (C- α, C- β, C- γ, C- δ ).¹¹⁹Sn NMR (CDCl₃, ppm): 65

Diphenylstannanediyl bis[3-(¹H-indol-3-yl)propanoate 6: (Yield: 70.32 %). M.p: 128-131 °C, Elemental analysis, % Calculated (Found), for C₃₄H₃₀N₂O₄Sn: C, 62.89 (62.84); H, 4.66 (4.70); N, 4.31 (4.35). FT-IR (cm^-1): ν_as (COO) 1587, ν_s (COO) 1392, Δν (195). ¹H-NMR (ppm): 2.14 (t, H_2,2’,³J=7Hz), 2.45 (t, H_3,3’, ³J=7Hz), 10.58 (N-H), 6.85 (s, H_12,12’), 7.06-7.38 (m, Ar-H_6-9), 7.43-7.52 (m, 6H), 7.81-7.85 (m, 4H).¹³C-NMR (ppm): 197.1 (C-1), 42.3 (C-2), 39.1 (C-3), 108.9 (C-4), 122.4 (C-12) 135.5, 125.5, 126.3, 128.7, 127.3 134.1 (Ar-C), 139.2 (C- α ), 137.0 (C- β ), 128.3 (C- γ ), 133.2 (C- δ ), ¹¹⁹Sn NMR (CDCl₃, ppm): -197

Cytotoxic activity

Cytotoxic potential of compounds 1-6 was performed by MTT-Method [15], to evaluate the effect of compounds on the growth of cell. The human ovarian carcinoma cell line (A2780) was chosen for this purpose. This method is based on principle of reduction of yellow colored tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to purple formazan product, by the mitochondrial dehydrogenase of metabolically active cells. MTT solution was prepared by dissolving it at concentration of 5 mg/ml in phosphate buffer saline. Then 20 μL of this solution was added to each of 96 well plates containing 100 μL of culture medium and was incubated at 37ºC for 3 to 4 hr. The medium was aspirated carefully without disruption of purple coloured formazan crystals. The resulting purple colour zone was solubilized in 100 μL of DMSO and quantified by spectrophotometer. Micro ELISA plate reader was used to read the plate at wavelength of 590 nm to determine IC₅₀ value.

Results and Discussion

FT-IR Spectroscopy

FT-IR spectra of ligand and compounds 1-6 were recorded in the range of 4000-250 cm^-1. Asymmetric and symmetric stretching vibrations (-COO) are very useful in describing structure of organotin(IV) carboxylates. The ν_asym (COO) stretch appeared in the range of 1575-1618 cm^-1, while vibrations observed in region of 1408-1387 cm^-1 are characteristic of ν_sym (COO). It is reported in literature [16] that difference Δν (COO) above 200 cm^-1 suggests a monodentate coordination, while difference below 200 cm^-1 propose a bidentate coordination. In all synthesized compounds, the Δν value comes out to be less than 200 cm^-1 which suggest a chelating or bridging mode of coordination [17,18], therefore, tri- and diorganotin(IV) complexes exhibits five and six coordinated geometry, respectively in solid state.

Multinuclear NMR Spectroscopy

¹H, ¹³C and ¹¹⁹Sn NMR of compounds 1-6 were recorded in deuterated chloroform. In ¹H and ¹³C NMR spectra new signals for alkyl and aryl groups in corresponding region confirms complexation. In ¹H NMR, ²J[¹¹⁹Sn-¹H] coupling constant values obtained for complexes 1 and 4 depicted a tetrahedral geometry in solution which is in accordance with ¹J[¹¹⁹Sn-¹³C]. Also ¹J [¹¹⁹Sn-¹³C] coupling constant obtained for compound 2 confirms a tetrahedral environment around tin. Unfortunately, couplings were not observed for phenyl complexes in either ¹H or ¹³C NMR due to complex pattern; however ipso carbon gave a signal at 139.5 and 139.2 ppm for compound 3 and 6, respectively. N-H proton of indole unit appeared as a singlet in all complexes which propose that N atom of indolyl unit does not coordinate to Sn centre. It is apparent from these observations that complexes 1-6 adopt four coordinated geometry in solution state (NMR numbering is given as Scheme 2).

Scheme 2: Complexes 1-6 adopt four coordinated geometry in solution state.

To further confirm solution state geometry, ¹¹⁹Sn NMR spectra of complexes were recorded in deuterated chloroform.¹¹⁹Sn chemical shift of organotin(IV) compounds cover a range of ± 600 ppm [19]. It is reported that in alkyltin carboxylates, tetra-coordinated tin has δ (¹¹⁹Sn) values ranging from about +200 to -60 ppm, and pentacoordinated tin from -200 to -400 ppm [20]. A sharp signal appearing at -10, -29, -45, and 65 ppm indicates a 4-coordinated geometry in solution for compounds 1, 2, 4 and 5, respectively. For triphenyltin complexes, chemical shift in the range of -180 to -260 is an indicative of tetrahedral geometry [21]. In present case δ value of -221 and -197 ppm for complexes 3 and 6, respectively confirms a tetrahedral configuration in solution. Thus, ¹¹⁹Sn NMR results are in good congruence with ¹H and ¹³C NMR results.

Evaluation of DNA binding parameters

DNA binding studies of sodium 3-(¹H-indol-3-yl) propanoate (L-salt) and its organotin(IV) derivatives (complexes 1, 2 and 3) were carried out by using UV-vis spectroscopy and viscometry. Solutions of ligand salt and metal complexes were prepared in DMSO.

Viscosity measurement

The viscosity of DNA is sensitive to its length change. Mode of DNA binding to a compound can be well assigned by relative viscosity measurements which have been proved to be most reliable and least ambiguous technique. In the case of classic intercalation, DNA base pairs are separated in order to accommodate the bound compound, resulting in the lengthening of the DNA helix and consequently increased DNA viscosity [22-26]. On the other hand, the binding of a compound exclusively in DNA grooves by means of partial and/or non-classic intercalation, under same conditions, (e.g., netropsin, distamycin), causes a bend or kink in the DNA helix, reducing its effective length and, as a result, the DNA solutions viscosity is decreased or remains unchanged, i.e. groove binders and electrostatic interaction do not lengthen the DNA molecules [27-29].

Different sets of solution were prepared with a fixed DNA concentration (4.75 × 10^-4 M) and varying the concentration of ligand salt and complexes. The values of the relative viscosity (η/η₀)^1/3 were calculated from (t/t_o)^1/3 ratio for all samples, where t, t_o, η and η_{₀} represent the time and viscosity of the DNA solution with and without compound, respectively. A plot of (η/η₀)^1/3 versus the [compound]/[DNA] ratio shows that the viscosity increases with increasing concentration of the compounds, which suggests an intercalation mode of binding for ligand and all compounds.

Electronic absorption spectra

The absorption spectra of ligand salt and compounds 1, 2 and 3 in the absence and presence of different DNA concentrations are given as Figures 1-5. Two transitions bands were observed due to π - π* transitions. In all the compounds, both peaks showed hypochromism which suggest the intercalative mode of interaction between complexes and DNA. Decrease in transition probability as a consequence of interaction of partly filled π* orbital of ligand with π orbital of DNA base pairs may be the possible reason of hypochromism. There is only slight red shift of 1-2 nm, thus lack of any appreciable red shift in all compounds suggests the weak interactions with DNA [30].

Figure 1: Effects of the increasing amount of ligand and compounds on the relative viscosity of CT-DNA at 25 ± 0.1°C. [DNA]=4.75 × 10^-4 M.

Figure 2: Absorption spectra of 0.1 mM ligand salt in the absence (a) and presence of 6.4 μM (b), 9.1 μM (c), 11.9 μM (d), 14.3 μM (e) 16.5 μM (f) 18.6 μM (g) 20.5 μM (h), 22.3 μM (i) and 24.8 μM (j) DNA. The arrow direction indicates increasing concentrations of DNA. The inside graph is the plot of A_o/(A-A_o) vs. 1/[DNA] for the determination of the binding constant and Gibb’s free energy of the L salt-DNA adduct.

Figure 3: Absorption spectra of 0.1 mM compound 1 in the absence (a) and presence of 6.4 μM (b), 9.1 μM (c), 11.9 μM (d), 14.3 μM (e) 16.5 μM (f) 18.6 μM (g) and 20.5 μM (h) DNA. The arrow direction indicates increasing concentrations of DNA. The inside graph is the plot of A_o/(A - A_o) vs. 1/[DNA] for the determination of the binding constant and Gibb’s free energy of the (CH₃)3SnL–DNA adduct.

Figure 4: Absorption spectra of 0.1 mM compound 2 in the absence (a) and presence of 6.4 μM (b), 9.1 μM (c), 11.9 μM (d), 14.3 μM (e) 16.5 μM (f) and 18.6 μM (g) DNA. The arrow direction indicates increasing concentrations of DNA. The inside graph is the plot of A_o/(A-A_o) vs. 1/[DNA] for the determination of the binding constant and Gibb’s free energy of the (C₄H₉)₃SnL–DNA adduct.

Figure 5: Absorption spectra of 0.1 mM compound 3 in the absence (a) and presence of 6.4 μM (b), 9.1 μM (c), 11.9 μM (d), 14.3 μM (e) 16.5 μM (f) 18.6 μM (g) 20.5 μM (h) and 22.3 μM (i) DNA. The arrow direction indicates increasing concentrations of DNA. The inside graph is the plot of A_o/(A-A_o) vs. 1/[DNA] for the determination of the binding constant and Gibb’s free energy of the (C₆H₅)₃SnL–DNA adduct.

Benesi-Hildebrand equation [19] was used to calculate binding constant of these complexes.

A₀/(A-A₀)=Ɛ_G/(ƐH-G-Ɛ_G)+ Ɛ_G/(ƐH-G-Ɛ_G) × 1/K[DNA] (1)

In equation 1, K is the association constant, A₀ and A are the absorbance of the compound and its complex with DNA, respectively, while Ɛ_G and Ɛ_H-G are the absorption coefficients of the compound and the compound-DNA complex, respectively.

The binding constants were calculated from the intercept-to-slope ratios of straight line graphs obtained from A₀/ (A-A₀) vs. 1/[DNA] plots. A comparison in binding constants suggest that compound 2 forms most stable complex with DNA as compared to ligand salt, complexes 1 and 3. Gibb’s free energy (ΔG) was calculated by using following equation [31].

ΔG= -RTlnK

Where, R=8.314 JK^-1mol^-1(general gas constant), T=298 K (room temperature). Negative value of ΔG in all compounds showed that compound–DNA adduct formation is a spontaneous process.

Cytotoxic activity

Cellular viability of tumor cell line A2780 was tested with the ligand and complexes 1-6, by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay [15].

Cis-platin was used as a reference drug. The results of cytotoxic activities were determined as IC₅₀ which corresponds to the minimum concentration of compounds in μgmL^-1 that inhibits proliferation rate of the tumor cells by 50% in comparison to control untreated cells. All the tri- and diorganotin(IV) compounds showed good cytotoxic activities as compared to cis-platin and reported organotin compounds [32-34] and exhibited lower IC₅₀ values (Table 1). The biological activity of organotin complexes is influenced by the nature of the ligand environment, organic groups attached to the tin, compound structure, toxicity, and potential mechanism of action. The function of the ligand is to support the transport of the active organotin moiety to the site of the action where it is released by hydrolysis [35]. The anionic ligand also plays an important role in determining the degree of the activity of organotin compounds. As all complexes showed tetrahedral geometry in solution and showed significant activity, our results are consistent with the literature that states species generating a tetrahedral geometry in solution are more active [36]. The coordination number of the triorganotin derivatives are usually four in non-coordinating solvents while for diorganotin derivatives it vary from 4 to 6 due to fluxional behaviour/size of the ligands attached. As for as DMSO is concerned, it is a coordinating solvent and it might be possible that DMSO could change the coordination number of the complex to 5 or 6. However, in biological system when the complex reaches the target site having sulphur or nitrogen as donor for coordination, DMSO can vacate the site as a weak/hard donor [37,38].

Table 1: IC₅₀ values (μg/ml) of compounds 1-6 tested against human ovarian tumor cell lines A2780^a.

Activity and selectivity of complexes may also be related to the finding of recently reported organotin(IV) complexes against various other cell lines (i.e. Human lung tumorA549, hepatocellular liver carcinom HepG2, human colon cancer HCT 116, breast cancer MCF-7, leukemia K562 and normal cell line (3T3-L1) [39-41]. Additionally orientational and substitutional effects may also be considered in this connection.

Conclusion

It has been concluded that sodium salt of 3-(¹H-indol-3-yl) propanoic acid acts as bidentate and complexes exhibited 5 or 6 coordinated geometry in solid state. While multinuclear NMR (¹H,¹³C,¹¹⁹Sn) confirmed the tetrahedral geometry in solution. The complexes bind effectively with DNA and possessed good antitumor activity against ovarian carcinoma, at a concentration of 0.1 mM, thereby increasing their therapeutic value. Organotin(IV) complexes were found more active as compared to cis-platin, while triorganotin(IV) complexes are shown to possess greater cytotoxicity in comparison to diorganotin(IV) compounds.

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