Novel antiacanthamoebic compounds belonging to quinazolinones

European Journal of Medicinal Chemistry 182 (2019) 111575

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Novel antiacanthamoebic compounds belonging to quinazolinones Ayaz Anwar a, 1, Muhammad Saquib Shahbaz b, 1, Syed Muhammad Saad b, c, Kanwal b, Khalid Mohammed Khan b, d, *, Ruqaiyyah Siddiqui a, Naveed Ahmed Khan a, ** a

Department of Biological Sciences, School of Science and Technology, Sunway University, Subang Jaya, 47500, Selangor, Malaysia H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan c Department of Chemistry, University of Karachi, Karachi, 75270, Pakistan d Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 March 2019 Received in revised form 29 July 2019 Accepted 30 July 2019 Available online 30 July 2019

We report one-pot synthesis of a series of new 3-aryl-8-methylquinazolin-4(3H)-ones (QNZ) and their antimicrobial activity against Acanthamoeba castellanii belonging to T4 genotype. A library of fifteen synthetic derivatives of QNZs was synthesized, and their structural elucidation was performed by using nuclear magnetic resonance (NMR) spectroscopy and electron impact mass spectrometry (EI-MS). Elemental analyses and high-resolution mass spectrometry data of all derivatives were found to be in agreeable range. Amoebicidal assays performed at concentrations ranging from 50 to 100 mg/mL revealed that all derivatives of QNZ significantly decreased the viability of A. castellanii and QNZ 2, 5, 8, and 13 were found to have efficient antiamoebic effects. Field emission scanning electron microscopy (FESEM) imaging of amoeba treated with compounds 5 and 15 showed that these compounds cause structural alterations on the walls of A. castellanii. Furthermore, several QNZs inhibited the encystation and excystationas as well as abolished A. castellanii-mediated host cells cytopathogenicity in human cells. Whereas, these QNZs showed negligible cytotoxicity when tested against human cells in vitro. Hence, this study identified potential lead molecules having promising properties for drug development against A. castellanii. A brief structure-activity relationship is also developed to optimize the hit of most potent compounds from the library. To the best of our knowledge, it is first of its kind medicinal chemistry approach on a single class of compounds i.e., quinazolinone against keratitis and brain infection causing free-living amoeba, A. castellanii. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Quinazolinone Synthesis Acanthamoeba Antiamoebic FESEM

1. Introduction Acanthamoeba species are among most abundant free-living amoebae widely distributed in water and soil [1]. They survive in two phenotypic forms; reproductive, opportunistic pathogenic trophozoite state, and resistant, dormant, double-walled protected cysts [2]. The pathologies associated with Acanthamoeba species are blinding keratitis, and fatal brain infection, granulomatous amoebic encephalitis [3]. Despite the morbidity and mortality, there is no definite course of action to treat Acanthamoeba

* Corresponding author. Department of Biological Sciences, School of Science and Technology, Sunway University, Subang Jaya, 47500, Selangor, Malaysia. ** Corresponding author. E-mail addresses: [email protected] (K.M. Khan), [email protected] (N.A. Khan). 1 Both authors contributed equally. https://doi.org/10.1016/j.ejmech.2019.111575 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

infections [4]. The recommended drugs against Acanthamoeba infections have poor specificity and ineffectiveness against cysts which results in side effects and recurrence [5]. A vast number of compounds ranging from synthetic, natural, and nanomaterials have shown in vitro potential against Acanthamoeba, but the prognosis remains strikingly poor without the approval or interest of pharmaceutical industries [6]. Recently, high throughput assays and computational studies have identified promising synthetic targets which may serve as lead compounds for drug development against free-living amoebae [7e10]. Based on our interest in drug development against neglected microbes, the objective of this study was to evaluate the effects of quinazolinone derivatives against Acanthamoeba [11e13]. Heterocyclic compounds of class quinazoline represent promising scaffolds in medicinal chemistry [14]. Several studies have been reported on the biological activity of quinazoline analogues, including their antibacterial, fungicidal, antiviral, antileishmanial,

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A. Anwar et al. / European Journal of Medicinal Chemistry 182 (2019) 111575

and anti-trauma activities [15e17]. They are used in pharmaceuticals and agrochemicals [18], for example, fluquinconazole (Fig. 1) a known fungicide for the regulation of agriculture diseases [19]. Different derivatives of quinazolines and quinazolinones have been previously synthesized and reported as antileishmanial agents (Fig. 2) [12,13]. Keeping in mind the antiparasitic aspect of this moiety, we evaluated antiacanthamoebic activity of synthetic quinazolinone analogs. In this report, for the first time, we present a simple one-pot synthesis of chemically versatile quinazolinone derivatives, and studied their antiacanthamoebic potential. In this library, fifteen analogues of 3-aryl-8-methylquinazolin-4(3H)-ones were synthesized (Table 1), and a brief structure-activity relationship is developed to optimize the potent lead compound for further studies. 2. Experimental section 2.1. Materials and methods All reagents used in this study were commercially procured from Sigma until stated otherwise. HPLC grade solvents were used for synthesis and purification of compounds. 2.2. Synthesis of 3-aryl-8-methylquinazolin-4(3H)-ones A library of fifteen analogs of 3-aryl-8-methylquinazolin-4(3H)ones was synthesized via one pot reaction of 2-amino-3methylbenzoic acid (1 mmol) with triethoxymethane (3 mmol) and different substituted anilines (1 mmol) in acidic medium using acetic acid (Scheme 1). The reaction mixture was boiled and kept on reflux, while the reaction progress was examined with thin layer chromatography (TLC). After the complete absence of the starting materials, the reaction mixture was poured in water, precipitate formed were filtered, thoroughly washed with ultrapure water and dried under vacuum. The solid was crystallized from ethanol. The structures of these synthetic derivatives were elucidated by 1H NMR, 13C NMR, and EI-MS. Elemental analyses and high resolution EI-MS data of all compounds were found to be in consistent range. Based on the TLC and spectroscopic analyses, all compounds were found to contain no impurities. 2.3. Characterization of 3-aryl-8-mehtylquinazolin-4(3H)-ones 2.3.1. 3-(40 -chlorophenyl)-8-methylquinazolin-4(3H)-one (1) Yield: 76%; Light Grey Solid; m.p. 225e227  C; Rf: 0.86 (ethyl acetate/hexanes, 3:7); 1H NMR: (400 MHz, DMSO‑d6): dH 8.36 (s, 1H), 8.04 (d, J ¼ 7.6 Hz, 1H), 7.75 (d, J ¼ 7.2 Hz, 1H), 7.64 (d, J ¼ 8.8 Hz, 2H), 7.59 (d, J ¼ 8.8 Hz, 2H), 7.49 (t, J ¼ 7.6 Hz, 1H), 2.57 (s, 3H); 13C NMR: (100 MHz, DMSO‑d6): dC 160.1, 146.0, 145.9, 136.4,

Fig. 2. Rationale of current study.

135.5, 135.0, 133.3, 129.4, 129.1, 126.9, 124.0, 121.7, 17.0; EI-MS: m/z (rel. abund. %), 272 [Mþþ2] (30), 270 [M]þ (100), 242 (5), 244 (2), 111 (8), 105 (12); Anal. Calcd for C15H11ClN2O: C, 66.55; H, 4.10; Cl, 13.10; N, 10.35; O, 5.91; found: C, 66.51; H, 4.13; Cl, 13.12; N, 10.33; O, 5.94. 2.3.2. 3-(30 -fluorophenyl)-8-methylquinazolin-4(3H)-one (2) Yield: 70%; Off White Solid; m.p. 129e131  C; Rf: 0.88 (ethyl acetate/hexanes, 3:7); 1H NMR: (400 MHz, DMSO‑d6): dH 8.38 (s, 1H), 8.05 (d, J ¼ 7.6 Hz, 1H), 7.76 (d, J ¼ 7.6 Hz, 1H), 7.64 (dd, J ¼ 8.0 Hz, 1H), 7.54 (d, J ¼ 10.0 Hz, 1H), 7.50 (t, J ¼ 7.6 Hz, 1H), 7.42 (dd, J ¼ 8.8 Hz, 1H), 7.38 (d, J ¼ 9.2 Hz, 1H), 2.57 (s, 3H); 13C NMR: (125 MHz, DMSO‑d6): dC 162.8, 160.9, 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; EI-MS: m/z (rel. abund. %), 254 [M]þ (100), 226 (11), 105 (15), 95 (14); Anal. Calcd for C15H11FN2O: 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, 11.06; O, 6.27. 2.3.3. 3-(30 -bromophenyl)-8-methylquinazolin-4(3H)-one (3) Yield: 67%; Off White Solid; m.p. 132e135  C; Rf: 0.94 (ethyl acetate/hexanes, 3:7); 1H NMR: (400 MHz, DMSO‑d6): dH 8.37 (s, 1H), 8.04 (d, J ¼ 7.6 Hz, 1H), 7.85 (s, 1H), 7.75 (d, J ¼ 7.6 Hz, 1H), 7.73 (d, J ¼ 9.2 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.54 (t, J ¼ 7.6 Hz, 1H), 7.49 (t, J ¼ 7.6 Hz, 1H), 2.57 (s, 3H); 13C NMR: (125 MHz, DMSO‑d6): dC 160.6, 146.5, 146.3, 139.4, 136.0, 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. abund. %), 316 [Mþþ2] (91), 314 [M]þ (100), 286 (5), 288 (5), 155 (8), 157 (7), 105 (17); Anal.

Fig. 1. Core structure of heterocyclic compound Quinazoline and 4(3H)-Quinazolinone. Chemical structure of known fungicide compound of quinazolinone class Fluquinconazole.

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Table 1 Structures, names, chemical formulae, molecular weights and % yields of 3-Aryl-8-methylquinazolin-4(3H)-ones. No.

IUPAC Name

Mol. Formula

Mol. Weight

Yield (%)

1

Structure

3-(4-Chlorophenyl)-8-methylquinazolin-4(3H)-one

C15H11ClN2O

270.71

76

2

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

(continued on next page)

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A. Anwar et al. / European Journal of Medicinal Chemistry 182 (2019) 111575

Table 1 (continued ) No.

IUPAC Name

Mol. Formula

Mol. Weight

Yield (%)

9

Structure

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

Calcd for C15H11BrN2O: C, 57.16; H, 3.52; Br, 25.35; N, 8.89; O, 5.08; found: C, 57.12; H, 3.55; Br, 25.38; N, 8.86; O, 5.05. 2.3.4. 3-(30 -methylthiophenyl)-8-methylquinazolin-4(3H)-one (4) Yield: 65%; Grey Solid; m.p. 128e130  C; Rf: 0.76 (ethyl acetate/ hexanes, 3:7); 1H NMR: (400 MHz, DMSO‑d6): dH 8.36 (s, 1H), 8.04 (d, J ¼ 8.0 Hz, 1H), 7.75 (d, J ¼ 7.2 Hz, 1H), 7.50 (t, J ¼ 8.0 Hz, 1H), 7.49

(t, J ¼ 7.6 Hz, 1H), 7.43 (s, 1H), 7.39 (d, J ¼ 8.0 Hz, 1H); 7.30 (d, J ¼ 7.6 Hz, 1H), 2.57 (s, 3H), 2.50 (s, 3H); 13C NMR: (100 MHz, DMSO‑d6): dC 160.1, 146.1, 146.0, 139.6, 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: m/z (rel. abund. %), 282 [M]þ (100), 267 (5), 254 (3), 123 (4), 105 (15); Anal. Calcd for C16H14N2OS: 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, 5.62; S, 11.34.

A. Anwar et al. / European Journal of Medicinal Chemistry 182 (2019) 111575

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Scheme 1. One pot synthetic route to 3-aryl-8-methylquinazolin-4(3H)-ones.

2.3.5. 3-(40 -fluorophenyl)-8-methylquinazolin-4(3H)-one (5) Yield: 68%; Ash Grey Solid; m.p. 210e212  C; Rf: 0.78 (ethyl acetate/hexanes, 3:7); 1H NMR: (400 MHz, DMSO‑d6): dH 8.35 (s, 1H), 8.04 (d, J ¼ 8.0 Hz, 1H), 7.75 (d, J ¼ 7.2 Hz, 1H), 7.62 (dd, J ¼ 9.0 Hz, J ¼ 5.2 Hz, 2H), 7.49 (t, J ¼ 7.6 Hz), 7.42 (t, J ¼ 8.8 Hz, 2H), 2.57 (s, 3H); 13C NMR: (100 MHz, DMSO‑d6): dC 163.0, 160.5, 160.2, 146.1, 135.5, 135.0, 133.9, 129.8, 126.9, 124.0, 121.8, 116.1, 17.0; EI-MS: m/z (rel. abund. %), 254 [M]þ (100), 225 (8), 144 (4), 104 (9); Anal. Calcd for C15H11FN2O: C, 70.86; H, 4.36; F, 7.47; N, 11.02; O, 6.29; found: C, 70.85; H, 4.33; F, 7.49; N, 11.05; O, 6.27.

2.3.10. 3-(40 -bromophenyl)-8-methylquinazolin-4(3H)-one (10) Yield: 84%; White Solid; m.p. 234e237  C; Rf: 0.84 (ethyl acetate/hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.35 (s, 1H), 8.04 (d, J ¼ 7.2 Hz, 1H), 7.78 (d, 2H, J ¼ 8.4 Hz), 7.75 (d, 1H, J ¼ 9.0 Hz), 7.53 (d, 2H, J ¼ 8.7 Hz), 7.50 (t, J ¼ 8.7 Hz, 1H), 2.57 (s, 3H); 13C NMR: (125 MHz, DMSO‑d6): dC 160.0, 146.2, 146.0, 136.8, 135.5, 135.0, 132.1, 129.7, 126.9, 124.0, 121.8, 121.7, 17.0; EI-MS: m/z (rel. abund. %), 314 [M]þ (100), 316 [Mþþ2] (93), 235 (7), 206 (6); Anal. Calcd for C15H11BrN2O: C, 57.16; H, 3.52; Br, 25.35; N, 8.89; O, 5.08; found: C, 57.18; H, 3.56; Br, 25.34; N, 8.86; O, 5.05.

2.3.6. 3-(30 ,50 -dimethylphenyl)-8-methylquinazolin-4(3H)-one (6) Yield: 74%; Off White Solid; m.p. 98e103  C; Rf: 0.86 (ethyl acetate/hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.32 (s, 1H), 8.03 (d, J ¼ 7.2 Hz, 1H), 7.74 (d, J ¼ 7.2 Hz, 1H), 7.49 (t, J ¼ 7.8 Hz, 1H), 7.14 (s, 1H), 7.12 (s, 2H), 2.57 (s, 3H), 2.3 (s, 6H); 13C NMR: (100 MHz, DMSO‑d6): dC 160.1, 146.1, 138.5, 137.4, 135.4, 134.9, 130.0, 126.8, 124.9, 124.7, 124.0, 121.8, 20.7, 17.0; EI-MS: m/z (rel. abund. %), 264 [M]þ (100), 249 (12), 234 (1), 105 (57); Anal. Calcd for C17H16N2O: C, 77.25; H, 6.10; N, 10.60; O, 6.05; found: C, 77.29; H, 6.06; N, 10.63; O, 6.08.

2.3.11. 3-(20 ,50 -dimethoxyphenyl)-8-methylquinazolin-4(3H)-one (11) Yield: 81%; Dark Grey Solid; m.p. 133e135  C; Rf: 0.52 (ethyl acetate/hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.20 (s, 1H), 8.02 (d, J ¼ 9.0 Hz, 1H), 7.74 (d, J ¼ 7.2 Hz, 1H), 7.48 (t, J ¼ 7.5 Hz, 1H), 7.19 (d, J ¼ 9.0 Hz, 1H), 7.12 (d, J ¼ 3.0 Hz, 1H), 7.09 (dd, J ¼ 8.7 Hz, J ¼ 3.0 Hz, 1H),3.74 (s, 3H), 3.70 (s, 3H) 2.57 (s, 3H); 13C NMR: (125 MHz, DMSO‑d6): dC 159.8, 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, 56.2, 55.6, 17.0; EI-MS: m/z (rel. abund. %), 296 [M]þ (38), 265 (100), 250 (5); Anal. Calcd for C17H16N2O3: 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.

2.3.7. 3-(40 -iodophenyl)-8-methylquinazolin-4(3H)-one (7) Yield: 63%; Ash Grey Solid; m.p. 231e233  C; Rf: 0.86 (ethyl acetate/hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.35 (s, 1H), 8.04 (d, J ¼ 7.2 Hz, 1H), 7.93 (d, J ¼ 8.4 Hz, 2H), 7.75 (d, J ¼ 7.2 Hz, 1H), 7.49 (t, J ¼ 7.5 Hz, 1H), 7.37 (d, J ¼ 8.4 Hz, 2H), 2.57 (s, 3H); 13C NMR: (100 MHz, DMSO‑d6): dC 160.0, 146.0, 145.8, 137.9, 137.3, 135.5, 135.0, 129.7, 126.9, 124.0, 121.7, 94.9, 17.0; EI-MS: m/z (rel. abund. %), 362 [M]þ (100), 235 (14), 220 (7), 105 (28); Anal. Calcd for C15H11IN2O: C, 49.75; H, 3.06; I, 35.04; N, 7.73; O, 4.42; found: C, 49.73; H, 3.09; I, 35.01; N, 7.71; O, 4.45. 2.3.8. 3-(20 -iodophenyl)-8-methylquinazolin-4(3H)-one (8) Yield: 69%; Beige Solid; m.p. 162e165  C; Rf: 0.72 (ethyl acetate/ hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.24 (s, 1H), 8.07 (d, J ¼ 7.5 Hz, 1H), 8.05 (d, J ¼ 7.2 Hz, 1H), 7.77 (d, J ¼ 7.2 Hz, 1H), 7.61 (m, 2H), 7.51 (t, J ¼ 7.8 Hz, 1H), 7.34 (m, 1H), 2.59 (s, 3H); 13C NMR: (100 MHz, DMSO‑d6): dC 159.6, 146.2, 146.1, 140.3, 139.2, 135.7, 135.2, 131.1, 129.7, 129.5, 127.0, 124.1, 121.9, 99.5, 17.1; EI-MS: m/z (rel. abund. %), 362 [M]þ (32), 235 (100), 220 (1), 105 (5); Anal. Calcd for C15H11IN2O: C, 49.75; H, 3.06; I, 35.04; N, 7.73; O, 4.42; found: C, 49.77; H, 3.04; I, 35.03; N, 7.76; O, 4.40. 2.3.9. 3-(20 ,40 -difluorophenyl)-8-methylquinazolin-4(3H)-one (9) Yield: 83%; Off White Solid; m.p. 181e185  C; Rf: 0.78 (ethyl acetate/hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.38 (s, 1H), 8.04 (d, J ¼ 7.8 Hz, 1H), 7.80 (m, 1H), 7.75 (d, J ¼ 9.0 Hz, 1H), 7.62 (m, 1H), 7.52 (t, J ¼ 7.5 Hz, 1H), 7.36 (m, 1H), 2.58 (s, 3H); 13C NMR: (100 MHz, DMSO‑d6): dC 146.0, 135.7, 135.4, 131.4, 131.2, 127.2, 124.0, 112.5, 112.4, 112.1, 105.2, 105.1, 105.0, 104.7, 17.0; EI-MS: m/z (rel. abund. %), 372 [M]þ (100), 257 (5), 253 (68), 220 (7); Anal. Calcd for C15H10F2N2O: C, 66.17; H, 3.70; F, 13.96; N, 10.29; O, 5.88; found: C, 66.15; H, 3.72; F, 13.98; N, 10.27; O, 5.85.

2.3.12. 3-(30 -chlorophenyl)-8-methylquinazolin-4(3H)-one (12) Yield: 63%; Off White Solid; m.p. 125e129  C; Rf: 0.82 (ethyl acetate/hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.38 (s, 1H), 8.05 (d, J ¼ 7.8 Hz, 1H), 7.76 (d, 1H, J ¼ 7.5 Hz), 7.73 (s, 1H), 7.60 (m, 3H), 7.50 (t, J ¼ 7.8 Hz, 1H), 2.57 (s, 3H); 13C NMR: (125 MHz, DMSO‑d6): dC 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, 121.7, 17.0; EI-MS: m/z (rel. abund. %), 270 [M]þ (100), 272 [Mþþ2] (35), 235 (3); Anal. Calcd for C15H11ClN2O: C, 66.55; H, 4.10; Cl, 13.10; N, 10.35; O, 5.91; found: C, 66.51; H, 4.12; Cl, 13.11; N, 10.37; O, 5.93. 2.3.13. 3-(20 -methylphenyl)-8-methylquinazolin-4(3H)-one (13) Yield: 78%; Off White Solid; m.p. 132e135  C; Rf: 0.80 (ethyl acetate/hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.28 (s, 1H), 8.05 (d, J ¼ 7.8 Hz, 1H), 7.76 (d, J ¼ 7.5 Hz, 1H), 7.50 (t, J ¼ 7.8 Hz, 1H), 7.44 (m, 4H), 2.58 (s, 3H), 2.09 (s, 3H); 13C NMR: (100 MHz, DMSO‑d6): dC 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, 121.8, 120.8, 17.1, 17.0; EI-MS: m/z (rel. abund. %), 250 [M]þ (71), 233 (100), 132 (9); Anal. Calcd for C16H14N2O: C, 76.78; H, 5.64; N, 11.19; O, 6.39; found: C, 76.75; H, 5.67; N, 11.18; O, 6.37. 2.3.14. 3-(40 -methoxyphenyl)-8-methylquinazolin-4(3H)-one (14) Yield: 84%; Light Grey Solid; m.p. 152e155  C; Rf: 0.62 (ethyl acetate/hexanes, 3:7); 1H NMR: (300 MHz, DMSO‑d6): dH 8.32 (s, 1H), 8.03 (d, J ¼ 7.8 Hz, 1H), 7.74 (d, J ¼ 7.2 Hz, 1H), 7.48 (t, J ¼ 7.8 Hz, 1H), 7.45 (d, 2H, J ¼ 9.0 Hz), 7.10 (d, 2H, J ¼ 9.0 Hz), 3.82 (s, 3H), 2.57 (s, 3H); 13C NMR: (100 MHz, DMSO‑d6): dC 160.4, 159.2, 146.4, 146.1, 135.4, 134.8, 130.3, 128.6, 126.8, 124.0, 121.8, 114.3, 55.4, 17.0; EI-MS: m/z (rel. abund. %), 266 [M]þ (100), 251 (14), 235 (3); Anal. Calcd for C16H14N2O2: C, 72.16; H, 5.30; N, 10.52; O, 12.02; found: C, 72.18; H,

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5.33; N, 10.50; O, 12.00. 2.3.15. 3-(40 -methylphenyl)-8-methylquinazolin-4(3H)-one (15) Yield: 85%; Off White Solid; m.p. 118e120  C; Rf: 0.80 (ethyl acetate/hexanes, 3:7); 1H NMR: (400 MHz, DMSO‑d6): dH 8.32 (s, 1H), 8.04 (d, J ¼ 8.0 Hz, 1H), 7.74 (d, J ¼ 7.2 Hz, 1H), 7.48 (t, J ¼ 7.6 Hz, 1H), 7.41 (d, J ¼ 8.4 Hz, 2H), 7.36 (d, J ¼ 8.0 Hz, 2H), 2.57 (s, 3H), 2.38 (s, 3H); 13C NMR: (125 MHz, DMSO‑d6): dC 160.2, 146.2, 146.1, 138.2, 135.4, 135.0, 134.9, 129.6, 127.1, 126.8, 124.0, 121.8, 20.6, 17.0; EI-MS: m/z (rel. abund. %), 250 [M]þ (100), 235 (9), 220 (1); Anal. Calcd for C16H14N2O: C, 76.78; H, 5.64; N, 11.19; O, 6.39; found: C, 76.73; H, 5.69; N, 11.18; O, 6.40. 2.4. A. castellanii cultures A clinical strain of A. castellanii (ATCC 50492) T4 genotype, was cultured and maintained in 10 mL growth medium (PYG) composed of 0.75% w/v proteose peptone, 0.75% w/v extract of yeast, and 1.5% w/v glucose at 30  C in 75-cm2 culture flasks [20]. For amoebicidal assays, old PYG medium was replaced with fresh phosphate buffer saline (PBS) and adherent A. castellanii trophozoites were obtained by keeping culture flasks on ice for 15 min and subsequent gentle tapping to ensure the detachment of trophozoites. Finally, A. castellanii trophozoites were collected in a 50 mL conical tube and were centrifuged at 3000g for 5 min. The obtained pellet was re-suspended in 1 mL PBS, while the enumeration of A. castellanii was carried out by using a hemocytometer. 2.5. Amoebicidal assay The trophocidal activity of QNZs was determined by treating 5  105 healthy A. castellanii trophozoites per well with 100 and 50 mg per mL of QNZs and their respective controls in 24-well plates in RPMI-1640 medium. The plates were incubated at 30  C for 24 h as previously described [20]. Following the incubation time, the viable cells were identified by Trypan blue staining with 0.1% Trypan blue aqueous solution for 5 min. The number of live cells (nonstained) A. castellanii were tallied using a hemocytometer. Untreated A. castellanii were used as negative control, while chlorhexidine was used as a positive control. 2.6. Field emission scanning electron microscopy To determine the morphological effects on A. castellanii after treatment with QNZs, ultramicroscopic FESEM analysis was performed. A. castellanii untreated or treated with representative QNZs which showed either potent or low antiamoebic effects such as compounds 5 and 15 were fixated on glass cover slips using 2.5% glutaraldehyde solution prepared in PBS. The cover slips were kept at 4  C for 2 h to ensure complete infiltration. The samples were then dehydrated with a gradient concentrations of ethanol solutions ranging from 50 to 100%. After air drying of samples, they were sputtered with platinum, and imaging was carried out at FESEM instrument (Hitachi SU8010). Extensive images were recorded at different scales for each sample. The images presented here are representatives of various images. 2.7. Encystation assay To test the efficacy of QNZs in inhibiting the differentiation of A. castellanii trophozoites into cysts encystation assay was carried out. Briefly, 5  105 A. castellanii trophozoites suspended in PBS were treated with 100 mg/mL of QNZs in 1.5 mL centrifuge tubes at room temperature for 15 min. Simultaneously, encystation medium consisting of MgCl2 (50 mM) and glucose (10%) was added in 24-

well plates. After 15 min, QNZs treated or untreated A. castellanii were added in above 24-well plates containing encystation media. The cells were incubated at 30  C for 72 h to trigger the encystation [21]. After routine observation of plates, when mature cysts are formed, each well was treated with sodium dodecyl sulfate (SDS) aqueous solution (0.25%) to decompose the trophozoites and only the SDS resistant mature cysts were enumerated using a hemocytometer. 2.8. Excystation assay The non-nutrient agar plates were prepared by making bacteriological agar aqueous solution (1.5%). The agar was autoclaved, then poured on the petri plates and followed by the air drying inside biosafety cabinet. A. castellanii cysts were prepared by inoculating 1  106 trophozoites on non-nutrient agar plates. The inoculated plates were then incubated at 30  C for up to 2 weeks with routine observation under light microscope, till the mature cysts were formed. Cysts were scraped from plates using cell scraper with PBS, enumerated, and stored at 4  C. For excystation assay, 1  105 pre-formed cysts were incubated with 100 mg/mL QNZs and controls in growth medium PYG for 72 h [22]. Finally, cysts reformed into trophozoites were counted by using hemocytometer. 2.9. HaCaT keratinocyte cells culture HaCaT cells were routinely cultured in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% of each fetal bovine serum and Nu-serum, in 75-cm2 culture flasks. The growth medium was also complemented with 2 mM glutamine, 1 mM pyruvate, penicillin, streptomycin (100 units/mL and 100 mg/mL, respectively), vitamins and non-essential amino acids and vitamins [23]. After formation of confluent, uniform mono-layer (24e48 h), old media was aspirated, and cells were detached by using 2 mL trypsin. The trypsinized cell suspension was centrifuged at 2500g for 5 min. The obtained pellet was then re-suspended in 25 mL fresh cell growth media. Each well of a 96-well plate was seeded with 200 mL of above cell suspension and the plates were incubated in a 5% CO2 incubator with 95% humidity (at 37  C for 24 h) until the uniform monolayer of cells was observed under light microscope. 2.10. A .castellanii-mediated host cells cytotoxicity To determine whether QNZs can reduce the cytopathogenicity of A. castellanii, host cells cytotoxicity assays were performed as reported previously [24]. 5  105 A. castellanii were pre-treated with 100 mg/mL QNZs and negative and positive controls for 2 h in 30  C incubator. Next, these cultures were centrifuged at 3000g for 5 min. The supernatants were aspirated, and the cells pellet was re-suspended in 200 mL of fresh RPMI-1640 to remove excess compounds and any intracellular toxins. These suspensions were then incubated with HaCaT cells monolayers formed in 96-well plates at 37  C in a 5% CO2 incubator with 95% humidity. After incubation of 24 h, the supernatants were collected from each well and lactate dehydrogenase (LDH) was measured at 492 nm on microplate reader using LDH cytotoxicity detection kit (Invitrogen) as described previously [25]. LDH is released from damaged cells only, therefore healthy, untreated cells were the negative control, while cells completely disintegrated by using 0.1% Triton X-100 were taken as the positive control for all cytotoxicity assays. The percent cell cytotoxicity was calculated by using following equation:

A. Anwar et al. / European Journal of Medicinal Chemistry 182 (2019) 111575

% cell cytotoxicity ¼ (LDH released by cells with sample treatment eLDH measured in untreated cells) / (Total LDH released by Triton X-100 treated cells - LDH measured in untreated cells)  100

2.11. Cytotoxicity assay The cytotoxicity of QNZs on human normal cells (HaCaT keratinocytes) was also determined by LDH cytotoxicity assay, as reported previously [25]. Shortly, 100 mg/mL of QNZs and respective controls were incubated with uniform monolayer of HaCaT cells in a 96-well plates for 24 h (at 37  C in a 5% CO2 incubator). Following the incubation time, supernatants were collected, and cytotoxicity was calculated by determination of LDH enzyme as mentioned above. 2.12. Statistical analysis All of the presented results are representatives of several experiments performed in duplicate and are represented as the mean ± standard error. Student's T-test was performed for statistical analysis. The correlation and significance were measured on Microsoft Excel worksheets. The threshold level of significance was P < 0.05, using two-sample T-test and two-tailed distribution. * corresponds to P < 0.05, while **P < 0.01, and ***P < 0.001 respectively. 3. Results and discussion 3.1. Chemistry 3.1.1. General synthesis of QNZs (1e15) A library of 3-aryl-8-methylquinazolin-4(3H)-ones was synthesized via one pot reaction of 2-amino-3-methylbenzoic acid, triethoxymethane, and several substituted anilines in equimolar quantities under acidic medium (Scheme 1). The heterocyclic ring formation occur due to increased stability. The reaction mixture was refluxed till the complete consumption of starting material. The advancement in reaction was monitored through TLC analysis. The aqueous work up of reaction removed excess acid and the product was precipitated out. The products were then crystallized from ethanol for purification. The original spectra are presented in supplementary information. 3.2. Spectral characterization of a representative QNZ (5) 3.2.1. 1H NMR spectroscopy The 1H NMR spectrum of compound 5 (most active) was recorded, by using an instrument of 400 MHz in DMSO‑d6. H-2 of

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the quinazoline ring resonated at dH 8.35 as a sharp singlet and was the most downfield signal of the spectrum due to presence of two nitrogen atoms. All other aromatic protons were resonated in the range of dH 8.04e7.42. A doublet of H-5 was appeared at dH 8.04 with coupling constant value of J ¼ 8.0 Hz showing coupling with the neighboring proton 6. H-7 resonated at dH 7.75 as a doublet with J ¼ 7.2 Hz showing ortho coupling with H-6. H-2ʹ and H-6ʹ appeared as doublet of doublets at dH 7.62 (J2ʹ,3ʹ/ 6ʹ,5ʹ ¼ 9.0 Hz, J2ʹ,4ʹF/6ʹ,4ʹF ¼ 5.2 Hz) showing ortho coupling with H-3ʹ/ H-5ʹ and meta coupling with 4ʹ-F. A triplet of H-6 resonated at dH 7.49 (J6(5,7) ¼ 7.6 Hz) showing coupling with H-5 and -7, respectively. While H-3ʹ and H-5ʹ resonated at dH 7.42 as a triplet J30 (20 ,40 F)/ 50 (60 ,40 F) ¼ 8.8 Hz). The most upfiled signal of CH3 protons appeared at dH 2.57 as a singlet (Fig. 3). 3.2.2. Mass spectrometry The recorded EI-MS spectra of compound 5 depicted the Mþ at m/z 254, in consonance with C15H11FN2O (254.09) and was also the base peak. The ion obtained at m/z 249 was due to the removal of methyl group from molecule. The fragment at m/z 145 was acquired by the reason of quinazolinone. The key fragments are presented in Fig. 4. 3.3. Biology 3.3.1. Antiacanthamoebic and cytotoxic activity of QNZs A. castellanii is an opportunistic protist which is the causative agent of infections of eyes and brain. The risk factor for more prevailing pathology i.e., Acanthamoeba keratitis is practice of unhygienic contact lens handling and ineffective contact lens solution [26]. Currently, there are only a few identified molecular pathways to target A. castellanii which is one of the key limitations in development of effective therapeutics. Furthermore, the extended usage of recommended drugs (including a mixture of biguanides, azoles, amidines and antibiotics) can cause host cells cytotoxicity while their ineffectiveness against resistant cysts is prone to recurrence of the infection [27]. Hence, there is an urgent requisite for the development of novel and effective chemotherapeutics against A. castellanii infections. Heterocyclic compounds have proven to be promising drug candidates [28,29]. Among heterocyclic compounds, quinazoline and its derivatives have been thoroughly studied against fungi and bacteria [30,31]. Alho et al., have identified quinoxalinones; another structural variant of sixmembered heterocyclic rings with two nitrogen atoms as potent lead compounds against apicomplexa and mastigophora parasites [10]. In another study, 7-nitroquinoxalin-2-ones were found to be effective against Trypanosoma cruzi with a possible inhibition of the trypanothione reductase enzyme [32]. In current study, we synthesized a library of fifteen variants of 3-aryl-8-methylquinazolin4(3H)-ones and determined their antiamoebic activity against

Fig. 3. 1H NMR chemical shifts of compound 5.

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Fig. 4. Key fragmentation pattern (EI-MS) of compound 5.

A. castellanii. Current report is a valid example of medicinal chemistry aspect subjected to antiparasitic study with quinazolinones being tested against free-living amoeba A. castellanii for the first time. 3.3.2. QNZs exhibited significant antiamoebic effects against A. castellanii Amoebicidal assay performed at 100 and 50 mg/mL of QNZs revealed that almost all of fifteen compounds reduced the number of viable A. castellanii trophozoites at 100 mg/mL. Among these compounds, 5, 7, and 8 produced striking antiamoebic effects at 50 mg/mL, while additionally compound 2 exhibited potent results at 100 mg/mL (Fig. 5). The level of significance was calculated with respect to solvent control methanol. At higher concentration i.e., 100 mg/mL, only one compound 6 showed ineffectiveness. At these

concentrations, almost all of compounds showed more than 50% inhibition which corresponds to the IC50. The 100 mg/mL concentration of compound 2, 5 and 8, caused more than 80% inhibition, while at 50 mg/mL more than 60% inhibition was observed with compounds 5, 7, and 8, hence these data provide more quantitative picture of the antiamoebic assay. The QNZs treated A. castellanii were also subjected to FESEM analysis for the evaluation of surface alteration. FESEM images showed that A. castellanii treated with compounds 5 and 15 triggered surface disintegration and pores formation which might have resulted in high antiamoebic effects (Fig. 6).

3.3.3. QNZs inhibited encystation and excystation of A. castellanii Encystation of A. castellanii is the process of conversion of trophozoites into cysts which contributes to the resistance against

Fig. 5. Amoebicidal assay against A. castellaniiat (a) 100 mg per mL, and (b) 50 mg per mL. The viability of amoeba was determined after amoebicidal assay as described in the materials and methods section. Briefly, A. castellanii trophozoites were incubated with QNZs, negative and positive controls at 30  C for 24 h. Next, the viability was measured by Trypan blue exclusion assay. The results are presented as the mean ± standard error of various experiments performed in duplicate. * represents P < 0.05, ** represents P < 0.01, while *** represents P < 0.001. P values were obtained using two-sample T test and two-tailed distribution.

Fig. 6. FE-SEM images of A. castellanii with and without treatment with QNZs. A. castellanii were fixed on glass cover slips by using glutaraldehyde. Followed by fixation, the samples were washed with ethanol and images were recorded on Field-emission scanning electron microscope (FE-SEM) (Hitachi SU8010) instrument. (a) A. castellanii control. (b) A. castellanii treated with 100 mg per mL of compound 5. (c) A. castellanii treated with 100 mg per mL of 15. Control showed integrated A. castellanii wall, whereas treated amoeba showed disintegration and pores.

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drugs A. castellanii are transformed into cysts upon exposure to harsh conditions including treatment with drugs, change in pH and temperature, and nutrients deprivation. Encystation assays were performed to test whether these QNZs can cause inhibition of this morphological transformation. Fig. 7a describes that nine out of fifteen compounds inhibited encystation at 100 mg/mL with compounds 8, 9 and 13 being most effective. On the other hand, cyst is the most resistant form of A. castellanii which is one of the main reasons for recurrence of Acanthamoeba keratitis even after cornea transplant surgery [33]. Most of the drugs used currently against A. castellanii infections have shown limited potency against cysts. The effects of QNZs were also evaluated against excystation by application of 100 mg/mL with pre-formed cysts of A. castellanii. The results showed that most of the fifteen tested compounds inhibited excystation with compound 5 showed same effects as positive control chlorhexidine (CHX) (Fig. 7b). These results show potential of QNZs as suitable alternative for drug development against A. castellanii.

CHX

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3.3.4. QNZs exhibited minimal damage against human keratinocytes and reduced Acanthamoeba-mediated host cell cytotoxicity To evaluate the cytotoxicity of QNZs against human cells, LDH assays were performed. The results of cytotoxicity determination showed that QNZs produced minimal cytotoxic effects (Fig. 8a). Compound 3 and 8 exhibited 11 and 8% cytotoxicity, respectively, while the remaining thirteen compounds showed no toxicity at 100 mg/mL. The cytotoxic effects were studied against human cells and suggest that these QNZs are bio-safe and can further be assessed for in vivo studies. These QNZs also reduced the host cells cytotoxicity of A. castellanii. The pretreatment of amoeba with QNZs showed significant decline in their cytopathogenicity against normal human cells. Fig. 8b shows that untreated A. castellanii triggered 80% toxicity against HaCaT cells, while chlorhexidine treated amoeba showed protection of cells against amoeba. All fifteen compounds tested at 100 mg/mL significantly reduced the host cells cytopathogenicity. Notably, compounds 5, 7, 8 and 13 produced most potent effects which completely abolished the host cells toxicity.

CHX

Fig. 7. Depiction of the results of (a) encystation. QNZs inhibited A. castellanii encystation. A. castellanii (5  105) were inoculated in PBS in the presence of QNZs and respective controls at 100 mg per mL with encystation media and incubated at 30  C for 72 h. Next, 0.25% sodium dodecyl sulfate (SDS) was added and incubated at room temperature for 10 min to lyse A. castellanii trophozoites followed by enumeration of amoebae cysts using a hemocytometer. (b) Excystation assays was performed by incubating 100 mg per mL QNZs with A. castellanii cysts (1  105) in PYG at 30  C for 72 h. After this period, amoebae were counted using a hemocytometer. The results are presented as the mean ± standard error of various experiments performed in duplicate. * represents P < 0.05, ** represents P < 0.01, while *** represents P < 0.001. P values were obtained using two-sample T test and two-tailed distribution.

Fig. 8. (a) QNZs did not exhibit cytotoxicity against HaCaT cells at 100 mg per mL. These compounds and the respective controls were incubated with HeLa cells monolayer for 24 h at 37  C in a 5% CO2 incubator. Following this incubation, cell-free supernatant was collected, and cytotoxicity was determined using Lactate dehydrogenase (LDH) assay kit (Invitrogen). The negative control values for cytotoxicity assays were obtained by incubating cells with RPMI-1640 alone, and positive control values were obtained by 100% cell death using 0.1% Triton X-100. (b) Pretreatment of 100 mg per mL of QNZs abolished A. castellanii-mediated host cells cytotoxicity. Briefly, amoebae (1  105) were incubated at 30  C with QNZsand respective controls for 2 h in RPMI-1640 and then incubated with HaCaT cells for 24 h at 37  C in a 5% CO2 incubator as described in materials and methods section. Next, cell-free supernatant was collected, and cytotoxicity was determined using Lactate dehydrogenase (LDH) assay kit. The results are presented as the mean ± standard error of various experiments performed in duplicate.

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3.3.5. Structure-activity relationship Limited structure-activity relationship against A. castellanii trophozoites indicates that the variation in activity was observed based on different substituent present on QNZ ring and their respective positions on the aryl part. The presence of one fluorine, iodine, and methyl groups on aryl ring of compounds 2, 8, and 13 exhibited pronounced antiacanthamoebic activity against pathogenic trophozoite stage. However, the addition of another number of these groups reduced the effects. Notably, mono substitution at ortho position showed more activity as in compound 8 and 13

bearing iodo and methyl groups, respectively. Shifting the position of these substituents to para in compounds 7 and 15 displayed a remarkable regression in activity. Similarly, compound 6 with two methyl substituents at meta positions, exhibited lower activity as compared to compound 13 (Fig. 9). Interestingly, among fluoro substituted derivatives, compound 9 with two fluoro groups at ortho and para positions was most active as compared to its mono substituted derivatives. The mono substituted compounds 2 and 5 having fluoro group at meta and para positions were less active. Mono methoxy substituted compound 14 also exhibited good

Fig. 9. Structure activity relationship between iodo and methyl substituted QNZs.

Fig. 10. Structure activity relationship between fluoro and methoxy substituted QNZs.

A. Anwar et al. / European Journal of Medicinal Chemistry 182 (2019) 111575

antiacanthamoebic activity as compared to its dimethoxy counterpart 11. It was also noted that bromo and chloro substituted QNZs were less active (Fig. 10). Based on the observed effects of substituents present on the aryl part of QNZs, it can be concluded that iodo group at ortho position, fluoro groups at ortho and para positions, methyl group at ortho position, and methoxy group at para position caused highest antiamoebic activity. 4. Conclusions QNZs showed significant antiacanthamoebic effects especially compounds 2, 5, 8, and 13. These compounds exhibited potent antiamoebic activity, along with the inhibition of encystation and excystation in A. castellanii. Furthermore, these compounds significantly reduced the Acanthamoeba-mediated host cells cytopathogenicity. Interestingly, minimal cytotoxicity was observed by these compounds, when tested against human normal cells. Therefore, this study exhibits potential ability of QNZs for the development of effective antiacanthamoebic agents. These results are estimated to be a key advancement in the development of efficient drug leads against free-living amoeba A. castellanii. The determination of their mode of action and in vivo performance are included in our future investigation, along with testing more derivatives of QNZs for extensive SAR. Author contributions statement A.A. conducted the experiments on Acanthamoeba and wrote the first draft of manuscript. M.S.S. and S.M.S. synthesized the compounds under the supervision of K.M.K. M.S.S. and K. characterized the structures. R.S. and N.A.K conceived the idea and supervised all biological studies. K.M.K., R.S., and N.A.K. corrected and finalized the manuscript which is submitted with the consent of all authors. Conflicts of interest Authors declare no competing interests. The manuscript was submitted by the approval of all authors. Data availability Data will be provided upon request on case to case basis. Acknowledgements This work is supported by Sunway University, Malaysia (University Research Award INT-SST-DBS-2019-03) and the Pakistan Academy of Sciences for providing financial support Project No. (59/PAS/440). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111575. References [1] F. Marciano-Cabral, G. Cabral, Acanthamoeba spp. as agents of disease in humans, Clin. Microbiol. Rev. 16 (2003) 273e307. [2] N.A. Khan, Acanthamoeba: biology and increasing importance in human health, FEMS Microbiol. Rev. 30 (2006) 564e595. [3] G.S. Visvesvara, H. Moura, F.L. Schuster, Pathogenic and opportunistic freeliving amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappiniadiploidea, FEMS Immunol. Med. Microbiol. 50 (2007) 1e26. [4] J. Lorenzo-Morales, N.A. Khan, J. Walochnik, An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment, Parasite 22 (2015).

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