Arch. Pharm. Res. DOI 10.1007/s12272-015-0686-4

RESEARCH ARTICLE

Novel aryl carbamate derivatives of metronidazole as potential antiamoebic agents Faisal Hayat1 • Hussain Mustatab Wahedi1 • Seonghyeok Park1 • Saba Tariq2 Amir Azam2 • Dongyun Shin1

•

Received: 10 October 2015 / Accepted: 16 November 2015 Ó The Pharmaceutical Society of Korea 2015

Abstract A series of novel aryl carbamate derivatives of metronidazole (MNZ) were designed, synthesized, and screened for antiamoebic activity. As compared to MNZ, most of the derivatives exhibited moderate to excellent activity against the HM1:IMSS strain of Entamoeba histolytica. Compounds 7, 14, 16, 19, and 21 exhibited the most promising antiamoebic activity with IC50 values of 0.24, 0.08, 0.26, 0.26, and 0.15 lM, respectively, compared to that of MNZ (1.78 lM). Moreover, from the toxicological studies of these compounds on human melanocytes, the melan-a cell line revealed that the potent compounds are nontoxic at concentrations ranging from 2.5 to 50 lM. Keywords Metronidazole
Aryl carbamate
Structureactivity relationship
Entamoeba histolytica
Antiamoebic

Introduction Amoebiasis is a disease of the human gastrointestinal tract caused by Entamoeba histolytica, a protozoan parasite which is capable to penetrate the intestinal mucosa, causing amoebic colitis and spread via portal circulation to other organs, typically to the liver; it induces amoebic liver abscess, the most common extra intestinal manifestation of invasive amoebiasis (Stanley 2003; Oku et al. 2012; Petri & Dongyun Shin [email protected] 1

College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon 406-799, South Korea

2

Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, India

and Haque 2013). This condition constitutes a medical emergency, and it may lead to death (Stanley 2003). Entamoeba histolytica produces considerable amounts of cysteine proteinases (CPs), and this class of enzymes is responsible for the pathology induced by E. histolytica (Moncada et al. 2003). Infection by this parasite is a major cause of morbidity worldwide, causing approximately 50 million cases of amoebiasis and 100,000 deaths annually (Stanley 2003; Ralston and Petri 2011). The highest prevalence of E. histolytica infections is observed in countries dominated by poor sanitary conditions, particularly in Mexico, India, Central and South America as well as tropical regions of Asia and Africa (Ravdin and Stauffer 2005). Various structurally different compounds have been identified as potent antiamoebic agents, and some of them have been utilized in medical practice. Based on their structural features, they can be classified into several groups, such as azole (e.g., metronidazole or MNZ, tinidazole, and ornidazole), quinoline (e.g., iodoquinol), ester (e.g., diloxanide furoate), and some carbamate derivatives (Ordaz-Pichardo et al. 2005; Azam and Agarwal 2007; Singh et al. 2009; Salahuddin et al. 2012) (Figs. 1, 2). Among these compounds, MNZ, known to be a highly effective amoebicide and is considered to be the drug of choice for treating amoebiasis (Townson et al. 1994). However, recent studies have shown that this drug exhibits several toxic effects such as genotoxicity, gastric mucus irritation, and spermatozoid damage (Purohit and Basu 2000; El-Nahas and El-Ashmawy 2004). Furthermore, the treatment of several intestinal protozoan parasites has been reported to fail because they exhibit drug resistance (Abboud et al. 2001; Petri 2003). Therefore, it is imperative to constantly survey new molecules for the treatment of amoebiasis and to decrease or replace the use

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Fig. 1 Metronidazole-based antiamoebic compounds

et al. 2009; Fe´rriz et al. 2010), antimalarial (Verma et al. 2015; Mata et al. 2012), and antiamoebic activities (OrdazPichardo et al. 2005) as well as K channel blockers (Fluxe et al. 2006) and FAAH inhibitors (Vacondio et al. 2011). Carbamate bearing molecules play an important role in modern drug discovery and medicinal chemistry. In recent years, carbamate derivatives have attracted significant attention, owing to their application in drug design and discovery. In view of these considerations and our continuous effort toward the development of novel antiamoebic agents, herein, we report the synthesis and biological activity of new aryl carbamate derivatives of MNZ (5–23), bearing sulfonate (–SO2–) and ether (–O–) linkages, based on previously published antiamoebic agents (Azam and Agarwal 2007; Singh et al. 2009). Their structures are shown in Figs. 1 and 2. Figure 3 shows the novel antiamoebic agents designed herein.

Materials and methods General information

Fig. 2 para-Substituted aniline-based antiamoebic compounds

of MNZ and other toxic drugs. The side chains attached to MNZ facilitate further modification for the synthesis of novel molecules, which might exhibit better antiamoebic activity and lesser toxicity toward the host. In our previous study, some MNZ analogs exhibiting significant antiamoebic activity and very less cytotoxicity have been reported (Athar et al. 2005; Abid et al. 2008; Salahuddin et al. 2012). The study of carbamates has become of much interest on account of their diverse biological properties such as antiHIV (Solyev et al. 2012), antifungal (Kus and Altanlar 2003), anticancer (Janganati et al. 2014), anticonvulsant (Sadek et al. 2013), antitumor (Shreder et al. 2012), antimicrobial (Rogers et al. 2011), antituberculosis (Meng

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The melting points were recorded on automated melting point system (Optimelt) and are uncorrected. Proton and carbon magnetic resonance spectra (1H NMR and 13C NMR, respectively) were recorded on a Bruker 600 (1H NMR at 600 MHz and 13C NMR at 150 MHz) spectrometer with solvent resonance as the internal standard (1H NMR: DMSO-d6 at 2.51 ppm; 13C NMR: DMSO-d6 at 39.9 ppm). 1H NMR data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants (Hertz), and integration. Mass spectra were obtained using Agilent Q-TOF with an ESI source. Analytical thin layer chromatography (TLC) was performed on Sorbent Technologies 0.20 mm Silica G TLC plates. Visualization was accomplished with UV light, KMnO4, and/or aqueous ceric ammonium nitrate solution followed by heating. Purification of the reaction products was carried out by N

N NO2

N

H N

O O

NO2

N

H N

O O O S O R

O

O R

Fig. 3 General structures of the designed aryl carbamate derivatives of metronidazole

Novel aryl carbamate derivatives of metronidazole as potential antiamoebic agents

flash column chromatography using silica gel (40-63 lm) purchased from ZEOprepÒ. Unless otherwise noted, all reactions were carried out under an atmosphere of dry nitrogen in oven-dried glassware under magnetic stirring. Yield refers to isolated yield of analytically pure material unless otherwise noted. All reagents were purchased from TCIÒ or Sigma AldrichÒ and used without further purification. Organic solvents were of reagent grade or were purified before use by standard procedures.

Synthesis Synthesis of 2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethyl carbonochloridate (2) MNZ (1 mmol) was dissolved in 30 mL acetone and then 2–3 drops of triethyl amine were added. Now the resulting mixture was stirred at rt for 10 min. After 10 min stirring, triphosgene (0.5 mmol) was added over a period of 15 min at 0 °C. All the processes were carried out in high vacuum fuming hood. After completion of addition, the reaction mixture was allowed to room temperature and further stirred for 12–14 h under inert atmosphere (N2). After the completion of reaction (TLC), the reaction mixture was filtered to remove the white residue and the resulting mixture was concentrated on rotavapor. The desire product was obtained as green oil which was used for the next step without purification.

Synthesis of 2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethyl (4-hydroxyphenyl)carbamate (4) A mixture of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethylcarbonochloridate (1 mmol), 4-aminophenol (1 mmol) and anhydrous potassium carbonate (1.5 mmol) in 25 mL acetone was stirred at reflux temperature for 5–6 h. The progress of the reaction was measured by TLC. After completion of reaction, the resulting mixture was allowed to cool at room temperature and poured into water and extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium bicarbonate and brine solution and dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the crude product was purified by flash column chromatography using hexane/ethyl acetate as an eluent. Brown solid (62 %), mp176–178 °C: 1H NMR (DMSOd6, 600 MHz) d 9.32 (s, 1H), 9.15 (s, 1H), 8.06 (s, 1H), 7.15 (s, 2H), 6.68 (d, J = 8.4 Hz, 2H), 4.53 (t, J = 4.8 Hz, 2H), 4.42 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 153.5, 152.1, 138.9, 133.5, 131.8,

130.5, 121.0, 115.9, 115.6, 115.5, 62.4, 62.0, 45.9, 14.5, 14.3. General procedure for the preparation of compounds (5–13) 2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl (4-hydroxyphenyl) carbamate (1 mmol) and triethyl amine (1.5 mmol) were dissolved in 10 mL DMF under nitrogen gas atmosphere. The resulting mixture was stirred for a period of 15 min and then arylsulfonyl chloride (1 mmol) was added slowly to the mixture. The reaction mixture was further stirred for 30 min. After the reaction was complete, the reaction mixture was poured on to ice water and extracted with ethyl acetate (3 9 20 mL). The combined ethyl acetate extracts were then washed with water (20 mL), brine (20 mL) and dried over anhydrous sodium sulfate. The volatiles were removed under reduced pressure and the resulting mixture was purified over a silica gel column using hexane/ethyl acetate as an eluent.

4-(((2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethoxy)carbonyl)amino)phenyl naphthalene-1-sulfonate (5) White solid (72 %), mp 153–154 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.75 (s, 1H), 8.65 (d, J = 7.8 Hz, 1H), 8.42 (d, J = 8.4 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 6.0 Hz, 1H), 8.03 (s, 1H), 7.92 (t, J = 7.8 Hz, 1H), 7.80 (t, J = 5.7 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.26 (d, J = 7.8 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 4.57 (t, J = 5.4 Hz, 2H), 4.43 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13 C NMR (DMSO-d6, 150 MHz) d 153.3, 152.0, 144.2, 138.9, 138.3, 136.8, 134.2, 133.5, 131.8, 130.0, 129.9, 129.8, 128.0, 125.0, 124.5, 122.5, 119.9, 62.8, 62.5, 45.7, 14.3; HRMS (ESI) m/z calcd for C23H20N4O7S [(M ? H)?], 497.1125: found, 497.1116.

4-(((2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethoxy)carbonyl)amino)phenyl benzenesulfonate (6) White solid (58 %), mp 174–176 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.80 (s, 1H), 8.05 (s, 1H), 7.85–7.81 (m, 3H), 7.69–7.66 (m, 2H), 7.35 (d, J = 7.8 Hz, 2H), 6.39 (d, J = 9.0 Hz, 2H), 4.59 (t, J = 4.8 Hz, 2H), 4.46 (t, J = 5.1 Hz, 2H), 2.46 (s, 3H); 13C (DMSO-d6, 150 MHz) d 153.3, 152.0, 144.2, 138.3, 135.4, 134.7, 133.5, 130.0, 128.6, 122.9, 119.8, 62.8, 45.7, 14.4; HRMS (ESI) m/z calcd for C19H18N4O7S [(M ? H)?], 447.0969: found, 447.0990.

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4-(((2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethoxy)carbonyl)amino)phenyl 4-(trifluoromethyl) benzenesulfonate (7) Light yellow solid (52 %), mp 180–183 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.83 (s, 1H), 8.09–8.05 (m, 5H), 7.38 (d, J = 8.4 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H), 4.60 (t, J = 4.8 Hz, 2H), 4.46 (t, J = 5.1 Hz, 2H), 2.46 (s, 3H); 13 C NMR (DMSO-d6, 150 MHz) d 153.3, 152.0, 144.2, 138.9, 138.6, 138.5, 134.8, 134.6, 134.5, 133.5, 129.8, 127.4, 124.5, 122.9, 122.7, 119.9, 62.8, 45.7, 14.3; HRMS (ESI) m/z calcd for C20H17F3N4O7S [(M ? H)?], 515.0843: found, 515.0830. 4-(((2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethoxy)carbonyl)amino)phenyl4-(tertbutyl)benzenesulfonate (8) Light brown solid (53 %), mp 169–171 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.80 (s, 1H), 8.05 (s, 1H), 7.78 (d, J = 7.7 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 7.8 Hz, 2H), 6.95 (d, J = 6.6 Hz, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.46 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H), 1.32 (s, 9H); 13C NMR (DMSO-d6, 150 MHz) d 158.6, 153.3, 152.0, 144.3, 138.9, 138.2, 133.5, 132.0, 128.5, 127.0, 122.9, 119.8, 119.9, 62.8, 45.7, 31.1, 14.4; HRMS (ESI) m/z calcd for C23H26N4O7S [(M ? H)?], 503.1595: found, 503.1562. 4- (((2-(2-Methyl-5-nitro-1H-imidazol-1-yl) ethoxy) carbonyl) amino) phenyl 3, 5-dichlorobenzenesulfonate (9) Yellow solid (63 %), mp148–149 °C: 1H NMR (DMSOd6, 600 MHz) d 9.85 (s, 1H), 8.17 (t, J = 1.8 Hz, 1H), 8.05 (s, 1H), 7.89 (d, J = 1.8 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.05 (d, J = 6.6 Hz, 2H), 4.60 (t, J = 5.4 Hz, 2H), 4.47 (t, J = 5.4 Hz, 2H), 2.47 (s, 3H); 13C NMR (DMSOd6, 150 MHz) d 153.3, 152.0, 143.9, 138.9, 138.6, 137.6, 136.0, 135.2, 133.5, 128.5, 127.1, 123.0, 120.0, 62.8, 45.7, 14.4; HRMS (ESI) m/z calcd forC19H16Cl2N4O7S [(M ? H)?], 515.0190: found, 515.0151. 4-(((2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethoxy)carbonyl)amino)phenyl4-chlorobenzenesulfonate (10) Light brown solid (62 %), mp 165–167 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.82 (s, 1H), 8.05 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 7.8 Hz, 2H), 6.97 (d, J = 9.0 Hz, 2H), 4.60 (t, J = 5.1 Hz, 2H), 4.46 (t, J = 5.1 Hz, 2H), 2.47 (s, 3H); 13 C NMR (DMSO-d6, 150 MHz) d 153.3, 152.0, 144.1, 140.4, 138.9, 138.4, 133.5, 130.6, 130.4, 123.0, 119.9,

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62.8, 45.7, 14.4; HRMS (ESI) m/z calcd for C19H17 ClN4O7S [(M ? H)?], 481.0579: found, 481.0533. 4-(((2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethoxy)carbonyl)amino)phenyl 4-iodobenzenesulfonate (11) Light brown solid (64 %), mp 172–173 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.82 (s, 1H), 8.06 (s, 1H), 8.05 (d, J = 1.8 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 7.8 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 4.60 (t, J = 4.2 Hz, 2H), 4.46 (t, J = 5.1 Hz, 2H), 2.47 (s, 3H); 13 C NMR (DMSO-d6, 150 MHz) d 153.3, 152.0, 144.1, 139.1, 138.9, 138.4, 134.2, 133.5, 130.0, 122.9, 119.9, 104.5, 62.8, 45.7, 14.4; HRMS (ESI) m/z calcd for C19H17IN4O7S [(M ? H)?], 572.9935: found, 572.9895. 4-(((2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethoxy)carbonyl)amino)phenyl 4-methylbenzenesulfonate (12) Yellow solid (82 %), mp 152–153 °C: 1H NMR (DMSOd6, 600 MHz) d 9.79 (s, 1H), 8.05 (s, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H), 6.93 (d, J = 4.8 Hz, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.46 (t, J = 5.1 Hz, 2H), 2.46 (s, 3H), 2.43 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 153.3, 152.1, 138.9, 133.5, 130.5, 121.0, 115.5, 62.4, 49.0, 45.9, 14.3; HRMS (ESI) m/z calcd for C20H20N4O7S [(M ? H)?], 461.1125: found, 461.1089. 4-(((2-(2-Methyl-5-nitro-1H-imidazol-1yl)ethoxy)carbonyl)amino)phenyl 2,4,6triisopropylbenzenesulfonate (13) White solid (53 %), mp 111–112 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.79 (s, 1H), 8.03 (s, 1H), 7.38 (d, J = 7.8 Hz, 3H), 7.34 (s, 1H), 6.90 (d, J = 9.0 Hz, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.45 (t, J = 5.1 Hz, 2H), 3.90–3.85 (m, 2H), 3.01–2.96 (m, 1H), 2.45 (s, 3H), 1.23 (d, J = 7.2 Hz, 6H), 1.12 (d, J = 7.2 Hz, 12H); 13C NMR (DMSO-d6, 150 MHz) d 155.0, 153.3, 152.0, 151.1, 138.9, 138.3, 133.5, 129.1, 124.5, 123.0, 119.9, 62.8, 45.7, 33.8, 29.8, 24.6, 23.7, 14.3 HRMS (ESI) m/z calcd for C28H36N4O7S [(M ? H) ?], 573.2377: found, 573.2334.

General procedure for the preparation of compounds (14–23) 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-hydroxyphenyl)carbamate (1 mmol) was dissolved in 10 mL anhydrous DMF and then was poured slowly into 25 mL

Novel aryl carbamate derivatives of metronidazole as potential antiamoebic agents

flask containing a suspension of potassium carbonate (1.2 mmol) in 5 mL DMF under nitrogen gas atmosphere. The resulting mixture was stirred for a period of 10 min and then Ar-benzyl bromides (1 mmol) was added slowly to the mixture. The reaction mixture was further stirred for 5–6 h. After the reaction was complete, the reaction mixture was poured on to ice water and extracted with ethyl acetate (3 9 20 mL). The combined ethyl acetate extracts were then washed with water (20 mL), brine (20 mL) and dried over anhydrous sodium sulfate. The volatiles were removed under reduced pressure and the resulting mixture was purified over a silica gel column using hexane/ethyl acetate as an eluent. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl(4-((4fluorobenzyl)oxy)phenyl)carbamate (14) Yellow solid (32 %), mp169–170 °C: 1H NMR (DMSOd6, 600 MHz) d 9.48 (s, 1H), 8.06 (s, 1H), 7.49 (t, J = 6.0 Hz, 2H), 7.28 (s, 2H), 7.22 (t, J = 8.1 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H), 5.03 (s, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.41 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 162.9, 161.3, 153.5, 152.1, 138.9, 133.8, 133.5, 130.4, 130.3, 120.6, 115.7, 115.5, 115.4, 69.0, 62.5, 45.8, 14.3; HRMS (ESI) m/z calcd for C20H19FN4O5 [(M ? H)?], 415.1412: found, 415.1410. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl (4(benzyloxy)phenyl)carbamate (15)

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-((2nitrobenzyl)oxy)phenyl)carbamate (17) Light brown solid (42 %), mp 185–186 °C:1H NMR (DMSO-d6, 600 MHz) d 9.50 (s, 1H), 8.12 (d, J = 7.8 Hz, 1H), 8.06 (s, 1H), 7.79 (d, J = 4.4 Hz, 2H), 7.64-7.61 (m, 1H), 7.29 (s, 2H), 6.94 (t, J = 9.0 Hz, 2H), 5.41 (s, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.44 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 153.5, 147.9, 138.9, 134.3, 133.5, 129.7, 129.5, 125.2, 120.6, 115.4, 67.0, 62.5, 60.2, 45.8, 14.4; HRMS (ESI) m/z calcd for C19H19FN2O [(M ? H)?], 442.1357: found, 442.1349. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-((3iodobenzyl)oxy)phenyl)carbamate (18) Brown solid (25 %), mp 174–176 °C: 1H NMR (DMSOd6, 600 MHz) d 9.49 (s, 1H), 8.06 (s, 1H), 7.81 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.28 (s, 2H), 7.20 (t, J = 7.8 Hz, 1H), 6.94 (d, J = 9.0 Hz, 2H), 5.03 (s, 2H), 4.59 (t, J = 4.8 Hz, 2H), 4.44 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 152.1, 140.3, 138.9, 136.8, 136.4, 133.5, 131.0, 127.3, 120.5, 115.4, 95.2, 79.6, 79.4, 79.2, 68.7, 62.5, 60.2, 45.8, 14.4; HRMS (ESI) m/z calcd for C20H19IN4O5 [(M ? H)?], 523.0473: found, 523.0447. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-((4cyanobenzyl)oxy)phenyl)carbamate (19)

Light yellow solid (17 %), mp 103–105 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.47 (s, 1H), 8.06 (s, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.39 (t, J = 6.6 Hz, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.28 (s, 2H), 6.94 (t, J = 9.0 Hz, 2H), 5.05 (s, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.44 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 152.1, 138.9, 137.6, 133.5, 128.8, 128.2, 128.0, 120.6, 115.3, 79.6, 79.4, 79.1, 69.8, 45.8, 14.3; HRMS (ESI) m/z calcd for C20H20N4O5 [(M ? H)?], 397.1506: found, 397.1477.

Yellow solid (14 %), mp 108–110 °C: 1H NMR (DMSOd6, 600 MHz) d 9.49 (s, 1H), 8.06 (s, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 7.8 Hz, 2H), 7.29 (s, 2H), 6.94 (d, J = 9.0 Hz, 2H), 5.15 (s, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.44 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 152.1, 143.5, 133.5, 132.8, 128.4, 120.6, 119.2, 115.4, 110.8, 68.8, 62.5, 45.8, 14.4;HRMS (ESI) m/z calcd for C21H19N5O5 [(M ? H)?], 422.1459: found, 422.1430.

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl(4-((3,4difluorobenzyl)oxy)phenyl)carbamate (16)

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-((2methylbenzyl)oxy)phenyl)carbamate (20)

Light brown solid (12 %), mp 177–179 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.49 (s, 1H), 8.06 (s, 1H), 7.53–7.50 (m, 1H), 7.48–7.43 (m, 1H), 7.29 (bs, 3H), 6.94 (t, J = 9.0 Hz, 2H), 5.04 (s, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.44 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSOd6, 150 MHz) d 152.1, 133.5, 124.9, 120.5, 118.0, 117.9, 117.2, 117.1, 115.4, 68.5, 62.5, 45.8, 14.3; HRMS (ESI) m/ z calcd for C20H18F2N4O5 [(M ? H)?], 433.1318: found, 433.1280.

White solid (11 %), mp 143–144 °C: 1H NMR (DMSO-d6, 600 MHz) d 9.48 (s, 1H), 8.06 (s, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.29 (s, 2H), 7.26-7.19 (m, 3H), 6.96 (d, J = 9.0 Hz, 2H), 5.03 (s, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.44 (t, J = 5.1 Hz, 2H), 2.49 (s, 3H), 2.23 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 138.9, 136.9, 135.5, 133.5, 130.5, 128.9, 128.4, 126.1, 120.6, 115.3, 68.5, 62.5, 45.8, 18.9, 14.4; HRMS (ESI) m/z calcd for C21H22N4O5 [(M ? H)?], 411.1663: found, 411.1628.

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2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-((3chlorobenzyl)oxy)phenyl)carbamate (21) Yellow solid (19 %), mp 162–163 °C: 1H NMR (DMSOd6, 600 MHz) d 9.49 (s, 1H), 8.06 (s, 1H), 7.50 (s, 1H), 7.44–7.39 (m, 3H), 7.30 (s, 2H), 6.96 (d, J = 9.0 Hz, 2H), 5.08 (s, 2H), 4.59 (t, J = 4.8 Hz, 2H), 4.44 (t, J = 4.5 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 154.2, 153.5, 152.1, 140.2, 138.9, 133.5, 132.5, 130.8, 130.7, 128.1, 127.7, 127.6, 126.6, 120.5, 115.4, 68.8, 62.5, 45.8, 14.3; HRMS (ESI) m/z calcd for C20H19ClN4O5 [(M ? H)?], 431.1117: found, 431.1099. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-((4(trifluoromethyl) benzyl) oxy) phenyl) carbamate (22) Yellow solid (22 %), mp 174–176 °C: 1H NMR (DMSOd6, 600 MHz) d 9.49 (s, 1H), 8.06 (s, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.30 (s, 2H), 6.95 (d, J = 9.0 Hz, 2H), 5.18 (s, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.44 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 153.5, 152.1, 142.6, 138.9, 133.5, 128.3, 125.7, 123.8, 120.6, 115.4, 68.9, 62.5, 45.8, 14.3; HRMS (ESI) m/z calcd for C21H19F3N4O5 [(M ? H)?], 465.1380: found, 465.1358. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-((4chlorobenzyl)oxy)phenyl)carbamate (23) Brown solid (22 %), mp 179–180 °C: 1H NMR (DMSOd6, 600 MHz) d 9.48 (s, 1H), 8.06 (s, 1H), 7.47-7.44 (m, 4H), 7.28 (s, 2H), 6.93 (d, J = 9.0 Hz, 2H), 5.05 (s, 2H), 4.59 (t, J = 5.1 Hz, 2H), 4.44 (t, J = 5.1 Hz, 2H), 2.48 (s, 3H); 13C NMR (DMSO-d6, 150 MHz) d 153.5, 152.1, 138.9, 136.7, 133.5, 132.7, 129.9, 128.8, 120.5, 115.4, 68.9, 62.5, 45.8, 14.3; HRMS (ESI) m/z calcd for C20H19ClN4O5 [(M ? H)?], 431.1117: found, 431.1092.

dilutions were made in the wells of a 96-well microtiter plate. The following controls were included in each plate: metronidazole as a standard amoebicidal drug, control wells (culture medium plus amoebae) and a blank (culture medium only). All the experiments were carried out in triplicate at each concentration and repeated thrice. The amoeba suspension was prepared from a confluent culture by pouring off the medium at 37 °C and adding 5 mL of fresh medium, chilling the culture tube on ice to detach the organisms from the side of the flask. The number of amoeba/mL was estimated with a haemocytometer, using the Trypan blue exclusion assay to confirm viability. The suspension was diluted to 105 organism per mL in fresh medium and 170 mL of this suspension was added to the test and control wells in the plate such that an inoculum of 1.7 9 104 organisms/well was achieved to ensure confluency but no excessive growth in control wells. Plates were sealed and gassed for 10 min with nitrogen before incubation at 37 °C for 72 h. After incubation, the growth of amoeba in the plate was checked with a low power microscope. The culture medium was removed by inverting the plate and shaking gently. The plate was then immediately washed with prewarmed (37 °C) 0.9 % (w/v) sodium chloride solution. This procedure was completed as quickly as possible to ensure the plate did not cool, in order to prevent the detachment of amoebae. The plate was allowed to dry at room temperature and the amoebae were fixed with chilled (-20 °C) 100 % methanol and when dried, stained with 0.5 % aqueous eosin for 15 min. The stained plate was washed 3 times with distilled water and allowed to dry before 200 mL 0.1 N sodium hydroxide was added to each well to dissolve the protein and release the dye. The optical density of the resulting solution was determined at 490 nm with a microplate reader. The % inhibition of amoebal growth was calculated taking into account the controls and then plotted against the logarithm of the compound concentration. Linear regression analysis was used to determine the best fitting line from which the IC50 value was found (Table 1).

Pharmacological procedures MTT assay In vitro antiamoebic assay All the compounds (5–23) were screened in vitro for antiamoebic activity against HM1:IMSS strain of E. histolytica by microdilution method (wright et al. 1988). E. histolytica trophozoites were cultured in a 96-well microtiter plate suspended in Diamond TYIS- 33 growth medium (Diamond et al. 1978). The test compounds (1 mg) were dissolved in DMSO (40 lL, concentration at which no inhibition of amoeba growth occurred) (Gillin et al. 1982; Keene et al. 1986). The stock solutions (1 mg/mL) of the compounds were freshly prepared and twofold serial

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Melan-a cells were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum, 200 nM 12-otetradecanoylphorbol-13-acetate (TPA, Sigma-Aldrich, St. Louis, MO, USA),100 mg/ml gentamycin, 100 IU/ml penicillin and 100 mg/ml streptomycin (Vitrocell) at 37 °C in a humidified atmosphere with 5 % CO2.Only viable cells were used in the assay. Cell viability was determined by excluding the stained cells, as well as by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay (MTT, Sigma).Exponentially growing cells were plated in 6-well culture plates (2.5–6.5 9 104 cells per well) and

Novel aryl carbamate derivatives of metronidazole as potential antiamoebic agents Table 1 In vitro antiamoebic activity against the HM1:IMSS strain of E. histolytica and cytotoxicity profile of the aryl carbamate derivatives of metronidazole (5–23)

(5–13)

Compound

R

(14–23)

Antiamoebic activity IC50 (lM) ±SD

a

Cytotoxicity profile

incubated for 48 h before the addition of drugs. Stock solutions of compounds were initially dissolved in 20 % (v/v) DMSO and further diluted with fresh complete medium. The growth-inhibitory effects of the compounds were measured using standard tetrazolium MTT assay. After 48 h of incubation at 37 °C, the medium was removed and 2 mL of MTT reagent (2.5 mg/ml) in serum free medium was added to each well. The plates were incubated at 37 °C for 4 h. At the end of the incubation period, the medium was removed and 1 ml of absolute DMSO was added to each well for complete solubilization of the generated formazan. The contents were subsequently transferred to 96-well plates, and the absorbance was determined at 570 nm with the aid of a microplate reader (Bio-Rad, USA) (Rossato et al. 2014).

IC50 (lM) ±SDa

5

0.80

0.003

N.Db

N.Db

Result

6

0.56

0.007

N.Db

N.Db

7

0.24

0.007

[50

0.455

8

3.15

0.006

N.Db

N.Db

9

0.39

0.008

N.Db

N.Db

10

0.66

0.006

N.Db

N.Db

b

N.Db

Scheme 1 lists the synthetic pathway leading to the target compounds (5–23). First, MNZ was treated with triphosgene, affording the corresponding chloroformate 2. The reaction was conducted in the presence of various solvents using 2–3 drops of triethylamine (TEA) as the catalyst at different temperature ranges under anhydrous conditions. When the reactions were conducted using anhydrous acetone as the solvent and TEA as the base at room temperature under nitrogen, followed by stirring for 12–14 h, the desired products were obtained with the best yield. Second, after acetone was evaporated, the crude product was used for the next step without purification. Third, chloroformate 2 was reacted with 4-aminophenol 3 in dry acetone in the presence of anhydrous potassium carbonate at reflux temperature to afford the corresponding 2-(2-methyl-5-nitro1H-imidazol-1-yl)ethyl (4-hydroxyphenyl) carbamate 4 in a quantitative yield. Sulfonate compounds (5–13) were synthesized by the reaction between 2-(2-methyl-5-nitro1H-imidazol-1-yl)ethyl (4-hydroxyphenyl)carbamate 4 and commercially available sulfonyl chlorides. For this purpose, carbamate 4 was treated with various arylsulfonyl chlorides in presence of TEA as the base and DMF as the solvent. The reactions were completed within 2 h. The yield was further improved when anhydrous DMF was used under nitrogen, and the products were isolated within 15 min in moderate-to-good yields (50–80 %). For the synthesis of benzyloxy derivatives (14–23) from carbamate 4, O-alkylation was conducted in three different solvents (acetone, THF, and DMF) at different temperature ranges by using K2CO3 as the base. The desired products were generated in good yield when the reactions were conducted using anhydrous DMF as the solvent and K2CO3 as the base at room temperature, and the reactions were

11

1.38

0.011

N.D

12

5.46

0.014

N.Db

N.Db

13

6.34

0.010

N.Db

N.Db

14

0.08

0.004

[50

0.538 b

N.Db

15

7.11

0.006

N.D

16

0.26

0.004

[50

0.320

17

3.59

0.005

N.Db

N.Db

18

6.06

0.006

N.Db

N.Db

19

0.26

0.004

[50

1.18

20

3.21

0.006

N.Db

N.Db

21

0.15

0.006

[50

0.603 b

N.Db

22

6.81

0.007

N.D

23

0.37

0.006

N.Db

N.Db

1.78

0.003

[50

0.201

Metronidazole a

S.D (Standard deviation) the value obtained in at least three separate assays conducted in triplicate

b

N.D Not done

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F. Hayat et al.

Scheme 1 Synthesis of the aryl carbamate derivatives of metronidazole (5–23). Reagents and Conditions: (i) Triphosgene, TEA, acetone, rt, overnight (ii) K2CO3, acetone, reflux, 5 h. (iii) Arylsulfonyl chloride, TEA, DMF, 15 min. (iv) Substituted benzyl bromide, K2CO3, DMF, 2 h

completed within 2 h. Each compound synthesized was characterized by 1H NMR, 13C NMR, and high-resolution MS. The synthesized compounds (5–23) were screened in vitro for antiamoebic activity against the HM1:IMSS strain of E. histolytica by the microdilution method (Wright et al. 1988). All experiments were conducted in triplicate at each concentration level and repeated three times. Their antiamoebic effect was compared to MNZ, the most widely used medication for amoebiasis; it exhibited a 50 % inhibitory concentration (IC50) of 1.78 lM in our experiments. The cytotoxicity of the active compounds was studied by the MTT assay on normal melan-a (human melanocytes) cell line. Table 1 summarizes the results of antiamoebic activity and cytotoxicity.

Discussion Antiamoebic activity All synthesized compounds were categorized in two series. The first series of compounds were sulfonate derivatives (5–13), and the second series of compounds were

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benzyloxy derivatives (14–23), both of which contained 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl (4-hydroxyphenyl)carbamate (4) as the common moiety. The results showed that both series of compounds exhibit IC50 values ranging from 0.24 to 6.34 and 0.08 to 6.81 lM, respectively. Among them, 5-7, 9–11, 14, 16, 19, 21, and 23 inhibited the growth of trophozoite more strongly than standard MNZ. Compounds 7, 14, 16, 19, and 21 exhibited IC50 values of 0.24, 0.08, 0.26, 0.26, and 0.15 lM, respectively; they are the most promising compounds among the whole series of compounds (5–23). In terms of the structure–activity relationship (SAR) of arylsulfonyl derivatives (5–13), based on phenyl analog 6 (IC50 = 0.56 lM), substitution with a bulky or an electrondonating group at the para position such as tert-butyl (8, IC50 = 3.15 lM), methyl (12, IC50 = 5.46 lM), and isopropyl (13, IC50 = 6.34 lM) led to reduction in the inhibitory activity, whereas small-sized or electronwithdrawing groups such as trifluoromethyl (7, IC50 = 0.24 lM), chloro (10, IC50 = 0.66 lM), and iodo (11, IC50 = 1.38 lM) retained antiamoebic activity. With the result of partial SAR of arylsulfonates, phenyl group substituents of benzylether series (14–23) were narrowed to small-sized and electron-withdrawing ones. As

Novel aryl carbamate derivatives of metronidazole as potential antiamoebic agents Fig. 4 Measurement of cell viability after 48 h by the MTT assay

compared to the unsubstituted phenyl derivative 15 (IC50 = 7.11 lM), all compounds exhibited better inhibitory activity. Among them, 4-fluoro (14, IC50 = 0.08 lM), 3,4-difluoro (16, IC50 = 0.26 lM), 4-cyano (19, IC50 = 0.26 lM), 3-chloro (21, IC50 = 0.15 lM), and 4-chloro (23, IC50 = 0.37 lM) compounds exhibited strong antiamoebic activity of greater than ten times, although no improvement in the inhibitory activity was observed for the 2-nitro, 3-iodo, and 4-trifluoromethyl substituted compounds. Cytotoxicity profile For examining the effect of selected antiamoebic compounds 7, 14, 16, 19, and 21 on cell proliferation, their cytotoxicity on normal melan-a (human melanocytes) cell line was investigated. A subconfluent population of melana cells was treated with increasing concentrations of compounds, and the number of viable cells was measured after 48 h by the MTT cell viability assay (Rossato et al. 2014). The concentration range for the tested compounds was 2.5–50 lM. As shown in Fig. 4, all compounds exhibited [80 % viability at concentrations ranging from 2.5 to 50 lM. Table 1 lists the IC50 values of cytotoxicity along with the standard deviation values of compounds 7, 14, 16, 19, and 21.

Conclusion In summary, we designed and synthesized a novel series of aryl carbamate derivatives of metronidazole based on previously published results (Azam and Agarwal 2007; Singh et al. 2009) with the aim of surveying novel potent, low-toxicity antiamoebic agents. Two series of compounds, arylsulfonates and benzyl ethers, were synthesized in four sequential steps. They were further evaluated for their

antiamoebic activity against the HM1:IMMS strain of Entamoeba histolytica. The in vitro antiamoebic activity results showed that out of 19 compounds, five compounds 7, 14, 16, 19, and 21 exhibited most promising antiamoebic activity (IC50 = 0.24, 0.08, 0.26, 0.26, and 0.15 lM, respectively) compared to the reference drug metronidazole (IC50 = 1.78 lM). The MTT assay revealed that all tested compounds, i.e., 7, 14, 16, 19, and 21, are nontoxic at concentrations ranging from 2.5 to 50 lM. The partial structure–activity relationship also revealed that small, electron-withdrawing substituents on the terminal phenyl ring improve antiamoebic activity. Acknowledgments This research work was supported by the Pioneer Research Center Program through the NRF funded by the Ministry of Science, ICT &Future Planning (2014M3C1A3001556), and the Korea HealthTechnology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (grant no. HI14C1135) and Department of Chemistry, Jamia Millia Islamia (A Central University), New Delhi, India. Compliance with ethical standards Conflict of Interest

The authors declare no conflict of interest.

References Abboud P, Leme´e V, Gargala G, Brasseur P, Ballet JJ, Borsa-Lebas F, Caron F, Favennec L (2001) Successful treatment of metronidazole and albendazole-resistant giardiasis with nitazoxanide in a patient with acquired immunodeficiency syndrome. Clin Infect Dis 32:1792–1794 Abid M, Agarwal SM, Azam A (2008) Synthesis and antiamoebic activity of metronidazole thiosemicarbazone analogues. Eur J Med Chem 43:2035–2039 Athar F, Husain K, Abid M, Agarwal SM, Coles SJ, Hursthouse MB, Maurya MR, Azam A (2005) Synthesis and anti-amoebic activity of Gold(I), Ruthenium(II), and Copper(II) complexes of metronidazole. Chem Biodivers 2:1320–1330 Azam A, Agarwal SM (2007) Targeting amoebiasis: Status and new developments. Curr Bioact Compd 3:121–133

123

F. Hayat et al. Diamond LS, Harlow DR, Cunnick CC (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72:431–432 El-Nahas FA, El-Ashmawy IM (2004) Reproductive and cytogenetic toxicity of metronidazole in male mice. Basic Clin Pharmacol Toxicol 94:226–231 Fe´rriz JM, Va´vrova´ K, Kunc F, Imramovsky´ A, Stolarˇ´ıkova´ J, Vavrˇ´ıkova´ E, Vinsˇova´ J (2010) Salicylanilide carbamates: Antitubercular agents active against multidrug resistant Mycobacterium tuberculosis strains. Bio Med Chem 18:1054–1061 Fluxe A, Wu S, Sheffer JB, Janusz JM, Murawsky M, Fadayel GM, Fang B, Hare M, Djandjighian L (2006) Discovery and synthesis of tetrahydroindolone-derived carbamates as Kv1.5 blockers. Bio Med Chem Lett 16:5855–5858 Gillin FD, Reiner DS, Suffness M (1982) Bruceantin, a potent amoebicide from a plant, Brucea antidysenterica. Antimicrob Agents Chemother 22:342–345 Janganati V, Penthala NR, Madadi NR, Chen Z, Crooks PA (2014) Anticancer activity of carbamate derivatives of melampomagnolide B. Bio Med Chem Lett 24:3499–3502 Keene AT, Harris A, Phillipson JD, Warhurst DC (1986) In vitro amoebicidal testing of natural products; part I. Methodology. Planta Med 52:278–284 Kus C, Altanlar N (2003) Synthesis of some new benzimidazole carbamate derivatives for evaluation of antifungal activity. Turk J Chem 27:35–39 Mata G, do Rosa´rio VE, Iley J, Constantino L, Moreira R (2012) A carbamate-based approach to primaquine prodrugs: antimalarial activity, chemical stability and enzymatic activation. Bio Med Chem 20:886–892 Meng Q, Luo H, Liu Y, Li W, Zhang W, Yao Q (2009) Synthesis and evaluation of carbamate prodrugs of SQ109 as antituberculosis agents. Bio Med Chem Lett 19:2808–2810 Moncada D, Keller K, Chadee K (2003) Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function. Infect Immun 71:838–844 Oku E, Nomura K, Nakamura T, Morishige S, Seki R, Imamura R, Hashiguchi M, Osaki K, Mizuno S, Nagafuji K, Okamura T (2012) Amebic colitis and liver abscess complicated by high serum procalcitonin in acute myeloid leukemia. Kansenshogaku Zasshi 86:773–777 Ordaz-Pichardo C, Shibayama M, Villa-Trevin˜o S, Arriaga-Alba M, Angeles E, de la Garza M (2005) Antiamoebic and toxicity studies of a carbamic acid derivative and its therapeutic effect in a hamster model of hepatic amoebiasis. Antimicrob Agents Chemother 49:1160–1168 Petri WA (2003) Therapy of intestinal protozoa. Trends Parasitol 19:523–526 Petri WA, Haque R (2013). Chapter 9-Entamoeba histolytica brain abscess. Handbook of Clinical Neurology, pp 147–152 Purohit V, Basu AK (2000) Mutagenicity of nitroaromatic compounds. Chem Res Toxicol 13:673–692 Ralston KS, Petri WA (2011) The ways of a killer: how does Entamoeba histolytica elicit host cell death. Essays Biochem 91:193–210

123

Ravdin JI, Stauffer MM (2005) Entamoeba histolytica (Amoebiasis). In: Mendell GL, Benneth JE, Dolin R (eds) Principles and practice of infectious diseases, 6th edn. P. A. Churchill Livingstone, Philadelphia Rogers SA, Lindsey EA, Whitehead DC, Mullikin T, Melander C (2011) Synthesis and biological evaluation of 2-aminoimidazole/carbamate hybrid anti-biofilm and anti-microbial agent. Bio Med Chem Lett 21:1257–1260 Rossato FA, Zecchin KG, La Guardia PG, Ortega RM, Alberici LC, Costa RA, Catharino RR, Graner E, Castilho RF, Vercesi AE (2014) Fatty acid synthase inhibitors induce apoptosis in nontumorigenic melan-a cells associated with inhibition of mitochondrial respiration. PLoS ONE 9(6):e101060 Sadek B, Shehab S, Wie˛cek M, Subramanian D, Shafiullah M, Kiec´Kononowicz K, Adem A (2013) Anticonvulsant properties of histamine H3 receptor ligands belonging to N-substituted carbamates of imidazopropanol. Bio Med Chem Lett 17:4886–4891 Salahuddin A, Agarwal SM, Avecilla F, Azam A (2012) Metronidazole thiosalicylate conjugates: synthesis, crystal structure, docking studies and antiamoebic activity. Bio Med Chem Lett 22:5694–5699 Shreder KR, Lin ECK, Wu J, Cajica J, Amantea CM, Hu Y, Okerberg E, Brown HE, Pham LM, Chung DM, Fraser AS, McGee E, Rosenblum JS, Kozarich JW (2012) Synthesis and structure– activity relationship of (1-halo-2-naphthyl) carbamate based inhibitors of KIAA1363 (NCEH1/AADACL1). Bio Med Chem Lett 22:5748–5751 Singh S, Bharti N, Mohapatra PP (2009) Chemistry and biology of synthetic and naturally occurring antiamoebic agents. Chem Rev 109:1900–1947 Solyev PN, Shipitsin AV, Karpenko IL, Nosik DN, Kalnina LB, Kochetkov SN, Kukhanova MK, Jasko MV (2012) Synthesis and Anti-HIV properties of new carbamate prodrugs of AZT. Chem Biol Drug Des 80:947–952 StanleyJr SL (2003) Amoebiasis. Lancet 361:1025–1034 Townson SM, Boreham PFL, Upcroft P, Upcroft JA (1994) Resistance to the nitro heterocyclic drugs. Acta Trop 56:173–194 Vacondio F, Silva C, Lodola A, Carmi C, Rivara S, Duranti A, Tontini A, Sanchini S, Clapper JR, Piomelli D, Tarzia G, Mor M (2011) Biphenyl-3-yl alkylcarbamates as fatty acid amide hydrolase (FAAH) inhibitors: steric effects of N-alkyl chain on rat plasma and liver stability. Eur J Med Chem 46:4466–4473 Verma A, Wong DM, Islam R, Tong F, Ghavami M, Mutunga JM, Slebodnick C, Li J, Viayna E, Lam PC-H, Totrov MM, Bloomquist JR, Carlier PR (2015) Oxoisoxazole-2(3H)-carboxamides and isoxazol-3-yl carbamates: resistance-breaking acetylcholinesterase inhibitors targeting the malaria mosquito, Anopheles gambiae. Bio Med Chem 23:1321–1340 Wright CW, O’Neill MJ, Phillipson JD, Warhurst DC (1988) Use of microdilution to assess in vitro antiamoebic activities of Brucea javanica fruits, Simarouba amara stem, and a number of quassinoids. Antimicrob Agents Chemother 32:1725–1729