Antimicrobial activity of organotin(IV) complexes with the ligand benzil bis(benzoylhydrazone) and 4,4′-bipyridyl as coligand

Journal of Inorganic Biochemistry 105 (2011) 600–608

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Journal of Inorganic Biochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o

Antimicrobial activity of organotin(IV) complexes with the ligand benzil bis(benzoylhydrazone) and 4,4′-bipyridyl as coligand Elena López-Torres a,⁎, Franca Zani b,⁎⁎, M. Antonia Mendiola a a b

Departamento de Química Inorgánica, Universidad Autónoma de Madrid, C/ Francisco Tomás y Valiente 7, 28049 Madrid, Spain Dipartimento Farmaceutico, Università degli Studi di Parma, Viale G. P. Usberti 27/A, 43124 Parma, Italy

a r t i c l e

i n f o

Article history: Received 22 October 2010 Received in revised form 19 January 2011 Accepted 20 January 2011 Available online 3 February 2011 Keywords: Hydrazones Organotin(IV) complexes Antimicrobial activity Crystal structure

a b s t r a c t Several methyltin(IV) and butyltin(IV) complexes with the ligand benzil bis(benzoylhydrazone) and 4,4′bipyridyl as coligand were synthesised and characterized by elemental analysis and by IR, 1H, 13C and 119Sn NMR spectroscopies. Some of them were also analyzed using single crystal X-ray diffraction. The title compounds were evaluated for their in vitro antimicrobial properties. All buthyltin complexes showed significant inhibition of Gram positive bacteria, resulting Bacillus subtilis, Sarcina lutea and both methicillinsusceptible and methicillin-resistant Staphylococcus epidermidis the most sensitive strains. Furthermore, they were able to inhibit the growth of Gram negative bacteria, especially Proteus vulgaris, whereas no activity was exhibited against fungi. All methyltin complexes were devoid of antimicrobial properties. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Organotin complexes are among the most widely used organometallic species, and although they have an impact on the environment [1], they are well known for their pharmacological properties such as antifungal, antibacterial, biocidal and cytotoxic agents [2,3]. The biological activity and possible antibacterial/ antitumor applications of numerous organotin(IV) complexes have been described in the literature [4–14]. Within main-group metal compounds, they appear to exhibit the most potent antitumor activities, in some cases being more effective than cisplatin in in vitro tests [15,16]. For known cytotoxic organotin(IV) complexes, those with biologically active ligands, for example, carboxylates [3,17–19] or hydroxamic acids [20–23], have attracted particular interest. A significant amount of the work carried out on these Sn(IV) compounds was in relation to their antibacterial activity against a wide range of both Gram negative and Gram positive bacteria. While the results vary considerably depending on the different substitution patterns at the tin centre, a trend in the activity emerges inclining towards Gram positive bacteria, where a greater antibacterial effect is observed compared to Gram negative bacteria [24,25], which has been attributed to differences in the structures of the cell walls. The mode of action of organotin(IV) compounds has been associated with the inhibition of bacterial processes related to enzyme production and membrane functions: they include effects on energy transduction, solute transport, retention and oxidation of substrates.

Schiff bases still play an important role as ligands in metal coordination chemistry even after almost a century since their discovery, and their organotin(IV) complexes have received increasing attention owing both to their antitumor activities and potential applications in biotechnology [26–30]. Within the Schiff bases hydrazones are of particular interest due to they find applications in catalysis [31–33], cancer and metal chelating therapy [34,35] or by their fascinating structural properties [36,37]. For several years we have been interested in the coordinating behaviour of ligands that can bind to metals in a tetradentate chelate mode and we have established that the structures of the complexes depend both on the metal preferences and the reaction conditions [38–41]. In order to find new pharmacologically active molecules, here we continue with the systematic study of the coordinating behaviour of the ligand benzil bis(benzoylhydrazone), which was previously reacted with divalent metals to yield from monomers to trinuclear helicates [42] and with some SnR2Cl2 [43]. In general, the biological activity of organotin compounds is greatly influenced by the structure of the molecule and conceivably by the nuclearity of the complexes [44,45]. In this paper we report the synthesis of several complexes containing different numbers of 4,4bipyridyl units and the evaluation of their in vitro activity against many Gram positive and Gram negative bacteria as well as some fungi. 2. Experimental 2.1. Measurements

⁎ Corresponding author. Fax: + 34 4974833. ⁎⁎ Corresponding author. Fax: + 39 521 905006. E-mail addresses: [email protected] (E. López-Torres), [email protected] (F. Zani). 0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2011.01.014

Microanalyses were carried out using a LECO CHNS-932 Elemental Analyzer. IR spectra in the 4000–400 cm− 1 range were recorded as KBr

E. López-Torres et al. / Journal of Inorganic Biochemistry 105 (2011) 600–608

pellets on a Jasco FT/IR-410 spectrophotometer. Fast atom bombardment mass spectra (MS (FAB)) were recorded on a VG Auto Spec instrument using Cs as the fast atom and m-nitrobenzylalcohol (m-NBA) as the matrix. Electrospray mass spectrometry (ESI) experiments were performed with an ion trap instrument LCQ Deca XP plus (Thermo Instruments). An ESI source was used in positive ionization mode. The instrumental parameters were set as follows: mass range scanned from m/z 500–2000; Source Voltage (kV): 4.5; Seath gas flow rate: 11; Capillary Temperature (°C): 250; Capillary Voltage (V): 38 and Tube Lens Voltage (V): 30. 1H and 13C NMR spectra were recorded on a Bruker AMX-300 spectrometer using CDCl3 and DMSO-d6 as solvent and TMS (Tetramethylsilane) as internal reference. 119Sn NMR spectra were recorded in the same spectrometer and the chemical shifts are reported relative to Sn(Me)4 as internal reference. 13C CP/MAS (Cross Polarization Magic Angle Spinning) NMR spectra were recorded at 298 K in a Bruker AV400WB spectrometer equipped with a 4 mm MAS NMR probe and obtained using a cross-polarization pulse sequence. The external magnetic field was 9.4 T, the sample was spun at 10–14 kHz and the spectrometer frequencies were set to 100.61 MHz. For the recorded spectra a contact time of 4 ms were used and recycle delays of 4 s were used. Chemical shifts are reported relative to TMS, using the CH group of adamantane as a secondary reference (29.5 ppm). 119Sn CP/MAS NMR spectra were obtained in the same spectrometer using spinning rates of 10–14 kHz, pulse delays of 30 s, contact times of 8 ms and TPPM high power proton decoupling. Chemical shifts are reported relative to Sn(Me)4, using tin(IV) oxide as a secondary reference. 2.2. Materials All chemicals were purchased from Aldrich and used as received. 2.3. Synthesis of the compounds Benzil bis(benzoylhydrazone), LH2. The ligand was prepared following a previously reported procedure [42]. Selected spectroscopic data: 1H NMR (300 MHz, CDCl3, ppm): 9.01 (s (singlet), 2H, NH), 7.83 (m (multiplet), 4H, Ph), 7.40–7.22 (m, 16H, Ph). 13C CP/MAS NMR (300 MHz, ppm): 164.4 (CO), 147.5 (CN), 133.8, 132.3, 128.3, 123.8 (Ph). IR (KBr, cm− 1): 3232 m (medium), 3178 m ν(NH); 3063w (weak) ν(CH); 1674 s (strong), 1643 s ν(CO), 1604w ν(CN), 694 s δ(Ph). [SnMe 2 L] 1. The complex was synthesised following the procedure previously reported [43]. Selected spectroscopic data: 1 H NMR (300 MHz, CDCl3, ppm): 8.08 (d (doublet), 4H, Ph), 7.54–7.22 (m, 16H, Ph), 0.98 (s, 6H, Me, 2JSn,H = 94.0Hz). 13C NMR (300 MHz, CDCl3, ppm): 173.5 (CO), 148.7 (CN), 134.9, 132.2, 131.3, 130.8, 129.4, 128.6, 128.0, 127.4 (Ph), 4.9 (Me). 13C CP/MAS NMR (300 MHz, ppm): 172.5 (CO), 150.1 (CN), 134.5, 130.8, 129.4, 127.4 (Ph), 4.3 (Me). 119Sn NMR (300 MHz, CDCl3, ppm): − 261. 119Sn CP/ MAS NMR (300 MHz, ppm): − 270. IR (KBr, cm− 1): 3050w ν(CHPh), 2973w, 2938w ν(CHMe), 1600w ν(CN), 1585 m ν(CO), 712 s, 688 m δ(CHPh). MS (FAB+): m/z (%) = 595.1 [Calcd. 595.1] (100) [M+H]+, 578.2 [Calcd. 579.0] (15) [M−Me]+. [(SnMe2L)2(bipy)] 2. 14 mg (0.09 mmol) of 4,4′-bipyridyl (bipy) is dissolved in 1 mL of dichloromethane and added to a solution of 50 mg (0.08 mmol) of [SnMe2L] 1 in 5 mL of dichloromethane. The orange solution was stirred at room temperature for 6 h. Evaporation of the mother liquor gave pale orange crystals suitable for X-ray diffraction. Yield: 47 mg, 83%. C70H60N10O4Sn2 (1341.90): calcd. C 62.60, H 4.51, N 10.44; found C 62.41, H 4.48, N 10.54. 1H NMR (300 MHz, CDCl3, ppm): 8.75 (d, 4H, bipy), 8.08 (d, 8H, Ph), 7.54 (d of d, 4H, bipy), 7.44–7.28 (m, 32H, Ph), 0.98 (s, 12H, Me, 2JSn,H = 94.0Hz). 13C NMR (300 MHz, CDCl3, ppm): 173.5 (CO), 150.7, 148.7 (CN), 145.6 (bipy), 134.9, 132.2, 131.3, 130.9, 129.5, 128.7, 128.1, 127.5 (Ph), 121.5 (bipy), 5.1 (Me). 13C CP/ MAS NMR (300 MHz, ppm): 172.6 (CO), 149.0 (CN), 145.7 (bipy), 137.3, 136.1, 134.9, 132.2, 129.0, 128.1 (Ph), 121.3 (bipy), 15.6, 11.7 (Me).

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119 Sn NMR (300 MHz, CDCl3, ppm): −263. 119Sn CP/MAS NMR (300 MHz, ppm): −441. IR (KBr, cm− 1): 3060w ν(CHAr), 2976w, 2938w ν(CHMe), 1604 m ν(CN), 1587 m ν(CO), 713 s, 689 m δ(CHAr). MS (FAB+): m/z (%) = 595.1 (100) [M+H]+, 578.2 (15) [M−Me]+. MS (ESI+): m/z= 595.1 [Calcd. 595.1] [M+H]+, 578.2 [Calcd. 579.0] [M− Me]+. [SnMe2L(bipy)] 3. To a mixture of 100 mg (0.22 mmol) of LH2 and 36 mg (0.22 mmol) of 4,4′-bipyridyl in 5 mL of dichloromethane with 4 drops of Et3N was added a solution of 52 mg (0.22 mmol) of SnMe2Cl2 in 2 mL of the same solvent. The orange solution was stirred at room temperature for 6 h. The solution was evaporated to dryness and 20 mL of diethyl ether was added. The white precipitate corresponding to Et3N.HCl was discarded and the orange solution was allowed to evaporate slowly until a pale orange solid was obtained in good yield (135 mg, 80%). C40H34N6O2Sn (748.98): calcd. C 64.09, H 4.57, N 11.22; found C 64.37, H 4.68, N 11.13. 1H NMR (300 MHz, CDCl3, ppm): 8.76 (m, 4H, bipy) 8.08 (d, 4H, Ph), 7.55 (d, 4H, bipy), 7.43–7.31 (m, 16H, Ph), 0.98 (s, 12H, Me, 2 JSn,H = 94.6 Hz). 13C NMR (300 MHz, CDCl3, ppm): 150.6, 148.0 (CN), (bipy), 134.9, 132.1, 131.2, 130.8, 129.3, 128.6, 128.0, 127.4 (Ph), 121.4 (bipy), 5.1 (Me). 13C CP/MAS NMR (300 MHz, ppm): 172.8 (CO), 149.7 (CN), 145.6 (bipy), 137.0, 136.7, 134.5, 132.7, 129.6, 128.6 (Ph), 121.8 (bipy), 4.5 (Me). 119Sn NMR (300 MHz, CDCl3, ppm): − 267 119Sn CP/MAS NMR (300 MHz, ppm): − 448. IR (KBr, cm− 1): 3061w ν(CHAr), 2965w, 2916w ν(CHMe), 1603w ν(CN), 1587 m ν(CO), 711 s, 689 s δ(CHAr). MS (FAB+): m/z (%) = 595.1 (100) [M+H]+, 578.2 (15) [M−Me]+. MS (ESI+): m/z = 595.1 [Calcd. 595.1] [M+H]+, 578.2 [Calcd. 579.0] [M−Me]+. Recrystallisation in methanol gave orange crystals of [SnMe2L(MeOH)] 4. [SnMe2L(MeOH)] 4. To a mixture of 100 mg (0.22 mmol) of LH2 in 5 mL of methanol with 4 drops of Et3N was added a solution of 52 mg (0.22 mmol) of SnMe2Cl2 in 2 mL of the same solvent. The orange solution was refluxed for 24 h. After cooling to room temperature an orange solid appeared, which was filtered off, washed with cold methanol and dried in vacuum. C31H30N4O3Sn (624.94): calcd. C 59.09, H 4.84, N 8.96; found C 59.22, H 4.86, N 9.04. 1H NMR (300 MHz, CDCl3, ppm): 8.08 (d, 4H, Ph), 7.46–7.30 (m, 16H, Ph), 3.72 (s, 3H, MeOH), 0.98 (s, 6H, Me, 2JSn,H = 96.0 Hz). 13C CP/MAS NMR (300 MHz, ppm): 172.2 (CO), 150.9 (CN), 133.2, 131.5, 129.7, 127.7 (Ph), 58.9 (MeOH), 6.2 (Me). 119Sn CP/MAS NMR (300 MHz, ppm): − 416. IR (KBr, cm− 1): 3433 m ν(OH), 3057w ν(CHPh), 2962w, 2923w ν(CHMe), 1602w ν(CN), 1588 m ν(CO), 712 s, 691 m δ(CHPh). [SnMe2L(bipy)2] 5. The complex was synthesised using the same procedure used for the obtaining of 3 but using 72 mg (0.44 mmol) of 4,4′-bipyridyl. Yield: 148 mg, 73%. C50H42N8O2Sn (905.05): calcd. C 66.29, H 4.68, N 12.38; found C 66.47, H 4.84, N 12.23. 1H NMR (300 MHz, CDCl3, ppm): 8.75 (d, 8H, bipy), 8.08 (d, 4H, Ph), 7.54 (d, 8H, bipy), 7.44–7.32 (m, 16H, Ph), 0.98 (s, 6H, Me, 2JSn,H = 93.4 Hz). 13 C NMR (300 MHz, CDCl3, ppm): 150.6, 148.0 (CN), 145.6 (bipy), 134.9, 132.1, 131.2, 130.8, 129.3, 128.6, 128.0, 127.4 (Ph), 121.4 (bipy), 5.1 (Me). 13C CP/MAS NMR (300 MHz, ppm): 173.1 (CO), 149.5 (CN), 146.0 (bipy), 137.3, 136.3, 133.1, 129.7, 128.5 (Ph), 121.3 (bipy), 4.3 (Me). 119Sn NMR (300 MHz, CDCl3, ppm): − 267. 119Sn CP/MAS NMR (300 MHz, ppm): −705. IR (KBr, cm− 1): 3061w ν(CHAr), 2962w, 2917w ν(CHMe), 1601w ν(CN), 1588 m ν(CO), 711 s, 691 s, 683 m δ(CHAr). MS (FAB+): m/z (%) = 595.1 (100) [M+H]+, 578.2 (10) [M− Me]+. MS (ESI+): m/z = 595.1 [Calcd. 595.1] [M+H]+, 578.2 [Calcd. 579.0] [M−Me]+. [SnBu2L] 6. The complex was synthesised as reported previously [43]. 1H NMR (300 MHz, CDCl3, ppm): 8.10 (d, 4H, Ph), 7.47–7.26 (m, 16H, Ph), 1.74 (t (triplet), 4H, Bu), 1.55 (q (quartet), 4H, Bu), 1.32 (sx (sextet), 4H, Bu), 0.83 (t, 6H, Bu). 13C NMR (300 MHz, CDCl3, ppm): 173.7 (CO), 149.0 (CN), 135.1, 132.5, 131.2, 130.7, 129.2, 128.7, 128.0, 127.5 (Ph), 27.6, 26.2, 25.4, 13.6 (Bu). 13C CP/MAS NMR (300 MHz, ppm): 174.7 (CO), 151.5 (CN), 136.7, 134.3, 131.7, 129.1 (Ph), 28.1, 27.5, 25.2, 14.5 (Bu). 119Sn NMR (300 MHz, CDCl3, ppm): − 284. 119Sn

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CP/MAS NMR (300 MHz, ppm): − 273. IR (KBr, cm− 1): 3057w ν(CHPh), 2955 m, 2924 m, 2872 m, 2857 m ν(CHBu), 1598w ν(CN), 1588 m ν(CO), 712 s, 687 s δ(CHPh). MS (FAB+): m/z (%) = 679.2 (100) [M+H]+, 620.9 (75) [M−Bu]+, 568.1 (20) [M−2Bu+H]+. MS (ESI+): m/z = 679.2 [Calcd. 679.2] [M+H]+, 620.9 [Calcd. 620.1] [M− Bu]+, 562.8 [Calcd. 563.0] [M−2Bu+H]+. [SnBu2L(bipy)] 7. The complex was synthesised using the same procedure used for the obtaining of 3 but using 70 mg (0.22 mmol) of SnBu2Cl2. Single crystals were obtained from a solution in dichloromethane. Yield: 128 mg, 68%. C46H46N6O2Sn (833.08): calcd. C 66.26, H 5.56, N 10.09; found C 66.50, H 5.61, N 10.02. 1H NMR (300 MHz, CDCl3, ppm): 8.75 (d, 4H, bipy), 8.09 (d of d, 4H, Ph), 7.54 (d of d, 4H, bipy), 7.48–7.24 (m, 16H, Ph), 1.74–1.65 (m, 4H, Bu), 1.37–1.24 (m, 4H, Bu), 1.01–0.89 (m, 4H, Bu), 0.82 (t, 6H, Bu). 13 C NMR (300 MHz, CDCl3, ppm): 173.7 (CO), 150.8, 148.9 (CN), 145.5 (bipy), 135.0, 132.4, 131.1, 130.7, 129.2, 128.7, 128.0, 127.4 (Ph), 121.4 (bipy), 27.6, 26.2, 25.4, 13.6 (Bu). 13C CP/MAS NMR (300 MHz, ppm): 175.0 (CO), 151.9 (CN), 145.9 (bipy), 136.9, 136.5, 134.5, 132.5, 129.9, 128.0 (Ph), 122.0 (bipy), 28.1, 27.4, 25.3, 14.6 (Bu). 119Sn NMR (300 MHz, CDCl3, ppm): − 286. 119Sn CP/MAS NMR (300 MHz, ppm): − 427. IR (KBr, cm− 1): 3062 m, 3032 m ν(CHAr), 2955 s, 2922 s, 2869 m, 2855 m ν(CHBu), 1590 s ν(CN), 1568 m ν(CO), 737 m, 713 s, 691 s, 679 s δ(CHAr). MS (FAB+): m/z (%) = 679.2 (100) [M+H]+, 620.9 (75) [M−Bu]+, 568.1 (17) [M−2Bu+ H]+. MS (ESI+): m/z = 679.2 [Calcd. 679.2] [M+H]+, 620.9 [Calcd. 620.1] [M−Bu]+, 562.8 [Calcd. 563.0] [M−2Bu+H]+. [SnBu2L(bipy)2] 8. The complex was synthesised using the same procedure used for the obtaining of 5 but using 70 mg (0.22 mmol) of SnBu2Cl2. Yield: 157 mg, 71%. C56H54N8O2Sn (989.15): calcd. C 67.94, H 5.50, N 11.33; found C 68.11, H 5.39, N 11.39. 1H NMR (300 MHz, CDCl3, ppm): 8.75 (d, 8H, bipy), 8.09 (d, 4H, Ph), 7.54 (d of d, 8H, bipy), 7.46–7.34 (m, 16H, Ph), 1.83 (t, 4H, Bu), 1.41 (q, 4H, Bu), 1.30 (sx, 4H, Bu), 0.96 (t, 6H, Bu). 13C NMR (300 MHz, CDCl3, ppm): 151.3, 150.7 (CN), 145.5 (bipy), 130.7, 129.5, 129.2, 128.7, 128.0, 127.54 (Ph), 121.4 (bipy), 27.6, 26.9, 26.3, 13.4 (Bu). 13C CP/ MAS NMR (300 MHz, ppm): 174.9 (CO), 152.2 (CN), 146.1 (bipy), 137.6, 136.6, 134.3, 132.0, 129.7, 128.1 (Ph), 121.1 (bipy), 28.9, 27.6, 25.9, 15.3 (Bu). 119Sn NMR (300 MHz, CDCl3, ppm): − 283. 119Sn CP/ MAS NMR (300 MHz, ppm): − 694. IR (KBr, cm− 1): 3061w, 3037w ν(CHAr), 2957 m, 2922 m, 2870w, 2856w ν(CHBu), 1602 m ν(CN), 1590 m ν(CO), 713 s, 691 m, 681 m δ(CHAr). MS (FAB+): m/z (%) = 679.2 (100) [M+H]+, 620.9 (75) [M−Bu]+, 568.1 (20) [M−2Bu+ H]+. MS (ESI+): m/z = 679.2 [Calcd. 679.2] [M+H]+, 620.9 [Calcd. 620.1] [M−Bu]+, 562.8 [Calcd. 563.0] [M−2Bu+H]+.

factors are contained in the SHELXTL 6.10 program library. Main crystallographic and refinement data are summarised in Table 1. CCDC numbers 755426, 794110, 794112, 755427 and 794111 for complexes 1, 2, 4, 6 and 7.CH2Cl2 respectively, contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.cdcc.cam.ac.uk/data_request/cif. 2.5. Antimicrobial activity The in vitro antimicrobial activity of the title compounds was examined by the broth dilution method [50], using ampicillin and miconazole as standard drugs for the comparison of antibacterial and antifungal activities, respectively. Gram positive bacteria (Bacillus megaterium BGSC 7A2, Bacillus subtilis ATCC 6633, Bacillus thuringiensis var. kurstaki BGSC 4D1, Sarcina lutea ATCC 9341, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228 and clinical isolates of Staphylococcus haemolyticus, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium and both methicillin-resistant S. aureus and S. epidermidis strains), Gram negative bacteria (Enterobacter cloacae ATCC 23355, Escherichia coli ATCC 8739, Haemophilus influenzae ATCC 19418, Proteus vulgaris ATCC 13315, Salmonella typhimurium ATCC 14028, Serratia marcescens ATCC 8100, a clinical isolate of Klebsiella pneumoniae), yeasts (Candida tropicalis ATCC 1369, Saccharomyces cerevisiae ATCC 9763) and mould (Aspergillus niger ATCC 6275) were screened. All strains were obtained from the collection of the Laboratory of Dipartimento di Patologia e Medicina di Laboratorio, Università degli Studi di Parma, Italy. Stock drug solutions were prepared in dimethyl sulfoxide and diluted in the media to get graded concentrations ranging from 1600 μg mL− 1 to 0.0015 μg mL− 1. Haemiphilus test medium, Mueller Hinton broth and Sabouraud liquid medium (Oxoid, Basingstoke, UK) were chosen as nutrient media to grow H. influenzae, other bacteria and fungi, respectively. Inocula were obtained from the broth cultures in the log phase of growth and were adjusted to 5 × 105 CFU mL− 1 for bacteria and 1 × 103 CFU mL− 1 for fungi. After incubation at 37 °C for 24 h (bacteria) and at 30 °C for 48 h (fungi), the lowest concentration of the drug inhibiting the growth of the tested organisms was recorded as the minimal inhibitory concentration (MIC, μg mL− 1). Assay tubes containing only inoculated media were kept as negative control and likewise dimethyl sulfoxide controls were conducted Table 1 Crystal data and structure refinement for 2, 4 and 7.CH2Cl2.

2.4. Crystallography Data for complexes 2, 4 and 7.CH2Cl2 were acquired using a Bruker AXS Kappa Apex-II diffractometer equipped with an Apex-II CCD area detector using a graphite monochromator (Mo K α radiation, λ = 0.71073 Å). The substantial redundancy in data allows empirical absorption corrections (SADABS) [46] to be applied using multiple measurements of symmetry-equivalent reflections. The raw intensity data frames were integrated with the SAINT program, which also applied corrections for Lorentz and polarization effects [47]. The software package SHELXTL version 6.10 was used for space group determination, structure solution and refinement. The structures were solved by direct methods (SHELXS-97) [48], completed with difference Fourier syntheses, and refined with full-matrix least squares using SHELXL-97 minimizing ω(F20 − F2c ). Weighted R factors (Rw) and all goodness of fit S are based on F2; conventional R factors (R) are based on F [49]. All non-hydrogen atoms were refined with anisotropic displacement parameters and the hydrogen atoms were positioned geometrically, except the H of the OH group of the methanol in complex 4, which was located by difference maps and refined isotropically. All scattering factors and anomalous dispersion

Formula M Crystal system Space group a/Å b/Å c/Å α/° β/° γ/° U/Å3 Z Dc/Mgm− 3 Absorption coefficient mm− 1 F(000) Goodness of fit on F2 Reflections collected Independent reflections

2

4

7.CH2Cl2

C70H60N10O4Sn2 1342.66 Monoclinic P2(1)/c 21.394(2) 7.4343(8) 20.2054(17) 90 92.508(3) 90 3210.5(5) 2 1.389 0.834

C31H30N4O3Sn 625.28 Monoclinic P2(1)/n 18.833(5) 8.022(2) 19.264(4) 90 94.269(9) 90 2902.4(12) 4 1.431 0.918

C47H48N6O2Cl2Sn 918.50 Monoclinic C2/c 16.923(2) 23.4102(18) 12.9262(16) 90 115.997(3) 90 4602.8(9) 4 1.325 0.714

1364 1.045 34,148 7599 [R(int) = 0.0573] 0.0320, 0.0702

1272 1.161 45,183 5099 [R(int) = 0.0825] 0.0345, 0.0810

1888 1.054 16,904 4175 [R(int) = 0.0468] 0.0653, 0.1895

− 0.584, 0.420

− 0.648, 1.696

Final R1 and wR2 [I N 2σ(I)] Residual electron density − 0.677, 0.556 (min, max) (eÅ− 3)

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simultaneously. The amount of dimethyl sulfoxide never exceeded 1% v/v and no influence of the solvent on the growth of the tested microorganisms was detected. All tubes not showing visible growth were subcultured in fresh media and incubated at 37 °C for 24 h (bacteria) and at 30 °C for 48 h (fungi). The highest dilutions showing 99% inhibition were taken as minimal bactericidal concentrations (MBC, μg mL− 1) or minimal fungicidal concentrations (MFC, μg mL− 1). Each compound was assessed in triplicate and at least three independent experiments were performed.

3. Results and discussion 3.1. Synthesis Complexes 1 and 6 were obtained as described previously [43]. The complexes containing 4,4′-bipyridyl were synthesised by mixing the stoichiometric amounts of the ligand and bipy in the presence of Et3N and adding the corresponding organotin(IV) chloride, except complex [(SnMe2L)2(bipy)] 2, which can be exclusively synthesised by treating [SnMe2L] 1 with an excess of 4,4′-bipy. The analogous butyl derivative [(SnBu2L)2(bipy)] cannot be synthesised, probably due the higher steric hindrance imposed by the butyl groups. The reactions were carried out in dichloromethane at room temperature in the presence of Et3N (Schemes 1 and 2), which is essential to induce the ligand deprotonation, except in the synthesis of complex 2 that was obtained from [SnMe2L] 1, in which the ligand is already deprotonated. As a result a white crystalline material, Et3N. HCl, is formed, but can be eliminated due to its insolubility in diethyl ether.

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Elemental analysis of the complexes confirms the ligand:bipy:tin ratio, as well as the absence of Cl−. Mass spectra of all the complexes only show the peak corresponding to [SnR2L+H]+, as well as fragments corresponding to the loss of the organic groups, but peaks containing bipyridyl ligands cannot be observed. For all the peaks the found and calculated isotopic patterns are identical. 3.2. Crystal structures The crystal structures of complexes 1 and 6 have been previously described [43]. Both are isoestructural and a picture of complex 1 is shown in Fig. 1 for clarity. In all the complexes the ligand is doubly deprotonated and behaves as a N2O2 tetradentate chelate, coordination mode that leads to the formation of three five-membered chelate rings that confer high stability to the compounds. The crystal structure of complex 2 consists of two SnMe2L units linked by a bidentate 4,4-bipyridyl ligand (Fig. 2). The coordination environment of each tin atom is N3O2C2 in a distorted pentagonal bipyramid, with the methyl groups in the axial positions. The complex is centrosymmetric with the inversion point located in the middle of the C(9)–C(9#) bond of the bipy ligand. The bipyridyl rings are disordered over two positions, but in Fig. 2 only one of them is shown. The ligands can be considered planar, with a maximum deviation from the least-squares plane of 0.0832 Å for O(1). The aromatic rings belonging to the hydrazone moieties form dihedral angles with this plane of 3.20 and 18.96° for C(41)–C(46) and C(11)–C(16) respectively, while for the phenyls of benzil are 59.58° for C(21)–C(26) and 54.11° for C(31)–C(36). The bipyridyl is planar and is canted 26.73° with respect to the ligand plane. The Sn―O and Sn―C bond distances are within the normal range found in other tin complexes, as well as

Scheme 1. Reactions with SnMe2Cl2.

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Scheme 2. Reactions with SnBu2Cl2.

the Sn―N bonds with L (Table 2). It should be noticed that the bond distance with the bipy is considerably longer, which might induce its release in solution. There are CH…CH interactions between C(7A) and C(8A), leading to infinite chains running along the a axis. The crystal structure of complex 4 is made up of [SnMe2L(MeOH)] units (Fig. 3) in which the metal centre is in a N2O3C2 distorted

pentagonal bipyramid environment, with the methyl groups in the axial positions. In this complex the ligand is less planar than in complex 2 and the oxygen atoms O(1) and O(2) are 0.2146 Å under and 0.1326 Å above the least-squares plane. The phenyl rings of the hydrazone units are coplanar with this plane, while the aromatic rings from benzil form dihedral angles of 60.00° and 53.11° for C(21)–C(26)

Fig. 1. Molecular structure of complex [SnMe2L] 1. Thermal ellipsoids at 50% probability. Hydrogen atoms of the methyl groups have been omitted for clarity.

E. López-Torres et al. / Journal of Inorganic Biochemistry 105 (2011) 600–608

605

Fig. 2. Molecular structure of complex [SnMe2L]2(bipy) 2. Thermal ellipsoids at 50% probability. Hydrogen atoms have been omitted for clarity.

and C(31)–C(36) respectively. The bond distances involving the tin atom are within the range expected, but Sn―O bond with the methanol molecule is significantly longer than the bonds with the hydrazone ligand (Table 2). The molecules are forming dimers through hydrogen bonds between the methanol and O(1). The crystal structure of complex 7 is formed by molecules of [SnBu2L(bipy)] which crystallise with one CH2Cl2 molecule disordered over two positions (Fig. 4). The coordination sphere of the tin atom is N3O2C2 in a distorted capped octahedral arrangement, with the oxygen and carbon atoms forming the equatorial plane in trans disposition. The butyl groups are anti and pushed away from the bipyridyl. In contrast to complex 2, the bipy is not planar and the rings form a dihedral angle of 31.27°. In the bis(hydrazone) ligand, the mean deviation from the least-squares plane is for O(1) (0.0851 Å) and the rings corresponding to the hydrazone kernels are coplanar. By contrast, the phenyl rings from benzil form dihedral angles of 69.97°. As occurs in complex 2, the Sn–Nbipy bond distance is much longer than Sn–NL (Table 2). The dichloromethane molecule is located in front of the non-coordinate N atom of the bipy ring. There is an extended network of CH3…Nbipy interactions, leading a 2D network in the ab plane.

3.3. IR spectroscopy In all the complexes, coordination of the carbonyl group to the metal is confirmed by the shift of the ν(CO) band, at 1643 cm− 1 in the free ligand, to lower frequencies. By contrast, the ν(CN) bands are not very shifted, they appear close to that in the free ligand (1604 cm− 1) but the crystal structures of complexes 1, 2, 4, 6 and 7 confirm that the ligand is tetradentate N2O2. Concerning the bond Table 2 Selected bond distances of complexes 1 [43], 2, 4, 6 [43] and 7.CH2Cl2.

Sn(1)–O(1) Sn(1)–O(2) Sn(1)–O(3) Sn(1)–N(2) Sn(1)–N(3) Sn(2)–N(5) Sn(1)–C(3) Sn(1)–C(5) Sn(1)–C(6)

1

2

4

6

7.CH2Cl2

2.2675(19) 2.2960(19)

2.2517(17) 2.2794(17)

2.283(5) 2.290(5)

2.295(5)

2.238(2) 2.246(2)

2.308(2) 2.315(2) 2.502(2)

2.325(3) 2.243(3) 2.435(3) 2.303(3) 2.288(3)

2.240(7) 2.237(6)

2.324(5) 2.561(8)

2.106(3) 2.117(3)

2.111(3) 2.123(3)

2.104(4) 2.098(4)

2.134(7) 2.128(8)

2.129(7)

distances found in the crystal structures it can be observed that while CO bonds change considerably with respect to those of the free ligand, CN bond distances are very similar, explaining the small shifts observed in the IR spectra. Additional ν(CN) and δ(Ar) bands corresponding to the bipyridyl can be observed in complexes 2, 3, 5, 7 and 8, confirming its presence.

3.4. NMR spectroscopy The 1H NMR spectra of the complexes confirm the ligand deprotonation, due to the loss of the signal corresponding to the NH groups, as well as the presence of the organic groups bonded to the tin. In the complexes containing bipyridyl the position of the signals, identical to those of complexes [SnR2L], suggests that the bipy is released in solution. In addition, the position of the signals of the 4,4′-bipy correlates with the ones found for the commercial product. Nevertheless, the number of bipyridyl units can be confirmed from the integrals. The satellites corresponding to the coupling with tin can only be observed in the spectrum of the methyl derivatives. Substitution of 2 JSn,H value into the corresponding Lockhart–Manders equation (empirical relationship between the coupling constants and the C–Sn–C angle) [51] gives angles of about 154° for all the complexes, which are close to the value found in the crystal structure of 1, and differs significantly from that observed in the crystal structures of 2 (177.70°), confirming the loss of the bipy in solution. Due to the release of the bipyridyl in solution, 13C CP/MAS NMR spectra of all the complexes were recorded. Owing to the lower resolution of the solid state spectra, the signals of the C N group of the bis(hydrazone) and the bipy cannot be distinguished and appear as a sole signal, whose position suggests coordination of both ligands. The signal attributable to the CO bond is clearly downfield shifted with respect to the free ligand, indicating coordination of these groups to the tin. The bigger movement of the signals corresponding to the CO than the ones belonging to the CN bonds, as happens with the IR spectra, can also be explained with the X-ray diffraction data. The signals belonging to the organic groups are also observed in all the complexes, but the satellites corresponding to the 1JSn,C cannot be observed in any spectrum, so the corresponding Lockhart–Manders equation could not be used to get the value of the C–Sn–C angle. The 119Sn chemical shift of tin complexes appears to depend not only on coordination number, but is also very sensitive to the type of donor atoms bonded to the metal ion, so it is a useful tool to

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Fig. 3. Molecular structure of complex [SnMe2L(MeOH)] 4. Thermal ellipsoids at 50% probability. Hydrogen atoms, except of the methanol, have been omitted for clarity.

determine the chemical environment of the tin atom. 119Sn NMR spectra were acquired, both in solution and in the solid state. Holecek established for n-butyl derivatives that four-coordinate compounds have δ(119Sn) values in solution ranging from δ +200 to −60 ppm, five-coordinate compounds from δ −90 to − 190 ppm and sixcoordinate compounds from δ −210 to −400 ppm [52]. The values found in solution for all the complexes correspond to six-coordinate species, making clear once more the loss of the bipy. The chemical shifts found in the solid state for 1 (−270) and 6 (−284) agree with six-coordinate compounds; for complexes 2 (−441), 3 (−448), 4 (−416) and 7 (− 427) correlate well with seven-coordinate species, while for 5 (−705) and 8 (−694) correspond to eight-coordinate

complexes, confirming the environments found in the crystal structures of 1, 2, 4, 6 and 7.CH2Cl2.

3.5. Antimicrobial activity Biological studies of the metal complexes were carried out in vitro for antimicrobial activity against bacteria and fungi. The results are reported in Table 3 as minimal inhibitory concentrations (MICs). The effects of benzil bis(benzoylhydrazone) ligand, 4,4′-bipyridyl coligand and of organotin precursors SnMe2Cl2 and SnBu2Cl2 are also included for comparison purposes.

Fig. 4. Molecular structure of complex [SnBu2L(bipy)] 7.CH2Cl2. Thermal ellipsoids at 50% probability. Hydrogen atoms and the CH2Cl2 molecule have been omitted for clarity.

E. López-Torres et al. / Journal of Inorganic Biochemistry 105 (2011) 600–608 Table 3 Antimicrobial activity, expressed as MIC (μg mL− 1) and, in brackets, as MBC (μg mL− 1) and MFC (μg mL− 1). Compound

1 2 3 4 5 6 7 8 LH2 4,4′-Bipyridyl SnMe2Cl2 SnBu2Cl2 Ampicillin Miconazole

Bacteriaa

Fungib

BS

SA

EC

HI

SC

CT

AN

800 (1600) N 1600 1600 (N 1600) N 1600 1600 (N 1600) 12 (50) 12 (100) 6 (50) N 1600 N 1600 25 (100) 3 (6) 0.007 (0.15) –

N1600

N1600

N 1600

N 1600

N 1600

N1600

N1600 N1600

N1600 N1600

N 1600 N 1600

N 1600 N 1600

N 1600 N 1600

N1600

N1600 N1600

N1600 N1600

N 1600 N 1600

N 1600 N 1600

N 1600 N 1600

N1600 N1600

100 (400) 50 (100) 25 (50) N1600 N1600 200 (800) 6 (25) 0.07 (1.5) –

100 (400) 100 (800) 50 (800) N1600 N1600 400 (1600) 6 (100) 3 (25) –

100 (400) 100 (800) 100 (400) N 1600 N 1600 100 (800) 25 (200) 0.07 (0.3) –

400 (1600) N 1600

1600 (N1600) N 1600

400 (1600) N1600

N 1600

N 1600

N1600

N 1600 N 1600 400 (1600) 25 (400) –c

N 1600 N 1600 N 1600

N1600 N1600 N1600

400 (N1600) –

25 (400) –

12 (25)

6 (25)

3 (12)

MIC, minimal inhibitory concentration. MBC, minimal bactericidal concentrations. MFC, minimal fungicidal concentrations. a Gram positive bacteria: Bacillus subtilis ATCC 6633 (BS) and Staphylococcus aureus ATCC 6538 (SA); Gram negative bacteria: Escherichia coli ATCC 8739 (EC) and Haemophilus influenzae ATCC 19418 (HI). b Yeasts: Saccharomyces cerevisiae ATCC 9763 (SC) and Candida tropicalis ATCC 1369 (CT); mould: Aspergillus niger ATCC 6275 (AN). c Not tested.

All butyltin complexes 6–8 exhibited very good antibacterial properties against Gram positive B. subtilis (MICs 6–12 μg mL− 1). They also inhibited, to a lesser degree, the development of Gram positive S. aureus and Gram negative E. coli and H. influenzae at concentrations of 25–100 μg mL− 1. All methyltin complexes 1–5, LH2 ligand and 4,4′-bipyridyl coligand were devoid of antibacterial effect up the concentration of 1600 μg mL− 1, except compounds 1, 3 and 5 against B. subtilis cells. On the contrary, starting alkyltin compounds displayed inhibition at concentrations lower than those of the corresponding complexes: SnBu2Cl2 was, among all, the most active substance (MICs 3–25 μg mL− 1). No antifungal activity could be detected towards yeasts and mold, with the exception of parent SnBu2Cl2, that inhibited the growth of S. cerevisiae and A. niger at 25 μg mL− 1, and complex 6. None of the studied compounds caused excellent inhibition as ampicillin and miconazole control drugs. The antibacterial effectiveness of complexes 6–8 was further evaluated against a wide spectrum of selected Gram positive species, including bacilli, Sarcina, staphylococci and streptococci, and Gram negative ones (Table 4). Results demonstrated again the better inhibition of Gram positive than Gram negative strains, with MIC values in the range 12–50 μg mL− 1 and 25–100 μg mL− 1, respectively. This behaviour is in agreement with the data of Table 3 and with results reported in the literature for other organotins(IV) [24,25]. Compound 8 was found to be the most active complex. In addition, starting SnBu2Cl2, used for comparison, was more potent than its butyltin complexes towards both Gram positive (MICs 1.5–6 μg mL− 1) and Gram negative (MICs 6–25 μg mL− 1) bacteria. On the other hand, it is noteworthy to study the ability of the compounds under investigation to inhibit the development of methicillin-resistant staphylococci.

607

On the whole, B. subtilis, S. lutea and both methicillin-susceptible and methicillin-resistant S. epidermidis strains were the most sensitive Gram positive bacteria, while P. vulgaris was the most sensitive among Gram negative ones. With the aim to detect the kind of antimicrobial activity, the highest dilutions showing 99% inhibition of bacterial and fungal cells were recorded as minimal bactericidal concentrations (MBCs) and minimal fungicidal concentrations (MFCs), respectively. MBC values presented in Tables 3 and 4 range from 6 to N1600 μg mL− 1, whereas MFCs from 400 to N1600 μg mL− 1 (Table 3). In all cases the antimicrobial compounds act in a bacteriostatic and fungistatic manner, being their minimal bactericidal and fungicidal concentrations twofold, or more, higher than the corresponding minimal inhibitory ones. The investigation on the structure–activity relationships shows a relation between the antibacterial activity and the coordination environment of the tin atom. As expected, SnBu2Cl2 and its complexes exhibit better inhibitory properties than SnMe2Cl2 and its derivatives, owing to the more lipophilic character of butyl group that increases the ability of compounds to penetrate the cell membrane and to get the target sites. As concerns the complexes, their effectiveness against all the tested microorganisms is comparatively lower than that of the corresponding alkyltin precursors. This behaviour is presumably due to the condensation of the parent inhibitor compounds with ligand and coligand both inactive. It follows that only butyl complexes 6– 8 are antibacterial agents. Curiously, among these, the incorporation in compound 6 of one (compound 7) or two (compound 8) 4,4′bipyridyl moieties, devoid of activity, results in enhanced inhibitory effect. So, according to the data shown in Tables 3 and 4, compound 8 was the most potent antibacterial complex.

Table 4 Antibacterial activity (expressed as MIC and, in brackets, as MBC, both in μg mL− 1) of the most active compounds against several Gram positive and Gram negative strains. Microorganism

Compounds 6

Gram positive bacteria Bacillus megaterium

50 (400) Bacillus thuringiensis var. kurstaki 50 (800) Sarcina lutea 12 (200) Staphylococcus aureus methicillin-resistant 50 (400) Staphylococcus epidermidis 12 (100) Staphylococcus epidermidis methicillin- 12 resistant (200) Staphylococcus haemolyticus 50 (200) Streptococcus agalactiae 25 (200) Streptococcus faecalis 50 (200) Streptococcus faecium 50 (200) Gram negative bacteria Enterobacter cloacae 100 (400) Klebsiella pneumoniae 100 (1600) Proteus vulgaris 25 (400) Salmonella typhimurium 50 (400) Serratia marcescens 100 (400)

7

8

SnBu2Cl2

25 (200) 50 (200) 12 (100) 50 (200) 12 (100) 12 (100) 25 (100) 25 (200) 50 (200) 50 (200)

25 (200) 25 (200) 12 (100) 25 (200) 12 (100) 12 (100) 25 (100) 25 (100) 25 (200) 25 (200)

3 (6) 6 (50) 1.5 (25) 6 (25) 3 (25) 3 (25) 3 (25) 3 (25) 3 (12) 3 (50)

100 (1600) 100 (1600) 25 (400) 50 (1600) 100 (1600)

100 (800) 100 (800) 25 (400) 50 (800) 50 (800)

12 (200) 25 (200) 6 (50) 6 (25) 12 (200)

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Since structural studies concerning complexes 7 and 8 showed that the bipyridyl moiety was not bonded to tin but released in chloroform or dimethyl sulfoxide solution, our attention was focused on the behaviour of these complexes in the antibacterial test conditions for a more precise evaluation of the detected activity. So, different mixtures were prepared in dimethyl sulfoxide by combining complex 6 and bipyridyl coligand, in the same amounts that appear in complexes 7 and 8. Then additional experiments were performed to study their antimicrobial activity in aqueous culture medium against B. subtilis, S. aureus and E. coli selected bacteria, simultaneously with the corresponding complexes in order to compare them. Mixtures mimicking complex 7 exhibited identical MIC values than 7, while the interaction between compound 6 and coligand mimicking complex 8 exhibited inhibitory activity (MICs 25, 50 and 100 μg mL− 1 towards B. subtilis, S. aureus and E. coli, respectively) lower than that of the corresponding complex. These results prevent a rational assessment of the data concerning complex 7, but clearly suggest that, in the experimental conditions, complete disruption of the complex 8 with the formation of compound 6 and bipyridyl coligand did not take place. 4. Conclusions Some organotin(IV) complexes containing benzil bis(benzoylhydrazone) and different numbers of bipyridyl units have been synthesised and fully characterized. In all the complexes the bis (hydrazone) ligand is doubly deprotonated and behaves as N2O2 tetradentate chelate. The in vitro antimicrobial activity against bacteria and fungi was evaluated. Methyl derivatives are devoid of antimicrobial properties, whereas all butyltin(IV) complexes show a good activity against bacteria that increases with the number of bipyridyl units. Supplementary materials related to this article can be found online at doi:10.1016/j.jinorgbio.2011.01.014. Acknowledgements ELT and MAM thank César J. Pastor for the crystal measurements and to MICINN, Instituto de Salud Carlos III, for funding (Project PS09/ 00963). References [1] B.A. Buck-Koehntop, F. Porcelli, J.L. Lewin, C.J. Cramer, G. Veglia, J. Organomet. Chem. 691 (2006) 1748–17558 and references therein. [2] D. Kovala-Demertzi, P. Tairidou, U. Russo, M. Gielen, Inorg. Chim. Acta 239 (1995) 177–183. [3] M. Gielen, M. Biesemans, R. Willen, Appl. Organomet. Chem. 19 (2005) 440–450. [4] L. Pellerito, L. Nagy, Coord. Chem. Rev. 224 (2002) 111–150. [5] A.J. Crowe, Antitumor activity of tin compounds, in: S.P. Fricker (Ed.), Metal Compounds in Cancer Therapy, Chapman & Hall, London, 1994, pp. 147–179. [6] M. Gielen, Coord. Chem. Rev. 151 (1996) 41–51. [7] M. Gielen, E.R.T. Tiekink (Eds.), Metallotherapeutic Drug and Metal-Based Diagnostic Agents: 50Sn Tin Complexes and Their Therapeutic Potential, Wiley, New York, 2005. [8] M. Jain, S. Nehra, P.C. Trivedi, R.V. Singh, Met.-Based Drugs 9 (2002) 53–60. [9] M. Jain, S. Gaur, V.P. Singh, R.V. Singh, Appl. Organomet. Chem. 18 (2004) 73–82.

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