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

Abstract

Background

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.

Results

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.

Conclusion

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.

1 Background

Quinazolinone nucleus is an interesting molecule among the most important classes of an aromatic bicyclic compound with two nitrogen atoms in structure [1]. It consists of an aromatic benzo pyrimidine system made up of two fused six-member simple aromatic ring benzene and pyrimidine ring [2].

Recently, many efforts have been focused by researchers on the modification of quinazolinone ring for development of pharmaceutical and clinical compound. A brief survey about biological importance of quinazolinone and their derivatives revealed that large number of publications began to appear after 1960s [3, 4]. Most of quinazolinone derivative which have been identified consist of wide range of antioxidant, antiviral, anticonvulsant, anti-inflammatory, antitubercular, antimicrobial, and analgesic activity [5]. Nowadays, quinazolin-4(1H)-one nucleus has attracted the attention of medicinal chemists due to their clinical uses.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed for the treatment of acute and chronic inflammation, pain, and fever [6]. However, long-term clinical usage of NSAIDs is associated with significant side effects such as gastro-intestinal lesions, 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.

2 Methods

2.1 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 (¹HNMR) 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.

2.2 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.

2.3 Experimental procedure

2.3.1 Chemical synthesis and characterization

General procedure for the synthesis of compounds

Isatoic anhydride (1 mmol) and substituted aniline (1.1 mmol) were mixed in a sufficient mixture of equal volumes of ethanol and water in a round bottom flask. Then, 5 ml of acetic acid was added to the mixture and stirred for approximately 20–30 min to dissolve the 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.

Fig. 1

Preparation of 2,3-disustituted quinazolin-4(1H)-one derivative

Table 1 Chemical structures, names, and abbreviations of the synthesized compounds

2.3.2 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:

% inhibition = 100 × [A_t/A_c−1]

where A_t is the absorbance of test sample, and A_c is the absorbance of control.

2.3.3 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 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:

$\%\mathrm{inhibition}=\frac{\mathrm{Mean}\;\mathrm{paw}\;\mathrm{of}\ \mathrm{inflammation}\ \mathrm{of}\ \mathrm{control}\hbox{--} \mathrm{Mean}\;\mathrm{paw}\;\mathrm{of}\ \mathrm{inflammation}\ \mathrm{of}\ \mathrm{test}}{\mathrm{Mean}\;\mathrm{paw}\;\mathrm{of}\ \mathrm{inflammation}\ \mathrm{of}\ \mathrm{control}}\times 100$

2.3.4 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]:

% inhibition = (1−W_t/W_c) × 100

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.

3 Results

3.1 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.

3.1.1 Synthesis of 3-(3,4-dichlorophenyl)-2-(2-nitrophenyl)-2,3-dihydroquinazolin-4(1 h)-one

Compound G1

The reaction provided 50% yield. TLC monitored by (pet ether, ethyl acetate, methanol) (4:1:1) and R_f value was (0.30). Recrystallized using hot ethanol and melting point found to be in the range of 201–205 °C.

IR (KBr): ν cm–¹ 3309 (N-H stretching), 3089 (C-H stretching), 1633 (C=O stretching)), 1614 (C=C stretching), 1529 (NO₂ stretching), 1126 (C-N stretching), 825 (C-Cl stretching) (Fig. 2).

Fig. 2

IR spectrum of compound 1 [G1]

¹HNMR (400 MHz, DMSO-d6) δppm 8.295 (s, 1H, N-H), 8.155-8.152 (quartet, J = 7.6 Hz, 6.4 Hz, 1.2 Hz, 1H, Ar-H), 7.820 (s, 1H, Ar-H), 7.742 (s, 1H, Ar-H), 7.650 (s, 1H, Ar-H), 7.598 (s, 1H, Ar-H), 7.328 (s, 1H, Ar-H), 7.318- 7.314 (d, J = 1.6 Hz, 2H, Ar-H), 6.782 (s, 1H, C-H), 6.767 (s, 1H, Ar-H), 6.650-6.645 (d, J = 2 Hz, 2H, Ar-H) (Fig. 3).

Fig. 3

1HNMR spectrum of compound 1 [G1]

Mass spectrum shows the formation of molecular ion peak at m/z = 414.1 (C₂₁H₁₃Cl₂N₃O₃–414.24) (Fig. 4).

Fig. 4

MASS spectrum of compound 1 [G1]

3.1.2 Synthesis of 3-(4-bromo-2-methylphenyl)-2-(2-nitrophenyl)-2,3-dihydroquinazolin-4-(1H)-one

Compound G2

The reaction provided 76.99% yield. The TLC monitored by (pet ether 3:3 n-Hexane) and R_f value was (0.28). Recrystallized using hot ethanol and melting point found to be in the range of 230–231 °C.

IR (KBr): ν cm–¹ 3197 (N-H stretching), 3097 (C-H stretching), 3001 (Ar- H stretching), 1666 (C=C stretching) 1373 (CH₃ stretching), 1352 (NO₂ stretching), 1180 (C-N stretching), 698 (C-Br- stretching) (Fig. 5).

Fig. 5

IR spectrum of compound 2 [G2]

¹HNMR (400 MHz, DMSO-d6) δppm 8.275 (s, 1H, Ar-H), 7.872- 7.857 (d, J = 6 Hz, 2H, Ar-H), 7.742 (s, 1H, Ar-H), 7.460 (s, 1H, Ar-H), 7.328 (s, 2H, Ar-H), 7.272 (s, 1H, Ar-H), 6.773-6.751 (d, J = 8.8 Hz, 2H, Ar-H), 6.671 (s, 1H, Ar-H), 6.206 (s, 1H, C-H), 2.321 (s, 3H, CH₃) (Fig. 6).

Fig. 6

1HNMR spectrum of compound 2 [G2]

MS: Mass spectrum shows the formation of molecular ion peak at m/z = 439.5 (C₂₁H₁₆BrN₃O₃-438.27) (Fig. 7).

Fig. 7

MASS spectrum of compound 2 [G2]

3.1.3 Synthesis of 3-(4-bromophenyl)-2-(2–nitrophenyl)-2,3-dihydroquinazolin-4-(1H)-one

Compound G3

The reaction provided 39.26% yield. The TLC monitored by (pet ether 3:2 n-hexane) and R_f value was (0.55). Recrystallized using hot ethanol and melting point found to be in the range of 227–228 °C.

IR (KBr): ν cm–¹ 3331 (N-H stretching), 3089 (C-H stretching), 3082 (Ar-H stretching), 1768(C=O stretching), 1614(C=C stretching), 1519 (NO₂ stretching), 1176 (C-N stretching), 717 (C-Br-stretching) (Fig. 8).

Fig. 8

IR spectrum of compound 3 [G3]

¹HNMR (400 MHz, DMSO-d6) δppm 8.277- 8.273 (triplet, J = 1.6 Hz, 1.2 Hz, 1H, N-H), 8.139 (s, 1H, Ar-H), 7.739-7.736 (d, J = 1.2 Hz, 1H, Ar-H), 7.633 (s, 1H, Ar-H), 7.556-7.552 (d, J = 1.6 Hz, 1H, Ar-H), 7.542-7.539 (d, J = 1.2 Hz, 2H, Ar-H), 7.318-7.316 (d, J = 0.8 Hz, 2H, Ar-H), 7.283-7.279 (d, J = 1.6 Hz, 1H, Ar-H), 7.270- 7.266 (d, J = 1.6 Hz, 1H, Ar-H), 6.805 (s, 1H, Ar-H), 6.761 (s, 1H, Ar-H), 6.565-6.559 (d, J = 2.4 Hz, 1H, C-H) (Fig. 9).

Fig. 9

1HNMR spectrum of compound 3 [G3]

MS: Mass spectrum shows the formation of molecular ion peak at m/z = 425.1 (C₂₀H₁₄BrN₃O₃-424.24) (Fig. 10).

Fig. 10

MASS spectrum of compound 3 [G3]

3.2 Pharmacological methods for screening of synthesized compounds

3.2.1 In vitro anti-inflammatory activity

Egg albumin protein denaturation

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%).

Table 2 Effect of test compounds on egg albumin protein denaturation

3.2.2 Anti-inflammatory activity

Carrageenan-induced rat paw edema

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).

Fig. 11

Percentage inhibition of test compounds on carrageenan-induced rat paw oedema

Table 3 Effect of test compounds on carrageenan induced rat paw edema

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).

Table 4 Influence of derivatives on cotton pellet granuloma pouch method

4 Discussion

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 inhibiting the denaturation of proteins, and its effect was comparable to that of diclofenac (Table 2).

The present findings exhibited a concentration-dependent 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 anti-inflammatory 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.

5 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.