Synthesis and Evaluation of Anti-inflammatory Activity of Some Chalcone Hydrazide Derivatives

Aim: Chalcones are part of the selected group of chemical compounds related with diverse pharmacological activities such as antimicrobial, antiviral, antitumor, convulsion, anxiety, antiinflammatory, andanalgesic. The aim of this study was to synthesize chalcones hydrazide derivatives and to evaluate their anti-inflammatory activity in the model of carrageenan induced edema in rats. Methods: The chalcone hydrazide derivatives were prepared by treating Isonicotinyl and Nicotinyl hydrazide with respective parent chalconeand environmentally friendly solvents with 78–85% yields in approximately 6 hrs. These compounds were characterized by 1H NMR, 13C NMR and IR spectroscopy. The structures of synthesised derivatives were evaluated on biological activity at dose of 40 mg/kg and they showed anti-inflammatory protective effect by oral administration, this effect was time dependent. Results: All the derivatives were given satisfactory reaction yields that representing the efficiency of the employed synthetic route. In carrageenan-induced paw edema model, CL-2derivative showed most significant percentage of inhibition (61.97%) as compared to reference standard Indomethacin. Conclusion: Compound CL-2 was showed significant anti-inflammatory activity due to presence of electron donating groups like –CH3 on ring A and electron withdrawing group like NO2 on ring B at 4 th position in isonicotinyl hydrazide derivative. All synthetic chalcone hydrazide derivatives may be considered as safer drugs for treating inflammatory conditions. Original Research Article Panaskar et al.; JPRI, 34(15B): 18-26, 2022; Article no.JPRI.84125 19


INTRODUCTION
Inflammation is an evolutionarily well preserved process characterized by the activation of immune and non-immune cells that protect the host from bacteria, viruses, toxins and infections by removing pathogens and promoting tissue repair and recovery.There are normally two types of inflammation: acute and chronic inflammation. Inflammatory diseases include a huge array of conditions that are primarily characterized by inflammation. Common examples include allergy, inflammatory bowel disease, coeliac disease, asthma, autoimmune diseases, glomerulonephritis, perfusion injury, hepatitis, and transplant rejection etc. In the world, the most significant cause of death is chronic inflammatory disease [1][2][3]. The World Health Organization (WHO) ranks chronic diseases as the greatest threat to human health and further anticipated to increase persistently [4,5].
Steroidal and Non-steroidal anti-inflammatory drugs known as NSAIDs are generally used in the management of acute and chronic pain of several etiologies, including cancer associated pain as well as arthritis. In the treatment of mild to moderate pain these drugs are used individually and in case of severe pain are used along with opioid analgesics or adjuvant analgesic drugs. For decades, physicians trusted on steroids to suppress immune response. Steroids are important anti-inflammatory agents, but reported to have severe adverse effects like enlargement of the heart, liver cancer, weight gain, etc. NSAIDs are non-selective cyclooxygenase enzymes (isoenzymes 1 and 2) inhibitors; pose a potentially serious risk with its acute and chronic use like gastrointestinal ulceration, hematologic toxicity, nephrotoxicity etc. The severity of NSAIDs adverse effect is experiencing more in the immuno-compromised patients suffering from life-threatening illness like cancer, HIV/AIDS etc. This is due to the occurrence of gastrointestinal bleeding and the masking of opportunistic infections related to the antipyretic effects of NSAIDs pose particular risk and might even cause fataldifficulties in patients who are thrombocytopenic,neutropenic, or else immuno-compromised. The ability of researchers to better understand the underlying cause of disease, identify group of patients, through accurate medicine, who will respond better to certain treatments, can potentially lead to new and innovative medicines [6].
Current pharmacotherapy for inflammatory diseases reported limited therapeutic outcomes and are associated with many deleterious side effects. This indicates the need to develop new drugs for the treatment of inflammatory diseases with improved therapeutic outcome. Although the exact mechanisms of action of chalcone hydrazide analogues remain unknown, a study in inflammation indicated that chalcone can inhibit the action of COX-2. On this background, the main objective of the present study was to synthesize novel chalcone hydrazide derivativesand evaluated theiranti-inflammatory activityin the carrageenan induced paw edema model in rat.

Animals
Male Wistar rats (200-250 gm) was procured from Lacsmi Biopharms Pvt. Ltd and housed under standard laboratory condition of 12:12 h light/dark cycle in a temperaturecontrolled (22 ± 2ºC) environment with ad libitum access to rodent chow and water. All experimental protocols were approved by Institutional Animal

Synthesis
Step 1: A Methanolic solution of substituted benzophenone and substituted Benzaldehyde were prepared, then mix both the solution and stir on magnetic stirrer. In this solution add 5-6 drops of saturated solution of NaOH and continue the stirring for 30 min. A parent Chalcones were obtained by this procedure.Completion of reaction was checked by thin layer chromatography using mobile phase Chloroform: Methanol (70:30).
Step 2: A Methanolic solution of chalcone was prepared then add Methanolic solution of appropriate hydrazide such as Nicotinyl hydrazide or Isonicotinyl hydrazide, maintained the acidic medium using acetic acid and solution was refluxed for about 6hrs. Completion of reaction was checked by thin layer chromatography using mobile phase Chloroform: Methanol (90:10).

Physicochemical Studies and Characterization of Synthesized Compounds
Completion of reaction was checked by using thin-layer chromatographyusing Merck silica gel 60 F-254 plates had layer thickness about 0.25 mm and detection was performed in UV lamp and all solvents were distilled before use. All of the derivatives were characterized by IR, 1H, and 13C NMR spectra recorded with Bruker WM-300 in deuterated DMSO at 400 and 100 MHz, respectively using tetra methyl silane (TMS) as the internal standard. All chemical shifts are described on the δ scale.

Anti-inflammatory Activity
Anti-inflammatory potency was analyzed using carrageenan-induced paw edema in rats. The rats were divided into 09 groups consisting six in each group. The first control group was given vehicle DMSO per oral 1 ml/100 gm body weight whereas third group served as reference standard received Indomethacin 20 mg/kg orally. The test compounds Chalcone derivatives were administered to groups 4 th to 9 th at the dose of 40 mg/kg per oral. The vehicle used for the preparation of the test compound was DMSO.

Scheme 1. Synthetic scheme for chalcone hydrazide derivatives
After thirty minutes of above treatment, carrageenan solution 0.1 ml (1% w/v carrageenan dissolved in normal saline) injected into sub-plantar region of rat's left hind paw to induce inflammation. The group 2 nd received only carrageenan injection and served as induction control (Negative control) group.
Digital Plethysmometer was used to record the paw volume of control, reference standard and test compound treated groups and degree of paw edema measured at the interval of 1, 2, 3, 4 hours after carrageenan injection [21]. The percentage inhibition of edema was calculated using following formula.
Where, V0: is paw volume at 0 hours and VT: is paw volume of respective time interval.

Physicochemical Characterization of Chalcone hydrazide Derivatives
The Table 1 shows the physicochemical characterization of the derivatives examined in this study (Table 1). All the derivatives were properly separated from the reaction mixture and given satisfactory reaction yields, that representing the efficiency of the employed synthetic route. The Characterization of synthesized derivatives was carried out by using Infrared spectroscopy (IR), Proton NMR (H1 NMR) and Carbon NMR (C13 NMR) for structure elucidation.

Anti-inflammatory Activity
The effect of Chalcone hydrazide derivatives on carrageenan-induced paw edema in rats are summarized in Table 2 and Table 3 and Fig. 1. Results showed the significant (p< 0.001) increase in paw edema in group-II when compared to vehicle treated control group indicating induction of acute inflammation from 1 hour upto 4 hours.

CONCLUSION
In conclusion, Chalcone hydrazide derivatives (CL-1 to CL-6) were synthesized, employing shorter reaction times than the earlier reported in the literature. The spectroscopic characterizations of the six derivatives are in Carragenan + CL-5 Carragenan + CL-6 agreement with the structures expected. These chalcones hydrazide derivatives showed an antiinflammatory protective effect when administered orally route. From the result CL-2 compound (40 mg/kg) showed significant percentage of inhibition against carrageenan induced paw edema which is comparable with reference standard indomethacin. This anti-inflammatory activity was due to presence of electron donating group like -CH 3 on ring A and electron withdrawing group like NO 2 on ring B at 4 th position in isonicotinyl hydrazide derivative.
On the basis of result, it can be concluded that chalcone hydrazide derivatives exhibited antiinflammatory activity. All synthetic chalcone hydrazide derivatives may be considered as safer drugs for treating inflammatory conditions.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
The present study did not involve Patients.

ETHICAL APPROVAL
This project was approved by the Animal Ethics Committee from Dr. D. Y. Patil College of Pharmacy, Akurdi, Pune (DYPCOP/IAEC/ 2021/08).