In vivo assessment of newly synthesized achiral copper(ii) and zinc(ii) complexes of a benzimidazole derived scaffold as a potential analgesic, antipyretic and anti-inflammatory

Article abstract of DOI:10.1039/c5ra25071d

Two new complexes of copper(ii), [CuL₂], and zinc(ii), [ZnL₂], with a tridentate -ONN′- Schiff base ligand (L), a bioactive scaffold derived from 2-aminobenzimidazole and 2-hydroxy-3-methoxybenzaldehyde, were synthesized and characte

Full text of DOI:10.1039/c5ra25071d

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In vivo assessment of newly synthesized achiral copper(II) and
zinc(II) complexes of benzimidazole derived scaffold as a potential
analgesic, antipyretic and anti-inflammatory
Mohamed F. Al Ajmi,a Afzal Hussain,a Ali Alsalme,b and Rais Ahmad Khanb*
aDepartment of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh 11451, KSA
bDepartment of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh11451, KSA.
Abstract
Two new complexes of copper(II), [CuL2] and zinc(II), [ZnL2] with tridentate –ONN′–
Schiff base ligand (L), a bioactive scaffold derived from 2ꢀaminobenzimidazole and 2ꢀ
hydroxyꢀ3ꢀmethoxybenzaldehyde were synthesized and characterized by using various
1
spectroscopic techniques viz, IR, H, 13C NMR, EPR, HRMS, elemental analysis and
purity by UPLC studies. Both the complexes are nonꢀ electrolyte in nature. The newly
synthesized compounds were screened for acetic acidꢀinduced analgesic and yeastꢀ
induced antipyretic activities in mice and carrageenanꢀinduced paw edema in rats (antiꢀ
inflammatory). The results showed that [CuL2] compound (at 100 mg/kg b.w) possessed
potent antiꢀinflammatory activity whereas [ZnL2] (at 50 mg/kg and 100 mg/kg b.w)
exhibited significant analgesic activity when compared with standard drugs. Both the
complexes have apparently moderate and nearly akin antipyretic activity.
* Author for Correspondence:
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh, 11451, KSA. Mobile +966 536745404, Email: raischem@gmail.com (Khan, RA)
1

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Introduction
Inflammation is a complex biological response of body tissues to harmful stimuli, such
as pathogens, damaged cells, or irritants.1 Inflammation is classified into two acute and
chronic. Acute inflammation can lead to progressive tissue damage by the noxious
stimulus (e.g. pathogens, chemical irritants). While, chronic inflammation can lead to a
multitude of diseases, viz, hay fever, periodontitis, atherosclerosis, rheumatoid arthritis,
osteoarthritis, gout, Alzheimer's disease and obesity and even can cause cancer (e.g.,
gallbladder carcinoma).2,3 Therefore, the treatment of inflammation is of paramount
importance. Nonꢀsteroidal antiꢀinflammatory drugs (NSAIDs) viz. Indomethacin,
ibuprofen, and naproxen have frequently used for the cure of the first streak of various
chronic inflammatory disease.4 The classical NSAIDs demonstrate their action by
confining the biosynthesis of prostaglandin, some of them are proꢀinflammatory, which is
fetched by inhibiting the rateꢀlimiting cyclooxygenase (COX) enzyme involved in the
inflammatory cascade5 and are responsible for the transformation of arachidonic acid to
prostaglandins.6,7
Among NSAIDS, imidazole and fused imidazole with sixꢀmembered rings,8 possess
central position due to its significant potential as therapeutics in clinical applications.9–12
One of the most privileged classes of heterocyclic molecules are benzimidazoles in
medicinal chemistry, encompassing a diverse range of biological activity.13
Benzimidazoles derivatives displayed potential antimicrobial, antiꢀinflammatory,
antituberculosis, antioxidant, antihypertensive, and anticancer, activities.14ꢀ21
Benzimidazoleꢀ5ꢀcarboxylic acid and its derivatives have been ascended as potent
agents for hepatitis C virus infections also.22 Various derivatives of benzimidazole are
used as drugs, few amongst them are Omeprazole (proton pump inhibitor), Pimobendan

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(ionodilator), Albendazole (inhibitor of Encephalitozoon Intestinal infection in AIDS
patients) and Mebendazole. The presence of benzimidazole ring provides an active
structural motif, which possesses peculiar structural features viz, steric, electronic as
well as brings planarity to the drug candidate that makes the molecule active on different
targets via hydrogen bonding and πꢀπ* stacking. Also, benzimidazoles have the
advantage to get easily functionalized for synthesis purpose and lead to quite stable and
biologically privileged molecules.
Schiff base compounds are privileged ligand in medicinal chemistry and have proven
therapeutic potential, due to –CH=N– functionality giving rise to strong activity and plays
an essential role.23 The remarkable biological activities, including antitumor,
antibacterial, antifungal, antimalarial, antiviral, antioxidant, antiꢀinflammatory, analgesic,
anticonvulsant, antiglycation, antihypertensive, antidepressant and lipid lowering
properties.24ꢀ27
The designing and synthesis of metal complexes with biologically active scaffold are
an imperative and lively research area in bioinorganic chemistry.28,29 Metal chelation
is one of the excellent routes to increase the lipophilic character of the organic moiety
that may facilitate the absorption of the drug from the GI (gastrointestinal) tract.30 In
fact, on coordination, drugs/ligands exhibited significantly improved bioactivity profiles,
while some inactive ligands may acquire pharmacological properties.31,32 Among metal
salts, Cu(II) and Zn(II) are the most important biometals, Cu(II) is present in all living
organisms and plays a crucial role in redox chemistry, growth and development.33 It is a
preꢀrequisite for the functioning of various enzymes and proteins, which are occupied in
respiration, energy metabolism, and DNA synthesis, especially cytochrome oxidase,
superoxide dismutase (SOD), ascorbate oxidase and tyrosinase.34 Moreover, Cu(II) and

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Zn(II) are coꢀfactors in metabolic processes involved in collagen and bone metabolism
via articular/connective tissue and the immune system34 and play a crucial role in the PG
synthesis.35 In particular, copper carboxylates drugs belong to an important class of antiꢀ
inflammatory and anticancer agents, some of them are commercially available drugs.36
Thus, in the present work, we have described the synthesis and characterization of 2ꢀ
aminobenzimidazole and oꢀvanillin derived Schiff base ligand and their achiral
copper(II), [CuL2] and zinc(II), [ZnL2] complexes. Those above plentiful pharmacological
activities of benzimidazoles and derivatives encouraged us to study the inꢀvivo
analgesic, antipyretic and antiꢀinflammatory activities of synthesized complexes.
Result and Discussion
Chemistry
Heterocyclic Schiff base ligand viz, (2ꢀ[(1HꢀBenzoimidazolꢀ2ꢀylimino)ꢀmethyl]ꢀ6ꢀ
methoxyꢀ phenol) has been synthesized with high purities and acceptable yields. The
orangeꢀyellow compound obtained is stable to air and moisture and was characterized
13
by elemental analysis, HRMS (LCꢀQTOF) spectrometry and IR and NMR (1H and C)
spectroscopy. Reaction of Schiff base viz, 2ꢀ[(1HꢀBenzoimidazolꢀ2ꢀylimino)ꢀmethyl]ꢀ6ꢀ
methoxyꢀphenol ligand (L) with M(NO3)2ꢁn(H2O), where M = Cu(II) or Zn(II) in 2:1
stoichiometry, led to the isolation of neutral mononuclear complexes [CuL2] and [ZnL2]
in which the ligand behaves as anionic with deprotonation of phenolic (OH) proton
and tridentate with [˗ONN′˗] donor set (Scheme 1).

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H
N
NH2
O
N
HO
O
H
N
N
N
HO
O
Cu(NO3)2.3H2O
Zn(NO3)2.6H2O
H
N
H
N
N
N
N
O
O
N
O
O
Zn
Cu
O
O
O
N
O
N
N
N
N
N
H
H
Scheme 1: Schematic representation of the synthesized ligand (L) and its [CuL2] and
[ZnL2] complexes.
The achiral nature of the complexes was studied first by the help of polarimeter, the
specific rotation [α]25 value (1 x 10ꢀ3 M solution of the compound in DMSO) was found
zero. To ascertain the achirality, we also checked with circular dichroism, which
confirms the polarimeter results by giving no spectra. The stability of the complexes was
evaluated by UV–Vis spectral analysis at different times in a mixture containing
H2O/DMSO in 95%: 5% ratio and the absorbance, as well as wavelength, were remain
unaffected, even after 12 h. Thus, results indicate that the complexes are quite stable in
solution. Both complexes were characterized by analytical and spectroscopic
techniques and the purity of ligand, and the complexes are checked with the help of

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UPLC technique, which confirmed the purity of the synthesized materials to ~96 ± 2% of
retention time 7.827 min [L], 5.633 min [CuL2] and 6.747 min [ZnL2]. Further, the HRꢀ
MS exhibited molecular ion peak at m/z = 595.1 for [CuL2] and m/z = 597.1 for [ZnL2]
which corresponded to the predicted molecular weight of the [M+2H]+ and [M+H]+ ions,
respectively. The isotopic patterns of these signals fits well with the theoretical isotopic
distributions).
1
The significant IR vibrational bands and the H chemical shift values of the free ligand
and its complexes are listed in the experimental section (Fig. S1ꢀS8, see electronic
supplementary material). As shown, during metal complexation, the ligand behaves as
tridentate anionic forming two fourꢀmembered and one sixꢀmembered chelate rings
around the metal center. The stretching vibration ʋ(C=N) and the inꢀplane imidazole
deformation bands are shifted to higher wavenumbers that are consistent with the
implication of imine and imidazole nitrogen atoms in the coordination. In the ligand (L),
1H NMR spectrum, a broad singlet at δ = 12.98 ppm is observed for NH protons. A
characteristic signal of aldimine proton is found at δ = 9.61 ppm as a sharp singlet. A
multiplet at δ = 7.28ꢀ7.25 ppm, two doublets at δ = 7.18ꢀ7.17 and 7.10ꢀ7.08 ppm and
a triplet at δ = 6.98ꢀ6.94 ppm is assigned to aromatic ring protons. A sharp singlet at
δ= 3.91 ppm is assigned to the methyl protons. In [ZnL2] complex, the characteristic
signal for NH protons on complexation got deꢀshielded and shifted to the higher value of
14.51 ppm. Further, the coordination of the ligand to the metal center is evidenced by
the significant change of the aldimine signal to δ= 9.41 ppm. The aromatic ring protons
appear as multiplets between δ=7.58–6.94 ppm, the assigning of the respective peak is
difficult due to the merging of the aromatic peaks and at 6.63ꢀ6.61 observed triplet. The

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rest of the signals associated with the remaining protons appeared as expected. 13C
NMR spectra of the free ligand and the [ZnL2] compound exhibited carbon signals
supporting the 1H NMR assignments and by the proposed structures.
The EPR spectrum of [CuL2] in DMSO at LNT was recorded in the Xꢀband region (Fig
S9, see electronic supplementary material). The g factor is in respect to the standard
marker TCNE (g = 2.00277). The EPR spectrum of copper complex [CuL2] exhibited g║=
2.11 and g┴ = 2.06, values, respectively. EPR spectrum of the [CuL2] revealed axial
features (g║ > g┴ > 2.0023) and suggest a dx2ꢀy2 ground state. The measure of exchange
interaction between the copper centers in the polycrystalline compound can be given by
the geometric parameter “G” using the relation: G = (g║ ̶ 2.0023) / (g┴ ̶ 2.0023). The
exchange interaction may be negligible if G is greater than 4. A considerable exchange
interaction is indicated in the solid complex if G is less than 4.37 The value g║ > g┴
>2.0023 indicates dx–y or dz ground state are consistent with octahedral geometry. The
magnetic moment obtained for [CuL2] was 1.88 BM, consistent with values expected for
copper(II) with the spin S = 1/2.
Evaluation of Biological Activity in vivo
Tissue injury leads inflammation after a prolonged period of infection characterized by
edema, pain, tenderness and redness. These signs of inflammation are mediated by
various chemical mediators including prostaglandins (PGs) such as leukotrienes (LTs)
and hydroxyꢀeicosatetraenoic acids HETEs). Generally, these inflammatory mediators
are produced in two successive steps after incurring of any tissue injury in biological
systems. The first step involves the production of arachidonic acid by the action of
phospholipase A2 followed by the production of the individual inflammatory mediators
via the action of several enzymes as the second step. For example, generation of

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prostaglandins by action of Cyclooxygenase 1 (COX1) and cyclooxygenase 2 (COX2)
while production of leukotrienes by 5ꢀlipoxygenase and/or 12ꢀlipoxygenase enzymes.38 It
is documented that prostaglandins are responsible for vasodilation, capillary permeability
and pain during inflammatory response whereas LTs triggers capillary permeability,
chemotaxis of inflammatory mediators and extravasation of white blood cells leading to
sustenance of inflammation.39 Furthermore, these PGs are also the major mediators in
pain sensitization while the LTs are the primary mediators of inflammation
development.38 Thus all the chemical compounds or substances that inhibit inflammation,
pain and lower body temperature to the optimum, most probably show these effects by
inhibiting lipoxygenase enzyme.
Zinc (Zn) and Copper (Cu) are second and the third most important transition bioꢀ
metals after iron in all forms of the life.40,41 Both the bioꢀmetals participate in over 100
enzymes involved in metabolism, DNAꢀtranscription, stress management, immunity,
reproduction and nervous system.42 They are also essential for the expression of
important antioxidant molecules like superoxide dismutase, ceruplasmin, and
metallothionein that maintain redox homeostasis in living organisms.43 However, its
excessiveness, as well as deficiency both, have an adverse impact on the immune and
antioxidant response exacerbating any infection or inflammation of the living
organisms.44,45 Thus, the present study is aimed to investigate if the synthesized metal
[CuL2] and [ZnL2] complex have any antiꢀinflammatory, antipyretic and analgesic
properties in vivo.
Effects of [CuL2] and [ZnL2] on yeast-induced hyperthermia in mice

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Administration of [CuL2] in a dose of 100 mg/kg (but not 50mg/kg) i.p. to mice
significantly decreased body temperature a t 60, 90 and 120 min after injection (p
< 0.001, 0.001 and 0.01, respectively, n=6). Administration of [ZnL2] in a dose of 100
mg/kg (but not 50 mg/kg) i.p. to mice also significantly decreased body temperature at
60, 90 and 120 min after injection (p< 0.05 and 0.001, respectively, n=6). (Figure 1). On
the other hand, [CuL2] 100 mg/kg produced four folds the antipyretic activity of [CuL2]
50 mg/kg at 60 and 90 min while two folds at 120 min. However, statistical analysis of
these results revealed that they are not significantly different (Table 1).
Table 1: Percentage inhibition of hyperpyrexia, algesia, and inflammation induced by
each dose of the compounds relative to its zero time (pretreatment readings i.e. control).
Paracetamol was used as a standard (positive control) for both antipyretic and analgesic
experiments while diclofenac was used as a standard for the antiꢀinflammatory
experiment.
Test
Time
(min)
% inhibition of pyresis, algesia, and inflammation
Standard Predrug [CuL2] [CuL2] [ZnL2] [ZnL2]
(control)
50
100
50
100mg/kg
mg/kg mg/kg mg/kg
Antipyretic
activity
60
90
120
8.6
10.5
10.2
0
0
0
1.1
1
1.2
4.2
4
2.9
1.9
1.9
0.8
2.8
3.1
2.9
Analgesic
activity
30
60
120
51.8
62.4
65.9
0
0
0
13.1
23.2
26.4
13.0
36.8
25.9
11.9
29.8
26.3
37.6
53.5
48.5
Antiꢀ
180
65.4
0
36.3
56.1
30.6
46
inflammatory
activity
The compound [ZnL2] produced two folds the activity in 100 mg/kg then 50 mg/kg, but
none of these differences were statistically significant. Comparison of all the doses for

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[CuL2] with all the doses for [ZnL2] revealed that [CuL2] 100 mg/kg appears to be more
efficient in all the other treatments as antipyretic. However, the results are not
statistically significant.
Figure 1. Antipyretic effects of [CuL2] and [ZnL2] complexes at 60 min, 90 min, and 120
min after yeastꢀinduced hyperpyrexia. Rectal temperature after 60, 90 and 120 min were
compared to the rectal temperature before the administration of the compounds (preꢀ
drug temp.) i.e. [CuL2] and [ZnL2] in doses of 50 and 100 mg/kg i.p. Results have been
expressed in mean ±SD. ANOVA with Dunnett's test as post Hoc test was used to check
for any significance (n=6)
Effects of [CuL2] and [ZnL2] on hot plate-induced analgesia:
The synthesized molecule [CuL2] reduced analgesic activity significantly only after 120
min of the administration (p<0.05, n=6). Whereas, [ZnL2] (injected in doses of 50 and
100 mg/kg, i.p.) produced doseꢀ dependent analgesia at 30, 60 and 120 min after
injection (p<0.001, n=6). In the results, Figure 2 shows a comparison of different doses
of [CuL2] with those of [ZnL2]. The current findings revealed that [ZnL2] 100 mg/kg is
significantly more potent analgesic than 50 mg/kg and more potent than CuL2 all doses

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(p<0.001, table 1). [CuL2] 100 mg/kg in turn is significantly more potent than 50 mg/kg at
60 min (p<0.01, table 1).
Figure 2. Analgesic effect of [CuL2] and [ZnL2] complexes on hot plateꢀinduced algesia.
Mice reaction time to the hot plate at 30, 60 and 120 min after compounds’ treatment
was compared with preꢀdrug reaction time (reaction time prior to the administration of
the compounds). Results are plotted as mean ±SD. ANOVA with Dunnett's test as post
Hoc test was used to check for any significance (n=6).
Effects of [CuL2] and [ZnL2] on carrageenan-induced rat paw edema:
Administration of the proposed metal complex [CuL2] in doses of 50 and 100 mg/kg
produced a doseꢀdependent decrease in rat paw edema respectively (p<0.001,
p<0.0001, n=6). On the other hand, [ZnL2] produced a doseꢀdependent decrease in
paw edema when injected in doses of 50 and 100 mg/kg i.p. (p<0.05 and p<0.0001,
respectively, n=6). Hence, the antiꢀinflammatory effect of CuL2 at 100 mg/kg was

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significantly higher than that of 50 mg/kg and also as compared to both doses of ZnL2
(p<0.01, Table 1).
However, [ZnL2] showed stronger analgesic potential than [CuL2] (Figure 2) whereas
[CuL2] tends to be more potent antiꢀinflammatory than [ZnL2] (Figure 3). Hitherto,
there is no apparent statistically significant difference between [CuL2] and [ZnL2] on
the antipyretic activity. Intriguingly, [CuL2] at a dose of 100 mg/kg demonstrated the
antipyretic and antiꢀinflammatory activities but not an analgesic activity which might be
due to inhibition of 5ꢀlipoxygenase and/or 12ꢀlipoxygenase enzymes but might not
involve inhibition of cyclooxygenase enzyme due to lack of analgesic activity.
Figure 3. Effects of compounds on the carrageenanꢀinduced increase in paw edema in
rats. The increase in paw edema 3h after carrageenan administration in the compoundsꢀ
treated groups was compared with the carrageenan control group (not treated with
compounds). ANOVA test with Dunnett's test as post Hoc test was used to Check for
any significance, n=6.

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On the other hand, [ZnL2] (at doses of 50 and 100 mg/kg) possessed antipyretic,
analgesic and antiꢀinflammatory activities. These effects might be attributed to inhibition
of lipoxygenase enzyme with subsequent inhibition of arachidonic acid production or
inhibition of 5ꢀlipoxygenase, 12ꢀlipoxygenase, and cyclooxygenases leading to inhibition
of prostaglandins and leukotrienes.
Experimental Section
Material and Methods
2ꢀAminobenzimidazole, oꢀvanillin, Cu(NO3)2—3H2O, Zn(NO3)2—6H2O, Acetic acid, All
the solvents were used as purchased from the company SigmaꢀAldrich without further
purification. Infrared spectra were recorded as KBr pellets, using a Shimadzu IR
1
Affinityꢀ1 spectrometer with a resolution of 4 cm‾ . Elemental analysis (C, H, N) were
performed on a PerkinElmer 2400 Series II CHNS/O system. NMR spectra were
recorded at JEOLꢀ ECPꢀ400 spectrometer. The EPR spectrum of the copper complex
was acquired on a Varian E 112 spectrometer using Xꢀband frequency (9.1 GHz) at
liquid nitrogen temperature in the solid state. Circular dichroic spectra of both
complexes using Circular Dichroism (CD) Spectrometer with Stop FlowꢀApplied
PhotoPhysics Chirascan and JASCO Pꢀ1020 (Jasco International Co., Ltd, Tokyo,
Japan) digital polarimeter with a 10 cm optical length cell at 25 °C used for calculating
specific rotation. Ultraꢀperformance liquid chromatography (UPLC) experiments were
performed on a gradient system of Agilent Technologies (1290 Infinity) equipped with
infinity binary pump (G4220A), autosampler (G4226A), Thermostat column
compartment (G1316c), fraction collector (G1364C) and a photodiode array detector
(G4212A). Chemꢀstation software programs the above configuration of UPLC. The

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separations were performed on Eclipse C18 column (Agilent) 4.6 × 100 mm, 3.5 ꢂm
by using a gradient method.
All the animal based experiments were performed in compliance with the relevant laws
and institutional guidelines and have been approved and permitted by the by the
ethics committee of the College of Pharmacy, King Saud University, Riyadh, KSA.
Synthesis of Schiff base (L)
Solutions of equimolar amounts of 2ꢀaminobenzimidazole (0.133 g, 1.0 mmol) and
vanillin (0.152 g, 1.0 mmol) absolute ethanol with 2ꢀ3 drops of acetic acid were
refluxed for 4 h and the product obtained was filtered off and washed with hexane (3
x 5ml) and diethyl ether (3 x 5ml). Recrystallized from ethanol to obtain deep orange
color crystalline product. Yield: 0.188 g, 66 %; Anal. calc. for C15H13N3O2 (267.1):
C, 67.40; H, 4.90; N, 15.72, found: C, 67.36; H, 4.89; N, 15.70; HRMS m/z {in CHCl3,
observed (calcd)} for C15H13N3O2 + H+: 268.1, (268.1). FT IR (KBr pellets) cm–1:
1
3305, 1600, 1515, 1462, 1252, 735. H NMR (400MHz, DMSOꢀd6, ppm): 12.95 (br,
NH, 1H), 9.61 (s, N=CH, 1H), 7.28ꢀ7.25 (m, ArꢀH, 2H), 7.18ꢀ7.16 (d, ArꢀH, 2H), 7.09ꢀ
13
7.07 (d, ArꢀH, 2H), 6.98ꢀ6.94 (t, ArꢀH, 1H), 3.91(s, O(CH3), 3H), 2.16 (s, OH, 1H). C
NMR (100MHz, DMSOꢀd6, ppm): 167.6 (characteristic peak of Schiff base, N=CH),
151.6, 125.5, 123.2, 119.7, 118.9, 116.4 (ArꢀCs), 56.4 (OCH3).
Synthesis of copper(II) complex [CuL2]
To a methanolic (10 mL) solution containing Schiff base (267.1 mg, 1.0 mmol) was
added slowly methanolic solution (10 mL) of Cu(NO3)2ꢁ 3H2O (120.5 mg, 0.5 mmol)
and stirred for 5h. The precipitate filtered off and washed with hexane (3 x 5ml)
diethylether (3 x 5ml) and CHCl3 (3 x 5ml) and dried. Yield: 215 mg, 55%. Anal.
Calcd. for (C30H24N6O4Cu) (595.1): C, 60.45; H, 4.06; N, 14.10. found: C, 60.39; H,

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4.04; N, 14.07. HRMS m/z {in DMSO, observed (calcd)} for C30H24N6O4Cu: 595.1,
(595.1). FTꢀIR (KBr pellets) cm–1: 1609, 1535, 1455, 1254, 744. ꢂeff = 1.88 BM.
Synthesis of zinc (II) complex [ZnL2]
Similar procedure was adopted as for copper complex, using Zn(NO3)2ꢁ6H2O (148.7
mg, 0.5 mmol) Yield: 215 mg, 55%. Anal. Calcd. for (C30H24N6O4Zn) (597.1): C,
60.26; H, 4.05; N, 14.05. found: C, 60.19; H, 4.04; N, 14.03. HRMS, m/z {in DMSO,
observed (calcd)} for C30H24N6O4Zn + H+: 597.2, (597.9). FT IR (KBr pellets) cm–1:
1
1608, 1535, 1449, 1256, 741. H NMR (400 MHz, DMSOꢀd6, ppm): 14.51 (br, NH,
2H), 9.40 (s, N=CH, 2H), 7.58ꢀ7.52 (dd, ArꢀH, 4H), 7.19ꢀ6.94 (m, ArꢀH, 4H), 7.08ꢀ6.94
(t, ArꢀH, 4H), 6.65ꢀ6.61 (t, ArꢀH, 2H), 3.87 (s, O(CH3), 6H). 13C NMR (100 MHz,
DMSOꢀd6, ppm): 165.7 (characteristic peak of Schiff base, N=CH), 153.8, 150.5,
148.0, 142.6, 134.14, 122.43, 122.4, 123.5, 119.4, 119.2, 116.5, 111.2 (ArꢀCs), 55.9
(OCH3).
In Vivo Biological Studies
Antipyretic test (yeast induced hyperpyrexia in mice)
Hyperpyrexia was induced in mice by subcutaneous injection of (20 % Aqueous
suspension of brewer's yeast) of 20 ml/kg body weight (6 animals in each group) in
the back below the nape of the neck (Loux et al., 1972).46 The animals then fasted for
the duration of the experiment (approximately 26 h); water was made available ad lib.
Control temperatures were taken 24 h after the yeast injection to determine the
pyretic response to yeast. Rectal temperatures taken 1h before drug administration in
fevered animals served as a pre–drug control.
Analgesic Activity (Acetic acid induced writhing test i.e. algesia)

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Acetic acidꢀinduced writhing in the miceꢀThe test was carried out using the technique
of Siegmund et al.,47 as modified by Koster et al..48 Both the complexes (50 and 100
mg/kg body weight) were administered orally, to 16 h fasten mice, and divided into
groups of 6 animals each. One hour after treatment, the mice were injected
intraperitoneally with 0.2 ml of 3% acetic acid solution to induce the characteristic
writhing. The number of writhings occurring between 5 and 15 min after the acetic acid
injection in control and treated animals was recorded. The responses of compoundsꢀ
treated groups were compared with those of animals receiving in diclofenac sodium
(as a standard drug), 4 mg/kg, as well as with the control group.
Anti-inflammatory activity (Carrageenan-induced paw edema in rats)
Pedal inflammation in albino rats (8 to10 weeks old) of either sex weighing180ꢀ20 0 g
was carried out following the method described by Winter et al..49 An injection was
made of 0.0 5ml of 1% carrageenan sodium salt (BDH) into the right hind foot of each
rat under the plant araponeurosis. The test groups of rats were treated orally with 50
and 100 mg/kg 1h before the carrageenan injection. At the same time, the control
group was given 5 ml/kg of normal saline and the reference group was given 100
mg/kg of an aqueous solution of oxyphenbutazone. The measurements of foot
volume were done by the displacement technique using a plethysmometer (Apelex,
France) after +3 h after the injection of carrageenan. The inhibitory activity was
calculated according to the following formula:
Percent inhibition = 100 [1ꢀ(aꢀx)/(bꢀy)]
Where 'b' is the mean paw volume of control rats after carrageenan injection and 'y'
before the injection; whereas 'x' is the mean paw volume of treated rats before
injection and 'a' is the paw volume mean after carrageenan injection.

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Statistical analysis
Results are expressed as mean ±SEM. Oneꢀway ANOVA was used for a comparison
test of significant differences among groups followed by Dunnet’s multiple comparison
postꢀ tests. Level of significance (P<0.05) was taken for each test.
Conclusion
In this work, we have designed and synthesized new Cu(II) and Zn(II) complexes of 2ꢀ
[(1Hꢀ Benzoimidazolꢀ2ꢀylimino)ꢀmethyl]ꢀ6ꢀmethoxyꢀphenol Schiff base ligand derived
from the condensation of 2ꢀaminobenzimidazole and oꢀvanillin. Conventional
measurements characterized the ligand and complexes. The ligand behaves as a
neutral, tridentate (˗ONN′˗) donor yielding octahedral geometries of the metal
complexes. These synthesized complexes are achiral/nonꢀstereoꢀselective in nature.
Therefore, these synthesize NSAIDs will not cause side effects. The in vivo antiꢀ
inflammatory, analgesic and antipyretic activities of both the complexes are potent.
These studies suggested that a synergistic combination of ligand and metal ion was
critical in the design of potential NSAIDs. However the precise molecular mechanism(s)
of the antiꢀinflammatory, analgesic and antipyretic effects discovered in the present
study warrants further investigations.
Acknowledgement
The author would like to extend their sincere appreciation to the Deanship of Scientific
Research at King Saud University, Riyadh, Saudi Arabia for its funding this research
group No. RGPꢀ150.
References

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DOI: 10.1039/C5RA25071D
1 L FerreroꢀMiliani, O. H. Nielsen, P. S. Andersen and S. E. Girardin, Clin. Exp.
Immunol., 2007, 147, 227.
2 H. Ungefroren, S. Sebens, D. Seidl, H. Lehnert and R. Hass, Cell Communicat.
Signal., 2011, 9, 18.
3 K. C. S. Achar, K. M. Hosamani and H. R. Seetharamareddy, Eur. J. Med.
Chem., 2010, 45, 2048.
4 S. Kankala, R. K. Kankala, P. Gundepaka, N. Thota, S. Nerella and M. R.
Gangula, Bioorg Med. Chem. Lett., 2013, 23, 1306.
5 J.R. Vane, Nature, 1971, 231, 232.
6 J. Vane and R. Botting, Eds.; Clinical Significance and Potential of Selective
COXꢀ2 Inhibitors; Williams Harvey Press, 1998.
7 J. S. Carter, Expert Opin. Ther. Pat., 2000, 10, 1011.
8 P. Vicini, M. Incerti, L. Amoretti, V. Ballabeni, M. Ognolini and E. Barocelli, Il
Farmaco, 2002, 57, 363.
9 N. Terioglu and A. Gursoy, Eur. J. Med. Chem., 2003, 38, 781.
10 A. K. Gadad, M.N. Noolvi and R.K. Karpoormath, Bioorg. Med. Chem. 2004, 12,
5651.
11 A. Andreani, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi, Rambaldi, G. Linaz,
C. Bergamini and G. Farruggia, J. Med. Chem., 2005, 48, 3085.
12 A. Andreani, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi, M. Recanatini and V.
Garaliene, Bioorg. Med. Chem., 2000, 8, 2359.
13 Y. Bansal and O. Silakari, Bioorg. Med. Chem., 2012, 20, 6208.
14 L. Keurulainen, O. Salin, A. Siiskonen, J. M. Kern, J. Alvesalo, P. Kiuru, M.
Maass, J. YliꢀKauhaluoma and P. Vuorela, J. Med. Chem., 2010, 53, 7664.
15 A. Andreani, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi, M. Recanatini and V.
Garaliene, Bioorg. Med. Chem., 2000, 8, 2359.
16 D. J. Sheehan, C. A. Hitchcock and C. M. Sibley, Clin. Microbiol. Rev., 1999, 12,
40.
17 P. N. Preston, In the Chemistry of Heterocyclic Compounds, Benzimidazoles, and
Congeneric Tricyclic Compounds. John Wiley & Son, New York, 1980, p. 40.
18 D. A. Horton, G. T. Bourne and M. L. Smyth, Chem. Rev., 2003, 103, 893.

Page 19 of 22
RSC Advances
View Article Online
DOI: 10.1039/C5RA25071D
19 S. Y. Lin, Y. Isome, E. Stewart, J. F. Liu, D. Yohannes and Y. Libing, Tet. Lett.,
2006, 47, 2883.
20 R. K. Arora, N. Kaur, Y. Bansal and G. Bansal, Acta Pharmaceutica Sinica B,
2014, 4, 368.
21 M. Gabaa, P. Gabaa, D. Uppal, N. Dhingra, M. S. Bahia, O. Silakari and C.
Mohan, Acta Pharmaceutica Sinica B, 2015, 5, 337.
22 P. L. Beaulieu, M. Bos, Y. Bousquet, G. Fazal, J. Gauthier, J. Gillard, S. Goulet,
S. LaPlante, M.A. Poupart, S. Lefebvre, G. McKercher, C. Pellerin, V. Austel and
G. Kukolj, Bioorg. Med. Chem. Lett., 2004, 14, 119.
23 A. Kajal, S. Bala, S. Kamboj, N. Sharma and V. Saini, J. Catalysts, 2013, 2013,
ID 893512, 14 pages.
24 J. A. Makawana, J. Sun, and H.ꢀL. Zhu, Bioorg. Med.Chem. Lett., 2013, 23, 6264.
25 K. Sztanke, A. Maziarka, A. Osinka and M. Sztanke, Bioorg. Med. Chem., 2013,
21, 3648.
26 A. Kamal, G. B. Kumar, V. L. Nayak, V. S. Reddy, A. B. Shaik, Rajendera and
M. K. Reddy, Med. Chem. Commun., 2015, 6, 606.
27 S. Tabassum, A. Asim, R. A. Khan, F. Arjmand, D. Rajakumar, P. Balaji and M.
A. Akbarsha RSC Adv., 2015, 5, 47439.
28 T. W. Hambley, Science, 2007, 318, 1392.
29 M. J. Bloemink, J. Reedijk, in H. Sigel, Sigel (Eds.), A. Metal Ions in Biological
Systems, vol. 32, Marcel Dekker, New York, 1996, 641.
30 J. E. Weder, T. W. Hambley, B. J. Kennedy, P. A. Lay, D. MacLachlan, R.
Bramley, C. D. Delfs, K. S. Murray, B. Moubaraki, B. Warwick, J. R. Biffin and H.
L. Regtop, Inorg. Chem., 1999, 38, 1736.
31 R. S. Hoonur, B. R. Patil, D. S. Badiger, R. S. Vadavi, K. B. Gudasi, P. R.
Dandawate, M. M. Ghaisas, S. B. Padhye and M. Nethaji, Eur. J. Med. Chem.,
2010, 45, 2277ꢀ2282.
32 (a) M. R. Arveiller, V. Revelant, D. P. Huy, L. Maman, J. Fontagne, J. R. J.
Sorenson and J. P. Giroud, Agents Actions, 1990, 31, 65.; (b) M.
Konstandinidou, A. Kourounakis, M. Yiangou, L. Hadjipetrou, D. Kovalaꢀ
Demertzi, S. Hadjikakou and M. Demertzis, J. Inorg. Biochem., 1998, 70, 63.

RSC Advances
Page 20 of 22
View Article Online
DOI: 10.1039/C5RA25071D
33 M. C. Linder, Biochemistry of Copper. Plenum Press: New York, 1991.
34 C. Marzano, M. Pellei, F. Tisato and C. Santini, Anti-cancer Agents in Med.
Chem., 2009, 9, 185.
35 (a) E. D. Rosenstein and J. R. Caldwell, Rheum. Dis. Clin. North. Am., 1999,
25, 929; (b) O. A. ElꢀGammal, E. A. Elmorsy, and Y. E. Sherif, Spectrochim.
Acta A, 2014, 120, 332.
36 Y. Fujimoto, H. Nishida, K. Uchida, Y. Tsuji, S. Sakuma and T. Fujita, J. Clin.
Biochem. Nutr., 1993, 14, 45.
37 (a) M. V. Angelusiu, G. L. Almajan, T. Rosu, M. Negoiu, E. R. Almajan and
J. Roy, Eur. J. Med. Chem., 2009, 44, 3323; (b) J. E. Weder, C. T. Dillon, T. W.
Hambley, B. J. Kennedy, P. A. Lay, J. R. Biffin, H. L. Regtop and N. M. Davies,
Coord. Chem. Rev. 2002, 232, 95; (c) A. T. Florence and D. Attwood,
Physiochemical Principles of Pharmacy, Macmillan, London, 1998.
38 (a) P. F. Rapheal, E. Manoj and M. R. P. Kurup, Polyhedron, 2007, 26, 818; (b)
B. J. Hathaway Coord. Chem. Rev., 1970, 5, 143ꢀ207; (c) J. W. Akitt, N. N.
Greenwood and B. L. Khandelwal, J. Chem. Soc., Dalton Trans., 1972, 1226.
39 (a) H. Y. Chung, H. J. Kim, K. W. Kim, J. S. Choi and B. Y. Yu, Microsc. Res.
Tech., 2002, 59, 264; (b) C. J. Smith, C. M. Koboldt, J. Muhammad, B. S.
Zweifel, A. Shaffer, J. J. Talley, J. L. Masferrer, K. Seibert and P. Isakson, Proc.
Natl. Acad. Sci., 1998, 95, 13313; (c) B. Samuelsson, Science, 1983, 220, 568.
40 R. Milanino, U. Moretti, M. Concari, M. Marrella, G.P. Velo, Agents Actions 1988,
24, 365.
41 R. Milanino, A. Frigo, L.M. Bambara, M. Marrella, U. Moretti, M. Pasqualicchio,
Clin. Exp Rheum. 1993, 11, 271.
42 (a) S. Tuncer, A. Kamanli, E. Akcil, G. O. Kavas, B. Seckin, M. B. Atay, Biol.
Trace Elem. Res. 1999, 68, 137; (b) A. H. Shankar, A. S. Prasad, Am. J. Clin.
Nutr., 1998, 68, 447S.
43 S. Chibber, I. Hassan, M. Farhan, I. Naseem, J Photochem Photobiol B. 2011,
104(3), 387.
44 H. Ebaid, I. Hassan, S. Bashandy, N. A. Taha, A. Mahmood, S. Alomar, I.
Alhazza, A. Mashaly, A. Rady. Cent. Eur. J. Immunol. 2014, 39(4), 441.

Page 21 of 22
RSC Advances
View Article Online
DOI: 10.1039/C5RA25071D
45 P. I. Oteiza, M. S. Clegg, M. P. Zago, C. L. Keen, Free Radic. Biol. Med., 2000,
28, 1091.
46 J. J. Loux, P. D. DePalma and S. L. Yankell, Toxicol Appld Pharmacol, 1972, 22,
672.
47 E. Siegmund, R. Cadmus, G. A. Lu, Proc. Soc. Exp. Biol. Med., 1957, 95, 729.
48 R. Koster, M. Anderson and E. J. DeBeer, Fed Proc. 1959, 18, 412.
49 C. A. Winter, E. A. Risley and G. W. Nuss, Proc. Soc. Exp. Biol. Med., 1962, 111,
544.

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In vivo assessment of newly synthesized achiral copper(II) and
zinc(II) complexes of benzimidazole derived scaffold as a potential
analgesic, antipyretic and anti-inflammatory
Mohamed F. Al Ajmi,a Afzal Hussain,a Ali Alsalme,b and Rais Ahmad Khanb*
a
Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O.
Box 2457, Riyadh 11451, KSA
b
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh11451, KSA.
Graphical Abstract
Anti-pyretic, Analgesic and Anti-inflammatory
Activity (NSAIDs)
ZnL2 / CuL2 dose
* Author for Correspondence:
Department of Chemistry, College of Science, King Saud University, P.O. Box
2455, Riyadh 11451, KSA. Mobile +966 536745404, Email: raischem@gmail.com
(Khan, RA)