Dimeric diorganotin(IV) complexes with arylhydrazones of β-diketones: synthesis, structures, cytotoxicity and apoptosis properties

Abstract

Ten new dimeric diorganotin(IV) complexes of the type [R₂SnL]₂ (R = Me, Et, Bu, Ph or Oct, and H₂L = arylhydrazones of β-diketone) have been obtained by the reaction of the corresponding arylhydrazone of β-diketone with diorganotin(IV) dichloride under alkaline conditions. All the complexes were characterized by elemental analysis, IR and NMR (¹H, ¹³C, ¹¹⁹Sn) spectroscopies. Complexes 4 and 6 were also characterized by X-ray crystallography diffraction analysis, which revealed that the dimeric complexes have similar structures containing diorganotin(IV) skeletons formed by two weak but significant intermolecular Sn–O bonds. They were screened against the human tumor cell lines Hela, KB and HepG2. Complex 6 exhibited the highest in vitro cytotoxicity. The apoptosis induced by complexes 5, 6 and 9 was quantified using a flow cytometric assay. All the three organotin(IV) compounds induced apoptosis much more effectively than cisplatin, and the order of apoptosis induction is 6 > 5 > 9 > cisplatin. The results indicate that the apoptosis induction of the compounds correlates with the cytotoxicity. Moreover, quantification data of apoptosis in KB cells suggests that the mechanism of cell death might occur mainly by means of early apoptosis at more than 0.25 μM concentration of the complexes, which is beneficial for an optimal antitumor agent.

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1. Introduction

Arylhydrazone derivatives of β-diketones (ADDs) constitute a very important class of chelating agents with versatile biological activity.^1–7 The research on their coordination properties is mostly oriented towards modeling a biological function, namely as potential antitumor,⁸ analgesic,^1,2 antipyretic,² antibacterial^3–6 and antifungal⁷ drugs, although they also have been used as catalysts for many metal ions in catalytic chemistry.^9,10

Organotin(IV) complexes have been widely investigated because of their structural diversity and antitumor activity.^11–15 Different ligands have been reported to lead to a variety of organotin(IV) complexes, including monomeric and polymeric complexes with different coordination modes.^11–21 It was demonstrated that ADDs can form complexes with various metals which possess interesting structural and analytical properties.^21–25 However, the available data on organotin(IV) complexes of this type of ligands are still very limited. As far as we know, only one organotin(IV) complexes with ADDs have been reported, together with its in vitro antitumor activity.⁸

A number of organotin(IV) complexes show high in vitro and in vivo antitumour activity.^11,26 Although the mechanism of action of antitumour organotin compounds is not fully understood, it is thought that apoptosis plays an important role on mediating the in vitro antitumour activity of those compounds.^14,15,26,27

In order to further explore the antitumor properties of such a class of complexes, in this work we report the synthesis of ten new diorganotin(IV) complexes with substituted arylhydrazone derivatives of β-diketones, the structural characterization and the screening of the cytotoxicity and apoptosis properties.

2. Results and discussion

2.1. Synthesis and characterization of diorganotin(IV) complexes

The dimeric tin(IV) complexes 1–10 were prepared by reaction of the ligand with the corresponding diorganotin(IV) dichloride, in stoichiometric amounts. Sodium methoxide was used as a base for the deprotonation of the ligand as described in the experimental section (Scheme 1).

Scheme 1 Synthesis of diorganotin(IV) complexes with arylhydrazones of β-diketones.

Complexes 1–10 are solids, with different intense colors, and are soluble in polar organic solvents and stable under atmospheric conditions. The solids were recrystallized from dichloromethane/n-hexane and single crystals were formed by slow evaporation at room temperature for 4 and 6.

2.2. Spectral studies

In the IR spectra of the ligands, the stretching vibration bands of N–H and O–H appear at 3308–3473 and 3079–3099 cm⁻¹, respectively, and these bands disappear in the spectra of complexes 1–10. The disappearance of both ν_N–H and ν_O–H bands shows the deprotonation of the –NH and –OH groups^10,28 and their coordination to the central tin atom. The ν_C=O band of the ligands at 1631–1639 cm⁻¹ shifts to 1632–1705 cm⁻¹ in the spectra of the complexes 1–10, suggesting coordination of the carbonyl oxygen to the tin moiety.⁸ Two new bands at 543–590 and 438–508 cm⁻¹ are characteristic of Sn–O and Sn–N absorptions, respectively.^29–32 Strong bands in the 620–638 cm⁻¹ region are assigned to Sn–O–Sn bond vibrations.^33,34

In the ¹H NMR spectra of the free ligands (H₂L¹, H₂L² and H₂L³), the single resonance of the proton (–NHN=) is observed at 14.59, 14.17 and 14.30 ppm, respectively, and it is absent in the spectra of the complexes, thus indicating deprotonation of the –NHN= group and accounting for the N-ligand coordination to the tin. In the ¹H NMR spectra of complexes 1–10 there is no signal resonance for Ar–OH, which strongly suggests that the phenolic oxygen atoms participate in the coordination to the tin atom.

The ¹¹⁹Sn NMR spectra of complexes 1–8 and 10 show only one signal in the −155.4 to −349.4 ppm region, while that of 9 displays two signals (δ −115.6 and −338.8), probably due to the presence of isomers in solution.¹³ According to the literature,^20,35 the coordination number of the tin atoms for the ten organotin(IV) complexes should be five or six. This is in agreement with X-ray crystallography results of complexes 4 and 6 (see below) which disclose such a type of coordination with a weak bridging interaction.

2.3. X-ray diffraction analysis

The molecular structures of the complexes 4 and 6 were authenticated by single crystal X-ray diffraction analyses. The most relevant parameters of bond distances and angles are given in the legends of Fig. 1 and 2.

Fig. 1 Ellipsoid plot (30% probability level) of 4 with atom labelling scheme. Selected bond lengths (Å) and angles (°): Sn1–O1 2.0968(16), Sn1–O2 2.2815(19), Sn1–N1 2.2141(19), Sn1–O1a 2.610(2), Sn1–C12 2.110(3), Sn1–C14 2.144(13), N1–N2 1.315(3), N3–O4 1.213(3), N3–O5 1.226(4); C12–Sn1–C14 136.1(5), O1–Sn1–O2 151.49(7), O1–Sn1–N1 75.67(6), N1–Sn1–O2 76.08(7).

Fig. 2 Ellipsoid plot (30% probability level) of 6 with atom labelling scheme. Hydrogen atoms are omitted for clarity. Selected bond lengths (Å) and angles (°): Sn(1)–O(1) 2.1031(18), Sn(1)–O(1a) 2.669(2), Sn(1)–N(2) 2.201(2), Sn(1)–O(5) 2.275(2), Sn(1)–C(1A) 2.074(17), Sn(1)–C(1B) 2.114(3), C(1)–O(1) 1.338(3), N(2)–N(3) 1.309(3), N(1)–O(2) 1.214(4), N(1)–O(3) 1.217(3); C(1A)–Sn(1)–O(1) 102.0(5), C(1A)–Sn(1)–C(1B) 141.5(4), O(1)–Sn(1)–C(1B) 101.89(10), C(1A)–Sn(1)–N(2) 107.6(4), O(1)–Sn(1)–N(2) 76.11(7), O(5)–Sn(1)–N(2) 75.79(8), C(1B)–Sn(1)–N(2) 107.22(10), O(1)–Sn(1)–O(5) 151.86(7), C(1B)–Sn(1)–O(5) 87.70(10).

Complexes [Et₂Sn(L²)]₂ (4) and [Bu₂Sn(L²)]₂ (6) crystallize as binuclear species, each metal presenting a trigonal prismatic geometry (Scheme 1, Fig. 1 and 2). The β-diketone fragments act as tridentate units by means of the deprotonated aromatic OH group in ortho position, which bridge the tin(IV) atoms, one of the nitrogen atoms and one of the carbonyl oxygen atom. Thus, the tin atoms belong to three different metallacycles: the Sn₂O₂ core, which is the central planar ring of the molecule, and two fused six- and five-membered exo metallacycle rings, C₂N₂OSn and C₂NOSn, respectively.

The Sn–C bond lengths fall in a narrow range from 2.074(17) to 2.144(13) Å, typical for organotin(IV) derivatives. The C–Sn–C angles assume values of 136.1(5)° (4) and 141.5(4) (6). The Sn–O_carbonyl bond lengths of 2.2815(19) Å (4) and 2.275(2) Å (6) as well as the Sn–O_phenolate distances of 2.0968(16) Å (4) and 2.1031(18) Å (6), are in excellent agreement with those found in diorganotin(IV) complexes with hydrazone type ligands.^36–38 The Sn–N distances [2.2141(19) Å for 4, 2.201(2) Å for 6] are consistent with strong tin–nitrogen interactions, and they lie in the range 2.140(6)–2.327(2) Å already reported for this parameter.^39–41 These Sn–N bonds are unusual for organotin(IV) derivatives with ONO terdentate ligands.^39–42

2.4. In vitro antitumor activity assays

To analyze the cytotoxicity of the complexes (1–10) we incubated Hela, KB and HepG2 cells with a varying concentration of each complex, separately for 48 h and quantified the viable cells using the MTT assay as described by us previously.^14,15 The results demonstrated that most of the diorganotin(IV)–arylhydrazones complexes affected cell viability (Table 1) and induced cell apoptosis in KB cells in a concentration dependent manner (Fig. 3 and Table 2).

Table 1 The in vitro antitumor activity of the diorganotin(IV) complexes (1–10) against three human cancer cell lines (Hela, KB and HepG2) (n = 3)

Complex	R	R^1	R^2	IC50 (μM)
				Hela	KB	HepG2
1	Me	5-H	H	93.7	25.3	117.8
2	Me	5-NO2	H	29.9	23.9	41.4
3	Et	5-H	H	19.7	6.1	14.2
4	Et	5-NO2	H	8.2	2.6	5.3
5	Bu	5-H	H	2.1	0.3	4.4
6	Bu	5-NO2	H	0.3	0.2	1.7
7	Bu	5-H	OCH2CH3	0.5	0.4	6.3
8	Ph	5-H	H	1.6	1.2	4.2
9	Ph	5-NO2	H	1.3	0.9	2.9
10	Oct	5-NO2	H	8.4	5.5	59.9
cis-Platin				4.5 (ref. 43)	2.65 (ref. 44)	8.3 (ref. 43)

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Fig. 3 Apoptosis detection KB cells using the Annexin V assay after 24 h. The total percentage of apoptotic cells was considered as Q2 + Q4. Plot presents the fluorescence data of propidium iodide (PI) and Annexin V fluorescence in corresponding to (a) 16.0% (control), (b) 27.1% (0.10 μM, 6), (c) 41.6% (0.25 μM, 6), (d) 67.9% (1.0 μM, 6), (e) 84.7% (2.5 μM, 6), (f) 89.4% (10 μM, 6), (g) 64.6% (1.0 μM, 5), (h) 83.7% (10 μM, 5), (i) 21.9% (1.0 μM, 9), (j) 75.8% (10 μM, 9).

Table 2 Percentages of apoptosis of compounds (5, 6 and 9) against KB cells in different concentrations with cisplatin as positive control

Compd.	Conc. (μM)	Q2 (late apoptosis and necrotic cell, %)	Q4 (early apoptosis, %)	Q2+Q4 (total percentage)
control		9.3	6.7	16.0
5	1	12.8	51.8	64.6
	10	27.7	56.0	83.7
6	0.10	16.1	11.0	27.1
	0.25	21.1	20.5	41.6
	1.0	22.2	45.7	67.9
	2.5	17.5	67.2	84.7
	10	15.8	73.6	89.4
9	1	7.1	14.8	21.9
	10	2.8	73.0	75.8
cis-Platin^14	2.5	7.4	8.6	16.0
	10	10.2	8.0	18.2

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The IC₅₀ values for the active compounds against the three tumor cell lines lie between 0.2 and 8.4 μM except for 1, 2, 3 and 10 (Table 1). Complexes 5 and 9 are more active than cisplatin, the clinically widely used drug. Notably, [Me₂SnL¹]₂ (1) did not have any significant effect on cell viability. The following structure–activity relationships could be recognized: 1) with regard to the R group: the activity decreases in the order ⁿBu > Ph > Et > Oct > Me; 2) with regard to the R¹ substituent of the arylhydrazone aromatic ring: the dialkyltin(IV) derivatives with the nitro substituent are more active than the unsubstituted ones (2 > 1, 4 > 3, 6 > 5); hence, the antitumor activity can be determined by both lipophilicity and electronic effects of the ligands; the best combination is provided by the dibutyltin(IV) (R = ⁿBu) complex with the nitro-substituted (R¹ = NO₂) arylhydrazone ligand, namely [ⁿBu₂Sn(L²)]₂ (6); 3) with regard to the R² group: the dibutyltin(IV) derivative (7) with the ethyoxyl group showed a better activity on Hela tumor cells than complex 5 without it, but for KB and HepG2 cell, complex 7 showed a comparable or weaker activity than 5.

2.5. Cell apoptosis analysis by flow cytometry

Double staining with PI and annexin V-FITC can be used to distinguish between apoptotic and necrotic cell death. We selected three complexes (5, 6 and 9) as representatives to test by the flow cytometry assay if the mechanism of cell death is by means of early apoptosis, late apoptosis or necrosis.

On the basis of the antiproliferative study, all these diorganotin(IV) complexes are more sensitive to KB cells. KB cells were then selected for apoptosis detection using the Annexin V/PI binding assay after 24 h (Table 2 and Fig. 3). Annexin-V conjugated with the fluorochrome FITC serves as a marker for apoptotic cells because it has a strong binding affinity to phosphatidylserine (PS), which re-distributes from the inner to the outer layer of the plasma membrane in apoptotic cells. As shown in Fig. 3, the dual parametric dot plots show four quadrants and among them the lower left Q3 quadrant represents the viable cell population (Annexin-V negative and PI negative), the upper right Q2 represents apoptotic cells undergoing secondary necrosis at the last stage or dead cells (Annexin-V and PI double positive), and the lower right Q4 represents the early stage apoptotic cell population (Annexin-V positive and PI negative).

The apoptosis induced by the three dimeric organotin(IV) complexes was quantified using a flow cytometric assay. After 24 h of treatment of tin(IV) complexes, cells were double stained with annexinV-FITC and propidium iodide (PI). The results show that all the three organotin(IV) compounds induced apoptosis much more effectively than control or cisplatin (Table 2), and the order of apoptosis induction is 6 > 5 > 9 > cisplatin. The results indicate that the apoptosis induction of the compounds correlates with the cytotoxicity.

As the concentration of complex 6 increased from 0.10 to 10 μM, the Q4 cells increased from 11.0% to 73.6%, whereas the Q2 cells increased from 16.1% to 22.2%, and then decreased back to 15.8%, suggesting that more and more apoptotic cells progressed from the early stage to the late stage resulting in either death or secondary necrosis under the effect of complex 6 at concentrations of more than 0.25 μM. This confirms that complex 6 induced the cell apoptosis of KB cells.

From the data of the apoptosis detection of 5 and 9, we also can see that the percentages of early apoptosis (Q4) are bigger than that of late apoptosis and necrotic cell (Q2). Moreover, with the increasing concentrations of compounds, there have been marked increases in the ratio of early apoptosis. This result suggests that the mechanism of cell death might mainly by means of early apoptosis at more than 0.25 μM concentration of the complex, which is beneficial for an optimal antitumor agent.

3. Experimental

3.1. Materials and physical measurements

Me₂SnCl₂, Et₂SnCl₂, ⁿBu₂SnCl₂, Ph₂SnCl₂ and Oct₂SnCl₂ were purchased from Aldrich and used as received. All the other reagents used in the reactions were of analytical grade (Sinopharm Chemical Reagent Co., Ltd., China). The ligands (3-(2-hydroxyphenylhydrazo)pentane-2,4-dione (H₂L¹), 3-(2-hydroxy-5-nitrophenylhydrazo)pentane-2,4-dione (H₂L²) and 1-ethyoxyl-3-(2-hydroxyphenylhydrazo)pentane-2,4-dione (H₂L³)) were prepared according to the previous methods.⁴⁵ Elemental analyses were performed on a PE-2400-II elemental analyzer. IR spectra in the range 4000–400 cm⁻¹ were recorded on a Perkin Elmer FT-IR spectrophotometer with samples investigated as KBr discs. ¹H, ¹³C, ¹¹⁹Sn NMR spectra were recorded on a Bruker AM-400 spectrometer (400.0 MHz for ¹H, 100.0 MHz for ¹³C) and a Varian INOVA 600 spectrometer (223.6 MHz for ¹¹⁹Sn) at ambient temperature [δ values in ppm relative to Me₄Si (¹H, ¹³C) or Me₄Sn (¹¹⁹Sn)].

3.2. Synthesis of the dibutyltin(IV) complexes [R₂SnL]₂

3.2.1. Synthesis of [Me₂Sn(L¹)]₂ (1). The ligand HL¹ (1.0 mmol) was added to a methanol (20 ml) solution of sodium methoxide (1.0 mmol), and the mixture was stirred for 0.5 h, then Me₂SnCl₂ (1.0 mmol) was then added and the system stirred for 12 h at room temperature. The solution was filtered and evaporated to dryness, the solid was then recrystallized from dichloromethane–n-hexane. Red brown flaky crystals were obtained. Yield: 53.4%. Anal. Calc. for C₁₃H₁₆N₂O₃Sn (366.99): C 42.55, H 4.39, 7.63; found: C 42.15, H 4.33, N 7.15. IR (KBr): v = 1660 s, 1634w (C=O/C=N), 625 (Sn–O–Sn), 568m (Sn–O), 508w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 0.87 (s, 6H, Sn–CH₃), 2.52 (s, 3H, H-7), 2.57 (s, 3H, H-11), 6.74–6.81 (m, 2H, H-3, H-6), 7.16 (dt, 1H, H-5, ¹J = 7.5 Hz, ²J = 1.0 Hz), 7.59 (dd, 1H, H-4, ¹J = 8.0 Hz, ²J = 1.3 Hz). ¹³C NMR (CDCl₃): δ = 3.2 (Sn–C̲H₃), 28.0 (C-7), 30.1 (C-11), 116.1 (C-6), 118.2 (C-3), 118.4 (C-5), 130.0 (C-4), 133.2 (C-2), 137.8 (C-9), 157.1 (C-1), 191.3 (C-8), 198.1 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −177.2.

3.2.2. Synthesis of [Me₂Sn(L²)]₂ (2). Compound 2 was prepared analogously by following the method and conditions described for 1 but using HL² (1.0 mmol) and Me₂SnCl₂ (1.0 mmol). Red brown flaky crystals were obtained. Yield 51.0%. Anal. Calc. for C₁₃H₁₅N₃O₅Sn (411.99): C 37.90, H 3.67, N 10.20. Found: C 37.84, H 3.69, N 10.06. IR (KBr): v = 1670 s (C=O/C=N), 621 (Sn–O–Sn), 565w (Sn–O), 498w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 0.92 (s, 6H, Sn–CH₃), 2.53 (s, 3H, H-7), 2.64 (s, 3H, H-11), 7.62 (br, 3H, H-3, H-4, H-6). ¹³C NMR (CDCl₃): δ = 2.9 (Sn–C̲H₃), 27.8 (C-7), 30.7 (C-11), 113.4 (C-6), 114.0 (C-3), 115.7 (C-5), 134.4 (C-9), 142.9 (C-4), 147.8 (C-2), 156.9 (C-1), 191.3 (C-8), 198.1 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −155.4.

3.2.3. Synthesis of [Et₂Sn(L¹)]₂ (3). Compound 3 was prepared analogously by following the method and conditions described for 1 but using HL¹ (1.0 mmol) and Et₂SnCl₂ (1.0 mmol). Red brown flaky crystals were obtained. Yield 58.2%. Anal. Calc. for C₁₅H₂₀N₂O₃Sn (395.04): C 45.61, H 5.10, N 7.09. Found: C 45.62, H 5.13, N 7.07. IR (KBr): v = 1667 s, 1634w (C=O/C=N), 625 (Sn–O–Sn), 566w (Sn–O), 503w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 1.26 (t, 6H, Sn–CH₂CH̲₃, J = 8.0 Hz), 1.56 (m, 4H, Sn–CH̲₂CH₃), 2.52 (s, 3H, H-7), 2.60 (s, 3H, H-11), 6.73 (dt, 1H, H-3, ¹J = 8.0 Hz, ²J = 1.3 Hz), 6.82 (dd, 1H, H-5, ¹J = 8.0 Hz, ²J = 1.0 Hz), 7.15 (dt, 1H, H-6, ¹J = 8.0 Hz, ¹J = 1.5 Hz), 7.58 (dd, 1H, H-4, ¹J = 8.0 Hz, ¹J = 1.5 Hz). ¹³C NMR (CDCl₃): δ = 9.4 (Sn–CH₂C̲H₃), 16.2 (Sn–C̲H₂CH₃), 28.0 (C-7), 30.1 (C-11), 116.1 (C-6), 118.0 (C-3), 118.3 (C-5), 129.7 (C-4), 133.2 (C-9), 138.4 (C-2), 157.9 (C-1), 191.7 (C-8), 198.2 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −212.2.

3.2.4. Synthesis of [Et₂Sn(L²)]₂ (4). Compound 4 was prepared analogously by following the method and conditions described for 1 but using HL² (1.0 mmol) and Et₂SnCl₂ (1.0 mmol). Red brown flaky crystals were obtained. Yield 75.0%. Anal. Calc. for C₁₅H₁₉N₃O₅Sn (440.04): C 40.94, H 4.35, N 9.55. Found: C 41.04, H 4.38, N 9.62. IR (KBr): v = 1673 s, 1638w (C=O/C=N), 620 (Sn–O–Sn), 566w (Sn–O), 494w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 1.26 (t, 6H, Sn–CH₂CH̲₃, J = 7.5 Hz), 1.60 (m, 4H, Sn–CH̲₂CH₃), 2.53 (s, 3H, H-7), 2.66 (s, 3H, H-11), 7.60–7.62 (m, 3H, H-3, H-4 and H-6). ¹³C NMR (CDCl₃): δ = 9.2 (Sn–CH₂C̲H₃), 16.1 (Sn–C̲H₂CH₃), 27.8 (C-7), 30.7 (C-11), 113.2 (C-6), 113.3 (C-3), 115.7 (C-5), 134.4 (C-9), 143.5 (C-4), 147.7 (C-2), 157.5 (C-1), 195.1 (C-8), 197.6 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −193.6.

3.2.5. Synthesis of [Bu₂Sn(L¹)]₂ (5). Compound 5 was prepared analogously by following the method and conditions described for 1 but using HL¹ (1.0 mmol) and Bu₂SnCl₂ (1.0 mmol). Red brown bulk crystals were obtained. Yield 54.9%. Anal. Calc. for C₁₉H₂₃N₂O₃Sn (451.15): C 50.58, H 6.26, N 6.21. Found: C 50.68, H 6.22, N 6.12. IR (KBr): v = 1668 s (C=O/C=N), 625 (Sn–O–Sn), 560w (Sn–O), 503w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 0.88 (t, 6H, Sn–CH₂CH₂CH₂CH̲₃, J = 7.5), 1.31–1.36 (m, 4H, Sn–CH₂CH₂CH̲₂CH₃), 1.50–1.63 (m, 8H, Sn–CH̲₂CH̲₂CH₂CH₃), 2.52 (s, 3H, H-7), 2.58 (s, 3H, H-11), 6.72 (t, 1H, H-3, J = 7.3 Hz), 6.83 (d, 1H, H-5, J = 8.5 Hz), 7.15 (t, 1H, H-6, J = 8.5 Hz), 7.57 (d, 1H, H-4, J = 7.3 Hz). ¹³C NMR (CDCl₃): δ = 13.6 (Sn–CH₂CH₂CH₂C̲H₃), 23.0 (Sn–CH₂CH₂C̲H₂CH₃), 26.5 (Sn–CH₂C̲H₂CH₂CH₃), 26.7 (Sn–C̲H₂CH₂CH₂CH₃), 27.8 (C-7), 30.6 (C-11), 116.0 (C-6), 117.8 (C-3), 118.8 (C-5), 130.0 (C-4), 133.1 (C-9), 138.0 (C-2), 158.4 (C-1), 191.5 (C-8), 198.1 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −203.8.

3.2.6. Synthesis of [Bu₂Sn(L²)]₂ (6). Compound 6 was prepared analogously by following the method and conditions described for 1 but using HL² (1.0 mmol) and Bu₂SnCl₂ (1.0 mmol). Red brown bulk crystals were obtained. Yield 56.1%. Anal. Calc. for C₁₉H₂₇N₃O₅Sn (496.14): C 46.00, H 5.49, N 8.47. Found: C 45.98, H 5.45, N 8.39. IR (KBr): v = 1672 s, 1639w (C=O/C=N), 622 (Sn–O–Sn), 561w (Sn–O), 498w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 0.88 (t, 6H, Sn–CH₂CH₂CH₂CH̲₃, J = 7.5), 1.30–1.39 (m, 4H, Sn–CH₂CH₂CH̲₂CH₃), 1.50–1.67 (m, 8H, Sn–CH̲₂CH̲₂CH₂CH₃), 2.53 (s, 3H, H-7), 2.64 (s, 3H, H-11), 7.60–7.63 (m, 3H, H-3, H-4 and H-6). ¹³C NMR (CDCl₃): δ = 13.6 (Sn–CH₂CH₂CH₂C̲H₃), 23.6 (Sn–CH₂CH₂C̲H₂CH₃), 26.5 (Sn–CH₂C̲H₂CH₂CH₃), 26.7 (Sn–C̲H₂CH₂CH₂CH₃), 27.8 (C-7), 30.6 (C-11), 113.1 (C-6), 113.6 (C-3), 115.7 (C-5), 134.3 (C-9), 143.2 (C-4), 147.9 (C-2), 157.7 (C-1), 194.9 (C-8), 197.6 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −189.3.

3.2.7. Synthesis of [Bu₂Sn(L³)]₂ (7). Compound 7 was prepared analogously by following the method and conditions described for 1 but using HL³ (1.0 mmol) and Bu₂SnCl₂ (1.0 mmol). Red brown acicular crystals were obtained. Yield 49.6%. Anal. Calc. for C₂₀H₂₉N₃O₆Sn (526.17): C 49.92, H 6.28, N 5.82. Found: C 49.19, H 5.87, N 5.90. IR (KBr): v = 1705 s (C=O/C=N), 625 (Sn–O–Sn), 562w (Sn–O), 506w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 0.87 (t, 6H, Sn–CH₂CH₂CH₂CH̲₃, J = 7.0 Hz), 1.30–1.57 (m, 15H, Sn–CH̲₂CH̲₂CH̲₂CH₃ and R′–OCH₂CH̲₃), 2.57 (s, 3H, H-7), 3.87 (s, 2H, R′–OCH̲₂CH₃), 6.71 (dt, 1H, H-3, ¹J = 7.0 Hz, ²J = 1.5 Hz), 6.80 (d, 1H, H-5, J = 8.5 Hz), 7.14 (dt, 1H, H-6, ¹J = 7.0 Hz, ²J = 2.0 Hz), 7.63 (dd, 1H, H-4, ¹J = 8.0 Hz, ²J = 1.5 Hz). ¹³C NMR (CDCl₃): δ = 13.6 (Sn–CH₂CH₂CH₂C̲H₃), 14.6 (R′–OCH₂C̲H₃), 23.0 (Sn–CH₂CH₂C̲H₂CH₃), 26.6 (Sn–CH₂C̲H₂CH₂CH₃), 26.8 (Sn–C̲H₂CH₂CH₂CH₃), 29.1 (C-7), 52.1 (R′–OC̲H₂CH₃), 116.6 (C-6), 117.9 (C-3), 118.6 (C-5), 124.8 (C-4), 130.2 (C-9), 138.0 (C-2), 158.4 (C-1), 166.8 (C-10), 189.8 (C-8). ¹¹⁹Sn NMR (CDCl₃): –204.8.

3.2.8. Synthesis of [Ph₂Sn(L¹)]₂ (8). Compound 8 was prepared analogously by following the method and conditions described for 1 but using HL¹ (1.0 mmol) and Ph₂SnCl₂ (1.0 mmol). Orange powder was obtained. Yield 44.8%. Anal. Calc. for C₂₃H₂₀N₂O₃Sn (491.13): C 56.25, H 4.10, N 5.70. Found: C 55.98, H 4.25, N 5.62. IR (KBr): v = 1632 s (C=O/C=N), 638 (Sn–O–Sn), 543w (Sn–O), 470w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 2.50 (s, 3H, H-7), 2.78 (s, 3H, H-11), 6.72 (td, 1H, H-3, ¹J = 7.5 Hz, ²J = 1.0 Hz), 7.08 (d, 1H, H-5, J = 7.3 Hz), 7.23 (t, 1H, H-6, J = 7.5 Hz), 7.44–7.47 (m, 7H, Sn–C₆H̲₅), 7.59 (dd, 1H, H-4, ¹J = 8.0 Hz, ²J = 1.5 Hz), 7.76–7.79 (m, 3H, Sn–C₆H̲₅). ¹³C NMR (CDCl₃): δ = 129.3, 131.1, 136.0, 136.3, 137.5 (Sn–C₆H₅), 28.1 (C-7), 30.3 (C-11), 116.1 (C-6), 118.4 (C-3), 119.2 (C-5), 130.5 (C-4), 133.4 (C-9), 138.0 (C-2), 158.2 (C-1), 192.0 (C-8), 198.0 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −349.4.

3.2.9. Synthesis of [Ph₂Sn(L²)]₂ (9). Compound 9 was prepared analogously by following the method and conditions described for 1 but using HL² (1.0 mmol) and Ph₂SnCl₂ (1.0 mmol). Brown flaky crystals were obtained. Yield 52.2%. Anal. Calc. for C₂₃H₁₉N₃O₅Sn (536.12): C 51.53, H 3.57, N 7.84. Found: C 50.94, H 3.66, N 7.94. IR (KBr): v = 1684 s, 1640w (C=O/C=N), 623 (Sn–O–Sn), 562w (Sn–O), 444w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 2.53 (s, 3H, H-7), 2.86 (s, 3H, H-11), 7.48–7.50 (m, 7H, Sn–C₆H̲₅), 7.64–7.65 (m, 2H, H-3 and H-6) 7.74–7.77 (m, 3H, Sn–C₆H̲₅), 7.91 (d, 1H, H-4, J = 2.0 Hz).¹³C NMR (CDCl₃): δ = 129.6, 131.5, 135.8, 137.2 (Sn–C₆H₅), 28.0 (C-7), 31.0 (C-11), 113.6 (C-6), 114.2 (C-3), 115.9 (C-5), 134.6 (C-9), 142.6 (C-4), 148.0 (C-2), 157.3 (C-1), 195.7 (C-8), 197.5 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −115.6, –338.8.

3.2.10. Synthesis of [Oct₂Sn(L²)]₂ (10). Compound 10 was prepared analogously by following the method and conditions described for 1 but using HL² (1.0 mmol) and Oct₂SnCl₂ (1.0 mmol). Brown bulk crystals were obtained. Yield 21.9%. Anal. Calc. for C₂₇H₄₃N₃O₅Sn (608.36): C 53.60, H 7.02, N 6.80. Found: C 53.38, H 7.08, N 6.84. IR (KBr): v = 1678 s, 1639w (C=O/C=N), 621 (Sn–O–Sn), 590w (Sn–O), 439w (Sn–N) cm⁻¹. ¹H NMR (CDCl₃): δ = 0.85 (t, 6H, Sn–CH₂CH₂(CH₂)₅CH̲₃, J = 7.5 Hz), 1.20–1.29 (m, 20H, Sn–CH₂CH₂(CH̲₂)₅CH₃), 1.58–1.64 (m, 8H, Sn–CH̲₂CH̲₂(CH₂)₅CH₃), 2.53 (s, 3H, H-7), 2.64 (s, 3H, H-11), 7.60 (s, 2H, H-3, H-6), 7.63 (s, 2H, H-4). ¹³C NMR (CDCl₃): δ = 14.2, 22.7, 24.0, 24.6, 29.1, 29.2, 31.9, 33.4 (Sn–(C̲H₂)₇C̲H₃), 27.9 (C-7), 30.7 (C-11), 113.1 (C-6), 113.7 (C-3), 115.7 (C-5), 134.3 (C-9), 143.2 (C-4), 147.9 (C-2), 157.7 (C-1), 194.8 (C-8), 197.6 (C-10). ¹¹⁹Sn NMR (CDCl₃): δ = −189.5.

3.3. X-ray measurements

Suitable single crystals of the complexes 4 and 6 were mounted in glass capillaries for X-ray structural analysis. Diffraction data were collected at 298(2) K on a Bruker SMART CCD diffractometer with Mo Ka (λ = 0.71073 Å) radiation at room temperature. During the intensity data collection, no significant decay was observed. The intensities were collected for Lorentz-polarization effects and empirical absorption with the SADABS program. The structure was solved by direct methods using the SHELXL-97 program. All non-hydrogen atoms were found from the difference Fourier syntheses. The H atoms were included in calculated positions with isotropic thermal parameters related to those of the supporting carbon atoms but were not included in the refinement. All calculations were performed using the Bruker Smart program.⁴⁶ Crystallographic details are reported in Table 3.

Table 3 Experimental data for crystallographic analyses for compounds 4 and 6

Compound	4	6
Formula	C30H38N6O10Sn2	C38H54N6O10Sn2
M (g mol^−1)	880.04	992.25
Crystal system	Monoclinic	Triclinic
Space group	P21/c	P1̄
a (Å)	11.4930(16)	9.9310(8)
b (Å)	13.976(2)	11.1041(8)
c (Å)	11.1209(15)	11.1694(9)
α (deg)	90	98.9150(10)
β (deg)	90.011(3)	115.946(2)
γ (deg)	90	92.7020(10)
V (Å^3)	1786.4(4)	1084.98(15)
Z	2	1
ρ (g cm^−3)	1.636	1.519
μ (mm^−1)	1.459	1.210
F000	880	504
θ range (deg)	1.77 to 30.00	1.87 to 25.50
Index ranges	−16 ≤ h ≤ 16	−10 ≤ h ≤ 12
	−19 ≤ k ≤ 19	−10 ≤ k ≤ 13
	−15 ≤ l ≤ 14	−13 ≤ l ≤ 13
Nt	18 297	6425
N(Rint)	5192 (0.1039)	3987 (0.0384)
Restraints/parameters	27/243	18/3987
Completeness (%)	99.8	98.7
GOF on F^2	1.031	1.049
R1, wR2[I > 2σ(I)]	R1 = 0.0359, wR2 = 0.0815	R1 = 0.0291, wR2 = 0.0725
R Indices (all data)	R1 = 0.0514, wR2 = 0.0894	R1 = 0.0291, wR2 = 0.0733

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3.4. In vitro cytotoxic activity

The following cell lines were used for biological assays: human cervical carcinoma cell line (Hela), human nasopharyngeal carcinoma (KB), and human liver hepatocellular carcinoma (HepG2) cell lines. They were grown and maintained in RPMI-1640 medium (Zhejiang Tianhang Biological Technology Co., Ltd., China) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Biochemical reagent Co., Ltd., China), penicillin (100 U ml⁻¹), and streptomycin (100 mg ml⁻¹) at 37 °C in humidified incubators in an atmosphere of 5% CO₂.

The complexes were dissolved in DMSO at a concentration of 10 mM as stock solution, and diluted in culture medium at concentrations of 2.0, 1.0, 0.5, 0.25 and 0.125 μM as working solution. To avoid DMSO toxicity, the concentration of DMSO was less than 0.1% (v/v) in all experiments.⁴⁷

The cells harvested from the exponential phase were seeded equivalently into a 96-well plate, 24 h later added new culture medium to replace the previous and then the complexes were added to the wells to achieve final concentrations. Control wells were prepared by addition of culture medium. Wells without culture medium and cells were used as blanks. All experiments were performed in triplicate. The MTT assay was performed as described by Mosmann.⁴⁸ Upon completion of the incubation for 48 h, stock MTT dye solution (20 μl, 5 mg ml⁻¹) was added to each well. After 4 h incubation, DMSO (150 μl) was added to solubilize the MTT formazan. The OD of each well was measured on a microplate spectrophotometer at a wavelength of 492 nm. The IC₅₀ value was determined from plot of 50% viability against dose of compounds added.

3.5. Cell death by flow cytometry

KB cells were seeded in sterile twelve-well plates at density of 1 × 10⁶ and grown in 5% CO₂ at 37 °C. After 24 h incubation, cells were exposed to 5, 6, 9 and cisplatin for 24 h at concentrations of 0.1, 0.25, 1.0, 2.5 and 10 μM. Then the solutions with the compounds 5, 6, 9 or cisplatin were washed with cold PBS, harvested by trypsinisation, collected by centrifugation and washed two times with PBS. Re-suspended cells in 300 μl binding buffer and added 5 μl of Annexin V-FITC and 10 μl of PI (MultiSciences Biotech Co., Ltd., China) to cells, and then cells were incubated for 15 min at room temperature in the dark and then analyzed by flow cytometry (Beckman coulter flow cytometry).

4. Conclusions

Ten novel dimeric diorganotin(IV) complexes with arylhydrazones of β-diketones featuring a symmetrical centre were synthesized and characterized in detail. Cytotoxicity in three cancer cell lines (Hela, KB and HepG2) shows strong differences in the activity pattern dependent on both lipophilicity and electronic effects of the ligands. The dibutyltin(IV) (R = ⁿBu) complex with the nitro-substituted (R¹ = NO₂) arylhydrazone ligand, namely [ⁿBu₂Sn(L²)]₂ (6) showed the highest activity. These results suggest that the organo groups might influence the mode of action. Subsequently, three compounds with better activity but different substitutents (R or R¹) were selected for investigating the induction of apoptosis. The data of double staining indicate the apoptosis induction of the compounds correlates with the cytotoxicity. On account of these initial results, further investigations of their mechanism and mode of action towards the biological targets in tumor cell lines will be addressed.

Acknowledgements

This work has been supported by the National Natural Science Foundation of China (no. 81102311), the Fundamental Research Funds for the Central Universities of China (no. 2015TS131), and by the Fundação para a Ciência e a Tecnologia (project UID/QUI/00100/2013), Portugal.

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Footnote

† CCDC 1057981 (4) and 1057982 (6). For crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra06658a

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