untitled European Journal of Chemistry 8 (3) (2017) 317‐320 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2017 Atlanta Publishing House LLC ‐ All rights reserved ‐ Printed in the USA http://dx.doi.org/10.5155/eurjchem.8.3.317-320.1586 European Journal of Chemistry Journal webpage: www.eurjchem.com Synthesis of quinazolinones derivatives an antiproliferative agent against human lung carcinoma cells Satish Uttamrao Deshmukh 1, Kiran Ramesh Kharat 2, Gajanan Gulabrao Kadam 3 and Rajendra Pundlikrao Pawar 1,* 1 Department of Chemistry, Deogiri College, Aurangabad, 431005 (MS), India 2 Department of Biotechnology, Deogiri College, Aurangabad, 431005 (MS), India 3 Department of Chemistry, Shri Datta Arts, Commerce and Science College, Hadgaon, 431712 (MS), India * Corresponding author at: Department of Chemistry, Deogiri College, Aurangabad, 431005 (MS), India. Tel.: +91.0240.2367335. Fax: +91.0240.2367301. E‐mail address: rppawar@yahoo.com (R.P. Pawar). COMMUNICATION INFORMATION ABSTRACT DOI: 10.5155/eurjchem.8.3.317-320.1586 Received: 20 May 2017 Received in revised form: 31 July 2017 Accepted: 05 August 2017 Published online: 30 September 2017 Printed: 30 September 2017   2,3‐Dihydroquinazolin‐4(1H)‐one derivatives (3a‐p) were synthesized in excellent yields. These compounds were screened for antiproliferative activity against A549 cells and were found as potent cytotoxicity. Compounds A4, A8, A10 found to be more promising antiproliferative against the lung carcinoma A549 cells. IC50 values for compounds A4, A8 and A10 were found to be 8.6, 8.9 and 8.1 μg/L against A549 cells, respectively. KEYWORDS Ionic liquid Green chemistry 2‐Aminobenzamide Antiproliferative activity Lung carcinoma A549 cells 2,3‐Dihydroquinazolin‐4(1H)‐one Cite this: Eur. J. Chem. 2017, 8(3), 317‐320 1. Introduction Various quinazolinones were found as natural products and attracted a vast attention because of their wide range of biological activities. Previous reports of their derivatives showed significant effects in different pharmaceutical pro‐ ducts [1‐3]. Quinazolinone derivatives also reported as poten‐ tial antimicrobial [4], antimalarial [5], antitumor [6,7], anti‐ depressant [8], analgesic [9], CNS stimulant [10], tranquilizing [11] and anticancer agent [12]. Reaction of 2‐aminobezamide and aromatic aldehydes were widely used for the synthesis of quinazolinones derivatives. This is an efficient method for the synthesis of potentially active quinazolinone derivatives. Quinazolinone moieties are significant for biological activities in healthcare and medicines. In addition, quinazolinone heterocyclic is also utilized as a backbone skeleton of some drugs such as cloroqualone, afloqualone and quinethazone (Figure 1). Therefore a simple, mild, and versatile preparation method of quinazolinones in one‐pot reaction is still highly desirable. In continuation of our research devoted to the new methods for the preparation of heterocyclic compounds via multicomponent reactions, we describe the synthesis of quinazolinone derivatives using 2‐anthranilamides and aromatic substituted aldehydes in ethanol under reflux condition. Several methods has been used for cyclocondensation of 2‐ anthranilamides with aldehydes in the presence of various promoting agents, such as Lewis acids CuCl2.2H2O [13], Cerium (iv) sulphates tertrahydes [14], cyanuric chlorides [15], Amberlyst‐15 [16] and SnCl2 [17]. Reaction was also carried out by acids catalyzed one‐pot synthesis using PTSA [18], silica‐SO3H [19], acetic acid [20] and also in presence of alum [21] montmorillonite K‐10 [22], thiamine hydrochloride (VB1) [23]. Recently, nano‐catalyst metal‐CNTs [24], Wang‐OSO3H [25], basic ionic liquid [26] and [bmim] HSO4 [27], ceric ammonium nitrate [28] were also used in this transformation. Eventhough significant improvements and developments using various catalysts have been achieved; this protocol lacks implementation and limitations such as the use of excess of solvent, moisture sensitive, corrosive, expensive catalysts, tedious separation procedures for recovery of catalysts leads to toxic waste generation along with a long reaction time, high temperature and low yield of the desired products. 318 Deshmukh et al. / European Journal of Chemistry 8 (3) (2017) 317‐320 Figure 1. Some example of quinazolinone active heterocyclic compounds. Scheme 1 Herein, we report a mild and efficient protocol for the synthesis of, 2,3‐dihydroquinazolin‐4(1H)‐one derivatives in presence ionic liquid under mild condition (Scheme 1). 2. Experimental 2.1. Instrumentations 1H NMR spectra were recorded in solvent DMSO using Varian spectrometer instrument 1H (400 MHz) at 25 °C temperature. Chemical shifts were reported in δ ppm with tetramethylsilane as an internal standard (TMS, δ = 0.0). Infrared spectra were recorded on a FT‐IR spectrometer (Shimadzu FT‐IR 8300 spectrophotometer) in a range of 400‐ 4000 cm‐1. Mass spectra were recorded on VG‐7070H Micromass. Melting points taken are uncorrected. 2.2. Synthesis of the quinazolinones derivatives In a round bottom flask (10 mL), 2‐aminobenzamide (1 mmol) and substituted aldehydes 2a (1 mmol) were added. To it 1‐ethyl‐3‐methylimidazolium hydrogen sulphate (10 mol %) catalysts was added in ethanol (5 mL). The reaction mixture was heated under reflux for appropriate time. The progress of reactions was monitored by using TLC. After completion of reaction the product obtained was poured onto crushed ice. The solid obtained was filtered and recrystallized in hot ethanol to afford the pure product. 2‐(4‐Fluorophenyl)‐2,3‐dihydroquinazolin‐4(1H)‐one (3a): Color: White. M.p.: 203‐205 °C. FT‐IR (KBr, ν, cm‐1): 2915 (C‐ H), 1625 (C=C). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 5.77 (s, 1H, N‐H), 6.69 (d, 2H, Ar‐H), 7.19 (s, 1H, Ar‐H), 7.27 (t, 1H, C‐ H), 7.30 (dd, 4H, Ar‐H), 7.55 (d, 1H, Ar‐H), 8.56 (s, 1H, N‐H). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 66.42, 115.0, 115.51, 115.53, 118.0, 126.99, 130.50, 132.87, 139.25, 149.07, 162.06, 163.00. EI‐MS (m/z): 243 [M+1]+. 2‐(3‐Nitrophenyl)‐2,3‐dihydroquinazolin‐4(1H)‐one (3o): Color: White. M.p.: 205‐206 °C. FT‐IR (KBr, ν, cm‐1): 2914 (C‐ H), 1610 (C=C), 1456 (C‐Cl). 1H NMR (400 MHz, DMSO‐d6, δ, ppm): 5.73 (s, 1H, N‐H), 6.7 (d, 2H, Ar‐H), 7.17 (s, 1H, Ar‐H), 7.22 (t, 1H, C‐H), 7.4 (dd, 4H, Ar‐H), 7.6 (d, 1H, Ar‐H), 8.39 (s, 1H, N‐H). 13C NMR (100 MHz, DMSO‐d6, δ, ppm): 66.76, 78.11 79.28, 114.37, 118.11, 126.98, 128.14, 132.54, 133.74, 140.34, 146.57, 163.32. EI‐MS (m/z): 269 [M+1]+. 2.3. Cell culture conditions A lung carcinoma cell A549 were maintained in RPMI‐ 1640 containing 10 % v/v FBS (Life technologies Inc. USA). The cells sub‐cultured and incubated at 37 °C and 5% CO2 incubator. For all experiments, the cells were freshly grown by diluting the stock solution with D‐phosphate‐buffered saline (pH = 7.2) (Hi Media, India). 2.4. Determination of cell viability and IC50 The antiproliferative effect of compounds on Lung carci‐ noma cells was evaluated by Vybrant MTT Cell Proliferation Assay Kit (Life technologies Inc. USA). The procedure was adopted described previously [29]. Briefly, the cells were plated at ~1×104 cells in each well of 96 well plate in 100 μL of RPMI‐1640 medium. All the compounds (0, 5, 10, 25, 50 µg/mL) were added to each well separately. The standard reference drug Doxorubicin (10 uM) was used. Cell viability was determined after 24 hrs incubation in CO2 incubator at 37 °C. MTT (5 mg/mL in PBS) was added to each well and incubated for 4 hrs. The absorbance was recorded at 490 nm using a 96 well Multiscan Ascent (Thermo Inc.USA). The IC 50 was calculated by using formula 2.5. Statistical analysis Dose‐response curves were plotted to determine IC50 values using the GraphPad Prism. p‐values < 0.05 were considered significant. 3. Results and discussion Initially, we tried anthranilamide (1 mmol) and 4‐NO2 benzaldehyde (2a) (1 mmol) as the model reaction by using copper silicates catalyst in acetonitrile at room temperature; obtained product formation takes place in very low yield (Table 1, Entry 1). To improve the product yield the reaction condition like sonification and reflux condition of reaction were changed and the yield was increased up to 80‐82 % (Table 1, Entry 2‐3). The modified conditions for reactions along with the alteration in the temperature enhance the product yield (Table 1, Entry 4‐6). Deshmukh et al. / European Journal of Chemistry 8 (3) (2017) 317‐320 319 Table 1. Optimization of reaction conditions using various solvent and catalyst. Entry Solvent Catalyst Temp. (°C) Yield (%) 1 Acetonitrile Copper silicate RT 60 2 Acetonitrile Copper silicate Sonication 80 3 Acetonitrile Copper silicate 78 82 4 Ethanol Copper silicate RT 70 5 Ethanol Copper silicate Sonication 84 6 Ethanol Copper silicate 78 86 7 Acetonitrile Camphor sulphonic acid RT 68 8 Acetonitrile Camphor sulphonic acid Sonication 72 9 Acetonitrile Camphor sulphonic acid 78 74 10 Ethanol Camphor sulphonic acid RT 70 11 Ethanol Camphor sulphonic acid Sonication 74 12 Ethanol Camphor sulphonic acid 78 77 13 Acetonitrile 1‐Ethyl‐3‐methylimidazolium hydrogen sulphate RT 74 14 Acetonitrile 1‐Ethyl‐3‐methylimidazolium hydrogen sulphate Sonication 81 15 Acetonitrile 1‐Ethyl‐3‐methylimidazolium hydrogen sulphate 78 82 16 Ethanol 1‐Ethyl‐3‐methylimidazolium hydrogen sulphate RT 84 17 Ethanol 1‐Ethyl‐3‐methylimidazolium hydrogen sulphate Sonication 90 18 Ethanol 1‐Ethyl‐3‐methylimidazolium hydrogen sulphate 78 94 19 Ethanol 1‐Ethyl‐3‐methylimidazolium hydrogen sulphate, 15 % 78 94 20 Ethanol 1‐Ethyl‐3‐methylimidazolium hydrogen sulphate, 20 % 78 94 Table 2. Reaction of 2‐anthranilamide and various aldehydes for the preparation of 2,3‐dihydroquinazolinone. Entry Aldehydes (2) R1 R2 R3 2,3‐Dihydroquinazolinone (3a‐p) Code Time (min) Yield a (%) 1 4‐Fluorobenzaldehyde H H ‐F 3a A1 142 93 2 3‐Nitrobenzaldehyde H NO2 H 3b A2 155 89 3 4‐Hydroxy‐3‐methoxybenzaldehyde H OCH3 OH 3c Â3 163 84 4 1‐Naphthaldehyde H H H 3d A4 161 87 5 4‐Methylbenzaldehyde H H Me 3e A5 154 89 6 4‐Bromobenzaldehyde H H Br 3f A6 130 91 7 4‐Hydroxybenzaldehyde H H OH 3g A7 150 87 8 3‐Hydroxybenzaldehyde H OH H 3h A8 167 81 9 2‐Nitrobenzaldehyde NO2 H H 3i A9 168 86 10 2,4‐Dichlorobenzaldehyde Cl Cl H 3j A10 150 89 11 3‐Bromobenzaldehyde H Br H 3k A11 160 84 12 2,3‐Dichlorobenzaldehyde Cl Cl H 3l A12 150 87 13 Benzaldehyde H H H 3m A13 180 80 14 4‐Nitrobenzaldehyde H H NO2 3n A14 140 94 15 3‐Chlorobenzaldehyde H NO2 H 3o A15 120 93 16 2,4,6‐Trimethoxybenzaldehyde OCH3 OCH3 OCH3 3p A16 170 78 a Isolated yield. The use of camphor sulphonic acids in acetonitrile solvents with different reaction conditions has been carried out (Table 1, Entry 7‐9). It indicates, there is a scope to increase yield of product by changing the solvent as ethanol with different reaction conditions (Table 1, Entry 9‐12). Our interest in the area of developing green and sustainable method, has forced us to investigate an alternate milder method for the synthesis of 2,3‐dihydroquinazolin‐4(1H)‐one derivatives (Table 2). Herein, we report a green protocol for the synthesis of synthesis of 2,3‐dihydroquinazolin‐4(1H)‐one derivatives under reflux conditions by using cheap and readily available ionic liquids as catalyst under milder reaction conditions (Table 1, Entry 13). To change the reaction conditions with acetonitrile at sonifaction and reflux condition, good yield of product were obtained (Table 1, Entry 14‐15). The reaction solvent has been replaced to ethanol at room temperature reaction conditions gave the good yield of product (Table 1, Entry 16‐17). The best result was obtained at reflux conditions (Table 1, Entry 18). Increasing the amount of catalyst (15 mol%) did not improve the yield (Table 1, Entry 19‐20). Whereas in the absence of catalyst less amount product was obtained. 3.1. Biological activity 3.1.1. Determination of IC50 A lung carcinoma cells (A549) were treated with screening compounds and antiproliferative effect was evaluated by MTT assay. A dose dependant, decreases the growth of cancer cells with increasing concentration of the compounds. IC50 value of compounds in A549 cells are shown in Figure 2. The results obtained by MTT assay at 490 nm by addition of compounds to cancer cells and incubated for 24 h at 37 °C in CO2 incubator (5 % CO2) where a) A2, A3, A4, A5 compounds, b) A6, A7, A8, A9 compounds, c) A10, A11, A12, A13 and d) A14, A15, A16, Dox. The IC50 calculated by using the GraphPad Prism software. The MTT assay was used to evaluates the antiproliferative action of compounds at different concentrations (0, 5, 10, 25, 50 μg/L) on A549 cells. After 24 h exposure to compounds; approximately 2‐fold decrease in cell survival between the control (0 μg/L) and compounds treated cells has been reported (Figure 2). 320 Deshmukh et al. / European Journal of Chemistry 8 (3) (2017) 317‐320 (a) (b) (c) (d) Figure 2. Dose dependent effect of quinazolinones derivatives on the growth of cancer cells (A 549) where a) A2, A3, A4, A5 compounds, b) A6, A7, A8, A9 compounds, c) A10, A11, A12, A13 and d) A14, A15, A16, Dox. Compound A4, A8, A10 were found to be more promising antiproliferative against the lung carcinoma A549 cells. The IC50 values for compounds A4, A8 and A10 were found to be 8.133, 9.056 and 9.169 μg/L in A549 cells, respectively. Doxorobucin was used as a standard reference drug for this assay. 4. 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