INTRODUCTION:

In the field of medicinal chemistry, the family of heterocyclic compounds containing Nitrogen, Sulphur and Oxygen as hetero atoms in five and six membered ring structure plays an important role¹. In that, Oxazolone are heterocyclic compounds which performed an important role in the synthesis of several organic molecules including amino acid^2,3, amino alcohols, thiamine⁴, amides, peptides^5-7 and polyfunctional compound⁸. Oxazolone (4-ethoxylmethylene-2-phenyloxazol-5 one) is a classical haptenating agent that has historically been used for studying delayed type hypersensitive responses in skin⁹. Oxazolone exhibited promising photophysical as semiconductor activities^10-11. So, they are used as semiconductor devices such as electrophotographic photoreceptor and in non-linear optical materials^12-15.

Oxazolone are one of five membered heterocyclic compounds which are in three isomeric forms, one according to the location of the carbonyl groups and two according to the location of the double bond containing^16,17. Oxazolone is important synthons in synthesizing various drugs, oxazolone scaffold shows various biological activity such as anti-microbial^18,19, anti-diabetic²⁰, anti-viral²², anti-fungal, anti-cancer²³, cardioprotective²⁴, contact allergens²⁵, anti-inflammatory²⁶, anti-obesity²⁷, anti-depressant²⁸, anti-HIV²⁹, anti-angiogenic³⁰, anti-convulsant^31,32, sedative^33,34, tyrosinase inhibitor^35,36, fungicide and herbicide³⁷, and dyes³⁸.

These are used as synthesis for the construction of various alkaloid skeletons, immunomodulators and biosensors^39-42, photosensitive composition devices for proteins^43-45 Enzymes which are responsible for the conversion of arachidonic acid to prostaglandins. Anti-inflammatory are mediators that reduces production or activities of pro inflammatory cytokines and block immune cell trafficking into tissues, hence they may develop to treat inflammation. NSAIDs- non-steroidal anti-inflammatory drugs are a type of pain reliever. At prescription doses, these drugs also curb inflammation. Doctors use NSAIDs to treat many things that cause pain or inflammation, including arthritis. Inflammatory usually occurs when infectious microorganisms such as bacteria, viruses or fungi invade the body, residue in particular tissues and circulate in the blood^46-49. The innate immune system is the foremost defense mechanism against invading microorganism and cancer cells, involving the activity of various cells including macrophages, mast cells and dendritic cells. The adaptive immune systems involve the activity of more specialized cells such as B and T cells who are responsible for eradicating invading pathogens and cancer cells by producing specific receptors and antibodies. Numerous inflammatory mediators are synthesis and secreted during inflammatory responses of different types. Inflammatory substances are usually divided to two main categories: pro- and anti- inflammatory mediators. Nevertheless, some mediators such as interleukin (IL)- 12 possess both pro- and anti- inflammatory properties⁵⁰.

The anti-inflammatory mechanism mainly includes decreasing the release of histamine in mast cells, suppressing the activities of lipoxygenase, cyclooxygenase and phospholipase, and reducing the production of nitric oxide and reactive oxygen species, blocking the activation of the signal pathway, downregulating the expression of inflammatory factors and inhibiting the activities of elastase and complement. This mechanism can open up new avenues for the scientific community to develop or improve novel therapeutic approaches to tackle inflammatory diseases, such as arthritis, atherosclerosis, neuro inflammation, liver diseases, kidney diseases, diabetes, dermatitis, bowel diseases, cancer. The phytochemicals and their derivatives exert several bio activities including but not limited to anticancer, cardioprotective, anti-inflammatory, immune-regulatory and anti-obesity properties. They are strong antioxidants because of hydroxyl groups which play pivotal role in their anticancer, anti- inflammatory and cardio protective potential. They may play significant role in improving human health owing to anticarcinogenic, anti-stroke, and anti-atherosclerosis activities as several PAs have demonstrated biological activities against these diseases during invitro and in-vivo studies. This study was intended to “synthesis of novel oxazolone derivatives” by using different aromatic and aliphatic aldehydes like O-Anisaldehyde, Cinnamaldehyde, P-Chlorobenzaldehyde, 2-Chloro benzaldehyde and 4-Fluro benzaldehyde to find out the characterization by using FTIR and determine the NSAIDs activity. The properties of 5-(4H)-Oxazolone, systemic name: Oxazolone; Molecular formula: C₃H₃NO₂;^{Density: 1.3±0.1g/cm3; Refractive index: 1.548;}Molar refractivity: 18.5±0.3 cm³; Molar mass: 161.16g/mol.

Figure No 1: 5-(4H)-Oxazolone

MATERIALS AND METHODS:

Materials:

Glycine, cinnamaldehyde, 4-Chloro benzaldehyde and acetic anhydride was procured from Lobachemie Pvt Ltd. Benzoyl chloride and sodium acetate was obtained from Nice chemicals Pvt Ltd. 2-Fluro benzaldehyde, 4-Fluro benzaldehyde and O-Anisaldehyde was obtained from Kemphasol, Mumbai, and ethanol was purchased from Chanshu Hongsheng fine chemicals co ltd.

Methods:

General method for synthesis of oxazolone derivatives. Step 1: Preparation of Benzoyl glycine crystals:

· The compound was prepared by taking 0.75g of glycine is dissolved in 100ml of water.

· Add 2N of 30ml of sodium hydroxide and stirred vigorously with a magnetic stirrer for 30minutes until the solid was completely dissolved.

· Add 1.21ml of benzoyl chloride in five portions of glycine mixture, stirred continuously with a magnetic stirrer for 2 hours, maintain RPM nearly 500-550. Left the mixture overnight in refrigerator.

· Filter the solution by using Buchner funnel with help of vacuum pump.

· Washed with cold water and kept the crystals in hot air oven on further drying at 45ºC over 10-15 minutes. The crystals are separated.

Figure No 2: Chemical reaction of Preparation of benzoyl glycine crystals

Step 2: Synthesis of oxazolone derivatives:

· The five portions of 0.01mol of Benzoyl glycine taken in each round bottom flask.

· Add substituted aromatic and aliphatic aldehyde nearly 0.02mol in that each flask.

· Add 1gm anhydrous sodium acetate and 4g of Acetic anhydride in each of the flask.

· Reflux for 1hour on water bath with occasional stirring. Left the mixture overnight in refrigerator.

· Filter the solution, washed with cold water, recrystallized with ethanol and maintained room temperature.

· The crystals are separated.

Table No 1: Total quantity obtained of each crystals of the unknown compound of substituted aliphatic and aromatic aldehyde of oxazolone derivatives.

S No	Unknown product of oxazolone derivatives with substituted alipatic and aromatic aldehyde	Total quantity obtained at the end of the synthesis
1.	O-Anisaldehyde	2.67 gm
2.	2- Chlorobenzaldehyde	2.77 gm
3.	4-Chloro benzaldehyde	2.50 gm
4.	4-Fluro benzaldehyde	1.70 gm
5.	Cinnamaldehyde	3.45 gm

Fourier Transform-Infra Red (FT-IR) Studies:

The functional group of newly synthesized compounds were performed using Fourier Transform-Infra Red (FT- IR) spectrometer (JASCO FTIR 6300) by KBr disk method. A known weight of sample is mixed with KBr powder and compressed to 10-mm discs by hydraulic press at pressure of 150 bar for 30s at the scanning range and resolution of 400–4000cm-1and4cm-1. The spectra obtained the functional peaks of newly synthesized compounds such as P-Chlorobenaldehyde, 4-Fluro benaldehyde, Cinnamaldehyde, 2-Chloro benaldehyde, and O- Anisaldehyde in the corresponding regions.

Screening of in vitro anti-inflammatory activity by using Protein denaturation assay:

The in vitro anti-inflammatory activity of the synthesized compounds was studied using bovine serum albumin denaturation method^64,65. In brief, increasing concentrations of the test or reference compound (20, 40, 60, 80, 100µg/ml) were incubated with 0.5% w/v of bovine serum albumin at 37ºC for 20 min and the temperature was increased to keep the samples at 57 ºC for 30min. After cooling to room temperature, the turbidity was measured using UV-Visible spectrophotometer at 660 nm following addition of 2.5 ml of phosphate buffered saline. Diclofenac sodium was used as reference standard. The percentage inhibition of protein denaturation was calculated by using the following formula^50,51.

% Inhibition = (Absorbance of control – Absorbance of test)/Absorbance of Control x 100

Human red blood cell (HRBC) membrane stabilization assay:

The human red blood cell (HRBC) membrane stabilization method has been used as a method to study the in vitro anti-inflammatory activity of synthesized compounds^54-59.

Preparation of blood samples for membrane stabilization assays:

The blood sample was collected from human healthy volunteer who had not taken any NSAIDs for 2 weeks prior to the experiment and mixed with equal volume of Alsever solution (contains 2% dextrose + 0.8% sodium citrate + 0.5% citric acid + 0.42% NaCl). The blood samples were stored at 4°C for 24h before use. It was centrifuged at 2500rpm for 5min and the supernatant was removed. The cell suspension was washed with sterile saline solution (0.9% w/v NaCl) and centrifuged at 2500rpm for 5 min. This was repeated three times till the supernatant was clear and colorless and the packed cell volume was measured. The cellular component was reconstituted to a 10% suspension (v/v) with iso-saline and was used in the assay.

Hypotonicity-induced haemolysis:

The 1ml different concentrations of synthesized compounds, reference sample (20, 40, 60, 80, 100 µg/ml) and control were separately mixed with 1ml phosphate buffer, 2ml hyposaline and 0.5ml HRBC suspension. After incubation for 30min at 37°C the reaction mixture was centrifuged at 3000rpm for 10 min. The absorbance of the supernatant was assessed spectrophotometrically at 560nm. Diclofenac was used as a reference standard. The percentage of hemolysis was estimated by considering the percentage hemolysis of control as 100%. The percentage of protection/ percentage inhibition of hemolysis were evaluated using the formula^52,53

% Inhibition= 100 - Absorbance of test/Absorbance of Control x 100

RESULTS AND DISCUSSION:

During the synthesis of oxazolone analogous the end point for each step was identified by characterization method as well as invitro anti-inflammatory activity. Here are the results obtained for each step involved in the synthesis.

Fourier Transform-Infra Red (FT-IR) Studies

The functional group of newly synthesized compounds P-Chlorobenaldehyde, 4-Fluro benaldehyde, Cinnamaldehyde, 2-Chloro benaldehyde, and O- Anisaldehyde were performed using Fourier Transform-Infra Red (FT-IR) spectrometer (JASCO FTIR 6300) by KBr disk method (Figure No 4a-4e).

In-vitro anti-inflammatory activity by using Protein denaturation assay:

From the determination if invitro Anti-inflammatory activity by using egg albumin protein denaturation assay, concluded that all the synthesized oxazolone analogues had vast activity in pharmacological action. Firstly, the synthesized oxazolone analogues had compared with the standard Anti-inflammatory drug Diclofenac sodium to determines its potent activity. The test compounds 2-Chloro benzaldehyde had nearest IC50 value to the standard Diclofenac sodium when compared to all other test compounds. It produces better pharmacological effect as the chlorine atom present at ortho position. It had the highest zone of inhibition at the low concentration used to exhibit the high potency (Table No 4). The test compounds like P-Chlorobenzaldehyde, O-Anisaldehyde, 4-Fluro benzaldehyde produced the desired pharmacological effect according to their specific IC50 values. The compound Cinnamaldehyde have the less potency when compared to all other test compounds and standard. It produced their desired pharmacological effect where it contained the aliphatic chain in its complex structure, as it consists some specific features to be maintained like temperature, humidity to produced their product.

Table No 4: Effect of test compounds on egg albumin protein denaturation assay.

Compound No.	Test Compound	Concentration (µg/ml)	% Inhibition	^IC50 ^Value (µg/ml)
1.	P- Chlorobenzaldehyde	20	38.32	32.88
		40	56.51
		60	70.47
		80	85.57
		100	93.15
2.	4-Fluro benzaldehyde	20	37.52	44.69
		40	45.3
		60	47.38
		80	85.77
		100	88.52
3	Cinnamaldehyde	20	37.18	53.86
		40	45.57
		60	47.92
		80	48.39
		100	86.51
4.	2-Chloro	20	40.81	12.59

In-vitro anti-inflammatory activity by effect of test compounds on hypotonicity induced haemolysis of RBC membrane:

Here from the determination of invitro anti-inflammatory activity by using HRBC test concluded that all synthesized oxazolone compounds shows the highest action while using low concentration of test compounds. The test compounds 4-Fluro-benzaldehyde shows the less IC50 value, where it obtained low concentration of drug to produce the high efficacy due to the high electronegative atom fluorine is present at para- position (Table No 5). All other test compounds like P-Chlorobenzaldehyde, 2-Chloro benzaldehyde, O-Anisaldehyde produced their desired pharmacological effect. The test compounds Cinnamaldehyde (aliphatic compound) which binds to the receptor because its nature, it produces the good pharmacological effect. Hence it exhibits high IC50 values. All the test compounds were compared with the standard Diclofenac sodium to determine the potency, concentration, and pharmacological effect.

Table No 5: Effect of test compounds on hypotonicity induced haemolysis of RBC membrane

Compound No.	Test Compound	Concentration (µg/ml)	% Inhibition	^IC50 ^Value (µg/ml)
1.	P- Chlorobenaldehyde	20	42.25	35.61
		40	51.55
		60	61.97
		80	81.27
		100	92.54
2.	4-Fluro	20	46.90	22.80
		40	56.62
		60	68.03

DISCUSSION:

The anti-inflammatory activity was performed by using novel oxazolone derivatives (D1, D2, D3, D4, D5) and Diclofenac sodium (as standard) on the method of protein denaturation assay and HRBC membrane stabilization assay method. The anti-inflammatory activity was performed by measuring the percentage of inhibition. The synthesized oxazolone derivatives was compared with the standard drug Diclofenac sodium for anti-inflammatory activity by using the protein denaturation assay and HRBC membrane stabilization assay. All the synthesized oxazolone derivatives (O-Anisaldehyde, Cinnamaldehyde, 4-Chloro benzaldehyde, 4-Fluro benzaldehyde, 2- Chlorobenzaldehyde) having good anti-inflammatory activity as that of standard. The in-vitro anti-inflammatory activity of synthesized compounds was significant. In protein denaturation assay, particularly the compound 2-Chloro benzaldehyde having better NSAIDs activity with the best IC50value, among all other synthesized compounds when compared with the standard. In that, the other synthesized compounds like P-Chlorobenzaldehyde, 4- Fluro benzaldehyde, Cinnamaldehyde and O- Anisaldehyde also having good NSAIDs activity. In the same way, in HRBC assay the compound 4-Fluro benzaldehyde having better NSAIDs activity with the best IC50 value among all other compounds when compared with the standard. The other compounds like P-Chlorobenzaldehyde, Cinnamaldehyde, O-Anisaldehyde, 2-Chlorobenzaldehyde also having good NSAIDs activity obtained with their respective IC50 values.

CONCLUSION:

The screening of the synthesized novel oxazolone derivatives using different aliphatic and aromatic aldehydes like O-Anisaldehyde, Cinnamaldehyde, 4-Chloro benzaldehyde, 4-Fluro benzaldehyde and 2-Chloro benzaldehyde revealed that the better anti-inflammatory activity. The functional group that were present in the synthesized compounds were identified by using FT-IR spectroscopy. The novel synthesis of oxazolone derivatives shown significant differences from standard drugs especially aliphatic compound of Cinnamaldehyde shown major significant difference. The in-vitro anti-inflammatory activity of synthesized compounds was significant. In protein denaturation assay, particularly the compound 2-Chloro benzaldehyde having better NSAIDs activity with the best IC50 value among all other synthesized compounds when compared with the standard. In that, the other synthesized compounds like P-Chlorobenzaldehyde, 4-Fluro benzaldehyde, Cinnamaldehyde and O-Anisaldehyde also having good NSAIDs activity. In the same way, in HRBC assay the compound 4-Fluro benzaldehyde having the better NSAIDs activity with the best IC50 value among all other compounds when compared with the standard. The other compounds like P-Chlorobenzaldehyde, Cinnamaldehyde, O-Anisaldehyde, 2-Chlorobenzaldehyde also having good NSAIDs activity with their respective IC50 values.

DECLARATION OF COMPETING INTEREST:

The author does not declare any conflict of interest.

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Received on 02.05.2023 Modified on 08.06.2023

Asian J. Pharm. Res. 2023; 13(3):145-152.

DOI: 10.52711/2231-5691.2023.00028