Novel antiacanthamoebic compounds belonging to quinazolinones
Ayaz Anwar, Muhammad Saquib Shahbaz, Syed Muhammad Saad, Kanwal, Khalid Mohammed Khan, Ruqaiyyah Siddiqui, Naveed Ahmed Khan
PII: S0223-5234(19)30705-6
DOI: https://doi.org/10.1016/j.ejmech.2019.111575
Reference: EJMECH 111575
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 March 2019 Revised Date: 29 July 2019 Accepted Date: 30 July 2019
Please cite this article as: A. Anwar, M.S. Shahbaz, S.M. Saad, Kanwal, K.M. Khan, R. Siddiqui, N.A. Khan, Novel antiacanthamoebic compounds belonging to quinazolinones, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111575.
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1Novel antiacanthamoebic compounds belonging to Quinazolinones
2Ayaz Anwar1#, Muhammad Saquib Shahbaz2#, Syed Muhammad Saad2,3, Kanwal2, Khalid
3Mohammed Khan2,4,* Ruqaiyyah Siddiqui1, and Naveed Ahmed Khan1∗*
41Department of Biological Sciences, School of Science and Technology, Sunway University,
5Subang Jaya 47500, Selangor, Malaysia.
62H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
7Sciences, University of Karachi, Karachi 75270, Pakistan.
83Department of Chemistry, University of Karachi, Karachi75270, Pakistan
94Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
10
11
Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia
Corresponding Authors: **Naveed A. Khan, E-mail: [email protected]; Tel: +603-74918622 Ext. 7169; Fax: +603-56358630; *Khalid M. Khan, [email protected], [email protected], Tel.: 0092-21-34824910; Fax: 0092-21-34819018: # Both authors contributed equally.
12 ABSTRACT
13 We report one-pot synthesis of a series of new 3-aryl-8-methylquinazolin-4(3H)-ones
14(QNZ) and their antimicrobial activity against Acanthamoeba castellanii belonging to T4
15genotype. A library of fifteen synthetic derivatives of QNZs was synthesized, and their structural
16elucidation was performed by using proton nuclear magnetic resonance (1H-NMR) spectroscopy
17and electron impact mass spectrometry (EI-MS). Elemental analyses and high-resolution mass
18spectrometry data of all derivatives were found to be in agreeable range. Amoebicidal assays
19performed at concentrations ranging from 50 to100 µ g/mL revealed that all derivatives of QNZ
20significantly decreased the viability of A. castellanii and QNZ 2, 5, 8, and 13 were found to have
21efficient antiamoebic effects. Field emission scanning electron microscopy (FESEM) imaging of
22compounds 5 and 13 showed that these compounds cause structural alterations on the walls of A.
23castellanii. Furthermore, several QNZs inhibited the encystation and excystationas well as
24abolished A. castellanii-mediated host cells cytopathogenicity in human cells. Whereas, these
25QNZs showed negligible cytotoxicity when tested against human cells in vitro. Hence, this study
26identified potential lead molecules having promising properties for drug development against A.
27castellanii. A brief structure-activity relationship is also developed to optimize the hit of most
28potent compounds from the library. To the best of our knowledge, it is first of its kind medicinal
29chemistry approach on a single class of compounds i.e., quinazolinone against keratitis and brain
30infection causing free-living amoeba, A. castellanii.
31
32
Keywords: Quinazolinone; Synthesis; Acanthamoeba; Antiamoebic; FESEM.
33 INTRODUCTION
34 Acanthamoeba species are among most abundant free-living amoebae widely distributed
35in water and soil [1]. They survive in two phenotypic forms; reproductive, opportunistic
36pathogenic trophozoite state, and resistant, dormant, double-walled protected cysts [2]. The
37pathologies associated with Acanthamoeba species are blinding keratitis, and fatal brain
38infection, granulomatous amoebic encephalitis [3]. Despite the morbidity and mortality, there is
39no definite course of action to treat Acanthamoeba infections [4]. The recommended drugs
40against Acanthamoeba infections have poor specificity and ineffectiveness against cysts which
41results in side effects and recurrence [5]. A vast number of compounds ranging from synthetic,
42natural, and nanomaterials have shown in vitro potential against Acanthamoeba, but the
43prognosis remains strikingly poor without the approval or interest of pharmaceutical industries
44[6]. Recently, high throughput assays and computational studies have identified promising
45synthetic targets which may serve as lead compounds for drug development against free-living
46amoebae [7-10].
47Based on our interest in drug development against neglected microbes, the objective of this
48study was to evaluate the quinazolinone derivatives [11-13]. Heterocyclic compounds of class
49quinazoline represent promising scaffolds in medicinal chemistry [14]. Several studies have been
50reported on the biological activity of quinazoline analogues, including their antibacterial,
51fungicidal, antiviral, antileishmanial, and anti-trauma activities [15-17]. They are used in
52pharmaceuticals and agrochemicals [18], for example, fluquinconazole (Fig. 1) a known
53fungicide for the regulation of agriculture diseases [19].
54Different derivatives of quinazolines and quinazolinones have been previously synthesized
55and reported as antileishmanial agents (Fig. 2) [12,13]. Keeping in mind the antiparasitic aspect
56of this moiety, we had evaluated antiacanthamoebic activity of these synthetic analogs. In this
57report, for the first time, we present a simple one-pot synthesis of chemically versatile
58quinazolinone derivatives, and evaluation of their antiacanthamoebic potential. In this library,
59fifteen analogues of 3-aryl-8-methylquinazolin-4(3H)-ones were synthesized (Table 1), and a
60brief structure-activity relationship is developed to optimize the potent lead compound for further
61studies.
62EXPERIMENTAL SECTION
63Materials and Methods
64 All reagents used in this study were commercially procured from Sigma until stated
65otherwise. HPLC grade solvents were used for synthesis and purification of compounds.
66Synthesis of 3-aryl-8-methylquinazolin-4(3H)-ones
67 A library of fifteen analogs of 3-aryl-8-methylquinazolin-4(3H)-ones was synthesized via
68one pot reaction of 2-amino-3-methylbenzoic acid (1 mmol) with triethoxymethane (3 mmol)
69and different substituted anilines (1 mmol) in acidic medium using acetic acid (Scheme-1). The
70reaction mixture was boiled and kept on reflux, while the reaction progress was examined with
71thin layer chromatography (TLC). After the complete absence of the starting materials, the
72reaction mixture was poured in water, precipitate formed were filtered, thoroughly washed with
73ultrapure water and dried under vacuum. The solid was crystallized from ethanol. The structures
74of these synthetic derivatives were elucidated by 1H-NMR, 13C-NMR, and EI-MS. Elemental
75analyses and high resolution EI-MS data of all compounds were found to be in consistent range.
76Based on the TLC and spectroscopic analyses, all compounds were found to contain no
77impurities.
78Characterization of 3-aryl-8-mehtylquinazolin-4(3H)-ones
793-(4′-Chlorophenyl)-8-methylquinazolin-4(3H)-one (1)
80 Yield: 76%; Light Grey Solid; m.p. 225-227 ºC; Rf: 0.86 (ethyl acetate/hexanes, 3:7); 1H-
81NMR: (400 MHz, DMSO-d6): δH 8.36 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H),
827.64 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.49 (t, J = 7.6 Hz, 1H), 2.57 (s, 3H); 13C-
83NMR: (100 MHz, DMSO-d6): δC 160.1, 146.0, 145.9, 136.4, 135.5, 135.0, 133.3, 129.4, 129.1,
84126.9, 124.0, 121.7, 17.0; EI-MS: m/z (rel. abund. %), 272 [M++2] (30), 270 [M]+ (100), 242 (5),
85244 (2), 111 (8), 105 (12); Anal. Calcd for C15H11ClN2O: C, 66.55; H, 4.10; Cl, 13.10; N, 10.35;
86O, 5.91; found: C, 66.51; H, 4.13; Cl, 13.12; N, 10.33; O, 5.94.
873-(3′-Fluorophenyl)-8-methylquinazolin-4(3H)-one (2)
88 Yield: 70%; Off White Solid; m.p. 129-131 ºC; Rf: 0.88 (ethyl acetate/hexanes, 3:7); 1H-
89NMR: (400 MHz, DMSO-d6): δH 8.38 (s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H),
907.64 (dd, J = 8.0 Hz, 1H), 7.54 (d, J = 10.0 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.42 (dd, J = 8.8
91Hz, 1H), 7.38 (d, J = 9.2 Hz, 1H), 2.57 (s, 3H); 13C-NMR: (125 MHz, DMSO-d6): δC 162.8,
92160.9, 160.0, 146.0, 139.0, 135.5, 135.1, 130.8, 126.9, 124.1, 123.7, 121.7, 115.8, 115.2, 17.0;
93EI-MS: m/z (rel. abund. %), 254 [M]+ (100), 226 (11), 105 (15), 95 (14); Anal. Calcd for
94C15H11FN2O: C, 70.86; H, 4.36; F, 7.47; N, 11.02; O, 6.29; found: C, 70.83; H, 4.34; F, 7.44; N,
9511.06; O, 6.27.
963-(3′-Bromophenyl)-8-methylquinazolin-4(3H)-one (3)
97 Yield: 67%; Off White Solid; m.p. 132-135 ºC; Rf: 0.94 (ethyl acetate/hexanes, 3:7); 1H-
98NMR: (400 MHz, DMSO-d6): δH 8.37 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.85 (s, 1H), 7.75 (d, J =
997.6 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.49 (t, J
100= 7.6 Hz, 1H), 2.57 (s, 3H); 13C-NMR: (125 MHz, DMSO-d6): δC 160.6, 146.5, 146.3, 139.4,
101136.0, 135.6, 132.1, 131.5, 131.0, 127.4, 127.2, 124.6, 122.2, 121.9, 17.5; EI-MS: m/z (rel.
102abund. %), 316 [M++2] (91), 314 [M]+ (100), 286 (5), 288 (5), 155 (8), 157 (7), 105 (17); Anal.
103Calcd for C15H11BrN2O: C, 57.16; H, 3.52; Br, 25.35; N, 8.89; O, 5.08; found: C, 57.12; H, 3.55;
104Br, 25.38; N, 8.86; O, 5.05.
1053-(3′-Methylthiophenyl)-8-methylquinazolin-4(3H)-one (4)
106 Yield: 65%; Grey Solid; m.p. 128-130 ºC; Rf: 0.76 (ethyl acetate/hexanes, 3:7); 1H-NMR:
107(400 MHz, DMSO-d6): δH 8.36 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.50 (t,
108J = 8.0 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.43 (s, 1H), 7.39 (d, J = 8.0 Hz, 1H); 7.30 (d, J = 7.6
109Hz, 1H), 2.57 (s, 3H), 2.50 (s, 3H); 13C-NMR: (100 MHz, DMSO-d6): δC 160.1, 146.1, 146.0,
110139.6, 138.2, 135.4, 135.0, 129.5, 126.9, 125.9, 124.4, 124.0, 123.8, 121.8, 17.5, 14.4; EI-MS:
111m/z (rel. abund. %), 282 [M]+ (100), 267 (5), 254 (3), 123 (4), 105 (15); Anal. Calcd for
112C16H14N2OS: C, 68.06; H, 5.00; N, 9.92; O, 5.67; S, 11.36; found: C, 68.08; H, 5.04; N, 9.95; O,
1135.62; S, 11.34.
1143-(4′-Fluorophenyl)-8-methylquinazolin-4(3H)-one (5)
115 Yield: 68%; Ash Grey Solid; m.p. 210-212 ºC; Rf: 0.78 (ethyl acetate/hexanes, 3:7); 1H-
116NMR: (400 MHz, DMSO-d6): δH 8.35 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H),
1177.62 (dd, J = 9.0 Hz, J = 5.2 Hz, 2H), 7.49 (t, J = 7.6 Hz), 7.42 (t, J = 8.8 Hz, 2H), 2.57 (s, 3H);
11813C-NMR: (100 MHz, DMSO-d6): δC 163.0, 160.5, 160.2, 146.1, 135.5, 135.0, 133.9, 129.8,
119126.9, 124.0, 121.8, 116.1, 17.0; EI-MS: m/z (rel. abund. %), 254 [M]+ (100), 225 (8), 144 (4),
120104 (9); Anal. Calcd for C15H11FN2O: C, 70.86; H, 4.36; F, 7.47; N, 11.02; O, 6.29; found: C,
12170.85; H, 4.33; F, 7.49; N, 11.05; O, 6.27.
1223-(3′,5′-Dimethylphenyl)-8-methylquinazolin-4(3H)-one (6)
123 Yield: 74%; Off White Solid; m.p. 98-103 ºC; Rf: 0.86 (ethyl acetate/hexanes, 3:7); 1H-
124NMR: (300 MHz, DMSO-d6): δH 8.32 (s, 1H), 8.03 (d, J = 7.2 Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H),
1257.49 (t, J = 7.8 Hz, 1H), 7.14 (s, 1H), 7.12 (s, 2H), 2.57 (s, 3H), 2.3 (s, 6H); 13C-NMR: (100
126MHz, DMSO-d6): δC 160.1, 146.1, 138.5, 137.4, 135.4, 134.9, 130.0, 126.8, 124.9, 124.7, 124.0,
127121.8, 20.7, 17.0; EI-MS: m/z (rel. abund. %), 264 [M]+ (100), 249 (12), 234 (1), 105 (57); Anal.
128Calcd for C17H16N2O: C, 77.25; H, 6.10; N, 10.60; O, 6.05; found: C, 77.29; H, 6.06; N, 10.63;
129O, 6.08.
1303-(4′-Iodophenyl)-8-methylquinazolin-4(3H)-one (7)
131 Yield: 63%; Ash Grey Solid; m.p. 231-233 ºC; Rf: 0.86 (ethyl acetate/hexanes, 3:7); 1H-
132NMR: (300 MHz, DMSO-d6): δH 8.35 (s, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H),
1337.75 (d, J = 7.2 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.37 (d, J = 8.4 Hz, 2H), 2.57 (s, 3H); 13C-
134NMR: (100 MHz, DMSO-d6): δC 160.0, 146.0, 145.8, 137.9, 137.3, 135.5, 135.0, 129.7, 126.9,
135124.0, 121.7, 94.9, 17.0; EI-MS: m/z (rel. abund. %), 362 [M]+ (100), 235 (14), 220 (7), 105
136(28); Anal. Calcd for C15H11IN2O: C, 49.75; H, 3.06; I, 35.04; N, 7.73; O, 4.42; found: C, 49.73;
137H, 3.09; I, 35.01; N, 7.71; O, 4.45.
1383-(2′-Iodophenyl)-8-methylquinazolin-4(3H)-one (8)
139 Yield: 69%; Beige Solid; m.p. 162-165 ºC; Rf: 0.72 (ethyl acetate/hexanes, 3:7); 1H-
140NMR: (300 MHz, DMSO-d6): δH 8.24 (s, 1H), 8.07 (d, J = 7.5 Hz, 1H), 8.05 (d, J = 7.2 Hz, 1H),
1417.77 (d, J = 7.2 Hz, 1H), 7.61 (m, 2H), 7.51 (t, J = 7.8 Hz, 1H), 7.34 (m, 1H), 2.59 (s, 3H); 13C-
142NMR: (100 MHz, DMSO-d6): δC 159.6, 146.2, 146.1, 140.3, 139.2, 135.7, 135.2, 131.1, 129.7,
143129.5, 127.0, 124.1, 121.9, 99.5, 17.1; EI-MS: m/z (rel. abund. %), 362 [M]+ (32), 235 (100), 220
144(1), 105 (5); Anal. Calcd for C15H11IN2O: C, 49.75; H, 3.06; I, 35.04; N, 7.73; O, 4.42; found: C,
14549.77; H, 3.04; I, 35.03; N, 7.76; O, 4.40.
1463-(2′,4′-Difluorophenyl)-8-methylquinazolin-4(3H)-one (9)
147 Yield: 83%; Off White Solid; m.p. 181-185 ºC; Rf: 0.78 (ethyl acetate/hexanes, 3:7); 1H-
148NMR: (300 MHz, DMSO-d6): δH 8.38 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.80 (m, 1H), 7.75 (d, J
149= 9.0 Hz, 1H), 7.62 (m, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.36 (m, 1H), 2.58 (s, 3H); 13C-NMR: (100
150MHz, DMSO-d6): δC 146.0, 135.7, 135.4, 131.4, 131.2, 127.2, 124.0, 112.5, 112.4, 112.1, 105.2,
151105.1, 105.0, 104.7, 17.0; EI-MS: m/z (rel. abund. %), 372 [M]+ (100), 257 (5), 253 (68), 220
152(7); Anal. Calcd for C15H10F2N2O: C, 66.17; H, 3.70; F, 13.96; N, 10.29; O, 5.88; found: C,
15366.15; H, 3.72; F, 13.98; N, 10.27; O, 5.85.
1543-(4′-Bromophenyl)-8-methylquinazolin-4(3H)-one (10)
155 Yield: 84%; White Solid; m.p. 234-237 ºC; Rf: 0.84 (ethyl acetate/hexanes, 3:7); 1H-
156NMR: (300 MHz, DMSO-d6): δH 8.35 (s, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.78 (d, 2H, J = 8.4 Hz),
1577.75 (d, 1H, J = 9.0 Hz), 7.53 (d, 2H, J = 8.7 Hz), 7.50 (t, J = 8.7 Hz, 1H), 2.57 (s, 3H); 13C-
158NMR: (125 MHz, DMSO-d6): δC 160.0, 146.2, 146.0, 136.8, 135.5, 135.0, 132.1, 129.7, 126.9,
159124.0, 121.8, 121.7, 17.0; EI-MS: m/z (rel. abund. %), 314 [M]+ (100), 316 [M++2] (93), 235 (7),
160206 (6); Anal. Calcd for C15H11BrN2O: C, 57.16; H, 3.52; Br, 25.35; N, 8.89; O, 5.08; found: C,
16157.18; H, 3.56; Br, 25.34; N, 8.86; O, 5.05.
1623-(2′,5′-Dimethoxyphenyl)-8-methylquinazolin-4(3H)-one (11)
163 Yield: 81%; Dark Grey Solid; m.p. 133-135 ºC; Rf: 0.52 (ethyl acetate/hexanes, 3:7); 1H-
164NMR: (300 MHz, DMSO-d6): δH 8.20 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H),
1657.48 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 9.0 Hz, 1H), 7.12 (d, J = 3.0 Hz, 1H), 7.09 (dd, J = 8.7 Hz, J
166= 3.0 Hz, 1H),3.74 (s, 3H), 3.70 (s, 3H) 2.57 (s, 3H); 13C-NMR: (125 MHz, DMSO-d6): δC
167159.8, 153.0, 148.6, 146.8, 146.1, 135.5, 134.9, 126.8, 126.4, 123.9, 121.9, 115.4, 115.3, 113.4,
16856.2, 55.6, 17.0; EI-MS: m/z (rel. abund. %), 296 [M]+ (38), 265 (100), 250 (5); Anal. Calcd for
169C17H16N2O3: C, 68.91; H, 5.44; N, 9.45; O, 16.20; found: C, 68.94; H, 5.42; N, 9.44; O, 16.23.
1703-(3′-Chlorophenyl)-8-methylquinazolin-4(3H)-one (12)
171 Yield: 63%; Off White Solid; m.p. 125-129 ºC; Rf: 0.82 (ethyl acetate/hexanes, 3:7); 1H-
172NMR: (300 MHz, DMSO-d6): δH 8.38 (s, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.76 (d, 1H, J = 7.5 Hz),
1737.73 (s, 1H), 7.60 (m, 3H), 7.50 (t, J = 7.8 Hz, 1H), 2.57 (s, 3H); 13C-NMR: (125 MHz, DMSO-
174d6): δC 160.0, 146.0, 145.8, 138.8, 135.5, 135.1, 133.2, 130.7, 128.7, 127.7, 126.9, 126.4, 124.1,
175121.7, 17.0; EI-MS: m/z (rel. abund. %), 270 [M]+ (100), 272 [M++2] (35), 235 (3); Anal. Calcd
176for C15H11ClN2O: C, 66.55; H, 4.10; Cl, 13.10; N, 10.35; O, 5.91; found: C, 66.51; H, 4.12; Cl,
17713.11; N, 10.37; O, 5.93.
1783-(2′-Methylphenyl)-8-methylquinazolin-4(3H)-one (13)
179 Yield: 78%; Off White Solid; m.p. 132-135 ºC; Rf: 0.80 (ethyl acetate/hexanes, 3:7); 1H-
180NMR: (300 MHz, DMSO-d6): δH 8.28 (s, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H),
1817.50 (t, J = 7.8 Hz, 1H), 7.44 (m, 4H), 2.58 (s, 3H), 2.09 (s, 3H); 13C-NMR: (100 MHz, DMSO-
182d6): δC 159.8, 146.3, 136.8, 135.6, 135.4, 135.0, 130.7, 129.3, 128.2, 127.0, 126.9, 126.0, 124.0,
183121.8, 120.8, 17.1, 17.0; EI-MS: m/z (rel. abund. %), 250 [M]+ (71), 233 (100), 132 (9); Anal.
184Calcd for C16H14N2O: C, 76.78; H, 5.64; N, 11.19; O, 6.39; found: C, 76.75; H, 5.67; N, 11.18;
185O, 6.37.
1863-(4′-Methoxyphenyl)-8-methylquinazolin-4(3H)-one (14)
187 Yield: 84%; Light Grey Solid; m.p. 152-155 ºC; Rf: 0.62 (ethyl acetate/hexanes, 3:7); 1H-
188NMR: (300 MHz, DMSO-d6): δH 8.32 (s, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H),
1897.48 (t, J = 7.8 Hz, 1H), 7.45 (d, 2H, J = 9.0 Hz), 7.10 (d, 2H, J = 9.0 Hz), 3.82 (s, 3H), 2.57 (s,
1903H); 13C-NMR: (100 MHz, DMSO-d6): δC 160.4, 159.2, 146.4, 146.1, 135.4, 134.8, 130.3,
191128.6, 126.8, 124.0, 121.8, 114.3, 55.4, 17.0; EI-MS: m/z (rel. abund. %), 266 [M]+ (100), 251
192(14), 235 (3); Anal. Calcd for C16H14N2O2: C, 72.16; H, 5.30; N, 10.52; O, 12.02; found: C,
19372.18; H, 5.33; N, 10.50; O, 12.00.
1943-(4′-Methylphenyl)-8-methylquinazolin-4(3H)-one (15)
195 Yield: 85%; Off White Solid; m.p. 118-120 ºC; Rf: 0.80 (ethyl acetate/hexanes, 3:7); 1H-
196NMR: (400 MHz, DMSO-d6): δH 8.32 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H),
1977.48 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.57 (s, 3H), 2.38 (s,
1983H); 13C-NMR: (125 MHz, DMSO-d6): δC 160.2, 146.2, 146.1, 138.2, 135.4, 135.0, 134.9,
199129.6, 127.1, 126.8, 124.0, 121.8, 20.6, 17.0; EI-MS: m/z (rel. abund. %), 250 [M]+ (100), 235
200(9), 220 (1); Anal. Calcd for C16H14N2O: C, 76.78; H, 5.64; N, 11.19; O, 6.39; found: C, 76.73;
201H, 5.69; N, 11.18; O, 6.40.
202A. castellanii cultures
203 A clinical strain of A. castellanii (ATCC 50492) T4 genotype, was cultured and
204maintained in 10 mL growth medium (PYG) composed of 0.75% w/v proteose peptone, 0.75%
205w/v extract of yeast, and 1.5% w/v glucose at 30 ˚C in 75-cm2 culture flasks [20]. For
206amoebicidal assays, old PYG medium was replaced with fresh phosphate buffer saline (PBS) and
207adherent A. castellanii trophozoites were obtained by keeping culture flasks on ice for 15 min
208and subsequent gentle tapping to ensure the detachment of trophozoites. Finally, A. castellanii
209trophozoites were collected in a 50 mL conical tube and were centrifuged at 3000 × g for 5 min.
210The obtained pellet was re-suspended in 1 mL PBS, while the enumeration of A. castellanii was
211carried out by using a hemocytometer.
212Amoebicidal assay
213 The trophocidal activity of QNZs was determined by treating 5 x 105 healthy A.
214castellanii trophozoites per well with 100 and 50 µ g per mL of QNZs and their respective
215controls in 24-well plates in RPMI-1640 medium. The plates were incubated at 30 ˚C for 24 h as
216previously described [20]. Following the incubation time, the viable cells were differentiated by
217Trypan blue staining with 0.1% Trypan blue aqueous solution for 5 min. The number of live cells
218(non-stained) A. castellaniiwere tallied using a hemocytometer. Un-treated A. castellanii were
219used as negative control, while chlorhexidine was used as a positive control.
220Field emission scanning electron microscopy
221 To determine the morphological effects on A. castellaniiafter treatment with QNZs,
222ultramicroscopic FESEM analysis was performed. A. castellanii untreated or treated with
223representative QNZs which showed either potent or low antiamoebic effects such as compounds
2245 and 15 were fixated on glass cover slips using 2.5% glutaraldehyde solution prepared in PBS.
225The cover slips were kept at 4 ˚C for 2 h to ensure complete infiltration. The samples were then
226dehydrated with a gradient concentrations of ethanol solutions ranging from 50 to 100%. After
227air drying of samples, they were sputtered with platinum, and imaging was carried out at FESEM
228instrument (Hitachi SU8010). Extensive images were recorded at different scales for each
229sample. The images presented here are representatives of various images.
230Encystation assay
231 To test the efficacy of QNZs in inhibiting the differentiation of A. castellanii trophozoites
232into cysts encystation assay was carried out. Briefly, 5×105 A. castellanii trophozoites suspended
233in PBS were treated with 100 µ g/mL of QNZs in 1.5 mL centrifuge tubes at room temperature
234for 15 min. Simultaneously, encystation medium consisting of MgCl2 (50 mM) and glucose
235(10%) was added in 24-well plates. After 15 min, QNZs treated or untreated A. castellanii were
236added in above 24-well plates containing encystation media. The cells were incubated at 30 ˚C
237for 72 h to trigger the encystation [21]. After routine observation of plates, when mature cysts are
238formed, each well was treated with sodium dodecyl sulfate (SDS) aqueous solution (0.25%) to
239decompose the trophozoites and only the SDS resistant mature cysts were enumerated using a
240hemocytometer.
241Excystation assay
242 The non-nutrient agar plates were prepared bymaking bacteriological agar aqueous
243solution (1.5%). The agar was autoclaved, then poured on the petri plates and followed by the air
244drying inside biosafety cabinet. A. castellanii cysts were prepared by inoculating 1 x 106
245trophozoiteson non-nutrient agar plates. The inoculated plates were then incubated at 30 ˚C for
246up to 2 weeks with routine observation under light microscope, till the mature cysts were formed.
247Cysts were scraped from plates using cell scraper with PBS, enumerated, and stored at 4 ˚C. For
248excystation assay, 1 x 105 pre-formed cysts were incubated with 100 µ g/mL QNZs and controls
249in growth medium PYG for 72 h [22]. Finally, cysts reformed into trophozoites were counted by
250using hemocytometer.
251HaCaT keratinocyte cells culture
252 HaCaT cells were routinely cultured in Roswell Park Memorial Institute (RPMI)-1640
253supplemented with 10% of each fetal bovine serum and Nu-serum, in 75-cm2 culture flasks. The
254growth medium was also complemented with 2 mM glutamine, 1 mM pyruvate, penicillin,
255streptomycin (100 units/mL and 100 µg/mL, respectively), vitamins and non-essential amino
256acids and vitamins [23]. After formation of confluent, uniform mono-layer (24-48 h), old media
257was aspirated, and cells were detached by using 2 mL trypsin. The trypsinized cell suspension
258was centrifuged at 2500 × g for 5 minutes. The obtained pellet was then re-suspended in 25 mL
259fresh cell growth media. Each well of a 96-well plate was seeded with 200 µL of above cell
260suspension and the plates were incubated in a 5% CO2 incubator with 95% humidity (at 37 ˚C for
26124 h) until the uniform monolayer of cells was observed under light microscope.
262A. castellanii-mediated host cells cytotoxicity
263 To determine whether QNZs can reduce the cytopathogenicity of A. castellanii, host cells
264cytotoxicity assays were performed as reported previously [24]. 5 x 105 A. castellanii were pre-
265treated with 100 µ g/mL QNZs and negative and positive controls for 2 h in 30 ˚C incubator.
266Next, these cultures were centrifuged at 3000 x g for 5 minutes. The supernatants were aspirated,
267and the cells pellet was re-suspended in 200 µ L of fresh RPMI-1640 to remove excess
268compounds and any intracellular toxins. These suspensions were then incubated with HaCaT
269cells monolayers formed in 96-well plates at 37 ˚C in a 5% CO2 incubator with 95% humidity.
270After incubation of 24 h, the supernatants were collected from each well and lactate
271dehydrogenase (LDH) was measured at 492 nm on microplate reader using LDH cytotoxicity
272detection kit (Invitrogen) as described previously [25]. LDH is released from damaged cells
273only, therefore healthy, untreated cells were the negative control, while cells completely
274disintegrated by using 0.1% Triton X-100 were taken as the positive control for all cytotoxicity
275assays. The percent cell cytotoxicity was calculated by using following equation:
276 % cell cytotoxicity = (LDH released by cells with sample treatment –LDH measured in
277untreated cells) / (Total LDH released by Triton X-100 treated cells – LDH measured in
278untreated cells) × 100.
279Cytotoxicity assay
280 The cytotoxicity of QNZs on human normal cells (HaCaT keratinocytes) was also
281determined by LDH cytotoxicity assay, as reported previously [25]. Shortly, 100 µ g/mL of
282QNZs and respective controls were incubated with uniform monolayer of HaCaT cells in a 96-
283well plates for 24 h (at 37 ˚C in a 5% CO2 incubator). Following the incubation time,
284supernatants were collected, and cytotoxicity was calculated by determination of LDH enzyme
285as mentioned above.
286Statistical analysis
287 All of the presented results are representatives of several experiments performed in
288duplicate and are represented as the mean ± standard error. Student’s T-test was performed for
289statistical analysis. The correlation and significance were measured on Microsoft Excel
290worksheets. The threshold level of significance was P < 0.05, using two-sample T-test and two- 291tailed distribution. * corresponds to P < 0.05, while ** P < 0.01, and *** P < 0.001 respectively. 292RESULTS AND DISCUSSION 293CHEMISTRY 294General synthesis of QNZs (1-15) 295 A library of 3-aryl-8-methylquinazolin-4(3H)-ones was synthesized via one pot reaction 296of 2-amino-3-methylbenzoic acid, triethoxymethane, and several substituted anilines in 297equimolar quantities under acidic medium (Scheme 1). The cyclization and heterocyclic ring 298formation occur due to increased stability. The reaction mixture was refluxed till the complete 299consumption of starting material. The advancement in reaction was monitored through TLC 300analysis. The aqueous work up of reaction removed excess acid and the product was precipitated 301out. The products were then crystallized from ethanol for purification. The original spectra are 302presented in supplementary information. 303Spectral characterization of a representative QNZ (5) 3041H-NMR Spectroscopy 305 The 1H-NMR spectrum of compound 5 (most active) was recorded, by using an 306instrument of 400 MHz in DMSO-d6. H-2 of the quinazoline ring resonated at δH 8.35 as a sharp 307singlet and was the most downfield signal of the spectrum due to presence of two nitrogen 308atoms. All other aromatic protons were resonated in the range of δH 8.04-7.42. A doublet of H-5 309was appeared at δH 8.04 with coupling constant value of J = 8.0 Hz showing coupling with the 310neighboring proton 6. H-7 resonated at δH 7.75 as a doublet with J =7.2 Hz showing ortho 311coupling with H-6. 312 H-2ʹ and H-6ʹ appeared as doublet of doublets at δH 7.62 (J2ʹ,3ʹ/6ʹ,5ʹ = 9.0 Hz, J2ʹ,4ʹF/6ʹ,4ʹF = 3135.2 Hz) showing ortho coupling with H-3ʹ/H-5ʹ and meta coupling with 4ʹ-F. A triplet of H-6 314resonated at δH 7.49 (J6(5,7) = 7.6 Hz) showing coupling with H-5 and -7, respectively. While H-3ʹ 315and H-5ʹ resonated at δH 7.42 as a triplet J3′(2′,4′F )/5′(6′,4′F ) = 8.8 Hz). The most upfiled signal of 316CH3 protons appeared at δH 2.57 as a singlet (Fig. 3). 317Mass Spectrometry 318 The recorded EI-MS spectra of compound 5 depicted the M+ at m/z 254, in consonance 319with C15H11FN2O (254.09) and was also the base peak. The ion obtained at m/z 249 was due to 320the removal of methyl group from molecule. The fragment at m/z 145 was acquired by the reason 321of quinazolinone. The key fragments are presented in Fig. 4. 322BIOLOGY 323Antiacanthamoebic and cytotoxic activity of QNZs 324 A. castellanii is an opportunistic protist which is the causative agent of infections of eyes 325and brain. The risk factor for more prevailing pathology i.e., Acanthamoeba keratitis is practice 326of unhygienic contact lens handling and ineffective contact lens solution [26]. Currently, there 327are only a few identified molecular pathways to target A. castellanii which is one of the key 328limitations in development of effective therapeutics. Furthermore, the extended usage of 329recommended drugs (including a mixture of biguanides, azoles, amidines and antibiotics) can 330cause host cells cytotoxicity while their ineffectiveness against resistant cysts is prone to 331recurrence of the infection [27]. Hence, there is an urgent requisite for the development of novel 332and effective chemotherapeutics against A. castellanii infections. Heterocyclic compounds have 333proven to be promising drug candidates [28, 29]. Among heterocyclic compounds, quinazoline 334and its derivatives have been thoroughly studied against fungi and bacteria [30, 31]. Alho et al., 335have identified quinoxalinones; another structural variant of six- membered heterocyclic rings 336with two nitrogen atoms as potent lead compounds against apicomplexa and mastigophora 337parasites [10]. In another study, 7-nitroquinoxalin-2-ones were found to be effective against 338Trypanosoma cruzi with a possible inhibition of the trypanothione reductase enzyme [32]. In 339current study, we synthesized a library of fifteen variants of 3-aryl-8-methylquinazolin-4(3H)- 340ones and determined their antiamoebic activity against A. castellanii. Current report is a valid 341example of medicinal chemistry aspect subjected to antiparasitic study with quinazolinones 342being tested against free-living amoeba A. castellanii for the first time. 343QNZs exhibited significant antiamoebic effects against A. castellanii 344 Amoebicidal assay performed at 100 and 50 µ g/mL of QNZs revealed that almost all of 345fifteen compounds reduced the number of viable A. castellanii trophozoites at 100 µ g/mL. 346Among these compounds, 5, 7, and 8 produced striking antiamoebic effects at 50 µ g/mL, while 347additionally compound 2 exhibited potent results at 100 µ g/mL (Fig. 5). The level of significance 348was calculated with respect to solvent control methanol. At higher concentration i.e., 100 µ g/mL, 349only one compound 6 showed ineffectiveness. At these concentrations, almost all of compounds 350showed more than 50 % inhibition which corresponds to the IC50. The 100 µ g/mL concentration 351of compound 2, 5 and 8, caused more than 80 % inhibition, while at 50 µ g/mL more than 60 % 352inhibition was observed with compounds 5, 7, and 8, hence these data provide more quantitative 353picture of the antiamoebic assay. The QNZs treated A. castellanii were also subjected to FESEM 354analysis for the evaluation of surface alteration. FESEM images showed that A. castellanii 355treated with compounds 5 and 15 triggered surface disintegration and pores formation which 356 357 might have resulted in high antiamoebic effects (Fig. 6). 358 QNZs inhibited encystation and excystation of A. castellanii 359 Encystation of A. castellanii is the process of conversion of trophozoites into cysts which 360contributes for the resistance against drugs A. castellanii are transformed into cysts upon 361exposure to harsh conditions including treatment with drugs, change in pH and temperature, and 362nutrients deprivation. Encystation assays were performed to test whether these QNZs can cause 363inhibition of this morphological transformation. Figure 7a describes that nine out of fifteen 364compounds inhibited encystation at 100 µ g/mL with compounds 8, 9 and 13 being most 365effective. On the other hand, cyst is the most resistant form of A. castellanii which is one of the 366main reasons for recurrence of Acanthamoeba keratitis even after cornea transplant surgery [33]. 367Most of the drugs used currently against A. castellanii infections have shown limited potency 368against cysts. The effects of QNZs were also evaluated against excystation by application of 100 369µ g/mL with pre-formed cysts of A. castellanii. The results showed that most of the fifteen tested 370compounds inhibited excystation with compound 5 showed same effects as positive control 371chlorhexidine (Fig. 7b). These results show potential of QNZs as suitable alternative for drug 372development against A. castellanii. 373QNZs exhibited minimal damage against human keratinocytes and reduced Acanthamoeba- 374mediated host cell cytotoxicity 375 To evaluate the cytotoxicity of QNZs against human cells, LDH assays were performed. 376The results of cytotoxicity determination showed that QNZs produced minimal cytotoxic effects 377(Fig. 8a). Compound 3 and 8 exhibited 11 and 8% cytotoxicity, respectively, while the remaining 378thirteen compounds showed no toxicity at 100 µ g/mL. The cytotoxic effects were studied against 379human cells and suggest that these QNZs are bio-safe and can further be assessed for in vivo 380studies. These QNZs also reduced the host cells cytotoxicity of A. castellanii. The pretreatment 381of amoeba with QNZs showed significant decline in their cytopathogenicity against normal 382human cells. Fig. 8b shows that untreated A. castellanii triggered 80% toxicity against HaCaT 383cells, while chlorhexidine treated amoeba showed protection of cells against amoeba. All fifteen 384compounds tested at 100 µ g/mL significantly reduced the host cells cytopathogenicity. Notably, 385compounds 5, 7, 8 and 13 produced most potent effects which completely abolished the host 386cells toxicity. 387Structure-activity relationship 388 Limited structure-activity relationship against A. castellanii trophozoites indicates that 389the variation in activity was observed based on different substituent present on QNZ ring and 390their respective positions on the aryl part. The presence of one fluorine, iodine, and methyl 391groups on aryl ring of compounds 2, 8, and 13 exhibited pronounced antiacanthamoebic activity 392against pathogenic trophozoite stage. However, the addition of another number of these groups 393reduced the effects. Notably, mono substitution at ortho position showed more activity as in 394compound 8 and 13 bearing iodo and methyl groups, respectively. Shifting the position of these 395substituents to para in compounds 7 and 15 displayed a remarkable regression in activity. 396Similarly, compound 7 with two methyl substituents at meta positions, exhibited lower activity 397as compared to compound 13 (Fig. 9). Interestingly, among fluoro substituted derivatives, 398compound 9 with two fluoro groups at ortho and para positions was most active as compared to 399its mono substituted derivatives. The mono substituted compounds 2 and 5 having fluoro group 400at meta and para positions were less active. Mono methoxy substituted compound 14 also 401exhibited good antiacanthamoebic activity as compared to its dimethoxy counterpart 11. It was 402also noted that bromo and chloro substituted QNZs were less active (Fig. 10). Based on the 403observed effects of substituents present on the aryl part of QNZs, it can be concluded that iodo 404group at ortho position, fluoro groups at ortho and para positions, methyl group at ortho 405position, and methoxy group at para position caused highest antiamoebic activity. 406CONCLUSIONS 407 QNZs showed significant antiacanthamoebic effects especially compounds 2, 5, 8, and 40813. These compounds exhibited potent antiamoebic activity, along with the inhibition of 409encystation and excystationin A. castellanii. Furthermore, these compounds significantly reduced 410the Acanthamoeba-mediated host cells cytopathogenicity. Interestingly, minimal cytotoxicity 411was observed by these compounds, when tested against human normal cells. Therefore, this 412study exhibits potential ability of QNZs for the development of effective antiacanthamoebic 413agents. These results are estimated to be a key advancement in the development of efficient drug 414leads against free-living amoeba A. castellanii. The determination of their mode of action and in 415vivo performance are included in our future investigation, along with testing more derivatives of 416QNZs for extensive SAR. 417Acknowledgements 418 This work is supported by Sunway University, Malaysia (University Research Award 419INT-SST-DBS-2019-03) and the Pakistan Academy of Sciences for providing financial support 420Project No. (5-9/PAS/440). 421Author contributions statement 422 A.A. conducted the experiments on Acanthamoeba and wrote the first draft of 423manuscript. M.S.S. and S.M.S. synthesized the compounds under the supervision of K.M.K. 424M.S.S. and K. characterized the structures. R.S. and N.A.K conceived the idea and supervised all 425biological studies. K.M.K., R.S., and N.A.K. corrected and finalized the manuscript which is 426submitted with the consent of all authors. 427Competing interests 428 Authors declare no competing interests. The manuscript was submitted by the approval of 429all authors. 430Data availability 431 Data will be provided upon request on case to case basis. 432References 4331. F. Marciano-Cabral, G. Cabral, Acanthamoeba spp. as agents of disease in humans. Clin. 434 Microbiol. Rev.16 (2003) 273-307. 435 2. N.A. Khan, Acanthamoeba: biology and increasing importance in human health. FEMS 436 Microbiol. Rev.30 (2006) 564-595. 437 3. G.S. Visvesvara, H. Moura, F.L. Schuster, Pathogenic and opportunistic free-living 438 amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and 439 Sappiniadiploidea. FEMS Immun. Med. Microbiol.50 (2007) 1-26. 440 4. J. Lorenzo-Morales, N.A. Khan,J. Walochnik, An update on Acanthamoeba keratitis: 441 diagnosis, pathogenesis and treatment. Parasite.22 (2015). 442 5. 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López-Munoz, ESR, electrochemical, molecular 529 modeling and biological evaluation of 4-substituted and 1,4-disubstituted 7- 530 nitroquinoxalin-2-ones as potential anti-Trypanosoma cruzi agents. Spectrochim. Acta 531 Part A.78 (2011) 1004-1012. 532 33. L.A. Ficker, C. Kirkness, P. Wright, Prognosis for keratoplasty in Acanthamoebakeratitis. 533 Ophthalmology. 100 (1993) 105-110. 534Figure legends (Figures are attached separately) 535Fig. 1. Core structure of heterocyclic compound Quinazoline and 4(3H)-Quinazolinone. 536Chemical structure of known fungicide compound of quinazolinone class Fluquinconazole. 537Fig. 2. Rationale of current study 538Fig. 3. 1H-NMR chemical shifts of compound 5. 539Fig. 4. Key fragmentation pattern (EI-MS) of compound 5. 540Fig. 5. Amoebicidal assay against A. castellaniiat (a) 100 µ g per mL, and (b) 50 µ g per mL. The 541viability of amoeba was determined after amoebicidal assay as described in the materials and 542methods section. Briefly, A. castellanii trophozoites were incubated with QNZs, negative and 543positive controls at 30 ˚C for 24 h. Next, the viability was measured by Trypan blue exclusion 544assay. The results are presented as the mean ± standard error of various experiments performed 545in duplicate. * represents P < 0.05, ** represents P < 0.01, while *** represents P < 0.001. P 546values were obtained using two-sample T test and two-tailed distribution. 547Fig. 6. FE-SEM images of A. castellanii with and without treatment with QNZs. A. castellanii 548were fixed on glass cover slips by using glutaraldehyde. Followed by fixation, the samples were 549washed with ethanol and images were recorded on Field-emission scanning electron microscope 550(FE-SEM) (Hitachi SU8010) instrument. (a) A. castellanii control. (b) A. castellanii treated with 551100 µ g per mL of compound 5. (c) A. castellanii treated with 100 µ g per mL of 15. Control 552showed integrated A. castellanii wall, whereas treated amoeba showed disintegration and pores. 553Fig. 7. Depiction of the results of (a) encystation. QNZs inhibited A. castellanii encystation. A. 554castellanii (1 x 105) were inoculated in PBS in the presence of QNZs and respective controls at 555100 µ g per mL with encystation media and incubated at 30 ˚C for 72 h. Next, 0.25% sodium 556dodecyl sulfate (SDS) was added and incubated at room temperature for 10 min to lyse A. 557castellanii trophozoites followed by enumeration of amoebae cysts using a hemocytometer. (b) 558Excystation assays was performed by incubating 100 µ g per mL QNZs with A. castellanii cysts 559(1 x 105) in PYG at 30 ˚C for 72 h. After this period, amoebae were counted using a 560hemocytometer. The results are presented as the mean ± standard error of various experiments 561performed in duplicate. * represents P < 0.05, ** represents P < 0.01, while *** represents P < 5620.001. P values were obtained using two-sample T test and two-tailed distribution. 563Fig. 8. (a) QNZs did not exhibit cytotoxicity against HaCaT cells at 100 µ g per mL. These 564compounds and the respective controls were incubated with HeLa cells monolayer for 24 h at 37 565˚C in a 5% CO2 incubator. Following this incubation, cell-free supernatant was collected, and 566cytotoxicity was determined using Lactate dehydrogenase (LDH) assay kit (Roche). The 567negative control values for cytotoxicity assays were obtained by incubating cells with RPMI- 5681640 alone, and positive control values were obtained by 100% cell death using 0.1% Triton X- 569100. (b) Pretreatment of 100 µ g per mL of QNZs abolished A. castellanii-mediated host cells 570cytotoxicity. Briefly, amoebae (1 x 105) were incubated at 30 ˚C with QNZsand respective 571controls for 2 h in RPMI-1640 and then incubated with HaCaT cells for 24 h at 37 ˚C in a 5% 572CO2 incubator as described in materials and methods section. Next, cell-free supernatant was 573collected, and cytotoxicity was determined using Lactate dehydrogenase (LDH) assay kit 574(Roche). The results are presented as the mean ± standard error of various experiments 575performed in duplicate. 576Fig. 9. Structure activity relationship between iodo and methyl substituted QNZs. 577Fig. 10. Structure activity relationship between fluoro and methoxy substituted QNZs.
578
579
Scheme-1. One pot synthetic route to 3-aryl-8-methylquinazolin-4(3H)-ones.
580 Table 1. Structures, names, chemical formulae and molecular weights of tested 3-Aryl-8-
581
582
methylquinazolin-4(3H)-ones
No. Structure IUPAC Name Mol. Formula Mol.
Weight Yield
(%)
1.
3-(4-Chlorophenyl)-8- methylquinazolin-4(3H)-one
C15H11ClN2O
270.71
76
2. O
N F
N
Me
3-(3-Fluorophenyl)-8- methylquinazolin-4(3H)-one
C15H11FN2O
254.26
70
3.
3-(3-Bromophenyl)-8- methylquinazolin-4(3H)-one
C15H11BrN2O
315.16
67
4.
8-Methyl-3-(3-
(methylthio)phenyl)quinazolin-
4(3H)-one
C16H14N2OS
282.36
65
5.
3-(4-Fluorophenyl)-8- methylquinazolin-4(3H)-one
C15H11FN2O
254.26
68
6.
3-(3,5-Dimethylphenyl)-8-
methylquinazolin-4(3H)-one
C17H16N2O
264.32
74
7.
3-(4-Iodophenyl)-8-
methylquinazolin-4(3H)-one
C15H11IN2O
362.17
63
8.
3-(2-Iodophenyl)-8-
methylquinazolin-4(3H)-one
C15H11IN2O
362.17
69
9.
3-(2,4-Difluorophenyl)-8- methylquinazolin-4(3H)-one
C15H10F2N2O
272.25
83
10.
3-(4-Bromophenyl)-8- methylquinazolin-4(3H)-one
C15H11BrN2O
315.16
84
11.
3-(2,5-Dimethoxyphenyl)-8- methylquinazolin-4(3H)-one
C17H16N2O3
296.32
81
12.
3-(3-Chlorophenyl)-8- methylquinazolin-4(3H)-one
C15H11ClN2O
270.71
63
13.
8-Methyl-3-(o-
tolyl)quinazolin-4(3H)-one
C16H14N2O
250.30
78
14.
3-(4-Methoxyphenyl)-8- methylquinazolin-4(3H)-one
C16H14N2O2
266.29
84
15.
8-Methyl-3-(p-
tolyl)quinazolin-4(3H)-one
C16H14N2O
250.30
85
583
Fig. 1
Scheme 1
O
OH
NH2
+
O
O
O
+
NH2
R
Acetic acid
Refluxed at 120 °C
8h
O
N
N
R
4.50E+05
4.00E+05 3.50E+05
(a)
5.00E+05 4.50E+05 4.00E+05
(b)
3.00E+05 2.50E+05 2.00E+05 1.50E+05 1.00E+05 5.00E+04 0.00E+00
*
***
* *
***
**
***
*
*
*
*
***
* *
3.50E+05 3.00E+05 2.50E+05 2.00E+05 1.50E+05 1.00E+05 5.00E+04 0.00E+00
*
*
*
**
**
***
*
*
*
RPMI CHX 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MeOH
1 %
RPMI CHX 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MeOH
0.5 %
(a) (b) (c)
6.00E+05 5.00E+05 4.00E+05 3.00E+05 2.00E+05 1.00E+05 0.00E+00
9.00E+04 8.00E+04 7.00E+04 6.00E+04 5.00E+04 4.00E+04 3.00E+04 2.00E+04 1.00E+04 0.00E+00
*
**
**
**
***
**
*
*
(b)
*
PBS EM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MeOH
1 %
PBS EM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MeOH
1 %
100%
(a)
100% (b)
75%
75%
50%
50%
25%
0%
25%
0%
1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 MeOH
1 %
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 A. CHX Triton
castellanii X-100
Highlights
•Acanthamoeba is an opportunistic parasite that can cause keratitis and encephalitis.
•Quinazolinones hold promise in the drug development against infectious diseases.
•3-aryl-8-methylquinazolin-4(3H)-ones were synthesized by varying aniline derivatives.
•A series of fifteen new quinazolinone derivatives have been tested against A. castellanii for the first time.
•These quinazolinone derivatives exhibited potent anti-acanthamoebic activity.