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

Abstract

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 characterized using various spectroscopic techniques, viz, IR, ¹H and ¹³C NMR, EPR, HRMS, and elemental analysis and purity analysis using UPLC studies. Both the complexes are non-electrolytic by nature. The newly synthesized compounds were screened for acetic acid-induced analgesic and yeast-induced antipyretic activities in mice and carrageenan-induced paw edema in rats (anti-inflammatory). The results showed that the [CuL₂] compound (at 100 mg kg⁻¹ b.w) possessed potent anti-inflammatory activity whereas [ZnL₂] (at 50 mg kg⁻¹ and 100 mg kg⁻¹ b.w) exhibited significant analgesic activity when compared with standard drugs. Both the complexes have apparently moderate and nearly akin antipyretic activity.

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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 types: acute and chronic. Acute inflammation can lead to progressive tissue damage by the noxious stimulus (e.g. pathogens and chemical irritants). Meanwhile, chronic inflammation can lead to a multitude of diseases, viz, hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, osteoarthritis, gout, Alzheimer’s disease and obesity and can even cause cancer (e.g., gallbladder carcinoma).^2,3 Therefore, the treatment of inflammation is of paramount importance. Non-steroidal anti-inflammatory drugs (NSAIDs), viz., indomethacin, ibuprofen, and naproxen have frequently been used for the curing of the first streak of various chronic inflammatory diseases.⁴ The classical NSAIDs demonstrate their action by restricting the biosynthesis of prostaglandin. Some of them are pro-inflammatory, which is brought about by inhibiting the rate-limiting cyclooxygenase (COX) enzyme in the inflammatory cascade,⁵ and are responsible for the transformation of arachidonic acid to prostaglandins.^6,7

Among NSAIDS, imidazole and fused imidazole with six-membered rings⁸ possess a central position due to their significant potential as therapeutics in clinical applications.^9–12 One of the most privileged classes of heterocyclic molecules in medicinal chemistry are benzimidazoles, encompassing a diverse range of biological activity.¹³ Benzimidazole derivatives displayed potential antimicrobial, anti-inflammatory, antituberculosis, antioxidant, antihypertensive, and anticancer activities.^14–21 Benzimidazole-5-carboxylic acid and its derivatives have ascended as potent agents for treating hepatitis C virus infections also.²² Various derivatives of benzimidazole are used as drugs, a few amongst them are omeprazole (proton pump inhibitor), pimobendan (ionodilator), albendazole (inhibitor of Encephalitozoon Intestinal infection in AIDS patients) and mebendazole. The presence of the benzimidazole ring provides an active structural motif, which possesses peculiar structural features, viz., steric and electronic, as well as bringing planarity to the drug candidate, which makes the molecule active on different targets via hydrogen bonding and π–π* stacking. Also, benzimidazoles have the advantage of being easily functionalized for synthesis purposes and lead to quite stable and biologically privileged molecules.

Schiff base compounds are privileged ligands in medicinal chemistry and have proven therapeutic potential, due to –CH=N– functionality giving rise to strong activity and playing an essential role.²³ The remarkable biological activities include antitumor, antibacterial, antifungal, antimalarial, antiviral, antioxidant, anti-inflammatory, analgesic, anticonvulsant, antiglycation, antihypertensive, antidepressant and lipid lowering properties.^24–27

The design and synthesis of metal complexes with a biologically active scaffold is 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 pre-requisite for the functioning of various enzymes and proteins, which are involved in respiration, energy metabolism, and DNA synthesis, especially cytochrome oxidase, superoxide dismutase (SOD), ascorbate oxidase and tyrosinase.³⁴ Moreover, Cu(II) and Zn(II) are co-factors in metabolic processes involved in collagen and bone metabolism via articular/connective tissue and the immune system³⁴ and play a crucial role in PG synthesis.³⁵ In particular, copper carboxylate drugs belong to an important class of anti-inflammatory and anticancer agents, and some of them are commercially available drugs.³⁶

Thus, in the present work, we have described the synthesis and characterization of a 2-aminobenzimidazole and o-vanillin derived Schiff base ligand and its achiral copper(II), [CuL₂], and zinc(II), [ZnL₂], complexes. The above-mentioned plentiful pharmacological activities of benzimidazoles and their derivatives encouraged us to study the in vivo analgesic, antipyretic and anti-inflammatory activities of the synthesized complexes.

Results and discussion

Chemistry

A heterocyclic Schiff base ligand, viz, (2-[(1H-benzoimidazol-2-ylimino)-methyl]-6-methoxy-phenol) has been synthesized with high purity and acceptable yield. The orange-yellow compound obtained is stable in air and moisture and was characterized using elemental analysis, HRMS (LC-QTOF) spectrometry and IR and NMR (¹H and ¹³C) spectroscopy. Reaction of the Schiff base, viz, 2-[(1H-benzoimidazol-2-ylimino)-methyl]-6-methoxy-phenol ligand (L) with M(NO₃)₂·n(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 anionically with deprotonation of a phenolic (OH) proton and tridentate with an [–ONN′–] donor set (Scheme 1).

Scheme 1 Schematic representation of the synthesized ligand (L) and its [CuL₂] and [ZnL₂] complexes.

The achiral nature of the complexes was studied first with a polarimeter, and the specific rotation [α]²⁵ value (1 × 10⁻³ M solution of the compound in DMSO) was found to be zero. To ascertain the achirality, we also checked using circular dichroism, which confirmed the polarimeter results by giving no spectra. The stability of the complexes was evaluated using UV-Vis spectral analysis at different times in a mixture containing H₂O/DMSO in 95 : 5 ratio and the absorbance, as well as the wavelength, remained unaffected, even after 12 h. Thus, the results indicate that the complexes are quite stable in solution. Both complexes were characterized using analytical and spectroscopic techniques and the purity of the ligand and the complexes were checked using a UPLC technique, which confirmed the purity of the synthesized materials to be ∼96 ± 2% with retention times of 7.827 min [L], 5.633 min [CuL₂] and 6.747 min [ZnL₂]. Further, the HR-MS exhibited a 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.

The significant IR vibrational bands and the ¹H chemical shift values of the free ligand and its complexes are listed in the experimental section (Fig. S1–S8, see ESI†). As shown, during metal complexation, the ligand behaves as a tridentate anion, forming two four-membered and one six-membered chelate rings around the metal center. The stretching vibration ν(C=N) and the in-plane 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 an aldimine proton is found at δ = 9.61 ppm as a sharp singlet. A multiplet at δ = 7.28–7.25 ppm, two doublets at δ = 7.18–7.17 and 7.10–7.08 ppm and a triplet at δ = 6.98–6.94 ppm are assigned to aromatic ring protons. A sharp singlet at δ = 3.91 ppm is assigned to the methyl protons. In the [ZnL₂] complex, the characteristic signal for NH protons on complexation was de-shielded and shifted to a higher value of 14.51 ppm. Further, the coordination of the ligand to the metal center is evidenced by the significant change in 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 peaks is difficult due to the merging of the aromatic peaks and the observed triplet at δ = 6.63–6.61 ppm. The 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 the proposed structures.

The EPR spectrum of [CuL₂] in DMSO at LNT was recorded in the X-band region (Fig. S9, see ESI†). 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. The EPR spectrum of [CuL₂] revealed axial features (g_∥ > g_⊥ > 2.0023) and suggests a dx² − y² ground state. The measure of the 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 that the d_x−y or d_z ground states are consistent with octahedral geometry. The magnetic moment obtained for [CuL₂] was 1.88 BM, consistent with values expected for copper(II) with spin S = 1/2.

Evaluation of biological activity in vivo

Tissue injury leads to 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 hydroxy-eicosatetraenoic acids HETEs). Generally, these inflammatory mediators are produced in two successive steps after incurrence 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 individual inflammatory mediators via the action of several enzymes as the second step. For example, prostaglandins are generated by the actions of cyclooxygenase 1 (COX1) and cyclooxygenase 2 (COX2) while leukotrienes are produced by those of 5-lipoxygenase and/or 12-lipoxygenase enzymes.³⁸ It is documented that prostaglandins are responsible for vasodilation, capillary permeability and pain during the inflammatory response whereas LTs trigger capillary permeability, chemotaxis of inflammatory mediators and extravasation of white blood cells leading to the 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 and pain, and lower body temperature to the optimum, most probably show these effects by inhibiting lipoxygenase enzymes.

Zinc (Zn) and copper (Cu) are the second and third most important transition bio-metals after iron in all forms of life.^40,41 Both of the bio-metals participate in the activity of over 100 enzymes involved in metabolism, DNA-transcription, stress management, immunity, reproduction and the 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, both its excessiveness, as well as deficiency, have an adverse impact on the immune and antioxidant response, exacerbating any infection or inflammation of living organisms.^44,45 Thus, the present study aims to investigate if the synthesized metal [CuL₂] and [ZnL₂] complexes have any anti-inflammatory, antipyretic and analgesic properties in vivo.

Effects of [CuL₂] and [ZnL₂] on yeast-induced hyperthermia in mice

Administration of [CuL₂] in a dose of 100 mg kg⁻¹ (but not 50 mg kg⁻¹) i.p. to mice significantly decreased body temperature at 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⁻¹ (but not 50 mg kg⁻¹) i.p. to mice also significantly decreased body temperature at 60, 90 and 120 min after injection (p < 0.05, 0.05 and 0.001, respectively, n = 6) (Fig. 1). On the other hand, [CuL₂] at 100 mg kg⁻¹ produced four fold the antipyretic activity of [CuL₂] at 50 mg kg⁻¹ at 60 and 90 min, while it was two fold at 120 min. However, statistical analysis of these results revealed that they are not significantly different (Table 1).

Fig. 1 Antipyretic effects of [CuL₂] and [ZnL₂] complexes at 60 min, 90 min, and 120 min after yeast-induced hyperpyrexia. Rectal temperatures after 60, 90 and 120 min were compared to the rectal temperature before the administration of the compounds (pre-drug temp.), i.e. [CuL₂] and [ZnL₂] in doses of 50 and 100 mg kg⁻¹ i.p. Results have been expressed as the mean ± SD. ANOVA with Dunnett’s test as a post hoc test was used to check for any significance (n = 6).

Table 1 Percentage inhibition of hyperpyrexia, algesia, and inflammation induced by each dose of the compounds relative to the 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 anti-inflammatory experiment

Test	Time (min)	% inhibition of pyresis, algesia, and inflammation
		Standard	Predrug (control)	[CuL2] 50 mg kg^−1	[CuL2] 100 mg kg^−1	[ZnL2] 50 mg kg^−1	[ZnL2] 100 mg kg^−1
Antipyretic activity	60	8.6	0	1.1	4.2	1.9	2.8
	90	10.5	0	1	4	1.9	3.1
	120	10.2	0	1.2	2.9	0.8	2.9
Analgesic activity	30	51.8	0	13.1	13.0	11.9	37.6
	60	62.4	0	23.2	36.8	29.8	53.5
	120	65.9	0	26.4	25.9	26.3	48.5
Anti-inflammatory activity	180	65.4	0	36.3	56.1	30.6	46

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The compound [ZnL₂] produced two fold the activity at 100 mg kg⁻¹ than at 50 mg kg⁻¹, but none of these differences were statistically significant. Comparison of all the doses for [CuL₂] with all the doses for [ZnL₂] revealed that [CuL₂] at 100 mg kg⁻¹ appears to be more efficient than all the other treatments as an antipyretic. However, the results are not statistically significant.

Effects of [CuL₂] and [ZnL₂] on hot plate-induced algesia

The synthesized molecule [CuL₂] reduced algesia significantly only after 120 min of the administration (p < 0.05, n = 6), whereas [ZnL₂] (injected in doses of 50 and 100 mg kg⁻¹, i.p.) produced dose-dependent analgesia at 30, 60 and 120 min after injection (p < 0.001, n = 6). In the results, Fig. 2 shows a comparison of different doses of [CuL₂] with those of [ZnL₂]. The current findings revealed that [ZnL₂] at 100 mg kg⁻¹ is a significantly more potent analgesic than at 50 mg kg⁻¹ and is more potent than CuL₂ at all doses (p < 0.001, Table 1). [CuL₂] at 100 mg kg⁻¹ in turn is significantly more potent than at 50 mg kg⁻¹ at 60 min (p < 0.01, Table 1).

Fig. 2 Analgesic effect of [CuL₂] and [ZnL₂] complexes on hot plate-induced algesia. Reaction time of mice to the hot plate at 30, 60 and 120 min after compounds’ treatment was compared with pre-drug reaction time (reaction time prior to the administration of the compounds). Results are plotted as mean ± SD. ANOVA with Dunnett’s test as a 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⁻¹ produced a dose-dependent decrease in rat paw edema respectively (p < 0.001 and p < 0.0001, n = 6). On the other hand, [ZnL₂] produced a dose-dependent decrease in paw edema when injected in doses of 50 and 100 mg kg⁻¹ i.p. (p < 0.05 and p < 0.0001, respectively, n = 6). Hence, the anti-inflammatory effect of CuL₂ at 100 mg kg⁻¹ was significantly higher than that at 50 mg kg⁻¹ and also when compared to both doses of ZnL₂ (p < 0.01, Table 1).

However, [ZnL₂] showed stronger analgesic potential than [CuL₂] (Fig. 2), whereas [CuL₂] tends to be a more potent anti-inflammatory than [ZnL₂] (Fig. 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⁻¹ demonstrated the antipyretic and anti-inflammatory activities but not an analgesic activity, which might be due to inhibition of 5-lipoxygenase and/or 12-lipoxygenase enzymes but not inhibition of the cyclooxygenase enzyme due to a lack of analgesic activity.

Fig. 3 Effects of compounds on the carrageenan-induced increase in paw edema in rats. The increase in paw edema 3 h after carrageenan administration in the compound-treated groups was compared with the carrageenan control group (not treated with compounds). ANOVA test with Dunnett’s test as a post hoc test was used to check for any significance, n = 6.

On the other hand, [ZnL₂] (at doses of 50 and 100 mg kg⁻¹) possessed antipyretic, analgesic and anti-inflammatory activities. These effects might be attributed to inhibition of the lipoxygenase enzyme with subsequent inhibition of arachidonic acid production or inhibition of 5-lipoxygenase, 12-lipoxygenase, and cyclooxygenases leading to inhibition of prostaglandins and leukotrienes.

Experimental section

Material and methods

2-Aminobenzimidazole, o-vanillin, Cu(NO₃)₂·3H₂O, Zn(NO₃)₂·6H₂O, acetic acid, and all the solvents were used as purchased from the company Sigma-Aldrich without further purification. Infrared spectra were recorded with KBr pellets, using a Shimadzu IR Affinity-1 spectrometer with a resolution of 4 cm⁻¹. Elemental analyses (C, H, and N) were performed on a PerkinElmer 2400 Series II CHNS/O system. NMR spectra were recorded using a JEOL-ECP-400 spectrometer. The EPR spectrum of the copper complex was acquired on a Varian E 112 spectrometer using X-band frequency (9.1 GHz) at liquid nitrogen temperature in the solid state. Circular dichroic spectra of both complexes were obtained using a Circular Dichroism (CD) Spectrometer with Stop Flow-Applied PhotoPhysics Chirascan and a JASCO P-1020 (Jasco International Co., Ltd, Tokyo, Japan) digital polarimeter with a 10 cm optical length cell at 25 °C used for calculating specific rotation. Ultra-performance liquid chromatography (UPLC) experiments were performed on a gradient system from Agilent Technologies (1290 Infinity) equipped with an infinity binary pump (G4220A), autosampler (G4226A), Thermostat column compartment (G1316c), fraction collector (G1364C) and a photodiode array detector (G4212A). Chem-station software was used to program the above configuration of UPLC. The separations were performed on an Eclipse C18 column (Agilent), 4.6 × 100 mm, 3.5 μm, 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 ethics committee of the College of Pharmacy, King Saud University, Riyadh, KSA.

Synthesis of Schiff base (L)

Solutions of equimolar amounts of 2-aminobenzimidazole (0.133 g, 1.0 mmol) and vanillin (0.152 g, 1.0 mmol) in absolute ethanol with 2–3 drops of acetic acid were refluxed for 4 h and the product obtained was filtered off and washed with hexane (3 × 5 ml) and diethyl ether (3 × 5 ml). The product was recrystallized from ethanol to obtain a deep orange colored 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: 3305, 1600, 1515, 1462, 1252, 735. ¹H NMR (400 MHz, DMSO-d₆, ppm): 12.95 (br, NH, 1H), 9.61 (s, N=CH, 1H), 7.28–7.25 (m, Ar-H, 2H), 7.18–7.16 (d, Ar-H, 2H), 7.09–7.07 (d, Ar-H, 2H), 6.98–6.94 (t, Ar-H, 1H), 3.91(s, O(CH₃), 3H), 2.16 (s, OH, 1H). ¹³C NMR (100 MHz, DMSO-d₆, ppm): 167.6 (characteristic peak of Schiff base, N=CH), 151.6, 125.5, 123.2, 119.7, 118.9, 116.4 (Ar-Cs), 56.4 (OCH3).

Synthesis of copper(II) complex [CuL₂]

To a methanolic (10 ml) solution containing the Schiff base (267.1 mg, 1.0 mmol) was added slowly a methanolic solution (10 ml) of Cu(NO₃)₂·3H₂O (120.5 mg, 0.5 mmol) and it was stirred for 5 h. The precipitate was filtered off and washed with hexane (3 × 5 ml) diethylether (3 × 5 ml) and CHCl₃ (3 × 5 ml) 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, 4.04; N, 14.07. HRMS m/z {in DMSO, observed (calcd)} for C₃₀H₂₄N₆O₄Cu: 595.1, (595.1). FT-IR (KBr pellets) cm⁻¹: 1609, 1535, 1455, 1254, 744. μ_eff = 1.88 BM.

Synthesis of zinc(II) complex [ZnL₂]

A similar procedure was adopted as for the copper complex, using Zn(NO₃)₂·6H₂O (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: 1608, 1535, 1449, 1256, 741. ¹H NMR (400 MHz, DMSO-d₆, ppm): 14.51 (br, NH, 2H), 9.40 (s, N=CH, 2H), 7.58–7.52 (dd, Ar-H, 4H), 7.19–6.94 (m, Ar-H, 4H), 7.08–6.94 (t, Ar-H, 4H), 6.65–6.61 (t, Ar-H, 2H), 3.87 (s, O(CH₃), 6H). ¹³C NMR (100 MHz, DMSO-d₆, 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 (Ar-Cs), 55.9 (OCH₃).

In vivo biological studies

Antipyretic test (yeast induced hyperpyrexia in mice). Hyperpyrexia was induced in mice by subcutaneous injection of a 20% aqueous suspension of brewer’s yeast of 20 ml per 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 1 h before drug administration in fevered animals served as a pre-drug control.

Analgesic activity (acetic acid induced writhing test, i.e. algesia). Acetic acid-induced writhing in the mice – the 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 per kg body weight) were administered orally, to 16 h fasted mice, which were 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 compound-treated groups were compared with those of animals receiving diclofenac sodium (as a standard drug), 4 mg kg⁻¹, as well as with the control group.

Anti-inflammatory activity (carrageenan-induced paw edema in rats). Pedal inflammation in albino rats (8 to 10 weeks old) of either sex weighing 180–200 g was carried out following the method described by Winter et al.⁴⁹ An injection was made of 0.05 ml of 1% carrageenan sodium salt (BDH) into the right hind foot of each rat under the plantar aponeurosis. The test groups of rats were treated orally with both complexes with 50 and 100 mg kg⁻¹ 1 h before the carrageenan injection. At the same time, the control group was given 5 ml kg⁻¹ of normal saline and the reference group was given 100 mg kg⁻¹ of an aqueous solution of oxyphenbutazone. The measurements of foot volume were done using the displacement technique using a plethysmometer (Apelex, France) 3 h after the injection of carrageenan. The inhibitory activity was calculated according to the following formula:

Percent inhibition = 100[1 − (a − x)/(b − y)]

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 mean paw volume after carrageenan injection.

Statistical analysis

Results are expressed as mean ± SEM. One-way ANOVA was used for a comparison test of significant differences among groups followed by Dunnet’s multiple comparison post-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 a 2-[(1H-benzoimidazol-2-ylimino)-methyl]-6-methoxy-phenol Schiff base ligand derived from the condensation of 2-aminobenzimidazole and o-vanillin. 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/non-stereo-selective in nature. Therefore, these synthesized NSAIDs will not cause side effects. The in vivo anti-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 anti-inflammatory, analgesic and antipyretic effects discovered in the present study warrants further investigation.

Acknowledgements

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University, Riyadh, Saudi Arabia for its funding of this research group, No. RGP-150.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25071d

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