Synthesis and biological applications of ionic triphenyltin(IV) chloride carboxylate complexes with exceptionally high cytotoxicity

Abstract

The reaction of N-phthaloylglycine (P-GlyH), N-phthaloyl-L-alanine (P-AlaH), and 1,2,4-benzenetricarboxylic 1,2-anhydride (BTCH) with triethylamine led to the formation of the corresponding ammonium salts [NHEt₃][P-Gly] (1), [NHEt₃][P-Ala] (2) and [NHEt₃][BTC] (3) in very high yields. The subsequent reaction of 1–3 with triphenyltin(IV) chloride (1 : 1) yielded the compounds [NHEt₃][SnPh₃Cl(P-Gly)] (4), [NHEt₃][SnPh₃Cl(P-Ala)] (5), and [NHEt₃][SnPh₃Cl(BTC)] (6), respectively. The molecular structure of 4 was determined by X-ray diffraction studies. The cytotoxic activity of the ammonium salts (1–3) and the triphenyltin(IV) chloride derivatives (4–6) were tested against human tumor cell lines from five different histogenic origins: 8505C (anaplastic thyroid cancer), A253 (head and neck cancer), A549 (lung carcinoma), A2780 (ovarian cancer) and DLD-1 (colon cancer). Triphenyltin(IV) chloride derivatives (4–6) show very high activity against these cell lines while the ammonium salts of the corresponding carboxylic acids (1–3) are totally inactive. The most active compound is 4 which is 50 times more active than cisplatin. Compound 4 is found to induce apoptosis via extrinsic pathways on DLD-1 cell lines, probably by accumulation of caspases 2, 3 and 8. Furthermore, compound 4 seems to cause disturbances in G1 and G2/M phases in cell cycle of DLD-1 cell line.

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1. Introduction

The fight against cancer is one of the primary targets concerning medicinal chemistry, in which bioorganometallic chemistry has become an interesting and fruitful research field.¹ The initial efforts in the evaluation of platinum-based anticancer drugs, have been shifted to non-platinum metal-based agents.^2–14 Many different metals, for example Ti, Ga, Ge, Pd, Au, Co, Ru and Sn have been already used for these purposes and have helped in the minimization of the side effects associated with the use of platinum compounds as anticancer drugs.^2–11 In this context, many organotin(IV) complexes have shown interesting in vivo anticancer activity as new chemotherapy agents.^15–20 However, the possible application of the synthesized organotin derivatives was very limited by their poor water solubility.⁷ Thus, solubility seems to be the one of the main problems for the use of tin(IV) in anticancer drugs, however, with a rational design and functionalization of the corresponding complexes, some new tin(IV) complexes with higher water solubility have been reported.^21–23 Additional interest, due to solubility reasons, have been paid to the synthesis and evaluation of the cytotoxicity of organotin(IV) compounds containing carboxylate ligands.^24–31 In addition, coordination of carboxylates to organotin moieties offers the possibility of studying the variations of the coordination mode from monodentate or chelate (symmetric or asymmetric) to bridging which may give rise to oligomeric or polymeric structures.³² The moieties R_nSn^(4–n)+ (n = 2 or 3) may bind to membrane proteins or glycoproteins, or to cellular proteins; e.g. to hexokinase, ATPase, acetylcholinesterase of human erythrocyte membrane or to skeletal muscle membranes²⁶ or may interact directly with DNA.³³

Following our research on the synthesis, characterization and cytotoxic properties of metal-based anticancer drugs,^34–43 and using carboxylato ligands which have shown interesting biological properties when bound to metals,^31,38,39 here we present the synthesis, characterization and the study of the cytotoxicity of three different ionic triphenyltin(IV) chloride complexes containing carboxylate ligands which present, according to their ionic nature, higher solubility in polar solvents. These compounds represent the first examples of triethylammonium salts of triphenyltin(IV) carboxylates which have shown an extremely high cytotoxic activity.

The carboxylic acids used in this study (Fig. 1) have been previously used in the synthesis of different neutral triorganotin(IV) carboxylates,^44–52 however, the synthesis and characterization of more convenient ionic tin(IV) complexes containing these ligands have not yet been reported and their cytotoxicity has not been studied. Thus, as numerous reports show that there is not a single or a well-defined number of pathways for the interaction of tin compounds with the membrane or constituents within the cell^53–56 and many other studies indicate the possible mechanisms of action of tin compounds, the study of the biological properties and anticancer mechanisms of this new group of anionic organotin carboxylates, which may give rise to intermolecular interactions with an effect on the overall and the local tin environment and in the final cytotoxicity, is very useful for gaining novel insights on the cellular action of organotin complexes.

Fig. 1 Carboxylic acids used in the study.

2. Experimental

2.1 General manipulations

All experiments were performed under an atmosphere of dry nitrogen using standard Schlenk techniques. Solvents and triethylamine were distilled from the appropriate drying agents and degassed before use. NMR spectra were recorded on a Bruker AVANCE DRX 400 spectrometer. ¹H NMR (400.13 MHz): internal standard solvent, external standard TMS; ¹³C{¹H} NMR (100.6 MHz): internal standard solvent, external standard TMS; ¹¹⁹Sn{¹H} NMR (149.2 MHz): internal standard solvent, external standard tetramethyltin. The splitting of proton resonances in the reported ¹H NMR spectra are defined as s = singlet, d = doublet, t = triplet, q = quadruplet, br = broad and m = multiplet. IR spectra: KBr pellets were prepared in a nitrogen-filled glove box and the spectra were recorded on a Perkin-Elmer System 2000 FTIR spectrometer in the range 350–4000 cm⁻¹. ESI MS were recorded with a FT-ICR MS Bruker-Daltonics (APEX II, 7 Tesla, MASPEC II), and solutions of ca. 1 mg mL⁻¹ of the compounds in a mixture of dry CHCl₃–CH₃CN (1 : 1) were injected. All solvents were purified by distillation, dried, saturated with nitrogen and stored over potassium mirror. N-phthaloylglycine (P-GlyH), N-phthaloyl-L-alanine (P-AlaH), and 1,2,4-benzenetricarboxylic 1,2-anhydride (BTCH) and triphenyltin(IV) chloride were purchased from Alfa Aesar or Acros Organics. All the commercial reagents were used directly, without further purification.

2.2 Synthesis of [NHEt₃][P-Gly] (1)

A solution of NEt₃ (1.76 mL, 12.19 mmol) in THF (20 mL) was added dropwise over 15 min to a solution of P-GlyH (2.50 g, 12.19 mmol) in THF (130 mL) at room temperature. The reaction mixture was stirred under reflux overnight. The solvent was then evaporated under reduced pressure and the resulting white solid was washed twice with hexane (40 mL) to give a white solid characterized as 1. Yield: 3.32 g, 89%. FT-IR (KBr): 3491 (s) (υ NH), 1720 (s) (υ CO) 1616 (s) (υ_a COO⁻), 1383 (s) (υ_s COO⁻); ¹H NMR (400 MHz, CDCl₃, 25 °C): δ 1.25 (t, 9H, ³J(¹H-¹H) = 7.6 Hz, CH₃ of Et), 3.01 (q, 6H, ³J(¹H-¹H) = 7.6 Hz, CH₂ of Et), 4.28 (s, 2H, CH₂ of P-Gly), 7.67 (m, 2H, aromatic H in P-Gly), 7.83 (m, 2H, aromatic H in P-Gly), 14.31 (br, 1H, NHEt₃); ¹³C{¹H} NMR (100.6 MHz, CDCl₃, 25 °C): δ 8.5 (CH₃ of Et), 41.3 (CH₂ of P-Gly), 44.9 (CH₂ of Et), 123.1, 133.5 (aromatic CH of P-Gly) 132.7 (aromatic ipso-C of P-Gly), 168.3 (CO of P-Gly), 172.3 (COO); ESI-MS (negative)(m/z): 203.8 [M⁺-NHEt₃], 159.9 [M⁺-NHEt₃-COO]; ESI-MS (positive)(m/z): 102.1 [M⁺-(P-Gly) + H]; elemental analysis for C₁₆H₂₂N₂O₄ (306.4): calc. C, 62.73; H, 7.24; N, 9.14, found C, 62.40; H, 7.19; N 9.09%.

2.3 Synthesis of [NHEt₃][P-Ala] (2)

The synthesis of 2 was carried out in an identical manner to 1. NEt₃ (1.65 mL, 11.46 mmol) and P-AlaH (2.50 g, 11.46 mmol). Yield: 3.41 g, 93%. FT-IR (KBr): 3460 (s) (υ NH), 1710 (s) (υ CO) 1650 (s) (υ_a COO⁻), 1390 (s) (υ_s COO⁻); ¹H NMR (400 MHz, CDCl₃, 25 °C): δ 1.25 (t, 9H, ³J(¹H-¹H) = 7.2 Hz, CH₃ of Et), 1.56 (d, 3H, ³J(¹H-¹H) = 7.6 Hz, CH₃ of P-Ala), 3.08 (q, 6H, ³J(¹H-¹H) = 7.6 Hz, CH₂ of Et), 4.73 (q, 1H, ³J(¹H-¹H) = 7.2 Hz, CH of P-Ala), 7.67 (m, 2H, aromatic H in P-Ala), 7.79 (m, 2H, aromatic H in P-Ala), 11.88 (br, 1H, NHEt₃); ¹³C{¹H} NMR (100.6 MHz, CDCl₃, 25 °C): δ 8.9 (CH₃ of Et), 16.3 (CH₃ of P-Ala) 41.9 (CH of P-Ala), 44.8 (CH₂ of Et), 123.7, 133.2 (aromatic CH of P-Ala) 132.9 (aromatic ipso-C of P-Ala), 168.9 (CO of P-Ala), 172.5 (COO); ESI-MS (negative)(m/z): 217.9 [M⁺-NHEt₃], 174.2 [M⁺-NHEt₃-COO]; ESI-MS (positive)(m/z): 102.1 [M⁺-(P-Ala) + H]; elemental analysis for C₁₇H₂₄N₂O₄ (320.4): calc. C, 63.73; H, 7.55; N, 8.74, found C, 63.55; H, 7.39; N 8.79%.

2.4 Synthesis of [NHEt₃][BTC] (3)

The synthesis of 3 was carried out in an identical manner to 1. NEt₃ (1.88 mL, 13.01 mmol) and BTCH (2.50 g, 13.01 mmol). Yield: 3.09 g, 81%. FT-IR (KBr): 3435 (s) (υ NH), 1778, 1715 (s) (υ CO) 1615 (s) (υ_a COO⁻), 1351 (s) (υ_s COO⁻); ¹H NMR (400 MHz, CDCl₃, 25 °C): δ 1.39 (t, 9H, ³J(¹H-¹H) = 7.2 Hz, CH₃ of Et), 3.23 (q, 6H, ³J(¹H-¹H) = 7.2 Hz, CH₂ of Et), 7.99 (d, 1H, ³J(¹H-¹H) = 8.0 Hz, aromatic H in BTC), 8.58 (d, 1H, ³J(¹H-¹H) = 8.0 Hz, aromatic H in BTC), 8.64 (s, 1H, aromatic H in BTC), 13.33 (br, 1H, NHEt₃); ¹³C{¹H} NMR (100.6 MHz, CDCl₃, 25 °C): δ 8.6 (CH₃ of Et), 45.6 (CH₂ of Et), 125.1, 126.8, 137.2 (aromatic CH of BTC), 145.5 (aromatic C–COO of BTC), 162.8, 162.9 (C–CO of BTC), 169.3 (COO of BTC), 170.7, 170.8 (C=O of anhydride of BTC); ESI-MS (negative)(m/z): 190.8 [M⁺-NHEt₃], 164.9 [M⁺-NHEt₃-O], 146.9 [M⁺-NHEt₃-COO]; ESI-MS (positive)(m/z): 102.1 [M⁺-BTC + H]; elemental analysis for C₁₅H₁₉NO₅ (293.3): calc. C, 61.42; H, 6.53; N, 4.78, found C, 61.21; H, 6.44; N 4.79%.

2.5 Synthesis of [NHEt₃][SnPh₃Cl(P-Gly)] (4)

A solution of [NHEt₃][P-Gly] (1) (0.79 g, 2.59 mmol) in THF (20 mL) was added dropwise over 15 min to a solution of SnPh₃Cl (1.00 g, 2.59 mmol) in THF (80 mL) at room temperature. The reaction mixture was stirred under reflux overnight. The solvent was then evaporated under reduced pressure and the resulting white solid was washed twice with hexane (40 mL) to give a white solid characterized as 4. The product is totally soluble in EtOH, DMSO, CHCl₃, CH₂Cl₂,·CH₃CN and in mixtures EtOH–H₂O and is partially soluble in toluene. Crystallization in toluene afforded crystals suitable for X-ray diffraction analysis. Yield: 1.32 g, 74%. FT-IR (KBr): 3436 (s) (υ NH), 1718 (s) (υ CO) 1623 (s) (υ_a COO⁻), 1370 (s) (υ_s COO⁻), 457 (m) (υ Sn–O); ¹H NMR (400 MHz, CDCl₃, 25 °C): δ 1.05 (t, 9H, ³J(¹H-¹H) = 7.2 Hz, CH₃ of Et), 2.70 (q, 6H, ³J(¹H-¹H) = 7.6 Hz, CH₂ of Et), 4.15 (s, 2H, CH₂ of P-Gly), 7.35 (br m, 9H, m- and p-protons in SnPh₃), 7.66 (m, 2H, aromatic H in P-Gly), 7.73 (br m, 6H, o-protons in SnPh₃, ³J(¹H–Sn) = ca. 60 Hz); 7.80 (m, 2H, aromatic H in P-Gly), 11.62 (br, 1H, NHEt₃), ¹³C{¹H} NMR (100.6 MHz, CDCl₃, 25 °C): δ 8.4 (CH₃ of Et), 40.9 (CH₂ of P-Gly), 45.4 (CH₂ of Et), 123.2, 133.8 (aromatic CH of P-Gly), 128.6 (C-3 and C-5 of SnPh₃, ³J(¹³C–Sn) = 67.2 Hz), 129.4 C-4 of SnPh₃, ⁴J(¹³C–Sn) = 46.5 Hz), 132.4 (aromatic ipso-C of P-Gly), 136.4 (C-2 and C-6 of SnPh₃, ²J(¹³C–Sn) = 48.7 Hz), 140.4 (C-1 of SnPh₃, ¹J(¹³C–Sn) not observed), 167.9 (CO of P-Gly), 171.9 (COO); ¹¹⁹Sn{¹H} NMR (149.2 MHz, CDCl₃, 25 °C): δ −144.5; ESI-MS (negative)(m/z): 590.02 [M⁺-NHEt₃]; elemental analysis for C₃₄H₃₇ClN₂O₄Sn (691.8): calc. C, 59.03; H, 5.39; N, 4.05, found C, 58.59; H, 5.29; N 4.09%.

2.6 Synthesis of [NHEt₃][SnPh₃Cl(P-Ala)] (5)

The synthesis of 5 was carried out in an identical manner to 4. [NHEt₃][P-Ala] (2) (0.83 g, 2.59 mmol) and SnPh₃Cl (1.00 g, 2.59 mmol). Yield: 1.42 g, 78%. FT-IR (KBr): 3447 (s) (υ NH), 1713 (s) (υ CO) 1618 (s) (υ_a COO⁻), 1389 (s) (υ_s COO⁻), 455 (m) (υ Sn–O); ¹H NMR (400 MHz, CDCl₃, 25 °C): δ 1.22 (t, 9H, ³J(¹H-¹H) = 7.2 Hz, CH₃ of Et), 1.71 (d, 3H, ³J(¹H-¹H) = 7.2 Hz, CH₃ of P-Ala), 2.93 (q, 6H, ³J(¹H-¹H) = 7.6 Hz, CH₂ of Et), 4.99 (q, 1H, ³J(¹H-¹H) = 7.6 Hz, CH of P-Ala), 7.44 (br m, 9H, m- and p- protons in SnPh₃), 7.68 (m, 2H, aromatic H in P-Ala), 7.70 (br m, 6H, o- protons in SnPh₃, ³J(¹H–Sn) = ca. 52 Hz); 7.83 (m, 2H, aromatic H in P-Ala), 11.70 (br, 1H, NHEt₃), ¹³C{¹H} NMR (100.6 MHz, CDCl₃, 25 °C): δ 8.5 (CH₃ of Et), 16.0 (CH₃ of P-Ala) 45.6 (CH of P-Ala), 48.2 (CH₂ of Et), 123.3, 133.9 (aromatic CH of P-Ala), 129.0 (C-3 and C-5 of SnPh₃, ³J(¹³C–Sn) = 65.1 Hz), 130.2 (C-4 of SnPh₃, ⁴J(¹³C–Sn) = 13.6 Hz), 132.1 (aromatic ipso-C of P-Gly), 136.3 (C-2 and C-6 of SnPh₃, ²J(¹³C–Sn) = 48.7 Hz), 139.8 (C-1 of SnPh₃, ¹J(¹³C–Sn) not observed), 167.5 (CO of P-Ala), 178.1 (COO); ¹¹⁹Sn{¹H} NMR (149.2 MHz, CDCl₃, 25 °C): δ −94.0; ESI-MS (negative)(m/z): 604.03 [M⁺-NHEt₃]; elemental analysis for C₃₅H₃₉ClN₂O₄Sn (705.9): calc. C, 59.56; H, 5.57; N, 3.97, found C, 59.49; H, 5.48; N 3.98%.

2.7 Synthesis of [NHEt₃][SnPh₃Cl(BTC)] (6)

The synthesis of 6 was carried out in an identical manner to 4. [NHEt₃][BTC] (3) (0.76 g, 2.59 mmol) and SnPh₃Cl (1.00 g, 2.59 mmol). Yield: 1.16 g, 66%. FT-IR (KBr): 3433 (s) (υ NH), 1780, 1719 (s) (υ CO) 1616 (s) (υ_a COO⁻), 1362 (s) (υ_s COO⁻), 454 (m) (υ Sn–O); ¹H NMR (400 MHz, CDCl₃, 25 ºC): δ 1.33 (t, 9H, ³J(¹H-¹H) = 7.2 Hz, CH₃ of Et), 3.07 (q, 6H, ³J(¹H-¹H) = 7.2 Hz, CH₂ of Et), 7.46 (br m, 9H, m- and p- protons in SnPh₃), 7.75 (br m, 6H, o- protons in SnPh₃, ³J(¹H–Sn) = 61.1 Hz), 7.98 (d, 1H, ³J(¹H-¹H) = 7.9 Hz, aromatic H in BTC), 8.56 (d, 1H, ³J(¹H-¹H) = 7.9 Hz, aromatic H in BTC), 8.65 (s, 1H, aromatic H in BTC), 12.67 (br, 1H, NHEt₃); ¹³C{¹H} NMR (100.6 MHz, CDCl₃, 25 °C): δ 8.6 (CH₃ of Et), 45.9 (CH₂ of Et), 125.3, 127.1, 137.5 (aromatic CH of BTC), 128.9 (C-3 and C-5 of SnPh₃, ³J(¹³C–Sn) = 63.8 Hz), 130.1 (C-4 of SnPh₃, ⁴J(¹³C–Sn) = 13.6 Hz), 136.4 (C-2 and C-6 of SnPh₃, ²J(¹³C–Sn) = 48.8 Hz), 138.9 (C-1 of SnPh₃, ¹J(¹³C–Sn) not observed), 142.6 (aromatic C-COO of BTC), 162.5, 162.6 (C-CO of BTC), 168.7 (COO of BTC), 170.6, 170.8 (C=O of anhydride of BTC); ¹¹⁹Sn{¹H} NMR (149.2 MHz, CDCl₃, 25 °C): δ −95.0; ESI-MS (negative)(m/z): 576.99 [M⁺-NHEt₃]; elemental analysis for C₃₃H₃₄ClNO₅Sn (705.9): calc. C, 58.39; H, 5.05; N, 2.06, found C, 58.10; H, 4.97; N 2.19%.

2.8 Data Collection and Structural Refinement of 4

The data of 4 were collected with a CCD Oxford Xcalibur S (λ(Mo-Kα) = 0.71073 Å) using ω and φ scans mode. Semi-empirical from equivalents absorption corrections were carried out with SCALE3 ABSPACK.⁵⁷ The structure was solved by Patterson methods.⁵⁸ Structure refinement was carried out with SHELXL-97.⁵⁹ All non-hydrogen atoms were refined anisotropically, H atom bonded to nitrogen was localized and refined freely and the other hydrogen atoms were placed in calculated positions and refined with calculated isotropic displacement parameters. Table 1 lists crystallographic details. Crystallographic data for the structural analysis 4 have been deposited with the Cambridge Crystallographic Data Centre, CCDC-759465 (4).†

Table 1 Crystallographic data for 4

	4
Formula	C34H37ClN2O4Sn
Fw	691.80
T/K	130(2)
Cryst syst	Monoclinic
Space group	P21/n
a/pm	990.82(5)
b/pm	3066.76(9)
c/pm	1121.89(4)
α (°)	90
β (°)	107.661(5)
γ (°)	90
V (nm^3)	3.2483(2)
Z	4
Dc (Mg m^−3)	1.415
μ/mm^−1	0.908
F(000)	1416
Cryst dimens/mm	0.3 × 0.2 × 0.2
θ range (°)	2.66 to 28.28
hkl ranges	−13 ≤ h ≤ 13,
	−37 ≤ k ≤ 40,
	−14 ≤ l ≤ 14
Data/parameters	8049/350
goodness-of-fit on F^2	1.055
Final R indices [I > 2σ(I)]	R 1 = 0.0558,
	wR2 = 0.1264
R indices (all data)	R 1 = 0.0837,
	wR2 = 0.1352
largest diff. peak and hole/e Å^−3	1.769 and −1.353

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2.9. In vitro studies

2.9.1. Preparation of drug solutions. Stock solutions of investigated compounds (1–6) were prepared in dimethyl sulfoxide (DMSO, Sigma Aldrich) at a concentration of 20 mM, filtered through Millipore filter, 0.22 μm, before use, and diluted by nutrient medium to various working concentrations. Nutrient medium was RPMI-1640 (PAA Laboratories) supplemented with 10% fetal bovine serum (Biochrom AG) and penicillin/streptomycin (PAA Laboratories).

2.9.2. Cell lines and culture conditions. The cell lines 8505C, A253, A549, A2780 and DLD-1, included in this study, were kindly provided by Dr Thomas Mueller, Department of Hematology/Oncology, Martin Luther University of Halle-Wittenberg, Halle (Saale), Germany. Cultures were maintained as monolayer in RPMI 1640 (PAA Laboratories, Pasching, Germany) supplemented with 10% heat inactivated fetal bovine serum (Biochrom AG, Berlin, Germany) and penicillin/streptomycin (PAA Laboratories) at 37 °C in a humidified atmosphere of 5% (ν/ν) CO₂.

2.9.3. Cytotoxicity assay. The cytotoxic activities of the compounds were evaluated using the sulforhodamine-B (SRB, Sigma Aldrich) microculture colorimetric assay.⁶⁰ In short, exponentially growing cells were seeded into 96-well plates on day 0 at the appropriate cell densities to prevent confluence of the cells during the period of experiment. After 24 h, the cells were treated with serial dilutions of the studied compounds for 96 h. Final concentrations achieved in treated wells were 0–100 μM for 1–3 and 0.008, 0.02, 0.04, 0.08, 0.12, 0.16, 0.2, 0.3 and 0.4 μM for tin(IV) complexes (4–6). Each concentration was tested in triplicate on each cell line. The final concentration of DMSO solvent never exceeded 0.5%, which was non-toxic to the cells. The percentages of surviving cells relative to untreated controls were determined 96 h after the beginning of drug exposure. After 96 h treatment, the supernatant medium from the 96 well plates was thrown away and the cells were fixed with 10% TCA. For a thorough fixation, plates were then allowed to stand at 4 °C. After fixation, the cells were washed in a strip washer. The washing was carried out four times with water using alternate dispensing and aspiration procedures. The plates were then dyed with 100 μl of 0.4% SRB for about 45 min. After dying, the plates were again washed to remove the dye with 1% acetic acid and allowed to air dry overnight. 100 μL of 10 mM Tris base solutions were added to each well of the plate and absorbance was measured at 570 nm using a 96 well plate reader (Tecan Spectra, Crailsheim, Germany). The IC₅₀ value, defined as the concentrations of the compound at which 50% cell inhibition was observed, was estimated from the dose-response curves.

2.9.4 Apoptosis tests.
2.9.4.1 Trypan blue exclusion test. Apoptotic cell death was analyzed by trypan blue dye (Sigma Aldrich, Germany) on DLD-1 cell line. The cell culture flasks with 70% to 80% confluence were treated with IC₉₀ dose of 4 for 24 h. The supernatant medium with floating cells was collected after treatment and centrifuged to collect the dead and apoptotic cells. The cell pellet was resuspended in serum free media. Equal amounts of cell suspension and trypan blue were mixed and this was analyzed under a microscope. The cells which were viable excluded the dye and were colorless while those whose cell membrane was destroyed were blue. If the proportion of colorless cells is higher than the colored cells, then the death can be characterized as apoptotic.
2.9.4.2 DNA fragmentation assay. Determination of apoptotic cell death was performed by DNA gel electrophoresis. Briefly, DLD-1 cells were treated with the respective IC₉₀ dose of 4 for 24 h. Floating cells induced by drug exposure were collected, washed with PBS and lysed with lysis buffer (100 mM Tris-HCl pH 8.0; 20 mM EDTA; 0.8% SDS; all from Sigma Aldrich). Then they were treated with RNAse A at 37 °C for 2 h and proteinase K at 50 °C (both from Roche Diagnostics chemical company, Mannheim, Germany). DNA laddering was observed by running the samples on 2% agarose gel followed by ethidium bromide (Sigma Aldrich) staining.

2.9.5 Caspase 2, 3, 8 and 9 enzyme activity assay. Activity of caspases 2, 3, 8 and 9 was measured using the caspase substrate cleavage assay. After exposure to equitoxic IC₅₀ concentration of 4, DLD-1 cells were sampled 2 and 6 h for cleavage of caspases. To summarize, adherent cells were washed with cold PBS, collected with a cell scraper, and suspended in cell lyses buffer (50 mM Hepes pH 7.4, 1% Triton X100, all from Sigma-Aldrich). After incubation for 10 min on ice and centrifugation, protein concentrations of the supernatants were measured according to the method of Bradford (Bio-Rad Laboratories). Samples (50 μg protein extract respectively) were incubated on a microplate at 37 °C overnight in reaction buffer (50 mM Hepes pH 7.4, 0.1% CHAPS, 5 mM EGTA, 5% glycerol) containing 10 mM DTT (all from Sigma-Aldrich) and a specific substrate of caspases (2, Ac-VDVAD-pNA; 3, Ac-DEVD-pNA; 8, Ac-IETD-pNA; 9, Ac-LEHD-pNA, Axxora, Loerrach, Germany). Extinction of released p-nitroaniline was measured at 405 nm (Tecan Spectra, Crailsheim, Germany) and activity of caspases 2, 3, 8 and 9 was evaluated by OD ratio of treated/untreated samples.⁶¹

2.9.6 Cell cycle analysis. Cell cycle was assessed by flow cytometry using a fluorescence-activated cell sorter (FACS). For this assay, 1 × 10⁶ DLD-1 cells, were seeded in 25 cm² cell culture flasks, with 10 mL of medium. After 24 h of incubation, 4 was added at IC₉₀ concentration. Following 24 h of incubation, cells were harvested by mild trypsinization, collected by centrifugation, washed with PBS and both adherent and floating cells were resuspended in 100 μl of PBS and fixed with 2 mL of 70% ethanol at 4 °C for at least 1 h. The fixed samples were then centrifuged, the cell pellet was washed with 2 mL of staining buffer (PBS + 2% FCS + 0.01% NaN₃) and again centrifuged. The cell pellet was resuspended in 100 μL of RNase A (1 mg mL⁻¹) and incubated for 30 min at 37 °C. At the end of incubation the samples were treated with propidium iodide (20 μg/1 mL of staining buffer) and allowed to stand in the dark at least for 30 min before analysis. The fluorescence intensity was determined by a Facscalibur (Becton Dickinson, Heidelberg, Germany). Each analysis was done using ca. 1 × 10⁴ events.

3. Results and discussion

3.1 Synthesis and spectroscopic studies

The ammonium salts [NHEt₃][P-Gly] (1), [NHEt₃][P-Ala] (2) (Scheme 1) and [NHEt₃][BTC] (3) (Scheme 2) were prepared, in very high yields, by the reaction of N-phthaloylglycine (P-GlyH), N-phthaloyl-L-alanine (P-AlaH), or 1,2,4-benzenetricarboxylic 1,2-anhydride (BTCH) with triethylamine respectively. The NMR, mass and IR spectra showed that all the complexes, isolated as crystalline solids, were of high purity. In the ¹H NMR spectra of 1–3, a set of three signals consisting in a triplet at ca. 1.3 ppm, a quadruplet at ca. 3.2 and a broad signal between 11 and 13 ppm corresponding to protons of the triethylammonium cation were observed. For each compound, signals corresponding to the different carboxylate anions were recorded. For 1 one singlet at 4.28 ppm corresponding to the –CH₂– protons and two multiplets at 7.67 and 7.83 ppm assigned to the aromatic protons of the N-phthaloyl group were observed. In the case of 2, two multiplets (at 7.67 and 7.79 ppm) were also recorded for the phthaloyl group and a doublet at 1.56 and a quadruplet at 3.08 ppm were assigned for the CH–CH₃ moiety of the N-phthaloyl-L-alanine. In the ¹H NMR spectrum of compound 3, two doublets and one singlet between 7.9 and 8.7 ppm were assigned to the aromatic protons of the carboxylate, in addition to the signals corresponding to the cation. The ¹³C{¹H} NMR spectra of 1–3 showed the expected signals. The IR spectra of the ionic ligands present strong bands in two different regions between 1650–1610 and 1390–1350 cm⁻¹, which correspond to the asymmetric and symmetric vibrations, respectively, of the COO moiety. The difference between the asymmetric and symmetric vibrations of more than 200 cm⁻¹ in all cases, indicates monodentate coordination of the carboxylate ligand,⁶² in addition to these signals, a typical band at ca. 3450 cm⁻¹ assigned to the N–H vibration was observed in all the spectra.⁶³ The ESI-MS spectra of 1–3 recorded in negative mode showed the peaks of the anionic part of these molecules, while recorded in positive mode, presented a peak corresponding to the triethylammonium cation.

Scheme 1

Scheme 2

The subsequent reaction of 1–3 with triphenyltin chloride (1 : 1) yielded the compounds [NHEt₃][SnPh₃Cl(P-Gly)] (4), [NHEt₃][SnPh₃Cl(P-Ala)] (5) (Scheme 1) and [NHEt₃][SnPh₃Cl(BTC)] (6) (Scheme 2), respectively.

The organotin(IV) complexes 4–6 were characterized by multinuclear NMR spectroscopy, mass spectrometry, IR spectroscopy and elemental analysis. In the ¹H NMR spectra of all the compounds a set of two signals at ca. 7.3 and 7.7 ppm corresponding to the protons of the phenyl groups of the SnPh₃Cl moiety, was observed in addition to the signals corresponding to the carboxylate ligands, which presented similar spectral patterns to those observed in the ¹H NMR spectra of 1–3. ¹³C{¹H} NMR spectra for 4–6 showed the expected signals for the phenyl groups, as well as the signals corresponding to the different carboxylate ligands. A typical signal at low field (ca. 170 ppm) was observed in all the spectra and assigned to the carbon atom of the carboxylate group. Analogously to the IR spectra of the starting carboxylate 1–3, the spectra of 4–6 present strong bands between 1630–1610 and 1390–1360 cm⁻¹, which correspond to the asymmetric and symmetric vibrations, respectively, of the COO moiety. Again, the difference between the asymmetric and symmetric vibrations of more than 200 cm⁻¹ in all cases, indicates monodentate coordination of the carboxylate ligand,⁶² as was also confirmed, in the case of complex 4, by single crystal X-ray diffraction studies (see section 3.2). In addition, medium absorptions corresponding to the Sn–O stretching mode of vibration appeared at ca. 455 cm⁻¹. 4–6 were also characterized by ESI-MS observing, in all cases in the negative mode; the peaks corresponding to the anionic parts of 4–6 (see sections 2.2–2.7).

Stability of the synthesized complexes in d₆-DMSO and d₆-DMSO/D₂O solutions has been followed by NMR spectroscopy. After several hours in dry d₆-DMSO, the NMR spectra of the studied compounds suffered apparent no changes, indicating their relatively high stability. The analysis of the spectra of 4–6 in d₆-DMSO/D₂O show a very slow decomposition process which over 72 h leads to the formation of a mixture of tin-containing products which included Ph₃SnOSnPh₃, SnPh₃OH, SnPh₃L (L = P-Gly, P-Ala, BTC) and the free carboxylic acids, between other unidentified compounds.

3.2 Structural studies

Complex 4 (Fig. 2) crystallizes in the centrosymmetric monoclinic space group P2₁/n with four molecules in the unit cell.

Fig. 2 Molecular structure and atom-labeling scheme for 4 with thermal ellipsoids at 50% probability (hydrogen atoms, except at nitrogen, are omitted for clarity).

The molecular structure of 4 reveals that the central tin atom is pentacoordinated; presenting a almost ideal trigonal bipyramidal geometry with the Cl(1)–Sn(1)–O(1) angle of 177.23(8)° and O(1)–Sn(1)–C_phenyl and Cl(1)–Sn(1)–C_phenyl close to 90°. The Sn–O bond lengths Sn(1)–O(1) 223.1(3) and Sn(1)–O(2) 333.8(3) pm are quite different indicating monodentate coordination to tin of the carboxylate ligand. Weak interionic interactions (O(2)⋯H(2N) ca. 168 pm) between the hydrogen atom of the ammonium cation and the non-coordinated oxygen of the anion were observed. The bond length N(2)–H(1N) yielded reasonable distances of ca. 98 pm. The most interesting structural parameter of this complex is the bond length Sn(1)–Cl(1) of 256.5(2) pm which is in agreement with other Sn–Cl bonds in zwitterionic complexes and about ca. 10 pm longer than the expected for a tryaryltin(IV) chloride derivative,^64–68 indicating some repulsive effect of the carboxylate ligand on the Sn–Cl bond which elongates the distance between the two atoms. Selected bond lengths and angles for 4 are summarized in Table 2.

Table 2 Selected Bond Lengths (pm) and Angles (°) for 4

	4
Sn(1)–O(1)	223.1(3)
Sn(1)–O(2)	333.8(3)
Sn(1)–Cl(1)	256.5(2)
C(19)–O(1)	125.8(5)
C(19)–O(2)	123.4(6)
C(22)–O(3)	119.5(5)
C(23)–O(4)	120.4(6)
N(2)–H(1N)	98(2)
O(2)⋯H(2N)	168(2)
Cl(1)–Sn(1)–O(1)	177.23(8)
Cl(1)–Sn(1)–O(2)	141.45(9)
Cl(1)–Sn(1)–C(1)	91.0(2)
Cl(1)–Sn(1)–C(7)	92.7(1)
Cl(1)–Sn(1)–C(13)	91.2(2)
O(1)–C(19)–O(2)	125.3(5)

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3.3 In vitro studies

3.3.1 Cytotoxic studies. The in vitro cytotoxicities of triethylammonium carboxylates 1–3 as well as corresponding triphenyltin(IV) complexes 4–6 against human tumor cell lines 8505C anaplastic thyroid cancer, A253 head and neck tumor, A549 lung carcinoma, A2780 ovarian cancer and DLD-1 colon carcinoma were determined by using the SRB microculture colorimetric assay.⁶⁰ In addition, cytotoxicity of cisplatin have been included for comparison.

Triethylammonium salts of carboxylic acids were inactive against the investigated panel of tumor cell lines (IC₅₀ >100 μM). In this way is also proved that neither hypothetic activity of the free thriethylammonium cation nor the uncoordinated carboxylato ligand have influence on the activity of the studied triphenyltin(IV) complexes. The IC₅₀ values of the studied compounds and cisplatin are summarized in Table 3. The triphenyltin(IV) compounds showed a dose-dependent antiproliferative effect toward all the studied cancer cell lines (Fig. 3). Triphenyltin(IV) compounds present lower IC₅₀ values than those of cisplatin. The difference in activity of compounds 4–6 and cisplatin presents a maximum of up to 50 times, in cisplatin resistant cell line DLD-1. A similar efficiency was found against anaplastic thyroid cancer cisplatin resistant cell line 8505C, where triphenyltin(IV) compounds are almost up to 40 times more active than cisplatin. These values become lower in cisplatin sensitive A2780, A253 and A549 cell lines where triphenyltin(IV) compounds are only between 3.2 and 14.8 times more cytotoxic than cisplatin.

Fig. 3 Representative graphs show survival of 8505C, A253, A549, A2780 and DLD-1 cells grown for 96 h in the presence of increasing concentrations of triphenyltin(IV) complexes 4–6.

Table 3 IC₅₀ [μM] for the 96 h of action of investigated compounds and cisplatin on 8505C, A253, A549, A2780 and DLD-1 cells determined by SRB test

Compound	IC50/μM
	8505C	A253	A549	A2780	DLD-1
1–3	>100	>100	>100	>100	>100
4	0.129 ± 0.004	0.093 ± 0.003	0.102 ± 0.004	0.121 ± 0.002	0.103 ± 0.004
5	0.179 ± 0.003	0.139 ± 0.005	0.152 ± 0.003	0.170 ± 0.002	0.165 ± 0.003
6	0.241 ± 0.058	0.238 ± 0.002	0.236 ± 0.011	0.130 ± 0.003	0.210 ± 0.006
cisplatin	5.02 ± 0.23	0.81 ± 0.02	1.51 ± 0.02	0.55 ± 0.03	5.14 ± 0.12

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Triphenyltin(IV) complexes 4 and 5 bearing very similar moieties showed notable differences in the cytotoxic activity. From those results one can envisage that small structural changes, (replacement of H with Me group (4 to give 5)) led to a significant decrease of the final cytotoxic activity. On the other hand, triphenyltin(IV) compound substitution of the phthaloyl aminoacids by 1,2,4-benzenetricarboxylic-1,2-anhydride (6) led to further decreases in the cytotoxicity, except in A2780 cell line, when compared with 4 and 5. Thus, one can think that the incorporation of biocompatible phthaloyl aminoacids as carboxylato ligands may have a positive influence in the final cytotoxicity of the tin(IV) complexes when compared with other different carboxylato ligands.

3.3.2 Apoptosis studies.
3.3.2.1 Dye exclusion test and DNA laddering. Apoptosis and cell mediated cytotoxicity are characterized by a fragmentation of the genomic DNA. These DNA fragments have a length of about 180 base pairs or multiples of this number (360, 540, 720, …), this is, the characteristic DNA-length of a nucleosome (DNA-histone-complex). Endonucleases cleave selectively DNA at sites located between nucleosomal units (linker DNA). In agarose gel electrophoresis of these, DNA fragments are resolved to a distinctive ladder pattern. To test whether complex 4 induced cell death mediated by apoptosis, floating cells from DLD-1 colon carcinoma cell line after 24 h treatment with the IC₉₀ concentration were collected and analyzed by DNA laddering technique. In DLD-1 cell line with triphenyltin(IV) compound 4, typical DNA laddering was observed (Fig. 4). Furthermore, DLD-1 cells were exposed to IC₉₀ dose of complex 4, the ability to exclude trypan blue and detached cells were investigated. The treatment with IC₉₀ dose of 4 resulted in apoptotic cell death, in which floating cells showed the ability to exclude the blue dye (Fig. 4).

Fig. 4 Dye exclusion test (right) and DNA laddering (left) for DLD-1 cell line treated 24 h with 4.

3.3.2.2 Effect of 4 on the caspase 2, 3, 8 and 9 activities. In order to gain insights into the induced apoptosis mechanism by the most active triphenyltin(IV) complex 4, we analyzed whether caspases were involved as downstream effectors in the induced cell death. The upstream caspases 2, 8 and 9, and the downstream caspase 3 were used for the present study. DLD-1 colon carcinoma cell line was chosen for investigation of the activity of caspases 2, 3, 8 and 9. When treated with compound 4 for 2 h, only caspase 8 was found to be upregulated. Similarly to that, when cisplatin was used for the same time period also activation of only caspase 8 was observed (Fig. 5). The cells treated for 6 h with 4 show activation of caspase 2 and 8 (apoptosis activator) as well as caspase 3 (apoptosis executioner). On the other hand, treatment for 6 h with cisplatin showed activation of caspase 8 and 9 (apoptosis activators). As shown here, complex 4 is activating apoptosis faster than cisplatin within 6 h of action on DLD-1 cell line. Furthermore, complex 4 and cisplatin seem to express a different apoptosis pathway on DLD-1 cell line. In the case of cisplatin, apoptosis is triggered by both internal (intrinsic or mitochondrial pathway, caspase 9 dependent pathway) and external signals (extrinsic or death receptor pathway), while for triphenyltin(IV) complex 4 apoptosis is induced via extrinsic receptor pathway on DLD-1 cell line.

Fig. 5 Activity of caspases 2 (C2), 3 (C3), 8 (C8) and 9 (C9) on DLD-1 colon cancer cell line treated for 2 and 6 h with 4 and cisplatin.

3.3.2.3 Cell cycle perturbations. Cell cycle perturbations were analyzed on colon carcinoma DLD-1 cell line. The cells were treated with IC₉₀ concentration of 4 for 24 h (Fig. 6). When compared to control, compound 4 caused almost no changes S phases, however, a decrease at around 46 and 40% in the number of cells in G1 and G2/M phases, respectively, with an increase in the number of apoptotic cells (SubG1-peak), was observed. Those results indicate that apoptosis caused by 4 on colon carcinoma DLD-1 cell line may be due to disturbances caused in both G1 and G2/M phases in the cell cycle.

Fig. 6 Cell cycle analysis of DLD-1 cells untreated (control) and treated with the IC₉₀ concentration of 4 for 24 h.

4. Conclusions and outlook

Novel triethylammonium salts of triphenyltin(IV) chloride carboxylate complexes have been synthesized and structurally characterized. The complexes were tested for in vitro antiproliferative activity against five cell lines of different histogenic origin. The triphenyltin(IV) complexes exhibited much higher cytotoxicity than cisplatin, up to 50 times (4). Although it seems that the active species of these mixtures beside complexes 4–6 may be SnPh₃⁺ cations, based on the stability studies carried out for these compounds in aqueous solutions. In addition, a positive influence in the final cytotoxicity of the tin(IV) complexes has been observed with the incorporation of biocompatible phthaloyl aminoacids as ligands when compared with other different carboxylato ligands.

Complex 4 seems to induce apoptosis via extrinsic or death receptor pathway. The presented data clearly demonstrate a cell-cycle dependence of triphenyltin(IV) compound 4 which induced cell death in DLD-1 cell line. Following compound 4 exposure, cells undergo G1 and G2/M arrest and apoptosis is finally induced in these phases of the cell cycle. Thus, while the phases affected by the tin compounds are G1 (when organelles are being synthesized resulting in a great amount of protein synthesis and a high metabolic rate in the cell) and G2/M (when the protein kinase plays a quite important role), the action mechanism of these compounds seem to be related either to the interaction of SnPh₃⁺ moieties with protein kinases and DNA, as previously reported for other neutral triphenyltin(IV) carboxylates,^26,33 or to the possible binding to the phosphate groups in DNA,^69–71 changing the intracellular metabolism of the phospholipids of the endoplasmic reticulum.^72,73 However, further studies, already in progress in our laboratories, will try to clarify this fact.

Therefore, complex 4 seems to be a very promising candidate for further in vivo tests which will be carried in the near future.

Acknowledgements

We gratefully acknowledge financial support from the Ministerio de Educación y Ciencia, Spain (Grant no. CTQ2008-05892/BQU) the Universidad Rey Juan Carlos and Comunidad de Madrid (postdoctoral fellowship for S.G-R). We would also like to thank Ministerium für Wirtschaft und Arbeit des Landes Sachsen-Anhalt, Deutschland (Grant No. 6003368706) and BioSolutions Halle for cell culture facilities.

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Footnote

† CCDC reference number 759465. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c0mt00007h

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