Synthesis, characterization, and investigation of the anti-inflammatory effect of 2,3-disubstituted quinazoline-4(1H)-one

Quinazolin-4(1H)-one nucleus has attracted the attention of medicinal chemists due to their clinical uses. Modification of quinazolinone ring for the development of pharmaceutical and clinical compound for its anti-inflammatory potential. In vitro anti-inflammatory activity of the synthesized compounds was performed by using egg albumin protein denaturation assay, while in vivo anti-inflammatory activity was performed by using carrageenan-induced rat paw edema and cotton pellet-induced granuloma pouch model. In the present study, we synthesized a new series of 2,3-disubstituted quinazolin-4(1H)-one derivatives and evaluated their in vivo, in vitro anti-inflammatory effect. Their chemical structures are confirmed by FTIR, 1HNMR, and mass spectrum. Among all the synthesized compounds, G1 and G3 exhibit the significant anti-inflammatory activity by inhibiting release of inflammatory mediators like prostaglandin, histamine, and serotonin. in both in vivo and in vitro models as compared to compound G2. These synthesized compounds showed anti-inflammatory activity by inhibiting prostaglandins and COX enzymes. So, all test compounds may be used for both inflammation as well as inflammation-induced cancer therapy. Future various screening method related with inflammation and inflammation-induced cancer needs to be evaluated pre-clinically and clinically.

bleeding, and nephrotoxicity [7]; therefore, discovery of new safer anti-inflammatory agent presents a challenging goal.
SAR studies on quinazolinone ring system in various literatures suggest that substitution at positions 2 and 4 is very much important for better pharmacological activities [8]. The present work is to design novel substituted quinazolin-4-(1H)-one derivative and screened for their anti-inflammatory property.

Chemicals and apparatus
All the chemicals used were purchased from Alfa Aesar, Spectrochem Ltd and SD fine chemicals Ltd. Kandlakoya Village, Hyderabad, Telangana and Autonagar, Vanasthalipuram, Hayathnagar Mandal, Hyderabad, Telangana, India, respectively. Melting points of all the compounds were determined on an open capillary tube on super fit melting point apparatus.
The purity of all the final compounds was assessed by thin layer chromatography plates coated with silica Gel G (100-200 mesh size) to establish identity the reactants and products monitored in between reaction. Used petroleum ether:ethyl acetate:methanol 4:1:1 system as mobile phase. The spots were visualized by iodine vapor in iodine chamber and also using the UV-chamber. Solvents were distilled before use. Solvent extracts were dried over anhydrous sodium sulphate.
Infrared spectra were recorded using KBr on Shimadzu FTIR-Affinity spectrophotometer. Proton resonance magnetic spectra ( 1 HNMR) were recorded using BRUKER AVANCE II 400 NMR spectrometer, and chemical shifts were expressed in parts per million (δ ppm), down field from TMS as an internal standard. Mass spectra were recorded using mass spectrometer; the spectra were recorded on WATER, Q-TOF MICROMASS (ESI-MS) instrument.

Experimental animal
Wistar rats weighing 150-250 g were procured from animal house of AISSMS College of Pharmacy, Pune, Maharashtra, India, ref. No. COP/PN/2017-18/91-3 on dates 6 October 2017 (IAEC No. CPCSEA/IAEC/OP-03/ 01-2 K17). Animals are provided with standard pallet diet and water ad libitum. Animals were placed in the cages 5 days prior to the experiment and ascertained the health status and were exposed to alternate 12 h light and dark cycles.  reactants completely. Subsequently, substituted aldehyde (1 mmol) was added with condenser and refluxed for 1.5 h with continuous stirring. The reaction was monitored by thin layer chromatography using ethyl acetate and pet ether as the solvent system. After completion of the reaction, the content was poured into ice water. Solid mass or precipitate obtained was then filtered, washed with ice cold water, and recrystallized from hot ethanol to obtain pure 2,3-disustituted quinazolin-4(1H)-one derivative. The melting points of compounds have been taken on open capillary tube and are uncorrected. All synthesized compounds were solid in nature and soluble in dimethyl sulfoxide solvent ( Fig. 1) [9,10]. The chemical structures, names, and abbreviations of the synthesized compounds are presented in Table 1.

Egg albumin protein denaturation
Egg albumin (0.2 ml), phosphate buffer saline of pH 6.4 (2.8 ml), and varying concentrations (50, 100, 150, 200, and 250 μg/ml) of test compounds of G1, G2, and G3 (2 ml) were used to prepare the reaction mixture (5 ml). The mixtures were incubated at 37°C for 15 min and then heated at 70°C for 5 min. After cooling, the absorbance was measured at 660 nm by using the vehicle as blank. Diclofenac sodium at 50, 100, 150, 200, and 250 μg/ml concentrations was used as reference standard [11]. The percent inhibition of protein denaturation was calculated by following equation: where A t is the absorbance of test sample, and A c is the absorbance of control.

Carrageenan-induced rat paw edema model
Acute anti-inflammatory activity was performed by using carrageenan-induced rat paw edema model [12]. Animals were divided into twelve groups (n = 6), placed into different cages, and kept under standard conditions with free access to food and water.
Group 1 served as control received 1% CMC (carboxyl methyl cellulose). Group 2 served as positive Table 1 Chemical structures, names, and abbreviations of the synthesized compounds control received carrageenan. Group 3 served as standard received diclofenac sodium 20 mg/kg and suspended in 1% CMC [13]. Groups 4 to 12 were treated with test compounds G1, G2, and G3 with varying doses (5, 10, and 20 mg/kg), respectively. Edema was induced by injecting 0.1 ml of a 1% solution of carrageenan in saline into the sub plantar region of the right hind paw of the Wister rat [14]. The vehicle, test solutions, and diclofenac were administered 60 min prior to the injection of the phlogestic agent (carrageenan). The volume of paw edema was measured at 0, 1/2, 1, 2, 3, 4, 5, and 24 h after injection of carrageenan using plethysomometer [15].The % inhibition in edema volume was calculated for each animal group using the following formula: Mean paw of inflammation of control Â 100

Cotton pellet-induced granuloma model
Sub-acute anti-inflammatory activity was assessed by cotton pellet-induced granuloma in rat. Animals were divided into eight groups (n = 6) and kept under standard conditions with free access to food and water. The rats were anesthetized under light ether anesthesia, and an incision was made in the lumbar region by curved scissors, a subcutaneous tunnel was made, and a sterilized cotton pellet (100 ± 1 mg) was inserted using blunted forceps. Group 1 served as control received 1% CMC. Group 2 served as standard received diclofenac sodium 10 mg/kg [16]. Groups 3 to 8 received 10 and 20 mg/kg test compound (G1, G2, and G3) orally for seven consecutive days from the day of cotton pellet insertion. On the 8th day, under ether anesthesia cotton pellets were removed, dried, and weighed. The % inhibition was calculated by using the following formula [17]: where W t is the weight of the cotton pellet of the test solution, and W c is the weight of the cotton pellet of the control.

Synthesis and spectral data
All the compounds were found to be soluble in dimethyl sulphoxide (DMSO). The mobile phase used for thin layer chromatography was petroleum ether:ethyl acetate: methanol.   (Fig. 3).
MS: Mass spectrum shows the formation of molecular ion peak at m/z = 439.5 (C 21 H 16 BrN 3 O 3 -438.27) (Fig. 7).    (Fig. 9). MS: Mass spectrum shows the formation of molecular ion peak at m/z = 425.1 (C 20 H 14 BrN 3 O 3 -424.24) (Fig. 10). Table 2 presents G1, G2, and G3 that showed significant inhibition of protein denaturation, i.e., 40%, 41.66%, and 49.16%, respectively, at 250 μg/kg, which were comparable to that of the diclofenac-treated group (70%). Figure 11 presents percent inhibition in the anti-inflammatory activity of compound G1, G2, and G3 and was evaluated in carrageenan-induced rat paw edema model. The results of the study indicated, oral administration of compounds G1, G2, and G3 significantly (p < 0.01) decreased the carrageenan-induced rat paw edema volume after 2 h of carrageenan injection. The results of the study were comparable to that of diclofenac-treated animals ( Table 3).

Cotton-pellet induced granuloma
Groups treated with test compounds G1, G2, and G3 showed significant reduction in the weight of the cotton pellet when compared with the control group. Among these test compounds, G3 at a dose of 20 mg/kg showed about 50.68% inhibition in granuloma weight, which is comparable to that of diclofenac (54.20%) ( Table 4). Heat-induced denaturation of egg albumin proteins is as effective as native proteins in provoking delayed hypersensitivity like sickness and glomerulo-nephritis [18]. Moreover, it was already proved that conventional NSAIDs do not act only by the inhibition of endogenous prostaglandin production by blocking the COX enzyme but also by prevention of denaturation of proteins [19]. From the result obtained in the present study, the quinazolinone derivatives showed considerable anti-inflammatory activity by Values are expressed as mean ± SEM (n = 6) and ANOVA followed by Dunnett's test *p < 0.05, **p < 0.001 when compared with control inhibiting the denaturation of proteins, and its effect was comparable to that of diclofenac ( Table 2). The present findings exhibited a concentrationdependent inhibition of protein denaturation by quinazolinone derivatives (G1, G2, and G3) throughout the concentration range. Diclofenac sodium (at the concentration range of 50-250 μg/ml) was used as reference drug exhibited concentration-dependent inhibition of protein denaturation; however, the effect of diclofenac was found to be more significant (70% at 250 μg/ml) as compared to synthesized compounds. Among these, G2 and G3 showed prominent inhibition of protein denaturation 41.66% and 49.16% at 250 μg/ml concentration, respectively.
Carrageenan, a muco-polysaccharide, is a most commonly and well-studied phlogistic agent, producing edema in 3 h [20]. In the present study, quninzoline derivative showed promising anti-inflammatory activity; this may be due to reduction in prostaglandin synthesis via COX inhibition [21]. In the present study, quninzoline derivatives, i.e., G1, G2, and G3, showed dose-dependent inhibition of the second phase of carrageenan-induced rat paw edema, suggesting the inhibition of prostaglandins release [22]. Among all test compounds, G3 showed more significant inhibition as compared to G1 and G2 (G3 > G1 > G2). This suggests that the possible mechanism of the antiinflammatory action of the test compounds may be due to reduction in pro-inflammatory mediators like prostaglandins, histamine, and serotonin [5].
The cotton pellet-induced granuloma pouch method is widely used to assess the transudative and proliferative components of chronic inflammation [23]. In the present study, G3 at a dose of 10 mg/kg showed significant reduction in the cotton pallet weight 50.68% when compared with control, whereas animals treated with diclofenac depicted significant reduction in cotton pallet weight (54.20%). Newly synthesized compound of 2,3-disubstituted quinazolin-4(1H)-one derivatives may possess prominent ant-inflammatory action with minimum side effect over conventional quinazoline derivative.

Conclusion
A series of new 2,3-disubstituted quinazolin-4(1H)-one derivatives (G1, G2, and G3) were synthesized as well as characterized for their structure elucidation and screened for its anti-inflammatory (in vivo and in vitro).
Among these synthesized compounds, G1 and G3 exhibit more significant anti-inflammatory activity in both in vivo and in vitro models.
In future, this antagonism between inflammation and immunity also affects the outcome of cancer treatment and needs to be considered when designing new therapeutic approaches. Values are expressed as mean ± SEM (n = 6) and ANOVA followed by Dunnett's test *p < 0.05, **p < 0.001 when compared with the control group