Antibacterial dimeric copper(II) complexes with chromone-derived compounds

Abstract

A new series of six chromone-derived compounds and their Cu(II) complexes have been synthesized and characterized by their physical, spectral and analytical data. The ligands and their Cu(II) complexes were screened for their in vitro antibacterial activity against four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri) and two Gram-positive (Bacillus subtilis, Staphylococcus aureus) bacterial strains by agar-well diffusion method. The ligands were found to exhibit either no or low-to-moderate activities against one or more bacterial species whereas, the Cu(II) complexes exhibited moderate-to-high activity. The ligands which were inactive before complexation became active upon complexation with the Cu(II) metal ion and less active became more active.

Keywords::

• Chromone derivatives
• dimeric Cu(II) complexes
• antibacterial activity

Introduction

Metal chelation and its relationship with different biological processes represent a promising area of research in designing novel therapeutic methodologies to deal with global problem of ever increasing “bacterial resistance”. Many metal ions are known to play a significant role in different biological processes^1–3. For example, zinc(II) and copper(II) ions are the second- and third-most abundant transition metals present in humans. They are found either at the active sites or as structural components of enzymes or co-enzyme in biochemical processes⁴.

Chromones contain γ-pyrone nucleus fused with benzene ring. Molecules having chromone moiety are versatile compounds^5–10 with a reactive carbonyl group that show a high reactivity towards many nucleophiles allowing synthesis of a wide variety of biologically active compounds^11–14. In view of biological significance and their interesting structural behaviour, a novel class of chromones (1)–(6) and their Cu(II) complexes (7)–(12) have been prepared, characterized and tested for in vitro antibacterial activity against four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri) and two Gram-positive (Bacillus subtilis, Staphylococcus aureus) bacterial strains. The present metal based chromones constitute a new class of antibacterial agents that may serve as good candidates for issues of clinical drug resistance.

Material and methods

Experimental

All reagents and solvents were used as obtained from the supplier and recrystallized/redistilled as necessary. Thin-layer chromatography (TLC) was performed for checking the purity of the compounds. Infrared (IR; KBr disc) was recorded with a Hitachi Model 200-50 Fourier transform infrared spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded in dimethyl sulphoxide (DMSO)-d6 with Bruker AM 300 and AM 400 spectrometer (Rheinstetten–Forchheim, Germany) operating at 300 and 400 MHz, respectively. Mass spectra of the ligands were obtained by JEOL MS Route spectrometer using electron ionization (EI) mode. CHN analyses were carried out using Elemental Analyzer Flash EA 1112. Conductance of the metal complexes was measured on conductivity meter 4071, Jenway. Magnetic susceptibility measurements of the metal complexes in the solid state were performed on the Gouy’s balance at room temperature. Melting points were recorded on a Gallenkamp apparatus.

General procedure for the preparation of ligands (1)–(6)

To a stirred solution of 3,5-dichloroformylchromone (0.01 mole) in ethanol (30 ml) was an appropriate solution of hydrazide (0.01 mole) in ethanol (20 ml) was added, according to Scheme 1. The resultant mixture was heated under reflux for 3–4 h. The solid product thus formed during refluxing was collected by suction filtration. Thorough washing with hot ethanol followed by ether furnished the required product in pure form as a single spot on TLC. All compounds were prepared similarly and characterized as below:

Scheme 1.  Preparation of ligands (1)–(6) and their Cu(II) complexes (7)–(12).

(3,5-Dichloro-2-hydroxyphenyl)[1-(2,3,5,6-tetrafluorophenyl)-1H-pyrazol-4-yl]methanone (1)

Yield 68% as a light yellow powder; m.p. 91–93°C; IR (KBr, cm⁻¹): 3440 (dichlorohydroxyphenyl, OH), 1685 (C=O), 1040 (pyrazol, N-N), 620 (C-F); ¹H NMR (DMSO-d6, δ ppm): 8.32 (s, 1H, pyrazol), 8.81 (s, 1H, pyrazol), 7.86 (s, 1H, tetrafluorobenzene), 8.01 (s, 1H, dichlorohydroxyphenyl), 8.03 (s, 1H, dichlorohydroxyphenyl), 11.83 (s, 1H, OH); ¹³C NMR (DMSO-d6, ppm): 106.3, 116.9, 136.6, 136.6, 147.3, 147.4 (tetrafluorobenzene), 107.4, 128.7, 141.8 (pyrazole), 196.3 (C=O), 128.4, 129.3, 135.7, 125.7, 158.4 (C-O), 117.6 (dichlorohydroxyphenyl), EIMS (70 eV) m/z (%): 405.13 (M+, 68%); Anal. Calcd.: for C₁₆H₆C_l2F₄N₂O₂ (405.13): C, 47.43; H, 1.49; N, 6.91; Cl, 17.50; F, 18.76; Found: C, 47.35; H, 1.39; N, 6.93, Cl, 17.55%.

(3,5-Dichloro-2-hydroxyphenyl)[1-(2,5-difluorophenyl)-1H-pyrazol-4-yl]methanone (2)

Yield 75% as a yellowish powder; m.p. 75–76°C; IR (KBr, cm⁻¹): 3440 (dichlorohydroxyphenyl, OH), 1680 (C=O), 1041 (pyrazol, N-N), 620 (C-F); ¹H NMR (DMSO-d6, δ ppm): 8.33 (s, 1H, pyrazol), 8.07 (s, 1H, pyrazol), 7.68 (s, 1H, difluorobenzene), 7.68 (s, 1H, difuorobenzene), 7.71 (s, 1H, difluorobenzene), 7.96 (s, 1H, dichlorohydroxyphenyl), 8.01 (s, 1H, dichlorohydroxyphenyl), 11.82 (s, 1H, OH); ¹³C NMR (DMSO-d6, ppm): 107.1, 115.4, 118.6, 152.2, 152.4, 130.1 (difluorobenzene), 107.4, 128.7, 141.8 (pyrazol), 196.2 (C=O), 128.4, 129.3, 135.7, 125.7, 158.4 (C-O),117.6 (dichlorohydroxyphenyl); EIMS (70 eV) m/z (%): 369.15 (M+, 88%); Anal. Calcd.: for C₁₆H₈Cl₂F₂N₂O₂ (369.15): C, 52.01; H, 2.16; N, 7.58; Cl, 19.23. Found: C, 52.11; H, 2.09; N, 7.62; Cl, 19.19%.

(3,5-Dichloro-2-hydroxyphenyl)[1-(2,5-dichlorophenyl)-1H-pyrazol-4-yl]methanone (3)

Yield 72% as a yellowish powder; m.p. 102–103°C; IR (KBr, cm⁻¹): 3435 (dichlorohydroxyphenyl, OH), 1685 (C=O), 1040 (pyrazol, N-N), 620 (C-F); ¹H NMR (DMSO-d6, δ ppm): 8.53 (s, 1H, pyrazol), 8.76 (s, 1H, pyrazol), 7.91 (s, 1H, dichlorohydroxyphenyl), 8.02 (s, 1H, dichlorohydroxyphenyl), 7.13 (s, 1H, dichlorobenzene), 7.96 (s, 1H, dichlorobenzene), 8.82 (dichlorobenzene), 11.81 (s, 1H, OH); ¹³C NMR (DMSO-d6, δ ppm): 117.1, 132.4, 128.6, 131.2, 132.4, 144.1 (dichlorohydroxyphenyl), 107.4, 128.7, 141.8 (pyrazol), 196.3 (C=O), 128.4, 129.3, 135.7, 125.7, 158.3 (C-O), 117.6 (dichlorobenzene); EIMS (70 eV) m/z (%): 402.06 (M+, 79%); Anal. Calcd.: for C₁₆H₈Cl₄N₂O₂ (402.06): C, 47.75; H, 1.99; N, 6.96; Cl, 35.31. Found: C, 47.69; H, 2.06; N, 6.95; Cl, 35.35%.

(3,5-Dichloro-2-hydroxyphenyl)[1-(3-methylphenyl)-1H-pyrazol-4-yl]methanone (4)

Yield 75% as a bright yellow powder; m.p. 184–186°C; IR (KBr, cm⁻¹): 3435 (dichlorohydroxyphenyl, OH), 1685 (C=O), 1040 (pyrazol, N-N), 620 (C-F); ¹H NMR (DMSO-d6, δ ppm): 2.34 (s, 3H, CH3), 8.47 (s, 1H, pyrazol), 8.22 (s, 1H, pyrazol), 7.91 (s, 1H, dichlorohydroxyphenyl), 8.02 (s, 1H, dichlorohydroxyphenyl), 7. 8.1 (s, 1H, methylbenzene), 7.1 (d, 1H, methylbenzene), 7.22 (m, 1H, methylbenzene), 7.58 (d, 1H, methylbenzene), 11.83 (s, IH, OH); ¹³C NMR (DMSO-d6, δ ppm): 24.6 (CH3), 117.1, 132.4, 128.6, 131.2, 132.4, 144.1 (methylbenzene), 107.4, 128.7, 141.8 (pyrazol), 196.3 (C=O), 128.4, 129.3, 135.7, 125.7, 158.3 (C-O), 117.6 (dichlorohydroxyphenyl); EIMS (70 eV) m/z (%):347.19 (M+, 62%), Anal. Calcd.: for C₁₇H₁₂C_l2N₂O₂ (347.19): C, 58.75; H, 3.45; N, 8.06; Cl, 20.44. Found: C, 58.78; H, 3.51; N, 8.11; Cl, 20.52%.

4-[4-(3,5-Dichloro-2-hydroxybenzoyl)-1H-pyrazol-1-yl]benzoic acid (5)

Yield 72% as a dull yellow powder; m.p. 253–255°C; IR (KBr, cm⁻¹): 3435 (dichlorohydroxyphenyl, OH), 1782 (COOH), 1685 (C=O), 1040 (pyrazol, N-N), 620 (C-F); ¹H NMR (DMSO-d6, δ ppm): 8.47 (s, 1H, pyrazol), 8.22 (s, 1H, pyrazol), 7.91 (s, 1H, dichlorohydroxyphenyl), 8.02 (s, 1H, dichlorohydroxyphenyl), 8.3 (s, 1H, benzoic acid), 7.5 (d, 1H, benzoic acid), 8.13 (m, 1H, benzoic acid), 8.73 (d, 1H, benzoic acid), 11.825 (s, 1H, OH), 12.14 (s, IH, COOH); ¹³C NMR (DMSO-d6, ppm): 125.3, 129.4, 128.2, 131.2, 106.6, 140.1 (benzoic acid), 107.4, 128.7, 141.8 (pyrazol), 196.3 (C=O), 128.4, 129.3, 135.7, 125.7, 158.3 (C-O), 117.6 (dichlorohydroxyphenyl), 170.2 (COOH); EIMS (70 eV) m/z (%):377.18 (M+, 72%), Anal. Calcd.: for C₁₇H₁₀C_l2N₂O₄ (377.18): C, 54.08; H, 2.65; N, 7.42; Cl, 18.82. Found: C, 54.11; H, 2.69; N, 7.39; Cl, 18.77%.

(3,5-Dichloro-2-hydroxyphenyl)[1-(4-methoxyphenyl)-1H-pyrazol-4-yl]methanone (6)

Yield 70% as a yellowish powder; m.p. 160–162°C; IR (KBr, cm⁻¹): 3435 (dichlorohydroxyphenyl, OH), 1685 (C=O), 1040 (pyrazol, N-N), 620 (C-F); ¹H NMR (DMSO-d6, δ ppm): 3.82 (s, 3H, OCH₃), 8.46 (s, 1H, pyrazol), 8.25 (s, 1H, pyrazol), 7.91 (s, 1H, dichlorohydroxyphenyl), 8.07 (s, 1H, dichlorohydroxyphenyl), 7.68 (s, 1H, methoxylbenzene), 7.37 (d, 1H, methoxylbenzene), 6.98 (m, 1H, methoxylbenzene), 7.52 (d, 1H, methoxylbenzene), 11.83 (s, IH, OH); 13C NMR (DMSO-d6, δ ppm): 56.3 (OCH₃), 113.1, 131.4, 112.2, 161.5, 101.4, 141.4 (methoxylbenzene), 107.4, 128.7, 141.8 (pyrazol), 196.3 (C=O), 128.4, 129.3, 135.7, 125.7, 158.3 (C-O), 117.6 (dichlorohydroxyphenyl); EIMS (70 eV) m/z (%):363.19 (M+, 78%), Anal. Calcd.: for C₁₇H₁₂Cl₂N₂O₃ (363.19): C, 56.16; H, 3.30; N, 7.71; Cl, 19.55. Found: C, 56.09; H, 3.27; N, 7.65; Cl, 19.61%.

General procedure for the preparation of Cu(II) complexes (7)–(12)

To a hot magnetically stirred solution of an appropriate ligand (0.02 mol) in methanol (20 ml) was added a solution of copper(II) chloride (0.01 mol) in warm methanol (10 ml) and resultant mixture refluxed for 1 h. The solid thus formed during reflux was cooled and collected by suction filtration. Thorough washing with hot methanol followed by ether furnished the desired product. It was re-crystallized as the pure product from aqueous-methanol (30:70) by keeping at room temperature for 24 h.

Copper(II) complex of (3,5-dichloro-2-hydroxyphenyl)[1-(2,3,5,6-tetrafluorophenyl)-1H-pyrazol-4-yl]metha-none (7)

Yield 63% as a brownish powder; m.p. (decomp.) 194–196°C; IR (KBr, cm⁻¹): 1696 (C=O), 1335 (C-O), 1040 (pyrazol, N-N), 620 (C-F), 485 (M-O of C=O), 455 (M-O of deprotonated OH); UV (DMSO): λ_max (cm⁻¹); 12985; µ_eff 2.36 B.M.; molar conductance (34 Ohm⁻¹ cm² mol⁻¹); water content, 5.92%; Anal. Calcd.: for C₆₄H₂₈C_l8Cu₂F₁₆N₁₂O₈ (1807.67): C, 42.52; H, 1.56; N, 9.30; Cl, 15.71. Found: C, 42.63; H, 1.65; N, 9.43; Cl, 15.69%.

Copper(II) complex of (3,5-dichloro-2-hydroxyphenyl)[1-(2,5-difluorophenyl)-1H-pyrazol-4-yl]methanone (8)

Yield 59% as a dark green powder; m.p. (decomp.) 188–190°C; IR (KBr, cm⁻¹): 1695 (C=O), 1338 (C-O), 1041 (pyrazol, N-N), 620 (C-F), 490 (M-O of C=O), 465 (M-O of deprotonated OH); UV (DMSO): λ_max (cm⁻¹); 14150; µ_eff 2.42 B.M.; molar conductance (32 Ohm⁻¹ cm² mol⁻¹); water content, 0.49%; Anal. Calcd.: for C₆₄H₃₆C_l8Cu₂F₈N₁₂O₈ (1663.75): C, 46.20; H, 2.18; N, 10.10; Cl, 17.08 Found.: C, 46.41; H, 2.16; N, 11.24; Cl, 17.12%.

Copper(II) complex of (3,5-dichloro-2-hydroxyphenyl)[1-(2,5-dichlorophenyl)-1H-pyrazol-4-yl]methanone (9)

Yield 62% as a reddish brown powder; m.p. (decomp.) 214–217°C; IR (KBr, cm⁻¹): 1695 (C=O), 1335 (C-O), 1040 (pyrazol, N-N), 620 (C-F), 470 (M-O of C=O), 465 (M-O of deprotonated OH); UV (DMSO): λ_max (cm⁻¹); 16810; µ_eff 1.92 B.M.; molar conductance (36 Ohm⁻¹ cm² mol⁻¹); water content, 0.53%; Anal. Calcd.: for C₆₄ H₃₆C_l16Cu₂ N₁₂O₈ (1795.38): C, 42.81; H, 2.02; N, 9.36; Cl, 31.64 Found: C, 42.78; H, 2.00; N, 9.35; Cl, 31.59%.

Copper(II) complex of (3,5-dichloro-2-hydroxyphenyl)[1-(3-methylphenyl)-1H-pyrazol-4-yl]methanone (10)

Yield 64% as a brown powder; m.p. (decomp.) 310–312°C; IR (KBr, cm⁻¹): 1695 (C=O), 1335 (C-O), 1040 (pyrazol, N-N), 620 (C-F), 465 (M-O of C=O), 460 (M-O of deprotonated OH); UV (DMSO): λ_max (cm⁻¹); 11760; µ_eff 2.05 B.M.; molar conductance (35 Ohm⁻¹ cm² mol⁻¹); water content, 0.98%; Anal. Calcd.: for C₆₈H₅₂C_l8Cu₂N₁₂O₈ (1575.93): C, 51.83; H, 3.33; N, 10.67; Cl, 18.03 Found: C, 51.80; H, 3.30; N, 10.66; Cl, 17.99%.

Copper (II) complex of 4-[4-(3,5-dichloro-2-hydroxybenzoyl)-1H-pyrazol-1-yl]benzoic acid (11)

Yield 62% as a brownish powder; m.p. (decomp.) 240–243°C; IR (KBr, cm⁻¹): 1695 (C=O), 1337(C-O), 1040 (pyrazol, N-N), 620 (C-F), 460 (M-O of C=O), 445 (M-O of deptotonated OH); UV (DMSO): λ_max (cm⁻¹); 14620; µ_eff 1.95 B.M.; molar conductance (34 Ohm⁻¹ cm² mol⁻¹); water content, 0.01%; Anal. Calcd.: for C₆₈H₄₄C_l8Cu₂N₁₂O₁₆ (1695.86): C, 48.16; H, 2.62; N, 9.91; Cl, 16.75. Found: C, 48.14; H, 2.59; N, 9.91; Cl, 16.81%.

Copper (II) complex of (3,5-dichloro-2-hydroxyphenyl)[1-(4-methoxyphenyl)-1H-pyrazol-4-yl]methanone (12)

Yield 60% as a dark green powder; m.p. (decomp.) 309–311°C; IR (KBr, cm⁻¹): 1695 (C=O), 1335 (C-O), 1040 (pyrazol, N-N), 620 (C-F), 455 (M-O of C=O), 465 (M-O of deprotonated OH); UV (DMSO): λ_max (cm⁻¹); 12700; µ_eff 1.98 B.M.; Molar conductance (36 Ohm⁻¹ cm² mol⁻¹); water content, 0.01%; Anal. Calcd.: for C₆₈H₅₂C_l8Cu₂N₁₂O₁₂ (1639.93): C, 49.80; H, 3.20; N, 10.25; Cl, 17.32. Found: C, 49.68; H, 3.27; N, 10.34; Cl, 17.40%.

Antibacterial bioassay (in vitro)

Preliminary screening

The synthesized ligands (1)–(6) and their copper(II) complexes (7)–(12) were screened in vitro for their antibacterial activity against four Gram-negative (E. coli, P. aeruginosa, S. typhi and S. flexneri) and two Gram-positive (B. subtilis and S. aureus) bacterial strains by the agar-well diffusion method^15,16. The wells (6 mm in diameter) were dug in the media with the help of a sterile metallic borer with centres at least 24 mm apart. Two- to eight-hours-old bacterial inocula containing approximately 104–106 colony-forming units (CFU/ml) were spread over the surface of the nutrient agar using a sterile cotton swab. Appropriate amount of the test sample (1 mg ml⁻¹ in DMSO) was introduced into the respective wells. Other wells supplemented with DMSO and reference antibacterial drug, imipenam, served as negative and positive controls, respectively. The plates were incubated immediately at 37°C for 24 h. Activity was determined by measuring the diameter (mm) of zones showing complete inhibition. In order to clarify any participating role of DMSO in the biological screening, separate studies were carried out with pure DMSO and they showed no activity against any bacterial strains.

Minimum inhibitory concentration

Compounds exhibiting most significant antibacterial activity (greater than 16 mm) were selected for minimum inhibitory concentration (MIC) determinations by use of disc diffusion technique by employing discs containing 5, 10, 25, 50 and 100 μg ml⁻¹ of the compound under investigation and applying the reported protocol¹⁷.

Results and discussion

Chemistry

The ligands (1)–(6), were prepared by refluxing 3,5-dichloroformylchromone in equimolar quantities with the respective hydrazide for 3–4 h in ethanol (30–40 ml) as shown in Scheme 1. All synthesized ligands were characterized by their physical, spectroscopic (IR and 1H NMR), mass spectral and elemental analyses data. The metal complexes (7)–(12) were all air stable and prepared by the stoichiometric reaction of the copper(II) chloride with the ligand in a molar ratio (metal:ligand) of 1:2. The complexes were intensely coloured and amorphous solids which decomposed without melting. They were insoluble in common organic solvents such as ethanol, methanol, chloroform or acetone and soluble in aqueous-methanol, DMSO and N,N-dimethylformamide (DMF). Molar conductance (32–36 Ohm⁻¹ cm² mol⁻¹) of the complexes (7)–(12) in DMF (10⁻³M solution at 25°C), indicated that they are non-electrolytic in nature¹⁸. The elemental analysis data of the Cu(II) complexes agree well with the proposed composition of the compounds, which were found to be dimeric in nature with two hydrazine molecules bridging the two copper atoms through coordination. Efforts to grow suitable crystals of the metal complexes for x-ray diffraction studies were unsuccessful due to their poor solubility in common organic solvents.

IR, NMR and mass spectra

In the IR spectra of the ligands, the absence of characteristic bands at 1670 and 3315 cm⁻¹ assigned to formylo (HC=O) and amino (-NH₂) moieties, respectively, indicated¹⁹ the coordination of the starting material. The IR spectrum of all the ligands displayed ν(C=O) and ν(OH) at 1650–1685 and 3450 cm⁻¹. The IR spectrum of the ligand (5) exhibited ν(COOH) in the region at 2785 cm⁻¹, respectively. The comparison of the IR spectra of the ligands (1)–(6) with their metal complexes (7)–(12) revealed that the compounds are bidentately coordinated to the metal ions. In all the complexes, the band appearing at 1650–1670 cm⁻¹ due to the νC=O vibrations is shifted to higher frequency by 17–25 cm⁻¹, which is indicative of the involvement of the C=O in chelation. In addition to this, the band at 3450 cm⁻¹ attributed to ν(OH) in the ligands shifted to lower frequency at 1345 cm⁻¹ by 15–25 cm⁻¹ in its metal complexes indicating the deprotonation and coordination of the oxygen atom to the metal atom. These new bands were not present in the spectra of their corresponding ligands. Further conclusive evidence of the coordination of the ligands with the metal ions was established by the far IR spectra in which new bands appearing in the spectra of the metal complexes and not in the spectra of the ligands at 455–470 and 420–440 cm⁻¹ assigned to M-O (of carbonyl)²⁰ and M-O (of dichlorohydroxyphenyl) were observed. Moreover, bands at 615 cm⁻¹ were assigned to C-Cl in the spectra of all compounds.

Based on the experimental evidences available through this study, the complexes are proposed to have binuclear structures as shown below in Scheme 1. Interestingly, the presence of hydrazine molecule (N₂H₄) is shown by thermal analysis data that acts as a bridging link between the two copper atoms. Construction of molecular model reveals that the proposed structure for the complexes is justifiable. The ¹H and C¹³ NMR spectral data of ligands (1)–(6) along with their possible assignments is reported in the experimental part and all the protons and carbons were found in their expected region. These studies are well supported by their IR and ¹H NMR spectra.

Mass spectral data along with the fragments of the ligands under study are given. The molecular ion peaks (M⁺) was visible in this spectra. This data clearly indicate the formation of the ligands having the proposed structures. Fragmentation pattern of the representative ligands (4) and (6) are reproduced in Figure 1.

Figure 1.  Fragmentation patterns of ligands (4) and (6).

Magnetic susceptibility measurements

The room temperature magnetic moments of the solid Cu(II) complexes were found in the range 1.92–2.42 B.M. The values of complexes (7)–(12) were higher than the normal values (~1.63 B.M.) for Cu(II) ion, suggesting^21,22 that these complexes are not mononuclear. Thus the magnetic moment values support the proposed molecular structure of the complexes as binuclear²³.

Conductivity measurements

The conductance values in DMF at the concentration 10⁻³ mole dm⁻³ fall in the range 32–36 Ohm⁻¹ cm² mol⁻¹. The conductance values indicate the non-electrolytic nature of the complexes as there are no anions present in the lattice²⁴. However, slightly higher conductivity values are because of the binuclear nature of the complexes.

Water content and thermal analysis

Water contents of the Cu(II) complexes were determined by the Karl–Fischer titration method. The values do not equate with any whole number of water molecules suggesting that the water content is just the lattice water and the complexes are not completely dry.

The TG, DSC and DTA thermograms of respective Cu(II) complexes are shown as below. The relevant data obtained from the thermograms of all the metal complexes is given in Table 1. The TG-DTA curves of all the complexes showed three to five steps in their decomposition. In the first step of each complex, one out of two hydrazine molecules was lost between 100–156°C. The complexes begin to lose weight around 200–500°C; a sharp decrease in weight shows the loss of one of the ligands from the complexes. The DTA curves show different peaks in the range of 210–390°C. The endothermic peaks in these complexes in the range of 210–375°C are assigned to the loss of the ligands. Some of the representative thermograms are shown in Supplementary Figure S1

Table 1.  Thermal analysis data of Cu(II) complexes (7)–(12).

Compd.	Temp. range (°C)	Decomp. stages	TGA Wt. loss. (%) found	DTA temp., peak (°C)/thermal effect (exo/endo)	Evolved moiety
					Formula	Mass calcd. (%)
(7)	100–219	1	0.94	251 (exo)	NH3	16.9
	219–384	2	77.42	379 (exo)	C48H19Cl16Cu2F12N10O6	1399.3
	384–582	3	92.42	558 (exo)	C56H25Cl8Cu2F14N12O8	1670.2
(8)	100–210	1	0.12	249 (exo)	N2H4	32.6
	210–356	2	97.35	259 (endo)	C64H34Cl8Cu2F8N9O8	1619.3
(9)	100–200	1	0.09	252 (exo)	C6H4	75.3
	200–338	2	86.32	263 (endo)	C53H32Cl13Cu2F8N12O8	1549.3
	338–412	3	92.4	334 (exo)	C53H23C13Cu2F8N12O8	1558.9
(10)	100–217	1	1.7	216 (exo)	NH3	16.2
	217–403	2	67.22	225 (endo)	C46H38Cl6Cu2F8N10O5	1059.4
	403–733	3	93.23	326 (endo)	C61H44C18Cu2N11O8	1469.2
(11)	100–313	1	6.23	165 (endo)	C6H4O2	105.6
	313–407	2	81.43	173 (exo)	C54H44Cl6Cu2N11O13	1380.9
(12)	100–338	1	6.35	279 (exo)	C6H3O2	104.3
	3383–460	2	65.49	293 (endo)	C44H38Cl5CuN11O7	1073.9
	460-S849	3	9S8.87	326 (endo)	C68H50Cl8Cu2N12O11	1621.4

Electronic absorption spectra

The electronic absorption bands of the complexes under investigation are listed in experimental data. The d-d spectra are consistent with the distorted octahedral geometry of the complexes. In the absorption spectra, there is an intense broad band observed at 11,760–16,810 cm⁻¹ which is assigned to a 2e_g→2t_2g transition²⁵. Although three transitions are expected in this case, but they are very close in energy and often appear in the form of one broad envelop²⁶. The values of the electronic transitions for the Cu(II) complexes are specific to an axially deformed octahedral geometry²⁷. The band around 22,220 cm⁻¹ may be assigned to Cu-Cu linkage or bridging²⁸.

Biological activity

Antibacterial studies of the ligands (1)–(6) and their copper(II) complexes (7)–(12)

Antibacterial activities of the synthesized ligands (1)–(6) and their corresponding Cu(II) complexes (7)–(12) were determined against four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Shigella flexneri) and two Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacterial strains. The results of all the synthesized compounds were compared with those of the standard drug imipenam (Table 2). The ligands either exhibited no activity or have varying degree of inhibitory effects (low-to-moderate) against different tested strains. Among all the tested compounds, (1), (4), (5), (7), (8), (10), (11) and (12) were found to be active against Gram-negative and Gram-positive bacterial strains. In comparison to the ligands, the activity of the Cu(II) complexes against all the Gram-negative and Gram-positive species increased on coordination of the ligands with the Cu(II) except the compounds (3), (4), (6) and (9). Interestingly, five complexes (7), (8) and (10)–(12) were found to be more active than the ligands. These results substantiate our own findings and the findings of some other workers^29–31 that biologically inactive compounds become active and less active compounds become more active upon coordination/complexation. Such induction or enhancement in activity of the metal complexes can be explained on the basis of Overtone’s concept and chelation theory. According to Overtone’s concept of cell permeability, the lipid membrane that surrounds the cell favours the passage of only lipid soluble materials due to which liposolubility is an important factor that controls antimicrobial activity. On chelation, the polarity of the metal ion is reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups. Further, it increases the delocalization of π-electrons over the whole chelate ring and enhances the lipophilicity of the complex. This increased lipophilicity in turn enhances the penetration of the complexes into lipid membranes and blocking of metal binding sites on the enzymes of the microorganisms³². The metal complex may also be a vehicle for activation of the ligand as the cytotoxic agent. Moreover, coordination may lead to significant reduction of drug resistance^33–35. Apart from this, other factors such as solubility, conductivity and dipole moment as influenced by the presence of metal ions may also be among the possible reasons causing enhancement of the bactericidal activity of the metal complexes as compared to the uncomplexed compounds^36–39.

Table 2.  Antibacterial activity data of the prepared ligands (1)–(6) and their Cu(II) complexes (7)–(12).

Compd.	Inhibition zone (mm)
	Gram-negative			Gram-positive
	A	B	C	D	E	F
1	0	0	0	0	0	0
2	6.33	6.33	7	6.66	7.33	7.33
3	6.66	7.33	6.66	8.33	8.33	7.66
4	0	0	0	0	0	0
5	7	6.66	0	6.33	7	6.33
6	0	0	0	0	0	0
7	22	19	12	17	24	19
8	26	14	14	16	26	18
9	22	17	15	14	22	16
10	24	15	17	18	24	19
11	22	13	16	20	24	21
12	26	16	15	19	26	22
Standard	30	24	25	27	33	33

A: Escherichia coli; B: Pseudomonas aeruginosa; C: Salmonella typhi; D: Shigella flexneri; E: Bacillus subtilis; F: Staphylococcus aureus.

0: Absence of measurable inhibitory action; >9: weak; 9–16: moderate; >16: significant.

No activity observed against negative control.

Minimum inhibitory concentration

The data obtained after preliminary antibacterial screening showed that compounds (7), (8), (9), (10), (11) and (12) were the most active (above 80%) and their average inhibition values were 18.83, 19.00, 17.66, 19.5, 19.33 and 20.66 mm, respectively. These compounds were therefore, selected for MIC studies (Table 3). The MIC of these compounds was in the range 7.136 × 10⁻⁸ to 1.282 × 10⁻⁶ M. The compound (8) proved to be the most active. It inhibited the growth of B. subtilis at 1.282 × 10⁻⁶ M.

Table 3.  Minimum inhibitory concentration (M/ml) of the Selected Compounds (7), (8), (9), (10), (11) and (12) against selected bacterial strains.

No.	(7)	(8)	(9)	(10)	(11)	(12)
Gram-negative
Escherichia coli	—	2.389 × 10^−6	—	5.261 × 10^−7	—	4.295 × 10^−6
Pseudomonas aeruginosa	5.156 × 10^−8	—	—	—	—	—
Salmonella typhi	—	—	7.136 × 10^−8	—	—	—
Gram-positive
Staphylococcus aureus	—	—	—	—	2.417 × 10^−7	5.238 × 10^−7
Bacillus subtilis	3.324 × 10^−8	1.282 × 10^−6	—	1.294 × 10^−8	3.204 × 10^−8	6.647 × 10^−8

Declaration of interest

The authors report no conflict of interests and are responsible for the contents of the paper.