Synthesis, characterization, and biological activity of transition metals complexes with mefenamic acid (NSAIDs)

Article abstract of DOI:10.1134/S1070363215070282

A new series of transition metal complexes have been synthesized with [(2,3-dimethylphenyl)-amino]benzoic acid (Mefenamic acid) in 1: 1/1: 2 M: L ratio. The complexes have been characterized by elemental analysis, FT-IR and UV-Vis spectroscopy. Antifungal

Full text of DOI:10.1134/S1070363215070282

ISSN 1070-3632, Russian Journal of General Chemistry, 2015, Vol. 85, No. 7, pp. 1745–1751. © Pleiades Publishing, Ltd., 2015.
Synthesis, Characterization, and Biological Activity
of Transition Metals Complexes
with Mefenamic Acid (NSAIDs)1
S. Ramzana, S. Saleemb, B. Mirzab, S. Alia, F. Ahmedc, and S. Shahzadia
a Department of Chemistry, Quaid-i-Azam University, Islamabad, 45320 Pakistan
e-mail: drsa54@yahoo.com; sairashahzadi@hotmail.com
b Department of Biochemistry, Quaid-i-Azam University, Islamabad, 45320 Pakistan
c NESCOM, P.O. Box no. 2216, Islamabad, Pakistan
Received April 3, 2015
Abstract―A new series of transition metal complexes have been synthesized with [(2,3-dimethylphenyl)-
amino]benzoic acid (Mefenamic acid) in 1 : 1/1 : 2 M : L ratio. The complexes have been characterized by
elemental analysis, FT-IR and UV–Vis spectroscopy. Antifungal, antibacterial, antitumor, antioxidant (DPPH,
H2O2 induced DNA damage) activity of the complexes and DNA interaction with the complexes were studied.
The results revealed that the ligand and most of synthesized compounds did not demonstrate significant
antibacterial activity unlike [Ni(Mef)(H2O)Cl] that exhibited pronounced activity against F. Solani. Antitumor
activity of the products was higher than that of the free ligand.
Keywords: mefenamic acid, transition metals, complexes, IR, UV–Vis, conductance, antibacterial, antifungal,
antitumor, antioxidant activity, DNA interaction
DOI: 10.1134/S1070363215070282
INTRODUCTION
EXPERIMENTAL
According to numerous investigations binding of a
drug to a metal enhances its activity [1, 2]. Cationic
metal centers actively bind to negatively charged
biomolecules including proteins and nucleic acids.
Metal complexes demonstrate high pharmaceutical
potential [3, 4].
Chemicals and instrumentation. Most chemicals
used were of analytical grade purchased from Merck.
Mefenamic acid was provided by Ferozsons Ltd.
(Lahore). The solvents were purified according to the
conventional methods [13]. Melting points were
measured in open capillary tubes by Mitamura Rikero
Kogyo (Japan). IR spectra were recorded as KBr discs
in the range of 4000–400 cm–1 on a Perkin-Elmer
Spectrum-1000 FT-IR Spectrophotometer. CHN
analysis was carried out with a Perkin-Elmer 2400
Series II instrument. Electronic spectra were recorded
on a Lambda 2S Perkin Elmer spectrophotometer. Con-
ductance was measured by Metrohm 712 conductometer.
[2-(2,3-Dimethylphenyl)amino]benzoic acid (mefenamic
acid) is anthranilic acid derivative that demonstrates
non-steroidal anti-inflammatory, antipyretic, and
analgesic activity [5, 6]. It inhibits proliferation and
triggers apoptotic cell death of several human cancer
cell lines [7–9], acts as a neuro-protection in
Alzheimer disease [10] and demonstrates many other
biological sites of action [11, 12].
Synthesis of potassium salt of mefenamic acid.
10 mL of KOH (1 mmol) solution were added
Pharmacological importance of mefenamic acid
initiated our study of synthesis and biological activity
of its metal complexes (Fig. 1).
CH3
COOH
H3C
NH
Fig. 1. 2-[(2,3-dimethylphenyl)amino]benzoic acid (Mefenamic
acid).
1 The text was submitted by the authors in English.
1745

1746
RAMZAN et al.
dropwise upon stirring to the solution of mefenamic
acid (1 mmol) in ethanol (100 mL). The mixture was
stirred for 1 h at room temperature. The solvent was
removed in a rotary evaporator under reduced pressure.
The solid product was recrystallized in chloroform : n-
hexane (1 : 1).
Antifungal assay. Antifungal activity of free ligand
and metal complexes was studied against fungal strains
(Mucor species, Aspergillus flavus, Aspergillus
fumigatus and Fusarium solani) by using agar tube
dilution method [15]. Screw caped test tubes
containing Sabouraud dextrose agar (SDA) medium
(4 mL) were autoclaved at 121ºC for 15 min. Test
tubes were allowed to cool down to 50ºC and non
solidified SDA was loaded with 66.6 μL of the test
compound from the stock solution (12 mg/mL in
DMSO) to make final concentration 200 μL/mL. Test
tubes content was then allowed to solidify in slanting
position at room temperature. Each test tube containing
solidified media and test compound were then
inoculated with 4 mm diameter piece of inoculum from
a seven days old culture. The media supplemented
with DMSO and Terbinafine (200 μL/mL) were used
as negative and positive controls, respectively. Test
tubes were incubated at 28ºC for seven days and
fungal growth was determined by measuring linear
growth (mm). Growth inhibition was calculated with
reference to the negative control.
Synthesis of transition metal complexes I–VIII.
Potassium salt of mefenamic acid (1 mmol/2 mmol)
was dissolved in ethanol (100 mL) at room
temperature. The transition metal chloride (1 mmol)
dissolved in ethanol (50 mL) was added drop wise to
the above solution upon constant stirring. The reaction
mixture was refluxed for 6 h with continuous stirring.
KCl was filtered off and the solvent was evaporated in
rotary evaporator under reduced pressure. The solid
residue was recrystallized in chloroform : n-hexane
mixture (4 : 1).
.
nR-COOK + MClx mH2O
EtOH, Reflux 6 h
.
M(RCOOO)nClx mH2O + KCl,
n = 1, 2; x = 2, 3; m = 2, 4, 6; M = Cr(III), Fe(III), Ni(II),
Cu(II).
Antitumor assay. To detect antitumor activity of the
test compounds, potato disc antitumor assay was
performed [15]. The 48 h old culture of AT-10 strain
of Agrobacterium tumefaciens was used. Inoculums of
five concentrations (0.01, 0.1, 10, 100, 1000 ppm) of
each test sample containing bacterial culture were
prepared. Red skinned potatoes were surface sterilized
in 0.1% mercuric chloride solution and then under
complete aseptic conditions. Potato discs (0.5 cm
thickness) were obtained from these potatoes by using
sterile metallic cork borer. The potato discs were
placed in petri plates each containing 25 mL of 1.5%
autoclaved agar solution. The inoculum (50 μL) was
poured on each disc. The plates were sealed with
parafilm and incubated at 28ºC for 21 days. Number of
tumors was counted after staining with the lugol
solution (10% KI and 5% I2) with the help of
dissecting microscope.
Biological activity. All products were tested for
antibacterial, antifungal antioxidants, and potato disc
tumor activity.
Antibacterial assay. The free ligand and metal
complexes were tested for antibacterial activity against
bacterial strains: gram positive Staphylococcus aureus
(ATCC 6538), Bacillus subtilis (ATCC 6633) and
Micrococcus luteus (ATCC 10240); and gram negative
Escherichia coli (ATCC 15224) and Enterobacter
aerogenes (ATCC 13048) by using agar well diffusion
method [14]. Broth culture (0.75 mL) containing
approximately 106 colony forming units (CFU/mL) of
test strain was added to nutrient agar medium (75 mL)
at 45ºC, mixed well, and poured into a 14 cm sterile
petri plates. The medium was allowed to solidify and 8
mm wells were dug with a sterile metallic borer. One
hundred µL of the test sample (1 mg/mL in DMSO)
was poured in respective wells. In each plate DMSO
served as negative control and standard antibacterial
drugs Roxithromycin (1 mg/mL) and Cefexime (1 mg/mL)
served as a positive control. Triplicate plates of each
bacterial strain were prepared. The plates were
incubated at 37ºC for 24 h. Antibacterial activity was
determined by measuring the diameter of zones with
complete inhibition (mm). Growth inhibition was
calculated with reference to positive control.
Antioxidant assay. Antioxidant and prooxidant
effects of the synthesized compounds were studied in
vitro by using DNA protection assay [16].
DPPH assay. Radical scavenging activity of
compounds (conc. 100, 50, and 25 ppm) against stable
free radical DPPH was determined spectrophoto-
metrically [16]. Assay was carried out in triplicate.
Reaction mixture was prepared by adding 100 μL of
each test compound solution, 0.1 mM DPPH in ethanol
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 7 2015

SYNTHESIS, CHARACTERIZATION, AND BIOLOGICAL ACTIVITY
Table 1. Physical data of transition metal complexes of mefenamic acid
1747
Elemental analysis data
H
Comp.
no.
Yield,
%
C
N
Formula
MW
mp, ºC
found, calculated, found, calculated, found, calculated,
%
%
%
%
%
%
HL
I
Mefenamic acid
241.2 230–231
399.2 210–212
586.0 215–216
–
–
–
–
–
–
–
[Cr(Mef(H2O)2Cl2]
[Cr(Mef)2(H2O)Cl]
76
68
78
71
69
82
77
67
13.02
9.16
13.26
9.47
16.65
10.88
16.93
11.68
13.06
9.12
13.21
9.51
16.61
10.84
16.97
11.72
4.54
4.93
4.79
5.13
4.58
5.20
4.83
5.19
4.58
4.89
4.74
5.17
4.53
5.24
4.88
5.15
3.51
4.93
3.33
4.75
3.97
5.19
3.73
5.15
3.47
4.97
3.37
4.71
3.93
5.23
3.77
5.10
II
III
IV
V
[Fe(Mef)(H2O)3Cl]Cl 421.1 205–206
[Fe(Mef)2(H2O)Cl]
[Ni(Mef)(H2O)Cl]
[Ni(Mef)2]
589.9 175–176
352.4 216–218
VI
VII
VIII
539.2
90–91
[Cu(Mef)(H2O)2]Cl
[Cu(Mef)2]
375.3 204–206
544.1 154–155
solution and 0.9 mL of 0.1 mM Tris-HCl in capped
vials. DMSO was used as the negative control. Reac-
tion mixture was incubated in darkness for 30 min at
room temperature. Upon incubation absorbance of each
reaction mixture was measured spectrophotometri-
cally at 517 nm. Scavenging (%) of DPPH free radicals
for each concentration of each compound was calculated.
DNA interaction studies. The DNA solution was
made in distilled water which gave a ratio of UV
absorbances at 260 and 280 nm of 1.72, indicating that
the DNA was sufficiently free of proteins [18]. The
DNA concentration was determined at 260 nm by
using the formula: DNA conc. (μg/mL) = Absorbance
at 260 nm × dil. Factor × 50 μg/mL × ratio. Standard
ratio (1.8)
H2O2 induced DNA damage assay. To determine
the antioxidant/pro-oxidant potential of test com-
pounds DNA protection assay was performed [17].
The reaction was conducted in eppendorf tubes at total
volume of 15 μL containing 0.5 μg of pBR 322 DNA
in 3 μL of 50 mM phosphate buffer (pH 7.4), 3 μL of
2 mM FeSO4, and 5 μL of tested samples at various
concentrations (10, 100, 1000 ppm). Then, 4 μL of
30% H2O2 were added. Similarly DNA samples of the
reference containing the same content except the
compounds were prepared. All reaction mixtures were
incubated at 37ºC for 1 h. The mixtures were subjected
to 1% agarose gel electrophoresis. DNA bands (super-
coiled, linear and open circular) were stained with
ethidium bromide. Gels were documented (Gel Doc,
BioRad) and intensity of the bands was determined.
DNA protection was calculated based on the DNA
band corresponding to that of native in the presence
and absence of different concentrations (10, 100,
1000 ppm) of test compounds. Evaluations of anti-
oxidant or prooxidant effects on DNA were based on
percent of increase or loss of supercoiled monomer com-
pared to the control value. To avoid the effects of photo-
excitation of samples, experiments were done in darkness.
DNA solution of each compound was prepared in
DMF of 1 × 10–3 concentration. Solvent mixture was
prepared in the ratio water : DMF in which both the
compound and DNA soluble.
Phosphate buffer (0.1 M) was prepared by adding
14.19 g of Na2HPO4 and 15.6 g of NaH2PO4 in 1 L of
solvent mixture (BSM of pH 7.4). The assay was done
on Agilent chemstation UV–Vis spectrophotometer.
BSM was used as a blank and 50 µL of compound
were added to 2950 µL of BSM. Change in absorbance
upon addition of DNA was measured and spectra were
recorded until we got the maximum interaction.
RESULTS AND DISCUSSION
The complexes were solids soluble in common
organic solvents. Molar conductance of compounds
was measured in 0.001M ethanol solution, µS cm–1:
14.7 (Ligand), 40.1 (1), 27.6 (2), 29.9 (3), 25.9 (4),
18.2 (5), 55.3 (6), 25.9 (7), and 5.4 (8). Physical data
are presented in Table 1.
FT-IR spectroscopy. The NH band of mefenamic
acid at 3340 cm–1 did not exhibit significant shift upon
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 7 2015

1748
RAMZAN et al.
Table 2. IR data (cm–1) for the transition metals complexes
Table 3. UV–Vis data of transition metal complexes of
mefenamic acid
of mefenamic acid
ν(COO)
Comp.
no.
Electronic
transition
Comp.
no.
λ, cm–1 (nm)
Geometry
Octahedral
ν(N–H) ν(C=O)
Δν ν(M–O)
νas(COO) νs(COO)
4
I
17452 (573) A2g → 4T2g
21673 (461) A2g → 4T1g (F)
4
HL
3340
3344
3343
3332
3334
3345
3339
3350
3343
1730
1730
1756
1759
1729
1722
1734
1710
1723
1673
1612
1603
1609
1610
1576
1608
1566
1617
1422
1421
1490
1486
1448
1451
1413
1436
1452
251
–
34030 (294) A2g → 4T1g (P)
4
16125 (620) A2g → 4T2g
Octahedral
191 486
113 470
123 468
162 462
125 449
195 468
130 496
165 497
4
I
II
21009 (476) A2g → 4T1g (F)
4
36122 (277) A2g → 4T1g (P)
4
II
6
III
IV
16579 (603) A1g→5T1g
Octahedral
Octahedral
III
IV
V
22124 (452) A1g →4T2g (G)
6
16560 (604) A1g→5T1g
6
21746 (460) A1g →4T2g (G)
6
3
V
19987 (500) A2g→3T1g (F) Square planner
25125 (398) A2g→3T1g (P)
3
VI
VII
VIII
3
VI
19550 (512) A2g→3T1g (F) Square planner
24991 (400) A2g→3T1g (P)
3
2
VII
23678 (422) Eg → 2B1g
Square planner [21]
Square planner
2
VIII
24598 (406) Eg → 2B1g
complexation indicating no participation of the group
in the process (Table 2).
reference drug (Vincristine) and the ligand (Table 5)
[28–30].
IR data of the complexes demonstrated the
separation value (Δν) between νas(COO) and νs(COO)
to be < 200 cm–1 in the spectra of ligands suggesting
bidentate bonding for the transition metals
carboxylates [19, 20]. The new band in the range of
486–449 cm–1 in the spectra of products was assigned
to (M–O) bond formation.
Antioxidant assay. In the assay majority of the
products exhibited antioxidant concentration dependent
activity (Table 6). Most of the products enhanced the
Table 4. Antifungal activity of transition metals complexes
of mefenamic acid
UV–Vis spectra. UV–Vis absorption pattern of the
synthesized compounds was recorded in the region
200–800 nm (Table 3). Some bands in the spectra of
complexes I–VIII were typical for interligand and
charge transfer or n→π* transitions and had decreased
intensities. Sharp bands observed in the range 240–
270 nm could be assigned to charge transfer L→M or
n→π* transition [21].
Percentage growth inhibition
Comp.
no.
A. fumigatus A. flavus F. solani Mucor sp.
I
38.06452
59.85401 66.66667 14.28571
II
41.29032
27.0073 60.95238 13.60544
III
IV
50.32258
32.25806
37.41935
44.51613
43.22581
38.06452
42.33577 74.28571 23.80952
30.65693 57.14286 16.32653
39.41606 71.42857 22.44898
37.22628 55.2381 27.89116
41.60584 33.33333 4.081633
35.76642 57.14286 25.85034
Antimicrobial activity. The ligand and compounds
I–VIII did not demonstrate any activity against all five
bacterial strains tested [22–24]. Only compound V
demonstrated significant antifungal activity against
sF. solani [25–27] (Table 4).
V
VI
VII
VIII
Antitumor assay. All products exhibited
significant level of tumor inhibition as compared to the
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SYNTHESIS, CHARACTERIZATION, AND BIOLOGICAL ACTIVITY
1749
Table 5. Antitumor activity of transition metal complexes of
Table 6. DPPH free radical scavenging activity of transition
mefenamic acid
metals complexes of mefenamic acid
Percentage age tumor inhibition
Percentage scavenging, ppm
Comp.
no.
IC50,
µg/mL
Comp.
no.
IC50,
µg/mL
at concentrations, ppm
Remarks
1000
100
100
100
100
100
100
100
100
100 10 0.1
0.01
100
50
25
I
100.00 100.00 75.98 68.99 BDR
76.54 62.12 51.51 30.30 0.281
–36.2347 –11.1518 –5.72943 BDR Pro-oxidant
–35.6227 –16.2084 –3.41025 BDR Pro-oxidant
–10.8427 1.195779 6.837513 BDR Pro-oxidant
–11.6815 1.230694 5.395298 BDR Pro-oxidant
6.68587 1.413886 –1.42537 BDR Pro-oxidant
–31.9296 21.98066 39.45954 BDR Pro-oxidant
8.833381 11.17964 13.8137 >100 Antioxidant
62.71571 48.33388 40.24352 48.691 Antioxidant
I
II
II
III
IV
V
100.00 63.56 58.13
5.42 BDR
III
IV
V
90.15 89.39 70.45 60.60 BDR
100.00 96.89 77.51 56.58 BDR
66.47 56.60 40.03 33.33 0.661
49.11 48.71 44.37 44.37 0.935
72.78 65.28 57.39 49.50 0.024
VI
VII
VIII
VI
VII
VIII
DNA damage [31] at concentration 1000 ppm
(Table 7). The compounds II–IV and VI protected
plasmid DNA at all concentrations.
electronic states of chromophores with DNA bases and
indicated close proximity of the complex molecules to
DNA bases.
Interaction with DNA. Absorbance of the complex
IV was DNA concentration dependent (Figs. 2, 3). The
hypochromic effect was due to the interaction between
Binding constant, K, was calculated on the basis of
variations in the absorbance spectra of complexes upon
binding to DNA according to the equation reported
Wavelength, nm
Fig. 2. Absorption spectra of mefenamic acid at different concentrations of DNA.
2
↓
5
1
Wavelength, nm
Fig. 3. Absorption spectra of (1) compound IV at different concentrations of DNA, M × 10–5: (2) 1.64; (3) 3.23; (4) 4.76; and
(5) 6.25.
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1750
RAMZAN et al.
Table 7. DNA protection of transition metals complexes of
Table 8. Association constants and Gibbs free energies of
transition metals complexes of mefenamic acid
mefenamic acida
Comp. no.
Concentration Protection
Damage
Comp. no.
(K/M)–1
–∆G, kJ/mol
I
1000
100
10
–
+++
–
HL
III
58.24 × 103
–57.75 × 103
39.15 × 103
13.51 × 103
48.48 × 103
27.18
–
++
++
++
++
+
–
IV
26.20
23.56
26.73
II
1000
100
10
–
VII
VIII
–
–
III
IV
V
1000
100
10
++
++
++
++
++
++
–
–
CONCLUSIONS
–
FT-IR data indicated that mefenamic acid acted as
the bidentate ligand. The products did not exhibit
antibacterial activity against tested bacterial strains
probably due to their low lipophilicity. Only [Ni(Mef)·
(H2O)Cl] (V) demonstrated significant antifungal
activity against F. solani. All complexes were
remarkably antitumor active against AT10. The
compounds II–IV and VI protected the plasmid DNA
at all concentrations. The hypochromic effect in UV–
Vis spectra of products indicated the interaction among
electronic sites of chromophores with DNA bases.
–
1000
100
10
–
–
–
1000
100
10
+++
–
++
++
++
++
++
–
–
VI
VII
VIII
1000
100
10
–
ACKNOWLEDGMENTS
–
QAU is acknowledged for financial support as
URF.
-
1000
100
10
+++
–
++
++
–
–
1/[DNA], μM–1
1000
100
10
+++
++
–
–
++
a
(+) Weaklyeffective; (++) moderately effective; (+++) strongly
effective.
earlier [32] (Table 8). The plot of A0/(A – A0) vs.
1/[DNA] was contracted using the data. The intercept
to slope ratio yielded the binding constant (Fig. 4). The
accumulated data indicated high affinity of the
products with DNA.
Fig. 4. Plot of A0/(A – A0) vs. 1/[DNA] for determination of
binding constant of complex IV–DNA adduct.
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SYNTHESIS, CHARACTERIZATION, AND BIOLOGICAL ACTIVITY
1751
Bioassay Techniques for Drug Development,
Amsterdam: Harward Academic Publishers, 2001.
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