Optimization of the Synthesis of Diclofenac Derivatives with Hydrazone Structure and In Vitro Evaluation of the Anti-Inflammatory Potential

The aim of the study was to optimize the synthesis of diclofenac derivative with hydrazones structure in order to obtain higher yields and purity by variation of different parameters such as: ratio between reactants, solvent, catalyst, temperature, time of reaction and method used. The antiinflammatory effects of diclofenac derivatives were evaluated using in vitro assays: albumin denaturation and erythrocyte membrane stability. The obtained results showed that the effect of the tested derivatives is increasing with the concentration, the best results being obtained at the concentration of 125 μg/mL (albumin denaturation assay), respectively 111.11 μg/mL (erythrocyte membrane stability assay). The most active compound was 4d which showed the highest inhibition effect on albumin denaturation and an appreciable effect on erythrocyte membrane stability, in comparation with diclofenac, used as drug reference.


Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the oldest and most successful drugs known in modern medicine to decrease pain and inflammation by inhibiting prostaglandins synthesis, but unfortunately their use is associated with a number of serious side effects. NSAIDs, such as diclofenac, are indicated for improvement of all degrees inflammation associated with a large number of conditions, including arthritic disorders, acute musculoskeletal disorders and other painful conditions resulted from trauma. Using NSAIDs have been reported gastrointestinal bleeding, ulceration or perforations, which can be fatal, and can occur at any time during treatment, with or without a history of serious gastrointestinal disorders. If gastrointestinal bleeding or ulceration occurs to patients who receive diclofenac, treatment will be ended [1,2].
It is important to note that cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes play an important role in inflammation. The modifying the structure of the classical NSAIDs is a common practice which was used over the years in order to improve the biological effects of parent molecule. Hydrazone derivatives represent an attractive group of compounds with a broad spectrum of pharmacological and biological characteristics [3,4]. In order to improve the pharmacological profile of diclofenac, some hydrazone derivatives were synthesized by researchers [5,6].
The aim of this study was to optimize the method for synthesis of diclofenac derivatives with hydrazone structure in order to apply it for extending the diclofenac hydrazone derivatives library. The synthesized derivatives were evaluated for antiinflmmatory effects using in vitro assays.
In vitro anti-inflammatory assays are important tools to select the most active derivatives to be tested using in vivo assays, and so, the use of a high number of animals is avoided [7,8]. Proteins denaturation is a well -documented cause of inflammation and the anti-inflammatory drugs, such as

Synthesis
In order to optimize the synthesis of diclofenac derivatives different parameters were varied such as: the molar ratio between reagents, the solvent, temperature, the reaction time, the catalyst and the method used (classic or microwaves). The synthesis was monitored by thin layer chromatography (TLC) using different eluents and the structure of the derivatives was proved by infrared (IR), nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR) and high resolution mass spectrometry (HR-MS) [9,10].
The synthesis of the diclofenac derivatives with hydrazone structure was performed according to the Scheme 1.

Scheme 1.
The synthesis of diclofenac derivatives with hydrazone structure (4a-s)
Procedure: stock solutions of 1 mg/mL in DMSO were prepared using diclofenac and diclofenac derivatives (4a-s). From each stock solution a volume of 50 μL, 100 μL, 200 μL and 500 μL was measured and methyl alcohol was added to 1000 μL. Then, a sample of 30 μL from each dilution was added to 3 mL of 1% bovine serum albumin solution. The concentration of the tested derivatives in the obtained samples was 12.5 µg/mL, 25 µg/mL, 50 µg/mL and 125 µg/mL, respectively. A mixture of 30 μL of methanol and 3 mL of 1% bovine serum albumin was used as control. The samples and the https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (5) control were incubated for 20 min at 37°C, then 5 min at 72ºC, cooled at room temperature for 10 min and then 1 mL of phosphate buffered saline pH 7.2 was added. The turbidity of the samples and control was read at 416 nm against distilled water. The capacity of the tested derivatives to inhibit serum albumin denaturation was calculated using the following formula [11]: Denaturation inhibition (%) = [(Ac -As)/Ac] x 100 in which: Acthe absorbance value of the control; Asthe absorbance value of the sample. For each sample the effective concentration (EC50) was calculated by linear regression analysis and diclofenac (1 mg/mL) was used as the reference drug. All experiments were performed in triplicate.
Procedure: stock solutions of 1 mg/mL in DMSO were prepared using diclofenac and diclofenac derivatives (4a-s). From each stock solution, a volume of 100 μL, 200 μL and 500 μL was measured and 0.85% NaCl solution was added to 1000 μL. To each sample 1 mL of phosphate buffer, 2 mL of 0.36% NaCl solution and 0.5 mL of HRBC solution (10% v/v) were added. The concentration of the tested derivatives in the obtained samples was 22.22 µg/mL, 44.44 µg/mL and 111.11 µg/mL, respectively. The control was prepared by adding of 1 mL of 0.85% NaCl solution to a mixture of 1 mL of phosphate buffer, 2 mL of 0.36% NaCl solution and 0.5 mL of HRBC [12,13].
The samples and the control were incubated at 37°C for 30 min, then centrifuged at 3000 rpm for 20 min. The absorbance of the supernatant was read at 560 nm against distilled water. The capacity of the tested derivatives to preserve erythrocyte membrane expressed as stability (%), was calculated using the following formula [14]: Stability (%) = 100 -[(As/Ac) x 100] in which: Acthe absorbance value of the control; Asthe absorbance value of the sample. For each sample the effective concentration (EC50) was calculated by linear regression analysis and diclofenac (1 mg/mL) was used as the reference drug. All experiments were performed in triplicate.
It was observed that the highest yield (85%) was obtained using the following conditions: ratio between diclofenac and H2SO4 of 1 eq : 0.25 eq., heating at 80°C (reflux) for 3 h and ethanol as solvent (20 mL).
It was observed that the highest yield (90%) was obtained using he following conditions: ratio between diclofenac ethyl ester (2) and hydrazine hydrate 64% of 1 eq. : 20 eq., dioxane as solvent and heating at 105°C (reflux) for 6 h. Using these conditions the compound 3 was obtained in yield which is higher than the value of 72% which was reported by other researchers [5,6].  (-NH2) [19][20][21][22]. The influence of different parameters such as the ratio between 2-[(2,6-dichlorophenylamino)phenyl]acetohydrazide (diclofenac hydrazide) (3), 4-methyl-benzaldehyde and glacial acetic acid, as catalyst, the reaction temperature (65°C or 80°C), the solvent (methanol or ethanol), the time of reaction (between 30 min and 6 h) and the method used (classic or microwave) on the reaction yield is presented in Table 3.

Serum albumin denaturation assay
The generation of autoantigens in rheumatic and inflammatory disorders can cause tissue protein precipitation, thus leading to the aggravation of these diseases. It was proved that any compound exhibiting a protein degradation inhibition greater than 20% can be further evaluated as a potential anti-inflammatory agent. Many NSAIDs drugs such as aspirin, diclofenac, indomethacin, flufenamic acid, in addition to the ability to inhibit the synthesis of protaglandins (cyclooxygenase inhibition), also showed an appreciable capacity to prevent BSA denaturation [24].

Erytrocyte membrane stabilization test
It is known the stability of lysosomal mebrane is crucial for limiting the inflammation. By destroying of the lysosomal membrane, the released cellular constituents activate neutrophils, bactericidal enzymes and proteases, causing destruction of cells and tissue inflammation. The erythrocyte membrane, which is similar to the lysosomal one, is commonly used as assay for assessing the anti-inflammatory effect [25][26][27][28].

Conclusions
By variation of different synthesis parameters such as: the ratio between reagents, catalyst, the solvent, the temperature, the time of reaction and the method used (classic or microwave), many chemistry procedures were developed in order to increase the yield of synthesis and the purity of diclofenac derivatives. The derivatives 4d (R=3-OCH2CH3-4-OH) and 4g (R=4-OCH3), obtained by reaction of diclofenac hydrazide and 3-ethoxy-4-hydroxy-benzaldehyde and 4-methoxy-benzaldehyde respectively, showed the most intense effect on inhibition of serum albumin denaturation. In addition, compound 4d showed also a good stabilization effect on the erythrocyte membrane. An intense effect on stabilization of erythrocyte membrane, in reference to diclofenac, was also showed by derivative 4f (R = 2-Cl-5-CF3). The obtained results support that the chemical modulation of diclofenac structure had as result increasing the anti-inflammatory effects of some hydrazones derivatives, which opens new perspectives in the treatment of inflammatory diseases.