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

Article abstract of DOI:10.3109/14756366.2011.585135

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.

Full text of DOI:10.3109/14756366.2011.585135

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(2): 223–231
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.585135
RESEARCH ARTICLE
Antibacterial dimeric copper(II) complexes with
chromone-derived compounds
Zahid H. Chohan1, Mohammad S. Iqbal2, Syed K. Aftab3, and A. Rauf4
1Department of Chemistry, Bahauddin Zakariya University, Multan, Pakistan, 2Department of Chemistry, Forman
Christian College, Lahore, Pakistan, 3Department of Chemistry, University of Sargodha, Sargodha, Pakistan, and
4Department of Chemistry, e Islamia University of Bahawalpur, Bahawalpur, Pakistan
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
(Escherichia coli, Pseudomonas aeruginosa, Salmonella
Metal chelation and its relationship with different bio-
logical processes represent a promising area of research
in designing novel therapeutic methodologies to deal
with global problem of ever increasing “bacterial resis-
tance”. Many metal ions are known to play a significant
role in different biological processes1–3. For example,
zinc(II) and copper(II) ions are the second- and third-
most abundant transition metals present in humans.
ey 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 compounds5–10 with a reactive carbonyl group
that show a high reactivity towards many nucleophiles
allowing synthesis of a wide variety of biologically active
compounds11–14. 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
typhi, Shigella flexneri) and two Gram-positive (Bacillus
subtilis, Staphylococcus aureus) bacterial strains. e
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 neces-
sary. in-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.
Address for Correspondence: Zahid H. Chohan, Department of Chemistry, Bahauddin Zakariya University, Multan-60800, Pakistan.
E-mail: dr.zahidchohan@gmail.com
(Received 23 March 2011; revised 28 April 2011; accepted 28 April 2011)
223

224 Z. H. Chohan et al.
1
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.
1685 (C=O), 1040 (pyrazol, N-N), 620 (C-F); H 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, dichloroben-
zene), 7.96 (s, 1H, dichlorobenzene), 8.82 (dichlo-
robenzene), 11.81 (s, 1H, OH); 13C NMR (DMSO-d6, δ
ppm): 117.1, 132.4, 128.6, 131.2, 132.4, 144.1 (dichlo-
rohydroxyphenyl), 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%.
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. e resultant mixture was heated
under reflux for 3–4 h. e solid product thus formed dur-
ing refluxing was collected by suction filtration. orough
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:
(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),
1
1685 (C=O), 1040 (pyrazol, N-N), 620 (C-F); H NMR
(DMSO-d6, δ ppm): 2.34 (s, 3H, CH3), 8.47 (s, 1H, pyra-
zol), 8.22 (s, 1H, pyrazol), 7.91 (s, 1H, dichlorohydroxy-
phenyl), 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 (meth-
ylbenzene), 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 (dichlo-
rohydroxyphenyl); EIMS (70 eV) m/z (%):347.19 (M+,
62%), Anal. Calcd.: for C17H12Cl2N2O2 (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%.
(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),
1
1040 (pyrazol, N-N), 620 (C-F); H 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); 13
C
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 (70eV) m/z (%): 405.13
(M+, 68%); Anal. Calcd.: for C16H6Cl2F4N2O2 (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%.
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 C17H10Cl2N2O4 (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-(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),
1
1041 (pyrazol, N-N), 620 (C-F); H 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, dichlorohydroxy-
phenyl), 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-(4-methoxyphenyl)-1H-
pyrazol-4-yl]methanone (6)
(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),
Yield 70% as a yellowish powder; m.p. 160–162°C; IR (KBr,
cm−1): 3435 (dichlorohydroxyphenyl, OH), 1685 (C=O),
1
1040 (pyrazol, N-N), 620 (C-F); H NMR (DMSO-d6, δ
ppm): 3.82 (s, 3H, OCH3), 8.46 (s, 1H, pyrazol), 8.25 (s,
Journal of Enzyme Inhibition and Medicinal Chemistry

Chromone-derived compounds with antibacterial dimeric copper 225
Scheme 1. Preparation of ligands (1)–(6) and their Cu(II) complexes (7)–(12).
1H, pyrazol), 7.91 (s, 1H, dichlorohydroxyphenyl), 8.07
(s, 1H, dichlorohydroxyphenyl), 7.68 (s, 1H, methoxyl-
benzene), 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 (meth-
oxylbenzene), 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 (dichlorohy-
droxyphenyl); EIMS (70 eV) m/z (%):363.19 (M+, 78%),
Anal. Calcd.: for C17H12Cl2N2O3 (363.19): C, 56.16; H, 3.30;
© 2012 Informa UK, Ltd.

226 Z. H. Chohan et al.
N, 7.71; Cl, 19.55. Found: C, 56.09; H, 3.27; N, 7.65; Cl,
19.61%.
0.98%; Anal. Calcd.: for C68H52Cl8Cu2N12O8 (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%.
General procedure for the preparation of Cu(II)
complexes (7)–(12)
Copper (II) complex of 4-[4-(3,5-dichloro-2-
To a hot magnetically stirred solution of an appropriate
ligand (0.02 mol) in methanol (20 ml) was added a solu-
tion of copper(II) chloride (0.01 mol) in warm methanol
(10 ml) and resultant mixture refluxed for 1h. e solid
thus formed during reflux was cooled and collected by
suction filtration. orough washing with hot methanol
followed by ether furnished the desired product. It was
re-crystallized as the pure product from aqueous-metha-
nol (30:70) by keeping at room temperature for 24 h.
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): λmax (cm−1); 14620;
µeff 1.95 B.M.; molar conductance (34 Ohm−1 cm2 mol−1);
water content, 0.01%; Anal. Calcd.: for C68H44Cl8Cu2N12O16
(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-(2,3,5,6-tetrafluorophenyl)-1H-pyrazol-4-yl]metha-
none (7)
Copper (II) complex of (3,5-dichloro-2-hydroxyphenyl)
[1-(4-methoxyphenyl)-1H-pyrazol-4-yl]methanone
(12)
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): λmax (cm−1); 12985; µeff
2.36B.M.;molarconductance(34Ohm−1 cm2 mol−1);water
content, 5.92%; Anal. Calcd.: for C64H28Cl8Cu2F16N12O8
(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%.
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): λmax (cm−1); 12700;
µeff 1.98 B.M.; Molar conductance (36 Ohm−1 cm2 mol−1);
water content, 0.01%; Anal. Calcd.: for C68H52Cl8Cu2N12O12
(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%.
Copper(II) complex of (3,5-dichloro-2-hydroxyphenyl)
[1-(2,5-difluorophenyl)-1H-pyrazol-4-yl]methanone
(8)
Antibacterial bioassay (in vitro)
Preliminary screening
e 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 method15,16. e 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 approxi-
mately 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. e plates were incubated
immediately at 37°C for 24 h. Activity was determined by
measuring the diameter (mm) of zones showing com-
plete 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.
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): λmax (cm−1); 14150;
µeff 2.42 B.M.; molar conductance (32 Ohm−1 cm2 mol−1);
watercontent,0.49%;Anal.Calcd.:forC64H36Cl8Cu2F8N12O8
(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): λmax (cm−1); 16810;
µeff 1.92 B.M.; molar conductance (36 Ohm−1 cm2 mol−1);
water content, 0.53%; Anal. Calcd.: for C64 H36Cl16Cu2 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 deproto-
nated OH); UV (DMSO): λmax (cm−1); 11760; µeff 2.05 B.M.;
molar conductance (35 Ohm−1 cm2 mol−1); water content,
Minimum inhibitory concentration
Compounds exhibiting most significant antibacte-
rial activity (greater than 16 mm) were selected for
minimum inhibitory concentration (MIC) determina-
tions by use of disc diffusion technique by employ-
ing discs containing 5, 10, 25, 50 and 100 μg ml−1 of
Journal of Enzyme Inhibition and Medicinal Chemistry

Chromone-derived compounds with antibacterial dimeric copper 227
the compound under investigation and applying the
reported protocol17.
observed. Moreover, bands at 615 cm−1 were assigned to
C-Cl in the spectra of all compounds.
Basedontheexperimentalevidencesavailablethrough
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
Results and discussion
Chemistry
e ligands (1)–(6), were prepared by refluxing 3,5-
dichloroformylchromone in equimolar quantities with
the respective hydrazide for 3–4h 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.
e 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. e complexes were intensely coloured and amor-
phous solids which decomposed without melting. ey
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. e
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 hydra-
zine 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.
1
justifiable. e H 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. ese 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. e molecular ion peaks
(M+) was visible in this spectra. is data clearly indi-
cate the formation of the ligands having the proposed
structures. Fragmentation pattern of the representative
ligands (4) and (6) are reproduced in Figure 1.
Magnetic susceptibility measurements
e room temperature magnetic moments of the solid
Cu(II) complexes were found in the range 1.92–2.42 B.M.
e values of complexes (7)–(12) were higher than the
normal values (~1.63 B.M.) for Cu(II) ion, suggesting21,22
that these complexes are not mononuclear. us the
magnetic moment values support the proposed molecu-
lar structure of the complexes as binuclear23.
Conductivity measurements
IR, NMR and mass spectra
e conductance values in DMF at the concentration 10−3
mole dm−3 fall in the range 32–36 Ohm−1 cm2 mol−1. e
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.
In the IR spectra of the ligands, the absence of charac-
teristic bands at 1670 and 3315 cm−1 assigned to formylo
(HC=O) and amino (-NH2) moieties, respectively, indi-
cated19 the coordination of the starting material. e IR
spectrum of all the ligands displayed ν(C=O) and ν(OH)
at 1650–1685 and 3450 cm−1. e IR spectrum of the
ligand (5) exhibited ν(COOH) in the region at 2785 cm−1,
respectively. e comparison of the IR spectra of the
ligands (1)–(6) with their metal complexes (7)–(12)
revealed that the compounds are bidentately coordi-
nated 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 indicat-
ing the deprotonation and coordination of the oxygen
atom to the metal atom. ese 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 spec-
tra 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
Water content and thermal analysis
Water contents of the Cu(II) complexes were determined
by the Karl–Fischer titration method. e values do not
equate with any whole number of water molecules sug-
gesting that the water content is just the lattice water and
the complexes are not completely dry.
e TG, DSC and DTA thermograms of respective
Cu(II) complexes are shown as below. e relevant data
obtainedfromthethermogramsofallthemetalcomplexes
is given in Table 1. e TG-DTA curves of all the com-
plexes 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. e 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. e DTA curves show different peaks in the
range of 210–390°C. e endothermic peaks in these com-
plexes 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.
© 2012 Informa UK, Ltd.

228 Z. H. Chohan et al.
Figure 1. Fragmentation patterns of ligands (4) and (6).
Electronic absorption spectra
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. e values of the elec-
tronic transitions for the Cu(II) complexes are specific
to an axially deformed octahedral geometry27. e band
around 22,220 cm−1 may be assigned to Cu-Cu linkage or
bridging28.
e electronic absorption bands of the complexes under
investigation are listed in experimental data. e 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 2eg→2t2g transition25.
Journal of Enzyme Inhibition and Medicinal Chemistry

Chromone-derived compounds with antibacterial dimeric copper 229
comparison to the ligands, the activity of the Cu(II) com-
plexes 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. ese results
substantiate our own findings and the findings of some
other workers29–31 that biologically inactive compounds
become active and less active compounds become more
active upon coordination/complexation. Such induc-
tion 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
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. e results of all
the synthesized compounds were compared with those of
the standard drug imipenam (Table 2). e 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
Table 1. ermal analysis data of Cu(II) complexes (7)–(12).
Evolved moiety
TGA Wt. loss. (%)
found
DTA temp., peak (°C)/
thermal effect (exo/endo)
Compd.
Temp. range (°C)
100–219
219–384
384–582
100–210
210–356
100–200
200–338
338–412
100–217
217–403
403–733
100–313
313–407
100–338
3383–460
460-S849
Decomp. stages
Formula
Mass calcd. (%)
16.9
(7)
1
2
3
1
2
1
2
3
1
2
3
1
2
1
2
3
0.94
77.42
92.42
0.12
251 (exo)
379 (exo)
558 (exo)
249 (exo)
259 (endo)
252 (exo)
263 (endo)
334 (exo)
216 (exo)
225 (endo)
326 (endo)
165 (endo)
173 (exo)
279 (exo)
293 (endo)
326 (endo)
NH3
C48H19Cl16Cu2F12N10O6
C56H25Cl8Cu2F14N12O8
N2H4
1399.3
1670.2
32.6
(8)
(9)
97.35
0.09
C64H34Cl8Cu2F8N9O8
C6H4
1619.3
75.3
86.32
92.4
C53H32Cl13Cu2F8N12O8
C53H23C13Cu2F8N12O8
NH3
1549.3
1558.9
16.2
(10)
1.7
67.22
93.23
6.23
C46H38Cl6Cu2F8N10O5
C61H44C18Cu2N11O8
C6H4O2
1059.4
1469.2
105.6
(11)
(12)
81.43
6.35
C54H44Cl6Cu2N11O13
C6H3O2
1380.9
104.3
65.49
9S8.87
C44H38Cl5CuN11O7
C68H50Cl8Cu2N12O11
1073.9
1621.4
Table 2. Antibacterial activity data of the prepared ligands (1)–(6) and their Cu(II) complexes (7)–(12).
Inhibition zone (mm)
Gram-negative
Gram-positive
Compd.
A
0
B
0
C
0
D
0
E
0
F
0
1
2
3
4
6.33
6.66
0
6.33
7.33
0
7
6.66
8.33
0
7.33
8.33
0
7.33
7.66
0
6.66
0
5
6
7
6.66
0
0
6.33
0
7
6.33
0
0
0
0
7
8
9
10
11
12
Standard
22
26
22
24
22
26
30
19
14
17
15
13
16
24
12
14
15
17
16
15
25
17
16
14
18
20
19
27
24
26
22
24
24
26
33
19
18
16
19
21
22
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.
© 2012 Informa UK, Ltd.

230 Z. H. Chohan et al.
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
Pseudomonas aeruginosa
Salmonella typhi
Gram-positive
—
5.156×10−8
—
2.389×10−6
—
—
5.261×10−7
—
—
—
4.295×10−6
—
—
—
—
—
—
7.136×10−8
Staphylococcus aureus
Bacillus subtilis
—
—
—
—
—
2.417×10−7
3.204×10−8
5.238×10−7
6.647×10−8
3.324×10−8
1.282×10−6
1.294×10−8
6. Ellis GP. Chromenes, Chromanones and Chromones. John Wiley
and Sons, New York, (1977) p. 1
antimicrobial activity. On chelation, the polarity of the
metal ion is reduced to a greater extent due to the over-
lap of the ligand orbital and partial sharing of the posi-
tive 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. is 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. e 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 resistance33–35. Apart from this, other factors such
as solubility, conductivity and dipole moment as influ-
enced by the presence of metal ions may also be among
the possible reasons causing enhancement of the bacte-
ricidal activity of the metal complexes as compared to the
7. Staunton J., In: Barton D, Ollis WD, eds. Comprehensive Organic
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.
13. Chohan ZH, Youssoufi MH, Jarrahpour A, Ben Hadda T.
Identification of antibacterial and antifungal pharmacophore sites
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Minimum inhibitory concentration
e data obtained after preliminary antibacterial screen-
ing 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. ese compounds were there-
fore, selected for MIC studies (Table 3). e MIC of these
compounds was in the range 7.136× 10−8 to 1.282 × 10−6
M. e compound (8) proved to be the most active. It
inhibited the growth of B. subtilis at 1.282 × 10−6 M.
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Declaration of interest
18. Geary WJ. e use of conductivity measurements in organic
solvents for the characterization of coordination compounds.
Coord Chem Rev 1971;7:81–122.
e authors report no conflict of interests and are respon-
sible for the contents of the paper.
19. Nakamoto K. Infrared Spectra of Inorganic and Coordination
Compounds, 2nd edn. New York: Wiley Interscience 1970.
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