Antifungal, Insecticidal, Cytotoxicity, and Phytotoxicity Studies of Binuclear Ni(II) and Cu(II) Complexes of Diclofenac

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), is one of the most frequently used medicines for the treatment and management of pain and edema associated with rheumatoid arthritis, osteoarthritis, spondylitis, and many other inflammatory conditions. ¹ Other therapeutic applications include its use for the relief of pain associated with surgery, neoplastic diseases, and dysmenorrhea.^2–4 The antithrombotic effect is mediated mainly by COX-1, while anti-inflammatory effects are mediated by COX-2. Inflammation is the stimulus for overexpression of COX-2 in affected tissues, which leads to the production of the increased pain-mediating prostaglandins. COX-2 is the cytokine inflammatory inducible enzyme; however, the effect of diclofenac on COX-1 is low compared to the effect on COX-2.^5–8

Various metal ions, such as copper, cobalt, iron, nickel, and manganese, can take part in diverse functions and structures within biological systems.⁹ Organometallic complexation of drugs is an interesting area of research to explore the effects of metal complexes on the behavior of drugs, especially the NSAIDs.^10–13 It is known that several NSAIDs act via chelation or by inhibiting the activity of metalloenzymes. The role of copper complexation enhances the pharmacological profile of NSAID activity and reducing toxicity. Other pharmacological activities of copper complexes, and their potential as antiarthritic, antiulcer, anticancer, antidiabetic, and antiepileptic drugs, have been reported.^14–17 The Cu(II) complex of diclofenac has been found to have a far better anti-inflammatory activity than diclofenac.^18,19 Similarly, other metals, especially the binuclear complexes of diclofenac with nickel, produce pronounced pharmacological effects compared to diclofenac in free form.^19–21 In the current study, the aim was to investigate the unexplored and novel pharmacological effects of the previously synthesized binuclear copper and nickel—i.e., Cu(II) and Ni(II) complexes—with diclofenac to discover new therapeutic windows.

Materials and methods

Materials

All chemicals, solvents, and metal salts (obtained from E. Merck, Darmstadt, Germany) were of analytical grade and were used without further purification. The analysis of carbon, hydrogen, and nitrogen was carried out using a model EA 3000 elemental analyzer (EuroVector, Milan, Italy). Infrared absorption spectra were recorded on an FTIR-8400S spectrophotometer (Shimadzu, Columbia, MD). A minipress potassium bromide (KBr) disk was used to prepare a transparent disk of samples. The major and important peaks are reported in cm⁻¹. An Analyst 700 atomic absorption spectrometer (PerkinElmer, Shelton, CT) was used for the quantitative estimation of metal contents in complexes. Melting points were determined on a Reichert-Thermovar system from F.G. Bode Co. (Austria) by taking crystals of samples on the coverslip. A Nova-210 C digital pH/mV meter from Nova Scientific Inc. (Sturbridge, MA) was used to adjust the pH of the drugs with sodium hydroxide.

Fungal strains

Tests were performed on six fungal reference strains. Fungal strains included Trichophyton longifusus (clinical isolate), Candida albicans ATCC 2091, Aspergillus flavus ATCC 32611, Microspoum canis ATCC 11622, Fusarium solani ATCC 11712, and Candida glaberata ATCC 90030. They were maintained on agar slant at 4 °C. The strains were activated at 37 °C for 24 hr on Sabouraud glucose agar (SGA) prior to any screening.

Methods

The Cu and Ni complexes of diclofenac sodium were prepared according to the reported procedure.¹⁰ Thirty milliliters of 0.1 mol/100 mL aqueous solution of the metal ion were added to 60 mL of 0.1 mol/100 mL aqueous solution of the ligand (concentration of ligand/concentration of metal [CL/CM] = 2), maintaining the pH of the mixture in the range 5.5–6.5, by the addition of small aliquots of sodium hydroxide solution. The mixture was stirred for about 2 hr at room temperature, and then the precipitate was filtered off, washed with water, and dried in vacuum over silica gel for at least 48 hr. The analysis of all the solid compounds agreed with the literature and empirical formula:

M(D) ₂ · (H₂O) _x

where M and D are the metal and the diclofenate ions, respectively; x = 1 for Ni(II); and x = 2 for Cu(II).

MIC determination by macrodilution method

All the compounds (test and reference) were dissolved in dimethyl sulfoxide (DMSO) and added to the medium in order to obtain concentrations from 500 μg/mL to 1.0 μg/mL (for antifungal activity). Then, 1 × 10³ fungi/mL were added and the microplates were incubated at 30 °C for 48 hr in a laminar flow cabinet. The same volume of an actively growing culture of the test fungi was added to the different wells, and cultures were grown overnight in 100% relative humidity at 37 °C. The next morning, tetrazolium violet was added to all the wells. Growth was indicated by a violet color of the culture. The lowest concentration of the test solution that led to an inhibition of growth was taken as the minimum inhibitory concentration (MIC). The negative control acetone had no influence on the growth at the highest concentration used. Impenem, amphotericin B, and miconazole were used as controls for comparison.

Determination of cytotoxic activity

Brine shrimp (Artemia salina leach) eggs were hatched in a shallow rectangular plastic dish (22 × 32 cm) filled with artificial seawater, which was prepared with a commercial salt mixture and double-distilled water. An unequal partition was made in the plastic dish with the help of a perforated device. Approximately 50 mg of eggs was sprinkled into the large compartment, which was darkened, while the minor compartment was open to ordinary light. After two days, nauplii were collected by a pipet from the lighted side. A sample of the test compound was prepared by dissolving 20 mg of each compound in 2 mL of DMSO. From this, stock solutions of 500, 50, and 5 mg/mL were transferred to nine vials (three for each dilution were used for each test sample and LD₅₀ was the mean of three values), and one vial was kept as a control having 2 mL of dimethylformamide (DMF) only. The solvent was allowed to evaporate overnight. After two days, when the shrimp larvae were ready, 1 mL of seawater and 10 shrimps were added to each vial (30 shrimps/dilution) and the volume was adjusted with seawater to 5 mL per vial. After 24 hr, the number of survivors was counted.^21,22 Data were analyzed by a Finney computer program to determine the LD₅₀ values.²³

Determination of insecticidal activity

The insecticidal activity of the compounds was determined by direct contact application using filter paper.²⁴ Test compounds (1019 μg/cm²) were applied to filter papers (90 mm diam). After drying, each filter paper was placed in the separate petri dish along with 10 adults of each Tribolium castaneum, Callosobruchus analis, and Rhyzopartha dominica. Permethrin (235.71 μg/cm²) was used as a reference insecticide. All of these were kept without food for 24 hr, after which a mortality count was done.

Determination of phytotoxicity

Phytotoxic activity of the compounds was tested against the Lemna minor L.²¹ Three flasks for each 1000, 100, and 10 μg/mL were inoculated with stock solution of the compounds. To each flask, 20 mL medium and 10 plants, each containing a rosette of three fronds, were added. Paraquat was used as the reference growth inhibitor. All flasks were incubated in the growth cabinet for seven days, after which the growth regulation in percentage was calculated with reference to the negative control. IC₅₀ was calculated with a Finney computer program.

Results and discussion

Confirmation of the complexes

1. Physical characteristics. The complexes of Cu(II) and Ni(II) were prepared in aqueous or alcohol solutions according to the literature. All of the complexes were condensed with a 2:1 molar ratio. Their melting points were measured and their solubility checked in different solvents. The synthesis of complexes was confirmed physically by measuring their melting points. Copper and nickel complexes melt at 240 °C and 160 °C, respectively, compared to the ligand, with a melting point of 280 °C. The products were insoluble in water; slightly soluble in methanol, ethanol, and chloroform; and soluble in 0.1N HCl solutions. However, the ligand was freely soluble in water, methanol, ethanol, and 0.1N HCl solution. The color of the ligand (diclofenac sodium) was white, while that of the copper and nickel complexes was green and pale green, respectively.

Infrared spectral analysis

Table 1 - Selected infrared data for diclofenac metal complexes (cm^–1)*

Infrared spectra of the ligand and metal (copper and nickel) complexes were recorded in the range 4000–200 cm^–1 using KBr disks. The characteristic vibrational frequencies were identified by comparing spectra of complexes with the ligand. The complexation of metal with the ligand was confirmed by the change in the peaks. Tentative assignments were made on the basis of earlier publications,^10,20,25 and the important data are listed in Table 1. The absence of large systematic shifts of the υ(NH) bands in the spectra of all the complexes compared with those of the ligand indicates that there is no interaction between the NH group and the metal ions. The association of the carboxylic acid group to metal is proposed on the basis of magnitude of separation (Δυ values) of the υasym (COO) and υ_sym (COO) bands, and is compared with the ligand. The υ_asym (COO) and υ_sym (COO) bands of the ligand were at 1572 cm⁻¹ and 1402 cm⁻¹, respectively. The Δυ [υ_asym (COO) – υ_sym (COO)] value was 170 cm⁻¹. The υ_asym (COO) and υ_sym (COO) bands of the copper complex were at 1610 cm⁻¹ and 1415–1396 cm⁻¹, respectively. The Δυ [1610–1415] value was 195 cm⁻¹, which is greater than that of the ligand value, i.e., 170 cm⁻¹. The υ_asym (COO) and υ_sym (COO) bands of the Ni complex were at 1578 cm⁻¹ and 1400 cm⁻¹, respectively. The Δυ (1578–1400) value was 178 cm⁻¹.

All of these values were the same or very close to the reported figures.¹⁰ It was established that the carboxylato group can act as unidentate, bidentate, or as bridging ligand, and distinction between these binding states can be made from the frequency separation (Δυ = [υ_asym (COO) – υ_sym (COO)]) between the symmetric and the asymmetric stretching of this group.²⁶ By examining the symmetric and the asymmetric stretching of a large number of carboxylato complexes with known crystal structure, Deacon and Phillips²⁷ established the criteria that can be used to distinguish between the three binding states of the carboxylato complexes.