Synthesis, characterization and antimicrobial activity of a bidentate NS Schiff base of S-benzyl dithiocarbazate and its divalent complexes

Abstract The reaction of S-benzyl dithiocarbazate (SBDTC) with 2,4,5-trimethoxybenzaldehyde afforded a bidentate NS Schiff base 1 (benzyl-3-N-(2,4,5-trimethoxyphenylmethylenehydrazine carbodithioate), which on further reaction with M(II) (where M(II) = nickel(II), zinc(II), palladium(II) and copper(II)) in ethanol under reflux yielded bis-chelated inner complexes [ML2] 2–5 with deprotonated L. The ligand and its complexes were characterized by physicochemical techniques, viz., molar conductance, magnetic susceptibility measurement, IR, NMR, UV–Vis and mass spectroscopic techniques. The crystal structures of 1 and 5 were also determined by single-crystal X-ray crystallography. The crystal structure analysis showed that the ligand exists in its thione tautomeric form. In the complexes, each of the two deprotonated ligands chelated the metal ions through the β-nitrogen and the thione sulfur forming five-membered rings. The copper(II) complex (5) exhibited a square-planar geometry, where the two N2S2 chromophores are arranged trans. All the compounds showed strong antibacterial activity against S.-β-hemolyticus, Klebsiella pneumoni, and Escherichia coli. The compounds also showed strong antifungal activity against Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Candida albicans with the exception of the palladium(II) complex (4) which showed no activity, while all the compounds showed no activity against Fusarium vasinfectum.


Introduction
2,4,5-Trimethoxybenzaldehyde (2,4,5-TMBA) is a natural compound that is present in many plant roots, seeds, and leaves and was reported to be a significant inhibitor of cyclooxygenase-2 (COX-2) and to have antiobesity activity [1]. Sulfur compounds occur in almost all living creatures and have multitude of functions; biotin (Vitamin B7), thiamine, pantethine (Vitamin B complex), etc. taken as nutritional supplements or as food supplements cause many biological functions in the human body. S-benzyl dithiocarbazate (SBDTC), a nitrogen-sulfur donor, has been of immense interest in bioinorganic chemistry since its report [2]. Since then, a significant number of Schiff bases derived from SBDTC have been reported for their versatile coordination chemistry and increasingly important biological activities [3][4][5][6][7][8][9][10][11][12]. The antimicrobial activities of some chalcone and flavone derivatives of 2,4,5-and 2,4,6-trimethoxybenzaldehyde have been reported earlier [13,14]. SBDTC was also reported as a potential antimicrobial agent [10,15]. however, the coordination chemistry of Schiff bases derived from SBDTC with such aldehydes has not been reported. Considering the biological effects of SBDTC and 2,4,5-trimethoxybenzaldehyde, we report herein the antibacterial and antifungal activities of a bidentate Schiff base derived from condensation of SBDTC with 2,4,5-trimethoxy benzaldehyde and some of its bis-chelated complexes containing nickel(II), copper(II), zinc(II), and palladium(II).

Materials and instrumentation
All chemicals and solvents were of reagent grade and used without purification. Chemicals such as hydrazine hydrate (90%), carbon disulfide, potassium hydroxide, and benzyl chloride were purchased from Merck (India) while 2,4,5-trimethoxy benzaldehyde was obtained from himedia (India). The metal salts nickel(II) acetate tetrahydrate, copper(II) acetate monohydrate and zinc(II) acetate dihydrate were obtained from fluka Chemica (Switzerland), while palladium(II) nitrate hydrate was purchased from Sigma-Aldrich (USA). The solvents acetonitrile, chloroform, dichloromethane, DMSO, ethanol, and toluene were obtained from Active fine (Bangladesh). The biological reagents amoxicillin, nystatin, and nutrient agar (NA) were purchased from Sigma-Aldrich (USA). The micro-organisms (bacteria and fungi) of pure culture were primarily collected from the Department of Microbiology, University of Dhaka, the Institute of Nutrition and food Science (INfS), University of Dhaka and the Plant Pathology Laboratory of the Department of Botany, University of Rajshahi, and were further cultured at the Molecular Biology Laboratory, Institute of Biological Sciences, University of Rajshahi.
IR spectra (4000-400 cm −1 ) were obtained as KBr pellets from the Department of Chemistry, Rajshahi University of engineering & Technology (RUeT) using an IR Affinity 1S spectrophotometer, Shimadzu (Japan). 1 h-NMR spectra were recorded on a Bruker Ultra-Shield TM spectrometer (400 Mhz) in CDCl 3 using TMS as internal standard from 0 to 13 ppm at BCSIR, Dhaka (Bangladesh). Mass spectra (m/z: 0-1200) were obtained on a JeOL-JMS-D300 mass spectrometer from the Department of Applied Chemistry, University of Toyama (Japan). Magnetic susceptibility and molar conductance measurements were made on a magnetic susceptibility balance (Sherwood Scientific (UK) and an eCOSCAN CON5 conductivity/temperature meter (eutech Instruments, Singapore, Serial No. 101886) from the Department of Chemistry (RUeT). UV-vis spectra were recorded on a T60 UV-vis spectrophotometer (PG Instruments, UK) programmed with Win5 software, version 5.1, between 200 and 1100 nm using 10 −5 M solution in dichloromethane at the Department of Chemistry, RUeT.

X-ray crystal structure determination
Intensity measurements for the structures reported were carried out at a temperature of 173(1) K on a Rigaku R-AXIS RAPID diffractometer using filtered Cu-Kα radiation (λ = 1.54187 Å). All the structures were solved by direct methods [16] and successive fourier syntheses and refined by the full-matrix least-squares method based on F 2 with all observed reflections [17]. hydrogens were geometrically located and refined using the riding model. All calculations were performed using the Crystal Structure package [18] except for refinement for which SheLXL-97 was used [17]. Crystal data and details of refinement of the structures reported are summarized in table 1, while selected bond lengths and  angles are reported in table 2.   Table 1. Crystallographic data, experimental data, and structure refinement details for 1 and 5.

Preparation of the Schiff base (1)
The Schiff base was prepared following our previous report [19]

General method of preparation of 2-5
Complexes 2-5 were prepared by condensation of the Schiff base with nickel(II) acetate tetrahydrate, copper(II) acetate monohydrate, zinc(II) acetate dihydrate, and palladium(II) nitrate hydrate in ethanol in a 2 : 1 ligand to metal molar ratio under reflux for 0.25 to 1.5 h. The individual products were separated, washed with fresh ethanol, and dried in vacuum over anhydrous CaCl 2 . All the complexes were purified by crystallization from a mixture of chloroform and toluene (100 : 1; v/v). Only 5 was obtained as single-crystals suitable for X-ray diffraction analysis by crystallization from a mixture of hot dichloromethane and methanol (3 : 1 v/v). Physicochemical and spectroscopic characterization of the synthesized compounds are listed below:

Quantitative antimicrobial assay
The compound that showed the highest antibacterial activity (2) (inhibition diameter > 25 mm) was subjected to the broth dilution method for quantitative measurement of microbiostatic (inhibitory) activity. The serial dilution technique was followed using nutrient broth medium of Reiner [21] to determine the MIC (minimum inhibitory concentration) values of the test compound against S. β-hemolyticus, S. dysenteriae and E. coli. The selected compound was taken into different vials in a fixed amount (2 mg), and then broth medium 1 mL was added to each of the vials and agitated well to make sample solution whose concentration became 0.13 μML −1 . The standard antibiotic Amoxicillin solution 0.14 μML −1 was used for comparison. The lowest concentration that completely inhibited visible microbial growth was recorded as the minimum inhibitory concentration (MIC, μML −1 ). Amoxicillin was used as positive control for the bacteria.

Determination of stability constant and composition of the complexes
In order to determine the stability constant and composition of complexes in solution, 5 was considered as an example. The composition of the copper(II) complex was determined by Job's Method or continuous variation method at room temperature (25 °C) [22].

Preparation of 0.001 M Cu(OAc) 2 ·H 2 O
Cu(OAc)2·h2O (20 mg, 0001 M) was dissolved in 95% aqueous solution of DMSO and made up to 100 mL in a volumetric flask at room temperature.

Preparation of 0.001 M ligand solution (1)
The ligand (95 mg, 0.001 M) was dissolved in 95% aqueous solution of DMSO and made up to 100 mL in a volumetric flask at room temperature.

Selection of wavelength for photometric analysis
The wavelength selection of photometric measurement for the determination of stability constant of the complex and composition of ligand in the complex by continuous variation method was performed on a UV-visible spectrophotometer from 200 to 1100 nm for metal salt (Cu(OAc), ligand and Cu(II) complex using the scan spectrum. from the data (table 4), the wavelength 445 nm was selected for measuring the absorbance of test solutions by continuous variation.

Procedure for continuous variation method
The ligand to metal ratio (composition) in solution of 5 was determined by continuous variation method (Job's method) of equimolar solutions [22]. The metal salt solution (0.001 M) was gradually pipetted (9, 8, 7, ….0 mL) into 10 volumetric flasks (10 mL capacity) in which the ligand solution was previously added (1, 2, 3………10 mL), respectively, keeping the total volume constant. The aliquot was shaken for 1 h for the formation of the complex. All the measurements were made at 445 nm at room temperature. The data obtained (table 5) were plotted against mole fraction of the ligand (X L ) (figure 1) from which the mole fraction of the ligand at maximum absorbance was measured. The composition of the ligand in the complex (ML n ) was calculated using the formula n = (X L ) / (1 − X L ).

Syntheses and characterization
The Schiff base (1)

IR spectra
The ligand 1 has a thione group [-Nh-C(=S)-] with an adjacent imide proton that can exhibit tautomerism to the thiol form [23]. The IR spectrum of 1 does not show the ν(S-h) band at 2570 cm −1 , but exhibited a strong band at 3103 cm −1 for the ν (N-h), indicating that in solid state, the Schiff base is in the thione tautomeric form [19,21]. The absence of this band in the complexes indicates the presence of the deprotonated form and conjugation of the -C=N-N=C group [24][25][26]. Another strong band at 1097 cm −1 in 1 was assigned to ν(C=S), the absence of which in the complexes suggested coordination of the ligand in the thiolate form [23][24][25]. These clearly indicate extraction of the acidic Nh proton by acetate and nitrate ions of the metal salts, generating the anionic ligand during complexation. In addition, the ν(C=N) band observed at 1595 cm −1 in 1 shifts to lower wavenumbers in 2-5, as evidence for the coordination of 1 via the azomethine nitrogen [23][24][25]. 1 h NMR data are given in the experimental Section and the atom numbering scheme of the Schiff base is depicted in figure 2. The 1 h NMR spectrum of 1 in CDCl 3 showed a singlet at 10.18 ppm for the -Nh(C=S) proton [24,25]. This supports the presence of the thione form of the ligand even in solution.

NMR spectra
The absence of this band in 2-4 indicated deprotonation and the thiolate form of 1 in the complexes [23][24][25]. The methine proton observed at 8.19 ppm in the ligand shifts downfield after complexation (ca. 8.38-9.09 ppm), indicating coordination via the azomethine nitrogen [23][24][25]. The ligand showed two singlets at 3.81 and 3.89 ppm for the OCh 3 protons, where one of the methoxy groups overlaps at 3.81 ppm. Another singlet at 4.55 ppm and a multiplet at 7.27-7.41 ppm for SCh 2 and phenyl protons, respectively, of the benzyl group were observed in 1. All these bands showed slight variation upon complexation. The ligand showed two singlets at 6.42 and 7.77 ppm, tentatively assigned to phenyl protons of the trimethoxy benzaldehyde ring. These signals were shielded in the complexes.

Mass spectroscopic analyses
The

Crystal structures of 1 and 5
The molecular and crystal structures of 1 and 5 have been determined by single-crystal X-ray diffraction analysis. The ligand crystallizes in the monoclinic system with space group P2 1 /c. An ORTeP of the molecule is shown in figure 2 and table 2 lists a selection of bond distances and angles which are compared to the corresponding geometrical parameters measured in 5. In 1, the dithiocarbazate group adopts an EE configuration with respect to the C=N bond of the benzylidene group. The β-nitrogen and the thioketo sulfur are trans to the C(11)-N(2) bond. The molecule is in its thione tautomeric form with C(11)=S(1) bond length of 1.670(15) Å and the entire species has coplanar atoms with the exception of the S-benzyl phenyl ring, indicating electron delocalization within it. The crystal packing shows ligand molecules are connected by pairs of N-h⋯S hydrogen bonds (h⋯S = 2.496 Å, N-h⋯S angle = 171.57°) forming a dimer. C-h⋯π interactions are also observed in the crystal, linking the dimers. No appreciable π-π interaction among aromatic rings is present in the crystal packing.
The free ligand requires rotation about C(11)-N(2) by 180° to allow the N,S-chelating coordination to copper. The bis-chelated Cu(II) complex crystallizes in the monoclinic system with space group C2/c. Coordination bond lengths and angles are reported in table 2. An ORTeP drawing of 5 is depicted in figure 3. Copper(II) is located on a crystallographic twofold axis with a square-planar geometry. The two Schiff bases, in their deprotonated imino thiolate form, are coordinated to the metal center via the azomethine N(1) and thiolate S(1) in a trans-planar configuration. The molecule is also almost planar with the exception of the S-benzyl phenyl ring, indicating electron delocalization within it.
The crystal packing of 5 shows complexes arranged in the b-plane and the Cu(II) located on the crystallographic inversion center, leading to a trans configuration of the N,S-chelating ligands.

Antimicrobial screening
The Schiff base 1 and 2-5 were assayed for their in vitro antimicrobial activity against thirteen pathogenic micro-organisms by the disk diffusion method. Their corresponding test results are reported in table 3. As evident from the table, none of the tested compounds show bactericidal activity against S. aureus and P. aeruginosa. These results are comparable to the inactivity of octahedral Ni(II) and Cu(II) complexes against the latter [33]. In contrast, some square planar and octahedral complexes containing N,O-donor ligands of the same metals showed moderate-to-strong activity against these organisms [34][35][36]. On the other hand, all the tested compounds exhibited strong antibacterial activity against B. cereus, S. β-hemolyticus, K. pneumoni and E. coli (table 3), significantly higher than the flavone derivatives 2,4,5-trimethoxybenzaldehyde [13] and also better than previous reports [33][34][35][36]. however, although the Schiff base showed no activity against S. dysenteriae, its nickel(II) and copper(II) complexes (3 and 5) showed strong activity, comparable to the activity of oxo-bridged dimeric Ni(II) and Cu(II) complexes of square-planar geometry [35]. Similarly, all the complexes (except the palladium(II) complex) exhibited strong activity against S. lutea, compared to moderate activity of the ligand. The presence of metal centers has enhanced the antibacterial activity of 1. Unlike the antibacterial activity, the palladium(II) complex 4 showed no activity against fungi, while the remaining complexes exhibited strong antifungal activity against all the fungi (except f.v.), sufficiently stronger than the bischelated octahedral [ML 2 ] (M = Ni, Cu, Zn) complexes containing 3-hydrazinoquionoxaline-2-one and 12-diphenylethane-12-dione [36]. Although most of the compounds exhibited strong activity, only 2 was subjected to quantitative antibacterial assay against S. β-hemolyticus, K. pneumoni and E. coli using serial dilution method as described in the experimental Section. The MIC values of the nickel(II) complex against the test organisms (64, 32 and 128 μg/mL) indicate that it is more active than amoxicillin. The quantitative study of the other complexes against the test micro-organisms are still under investigation.

Composition study and stability constant of 5
The data obtained by continuous variation method (table 3) suggested that the metal to ligand ratio in 5 is 1 : 2 (n = 2.33). Thus, the general equation for the formation of the complex is:  Table 3 indicates that the stability constant (log β) of 5 varies from 5.07 to 8.38 in different solutions, which is maximum at absorbance of 1.931. however, these values are lower than the mixed ligand Cu(II) complexes of cetrizine and alanine [33]. Thus, further study is required for the determination of the stability constant. however, the stability constants of 2-4 were not determined as their metal salts have been depleted.

Conclusion
The ligand 1 afforded bis-chelated neutral complexes [ML 2 ] with Ni(II), Cu(II), Zn(II) and Pd(II) acting in a uninegative bidentate mode with its NS-donor set. The ligand exists predominantly in thione tautomeric form both in solution and in the solid state, with thiol form and subsequent deprotonation in the presence of metal center. Both the ligand and its complexes exhibited strong antimicrobial activities, even stronger than the standard drug amoxicillin. The biological activity of metal complexes depends on various factors, such as (i) number of chelate rings, (ii) number of bacterial cell walls, (iii) delocalization of π electrons through the molecule, (iv) compound polarity and (v) stability constant [33,37]. Thus, in order to determine the order of activity of the complexes, further study is required.

Supplementary data
CCDC 1434504 and 1057712 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2 1eZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data associated with this article can be found in the online version.