Synthesis, spectral characterization and antimicrobial evaluation of Schiff base Cr (III), Mn (III) and Fe (III) macrocyclic complexes

Article abstract of DOI:10.1016/j.ejmech.2012.03.025

A Schiff base ligand was synthesized by reacting 1,4-dicarbonyl-phenyl- dihydrazide and chromene-2,3-dione (2:2) and a series of metal complexes with this new ligand were synthesized by reaction with Cr (III), Mn (III), and Fe (III) metal salt in methanolic medium. The Schiff base ligand and its metal complexes have been characterized with the help of elemental analysis, conductance measurements, magnetic measurements and their structure configuration has been determined by various spectroscopic (electronic, IR, ¹H NMR, ¹³C NMR, GCMS) techniques. Electronic spectra of the complexes indicate that the geometry of the metal center was five coordinate square pyramidal. These metal complexes were also tested for their antimicrobial activities to assess their inhibiting potential.

Full text of DOI:10.1016/j.ejmech.2012.03.025

European Journal of Medicinal Chemistry 52 (2012) 269e274
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, spectral characterization and antimicrobial evaluation of Schiff base
Cr (III), Mn (III) and Fe (III) macrocyclic complexes
,
b
,
c
a
a
Gajendra Kumar a , Shoma Devi , Rajeev Johari , Dharmendra Kumar
*
a Department of Chemistry, Vardhaman College, Bijnor 246701, Uttar Pradesh, India
b Chemical Science Laboratory, Department of Applied Science, Bhagwant Institute of Technology, Muzaffarnagar 251315, Uttar Pradesh, India
c Department of Zoology, Vardhaman College, Bijnor 246701, Uttar Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A Schiff base ligand was synthesized by reacting 1,4-dicarbonyl-phenyl-dihydrazide and chromene-2,3-
dione (2:2) and a series of metal complexes with this new ligand were synthesized by reaction with Cr
(III), Mn (III), and Fe (III) metal salt in methanolic medium. The Schiff base ligand and its metal complexes
have been characterized with the help of elemental analysis, conductance measurements, magnetic
measurements and their structure configuration has been determined by various spectroscopic (elec-
tronic, IR, 1H NMR, 13C NMR, GCMS) techniques. Electronic spectra of the complexes indicate that the
geometry of the metal center was five coordinate square pyramidal. These metal complexes were also
tested for their antimicrobial activities to assess their inhibiting potential.
Received 10 November 2011
Received in revised form
5 March 2012
Accepted 13 March 2012
Available online 28 March 2012
Keywords:
Schiff base
Macrocyclic
Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
Complexes
Antimicrobial activity
Spectroscopic study
1. Introduction
metals at the active sites of numerous metallobiomolecules [11].
Metallorganic chemistry is becoming an emerging area of research
Macrocyclic compounds have attracted increasing interest
owing to their role in the understanding of molecular processes.
The role played by metals in a number of biological systems has
derived the research efforts of many scientists from a variety of
fields [1e3]. There are a number of important macrocyclic mole-
cules which show biological activities including antibacterial,
antifungal [4e13], antidiabetic [14], antitumor [15e17], anti-
proliferative [18,19], anticancer [20,21], herbicidal [22], anti-
inflammatory activities [5e7]. Schiff bases represent an important
class of compounds because they are utilized as starting materials
in the synthesis of industrial products [23]. Moreover, Schiff base is
regarded as a ligand [24]. Due to their capability to form complexes
with different transition metals Schiff bases can act as catalysts for
different reactions [25e29]. The cross linking agents can also be
derived from metal complexes with OeNe or eS ligand. For
example, the intra-coordination salt such as salicylates, anthrani-
lates and aliphatic or aromatic amines can form strong five or six
membered chelates rings, which are able to produce the metal
containing cross linking agents with required properties [30]. It is
well known fact that N atom plays a key role in the coordination of
due to the demand of new metal based antibacterial and antifungal
compounds [31,32]. Important characteristics that can be corre-
lated with good antimicrobial activities are the lipophilicity and
penetration of the complexes through the lipid membrane. A
survey of the literature reveals that no work has been carried out on
the synthesis of metal complexes with macrocyclic hydrazone
Schiff base derived from 1,4-dicarbonyl-phenyl-dihydrazide with
chromene-2,3-dione. The coordination abilities of Schiff base have
attracted our attention and aroused our interest in elucidating the
structure of Cr (III), Mn (III) and Fe (III) complexes.
The aim of the present work is to prepare, characterize the chem-
ical structure and evaluate their antibacterial and antifungal proper-
ties against various pathogenic strains of fungi viz. Rizoctonia sp.,
Aspergillus sp., and Penicillium sp., bacteria Staphylococcus aureus and
Bacillus subtilis (as gram positive bacteria) and Pseudomonas aerugi-
nosa, Escherichia coli and Salmonella typhi (as gram negative bacteria).
2. Chemistry
2.1. Reagents
* Corresponding author. Department of Chemistry, Vardhaman College, Bijnor
246701, Uttar Pradesh, India. Tel.: þ91 1342251081; fax: þ91 1342260020.
E-mail address: gaj.chem@gmail.com (G. Kumar).
The entire chemicals used, were of the analytical reagent grade,
chromene-2,3-dione, diethyl terephthalate and hydrazinhydrate
0223-5234/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.025

270
G. Kumar et al. / European Journal of Medicinal Chemistry 52 (2012) 269e274
procured from Acros and s.d.-fine, respectively. Metal salts were
purchased from Merck.
precipitate was filtered off, washed with methanol and dried under
vacuum over anhydrous CaCl2, yield 68e75% (Scheme 2).
2.2. Synthesis of 1,4-dicarbonyl-phenyl-dihydrazide
3. Pharmacology
A mixture of diethyl ester of terephthalic acid (2.22 g) and
hydrazine hydrate (98% 2 mL) in ethanol was refluxed for 4e5 h.
The reaction mixture was allowed to cool to the room tempera-
ture then, the cooled solution was poured onto ice cold water. The
dihydrazide of terephthalic acid was thus obtained, filtered and
recrystallized from ethanol [33].
3.1. Antifungal activity
The free ligand, its metal complexes, fungicide miconazole and
the control DMSO (dimethylsulfoxide) were screened for their
antifungal activity against various fungi viz. Rizoctonia sp., Asper-
gillus sp., and Penicillium sp. These species were isolated from the
infected organs of the host plants on potato dextrose agar (potato
250 g þ dextrose 20 g þ agar 20 g) medium. The culture of the fungi
was purified by single spore isolation technique [9]. The solution of
different concentrations 1, 1.5 and 2 mg/mL of each compound (free
ligand, its metal complexes and fungicide miconazole) in DMSO
were prepared for testing against spore germination. A drop of the
solution of each compound (each concentration) was kept sepa-
rately on glass slides. The conidia, fungal reproducing spores
(approx. 200) lifted with the help of an inoculating needle, were
mixed in every drop of each compound separately. Each treatment
was replicated thrice and a parallel DMSO solvent control set was
also run concurrently on separate glass slide. All the slides were
incubated in humid chambers at 25 ꢀC for 24 h. Each slide was
observed under the microscope for spore germination and percent
germination was finally calculated. The results were compared with
a standard fungicide miconazole at the same concentrations.
2.3. Synthesis of macrocyclic ligand
Chromene-2,3-dione (0.324 g, 2 mmol) in ethanol (20 mL) was
added to a solution of 1,4-dicarbonyl-phenyl-dihydrazide (0.388 g,
2 mmol) in ethanol (30 mL) containing a few drops of concentrated
HCl. The reaction mixture was refluxed for 3 h. The mixture was
cooled to room temperature and the solvent removed under
reduced pressure by rotavapour until a solid product was formed
that was washed with cold ethanol and dried under vacuum.
Melting point 145 ꢀC, yield 70e75% (Scheme 1).
2.4. Synthesis of the Cr (III), Mn (III) and Fe (III) complexes
A solution of trivalent metal salt (1.0 mmol) in methanol (8 mL)
was added to a hot solution (75 ꢀC) of macrocyclic ligand
(1.0 mmol) in ethanol (25 mL), and the reaction mixture was
refluxed for 2 h. The solution was concentrated under vacuum. The
Scheme 1. Formation of Schiff base ligand.
Scheme 2. Formation of Schiff base metal complex cation.

G. Kumar et al. / European Journal of Medicinal Chemistry 52 (2012) 269e274
271
3.2. Antibacterial activity
vibrations of aromatic moiety [35]. The various absorption band in the
range w1450e1590 cmꢁ1 may be assigned due to ѵ(C]C) aromatic
stretching vibrations of the aromatic ring [36]. The presence of the
absorption bands at 1408e1435, 1290e1324 and 1010e1035 cmꢁ1 in
theIR spectra of thenitratocomplexes suggests that the nitrategroups
are coordinated to the central metal ion in an unidentate fashion [37].
In IR spectra of the acetate complexes the appearance of three char-
acteristic bands in the ranges 1561e1559 cmꢁ1, 1370e1367 cmꢁ1 and
1775e1800 cmꢁ1 in the case of complexes was attributed to
nasym(COOꢁ), nsym(COOꢁ) and uncoordinated (COOꢁ), respectively,
indicating the participation of the carboxylate oxygen in the
complexes formation. The mode of coordination of carboxylate group
has often been deduced from the magnitude of the observed sepa-
ration between the nasym(COOꢁ) and nsym(COOꢁ). The difference
Antibacterial activities were evaluated using agar well diffusion
method [34]. The activity of the free ligand, its metal complexes and
standard drug Imipenem were studied against the S. aureus and
B. subtilis (as gram positive bacteria) and P. aeruginosa, E. coli and
S. typhi (as gram negative bacteria). Bacterial strains were obtained
from Microbial Type Collection and Gene Bank, Institute of Micro-
bial Technology (IMTECH) Chandigarh, India. The solution of 2 mg/
mL of each compound (free ligand, its metal complexes and stan-
dard drug Imipenem) in DMSO was prepared for testing against
bacteria. Centrifuged pelletes of bacteria from a 24 h old culture
containing approximately 104e106 CFU (colony forming unit) per
mL were spread on the surface of Muller Hinton Agar plates. Wells
were created in medium with the help of a sterile metallic bore and
nutrients agar media (agar 20 g þ beef extract 3 g þ peptones 5 g) in
1000 mL of distilled water (pH 7.0), autoclaved and cooled down to
45 ꢀC. Then it was seeded with 10 mL of prepared inocula to have
106 CFU/mL. Petri plates were prepared by pouring 75 mL of seeded
nutrient agar. The activity was determined by measuring the
diameter of the inhibition zone (in mm). The growth inhibition was
calculated according to reference [34].
between (nas
ꢁ
nsy) is around 191e193 cmꢁ1 which is greater than
190 cmꢁ1 indicating the monodentate coordination of the acetate ion
with the central metal ion [38]. The IR spectra of nitrato complexes
exhibits bands at 1280e1305 cmꢁ1
,
1040e1055 cmꢁ1 and
1430e1445 cmꢁ1 which can be assigned to NO2 symmetric stretching
n1); NeO stretching (n2); NO2 asymmetric stretching (n5); and out of
(
plane rocking, respectively. These frequencies are compatible with
monodentately coordinated nitro group [39], IR absorption band
w1353e1359 cmꢁ1 assigned the uncoordinated nitro group. The far
infrared spectra show bands in the region 425e450 cmꢁ1 corre-
4. Results and discussion
sponding to n(MeN) vibration [40e42]. The presence of bands in all
complexes in the region 425e450 cmꢁ1 originate from (MeN) azo-
methine vibrational mode and identify coordination of azomethine
nitrogen [43]. The band present in the range 300e325 cmꢁ1 may be
4.1. Mass spectra
The FAB mass spectra of Cr (III), Mn (III) and Fe (III) Schiff base
complexes have been recorded (Table 1). The molecular ion (Mþ)
peaks obtained from various complexes are as follows: (1) m/
z ¼ 640.62 C34H24N8O6 (Ligand), (2) m/z ¼ 798.97 [Cr(C34H24N8O6)
Cl]Cl2 (complex 1), (3) m/z ¼ 878.63 [Cr(C34H24N8O6)NO3](NO3)2
assigned due to
region 225e250 cmꢁ1 in all the nitrato complexes are assignable to
(MeO) stretching vibration [40,41].
n(MeCl) vibration [40e42]. The bands present in the
n
(complex 2), (4) m/z
¼
869.75 [Cr(C34H24N8O6)OAc](OAc)2
4.3. 1H NMR
(complex 3), (5) m/z ¼ 801.91 [Mn(C34H24N8O6)Cl]Cl2 (complex 4),
(6) m/z ¼ 881.58 [Mn(C34H24N8O6)NO3](NO3)2 (complex 5), (7) m/
z ¼ 872.69 [Mn(C34H24N8O6)OAc](OAc)2 (complex 6), (8) m/
z ¼ 802.82 [Fe(C34H24N8O6)Cl]Cl2 (complex 7), (9) m/z ¼ 882.48
A survey of literature reveals that the NMR spectroscopy has
been proved useful in establishing the structure and nature of many
Schiff base ligand and its metal complexes. The 1H NMR spectra of
Schiff base ligand (HL) was recorded in dimethylsulfoxide (DMSO-
d6) solution using Me4Si (TMS) as internal standard. The 1H NMR
spectra of the ligand shows broad signal at 9.4e12.1 ppm due to the
eNH [44], 2.1e2.8 ppm due to the eCH2e (cyclic) [10]. The multi-
plets in the region 6.54e8.76 ppm may be assigned to aromatic
proton [45,46].
[Fe(C34H24N8O6)NO3](NO3)2 (complex 8), (10) m/z
¼
873.60
[Fe(C34H24N8O6)OAc](OAc)2 (complex 9). This data is in good
agreement with the proposed molecular formula for these
complexes. In addition to the peaks due to the molecular ion, the
spectra exhibit peaks assignable to various fragments arising from
the thermal cleavage of the complexes.
13C NMRof the Schiffbaseligand, the signalappeared intheregion
113e158 are assigned to aromatic carbon. The signal at 172.8e165.6
and 144.3e142.3 ppm are due to C]N, and C]O respectively [10].
4.2. IR spectra
The IR spectral data of the Schiff base ligand show a medium
intensity absorption band at 3240 cmꢁ1 which is attributed to the
ѵ(NeH) stretching vibration. The high intensity band at
1613e1632 cmꢁ1 whichisattributed tothe ѵ(C]N) vibration. Another
strong band at 1735e1720 cmꢁ1 is a ѵ(C]O). The bands present in the
range w3010e3050 cmꢁ1 may be assigned due to ѵ(CeH) stretching
4.4. Electronic spectral studies, magnetic measurements and molar
conductance
The electronic spectra of Cr (III) complexes showed absorption
band in the region 8950e9310, 13,150e13,520, 17,550e18,450 and
Table 1
FAB mass spectral data of the trivalent chromium, manganese and iron complexes derived from diethyl terephthalate, hydrazinhydrate and chromene-2,3-dione.
Complexes
Mol. wt.
Molecular ion peak [M]þ
Important peak due to complex fragmentation
[Cr(C34H24N8O6)Cl]Cl2
798.97
878.63
869.75
801.91
881.58
872.69
802.82
882.48
873.60
[M]þ ¼ 797.9
[M]þ ¼ 877.6
[M]þ ¼ 868.7
[M]þ ¼ 800.9
[M]þ ¼ 880.5
[M]þ ¼ 871.6
[M]þ ¼ 801.8
[M]þ ¼ 881.4
[M]þ ¼ 872.6
157.9, 159.2, 317.25, 476.4, 634.5, 638.5, 690.5, 726.9
157.9, 159.2, 317.25, 476.4, 634.5, 638.5, 690.5, 753.5
157.9, 159.2, 317.25, 476.4, 634.5, 638.5, 690.5, 750.6
157.9, 159.2, 317.25, 476.4, 634.5, 638.5, 693.5, 729.9
157.9, 159.2, 317.25, 476.4, 634.5, 638.5, 693.5, 756.4
157.9, 159.2, 317.25, 476.4, 634.5, 638.5, 693.5, 753.5
157.9, 159.2, 317.25, 476.4, 634.5, 638.4, 694.4, 730.8
157.9, 159.2, 317.25, 476.4, 634.5, 638.3, 694.3, 757.3
157.9, 159.2, 317.25, 476.4, 634.5, 638.4, 694.4, 754.5
[Cr(C34H24N8O6)NO3](NO3)2
[Cr(C34H24N8O6)OAc](OAc)2
[Mn(C34H24N8O6)Cl]Cl2
[Mn(C34H24N8O6)NO3](NO3)2
[Mn(C34H24N8O6)OAc](OAc)2
[Fe(C34H24N8O6)Cl]Cl2
[Fe(C34H24N8O6)NO3](NO3)2
[Fe(C34H24N8O6)OAc](OAc)2

272
G. Kumar et al. / European Journal of Medicinal Chemistry 52 (2012) 269e274
Table 2
Analytical data of the trivalent chromium, manganese and iron complexes derived from diethyl terephthalate, hydrazinhydrate and chromene-2,3-dione.
Complexes
Mol. wt.
C
H
N
M
Colour
Yield
Conductance LM
C34H24N8O6
[Cr(C34H24N8O6)Cl]Cl2
640.62
798.97
878.63
869.75
801.91
881.58
872.69
802.82
882.48
873.60
63.72 (63.74)
51.08 (51.11)
46.45 (46.48)
55.20 (55.24)
50.90 (50.92)
46.28 (46.32)
54.98 (55.05)
50.79 (50.86)
46.25 (46.27)
54.95 (54.99)
3.78 (3.77)
3.00 (3.03)
2.78 (2.75)
3.80 (3.82)
2.98 (3.02)
2.72 (2.74)
3.75 (3.81)
2.99 (3.01)
2.70 (2.74)
3.75 (3.80)
17.45 (17.49)
13.98 (14.02)
17.50 (17.53)
12.85 (12.88)
13.95 (13.97)
17.45 (17.47)
12.81 (12.84)
13.92 (13.96)
17.44 (17.45)
12.85 (12.82)
6.48 (6.51)
5.88 (5.91)
5.95 (5.97)
6.83 (6.85)
6.18 (6.23)
6.25 (6.29)
6.92 (6.95)
6.18 (6.22)
6.40 (6.39)
Orange
Orange
Light yellow
Brown
Gray
Light gray
Light yellow
Reddish
68%
75%
72%
73%
71%
69%
74%
72%
71%
145 Uꢁ1
167 Uꢁ1
158 Uꢁ1
138 Uꢁ1
155 Uꢁ1
142 Uꢁ1
127 Uꢁ1
148 Uꢁ1
151 Uꢁ1
[Cr(C34H24N8O6)NO3]( NO3)2
[Cr(C34H24N8O6)OAc](OAc)2
[Mn(C34H24N8O6)Cl]Cl2
[Mn(C34H24N8O6)NO3]( NO3)2
[Mn(C34H24N8O6)OAc](OAc)2
[Fe(C34H24N8O6)Cl]Cl2
[Fe(C34H24N8O6)NO3]( NO3)2
[Fe(C34H24N8O6)OAc](OAc)2
Light yellow
4
27,380e27,780 cmꢁ1 attributed to B1g
/
4E1g
,
4B1g
/
4B2g
,
4.5. Antifungal activity
4B1g / 4A2g and 4B1g / 4Eg. The spectral bands are consistent with
that of five coordinated Cr (III) complexes [47]. On the bases of
spectral studies of these complexes, a five coordinated square
pyramidal geometry and C4v symmetry may be assigned for these
complexes [48,49]. The magnetic moment value for this complex
was found to be 3.68e4.93 B.M. at room temperature which is close
to the predicted values for three unpaired electrons in the metal
ions [50].
For the experimental data Table 3, it has been observed that the
ligand as well as its complexes shows a significant degree of anti-
fungal activity against Aspergillus sp., Rizoctonia sp. and Penicillium
sp. at 1, 1.5 and 2 mg/mL concentration. The effect is susceptible to
the concentration of the compound used for inhibition. The activity
is greatly enhanced at the higher concentration. DMSO (control)
has showed a negligible activity as compare to ligand and their
metal complexes. However, the metal complexes show better
activity than the ligand [52,53]. The complexes are highly effective
against Aspergillus sp. and show 75e99% activity at 2 mg/mL
concentration. [Cr(C34H24N8O6)OAc](OAc)2 is the only complex to
show 100% activity against Aspergillus sp. The antifungal activity of
the complexes varies in the following order of fungal species:
Aspergillus sp. > Penicillium sp. > Rizoctonia sp.
The antifungal experimental results of the compounds were
compared with the standard antifungal drugs Miconazole at the
same concentration. All the metal complexes exhibited greater
antifungal activity against Aspergillus sp. However, they show
slightly lesser activity against Rizoctonia sp. than standard drug
Miconazole. The Cr (III) and Fe (III) complexes are more effective
against Penicillium sp. than the standard drug. From the data it has
been also observed that the activity depends upon the type of
metal ion and varies in the following order of the metal ion:
Cr > Fe > Mn.
The absorption spectral bands of manganese (III) complexes
5
showed three spin allowed transitions: B1g
/ , / ,
5A1g 5B1g 5B2g
5B1g / 5Eg appearing in the ranges 12,250e12,550, 16,150e18,860
and 35,450e35,720 cmꢁ1, respectively consistent with a five coor-
dinated square pyramidal geometry and C4v symmetry for Mn (III)
complexes [48,49]. The magnetic moment values for these
complexes were found in the range 4.92e5.74 B.M expected for
square pyramidal manganese complexes [50].
The electronic spectra of the iron (III) complexes gave two bands
at 9850e9980, and 27,650e27,760 cmꢁ1, which could be assigned
6
6
to the transitions A1g
/ /
4T1g and A1g 4T2g, respectively, sug-
gesting a five coordinated square pyramidal geometry of Fe (III)
complexes [49,51] this idea also supported the C4v symmetry for
these complexes. The complexes show magnetic moment values in
the range 5.20e5.45 B.M [51].
The spectral characterization shows the formula for macrocyclic
complexes as [M(C34H24N8O6)X]X2] where M ¼ Cr (III), Mn (III), Fe
(III) and X ¼ Clꢁ, NO3ꢁ and CH3COOꢁ. The measurements of molar
conductance in DMSO are 125e160
U
ꢁ1. All complexes give satis-
4.6. Minimum inhibitory concentration (MIC) and minimum
bacterial concentration (MBC) of synthesized compounds
factory elemental analyses results as shown in Table 2. Various
attempts such as crystallization using mixtures of solvents, low
temperature crystallization were unsuccessful to obtain a single
crystal for X-ray crystallography. However, the analytical, spectro-
scopic and magnetic data enable us to predict the possible structure
of the synthesized complexes.
The antibacterial screening concentrations of the compounds to
be used were estimated from the minimum inhibitory concentra-
tion (MIC). Minimum inhibitory concentration is the lowest
concentration of an antimicrobial complex that will inhibit the
Table 3
Fungicidal screening data of the ligand and their corresponding metal complexes.
Compounds
% Inhibition of spore germination
Aspergillus sp. (mg/mL)
Penicillium sp. (mg/mL)
Rizoctonia sp. (mg/mL)
1.0
1.5
2.0
1.0
1.5
2.0
1.0
1.5
2.0
Ligand
55
75
58
88
80
68
86
82
70
87
52
63
92
69
93
85
72
88
86
75
90
59
69
94
85
99
89
78
96
90
87
96
92
34
68
60
79
72
61
72
72
63
73
59
44
75
64
82
75
65
75
74
66
76
72
48
88
80
92
80
72
80
81
78
84
77
59
58
50
75
65
60
62
66
63
68
7
63
65
58
78
69
62
55
70
67
78
88
63
70
68
85
75
65
72
78
67
78
88
[Cr(C34H24N8O6)Cl]Cl2
[Cr(C34H24N8O6)NO3](NO3)2
[Cr(C34H24N8O6)OAc](OAc)2
[Mn(C34H24N8O6)Cl]Cl2
[Mn(C34H24N8O6)NO3](NO3)2
[Mn(C34H24N8O6)OAc](OAc)2
[Fe(C34H24N8O6)Cl]Cl2
[Fe(C34H24N8O6)NO3](NO3)2
[Fe(C34H24N8O6)OAc](OAc)2
Miconazole (standard)

G. Kumar et al. / European Journal of Medicinal Chemistry 52 (2012) 269e274
273
Table 4
Bactericidal screening data of the ligand and their corresponding metal complexes (inhibition zone in mm).
Microorganism
Ligand
Complexes
Imipenem
1
2
3
4
5
6
7
8
9
Gram positive
Staphylococcus aureus
Bacillus subtilis
41
40
66
62
75
70
98
97
15
48
25
32
46
45
33
42
28
35
69
65
100
100
Gram negative
Escherichia coli
Salmonella typhi
Pseudomonas aeruginosa
12
12
na
14
68
45
48
42
32
72
66
65
12
16
13
22
30
24
40
42
38
44
48
40
25
32
28
38
42
70
00
100
100
Excellent activity (90e100% inhibition), good activity (60e70% inhibition), significant activity (30e50% inhibition), negligible activity (10e20% inhibition), na ¼ no activity,
size of well: 6 mm (diameter), complex 1 ¼ [Cr(C34H24N8O6)Cl]Cl2, 2 ¼ [Cr(C34H24N8O6)NO3](NO3)2, 3 ¼ [Cr(C34H24N8O6)OAc](OAc)2, 4 ¼ [Mn(C34H24N8O6)Cl]Cl2,
5
¼
[Mn(C34H24N8O6)NO3](NO3)2,
6
¼
[Mn(C34H24N8O6)OAc](OAc)2,
7
¼
[Fe(C34H24N8O6)Cl]Cl2,
8
¼
[Fe(C34H24N8O6)NO3](NO3)2,
9
¼
[Fe(C34H24N8O6)OAc](OAc)2,
Imipenem ¼ standard drug.
visible growth of microorganisms after overnight incubation. The
MIC of the chemical complexes was tested against bacterial strains
through a macrodilution tube method [54]. The MIC values for
ligand against B. subtilis, S. aureus, E. coli, S. typhi and P. aeruginosa
indicate that the ligand has moderate activity against S. typhi and
E. coli but no activity against P. aeruginosa. Ligand showed a signif-
icant activity towards B. subtilis and S. aureus. Antibacterial activity
of all the complexes towards gram positive and gram negative
bacteria is quite significant. Further to it, the ligand showed
moderate, and the complexes moderate to high activities as
compared to standard drug towards the all organism. The results of
the bactericidal study of the synthesized compounds are displayed
in Table 4.
The variation in the antimicrobial activity of different metal
complexes against different microorganisms depends on their
impermeability of the cell or the differences in ribosomes in
microbial cell [56]. The lipid membrane surrounding the cell favors
the passage of any lipid soluble materials and it is known that
lipophilicity is an important factor controlling antimicrobial
activity [57].
were 126, 126, 65, 65 and 65
15, 15 and 20 g/mL for the [Cr(C34H24N8O6)Cl]Cl2, 15, 15, 12, 10 and
10 g/mL for [Cr(C34H24N8O6)NO3](NO3)2, 10, 10, 12, 12 and 10 g/
mL for [Cr(C34H24N8O6)OAc](OAc)2, 62, 62, 40, 40 and 40 g/mL for
g/mL for
g/mL for
g/mL
g/mL [Fe(C34H24N8O6)
g/mL [Fe(C34H24N8O6)OAc](OAc)2,
g/mL for standard drug Imipenem. The values of
mg/mL for C34H24N8O6 (ligand) 15, 20,
m
m
m
m
[Mn(C34H24N8O6)Cl]Cl2, 50, 50, 32, 30 and 30
[Mn(C34H24N8O6)NO3](NO3)2, 40, 40, 32, 32 and 20
m
m
[Mn(C34H24N8O6)OAc](OAc)2, 30, 40, 25, 25 and 25
[Fe(C34H24N8O6)Cl]Cl2, 20, 30, 20, 25 and 25
NO3](NO3)2, 18,15,12,12 and 15
8, 8, 6, 6 and 6
m
m
m
m
MIC showed that the Cr complexes were found more potent as
compared to the other studied complexes.
Minimum bacterial concentration (MBC) is the lowest concen-
tration of antimicrobial compounds that will prevent the growth of
microorganism after subculture on the antibiotic free media [54].
Minimum bacterial concentration (MBCs) was determined by
In the present study, low activity of the some metal complexes is
due to their low lipophilicity, because of which penetration of the
complex through the lipid membrane was decreased and hence,
they could neither block nor inhibit the growth of the microor-
ganism. HPLC was used to analyze the lipophilicity of the
compounds.
spreading the 100 ml compound from one below MIC and MIC itself.
All tubes were incubated for 24 h at 37 ꢀC. The growth was observed
on each plate.
HPLC condition: RP-HPLC method flow rate of 1 mL/min, an
injection volume of 10
m
L, a column temperature of 25 ꢀC, the UV
4.7. Antibacterial activity
detection at 254 nm and a 25 min isocratic mobile phase methanol,
25 mmol KH2PO4, pH3 (20:80) was used to determine the lip-
ophilicity of the compounds [58]. Calibration curves were gener-
ated for the given complexes. Each data point was obtained from at
least two repeated measurements. Stock solutions of the complexes
were prepared as about 5 mg/mL in methanol, and then serial
dilution were made as required. Relative integrated peak area
versus injected amount of the compound for all the given
complexes. The calibration curves relative peak area versus
concentration of the compound showed good linearity (R2 ¼ 0.999).
The constant of calibration curves (relative peak areas versus
concentration of compound) of the complexes determined by linear
regression analysis are given in Table 5.
The ligand, its metal complexes and standard drug Imipenem
(C12H17N3O4S) were screened separately for their antibacterial
activity against the bacteria S. aureus and B. subtilis (as gram posi-
tive bacteria) and P. aeruginosa, E. coli and S. typhi (as gram negative
bacteria). The diffusion agar technique was used to evaluate the
antibacterial activity of the synthesized metal complexes [55]. From
the bactericidal activity, it is apparent that the complexes were
more toxic towards gram positive strains than gram negative
strains. The reason is the difference in the structure of the cell walls.
The walls of gram negative cells are more complex than those of
gram positive cells. The zones of inhibition (ZOI) values obtained
Table 5
The components of calibration curves (relative peak area vs. amount of compound) of the compounds determined by linear regression analysis.
Compounds
Slope
Intercept
Correlation coefficient (R2)
RSD for retention time (%)
RSD for peak area (%)
[Cr(C34H24N8O6)Cl]Cl2
16,807
67,696
6 ꢂ 106
16,825
76,184
6 ꢂ 106
16,770
71,940
6 ꢂ 106
60,133
15,223
31,897
55,709
17,277
24,222
28,982
16,200
25,260
0.999
0.999
0.999
0.998
0.999
0.999
0.998
0.999
0.999
0.115
0.126
0.099
0.114
0.113
0.114
0.114
0.115
0.114
1.41
1.54
1.43
1.05
1.25
1.30
1.41
1.44
1.42
[Cr(C34H24N8O6)NO3](NO3)2
[Cr(C34H24N8O6)OAc](OAc)2
[Mn(C34H24N8O6)Cl]Cl2
[Mn(C34H24N8O6)NO3](NO3)2
[Mn(C34H24N8O6)OAc](OAc)2
[Fe(C34H24N8O6)Cl]Cl2
[Fe(C34H24N8O6)NO3](NO3)2
[Fe(C34H24N8O6)OAc](OAc)2

274
G. Kumar et al. / European Journal of Medicinal Chemistry 52 (2012) 269e274
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The analytical data showed a single metal ion mononuclear five
coordinated square pyramidal geometry of the complexes
[M(C34H24N8O6)X]X2]. The ligand and their Cr, Mn and Fe
complexes were tested for their antimicrobial inhibition potential
against some pathogens studies reveals that, metal complexes have
more biological activity than free ligand. The process of chelation
increases the lipophilicity of the metal complexes, metal complexes
penetrate the bacterial membrane (Lipid membrane) more effec-
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[Cr(C34H24N8O6)OAc](OAc)2 shows best inhibition potential against
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6. Experimental protocols
The microanalysis of C, H and N were estimated by elemental
analyzer Perkin Elmer 2400 and the metal contents of Cr (III), Mn
(III) and Fe (III) were determined by atomic absorption spectro-
photometer (Perkin Elmer 5000). IR spectra was recorded on a FT-
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using Nujol Mull. 1H NMR and 13C NMR spectra (at room temper-
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recorded on JEUL JMS-AX-500 mass spectrometer, GCeMS analysis
was performed on a Shimadzu GCMS-QP5050A instrument, Indian
Institute of Petroleum Dehradun, India. Magnetic susceptibility
measurements were carried out at SAIF, IIT Roorkee, on vibrating
sample magnetometer (Model PAR 155). Electronic spectra in
DMSO were recorded on
a Hitachi 330 spectrophotometer
(1300e200 nm) at room temperature. The conductivity was
measured on digital conductivity meter (HPG system, G-3001). The
chromatographic apparatus consisted of a model waters 600 pump
with waters 600 controller, waters 2996 photodiode array detector,
a non polar C18 column, 15 cm ꢂ 4.6 mm I.D., 5
mm particles (all
from waters, Milford MA, USA).
Acknowledgment
We wish to express our cordial thanks to Dr. A.K. Bhatanagar,
(Scientist ‘G’) Indian Institute of Petroleum, (CSIR) Dehradun, India,
for fruitful discussion and suggestion for performing this research
work.
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