Structures, antimicrobial activity, DNA interaction and molecular docking studies of sulfamethoxazolyl-azo-acetylacetone and its nickel(II) complex

Polyhedron 99 (2015) 77–86

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Structures, antimicrobial activity, DNA interaction and molecular docking studies of sulfamethoxazolyl-azo-acetylacetone and its nickel(II) complex Dipankar Das a,1, Nilima Sahu a, Sudipa Mondal a, Suman Roy a, Paramita Dutta a, Suvroma Gupta b, Tapan Kumar Mondal a, Chittaranjan Sinha a,⇑ a b

Department of Chemistry, Inorganic Chemistry Section, Jadavpur University, Kolkata 700032, India Department of Biotechnology, Haldia Institute of Technology, Haldia, Purba Medinipur, West Bengal 721657, India

a r t i c l e

i n f o

Article history: Received 25 April 2015 Accepted 7 June 2015 Available online 30 June 2015 Keywords: SMX-N@N-acacH (HL) and [Ni(L)2(H2O)4] X-ray structures and theoretical computation DNA interaction Antibacterial properties Docking studies

a b s t r a c t 4-(Z)-((2-Hydroxy-4-oxopent-2-en-3-yl)diazenyl)-N-(5-methylisoxazol-3-yl)benzene sulfonamide (HL) and its nickel(II) complex [Ni(L)2(H2O)4] have been characterized by spectroscopic and single crystal X-ray diffraction measurements. Time dependent DFT computations have been used to explain the electronic spectra of the compounds. The interaction of CT DNA with [Ni(L)2(H2O)4] (Kb, 12.20  105 M1) is stronger than with HL (Kb, 6.09  105 M1). The antimicrobial activity of HL and the Ni(II) complex has been examined against Bacillus subtilis (ATCC 6633; IC50: 63.72 lg/ml (HL) and 81.49 lg/ml ([Ni(L)2(H2O)4])) and Escherichia coli (ATCC 8739; IC50: 77.25 lg/ml (HL) and 78.28 lg/ml ([Ni(L)2(H2O)4])). The in-silico test of HL with DHPS protein from E. coli helps in understanding the drug metabolism and has explained the drug–molecule interaction. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Sulfamethoxazole (SMX) is a useful antibiotic in the treatment of different microbial infections [1–8]. However, long use of SMX induces allergic reactions to skin, stomach, lungs, abdomen, kidney, liver, blood etc., [9–13] and hepato-toxicity, lymphadenopathy and haematological disorders [14]. The hypersensitivity is assumed to be the result of oxidative metabolites of SMX such as SMX-hydroxylamine (SMX-NHOH) and SMX-nitroso (SMX-NO) [15]. In order to overcome these difficulties and to improve drug activity, functionalized sulfonamides and their metal complexes [16,17] are in clinical trials. Azo dyes of sulfa drugs are well known for their antiseptic activity [18]. A large number of metal sulfonamide complexes are found to be more potent than the parent drugs [19]. Furthermore, azo compounds were reported to show a variety of biological activities, including antibacterial [20], antifungal [21], antiviral [22] and anti-inflammatory [23] activities. In this work, we have carried out coupling of the sulfamethoxazolyl-diazonium salt with acetylacetone to synthesize 4-(Z)-((2-hydroxy-4-oxopent-2-en-3yl)diazenyl)-N-(5-methylisoxazol-3-yl)benzene sulfonamide (HL),

⇑ Corresponding author. Fax: +91 2414 6584. E-mail address: [email protected] (C. Sinha). Present address: Greater Kolkata College of Engineering and Management, Ramnagar-II, Piyali Town, Baruipur, 24 Pgs(S), West Bengal 743387, India. 1

http://dx.doi.org/10.1016/j.poly.2015.06.027 0277-5387/Ó 2015 Elsevier Ltd. All rights reserved.

which has been used to synthesize the nickel(II) complex [Ni(L)2(H2O)4]. The complex was characterized by spectroscopic, thermal, magnetic and electrochemical data. The single crystal Xray diffraction technique was used to confirm the structures of HL and [Ni(L)2(H2O)4]. The electronic properties have been computed from DFT and TD-DFT data of optimized structures of HL and the Ni(II) complex. The interaction of HL and [Ni(L)2(H2O)4] with CTDNA were followed by spectroscopic data. The antimicrobial activities of these compounds were examined with Escherichia coli (Gram negative) and Bacillus subtilis (Gram positive) bacteria. The in-silico test of HL with DHPS protein (Protein Data Bank) from E. coli has been used to define the zone of interaction and efficiency of the drugs. The protein crystal structure of B. subtilis is not available in PDB and hence theoretical dealings could not be accounted. 2. Experimental 2.1. Materials Sulfamethoxazole (SMX) was purchased from Sigma–Aldrich Chemical Company and used without further purification. Acetylacetone and Ni(OAc)24H2O were purchased from Merck, India. The acetonitrile used for electrochemical studies was dried with CaH2 and distilled prior to use. The CT DNA was purchased from Sisco Research Laboratories, India, and dissolved in phosphate buffer (pH 7.4) containing 120 mM NaCl (AR grade, Merck,

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Germany). The purity of DNA was checked from the absorbance ratio A260/A280 [24]. The concentration of CT DNA in terms of nucleotide was determined by taking e260 = 6600 mol1 cm1. NaCl (0.500 M dm3) (AR grade, Merck, Germany) was used to maintain the ionic strength.

8.12 s cm2 M1 in DMF, supports a non-ionic complex. Spectra of both the ligand and complex are given as Supplementary material (Figs. S2–S5).

2.2. Physical measurements

The single crystals of HL (0.36  0.28  0.26 mm) and [Ni(L)2(H2O)4] (0.22  0.16  0.14 mm) were obtained by slow diffusion of a mixture of dichloromethane with a hexane solution (1:1, v/v) and dimethylformamide with a methanol solution (1:4, v/v), respectively. The relevant crystal structure data along with structure determination details are given in Table 1. Data were collected with a Bruker Smart Apex II CCD Area Detector at 296(2) K for HL and 273(2) K for [Ni(L)2(H2O)4]. Diffraction data were recorded with 2h in the range 1.43 6 h 6 25° (HL) and 2.70 6 h 6 25.64° [Ni(L)2(H2O)4]. A fine-focus sealed tube was used as the radiation source for the graphite-monochromatized Mo Ka radiation (k = 0.71073 Å). Data were corrected for Lorentz and polarization effects and an empirical absorption correction in the h k l range of 6 6 h 6 6; 12 6 k 6 12; 17 6 l 6 17 for HL and 9 6 h 6 9; 10 6 k 6 10; 18 6 l 6 18 for [Ni(L)2(H2O)4]. Multiscan absorption corrections were applied [27]. The structures were solved by direct methods with SHELXL-97 [28] and refined by fullmatrix least-squares techniques on F2 using the SHELXS-97 [28] program with anisotropic displacement parameters for all non-hydrogen atoms. ORTEP-3 [29] was used within WINGX [30] to prepare the figures and tables for publication. Hydrogen atoms were constrained to ride on their respective carbon atoms with isotropic displacement parameters equal to 1.2 times the equivalent isotropic displacement of their parent atom in all cases for the aromatic units.

The elemental analyses were carried out on Perkin Elmer model 2400 CHN/S elemental analyzers. Infrared spectra were obtained using a Perkin–Elmer RX-1 FTIR spectrophotometer (KBr disk, 4000–400 cm1). The UV–Vis-spectral data were recorded using a Perkin–Elmer Lambda 25 UV–Vis spectrophotometer. 1H NMR spectral data were collected in a suitable solvent using AC Brucker 300 and 500 MHz FT NMR spectrometers. The electrochemical studies were carried out with use of a computer controlled CH Instruments Electrochemical Workstation. All experiments were done at 298 K under an N2 atmosphere with a three electrode system, platinum and glassy carbon (GC) as working electrodes in MeCN. All potentials are referred to the saturated calomel electrode (SCE) and are uncorrected for junction contributions with n-tetra-butyl ammonium perchlorate, [nBu4N][ClO4] (TBAP), as the supporting electrolyte. The thermal analysis (TGA/DTG) was carried out under a nitrogen atmosphere with a heating rate of 12 °C/min (150 ml/min) using a Pyris Diamond TG/DTA analyzer made by Perkin Elmer (SINGAPORE) and a platinum crucible was used with alpha alumina powder as a reference. 2.3. Synthesis of [4-(Z)-((2-hydroxy-4-oxopent-2-en-3-yl)diazenyl)N-(5-methylisoxazol-3-yl)benzenesulfonamide] (HL) Sulfamethoxazole (0.5 g, 1.97 mmol) was dissolved in aqueousHCl (5% HCl solution, 15 ml) (Scheme 1) and was allowed to react with an aqueous solution of NaNO2 (1.0 g, 5 ml) solution at 0–5 °C, followed by coupling with acetylacetone (0.198 g, 1.97 mmol) in the presence of sodium acetate (2.0 g) to maintain the pH at 5.5– 6.5 in water, according to the literature procedure [25]. A bright yellow precipitate was obtained, which was filtered and dried at room temperature. It was then recrystallized by slow evaporation of a hot alcoholic solution and its purity was checked by TLC. Yield: 82%. M.P.: 205 ± 1 °C. Microanalytical data for HL; Anal. Calc. for C15H16N4O5S: C, 49.44; H, 4.43; N, 15.38; S, 8.80; Found: C, 49.34; H, 4.33; N, 15.31; S, 8.75%. FT-IR bands (KBr pellet, cm1): m(N@N), 1427; m(C@N), 1621; m(C@O), 1686; m(O–H), 3451; m(S–O), 1161. 1H NMR data (DMSO-d6, ppm): 6.12 (4H, s), 11.38 (6N-H, s), 7.70 (8,9H, d, J = 8.79 Hz), 7.83 (7,10H, d, J = 8.73 Hz), 13.44 (11-O-H, s), 2.27 (5-CH3, s), 2.42 (11-CH3, s), 2.41 (12-C H3, s). [M+H+], m/z: 365.09 (Supplementary materials, Fig. S1). 2.3.1. Preparation of the [Ni(L)2(H2O)4] complex To Ni(OAc)24H2O (0.060 g, 0.240 mmol) in a methanol solution (20 ml), the ligand HL (0.20 g, 0.55 mmol) was added with continuous stirring over 4 h and then the resulting solution was refluxed for 6 h. The color changed to bluish green. The solution was filtered and left undisturbed for a week; blue-green crystals deposited at the side of the beaker. These were collected by filtration, washed with MeOH–water (1:1 v/v). The complex was recrystallized from a DMF–methanol (1:4, v/v) mixture. Microanalytical data for [Ni(L)2(H2O)4]; Anal. Calc. for NiC30H3N8O14S2: C, 42.12; H, 4.24; N, 13.10; S, 7.50; Found: C, 42.05; H, 4.14; N, 13.01; S, 7.20%. FT-IR (KBr pellet, cm1): m(N@N), 1426; m(C@N), 1619; m(C@O), 1669; m(O–H), 3483; m(S–O), 1134. The charge neutrality in the complex was achieved by deprotonation of the sulfonamide group (–SO2N–) of both ligands [26]. The molar conductance of [Ni(L)2(H2O)4],

2.4. X-ray crystal structure analysis

2.5. Antibacterial studies of HL and [Ni(L)2(H2O)4] The antibacterial activities of HL and [Ni(L)2(H2O)4] have been examined. The compounds were dissolved in 100% DMSO and stored at 20 °C. Gram positive B. subtilis (ATCC 6633) and Gram negative E. coli (ATCC 8739), inoculated in a freshly prepared autoclaved LB broth from a 24 h old LA slant, were kept in a shaker overnight. The overnight culture was diluted with sterile LB to a final bacterial count of 1  104 cfu/ml. The diluted culture was distributed in a number of tubes and incubated in the absence as well as in the presence of various concentrations of the test compounds. All the tubes were incubated for 16–18 h at 37 °C under shaking conditions and the OD600 value was calculated using UV visible spectra. The OD600 value observed in the growth control (without any drug) was considered as a control with 100% growth for both the bacterial species. Compared to the control, the relative degree of bacterial growth inhibition was calculated for each set of ligand concentrations incubated under similar experimental conditions from a measurement of the OD value at 600 nm. The minimum inhibitory concentrations (IC50), i.e., the concentration of test compound required to inhibit the growth of bacteria by 50% was calculated from the % reduction of bacterial growth in comparison to the control. 2.6. Molecular docking studies of the ligand with DHPS of E. coli and ADMET Molecular docking was performed to explain how small molecules interact with proteins [31]. The PDB structure of 1AJ0 (DHPS of E. coli) was downloaded from the RCBS protein data bank, while the crystal structure of the protein of B. subtilis is not available. The enzyme 1AJ0 was co-crystallized with 2-amino-6-hydroxymethyl7,8-dihydro-3H-pteridin-4-one, sulfonamide and the sulfate ion. The amino acid chains of the proteins (1AJ0) were kept and the

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79

Scheme 1. Synthesis of HL.

Table 1 Crystal data and structure refinement for compound HL and [Ni(L)2(H2O)4].

Empirical formula Formula weight Crystal system Space group T (K) Wavelength (Å) a (Å) b (Å) c (Å) a (°) b (°) c (°) V (A3) Z Absorption coefficient (mm1) F(0 0 0) Dcalc (mg/m3) h range for data collection (°) Independent reflections Parameters Goodness-of-fit (GOF) on F2 Final R indices [I > 2r(I)]

HL

[Ni(L)2(H2O)4]

C15H16N4O5S 364.39 triclinic  P1

C30H36N8NiO14S2 855.50 triclinic  P1

296(2) 0.71073 5.4619(9) 10.8240(17) 14.512(2) 93.825(3) 100.753(3) 90.803(3) 840.7(2) 2 0.227

273(2) 0.71073 7.9356(3) 8.3947(3) 15.0695(7) 79.754(3) 88.968(3) 65.984(2) 900.70(6) 1 0.734

380 1.439 1.43–25.00

444 1.577 2.70–25.64

2940 231 1.038

3398 254 1.038

R1 = 0.0435a, wR2 = 0.1162b R1 = 0.0489a, wR2 = 0.1229b 0.36  0.28  0.26

R1 = 0.0538a, wR2 = 0.1264b R1 = 0.0830a, wR2 = 0.1411b 0.22  0.16  0.14

energy of the protein–ligand complex for analyzing the interaction between the ligand and the enzyme. Absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction was carried out with the ADMET descriptor module of the small molecules protocol of Discovery studio client. The drug-likeness of the compounds were also checked following Lipinski’s rule of five [33,34], using the ADMET module of the small molecule protocol of Discovery studio 4.0 software [32]. 2.7. Interaction of HL and [Ni(L)2(H2O)4] with calf thymus DNA 2.7.1. Preparation of solutions for DNA binding studies Stock solutions (5 ml) of both HL and [Ni(L)2(H2O)4] were prepared by dissolving HL (1.274 mg, 0.0035 mmol) in MeOH and [Ni(L)2(H2O)4] (2.749 mg, 0.0029 mmol) in DMSO. These solutions were diluted with Tris–HCl buffer for preparation of the working cell concentration for the experiment. An absorption spectral titration experiment was performed by keeping the concentration of HL or [Ni(L)2(H2O)4] constant with varying concentrations of CT-DNA. To eliminate the absorbance of DNA itself, an equal solution of CTDNA was added either to the HL or [Ni(L)2(H2O)4] solution or to the reference one.

R = RjFo  Fcj/RFo. wR = [Rw(F2o  F2c )/RwF40]1/2 are general but w are different, w = 1/[r2(F2o) + (0.0645P)2 + 0.4724P] for HL and w = 1/[r2(F2o) + (0.0582P)2 + 0.2103P] for HL and [Ni(L)2(H2O)4].

2.7.2. Preparation of calf thymus DNA The Tris–HCl buffer solution (pH 8.0) used in all the experiments involving CT-DNA was prepared using de-ionized and sonicated HPLC grade water (Merck). The CT-DNA used in the experiments was sufficiently free from protein. The concentration of DNA was determined with the help of its extinction coefficient e of 6600 mol1 cm1 at 260 nm [25]. Stock solutions of DNA were always stored at 4 °C and used within 4 days.

water molecules and co-crystallized ligand was removed and subsequently the missing atom types were repaired using the prepare protein module under the receptor ligand interaction of Discovery Studio 3.5 [32]. The prepared protein was saved as a PDB file and used for docking studies. The crystal structure of the ligand was saved as a MOL file, ligand preparation was carried out and the prepared ligand was used for docking. All docking calculations were carried out using CDOCKER and a grid box centered at the geometrical center of the co-crystallized ligand (sulfonamide for 1AJ0) was used. The x, y, z coordinates for the center of the grid box were 41.78, 8.05, 2.24 and -77.56, 85.05, 93.95 Å respectively. Finally most favorable pose was selected according to the minimum free

2.7.3. Absorption spectroscopic studies of HL and [Ni(L)2(H2O)4] in the presence of CT DNA Absorption spectroscopic studies were done on a spectrophotometer (Perkin Elmer, Lambda-25), using HL (8 lM) or [Ni(L)2(H2O)4] (2.32 lM) with increasing concentrations of CTDNA (0.558–2.75 lM). After each addition, the mixture of DNA and the compound was incubated at room temperature for 15 min and scanned at the 250–600 nm wavelength for the above compounds. The self-absorption of DNA was eliminated in each set of experiments. Each sample was scanned for a cycle number of 2, cycle time of 5 sec at a scan speed of 100 nm/min. A modified BenesiHildebrand [35] plot was used for the determination of the ground state binding constant between the compounds and CT-DNA. The binding constant Kb was determined by using the following relation:

R indices (all data) Crystal size (mm) a

b

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A0 =DA ¼ A0 =DAmax þ ðA0 =DAmax Þ  ð1=KÞ  ð1=Lt Þ where DA = A0  A, DAmax = maximum change in reduced absorbance, A0 = maximum absorbance of receptor molecules (without any DNA), A = reduced absorbance of receptor molecules (in the presence of DNA), Lt = DNA concentration. 2.8. Computational studies The X-ray crystallographic parameters of HL and [Ni(L)2(H2O)4] were used as input functions in the Density functional theory (DFT) calculations and the optimized geometries of HL and [Ni(L)2(H2O)4] were generated. The calculations have been carried out with the B3LYP-6-31G (d) basis set for C, H, N and O atoms and LanL2DZ [36] for Ni and S atoms, using the GAUSSIAN 09 program. The entire calculations, including graphical representations of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) data in the checkpoint files, were carried out using the GAUSSIAN 03 program package [37,38] with the aid of the GAUSSVIEW visualization program [39,40]. The vibrational frequency calculations were performed to ensure that the optimized geometries represent local minima and there are only positive eigen values. Vertical electronic excitations based on the B3LYP optimized geometries were computed for HL and the complexes using the time-dependent density functional theory (TD-DFT) formalism [41–43] in dichloromethane using a conductor-like polarizable continuum model (CPCM) [44–46]. GAUSS SUM was used to calculate the fractional contributions of various groups to each molecular orbital [47].

>1.2 V refers to water oxidation, whose residual small reduction peak is observed at 0.0 to 0.2 V. 3.2. Crystal structure description of HL and [Ni(L)2(H2O)4] The molecular structures of HL and [Ni(L)2(H2O)4], with the atom numbering scheme, are shown in Fig. 1a, and b, and selected bond parameters are listed in Table 2. Two aromatic groups, paraaminophenyl and methyl-oxazolyl, are bonded to the –SO2NH– (sulfonamide) function. The S@O bond length (1.444(3), 1.453(3) Å) is corroborated with literature data [35,49,50]. The N(3)–N(4) bond lengths in HL and [Ni(L)2(H2O)4] are 1.310(3) and 1.305(5) Å respectively, which matched well with the N–N bond lengths reported in the literature [35,51]. The bonding strength between azo-N(4) and acetyl acetone (C(17)–N(4), 1.319(3) Å) is stronger than that of azo-N(3) and sulfonamide-phenyl-C(11) (C(11)–N(3), 1.412(3) Å). The bond angle \O(2)–S(1)–O(1) is 104.07(18)°. Intramolecular and intermolecular hydrogen bonds, such as O(7)–(H7). . .N(2)–C(3) (2.004 Å), N(3). . .. . .H(5)–O(5)– C(14) (3.03 Å) and O(7)–(H7). . .O(3)–S(1) (2.07 Å), give rise to the formation of a 1D tape (Figs. 2 and 3) and 2D plane (Fig. 4). Relevant H-bonds are shown in the Supplementary material (Tables S1 and S2). Bond lengths and bond angles have been calculated from the optimized structures using the DFT computation technique and the metric parameters are elongated by a small amount, 0.01–0.2 Å (bond lengths) and 1°–2° (bond angles) (Table 2). 3.3. Antibacterial potential of HL and [Ni(L)2(H2O)4]

3. Results and discussion 3.1. The synthesis and formulation 4-(Z)-((2-Hydroxy-4-oxopent-2-en-3-yl)diazenyl)-N-(5-methylisoxazol- 3-yl)benzene sulfonamide (HL) has been synthesized by diazotization of sulfamethoxazole (SMX) followed by coupling of the diazonium ion (SMX-N@N-+) with acetylacetone (Hacac) in the presence of sodium acetate [25]. The reaction of HL with Ni(OAc)24H2O has isolated [Ni(L)2(H2O)4]. Microanalytical and spectroscopic (1H NMR, UV–Vis and FT-IR, see Supplementary materials, Figs. S1–S5) data have been used to characterize HL and the Ni(II) complex. The characteristic stretching vibrations of HL are m(N@N), 1415; m(C@O), 1621; m(S–O), 1161 cm1. The 1H NMR spectrum of HL (DMSO-d6) shows two resonances at 13.44 (d (OH)) and 11.38 m (d (NH)), three –Me signals (2.28, 5-CH3; 2.43, 12-CH3; 2.41 ppm, 11-CH3), the oxazolyl-H resonates at 6.12 ppm (4-H) and four phenyl Hs exhibit two doublets at 7.70 (8,9-H, J, 8.79 Hz) and 7.84 ppm (7,10-H, J, 8.73 Hz). The electronic spectra of HL in methanol and [Ni(L)2(H2O)4] in DMF show intense bands at 360 and 366 nm respectively, and these have been assigned to p–p⁄ transitions. The d-d band of [Ni(L)2(H2O)4] in DMF appears at 500 nm (Supplementary material, Fig. S6). The magnetic moment of 2.37 BM at 305 K supports a two electron (d8) paramagnetic nature of the complex. [Ni(L)2(H2O)4] starts to dehydrate at 82 °C (obs. = 8.41%, calc. = 8.42%) and finally decomposes at >250 °C, completing the process at 400 °C (Supplementary material, Figs. S7 and S8). Sulfamethoxazole decomposes within the temperature range 200–259 °C [48]. The cyclic voltammogram of the ligand (HL) and [Ni(L)2(H2O)4] in acetonitrile show azo reduction at 0.7 V (DE, 200 mV) and an oxidative irreversible response is noted for [Ni(L)2(H2O)4] only, which may be referred to the Ni(II) ? Ni(III) redox response [25] (Supplementary material, Fig. S9). A strong oxidative response at

HL and [Ni(L)2(H2O)4] have been examined against B. subtilis (ATCC 6633) and E. coli (ATCC 8739). The compounds exhibited inhibition of bacterial growth after 18–20 h of compound administration. The minimum inhibitory concentrations (IC50), i.e., the concentration of test compound required to inhibit the growth of bacteria by 50%, for HL and [Ni(L)2(H2O)4] have been determined and the results are presented in Figs. 5 and 6. From the results it is evident that HL and [Ni(L)2(H2O)4] produce a concentration dependant decrease in the growth of both Gram positive B. subtilis and Gram negative E. coli. The IC50 values for HL and [Ni(L)2(H2O)4] are 63.72 and 81.49 lg/ml respectively for gram negative E. coli (ATCC 8739). On the other hand these compounds show IC50 values of 77.25 and 78.28 lg/ml for gram negative E. coli (ATCC 8739) respectively. Metal ions are able to produce an antibacterial effect due to ‘‘oligodynamic action’’, as manifested from their use in the killing of microorganism [52,53]. Due to cost efficiency, SMX has been a centre of attraction for biomedical treatment; however its use has been restricted due to its toxicity. To resolve these problems, the antimicrobial potential of the sulfamethoxazolyl-azoacetylacetone (HL) ligand and its nickel(II) complex have been examined. 3.4. Molecular docking studies of the ligand with DHPS in the presence of E. coli Molecular modeling techniques, following computer aided molecular design, lead generation and optimization, and protein– drug interactions are recent topics in drug discovery and development in contemporary research for computational chemistry. Using calculated docking score, Log P data by molecular docking the library of molecules may be screened to evaluate the biomedical activity and ADMET (absorption, distribution, metabolism, excretion and toxicity) properties [54].

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Fig. 1. The molecular structure of the sulfamethoxazole-azo-acetylacetone ligand (L) (a) and [Ni(L)2(H2O)4] (b) with the atom numbering scheme. Hydrogen atoms are omitted for clarity.

The in vitro docking study has been carried out to understand the interaction of DHPS and the ligand by using the CDOCKER module of the receptor ligand interaction section available in Discovery studio client 4.0. A crystallographic structure with good resolution (2.0 Å) of the ligand bound DHPS of E. coli was downloaded from the RCSB protein data bank (http://www.rcsb.org/pdb/home/home.do) and used for the docking studies. Inside the binding sphere of DHPS of E. coli (PDB ID 1AJ0) there are 12 amino acid residues (Asn22, Ser61, Thr62, Arg63, Pro64, Phe190, Arg220, Lys221, Pro232, Arg235, Arg255, His257). Receptor ligand interaction analysis suggests that when the ligand binds with DHPS of E. coli, –SO2 and acetylacetonate group oxygen atoms together with oxazolyl moiety azo-N atoms form a total of six H-bonds with the protein residue (Figs. 7 and 8). The aromatic ring of the sulfonamide is involved in a p—p interaction with Arg63 (Table 3). The calculated binding energy of DHPS (PDB ID 1AJ0) and HL is 10529.58 kcal/mol, while the binding energy is 104.95 kcal/mol. The reference energy of HL is 32.27 kcal/mol and the protein energy is 10 457 kcal/mol. The CDOCKER energy and interaction energies are 10.61 and 37.23 a.u.

The drug likeness of the ligand has been checked following Lipinski’s rule of five [35]. ADMET modules of discovery studio client 4.0 have been used to check the ADMET (absorption, distribution, metabolism, excretion and toxicity) properties of the compounds. The predicted data show a good solubility level, moderate absorption stage, seven H-bond acceptors, two H-bond donors and follow Lipinski’s filter, and Log P is 1.955. The drug likeness and Ames prediction support the non-mutagenic character of HL (Supplementary material, Tables S3 and S4). 3.5. Interaction of HL and [Ni(L)2(H2O)4] with CT DNA: Absorption spectroscopic studies The intense absorption band around 263 nm of CT DNA refers to an intramolecular p–p⁄ transition. The interaction of both HL and [Ni(L)2(H2O)4] with CT DNA have been investigated by a spectrophotometric method. Addition of increasing amounts of CTDNA resulted in an increase in absorbency without any shift in the absorption maxima in the UV spectra of the compound. In general, an intercalative interaction of the aromatic chromophore and

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Table 2 Theoretical and experimental bond lengths (Å) and bond angles (°) of HL and [Ni(L)2(H2O)4]. Bond length (Å)

Bond angle (°)

Length type

Theoretical

Experimental

Angle type

Theoretical

Experimental

HL N(3)–N(4) C(11)–N(3) C(17)–N(4) O(1)–S(1) O(2)–S(1) N(1)–S(1) C(1)–N(1) C(1)–N(2)

1.333 1.409 1.339 1.623 1.624 1.693 1.393 1.349

1.310(3) 1.412(3) 1.320(3) 1.428(15) 1.424(15) 1.644(18) 1.405(3) 1.304(3)

N(3)–N(4)–C(17) N(4)–N(3)–C(11) O(2)–S(1)–O(1) N(2)–C(1)–N(1) N(2)–C(1)–C(4) N(1)–C(1)–C(4) C(16)–C(4)–C(1)

121.95 120.42 104.01 119.05 112.86 128.03 104.89

121.43(19) 119.34(18) 104.07(18) 117.74(18) 112.97(19) 129.28(19) 104.29(19)

[Ni(L)2(H2O)4] N(3)–N(4) Ni(1)–N(1) Ni(1)–O(7) Ni(1)–O(6) N(1)–C(3) N(2)–C(3) S(1)–O(3) S(1)–O(2) S(1)–N(2) O(1)–C(1) O(1)–N(1)

1.288 2.075 2.060 2.127 1.363 1.371 1.463 1.467 1.572 1.395 1.458

1.305(5) 2.029(3) 2.066(3) 2.099(3) 1.327(5) 1.378(5) 1.444(3) 1.453(3) 1.565(3) 1.347(5) 1.401(4)

N(3)–N(4)–C(11) N(4)–N(3)–C(8) O(3)–S(1)–O(2) O(3)–S(1)–N(2) O(2)–S(1)–N(2) O(3)–S(1)–C(5) O(2)–S(1)–C(5)

126.45 121.91 121.15 112.97 105.35 106.03 106.12

121.00(4) 119.00(4) 116.22(18) 106.69(17) 115.07(18) 106.34(18) 105.88(18)

Fig. 2. Formation of a 1D tape in [Ni(L)2(H2O)4] through both the occurrence of intra and inter hydrogen bonding interactions O(7)–(H7). . .N(2)–C3 and O(7)–(H7). . .O(3)–S1 along the b axis, generating a R22(12) cyclic motif.

the base pairs of DNA helix are reflected by hyperchromism and a red shift of the charge transfer band [55]. The spectral changes of both HL and [Ni(L)2(H2O)4] (Fig. 9a), observed in the presence of CT-DNA, can be illustrated in terms of groove binding [56]. It is reported that the aromatic ring of the molecule closely matches the helical turn of the CT-DNA groove and the aromatic ring of ligand interacts with DNA in Tris–HCl buffer through the formation of van der Waal’s contacts or hydrogen bonds in the DNA grooves. At the absorption maximum, we have calculated the binding constant for the both compounds HL and [Ni(L)2(H2O)4] (Fig. 9b) with DNA by using a modified Benesi–Hildebrand (BH) plot. The binding constant (Kb) of HL is 6.09  105 M1 and that of [Ni(L)2(H2O)4] is 12.20  105 M1. It may be indicative of an interaction with DNA through groove binding of the aromatic chromospheres and the base pairs of DNA. From the values of the binding constant (Kb) of HL and [Ni(L)2(H2O)4], the free energy (DG = 7.94 kcal and –8.35 kcal respectively) of the compound– DNA complex was calculated. Binding constants are measure of

Fig. 3. Intra molecular H-bonding of [Ni(L)2(H2O)4].

D. Das et al. / Polyhedron 99 (2015) 77–86

83

Fig. 4. Formation of 2D plane in SMX through the association of discrete HL monomeric units.

the interaction and shows a better interaction for the metal complex [Ni(L)2(H2O)4] compared to HL. 3.6. DFT computations

Fig. 5. The minimum inhibitory concentrations (IC50) of L and[Ni(L)2(H2O)4] in the presence of E. coli (ATCC 8739).

compound–DNA complex stability, while the free energy indicates the spontaneity of the compound–DNA binding. A negative free energy of the compound–DNA binding shows the spontaneity of

A structural agreement has been verified on comparing the bond distances and angles between the DFT optimized and X-ray determined structures of HL and [Ni(L)2(H2O)4] (Supplementary material Tables S5–7). To simplify the analysis of the DFT computation results, the structure of HL has been divided (Fig. 10) into benzene sulfonamide (BSN), methyloxazolyl (MOX) and azo-acetylacetone (AAA) (Supplementary material, Figs S10–12). The composition of the MOs and proposed electronic transitions in acetonitrile (CH3CN) solution (Supplementary material, Tables S8–S10) suggest that the azo function (AAA) is an electron acceptor while the methyloxazolyl (MOX) and benzene sulfonamide (BSN) either separately or jointly donate electrons; HOMO/HOMO-5?LUMO refers to BSN?AAA (367.21 nm; f, 0.8925); HOMO-2?LUMO refers to MOX?AAA and BSN?AAA (320.95 nm; f, 0.0510), HOMO-2?LUMO + 1 is MOX?BSN (277.29 nm; f, 0.0219). In the case of the selected a molecular orbital of [Ni(L)2(H2O)4], the HOMO is constituted of 33% BSN,

Fig. 6. The minimum inhibitory concentrations (IC50) of L and [Ni(L)2(H2O)4] in the presence of B. subtilis (ATCC 6633).

84

D. Das et al. / Polyhedron 99 (2015) 77–86

6 0.08

Absorbance

1

0.06

340

360

380

400

420

Wavelength Fig. 7. HL in the binding site of 1AJ0. Fig. 9a. Absorption spectral changes of [Ni(L)2(H2O)4] with increasing concentrations of CT DNA (0, 0.558, 1.11, 1.66, 2.21, 2.75 lM) respectively (1?6).

08% MOX and 59% AAA; the LUMO has a 21% contribution from BSN and 79% AAA. So, the HOMO?LUMO transition may be assigned to an intra-molecular charge transfer from BSN?AAA. In the b molecular orbitals of [Ni(L)2(H2O)4], the HOMO is

constituted of 32% BSN, 08% MOX and 60% AAA; the LUMO has a 21% contribution from BSN and 79% AAA (Fig. 10). So, the HOMO to LUMO transition may be assigned to an intra molecular charge transfer from BSN?AAA, MOX?AAA.

Fig. 8. HL in the cavity of DHPS of E. coli (PDB, id 1AJ0).

Table 3 Details of H-bonds and p–p interactions. Compound

HL

p–p interaction (Å)

Protein PDB id

H-bond (Å) No of Hbonds

Bond distance (Å)

End1

End2

No of p bonds

Distance

End1

End2

1AJ0

5

2.25 2.39

Arg63 Arg63

1

5.17

Arg63

Aromatic ring of the ligand

2.94 2.03

Arg63 Arg235

2.76 2.05

Arg235 Arg255

N atom of azo O atom of –OH of acetylacetone N atom of oxazole ring One of the O atoms of ASO2NH Other O atom of –SO2NH O atom of –C@O of acetylacetone

D. Das et al. / Polyhedron 99 (2015) 77–86

3.37 eV. Thus the HOMO to LUMO charge transfer is more preferable for intra charge transfer. The intensity of these transitions has been assessed from the oscillator strength (f). In CH3CN the calculated transition appears at 367 nm (f, 0.8925) for HL along with a large number of transitions in the UV region (<400 nm). The observed transitions are close to the calculated ones in HL. In [Ni(L)2(H2O)4], the electronic transition at 366 nm is due to a p?p⁄ transition. In CH3CN the calculated transition appears at 368 nm (f, 0.0018) for the a molecular orbital of [Ni(L)2(H2O)4]. The observed transitions are close to the calculated ones in the a molecular orbital of [Ni(L)2(H2O)4]. In CH3CN the calculated transition appears at 381.69 nm (f, 0.0163) for the b molecular orbital of [Ni (L)2(H2O)4].

40 35 30

A0/ A

85

25 20 15 5

4.0x10

5

8.0x10

6

1.2x10

6

1.6x10

6

2.0x10

4. Conclusion

1/ L t

Sulfamethoxazolyl-azo-acetylacetone (HL) and [Ni(L)2(H2O)4] have been characterized by single-crystal X-ray diffraction and other physicochemical data. The structure of the complex shows a hydrogen bonded supramolecular 1D tape and 2D plane. The interaction of HL and [Ni(L)2(H2O)4] with CT-DNA has been followed by an absorption spectroscopic study and the antibacterial activity has been examined against B. subtilis (ATCC 6633) and E. coli (ATCC 8739). This is preliminary information for our programed study towards the functionalization of sulfonamides and

Fig. 9b. Modified Benesi–Hildebrand plot for the determination of the ground state binding constant between CT DNA and the [Ni(L)2(H2O)4] complex.

In HL, the electronic transition at 360 nm is due to a p?p⁄ transition. A polar solvent stabilizes the occupied MOs more efficiently than the unoccupied MOs. Thus, the energy separation (DE) between the HOMO and LUMO increases in the CH3CN phase, being

HL

HOMO; E, -7.08 eV; BSN, 44; MOX, 23; AAA, LUMO; E, -3.4 eV; BSN, 21; AAA, 79% 33% [Ni(L)2(H2O)4]

HOMO; E, -6.3 eV; BSN, 33; MOX, 08; AAA, 59%

LUMO; E, -4.04 eV; BSN, 21; MOX, 00; AAA, 79 %

Spin density Fig. 10. Contour plots of the HOMO and LUMO of HL and [Ni(L)2(H2O)4].

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D. Das et al. / Polyhedron 99 (2015) 77–86

the complexation of the derivatives for exploration of less toxic efficient drugs. The in-silico test of HL with DHPS from E. coli helps to recognize the molecules for druglikeness and ADMET prediction. Acknowledgements Financial support from West Bengal DST, Kolkata (No. 228/1(10)/(Sanc.)/ST/P/S&T/9G-16/2012) and UGC, New Delhi (F. 31-123/2005 (S.R.) are thankfully acknowledged. Appendix A. Supplementary data CCDC 982339 and 982337; contains the supplementary crystallographic data for HL and the complex [Ni(L)2(H2O)4]. These data can be obtained free of charge via http://www.ccdc.cam.ac. uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 1223 336 033; or e-mail: [email protected]. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.poly.2015.06.027. References [1] J.-Y. Winum, A. Maresca, F. Carta, A. Scozzafava, C.T. Supuran, Chem. Commun. 48 (2012) 8177. [2] Z.H. Chohan, M.H. Youssoufi, A. Jarrahpour, T. Benhadda, Eur. J. Med. Chem. 45 (2010) 1189. [3] R.-J. Lu, J.A. Tucker, T. Zinevitch, O. Kirichenko, V. Konoplev, S. Kuznetsova, S. Sviridov, J. Pickens, S. Tandel, E. Brahmachary, Y. Yang, J. Wang, S. Freel, S. Fisher, A. Sullivan, J. Hou, S. Stanfield-Oakley, M. Greenberg, D. Bolognesi, B. Bray, B. Koszalka, P. Jeffs, A. Khasanov, Y.-A. Ma, C. Jeffries, C. Liu, T. Proskurina, T. Zhu, A. Chucholowski, R. Li, C. Sexton, J. Med. Chem. 50 (2007) 6535. [4] G. Xia, L. Liu, M. Xue, H. Liu, J. Yu, P. Li, Q. Chen, B. Xiong, X. Liu, J. Shen, Mol. Cell. Endocrinol. 358 (2012) 46. [5] K.G. Andanappa, S.M. Chanabasappa, N. Sudarshan, R. Anandkumar, Eur. J. Med. Chem. 35 (2000) 853. [6] X.L. Wang, K. Wan, C.H. Zhou, Eur. J. Med. Chem. 45 (2010) 4631. [7] M.E. Epstein, M. Amodio-Groton, N.S. Sadick, J. Am. Acad. Dermatol. 37 (1997) 365. [8] J.D. Smilack, Mayo Clin. Proc. 74 (1999) 730. [9] G.C. Slatore, A.S. Tilles, Immunol. Allergy Clin. North Am. 24 (2004) 477. [10] G. Choquet-Kastylevsky, T. Vial, J. Descotes, Curr. Allergy Asthma Rep. 2 (2002) 16. [11] A. Carr, A.S. Gross, J.M. Hoskins, R. Penny, A.D. Cooper, Aids. 8 (1994) 333. [12] M.F. Gordin, L.G. Simon, B.C. Wofsy, J. Mills, Ann. Intern. Med. 100 (1984) 495. [13] L.S. Walmsley, S. Khorasheh, J. Singer, O. Djurdjev, J. Acquir. Immunogenic Syndr. Human Retroviral 19 (1998) 498. [14] S. Mukesh, E.C. Alastair, Chem. Biol. Interact. 142 (2002) 155. [15] D.J. Naisbitt, J. Farrell, F.S. Gordon, L.J. Maggs, C. Burkhart, J.W. Pichler, M. Pirmohamed, K.B. Park, Mol. Pharmacol. 62 (2002) 628. [16] M.S. Iqbal, A.H. Khan, B.A. Loothar, I.H. Bukhari, Med. Chem. Res. 18 (2009) 31. [17] Z.H. Chohan, H.A. Shad, Appl. Organometal. Chem. 25 (2011) 591. [18] K. Lal, Indian J. Chem. 17A (1979) 313. [19] L. Goodman, A. Gilman, The Pharmacological Basis of Therapeutics, 4th Edn., MacMillan, New York, 1970. 1111. [20] P. Pathak, S.V. Jolly, P.K. Sharma, Orient. J. Chem. 16 (2000) 161.

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