Multiple heating rate kinetic parameters, thermal, X-ray diffraction studies of newly synthesized octahedral copper complexes based on bromo-coumarins along with their antioxidant, anti-tubercular and antimicrobial activity evaluation

Article abstract of DOI:10.1016/j.saa.2012.05.057

Series of new Cu(II) complexes were synthesized by classical thermal technique. The biologically potent ligands (L) were prepared by refluxing 6-brom 3-acetyl coumarin with aldehydes in the presence of piperidine in ethanol. The Cu(II) complexes have been synthesized by mixing an aqueous solution of Cu(NO₃)₂ in 1:1 molar ratios with ethanolic bidentate ligands and Clioquinol. The structures of the ligands and their copper complexes were investigated and confirmed by the elemental analysis, FT-IR, ¹H NMR, ¹³C NMR, mass spectral and powder X-ray diffraction studies respectively. Thermal behaviour of newly synthesized mixed ligand Cu(II) complexes were investigated by means of thermogravimetry, differential thermogravimetry, differential scanning calorimetry, electronic spectra and magnetic measurements. Dynamic scan of DSC experiments for Cu(II) complexes were taken at different heating rates (2.5-20 °C min^-1). Kinetic parameters for second step degradation of all complexes obtained by Kissinger's and Ozawa's methods were in good agreement. On the basis of these studies it is clear that ligands coordinated to metal atom in a monobasic bidentate mode, by OO and ON donor system. Thus, suitable octahedral geometry for hexa-coordinated state has been suggested for the metal complexes. Both the ligands as well as its complexes have been screened for their in vitro antioxidant, anti-tubercular and antimicrobial activities. All were found to be significant potent compared to parent ligands employed for complexation.

Full text of DOI:10.1016/j.saa.2012.05.057

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
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Multiple heating rate kinetic parameters, thermal, X-ray diffraction studies of
newly synthesized octahedral copper complexes based on bromo-coumarins
along with their antioxidant, anti-tubercular and antimicrobial activity evaluation
⇑
Ketan S. Patel, Jiten C. Patel, Hitesh R. Dholariya, Kanuprasad D. Patel
Chemistry Department, V.P. & R.P.T.P. Science College, Sardar Patel University, Vallabh Vidhyanagar 388 120, Gujarat, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
"
"
"
"
Mixed-ligand Cu(II) complexes
based on bromo-coumarins with
Clioquinol.
Octahedral geometry was confirmed
using electronic spectra and
magnetic measurement.
X-ray diffraction studies and multi-
heating-rate kinetic parameters
measurements of Cu(II) complexes.
Antioxidant, anti-tubercular and
antimicrobial studies of complexes.
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of new Cu(II) complexes were synthesized by classical thermal technique. The biologically potent
ligands (L) were prepared by refluxing 6-brom 3-acetyl coumarin with aldehydes in the presence of piper-
idine in ethanol. The Cu(II) complexes have been synthesized by mixing an aqueous solution of Cu(NO3)2
in 1:1 molar ratios with ethanolic bidentate ligands and Clioquinol. The structures of the ligands and their
copper complexes were investigated and confirmed by the elemental analysis, FT-IR, 1H NMR, 13C NMR,
mass spectral and powder X-ray diffraction studies respectively. Thermal behaviour of newly synthesized
mixed ligand Cu(II) complexes were investigated by means of thermogravimetry, differential thermo-
gravimetry, differential scanning calorimetry, electronic spectra and magnetic measurements. Dynamic
scan of DSC experiments for Cu(II) complexes were taken at different heating rates (2.5–20 °C minꢀ1).
Kinetic parameters for second step degradation of all complexes obtained by Kissinger’s and Ozawa’s
methods were in good agreement. On the basis of these studies it is clear that ligands coordinated to metal
atom in a monobasic bidentate mode, by OAO and OAN donor system. Thus, suitable octahedral geometry
for hexa-coordinated state has been suggested for the metal complexes. Both the ligands as well as its
complexes have been screened for their in vitro antioxidant, anti-tubercular and antimicrobial activities.
All were found to be significant potent compared to parent ligands employed for complexation.
Ó 2012 Elsevier B.V. All rights reserved.
Received 31 March 2012
Received in revised form 17 May 2012
Accepted 26 May 2012
Available online 4 June 2012
Keywords:
Clioquinol
Isoconversional method
Antioxidant
Anti-tubercular
Powder XRD
1. Introduction
atives, like a simplest naturally occurring phenolic substance
possessing fused benzene and a-pyrone rings. Naturally occurring
Interest in coumarin chemistry has flourished for many years;
basically from a result of the wide spread use of coumarin deriva-
tives. Coumarin was basic molecule found in family of many deriv-
as well as synthetic derivatives of coumarin compound have
importance in wide range of fields such as medicinal chemistry,
biochemistry, plant science and pharmacology [1]. A large number
of structurally novel coumarin derivatives have been ultimately re-
ported to show substantial cytotoxic and anti-HIV activity in vitro
and in vivo [2]. The coumarin exists in a variety of forms, due to the
various substitutions possible in their basic structure, which
⇑
Corresponding author. Tel.: +91 2692 230011; fax: +91 2692 235207.
E-mail addresses: ketan_patel1268@yahoo.com (K.S. Patel), drkdpatel64@
yahoo.co.in (K.D. Patel).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.05.057

K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
469
modulate their biological activity [3,4]. The coumarin derivatives
are known to have diverse applications as anti-HIV [5–8], antibac-
terial [9,10], anthelminthic [11], anti-inflammatory [12–14] and
antioxidant activities [15–14], anticoagulants, spasmolytics, anti-
cancer drugs or as plant growth regulating agents [18–20]. Their
complexation ability in respect to different metal ions has been
studied and discussed widely in a considerable number of investi-
gations [21–24]. It has been found that the binding of a metal to
coumarin moiety retains or even enhances its biological activity
[25–27]. In recent years the metal ions such as copper(II), iron(II),
iron(III) or platinum(II) exert wide biological activity, for example
against tumor cells. Also chromones, flavonoids and coumarins
have been known for similar properties. From above facts those
complexes of metals and ligands would be more active than the ba-
sic compounds. The biological activity of these complexes recently
described in the literature is similar to widely used carboplatin
[28–33]. Apart from the medicinal, biological and pharmacological
applications coumarins are also used as sweeteners, fixatives of
perfumes, additives in food, odour stabilizers in tobacco and an
odour masker in paints and rubber [33].
purification. Solvents employed were distilled, purified and dried
by standard procedures prior to use [53]. Clioquinol was purchased
from Agro Chemical Division, Atul Ltd., Valsad-India. The metal ni-
trates were used in hydrated form.
2.2. Physical measurements
All reactions were monitored by thin-layer chromatography
(TLC on alluminium plates coated with silica gel 60 F254
,
0.25 mm thickness, E. Merck, Mumbai-India) and detection of the
components were measured under UV light or explore in Iodine
chamber. Carbon, hydrogen and nitrogen were estimated by ele-
mental analyzer Perkin–Elmer, USA 2400-II CHN analyzer. Metal
ion analyses was carry out by the dissolution of solid complex in
hot concentrated nitric acid, further diluting with distilled water
and filtered to remove the precipitated organic ligands. Remaining
solution was neutralized with ammonia solution and the metal
ions were titrated against EDTA. 1H and 13C NMR measurements
were carried out on Advance-II 400 Bruker NMR spectrometer,
SAIF, Chandigarh. The chemical shifts were measured with respect
to TMS which used as internal standard and DMSO-d6 used as sol-
vent. Infrared spectra of solids were recorded in the region 4000–
400 cmꢀ1 on a Nicolet Impact 400D Fourier-Transform Infrared
Spectrophotometer using KBr pellets. The FAB mass spectrum of
the complex was recorded at SAIF, CDRI, Lucknow with JEOL SX-
102/DA-6000 mass spectrometer. Melting point of the ligands
and metal complexes were measured by open capillary tube meth-
od. Solid state magnetic susceptibility measurements were carried
out at room temperature using a Gouy’s magnetic susceptibility
balance with mercury tetrathiocyanato cobaltate(II) being used
as a reference standard (g = 16.44 ꢁ 10ꢀ6 c.g.s. units). Molar sus-
ceptibility was corrected using Pascal’s constant [54]. Thermal
decomposition (TG/DTG) analysis was obtained by a model Dia-
mond TG/DTA, Perkin–Elmer, USA. The experiments were per-
formed in N2 atmosphere at a heating rate of 20 °C minꢀ1 in the
temperature range 30–840 °C. DSC analyses were carried out using
Perkin–Elmer USA, Differncial Scanning Calorimetry (DSC-PYRIS-
1). DSC analyses of complexes were also evaluated from dynamic
scanning experiments at multiple heating rates of 2.5, 5, 10, 15
and 20 °C minꢀ1, respectively, with the best resolution and com-
parative results achieved at a scanning rate of 10 °C minꢀ1. The
samples sizes were ranged in mass from 3 to 8 mg were heated
in Al2O3 crucible. The electronic spectra were collected using
LAMBDA 19 UV/Vis/NIR spectrophotometer in the region 200–
1200 nm.
Clioquinol (5-chloro-7-iodo-8-hydoxyquinoline) belongs to the
quinoline class of compounds was first prepared in Germany by
Ciba–Geigy during the last century. Clioquinol was used as antibi-
otics for the period of 1950s to 1970s [34,35]. Moreover, Clioquinol
(CQ) is an antibiotic with metal-binding properties which has been
shown to have anticancer activity in a number of experimental
model systems [36,37]. Although Clioquinol has a long history of
use in humans, it was observed that the cause of an epidemic of
a
rare neurological disease (subacute myeloptic neuropathy
(SMON)) in Japan and was banned in many countries. Since that
time, others have pointed out that SMON was not seen in other
countries where Clioquinol was extensively used and have criti-
cized the epidemiological data that led to its banning [38,39]. Be-
cause of optimistic data in animal studies, Clioquinol has been
administered in clinical trials for Alzheimer’s disease without reap-
pearance of SMON [40,41], prompting careful re-evaluation of its
use as a therapeutic agent. Clioquinol was widely used as an anti-
biotic for the treatment of amoebic dysentery and skin infection
[42]. Regardless of it being a controversial compound, CQ can still
serve as a model compound from which analogues could be devel-
oped that exploit its copper binding potential but avoid its negative
associations. CQ is a lipophilic compound that is capable of forming
stable complexes with Cu(II) ions [43]. Furthermore, the complexes
of CQ with copper and zinc metal ions recognized for their biolog-
ical effects which are significantly allied with protein aggregation
and degeneration process in the brain [43], also these complexes
have been used as an antimicrobial agent since many years [44].
Coumarin and CQ used as capable applicant for biological aspects
[45]. Many reports were available on its credited [46].
Here the continuation of our earlier work [47,48], in present
communication we describe synthesis, characteristic, spectro-
scopic properties, powder X-ray diffraction study and thermal as-
pects of newly coumarin based mixed ligand Cu(II) complexes as
well as antioxidant, anti-tubercular and antimicrobial screening
of newly synthesized compounds. Kissinger [49,50] and Flynn–
Wall–Ozawa (FWO) [51,52] kinetic methods were employed to
evaluate the kinetic parameters i.e. activation energies and the
pre-exponential factor.
2.3. Crystallographic analysis
X-ray diffraction intensities were carried out with 0.0025
accuracy using XRD Diffractometer (powder), Xpert MPD, Philips,
Holland equipped with 2 kW power and Cu target X-ray tube used
as a source of wavelength 1.542 Å, while the data were accumulate
using JCPDF database. The detector used in the system was Xe-
filled counterate and 2° h measurement range of the instrument
is 3° to 136°. The system contains goniometer was operated on ver-
tical and horizontal mode with h–h and h–2h position respectively
with radius 130–230 mm.
2.4. Synthesis of 3-acetyl coumarin
2. Experimental
3-Acetyl coumarin was prepared according to the reported
method [55]. A mixture of 6-Bromo salicylaldehyde (0.1 mol,
12.2 g), ethyl acetoacetate (0.1 mol, 13.0 g) and 3–4 drop piperi-
dine were stirred for 10 min at room temperature in a 100 mL
round bottom flask. After 10 min it was heated for 30 min in water
bath. A yellow solid obtained was taken out and washed with cold
2.1. Materials
All reagents were of analytical reagent (AR) grade purchased
from Spectro Chem. Ltd., Mumbai-India and used without further

470
K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
ether. It was recrystallized from chloroform-hexane. Yield: 92%;
m.p. 119.5 °C.
2.5.3. Synthesis of 6-bromo-3-(3-(3-hydroxyphenyl)acryloyl)-2H-
chromen-2-one (L3)
L3 was synthesized by same method used for L1 by using 3-hy-
droxy benzaldehyde instead of 3-chloro benzaldehyde. Yield: 72%,
2.5. Synthesis of ligands (L1–L6)
m.p. 167–169 °C FT-IR (KBr. cmꢀ1):
m(C@O,
a,b-unsaturated ke-
tone), m(OAH) 3426, m(C@O, lactone carbonyl of coumarin) 1740.
The neutral bidentate ligands were synthesized using Claisen–
Schmidt condensation [56]. General procedure for synthesis of
the ligands (L) is shown in Scheme 1. The ligands were character-
ized using elemental analysis, FT-IR, Mass and NMR (1H and 13C)
spectroscopy, while the Mass (L2), NMR (L6) and FT-IR (L3) spectra
is given in the supplementary data (S1–S3).
1H NMR (DMSO-d6 400 MHz): d 6.77 (1H, d, J = 16, CH@CH– pro-
tons), d 6.92–8.14 (7H, m, aromatic protons), d 8.22 (1H, d, J = 16,
CH@CH– protons), d 8.56 (1H, s, C4-H). d 9.72 (1H, s, –OH). 13C
NMR (DMSO-d6 100 MHz): d 115.5, 117.9, 118.6, 120.2, 121.4,
124.8, 125.6, 130.2, 130.8, 134.5, 134.9, 135.4, 142.9 (13 different
types of aromatic carbons), 147.2(C-4), 152.2(C-9), 158.4(C-16, car-
bon attach to phenolic OH), 160.5 (C@O, lactone carbonyl of cou-
2.5.1. Synthesis of 6-bromo-3-(3-(3-chlorophenyl)acryloyl)-2H-
chromen-2-one (L1)
marin), 183.2 (C@O, a,b-unsaturated ketone). MS (ESI) m/z 370.0
[M+H]+, 372 [M+H]2+; elemental analysis found (%): C, 58.08; H,
2.82; calculated for C18H11BrO4 (371.18): C, 58.24; H, 2.99.
In a 100 ml round bottom flask 6-bromo 3-acetyl coumarin
(0.01 mol, 1.88 g) and 3-chloro benzaldehyde (0.015 mol) were ta-
ken in 15 mL of pyridine. Catalytic amount of piperidine (1.0 mL)
was added and the reaction mixture was stirred for 10 min at room
temperature. After clear solution obtained, the reaction mixture
was refluxed on oil bath. Completion of reaction was checked by
TLC using mobile phase Ethyl acetate:Hexane (7:3). After the com-
pletion of reaction, subsequently it was allowed to room tempera-
ture. Afterwards it was pour into ice-cold water and adjusts the pH
4–5 using diluted HCl. A solid product separated out was filtered
off, later on washed with cold ethanol and dried in air. It was
recrystallized from ethanol. Yield: 86%, m.p. 164–165 °C. FT-IR
2.5.4. Synthesis of 6-bromo-3-(3-(4-hydroxyphenyl)acryloyl)-2H-
chromen-2-one (L4)
L4 was synthesized by same method used for L1 by using 4-hy-
droxy benzaldehyde instead of 3-chloro benzaldehyde. Yield: 74%,
m.p. 158–160 °C FT-IR (KBr. cmꢀ1):
m
(C@O,
a,b-unsaturated ke-
tone) 1617,
m(C@O, lactone carbonyl of coumarin) 1734, m
(O–H)
3426. 1H NMR (DMSO-d6 400 MHz): d 6.88 (1H, d, J = 16, CH@CH–
protons), d 7.22 (2H, d, J = 7.8, CH@CH– protons), d 7.56 (2H, d,
J = 7.8, CH@CH– protons), d 7.48–8.11 (3H, m, three aromatic pro-
tons), d 8.20 (1H, d, J = 16, CH@CH– protons), d 8.58 (1H, s, C4-H), d
9.76 (1H, s, –OH). 13C NMR (DMSO-d6 100 MHz): d 114.9, 115.8,
118.1, 119.5, 124.2, 125.5, 127.3, 130.9, 134.7, 134.4, 142.6 (11 dif-
ferent types of aromatic carbons), 147.7(C-4), 152.8(C-9), 157.9(C-
17, carbon attach to phenolic OH), 159.2 (C@O, lactone carbonyl of
(KBr, cmꢀ1):
m(C@O, a,b-unsaturated ketone) 1627, m(C@O, lactone
carbonyl of coumarin) 1745. 1H NMR (DMSO-d6 400 MHz): d 6.88
(1H, d, J = 15.6, CH@CH– protons), d 7.74–8.03 (7H, m, aromatic
protons), d 8.16 (1H, d, J = 15.6 CH@CH– protons), d 8.59 (1H, s,
C4-H). 13C NMR (DMSO-d6 100 MHz): d 118.1, 119.8, 124.8, 125.5,
126.2, 126.7, 128.4, 130.1, 130.8, 133.6, 134.0, 134.7, 136.2,
142.6, (14 different types of aromatic carbons), 147.3(C-4),
152.6(C-9), 158.7 (C@O, lactone carbonyl of coumarin), 182.9
coumarin), 183.4 (C@O,
a,b-unsaturated ketone). MS (ESI) m/z
370.0 [M+H]+, 372.0 [M+H]2+; elemental analysis found (%): C,
58.09; H, 2.78; calculated for C18H11BrO4 (371.18): C, 58.24; H,
2.99.
(C@O, a
,b-unsaturated ketone). MS (ESI) m/z 390.0 [M+H]+, 392.1
[M+H]2+, 394.0 [M+H]4+; Elemental analysis found (%):C, 55.27;
H, 2.34; calculated for C18H10BrClO3 (389.63): C, 55.49; H, 2.59.
2.5.5. Synthesis of 6-bromo-3-(3-(3-hydroxy,4-
2.5.2. Synthesis of 6-bromo-3-(3-(4-chlorophenyl)acryloyl)-2H-
chromen-2-one (L2)
methoxyphenyl)acryloyl)-2H-chromen-2-one (L5)
L5 was synthesized by same method used for L1 by using 3-hy-
droxy,4-methoxybenzaldehyde instead of 3-chloro benzaldehyde.
L2 was synthesized by same method used for L1 by using 4-
chloro benzaldehyde instead of 3-chloro benzaldehyde. Yield:
Yield: 80%, m.p. 169–170 °C FT-IR (KBr. cmꢀ1):
m
(C@O,
a,b-unsatu-
76%, m.p. 164–165 °C FT-IR (KBr. cmꢀ1):
m
(C@O,
a
,b-unsaturated
rated ketone) 1618, m(C@O, lactone carbonyl of coumarin) 1735,
ketone) 1621,
m
(C@O, lactone carbonyl of coumarin) 1738. 1H
(C–O–C, asymmetric) 1240, (C–O–C, symmetric) 1042. 1H NMR
(DMSO-d6 400 MHz): d 3.79 (3H, s, –OCH3), d 6.82 (1H, d, J = 15.6,
CH@CH– protons), d 6.98–7.59 (6H, m, six aromatic protons), d
8.18 (1H, d, J = 15.6, CH@CH– protons), d 8.59 (1H, s, C4-H), d
11.10 (1H, s, –OH). 13C NMR (DMSO-d6 100 MHz): d 56.8 (C-18,
OCH3), 112.4, 117.5, 118.7, 120.4, 122.6, 127.7, 124.6, 125.7,
130.9, 134.8, 134.7, 142.5, 149.7 (13 different types of aromatic
carbons), 146.8(C-4), 147.5 (C-17, carbon attach to phenolic OH),
152.7(C-9), 159.6 (C@O, lactone carbonyl of coumarin), 183.7
NMR (DMSO-d6 400 MHz): d 6.81 (1H, d, J = 16, CH@CH– protons),
d 7.34 (2H, d, J = 7.8, CH@CH– protons), d 7.58 (2H, d, J = 7.8,
CH@CH– protons), d 7.78–8.08 (3H, m, three aromatic protons), d
8.12 (1H, d, J = 16, CH@CH– protons), d 8.59 (1H, s, C4-H). 13C
NMR (DMSO-d6 100 MHz): d 118.7, 119.4, 124.6, 125.1, 128.2,
129.7, 129.3, 133.1, 133.8, 130.2, 134.9, 142.6, (12 different types
of aromatic carbons), 147.6(C-4), 152.3(C-9), 159.7 (C@O, lactone
carbonyl of coumarin), 183.8 (C@O,
a,b-unsaturated ketone). MS
(ESI) m/z 390.0 [M+H]+, 392.0 [M+H]+2, 394.0 [M+H]4+; elemental
analysis found (%): C, 55.27; H, 2.41; calculated for C18H10BrClO3
(389.63): C, 55.49; H, 2.59.
(C@O, a
,b-unsaturated ketone). MS (ESI) m/z 400.0 [M+H]+, 402.0
[M+H]2+; elemental analysis found (%):C, 56.55; H, 3.03; calculated
for C19H13BrO5 (399.99): C, 56.88; H, 3.27.
Scheme 1. General procedure for synthesis of ligands (L).

K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
471
2.5.6. Synthesis of 6-bromo-3-cinnamoyl-2H-chromen-2-one (L6)
2.7. Antioxidant study
L6 was synthesized by same method used for L1 by using benz-
aldehyde instead of 3-chloro benzaldehyde. Yield: 79%, m.p. 166–
Ferric reducing antioxidant power (FRAP) was determine using
an adapted method [57,58]. The antioxidant potentials of the com-
pounds were examine by their reducing power of the TPTZ–Fe(III)
complex to TPTZ–Fe(II) complex for the total antioxidant capacity
of tested samples. This method was employed because of its sim-
ple, fast and also reproducible results can be obtained. Initially fol-
lowing solutions were prepared, (A) acetate buffer, 300 mM pH 3.6
(3.1 g sodium acetate trihydrate and 16 ml conc. acetic acid per L of
buffer solution), (B) 10 mM 2,4,6-tripyridyl-s-triazine in 40 mM
HCl, (C) 20 mM FeCl3ꢂ6H2O in distilled water, (D) 1 mM of ascorbic
acid dissolved in 100 mL distilled water. FRAP working solution
was prepared by mixing the above (A), (B) and (C) solutions in
169 °C FT-IR (KBr. cmꢀ1):
m(C@O,
a,b-unsaturated ketone) 1602,
m
(C@O, lactone carbonyl of coumarin) 1743, (C–O–C, asymmetric)
1242, (C–O–C, symmetric) 1040. 1H NMR (DMSO-d6 400 MHz): d
6.86 (1H, d, J = 16, CH@CH– protons), d 7.21–8.05 (8H, m, eight aro-
matic protons), d 8.25 (1H, d, J = 16, CH@CH– protons), d 8.59 (1H,
s, C4-H). 13C NMR (DMSO-d6 100 MHz): 118.1, 119.5, 124.1, 124.9,
126.8, 128.4, 129.2, 130.3, 134.5, 134.9, 135.7, 142.6 (12 different
types of aromatic carbons), 147.7(C-4), 152.4(C-9), 159.6 (C@O,
lactone carbonyl of coumarin), 183.7 (C@O,
a,b-unsaturated ke-
tone). MS (ESI) m/z 355.0 [M+H]+, 357.0 [M+H]2+; elemental analy-
sis found (%): C, 60.52; H, 3.03; calculated for C18H11BrO3 (355.18):
C, 60.67; H, 3.12.
the ratio of 10:1:1, respectively. A mixture of 40.0 lL, 0.5 mM sam-
ple solution and 1.2 mL FRAP reagent was incubated at 37 °C for
15 min. The working solution was necessary to use as freshly pre-
pared. The ascorbic acid was used as a standard antioxidant com-
pound and results were expressed with compared to ascorbic acid.
2.6. Synthesis of metal complexes
2.6.1. [Cu(L1)(CQ) (H2O)OH]ꢂ2H2O (C1)
An aqueous solution of Cu(NO3)2ꢂ3H2O salt (10 mmol) was
added into ethanolic solution of ligand (L1) (10 mmol) and subse-
quently an ethanolic solution of Clioqunol (10 mmol) was added
with continuous stirring. Then the pH was adjusted in between
4.5 and 6.0 by addition of diluted NH4OH solution. The resulting
solution was refluxed for 5 h and then heated over a steam bath
to evaporate up to half of the volume. The reaction mixture was
kept overnight at room temperature. A fine coloured product was
obtained in powder form. The obtained product was washed with
ether and dried over vacuum desiccators.
2.8. Anti-tubercular activity
Test compounds were evaluated for in vitro anti-tubercular
activity. The MICs were determined and interpreted for M. tubercu-
losis H37Rv according to the procedure of the approved micro dilu-
tion reference method of antimicrobial susceptibility testing
[59,60]. Compounds were taken at concentrations of 100, 50, 25
and 12 lg/mL in DMSO, 1.0 ml of each concentration was used
for the study. To this, 9.0 ml of Lowenstein–Jensen medium was
added. A sweep from M. tuberculosis H37RV strain culture was dis-
charged with the help of nichrome wire loop with a 3 mm external
diameter into a vial containing 4 ml of sterile distilled water. The
vial was shaken for 5 min. Then using nichrome wire loop suspen-
sion was inoculated on the surface of each of Lowenstein–Jensen
Complexes C2–C6 was prepared according to same method and
their physicochemical parameters are summarized in Table 1. The
synthetic protocol of complexes is shown in scheme 2, while FT-IR
spectrum of C3 is given in the supplementary data (S4).
Table 1
Analytical and physiochemical parameters of ligands and complexes.
Compounds empirical formula
Elemental analyses, % found (calc.)
Colour
M.p. (°C)
Yield (%)
Molecular weight
leff B.M.
C
H
N
Cu(II)
C1 C28H24BrCl2CuINO8
C2 C28H22BrCl2CuINO7
C3 C28H23BrClCuINO8
C4 C28H25BrClCuINO9
C5 C29H29BrClCuINO11
C6 C28H23BrClCuINO7
38.86(38.57)
40.73(40.42)
41.66(40.46)
40.75(40.52)
39.88(39.72)
41.56(41.78)
2.87(2.65)
2.69(2.43)
2.27(2.08)
3.05(2.76)
3.35(3.08)
2.42(2.60)
1.66(1.45)
1.70(1.47)
1.74(1.44)
1.70(1.41)
1.60(1.47)
1.58(1.80)
7.53(7.31)
7.70(7.42)
7.87(7.53)
7.70(7.44)
7.28(7.08)
8.02(8.19)
Greenish Yellow
Greenish Yellow
Greenish Yellow
Greenish Yellow
Greenish Yellow
Greenish Yellow
>350
>300
>350
>300
>350
>300
65
60
77
75
70
72
843.75
825.74
807.29
825.31
873.35
776.26
1.80
1.79
1.87
1.85
1.82
1.83
Scheme 2. General synthetic route of complexes (C).

472
K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
medium containing the test compounds. Further test media was
incubated for four weeks at 37 °C. Readings were taken at the
end of fourth week. The appearance of turbidity was considered
as bacterial growth and indicates resistance to the compound. Test
compounds were compared to reference drugs Isoniazid
3.1. Elemental analysis
The analytical and physiochemical data of the complexes are
summarized in Table 1. The complexes were colored, insoluble in
water and commonly organic solvents while soluble in DMSO as
well as stable in air. The structure of the complexes is assumed
according to the chemical reaction as shown below:
(MIC = 0.025
lg/mL), Streptomycin (MIC = 6.25
lg/mL) and Etham-
butol (MIC = 20
lg/mL). Lowenstein–Jensen medium containing
standard drugs as well as DMSO was inoculated with M. tuberculo-
sis H37RV strain. The anti-tubercular activity tests were run in
triplicate.
CuðNO3Þ2 ꢂ3H2OþLþCQ !½CuðLÞðCQÞðH2OÞOHꢃ:ꢁH2Oþ2 HNO3 þnH2O
ðwhere L¼L1;L2;L3;L4;L5;L6; x¼2;1;1;2;3;1 and n¼1;2;2;1;0;2Þ:
2.9. Antimicrobial activity
The in vitro antimicrobial activity of all the synthesized ligand
and corresponding Cu(II) complexes were screened for their anti-
bacterial against two Gram(+ve) Staphylococcus aureus, Bacillus sub-
tilis and two Gram(ꢀve) Escherichia coli, Pseudomonas aeruginosa,
where antifungal against Candida albicans, Aspergillus niger and
Aspergillus clavatus using the broth dilution method [61]. All the
ATCC culture was collected from institute of microbial technology,
Bangalore. 2% Luria broth solution was prepared in distilled water
while, pH of the solution was adjusted to 7.4 0.2 at room temper-
ature and sterilized by autoclaving at 15 lb pressure for 25 min.
The tested bacterial and fungal strains were prepared in the Luria
broth and incubated at 37 °C and 200 rpm in an orbital incubator
for overnight. Sample solutions were prepared in DMSO for con-
3.2. FT-IR spectra
The significant infrared spectral bands and their assignments
for the synthesized ligands and their complexes were recorded as
KBr disks and are presented in Table 2. The IR data of the free li-
gands and its metal complexes were carried out within the IR
range 4000–400 cmꢀ1. The vibrational frequencies of complexes
were comparing with their free ligand. The IR spectrum of the com-
pounds L3, L4 and L5 shows a strong OH stretching band in between
3460 and 3520 cmꢀ1, while spectra of the mixed-ligand Cu(II) com-
plexes reveals that a broad band in the region 3410–3450 cmꢀ1
due to stretching vibration of OH group which indicates formation
of complexes and other bands at ꢄ850 cmꢀ1 and ꢄ715 cmꢀ1 due to
the rocking and wagging vibration of the OH group respectively,
caused by the water molecule present in synthesized complexes
[62]. The IR spectrum of the free ligand shows a very stronger
bands at ꢄ1633 and ꢄ1598 cmꢀ1 due to stretching frequency of
C@N present in Clioquinol moiety. These bands were shifted to
lower frequencies in the complexes ꢄ30–38 cmꢀ1, which clearly
indicate that it has been affected upon complexation via metal
ions. The complexes of 8-hydroxyquinoline with divalent metals,
the observed band of C–O stretching frequency appeared at
ꢄ1120 cmꢀ1 region and this band faintly varies with the metal
[63]. In the free oxine molecule the C–O stretching frequency ob-
served at ꢄ1090 cmꢀ1, which is shifted to higher frequencies in
all the mixed ligand complexes with a strong absorption band at
ꢄ1110 cmꢀ1. This result indicates the coordination of 8-hydroxy-
quinoline in these complexes. In IR spectrum all the complexes
shows the bands at ꢄ549 and ꢄ456 cmꢀ1 due to the Cu–O and
Cu–N respectively. Mehmet Tumer et al. mentioned the weak band
around ꢄ550 cmꢀ1 and ꢄ468 cmꢀ1 were attributed to the Cu–O
and Cu–N stretching frequency [64,65]. The IR spectra of the cou-
marin derivatives shows ꢄ1610 and ꢄ1743 cmꢀ1 bands corre-
centration 200, 150, 100, 50, 25, 12 and 6 lg/mL. The standard
drug solution of streptomycin (antibacterial drug) and nystatin
(antifungal drug) were prepared in DMSO. Serial broth micro dilu-
tion was adopted as a reference method. Ten microliter solution of
test compound was inoculated in 5 mL Luria broth for each concen-
tration respectively and additionally one test tubes was kept as
control. Each of the test tubes was inoculated with a suspension
of standard microorganism to be tested and incubated at 35 °C
for 24 h. At the end of the incubation period, the tubes were exam-
ined for the turbidity. Turbidity in the test tubes indicated that
microorganism growth has not inhibited by the antibiotic con-
tained in the medium at the test concentration. The antimicrobial
activity tests were run in triplicate.
3. Result and discussion
The synthesized Cu(II) complexes were characterized by ele-
mental analysis, FTIR and mass (ESI–MS and FAB) spectra, while
geometry of the complexes were confirmed using electronic spec-
tra, magnetic moment, thermal properties and kinetic measure-
ments. However, ligands and its complexes have been screened
for their in vitro antioxidant, anti-tubercular and antimicrobial
activities.
sponding to a,b-unsaturated ketone and lactone carbonyl ketone,
respectively, on complexation these peaks shifted to a lower fre-
quency ꢄ1600 and ꢄ1730 cmꢀ1 due to complex formation.
Table 2
FT-IR data of synthesized compounds.
Compounds
m
(O–H) cmꢀ1(br)
m
(C@N) cmꢀ1(w)
a
,b-Unsaturated
m
(C@O) cmꢀ1(s) Lactone carbonyl
m
(C@O) cmꢀ1(s)
m
(Cu–N) cmꢀ1(w)
m
(Cu–O) cmꢀ1(w)
L1
L2
L3
L4
L5
L6
C1
C2
C3
C4
C5
C6
–
–
–
–
–
–
–
–
1555
1554
1558
1550
1556
1553
1614
1616
1620
1625
1623
1624
1602
1608
1612
1610
1605
1602
1739
1736
1741
1743
1741
1742
1742
1738
1740
1745
1741
1743
–
–
–
–
–
–
547
545
551
552
549
550
–
–
–
–
–
–
455
457
452
458
453
451
3464
3486
3482
3480
3420
3432
3423
3435
3440
3442
s = strong, w = weak, br = broad.

K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
473
3.3. Electronic spectra and magnetic measurement
netic moment data support the octahedral geometry of the all
complexes. The electronic spectra of C3 is given in Supplementary
material (S5).
Electronic spectral data along with magnetic susceptibility
measurements gave sufficient support to determine the geometry
of metal complexes. The electronic spectra of complexes were re-
corded in DMF solution with scan range 200–1200 nm. Usually
octahedral geometry was found for Cu(II) complexes [66,67], how-
ever the known preference for Jahn–Teller distortions in cases of
trigonal and octahedral geometry. A very weak low intense absorp-
tion band associated with d–d transition for Cu(II) complexes at
465, 532 nm was regular octahedral transition, its indicate the
octahedral geometry of complexes [68]. On the other hand, many
copper compounds show temperature dependent geometric dis-
tortions and single copper ions in a host lattice of regular symme-
try may reveal interesting spectroscopic properties [69].
3.4. Thermal studies of Cu(II) complexes
Thermal behaviour of the complexes was studied using TG, DTG
and DSC analysis. The thermal decomposition TG curves corre-
sponding to the complex (C3) is presented in Fig. 1, whereas data
for all complexes are given in Table 3. The thermal decomposition
occurs in four steps in air are observed. According to the mass
losses, the following degradation pattern might be proposed for
complex [Cu(L3)(CQ)(H2O)OH]ꢂH2O (C3) is represented in Scheme
3. All the compounds decompose with time respectively. Thermal
decomposition started by dehydration process and was accompa-
nied by endothermic effect between 70 and 100 °C due to loss of
one lattice water molecules in first step. The observed mass loss
was 2.58% which was nearly equal to theoretical value 2.23%. In
the second step, exothermic decomposition between 200 and
240 °C corresponds to loss of one coordinated water and one hy-
droxyl molecule. The observed mass loss was 4.53% which was
nearly equal to theoretical value 4.33%. Next two steps were also
exothermic and endothermic related with removal of coordinated
Clioquinol as well as ligands respectively. As temperature raise,
the intermediate complexes [Cu(L3)(CQ)] (340–400 °C) and
[Cu(CQ)] (510–610 °C) convert to CuO residue of fragments. The
observed mass loss for third and fourth stage was 38.07% (calc.
37.84%) and 42.59% (calc. 45.97%), respectively. The final solid
product of decomposition was CuO (obs. 12.03%; calc.9.63%)
accompanied by broad exothermic effect on above 600 °C.
In the electronic spectra of the complexes, the wide range bands
were observed due to either the
mophore or charge transfer transition arising from
p ?
p⁄ and n ? p⁄ of C@N chro-
p
electron
interactions between the metal and ligand, which involves either
a metal to ligand or ligand to metal electron transfer [70]. The elec-
tronic spectra of hexa coordinate Cu(II) complexes were shows D4h
or C4v symmetry in which the Eg and T2g level of 2D free ion term
will split into B1g, A1g, B2g and Eg levels respectively under the
influence of the distortion, which cause the two transitions such
as 2B1g ? 2B2g and 2B1g ? 2A1g. This promotes the distorted octahe-
dral Cu(II) complex which was usual in the d9 system [71,72]. The
electronic spectra of Cu(II) complexes (C1–C6) display three prom-
inent bands. Low intensity broad band in the region 16,900–
17,900 cmꢀ1 was assigned as 10 Dq band corresponding to
2Eg ? 2T2g transition [73]. In addition, there was a high intensity
band in the region 22,900–27,100 cmꢀ1. This band is due to sym-
metry forbidden ligand ? metal charge transfer transition [74].
The band above 27,100 cmꢀ1 was assigned as ligand band. There-
fore distorted octahedral geometry around Cu(II) ion was sug-
gested on the basis of electronic spectra [75], which was further
discovered by its magnetic moment value of 1.79–1.87 BM falls
within the range generally observed for octahedral Cu(II) com-
plexes [76,77]. In general, the electronic spectral data and mag-
DTG curves corresponding to the complex (C3) is represented in
Fig. 2. First step of decomposition was dehydration process with
endothermic effect on DTG curve at 86 °C, while increasing in tem-
perature of [Cu(L3)(CQ)(H2O)OH] and [Cu(L3)(CQ)] shows exother-
mic effect at 229 °C and 344 °C, respectively. Endothermic DTG
peak at 552 °C associated with elimination of coumarins. The final
residue predicted as copper oxide.
Fig. 1. TG curve of the complex [Cu(L3)(CQ)(H2O)OH]ꢂH2O.

474
K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
Table 3
Thermoanalytical results (TG and DTG) of metal complexes.
Complexes
C1
TG range °C
DTGmax °C
Mass loss% obs. (calc.)
46.77(46.55)
Assignment
70–390
96
Loss of two lattice water molecules
Loss of one –OH and H2O molecules
Removal of Clioquinol
225
335
570
>650
390–800
90–370
48.23
4.72
Removal of L1 ligand
Leaving CuO residue
C2
C3
C4
C5
C6
95
44.52(44.36)
Loss of one lattice water molecules
Loss of one –OH and H2O molecules
Removal of Clioquinol
240
346
560
>650
370–800
70–380
48.20
8.65
Removal of L2 ligand
Leaving CuO residue
86
45.37(45.31)
Loss of one lattice water molecules
Loss of one –OH and H2O molecules
Removal of Clioquinol
229
344
552
>650
380–800
70–360
42.590
12.26
Removal of L3 ligand
Leaving CuO residue
92
47.74(47.65)
Loss of two lattice water molecules
Loss of one –OH and H2O molecules
Removal of Clioquinol
242
352
545
>650
360–800
80–385
42.590
9.86
Removal of L4 ligand
Leaving CuO residue
98
45.98(45.90)
Loss of three lattice water molecules
Loss of one –OH and H2O molecules
Removal of Clioquinol
230
355
565
>650
385–800
85–380
48.96
5.21
Removal of L5 ligand
Leaving CuO residue
97
46.11(46.02)
Loss of one lattice water molecules
Loss of one –OH and H2O molecules
Removal of Clioquinol
235
350
564
>650
380–800
45.75
8.47
Removal of L6 ligand
Leaving CuO residue
Scheme 3. Thermal fragmentation of complex [Cu(L3)(CQ)(H2O)OH]ꢂH2O.
3.5. Kinetic measurement by isoconversion methods
exothermic peak temperature was shifted to higher temperatures
with increase in heating rate and according to Kissinger’s approach,
The isoconversion method was advanced tool for investigation
of the kinetics parameters at the start and end of the chemical
reaction. The kinetic parameters can easily interpreted by a com-
parison of measurements at different heating rates [78]. Multiple
dynamic DSC experiments of all complexes were carried out at
heating rates of 2.5, 5, 10, 15 and 20 °C minꢀ1, respectively, while
Fig. 3 shows DSC scans of the complexes C3 for second step decom-
position at different heating rates. An isconversional method,
which presumes the activation energy and pre-exponential factor
were both functions of the degree of heat flow, can be used to
investigate the multi-heating-rate scan data [79]. Similarly, this
approach was useful to estimate the order of reaction (n). More-
over, results obtained from multi heating rate dynamic scans
experiments were investigated using Kissinger’s [49,50] and Oza-
wa’s [51,52] approaches for determination of kinetic parameters.
The Kissinger’s approach work on the principle, the peak tempera-
ture is a function of the heating rate. In case of complex C3 the
the maximum reaction rate da a
/dt occurs at Tp, where d2 /dt2 = 0,
and the kinetic Eq. (1) can be expressed through the following first
order reaction
!
ꢀ
ꢁ
b
Tp2
AR
Ea
Ea
RTp
ln
¼ ln
ꢀ
ð1Þ
where Tp is the peak temperature of the DSC curve, b represents the
heating rate and R is the gas constant. This equation was employ
with a sensible estimation even to an nth order but it is not consid-
erable order. The reaction progress under appropriate conditions,
when thermal equilibrium was constantly maintained, Fig. 4 shows
a plot ln(b/Tp2) vs. 1/Tp gives a straight line with a slope equal to –
Ea/R which gives the values of activation energy while the values of
the pre-exponential factor A was found from the intercept.
The isoconversional Flynn Wall Ozawa method was the integral
method which provided a basic correlation between the activation

K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
475
Fig. 2. DTG curve of the complex [Cu(L3)(CQ)(H2O)OH]ꢂH2O.
Fig. 3. Dynamic scan DSC curve of complex C3.
Fig. 4. Kissinger’s approach to determine kinetic parameters for complex C3.
energy, the heating rate and isoconversion temperature shown in
Eq. (2),
in Fig. 5. The relationship of activation energy and conversion can
be measured through the whole reaction. The activation energy in
the starting decreases with rising value of conversion, getting a
least value at around 20% conversion. Then activation energy was
increase up to 60% conversion, prior to decreasing again at high con-
versions. From plots of each method, the experimental data sensibly
fine during most of the temperature range but deviate considerably
in the higher temperature area. Activation energy (Ea) shows a sim-
ilar fashion as A with conversion in Fig. 6, which recommended lin-
ear correlation between activation energy and pre-exponential
factor. The calculated results from the Kissinger’s method are
shown in Table 4. Ozawa’s method gives an average value of com-
plexes C3 91.824 kJ molꢀ1, which is a little bit lesser that obtained
from Kissinger’s method. Kissinger’s and Ozawa’s methods equally
show an outstanding linear correlation between kinetics parame-
ters based on the experimental figures.
0:4567Ea
log b ¼ ꢀ
þ A0
ð2Þ
RTi
where for each degree of conversion, A0 is a constant that can be ex-
pressed as Eq. (3),
ꢀ
ꢁ
AEa
gð ÞR
A0 ¼ log
ꢀ 2:315
ð3Þ
a
where Ea is the activation energy, A the pre-exponential factor, R the
gas constant, b the heating rate, the degree of conversion, g( ) is
the integral function of conversion and f( ) the differential function
of conversion, For each degree of conversion , the plot of log b vs.
a
a
a
a
1/T obtained at different heating rates should be a straight line
which gives a slope proportional to the corresponding activation
energy Ea and an intercept of the pre-exponential factor A shown

476
K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
3.7. X-ray diffraction studies
The feasible lattice dynamics of the final powdered compound
[Cu(L6)(CQ)(H2O)OH]ꢂH2O, structure was presumed on the basis
of X-ray powder diffraction studies. The observed inter planar
spacing values i.e. d-spacing were measured from the diffracto-
gram of the compound. Moreover, the Miller indices h, k and l were
assigned to each d value along with 2-Theta angles are given in Ta-
ble 5. The results show that the compound belongs to hexagonal
crystal system having unit cell parameters such as a = 4.9168,
b = 4.9168, c = 5.4089 with maximum deviation of 2-Theta = 0.025
and Alpha = 90, Beta = 90, Gama = 120 at the wavelength =
1.540598. We have tried to isolate single crystal of Cu(II) complex
for accurate X-ray crystal study but could not succeed to develop
single crystal, it might be due to polycrystalline nature of complex.
However, the structural information on the majority of inorganic
metal complexes is not available because of the powder or poly-
crystalline nature of these materials. Generally it is often difficult
to grow good quality single crystals of these inorganic complexes.
In such cases, the powder X-ray diffraction studies might be useful.
Even with some inherent limitations, this method yield valuable
information about the characteristics of the crystal w3x. Singh et
al. [80] were recently reported the X-ray powder diffraction studies
for structure of hexa-coordinated Tin(IV) complex in which the li-
gand adopts the most stereo-chemically favorable orientation. On
the basis of the above discussion, following structures shown in
Figs. 7 and 8 were proposed for the complexes.
Fig. 5. Kinetic parameters of complex C3 using Ozawa’s approach on.
3.8. Antioxidant studies
Antioxidant power was specifically the ability of transfer a sin-
gle electron for compound. The antioxidant capacity of complexes
C1–C6 was determined by a FRAP method. The FRAP results was ex-
pressed as an equivalent of standard antioxidant ascorbic acid
(mmol/100 g of dried compound). FRAP values indicate that all
the compounds have a ferric reducing antioxidant power. The com-
pounds C3 and C5 showed relatively high antioxidant activity while
compound C1 shows poor antioxidant power (Table 6). Among the
tested compounds hydroxyl group substituted derivatives possess
more ferric reducing power compared to that of chloro and meth-
oxy group substituted derivatives. Thus form the data obtained, the
potency order on the basis of various substitutions on p-position of
the phenyl ring is given as p-OH > p-OH-m-OCH3 > m-OH > p-
Cl > m-Cl. However, none of the compounds have been found more
potent than standard ascorbic acid.
Fig. 6. Linear relationship between a
, Ea and A for complex C3: Ozawa’s approach.
Table 4
Kinetic parameters of Cu(II) complexes by Kissinger’s approach.
3.9. Anti-tubercular activity
Heating rate °C minꢀ1
Tp °C for C3
Kissinger values for C3
Ea kJ molꢀ1
lnA
The anti-tubercular activities of all the synthesized compounds
were assessed against M. tuberculosis H37RV at 25, 50 and 100 lg/
20
15
10
5.0
2.5
229
222
213
202
189
94.098 0.5
13.14 0.1
mL. The minimum inhibitory concentrations of compounds com-
pared with the standard drugs isoniazid, streptomycin and etham-
butol, and are summarized in Table 6. Ligands show inhibition at
concentration 100
50
g/mL concentration while C3 and C4 complexes have shown
enhancement in activity with MIC of 25 g/mL. None of the tested
l
g/mL. Complexes C5 also exhibits activity at
l
l
3.6. FAB mass spectra
compounds have the inhibition more than standards.
The fast atom bombardment (FAB) mass spectra of the complex
C6 and its fragmentation scheme are given in⁄⁄ Supplementary
material (S6–S7). FAB mass spectrum reveals that isotropic peak
at m/z 757 of complex without water of crystallization, where as
several peaks observed at 740.82, 613.92, 561.16, 471.11, 391.20,
352.18, 326.16, 282.17, 147.97 and 146.07 m/z value. Thus the m/
z of all the fragments of complex with the relative intensity con-
firms the stoichiometry of the complex.
3.10. Antimicrobial bioassay
All the synthesized ligands and complexes were evaluated for
their antibacterial and antifungal studies. The antibacterial and
antifungal tests were carried out using the serial broth dilution
method. The in vitro antimicrobial activities of the investigated
compounds were screened against the bacterial species S. aureus,
B. subtilis, E. coli, P. aeruginosa and fungal species C. albicans,

K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
477
Table 5
Observed and calculated X-ray diffraction data of [Cu(L6)(CQ)(H2O)OH]ꢂH2O complex.
h
k
l
2 Theta (Obs.)
2 Theta (Calc.)
2 Theta (Diff.)
d (Obs.)
d (Calc.)
d (Diff.)
Intensity (Obs.)
0
1
1
1
2
1
0
0
0
0
0
1
1
0
1
2
1
2
16.4458
21.0783
26.4939
38.9913
44.7847
50.8907
16.375
20.845
26.622
39.440
45.763
50.104
0.708
0.233
ꢀ0.128
ꢀ0.4478
ꢀ0.9783
0.7867
5.39023
4.2149
3.3643
2.31002
2.02374
1.79285
5.40890
4.25807
3.34573
2.28291
1.98109
1.81913
ꢀ0.01860
ꢀ0.04317
0.01860
9.12
18.82
100.00
18.44
18.10
18.48
ꢀ0.02710
0.04265
ꢀ0.02628
Fig. 7. X-ray patterns of complex C6 powder samples.
A. Niger and A. Clavatus. The minimum inhibitory concentration
(MIC) values of the compounds are summarized in Table 6.
A relative study for MIC values of the ligands and their com-
plexes signify that complexes display higher antimicrobial activity
than the free ligands. In present investigation, the antimicrobial
activity of the ligands may be due to the heteroaromatic residues.
Compounds containing C@N group have improved antimicrobial
activity than C@C group. The growth of certain microorganisms
takes place even in the absence of oxygen. Hence, compounds con-
taining C@C group still capable of absorbing oxygen which are not
related with the growth of microorganisms. The greater activity of
the complexes can be clarified on the basis of Overtone’s concept
[81] and Tweedy’s chelation theory [82]. According to Overtone’s
concept of cell permeability, the lipid membrane surroundings in
the cell was permit only the lipid–soluble resources, which makes
liposolubility a key part that control the antimicrobial activity.
During complexation, the polarity of the metal ion will be decrease
up to a certain level due to the overlapping of the ligand orbital and
partial sharing of the positive charge of the Cu(II) ion with hetero
atoms. Furthermore, it enhances the delocalization of
p-electrons
around the entire complex ring and enhances the lipophilicity of
the complexes. This increased lipophilicity enhances the perme-
ation of the complexes into lipid membranes and blocking of the
metal binding sites in the enzymes of microorganisms. These com-
plexes also troubled the respiration process of the cell and hence
block the synthesis of proteins, which restricts additional growth
of the organism and as a result microorganisms died. The experi-
mental deviation in the activity of the Cu(II) complexes across
the different classes of organisms studied may be characteristic
Fig. 8. 3D Molecular structures of complex C6.

478
K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
Table 6
Antimicrobial, anti-tubercular and antioxidant results of compound.
Compounds
Minimum inhibition concentrationa of microorganisms (
l
g/mL)
Anti-tubercular activitya
Antioxidant activityb
Bacteria
Fungi
S. aureus
B. subtilis
E. coli
P. aeruginosa
C. albicans
A. Niger
A. Clavatus
L1
200
150
100
100
150
>200
>100
100
25
25
50
200
12
NT
>150
150
50
200
200
100
150
150
200
100
150
25
>25
>50
150
>6
200
150
100
150
150
200
100
100
25
>200
200
150
100
150
200
100
100
25
25
50
150
>12
NT
200
150
200
150
>150
200
150
100
50
200
>150
200
150
150
200
200
150
25
>100
100
100
>50
100
>100
100
>50
25
25
50
100
NT
0.025
20
NT
NT
NT
NT
NT
NT
311.70
337.23
407.02
345.74
387.44
302.56
NT
NT
NT
NT
NT
L2
L3
L4
50
L5
100
200
150
100
>25
25
50
150
12
NT
NT
6
NT
L6
C1
C2
C3
C4
25
25
25
C5
>50
200
12
NT
NT
12
NT
NT
>50
200
12
NT
NT
NT
12
NT
>50
200
12
NT
NT
NT
12
NT
C6
CQ
Isoniazide
Ethambutol
Streptomycin
Nystatin
Ascorbic acid
NT
NT
6
NT
NT
12
NT
NT
NT
NT
6
NT
6.25
NT
NT
NT
NT
500
NT = not tested.
a
Average value of triplicate results.
FRAP results expressed in mM of ascorbic acid per 100 g of sample i.e. mmol/100 g.
b
to differences in cell wall and/or membrane construction (Gram-
positive bacteria, Gram-negative bacteria and fungi). It is expected
that the more extensive hetero-aromatic ring system of Clioquinol
and the presence of the lipophilic group C@N would give better
lipophilicity on the Cu(II) complexes and permit it to penetrate
the cell wall and inhibit intracellular interactions. At the same
time, hydroxylated derivatives had excellent antibacterial activity,
it was unexpected that the other Cu(II) complexes were really
moderate against all of the microbial species tested. Although the
range of functionalities on the aromatic ring of the coumarin core
is significant only in the presence of a hydroxyl group on the aro-
matic ring shown antimicrobial activity for the subsequent Cu(II)
complexes. The role of the hydroxyl group in this activity is com-
plicated to find out but the metal complexes of other hydroxylated
derivatives of coumarin have been earlier shown to have excellent
antimicrobial activity. Examples contain Cu(II) and Ni(II) com-
plexes of 4-hydroxycoumarins [83,84]. In a previous study on the
antimicrobial activity of catechols, the position and number of hy-
droxyl groups on the aromatic ring were responsible for their rel-
ative toxicity towards microorganisms, with facts that increasing
hydroxylation results in an enhance in antimicrobial activity
[85]. The mechanism recommended being liable for catechol toxic-
ity to microorganisms contain enzyme inhibition by the oxidized
compounds. The results would be indicate that substitution of
the hydroxyl groups on the aromatic ring of the coumarin ligand
was also vital for giving antimicrobial activity onto the Cu(II)
complexes.
concentration while C3 and C4 complexes have shown enhance-
ment in activity with MIC of 25 lg/mL.
4. Conclusions
Present studies describe the synthesis of biological active cou-
marin derivatives (L1–L6) and their Cu(II) complexes (C1–C6). The
structures of the ligands were investigated and confirmed by the
elemental analysis, FT-IR, 1H NMR, 13C NMR, mass spectral studies.
The ligand coordinates to metal ion through phenolic OH via
deprotonation, eneamino nitrogen, lactone carbonyl oxygen and
a,b-unsaturated carbonyl oxygen. The octahedral geometry were
assigned for Cu(II) complexes on the basis of electronic, magnetic
moment and thermogravimetric analysis. On the basis of these
studies it is clear that ligands coordinated to metal atom in a
monobasic bidentate mode, by OAN and OAO donor system. X-
ray diffraction results show that the compound belongs to hexag-
onal crystal system with polycrystalline nature of complex which
was further conformed octahedral geometry. Activation energy
and pre-exponential factor calculated by isoconversional methods.
Kissinger’s and Ozawa’s methods equally show an outstanding lin-
ear correlation between kinetics parameters based on the experi-
mental figures and the reaction is first order. All the complexes
found antioxidant significant activity compared to parent ligands
employed for complexation. In vitro anti-tubercular and antimicro-
bial activity of all synthesized compounds show good results with
an enhancement of activity on complexation with metal ions. This
enhancement in the activity may be attributed to increased lipo-
philicity of the complexes. In review, the antimicrobial testing re-
sults reveal that complexes possess higher activity compared to
parent ligand. Anti-tubercular activity results show that the com-
plexation of ligand with Cu(II) metals has doubled its activity.
In fact, coordination, locking the polar electronegative atoms in
the inner core around the metal and confining the apolar residues
in an external lipophilic envelope, favor diffusion through bio-
membranes [86]. It is also suspected that factors such as solubility,
conductivity, dipole moment may be the possible reasons for the
increase in activity. Since all complexes have not shown enhanced
activity as compared to parent ligand were rationalized the fact
that, steric and pharmacokinetic factors also play a decisive role
in deciding the potency of an antimicrobial agent [87]. In review,
the antimicrobial testing results reveal that complexes possess
higher activity at lower concentration compared to parent ligand.
All ligands show inhibition concentration between 200 and
Acknowledgements
We are grateful to The Principal, and management of V.P. &
R.P.T.P. Science College, Vallabh Vidyanagar for providing infra-
structures and other obligatory facilities for research. We are
thankful to The Director, SICART, Vallabh Vidyanagar for FT-IR
100 l lg/mL
g/mL. Complexes C5 also exhibits activity at 50

K.S. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 96 (2012) 468–479
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