Organotin ( IV ) Complexes with 2-Acetylpyridine Benzoyl Hydrazones : Antimicrobial Activity

Reações de tricloreto de n-butilestanho, [BuSnCl 3 ], e tricloreto de fenilestanho, [PhSnCl 3 ], com 2-acetilpiridina benzoil hidrazona (H2AcPh), 2-acetilpiridina-para-cloro-benzoil hidrazona (H2AcpClPh) e 2-acetilpiridina-para-nitro benzoil hidrazona (H2AcpNO 2 Ph) produziram os complexos [BuSn(2AcPh)Cl 2 ] (1), [BuSn(2AcpClPh)Cl 2 ] (2), [BuSn(2AcpNO 2 Ph)Cl 2 ] (3), [PhSn(2AcPh)Cl 2 ] (4), [PhSn(2AcpClPh)Cl 2 ] (5) e [PhSn(2AcpNO 2 Ph)Cl 2 ] (6). H2AcpClPh revelou-se a hidrazona mais ativa contra Staphylococcus aureus e Candida albicans. Pela coordenação a atividade antibacteriana tanto do metal quanto das hidrazonas aumenta significativamente. Os complexos 2 e 5 revelaram-se os mais ativos como agentes antimicrobianos.


Introduction
Hydrazones are a versatile class of compounds which present a wide range of biological applications as antimicrobial, 1 antitubecular, 2 anticonvulsant, 3 antiinflammatory, 4 cytotoxic 5 and vasodilator 6 agents.Moreover, 9-acridanone-hydrazones proved to be active against Schistosoma mansoni 7 and quinoxaline-Nacylhydrazones showed trypanocidal activity. 8In addition, methyl pyrazinylketone isonicotinoyl hydrazones have been synthesized in an attempt to develop novel chelators with high affinity for iron for the treatment of iron overload disease. 9ydrazones also proved to be useful as sensitive analytical reagents for the determination of trace amounts of metal ions. 10,11Metal complexes with hydrazones have potential applications as catalysts, 12 luminescent probes, 13 and molecular sensors. 14Moreover, metal complexes with hydrazones present antimicrobial, [15][16][17] DNA-binding and cytotoxic activities. 17It has also been shown that metal complexes with hydrazones can be potent inhibitors of cell growth and DNA syntheses. 18in compounds present applications as PVC stabilizers, 19 for chemical vapor decomposition (CVD), 20 in non-linear optics 21 and in catalyses. 22Organotin complexes present innumerous pharmacological applications as antitumorals, 23 antimicrobials 24,25 and biocides. 26The cytotoxic activity of a variety of organotin complexes against tumor cell lines has been demonstrated by some of us. 27,280][31] In the present paper organotin(IV) complexes were prepared by reacting [Bu n SnCl 3 ] and [PhSnCl 3 ] with 2-acetylpyridine-benzoyl hydrazone (H2AcPh), 2-acetylpyridine para-chlorobenzoyl hydrazone (H2AcpClPh) and 2-acetylpyridine Experimental Apparatus Partial elemental analyses were performed on a Perkin Elmer CHN 2400 analyzer.Infrared spectra were recorded on a Perkin Elmer FT-IR Spectrum GX spectrometer using CsI pellets; an YSI model 31 conductivity bridge was employed for molar conductivity measurements; NMR spectra were obtained with a Bruker DPX-200 (200 MHz) spectrometer or with a Bruker DRX-400 Avance (400 MHz) spectrometer using deuterated dimethylsulfoxide (dmso-d 6 ) or deuterated chloroform (CDCl 3 ).The 1 H and 13 C NMR chemical shifts in ppm are reported from internal tetramethylsilane (tms) on the % scale.The 119 Sn NMR spectra were measured relative to Sn(CH 3 ) 4 .Bruker standard experiments were performed using decoupling sequences for 13 C and 119 Sn.Splitting patterns are designated as follows: s singlet; d doublet; t triplet; q quartet; m multiplet.

Syntheses of 2-acetylpyridine-benzoyllhydrazone (H2AcPh), 2-acetylpyridine-para-chloro-benzoyl hydrazone (H2AcpClPh) and 2-acetylpyridine-para-nitro-benzoyl hydrazone (H2AcpNO 2 Ph)
2-Acetylpyridine-benzoyl hydrazone (H2AcPh) and 2-acetylpyridine-para-chloro-benzoyl hydrazone (H2AcpClPh) were prepared as described in the literature. 32,332-Acetylpyridine-para-nitro-benzoyl hydrazone (H2AcpNO 2 Ph) was obtained by mixing equimolar amounts of 2-acetylpyridine with the para-nitrobenzoyl hydrazide in methanol with addition of three drops of acetic acid as catalyst.The reaction mixture was kept under reflux for 6 h.After cooling to room temperature the resulting solid was filtered off, washed with ethanol and ether and dried in vaccuum.The organotin(IV) complexes were obtained by refluxing an ethanol solution of the desired hydrazone with [Bu n SnCl 3 ] or [PhSnCl 3 ] in 1:1 ligand-to-metal molar ratio.The solids were washed with ethanol followed by diethylether and then dried in vaccuum.Crystals of complexes 1, 2, 3 and 5 were obtained from their ethanolic solutions and were stable in the air.Slow evaporation of 6 in 1:9 dmso:acetone solution gave crystals of [PhSn(2AcpNO 2 Ph)Cl 2 ] • dmso, (6a).• dmso (6a) were mounted on glass fibers and used for data collection.X-ray diffraction data collection was performed on a Oxford-Diffraction GEMINI diffractometer (LabCri) using graphite-Enhance Source MoK a radiation (l = 0.71069 Å) at 293(2) K. Data integration and scaling of the reflections were performed with the Crysalis suite. 34Final unit cell parameters were based on the fitting of all reflections positions.The structures were solved by direct methods using the program SHELXS-97 35 and refined by full-matrix least-squares techniques against F 2 using SHELXL-97. 36Positional and anisotropic atomic displacement parameters were refined for non hydrogen atoms, except for those involved in disorder systems for which isotropic ADP was used.Although some hydrogen atoms could be identified in a Fourier difference map, in the final model they were geometrically positioned and refined using a riding model.Criteria of a satisfactory complete analysis were the ratios of rms shift to standard deviation less than 0.001 and no significant features in final difference maps.Molecular graphics were obtained from ORTEP. 37 summary of the crystal's data, experimental details and refinement results are listed in Table 1.

In vitro antimicrobial activity
Antimicrobial activity was evaluated by minimum inhibitory concentration (MIC) using the macrodilution test. 38 (BHI) broth were subcultured for testing in the same medium and grown at 37 °C.Then the bacterial cells were suspended in BHI, according to Clinical and Laboratory Standards Institute (CLSI) guidelines, 39 to produce a suspension of about 10 5 CFU (colony forming units) mL -1 .C. albicans ATCC18804 stored in Sabouraud broth was sub-cultured for testing in the same medium and grown at 37 °C.Then the yeast cells were suspended according to the McFarland protocol 39 in saline solution to produce a suspension of about 10 3 cells mL -1 .For both the antibacterial and antifungal activity evaluations serial dilutions of the compounds, previously dissolved in dmso, were prepared in test tubes.A 24-h old inoculum (100 mL) was added to each tube.The MIC, defined as the lowest concentration of the test compound which inhibits the visible growth after 20 h, was determined after incubation at 37 °C.Tests using tetracycline (in the case of S. aureus) or fluconazole (in the case of C. albicans) as reference and dmso as negative control were carried out in parallel.The final dmso concentration never exceeded 1%.In all cases no inhibition was observed with 5% v/v dmso.All tests were performed in triplicates with full agreement between the results.6), in which an anionic hydrazone is attached to the metal centre along with a n-butyl or a phenyl group and the remaining coordination sites are occupied by two chloride ions.X-ray diffraction analysis confirms the structures of 1-4 and 6 (as its dmso solvate, [PhSn(2AcpNO 2 Ph)Cl 2 ] • dmso, 6a).

Infrared spectra
The n(N-H) and n(C=O) absorptions at 3180-3287 cm -1 and 1655-1676 cm -1 in the spectra of the uncomplexed hydrazones were not found in the spectra of complexes (1-6), in accordance with the presence of anionic ligands in the products.
The n(C=C) + n(C=N) composed mode observed at 1590-1460 cm -1 in the spectra of the hydrazones undergoes small shifts in the spectra of the complexes.However a new n(C=N) absorption due to the formation of a second C=N bond was observed at 1625-1632 cm -1 in the spectra of 1-6, indicating coordination of an anionic hydrazone.
][31] In addition, new absorptions at 495-475 cm -1 and 450-350 cm -1 have been attributed to n(Sn-N imine ) and n(Sn-C) respectively; absorptions at 230-215 cm −1 were assigned to n(Sn-N py ), 25,28,40 and bands in the 405-390 cm -1 range have been assigned to n(Sn-O).An absorption at 260-250 cm -1 has been attributed to n(Sn-Cl). 25,28,40Therefore, the infrared data for the complexes indicate coordination of the hydrazones through the N py -N-O chelating system, as confirmed by the X-ray structure determinations for 1-4 and 6a.

NMR spectra
The 1 H and 13 C NMR spectra of the hydrazones were recorded in dmso-d 6 as well as in CDCl 3 except in the case of H2AcpNO 2 Ph, which is insoluble in CDCl 3 .The spectra of complexes 1-6 were recorded only in CDCl 3 since some of them interact with dmso-d 6 .
The 1 H resonances were assigned on the basis of chemical shifts, multiplicities and coupling constants.The carbon type (C, CH) was determined by using distortionless enhancement by polarization transfer (DEPT135) experiments.The assignments of the protonated carbons were made by 2D heteronuclear multiple quantum coherence experiments (HMQC).A 119 Sn NMR study was performed for all complexes.
The NMR spectra of H2AcPh in CDCl 3 show only one signal for each hydrogen and each carbon, indicating the presence of only one configurational isomer.The signal of N3-H was observed at d 9.22, suggesting the presence of the E isomer.The spectra recorded after 24 h do not show any changes.0][31] The signals of N3-H at d 10.90 and d 15.80 were attributed to the E and Z isomers respectively (97% of E and 3% of Z).][31] H2AcpNO 2 Ph is soluble in dmso-d 6 but insoluble in CDCl 3 .In the 1 H and 13 C NMR spectra (dmso-d 6 ) of H2AcpNO 2 Ph only one signal has been observed for each hydrogen and each carbon.][31] The 1 H and 13 C NMR spectra of H2AcpClPh in dmso-d 6 show duplicated signals for the hydrogens, in accordance with the presence of two configurational isomers.0][31] Due to the low solubility of H2AcpClPh in dmso-d 6 only some of the signals of hydrogens could be attributed and no attribution was possible for the carbons of the Z isomer.
The spectra of this compound in CDCl 3 also show duplicated signals.However in this case the spectra are characteristic of the presence of the keto and enol forms. 41n fact no signal of N3-H was observed for the first isomer, according to the presence of the enol form.In addition, the signal of C-O was observed at d 167.Only one signal was observed for each hydrogen and each carbon in the spectra of complexes 1-6, suggesting the presence of only one configurational isomer.The signals of C=N, C=O and the pyridine carbons undergo significant shifts in the complexes indicating coordination through the N py -N-O chelating system.Hence the hydrazones adopt the E configuration in the complexes, as confirmed by crystal structure determinations of complexes 1-4 and 6a.Moreover, the signal of N3-H was not observed in the 1 H NMR spectra of the complexes, according to deprotonation of the hydrazones upon coordination.
In the spectra of complexes 1-3, new signals at d 2.35-1.05and at d 35.80-13.60 were attributed to the hydrogens and carbons, respectively, of the n-butyl group attached to the metal. 28In the spectra of complexes 4-6, the signals at d 8.33-7.59 and at d 136.62-129.08 were attributed to the hydrogens and carbons, respectively, of the phenyl group coordinated to tin. 40n the 119  The values of the 119 Sn signals are at the boundary between six-and seven-coordinated tin(IV) as reported in the literature. 42However, considering that the spectra were recorded in a non-coordinating solvent (CDCl 3 ), only six-coordinated species must be present in solution.The shielding effect on 119 Sn of the chloride ions could explain the lower frequencies observed for the 119 Sn signals with respect to that expected for a six-coordinated monorganotin(IV).    2 and 3.The distances and angles in all complexes are similar.In all complexes tin(IV) is coordinated to a tridentate anionic ligand, a n-butyl (1-3) or a phenyl (4 and 6a) group and two chloride ions trans to each other in an distorted octahedral environment.In the crystal structures of 3 and 4, two symmetrically independent molecules were found in the asymmetric unit (see Figure 2 and Figure 3).
In complexes 1-4 and 6a the N1-Sn-O1 angle of ca.144.3-146.7°deviates markedly from the ideal value of 180°, probably due to the spatial requirements of the ligand chelating system.By contrast, the Cl1-Sn-Cl2 and N2-Sn-C16 angles, which do not involve sterical hindrance, are approximately 167.3-171.4°and 172.5-179.1°(see Table 3).In all complexes the metal atom lies in the plane calculated for the N1N2O1C16 atoms.In structures 1-3 the distance of the tin atom to the calculated plane varies from 0.023Å to 0.059 Å; in structures 4 and 6a this distance is smaller (0.002 to 0.009 Å).
In all structures the hydrazone skeleton (C7(C15) N2N3C8O1) is planar, with medium deviation of the fitted atoms from the calculated plane ranging from 0.0055 to 0.0266 Å.In 1-4 and 6a the hydrazone adopts the E configuration in relation to the C7=N2 double bond.The angle between the pyridine and the hydrazone planes varies from 1.5(6) to 7.5(3)°, while the angle between the hydrazone and the phenyl planes varies in the 0.5(2) to 14.9(2)° range.Therefore the hydrazone ligand in all complexes is almost planar.Complexes 3 and 6a show the highest deviation from planarity for the angle between the hydrazone chain and the phenyl ring.
The molecular packings of all complexes show weak interactions involving Cl, H and O atoms with formation of molecular chains as shown in Figure 4. 1-4 show interactions between adjacent molecules involving Cl … H py .In 4 and 6a short Cl … H contacts are observed involving chlorides and hydrogens from the hydrazone phenyl ring.In 6a Cl … H interactions were observed as well, along with short H … O contacts involving the oxygens from dmso and from the nitro group and hydrogens from pyridine and dmso.

Antimicrobial activity
The minimum inhibitory concentrations (MICs) of the hydrazones and their organotin complexes against S. aureus and C. albicans are reported in Table 4.Among the hydrazones the antibacterial activity follows the order H2AcPh < H2AcpNO 2 Ph < H2AcpClPh, suggesting that the electron-withdrawing effect of the nitro and chloro groups probably favors the antibacterial effect.The calculated values of log P 45 were: 1.98 for H2AcPh, 2.58 for H2AcpClPh and 1.92 for H2AcpNO 2 Ph.Therefore the higher lipophilicity of H2AcpClPh probably accounts in part for its higher activity.
[Bu n SnCl 3 ] proved to be inactive against S. aureus while [PhSnCl 3 ] exhibited antibacterial activity in the assayed concentrations.The hydrazones showed antibacterial activity with MIC values similar to the phenyltin salt.Upon coordination the antibacterial activities of both the hydrazones and tin substantially increase.A synergistic effect involving tin and the hydrazone might be responsible for this enhancement.
The complex of H2AcPh with phenyltin is more active than its n-butyltin analogue, but the n-butyltin and phenyltin complexes with the other two hydrazones are equally active.The activities of the n-butyltin complexes with the different hydrazones follows the order [Bu n Sn(2AcPh)Cl 2 ] < [Bu n Sn(2AcpNO 2 Ph)Cl 2 ] < [Bu n Sn(2AcpClPh)Cl 2 ], which is the same order of activity of the free hydrazones.However, the activities of the phenyltin complexes are [PhSn(2AcPh)Cl 2 ] = [PhSn(2AcpNO 2 Ph)Cl 2 ] < [PhSn(2AcpClPh)Cl 2 ].The most active complexes were those with H2AcpClPh (2 and 5), with MIC values similar to that determined for tetracycline.
Among the hydrazones H2AcpClPh exhibited the highest activity against C. albicans.Upon coordination the antifungal activity of the hydrazones increased in complexes 3 and 6, but remained the same in the other cases.The antifungal activity increased in complexes 2, 3, 5 and 6 relative to the organotin salts.
Like in the case of the antibacterial activity, the best results were obtained for the organotin complexes with H2AcpClPh.The MIC values for complexes 2 and 5 (0.045 mmol mL -1 ) were the same, suggesting that the n-butyl or the phenyl group in the metal coordination sphere have the same effect.This value of MIC is of the same order of magnitude of that obtained for fluconazole.Likewise, the MIC values are the same for complexes 3 and 6.

Conclusions
Organotin compounds are known for their innumerous bioactivities but are also often rather toxic. 46,47Coordination of organotin salts with organic ligands could be an interesting strategy for activity improvement 48 or toxicity reduction.In the present work we demonstrated that coordination of organotin salts to hydrazones might improve the pharmacological profile of both metal and ligands. 49
Microanalyses and molar conductivity data suggest the formation of the following complexes: [Bu n Sn(2AcPh)Cl 2 ] (1), [Bu n Sn(2AcpClPh)Cl 2 ] (2), [Bu n Sn(2AcpNO 2 Ph)Cl 2 ] (3), [PhSn(2AcPh)Cl 2 ] (4), [PhSn(2AcpClPh)Cl 2 ] (5) and [PhSn(2AcpNO 2 Ph)Cl 2 ] ( 44.The resemblance in position of the carbon signals of H2AcpClPh with those of H2AcPh in the same solvent suggests that the enol isomer adopts the E configuration.As an example, the signal of C15 (acetyl) was observed at d 11.36, close to the signal for the same hydrogen at d 10.89 observed for the E isomer of H2AcPh in CDCl 3 .The keto isomer exhibits the signal of N3-H at d 15.83, characteristic of the Z configuration, and the signal of C=O at d 163.40.

Figures 2
Figures 2 and 3 are perspective views of [Bu n Sn(2AcPh)Cl 2 ] (1), [Bu n Sn(2AcpClPh)Cl 2 ] (2), [Bu n Sn(2AcpNO 2 Ph)Cl 2 ] (3), [PhSn(2AcPh)Cl 2 ] (4) and [PhSn(2AcpNO 2 Ph)Cl 2 ] • dmso (6a).Selected intramolecular bond distances and angles of the complexes are given in Tables2 and 3.The distances and angles in all complexes are similar.In all complexes tin(IV) is coordinated to a tridentate anionic ligand, a n-butyl (1-3) or a phenyl (4 and 6a) group and two chloride ions trans to each other in an distorted octahedral environment.In the crystal structures of 3 and 4, two symmetrically independent molecules were found in the asymmetric unit (see Figure2and Figure3).In complexes 1-4 and 6a the N1-Sn-O1 angle of ca.144.3-146.7°deviates markedly from the ideal value of 180°, probably due to the spatial requirements of the ligand chelating system.By contrast, the Cl1-Sn-Cl2 and N2-Sn-C16 angles, which do not involve sterical hindrance, are approximately 167.3-171.4°and 172.5-179.1°(see Table3).In all complexes the metal atom lies in the plane calculated for the N1N2O1C16 atoms.In structures 1-3 the distance of the tin atom to the calculated plane varies from 0.023Å to 0.059 Å; in structures 4 and 6a this distance is smaller (0.002 to 0.009 Å).In all structures the hydrazone skeleton (C7(C15) N2N3C8O1) is planar, with medium deviation of the fitted atoms from the calculated plane ranging from 0.0055 to 0.0266 Å.In 1-4 and 6a the hydrazone adopts the E configuration in relation to the C7=N2 double bond.The

Table 4 .
Minimum inhibitory concentrations (MIC) of the hydrazones and their organotin complexes against Staphylococcus aureus and Candida albicans