Tin thiocarbonohydrazone complexes: synthesis, crystal structures and biological evaluation

Three organotin thiocarbonohydrazone complexes have been synthesized, and growth inhibition assays indicated that complex 3 has the highest tumor cell selectivity.

Abstract

In this article, three organotin complexes formulated as [(Me)₂Sn(H₂L¹)] (1), [(Ph)₂Sn(H₂L¹)]·MeOH (2) and [(Me)₂Sn(HL²)(OAc)]₄(Me)₂O (3) (H₄L¹ = bis(2-hydroxybenzaldehyde) thiocarbohydrazone and H₂L² = bis(2-acetylpyrazine) thiocarbonohydrazone) have been synthesized and structurally characterized. Growth inhibition assays indicated that both the proligands and the three complexes are capable of showing anticancer activity against the human hepatocellular carcinoma HepG2 cells with H₂L² and complex 3 showing much higher cytotoxic potential. Subsequent toxicity studies on normal QSG7701cells showed that complex 3 has the highest tumor cell selectivity, and its IC₅₀ value on QSG7701 cells is 8.48 fold higher than that in HepG2 cells. In acute toxicity experiments, complex 3 produces a dose-dependent effect in NIH mice with a LD₅₀ value of 17.2 mg kg^–1.

Introduction

Thiosemicarbazones (TSCs) are well known for their coordination chemistry and for exhibiting a broad range of biological activities, such as antibacterial, antiviral, antifungal, antimalarial and anticancer activities.1–6 The anticancer activity of TSCs has been noticed from the antileukemic activity shown by 2-formyl pyridine thiosemicarbazones in a mouse model, and it was the first discovered representative of TSC compounds with potent anticancer activity.7 A typical representative of TSCs is 3-aminopyridine carboxaldehyde thiosemicarbazone (triapine), which is one of the most extensively studied TSCs for cancer chemotherapy and has been used in a number of clinical trials.8,9 Although its use was later discontinued because of its side-effects and ineffectiveness against specific cancer types,10–14 other promising TSCs like (E)-N′-(6,7-dihydroquinolin-8(5H)-ylidene)-4-(pyridin-2-yl)piperazine-1-carbothiohydrazide (COTI-2) entered clinical trials recently generating great interest in TSC compounds.15,16 TSCs are excellent chelating ligands for biologically-relevant transition metal ions, such as iron(ii/iii), tin(iv) and zinc(ii).17,18 The biological properties of TSCs are related to their coordination with metal ions. In many cases, their efficient in vivo activity is associated with a metal complex rather than the parent ligand.

Organotin compounds can display a variety of applications including all types of biological activities.19 Many organotin compounds have been discovered to exhibit potent anticancer properties.20 About 2000 tin-based compounds have been tested in the National Cancer Institute (NCI), which is the largest number ever tested among metal complexes.21 It has been reported that diorganotin(iv) compounds can exert important cytotoxic effects on tumor cell lines.22 The biological activity of organotin(iv) complexes is highly affected by the stereochemistry of the compound and the coordination number of the tin atom.21 The syntheses of diorganotin complexes with different TSC ligands could be a strategy for the exploration of new compounds with promising biological functions.

It was reported that the biological activities of TSCs are closely related to the parent aldehyde or ketone group, amino-terminal substitution and metal chelation ability.23 We have been engaged in the systematic investigation of tridentate TSCs and their metal complexes with different constituents, performing molecular design-based research to determine their biological activities at ultratrace levels with minimum side effects and low toxicity. 2-Hydroxybenzaldehyde TSC (or salicylaldehyde) and 2-acetylpyrazine TSC have been reported to form complexes with transition metals and show cytotoxic activity in tumor cells.17 Recently, we have paid close attention to the structurally related bis(2-hydroxybenzaldehyde) thiocarbonohydrazone or bis(2-acetylpyrazine) thiocarbono hydrazone and their organotin complexes, aiming to study their biological properties on a structural basis. Some early reported thiocarbonohydrazones can act as bridging ligands to build beautiful molecular architectures, but their biological activities have been relatively less discussed.24,25

Here, we describe the syntheses and characterization of the metal complexes formulated as [(Me)₂Sn(H₄L¹)] (1), [(Ph)₂Sn(H₄L¹)]·MeOH (2) and [(Me)₂Sn(H₂L²)(OAc)]₄·(Me)₂O (3) (H₄L¹ = bis(2-hydroxybenzaldehyde) dithiocarbohydrazone and H₂L² = bis(2-acetylpyrazine) dithiocarbonohydrazone) (Scheme 1). We discuss the structural features of the complexes, as well as their antiproliferative activity in human HepG2 cancer cell lines relative to their toxicity in normal hepatocyte QSG7701 cells. For complex 3, single-dose acute toxicity testing has been conducted.

Scheme 1. Synthetic scheme of complexes 1–3.

Experimental section

All reagents and solvents were used as purchased from commercial suppliers. Ligands H₄L¹ and H₂L² were synthesized according to the literature methods26,27 and confirmed by IR spectroscopy. Hepatocellular carcinoma HepG2 cells and normal hepatocyte QSG7701 cells were purchased from the Shanghai Institute of Biological Science, Chinese Academy of Science (Shanghai, China). Elemental analyses (C, H, N) were performed on a PerkinElmer 240 analyzer. The IR spectra were recorded in the range of 4000–400 cm^–1 on a Nicolet FT-IR 360 spectrometer using KBr pellets.

Synthetic procedures

Synthesis of complex 1

Complex 1 was previously reported in 2006.28 Here, we report another simple preparation method. A methanol solution containing (Me)₂SnCl₂ (0.044 g, 0.2 mmol) was added dropwise to a methanol solution (20 mL) of bis(2-hydroxybenzaldehyde) thiocarbonohydrazone (0.063 g, 0.2 mmol) and NaOAc (0.016 g, 0.2 mmol). After refluxing for 1 h with stirring, the resultant mixture was cooled to room temperature and then filtered. Yellow rod crystals suitable for X-ray studies were obtained by slow evaporation of a methanol solution of 1. Yield: 81%. Elemental Anal. Calcd (%) for C₁₇H₁₈N₄O₂SSn (1): C, 44.28; H, 3.94; N, 12.15. Found: C, 44.20; H, 3.78; N, 11.61. IR [KBr, ν (cm^–1)]: 3151 (NH), 1603 (C N), 1152 (N–N), 754 (C S). ESI-MS (m/z): 484.3 = [Na(Me)₂Sn(H₂L¹)]⁺ calc. mass = 484.1. ¹H NMR (300 MHz, DMSO-d₆ PPm): δ = 11.77(s, 1H, NH), 11.18(s, 1H, oH), 8.62(s, 1H, HC N), 8.25(s, 1H, HC N), 7.30(m, 4H, ph), 6.92(m, 2H, ph), 6.66(t, 2H, ph, J = 8.0 Hz), 0.90(s, 6H, CH₃).

Synthesis of complex 2

Complex 2 was prepared by a similar procedure to that described for 1, using (Ph)₂SnCl₂ (0.069 g, 0.2 mmol) instead of (Me)₂SnCl₂. Yield: 83%. Elemental Anal. Calcd (%) for C₂₈H₂₆N₄O₃SSn (2): C, 54.48; H, 4.25; N, 9.08. Found: C, 54.36; H, 4.31; N, 9.15. IR [KBr, ν (cm^–1)]: 3246 (NH), 1603 (C N), 1151 (N–N), 752 (C S). ESI-MS (m/z): 586.5 = [H(Ph)₂Sn(H₂L¹)]⁺ calc. mass = 586.3. ¹H NMR (300 MHz, DMSO-d₆ PPm): δ = 11.75(s, 1H, NH), 11.09(s, 1H, oH), 8.56(s, 1H, HC N), 8.29(s, 1H, HC N), 7.63(s, 10H, ph), 7.31(m, 4H, ph), 6.88(m, 2H, ph), 6.71(t, 2H, ph, J = 7.5 Hz).

Synthesis of complex 3

Complex 3 was synthesized by a similar procedure to that described for 1 by the reaction of (Me)₂SnCl₂ (0.044 g, 0.2 mmol) with the ligand of bis(2-acetylpyrazine) thiocarbonohydrazone (0.063 g, 0.2 mmol). Yield: 81%. Elemental Anal. Calcd (%) for C_17.5H₂₂N₈O_2.5SSn (3): C, 39.27; H, 4.14; N, 20.94. Found: C, 39.48; H, 4.35; N, 20.85. IR [KBr, ν (cm^–1)]: 3325 (NH), 1563 (C N), 1151 (N–N), 753 (C S). ESI-MS (m/z): 464.5 = [(Me)₂Sn(HL²)]⁺ calc. mass = 464.6. ¹H NMR (400 MHz, DMSO-d₆ PPm): δ = 9.35(d, 2H, Pz), 9.1(s, 1H, NH), 8.82(m, 2H, Pz), 8.67(d, 2H, Pz), 3.15(s, 3H, CH₃), 2.77(s, 3H, CH₃), 2.37(s, 3H, CH₃COO), 1.94(s, 6H, CH₃).

X-ray crystallographic study

Single crystals of compounds 1, 2 and 3 were sealed in a transparent glass tube for testing. Intensity data were collected at 296(2) K on a Bruker APEXII CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). Routine Lorentz and polarization corrections were performed. The structures were solved by direct methods, developed by successive difference Fourier syntheses, and refined by full-matrix least squares on F² using the SHELX program suite.29 Crystallographic and experimental details of crystal structure determination are given in Table 1, and the CCDC reference numbers are ; 1949907, ; 967523 and ; 1904269 for 1, 2 and 3, respectively.

Table 1. Crystallographic data for 1–3.

Complexes	1	2	3
Empirical formula	C17H18N4O2SSn	C28H26N4O3SSn	C17.5H22N8O2.5SSn
Formula weight	461.10	617.29	535.18
Crystal system	Monoclinic	Orthorhombic	Monoclinic
Space group	P21/c	P212121	C2/c
a (Å)	14.2062(6)	9.3571(4)	41.6903(15)
b (Å)	10.5143(4)	17.0983(8)	7.0301(2)
c (Å)	26.3152(11)	34.3432(16)	16.5254(6)
α/°	90.00	90.00	90.00
β/°	100.8310(10)	90.00	111.8870(10)
γ/°	90.00	90.00	90.00
V (Å3)	3860.6(3)	5494.6(4)	4494.3(3)
Z	8	8	8
D c (g cm–3)	1.587	1.492	1.582
μ (mm–1)	1.449	1.042	1.263
θ (°)	1.459–25.099	1.330∓25.094	2.106∓56.556
F (000)	1840	2496	2152
Index ranges	–16 ≤ h ≤ 16, –12 ≤ k ≤ 11, –26 ≤ l ≤ 31	–11 ≤ h ≤ 10, –19 ≤ k ≤ 20, –38 ≤ l ≤ 40	–52 ≤ h ≤ 53, –9 ≤ k ≤ 9,–19 ≤ l ≤ 22
Refl. collected/unique/Rint	19 646/6864/0.0492	28 871/9744/0.0627	13 775/5448/0.0199
R 1, wR2 [I ≥ 2σ(I)]	0.0275, 0.0692	0.0436, 0.0685	0.0252, 0.0592
R 1, wR2 (all data)	0.0356, 0.0735	0.0832, 0.0824	0.0331, 0.0654
GOF on F2	1.031	1.010	1.034

Cell culture

The HepG2 cells and QSG7701 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 IU mL^–1 penicillin and 100 μg mL^–1 streptomycin at 37 °C under a humidified atmosphere with 5% CO₂.27 The cells were harvested with 0.02% EDTA and 0.025% trypsin and then rinsed three times in phosphate-buffered saline (PBS). The resulting cell suspension was used in the following experiment.

Cytotoxicity assay

A colorimetric microculture assay (MTT assay) was carried out to evaluate cytotoxicity.27 Cells were plated into 96-well plates at a cell density of 1 × 10⁴ cells per well and allowed to grow in a CO₂ incubator. After 24 h, the medium was removed and replaced by fresh medium containing the tested compounds which were dissolved in DMSO at 0.01 M and diluted to various concentrations before the experiment, and the final concentration of DMSO is lower than 1%. After exposure for 24 h, the cells were incubated in 100 μL of MTT in media (0.5 mg ml^–1) for 4 h at 37 °C. Subsequently, the medium with MTT was removed, and 100 μL of DMSO was added to each well to dissolve the purple formazan crystals formed in viable cells. The absorbance at 570 nm was measured with a microplate reader (Bio-Tek ELX800, USA). The inhibitory percentage of each compound at various concentrations was calculated, and the 50% inhibitory concentrations (IC₅₀) were determined.

Single-dose acute toxicity testing

This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the Institutional Animal Care and Use Committee at Henan University (Kaifeng, China). A total of 80 male NIH mice (Laboratorial Animal Center of Henan, Zhengzhou, China), aged 5 weeks (weighing 18–22 g), were randomly divided into eight groups.30 The next day, 3 was injected into each mouse via enterocoelia. The doses were 30, 25.5, 21.7, 18.4, 15.7, 13.3, 11.3 and 9.6 mg kg^–1 for each group, respectively. The observation lasted for 14 days and the death rates were recorded, and then the LD₅₀ value, which is the dose corresponding to 50% animal death rate, was calculated.

Results and discussion

X-ray crystallography

Tin(iv) complexes 1, 2 and 3 were all studied by single crystal X-ray diffraction and their structures are depicted in Fig. 1. In complex 1 the coordination of tin(iv) was provided by the ONS-donor set of the Schiff base ligand and two methyl groups. The geometry of the five-coordinated tin(iv) atom can be described as distorted trigonal bipyramidal (with a τ value of 0.57). In complex 2 the five-coordinated tin(iv) atom adopts a distorted square pyramidal geometry (τ value 0.28) with the square plane defined by the tridentate ONS thiocarbonohydrazone and a benzene ring (containing C22), whereas the axial position is occupied by the Sn1–C16 bond. In complex 3 the seven-coordinated tin(iv) atom adopts a distorted pentagonal bipyramidal geometry, and the pentagonal plane is defined by the NNS thiocarbonohydrazone and the bidentate acetate group, whereas the apical positions are occupied by two trans-positioned methyl groups. The distortion from the ideal pentagonal bipyramidal geometry is evident and the axial C14–Sn1–C15 angle 157.686(109)° is far from linearity. The deviations may be related to the short bite angle of the acetate group which results in an O(2)–Sn(1)–O(1) angle of 52.084(69)° instead of the theoretical value of 72.

Fig. 1. Molecular structures of complexes 1 (a), 2 (b), and 3 (c) (the solvate molecules are omitted for clarity).

In vitro cytotoxicity

As shown in Fig. 2, complexes 1–3 exhibited significant antiproliferative activity against HepG2 cells, with complex 3 showing the highest cytotoxic potential (IC₅₀ = 3.83 ± 0.37 μM). In contrast to their ligands, the IC₅₀ value of complexes 1–2 are much lower than that of H₄L¹ (IC₅₀ = 54.36 ± 2.42 μM), while the ligand H₂L² exhibited a lower IC₅₀ value than complex 3. Complex 2 shows higher antiproliferative activity than complex 1, which may indicate that the phenyl diorganotin complex of the same ligand shows enhanced inhibitory activity than that of their corresponding methyl diorganotin derivatives.31 Complex 3 and its proligand show much higher inhibitory activity than complexes 1–2, which indicates that the NNS thiocarbonohydrazone ligand may show preferable biological activity than the ONS thiocarbonohydrazone system. Mitoxantrone (Mito) is a kind of antibiotic antitumor drug, which can be used as the reference compound for comparison. Among all these tested compounds, Mito displayed the lowest IC₅₀ value of 1.46. However, subsequent toxicity studies show that Mito and H₂L² exhibit very low IC₅₀ values against normal QSG7701 cells indicating their potent toxicity. H₄L¹ and complexes 1–3 all show much higher IC₅₀ values against normal QSG7701 cells. In comparison, complex 3 would be a potent anticancer drug candidate with a lower IC₅₀ value against HepG2 cells and a higher IC₅₀ value for normal QSG7701 cells (8.48 fold higher than in HepG2 cells).

Fig. 2. The cytotoxicity of the tested compounds against HepG2 cells and QSG7701 cells, compared with mitoxantrone. All the data are expressed as the mean ± standard deviation (SD) from three separate determinations.

Acute toxicity

In cytotoxicity experiments complex 3 exhibited evident antiproliferative activity against HepG2 cells with lower toxicity on normal QSG7701 cells. Its further biological evaluation in vivo has been conducted. Acute toxicity studies in animals are necessary for any pharmaceutical intended for human use. In this experiment, complex 3 produces dose-dependent effects in NIH mice (Fig. 3a), and the LD₅₀ value is 17.2 mg kg^–1 in NIH mice by intraperitoneal injection (95% confidence interval 15.0–19.8 mg kg^–1; Fig. 3b).

Fig. 3. (a) Dose-dependent effects of 3 on the NIH mice. (b) LD₅₀ value of 17.2 mg kg^–1 (95% confidence interval 15.0–19.8 mg kg^–1).

Conclusions

In summary, the organotin complexes bis(2-hydroxybenzaldehyde) thiocarbohydrazone and bis(2-acetylpyrazine) thiocarbonohydrazone have been synthesized and characterized. The in vitro cytotoxicity experiment on the proligands and the three complexes shows that the organotin complex bis(2-acetylpyrazine) thiocarbonohydrazone has the highest tumor cell selectivity. In acute toxicity experiments, complex 3 produces a dose-dependent effect in NIH mice with a LD₅₀ value of 17.2 mg kg^–1.

Conflicts of interest

There are no conflicts of interest to declare.