Novel Indole-Isoxazole Hybrids: Synthesis and In Vitro Anti ...

712

Send Orders for Reprints to [email protected] Letters in Drug Design & Discovery, 2017, 14, 712-717

RESEARCH ARTICLE ISSN: 1570-1808 eISSN: 1875-628X

Novel Indole-Isoxazole Hybrids: Synthesis and In Vitro Anti-Cholinesterase Activity

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BENTHAM SCIENCE

Fahimeh Vafadarnejada, Mina Saeedib,c, Mohammad Mahdavid, Ali Rafinejada, Elahe Karimpour-Razkenaric, Bilqees Sameeme, Mahnaz Khanavif and Tahmineh Akbarzadeha,c,* a

Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; c Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran; d Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Iran; eSchool of Pharmacy, International Campus (TUMS-IC), Tehran University of Medical Sciences, Tehran, Iran; fDepartment of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Letters in Drug Design & Discovery

b

A R T I C L E H I S T O R Y Received: August 22, 2016 Revised: October 11, 2016 Accepted: October 13, 2016 DOI: 10.2174/1570180813666161018124726

Abstract: Background: This work reports synthesis and in vitro cholinesterase inhibitory activity of novel indole-isoxazole hybrids. Method: The synthetic procedure was started from different ethyl 5-arylisoxazole-3-carboxylate derivatives. Hydrolysis and reaction of the later compound with tryptamine afforded the desired products in good yields. Conclusion: Among the synthesized compounds, N-(2-(1H-indol-3-yl)ethyl)-5-(2-chlorophenyl) isoxazole-3-carboxamide (4b) showed the best anti-cholinesterase activity.

Keywords: Acetylcholinesterase, Alzheimer’s disease, Butyrylcholinesterase, Heterocycles, Indole, Isoxazole. 1. INTRODUCTION Heterocycles are extremely important and vital compounds ubiquitous in a wide range of bioactive molecules and macromolecules. Moreover, hybridization of different heterocyclic scaffolds has been a versatile tool for the sophisticated drug discovery studies [1]. Among a wide range of heterocycles, isoxazoles attracted our attention due to their valuable biological activities [2]. Indole and its derivatives are also privileged scaffolds which have significant role in the development of bioactive agents such as anticancer activity against tumours of the central nervous system [3] and antiviral agents [4]. They can be synthesized through a variety of methods such as Fischer indole synthesis [5]. Recent developments consist of palladium-catalyzed cyclization reaction of N-aryl imines [6], metal-free C-H amination of N-Ts-2-alkenylanilines [7], palladium-catalyzed crosscoupling reaction of o-nitrobenzyl cyanides with boronic acids in the presence of Fe [8], gold(III)-catalyzed annulation of 2-alkynylanilines [9], and addition of N-tosylhydrazones to arynes [10].

*Address correspondence to this author at the Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences P.O. Box: 14155-6451, Tehran, Iran; Tel/Fax: +98-21-64122219, +98-2166461178; E-mail: [email protected]

1875-628/17 $58.00+.00

In this work, in continuation of our work on the synthesis of novel heterocycles [11-15] and anti-cholinesterase compounds [16-21] we focused on the synthesis of indoleisoxazole hybrids (Scheme 1) which have not been frequently considered in the literature. Also, all derivatives were evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity to search efficient anti-Alzheimer’s agents. Alzheimer’s disease (AD) is the main cause of dementia and is a progressive neurodegenerative disorder. It is characterized at a molecular level by protein misfolding and aggregation, mitochondrial abnormalities, and neuroinflammatory processes [22]. The exact cause of AD is still uncertain, however, reduced levels of acetylcholine in the hippocampus and cortex along with the amyloid β-peptide (Aβ) aggregation in the brain have been considered as the pathogenesis of AD [23]. Increasing acetylcholine levels by the action of acetylcholinesterase inhibitors (AChEI’s) have been proposed as the most important treatment pathway. The main AChE inhibitors include donepezil, rivastigmine, and galantamine which are mostly prescribed to ameliorate the symptoms of AD [24]. Butyrylcholinesterase (BChE) is another kind of cholinesterase in the brain of mammals. The interpretation of function of BChE has revealed that BChE activity increases when AChE activity decreases significantly in the brain of patients with AD [25]. Hence, ©2017 Bentham Science Publishers

Novel Indole-Isoxazole Hybrids: Synthesis and In Vitro Anti-cholinesterase Activity

Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6

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NH 2

O Ar

N

CO2 Et

KOH MeOH

1

O Ar

N

2

N H

CO2 H

3

EDCI, HOBt, CH 3CN rt

2

O O Ar

N

N H 4

1

NH

7

3 4

6 5

Scheme 1. Synthesis of indole-isoxazole hybrids 4.

recent studies have paid attention to BChE inhibitory (BChEI) activity and searching for new AChE/BChE inhibitors is highly in demand. 2. MATERIALS AND METHODS 2.1. General Information Melting points were taken on a Kofler hot stage apparatus and are uncorrected. 1H- and 13C-NMR spectra were recorded on a Bruker FT-500 (Germany), using TMS as an internal standard. The IR spectra were obtained on a Nicolet Magna FTIR 550 spectrometer (KBr disks). The elemental analysis was performed on an Elementar Analysensystem GmbH VarioEL CHNS mode (Germany). Compounds 1 and 2 were prepared according to the procedure described in the literature [26]. 2.2. Chemistry The Typical Procedure for the Synthesis of Indoleisoxazole Hybrids 4a-k; General Procedure A solution of compound 2 (1 mmol), 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDCI) (0.21 g, 1.1 mmol) and hydroxybenzotriazole (HOBt) (0.13 g, 1 mmol) in dry acetonitrile (10 mL) was stirred at room temperature for 30 min. Then, tryptamine 3 (0.16 g, 1 mmol) was added to the mixture and the reaction was continued at room temperature for 24 h. After completion of reaction, the solvent was reduced under vacuum at 40 °C and the residue was dissolved in dichloromethane (50%) and washed with sodium carbonate (10%). The organic phase was dried over Na2SO4 and the solvent was evaporated. The obtained compound 4a-k was completely pure. N-(2-(1H-Indol-3-yl)ethyl)-5-(4-fluorophenyl)isoxazole-3carboxamide (4a) Yield: 65%; M.p. 164-165 °C.- IR (KBr): ν = 3335, 3320, 3118, 3078, 2920, 2852, 1677, 1606, 1560, 1443 cm-1 - 1H NMR (500 MHz, DMSO) δ = 10.85 (s, 1H, NH), 8.94 (bs, 1H, NH), 8.02-8.01 (m, 2H, H2', H6'), 7.60 (d, J = 7.5 Hz, 1H, H4), 7.41 (t, J = 8.0 Hz, 2H, H3', H5'), 7.36 (s, 1H, isoxazole), 7.35 (d, J = 7.5 Hz, 1H, H7), 7.21 (s, 1H, H2), 7.08 (t, J = 7.5 Hz, 1H, H6), 7.00 (t, J = 7.5 Hz, 1H, H5), 3.58-3.56 (m, 2H, NCH2), 2.97 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ = 169.4, 163.4 (d, JC-F = 247.5 Hz), 159.8, 158.3, 136.2, 128.4, 127.2, 123.1, 122.7, 122.5, 121.0, 118.3, 116.5 (d, JC-F = 22.5 Hz), 111.5, 111.4, 99.8, 39.5, 24.9 - C20H16FN3O2 (349.3): calcd. C 68.76, H 4.62, N 12.03; found C 68.59, H 4.80, N 11.86.

N-(2-(1H-Indol-3-yl)ethyl)-5-(2-chlorophenyl)isoxazole-3carboxamide (4b) Yield: 81%; M.p. 145-146 °C. - IR (KBr): ν =3378, 3283, 2950, 2825, 1677, 1597, 1549, 1439 cm-1. - 1H NMR (500 MHz, DMSO) δ = 10.85 (s, 1H, NH), 9.0 (t, J = 7.0 Hz, 1H, NH), 7.96 (dd, J = 7.5, 1.5 Hz, 1H, H3'), 7.70 (dd, J = 7.5, 1.5 Hz, 1H, H6'), 7.60 (d, J = 7.5 Hz, 1H, H4), 7.59-7.54 (m, 2H, H4', H5'), 7.36 (d, J = 7.5 Hz, 1H, H7), 7.35 (s, 1H, isoxazole), 7.22 (s, 1H, H2), 7.08 (t, J = 7.5 Hz, 1H, H6), 7.00 (t, J = 7.5 Hz, 1H, H5), 3.59 (q, J = 7.0 Hz, 2H, NCH2), 2.99 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ =167.1, 159.4, 158.1, 136.3, 132.3, 131.0, 129.9, 128.3, 128.0, 127.2, 125.0, 122.7, 122.5, 121.0, 118.3, 111.5, 111.4, 103.8, 39.6, 24.9 - C20H16ClN3O2 (365.8): calcd. C 65.67, H 4.41, N 11.49; found C 65.81, H 4.31, N 11.26. N-(2-(1H-Indol-3-yl)ethyl)-5-(4-chlorophenyl)isoxazole-3carboxamide (4c) Yield: 77%; M.p.166-167 °C.- IR (KBr): ν =3337, 3281, 3121, 2922, 2890, 1665, 1607, 1559, 1489 cm-1 - 1H NMR (500 MHz, DMSO) δ = 10.84 (s, 1H, NH), 8.95 (t, J = 7.0 Hz, 1H, NH), 7.97 (d, J = 8.5 Hz, 2H, H3', H5'), 7.63 (d, J = 8.5 Hz, 2H, H2', H6'), 7.59 (d, J = 7.5 Hz, 1H, H4), 7.42 (s, 1H, isoxazole), 7.34 (d, J = 7.5 Hz, 1H, H7), 7.20 (s, 1H, H2), 7.07 (t, J = 7.5 Hz, 1H, H6), 6.99 (t, J = 7.5 Hz, 1H, H5), 3.56 (q, J = 7.0 Hz, 2H, NCH2), 2.97 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ =169.2, 159.8, 158.3, 136.2, 132.3, 135.5, 129.5, 127.6, 127.2, 125.2, 122.7, 121.0, 118.3, 111.5, 111.4, 100.5, 39.8, 24.9 - C20H16ClN3O2 (365.8): calcd. C 65.67, H 4.41, N 11.49; found C 65.81; H 4.27, N 11.34. N-(2-(1H-Indol-3-yl)ethyl)-5-(2,4-dichlorophenyl) isoxazole-3-carboxamide (4d) Yield: 78%; M.p.138-140 °C. - IR (KBr): ν = 3388, 337, 3176, 3078, 2926, 2857, 1674, 1594, 1551 cm-1- 1H NMR (500 MHz, DMSO) δ = 10.85 (s, 1H, NH), 9.05 (t, J = 7.0 Hz, 1H, NH), 7.98 (d, J = 8.5 Hz, 1H, H6'), 7.90 (d, J = 1.5 Hz, 1H, H3'), 7.65 (dd, J = 8.5, 1.5 Hz, 1H, H5'), 7.60 (d, J = 7.5 Hz, 1H, H4), 7.38 (s, 1H, isoxazole), 7.35 (d, J = 7.5 Hz, 1H, H7), 7.21 (s, 1H, H2), 7.08 (t, J = 7.5 Hz, 1H, H6), 6.99 (t, J = 7.5 Hz, 1H, H5), 3.57 (q, J = 7.0 Hz, 2H, NCH2), 2.98 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ = 166.2, 159.5, 158.0, 136.3, 136.1, 132.0, 131.1, 130.5, 128.3, 127.2, 124.0, 122.7, 122.6, 121.0, 118.3, 111.5, 111.4, 104.1, 39.5, 24.9 - C20H15Cl2N3O2 (400.2): calcd. C 60.01, H 3.78, N 10.50; found C 59.88, H 3.57, N 10.36.

714 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6

N-(2-(1H-Indol-3-yl)ethyl)-5-(4-bromophenyl)isoxazole-3carboxamide (4e) Yield: 65%; M.p.140-141 °C.- IR (KBr): ν = 3336, 3282, 3115, 3062, 2920, 2858, 1668, 1602, 1560 cm-1- 1H NMR (500 MHz, DMSO) δ = 10.84 (s, 1H, NH), 8.95 (t, J = 7.0 Hz, 1H, NH), 7.89 (d, J = 8.5 Hz, 2H, H3', H5'), 7.77 (d, J = 8.5 Hz, 2H, H2', H6'), 7.59 (d, J = 7.5 Hz, 1H, H4), 7.43 (s, 1H, isoxazole), 7.34 (d, J = 7.5 Hz, 1H, H7), 7.20 (s, 1H, H2), 7.07 (t, J = 7.5 Hz, 1H, H6), 6.99 (t, J = 7.5 Hz, 1H, H5), 3.56 (q, J = 7.0 Hz, 2H, NCH2), 2.97 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ = 169.3, 159.8, 158.2, 136.2, 132.4, 127.8, 127.2, 125.8, 125.5, 124.3, 122.7, 121.0, 118.3, 115.0, 111.4, 100.5, 39.5, 24.9 - C20H16BrN3O2 (410.2): calcd. C 58.55, H 3.93, N 10.24; found C 58.71, H 4.12, N 10.51. N-(2-(1H-indol-3-yl)ethyl)-5-(m-tolyl)isoxazole-3carboxamide (4f) Yield: 79%; M.p.154-155 °C.- IR (KBr): ν = 3431, 3313, 3116, 3075, 2926, 2850, 1667, 1563, 1449 cm-1- 1H NMR (500 MHz, DMSO) δ = 10.85 (bs, 1H, NH), 8.91 (t, J = 7.0 Hz, 1H, NH), 7.77 (s, 1H, H2'), 7.73 (d, J = 8.0 Hz, 1H, H6'), 7.59 (d, J = 7.5 Hz, 1H, H4), 7.44 (t, J = 8.0 Hz, 1H, H5'), 7.36-7.32 (m, 3H, H7, H4', isoxazole), 7.20 (d, J = 1.0 Hz, 1H, H2), 7.07 (td, J = 7.5, 1.0 Hz, 1H, H6), 6.99 (t, J = 7.5, 1.0 Hz, 1H, H5), 3.57 (q, J = 7.0 Hz, 2H, NCH2), 2.97 (t, J = 7.0 Hz, 2H, CH2), 2.40 (s, 3H, CH3). - 13C NMR (125 MHz, DMSO): δ = 170.5, 159.7, 158.4, 138.8, 136.2, 131.5, 130.3, 129.2, 127.2, 126.3, 126.2, 123.0, 122.7, 121.0, 118.3, 111.5, 111.4, 99.7, 39.5, 24.9, 20.9 - C21H19N3O2 (345.3): calcd. C 73.03, H 5.54, N 12.17; found C 72.90, H 5.68, N 12.31. N-(2-(1H-Indol-3-yl)ethyl)-5-(3-methoxyphenyl)isoxazole3-carboxamide (4g) Yield: 70%; M.p.170-171 °C.- IR (KBr): ν = 33431, 3314, 3118, 2964, 2926, 1664, 1560, 1438 cm-1- 1H NMR (500 MHz, DMSO) δ = 10.85 (bs, 1H, NH), 8.91 (t, J = 7.0 Hz, 1H, NH), 7.59 (d, J = 7.5 Hz, 1H, H4), 7.52-7.45 (m, 3H, H2', H5', H6'), 7.41 (s, 1H, isoxazole), 7.34 (d, J = 7.5 Hz, 1H, H7), 7.20 (d, J = 2.0 Hz, 1H, H2), 7.11 (dd, J = 8.0, 1.0 Hz, 1H, H4'), 7.07 (t, J = 7.5 Hz, 1H, H6), 6.99 (t, J = 7.5 Hz, 1H, H5), 3.84 (s, 3H, OCH3), 3.56 (q, J = 7.0 Hz, 2H, NCH2), 2.97 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ = 170.2, 159.8, 158.4, 136.2, 131.5, 130.6, 127.5, 127.2, 124.3, 122.7, 121.0, 118.3, 118.0, 116.8, 111.5, 111.4, 110.8, 100.2, 55.4, 39.8, 24.9 - C21H19N3O2 (345.3): calcd. C 69.79, H 5.30, N 11.63; found C 69.91, H 5.19, N 11.48. N-(2-(1H-Indol-3-yl)ethyl)-5-(4-methoxyphenyl)isoxazole3-carboxamide (4h) Yield: 76%; M.p.175-176 °C.- IR (KBr): ν = 3474, 3418, 3341, 3274, 2950, 2821, 1660, 1613, 1563, 1451 cm-1- 1H NMR (500 MHz, DMSO) δ = 10.84 (bs, 1H, NH), 8.89 (t, J = 7.0 Hz, 1H, NH), 7.88 (d, J = 9.0 Hz, 2H, H2', H6'), 7.59 (d, J = 7.5 Hz, 1H, H4), 7.34 (d, J = 7.5 Hz, 1H, H7), 7.217.20 (m, 2H, H2, isoxazole), 7.10 (d, J = 9.0 Hz, 2H, H3', H5'), 7.07 (t, J = 7.5 Hz, 1H, H6), 6.99 (t, J = 7.5 Hz, 1H, H5), 3.84 (s, 3H, OCH3), 3.56 (q, J = 7.0 Hz, 2H, NCH2), 2.97 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ = 170.4, 161.1, 159.7, 158.5, 136.2, 127.5, 127.2,

Vafadarnejad et al.

122.7, 121.5, 121.0, 119.0, 118.3, 114.7, 111.5, 111.4, 98.3, 55.4, 39.8, 24.9 - C21H19N3O2 (345.3): calcd. C 69.79, H 5.30, N 11.63; found C 69.51, H 5.44, N 11.80. N-(2-(1H-Indol-3-yl)ethyl)-5-(3,4-dimethoxyphenyl) isoxazole-3-carboxamide (4i) Yield: 85%; M.p.162-164 °C.- IR (KBr): ν = 3383, 3337, 3117, 2955, 2837, 1666, 1553, 1513 cm-1- 1H NMR (500 MHz, DMSO) δ = 10.84 (bs, 1H, NH), 8.88 (t, J = 7.0 Hz, 1H, NH), 7.59 (d, J = 7.5 Hz, 1H, H4), 7.50 (d, J = 8.5 Hz, 1H, H6'), 7.47 (s, 1H, H2'), 7.34 (d, J = 7.5 Hz, 1H, H7), 7.29 (d, J = 1.5 Hz, 1H, H2), 7.20 (s, 1H, isoxazole), 7.10 (d, J = 8.5 Hz, 1H, H5'), 7.07 (t, J = 7.5 Hz, 1H, H6), 6.99 (t, J = 7.5 Hz, 1H, H5), 3.86 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.56 (q, J = 7.0 Hz, 2H, NCH2), 2.97 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ = 170.6, 159.2, 158.5, 150.9, 149.1, 136.3, 127.2, 125.5, 122.7, 121.0, 119.0, 118.9, 118.3, 112.0, 111.6, 111.4, 109.1, 98.7, 55.8, 55.7, 39.5, 25.0 - C22H21N3O4 (391.4): calcd. C 67.51, H 5.41, N 10.74; found C 67.28, H 5.27, N 10.91. N-(2-(1H-Indol-3-yl)ethyl)-5-(3-nitrophenyl)isoxazole-3carboxamide (4j) Yield: 83%; M.p.187-188 °C.- IR (KBr): ν = 3405, 3319, 3138, 2935, 2850. 1679, 1621, 1524, 1445, 1347 cm-1- 1H NMR (500 MHz, DMSO) δ = 10.84 (bs, 1H, NH), 9.02 (t, J = 7.0 Hz, 1H, NH), 8.70 (d, J = 1.5 Hz, 1H, H2'), 8.40-8.36 (m, 2H, H3', H6'), 7.86 (td, J = 8.0, 1.5 Hz, 1H, H5'), 7.67 (d, J = 1.5 Hz, 1H, H2), 7.60 (d, J = 8.0 Hz, 1H, H4), 7.34 (d, J = 8.0 Hz, 1H, H7), 7.21 (s, 1H, isoxazole), 7.08 (t, J = 8.0 Hz, 1H, H6), 6.99 (t, J = 8.0 Hz, 1H, H5), 3.57 (q, J = 7.0 Hz, 2H, NCH2), 2.98 (t, J = 7.0 Hz, 2H, CH2). - 13C NMR (125 MHz, DMSO): δ = 168.1, 160.0, 158.1, 148.4, 136.3, 132.0, 131.9, 131.2, 127.7, 127.2, 125.3, 122.7, 121.0, 120.4, 118.3, 111.5, 111.4, 101.9, 39.5, 24.9 - C20H16N4O4 (376.3): calcd. C 63.82, H 4.28, N 14.89; found C 63.65, H 4.41, N 14.66. 2.3. Biological Assay Anti-AChE and anti-BChE activities were performed exactly based on our previous reports [16-21]. 2.4. Docking Study It was performed according to our previous reports [21] to find more insight into receptor-ligand interactions at molecular level using Auto dock tools. The receptor pdb text file (AChE) complexed with donepezil bearing PDB code (1EVE) and BChE PDB code 4AQD were retrieved from www.rcsb.org. Subsequently the complexed ligand, water was removed using Discovery Studio 4.1. Atomic coordinates of the ligand 4b were drawn with the help of Marvin Sketch 5.10.4, 2012. The structures of the ligand and receptor were converted into pdbqt files using Auto Dock tools. The Auto Dock parameters were set as follows: Grid Box size 60 × 60 × 60, co-ordinates x=138.8, y=123.6, z=38.7 and 0.375 Å as grid spacing distance. Other parameters were chosen as default. The calculated geometries were ranked in terms of free energy of binding and the best poses were chosen for further analysis. The molecular visualization


Table 1.

715

Synthesis and cholinesterase inhibitory activity of compounds 4. NH

O O

N

Ar

a


N H 4

Entry

Compound

Ar

AChEI Activity IC 50 (µM) a

BChEI Activity IC50 (µM)a

1

4a

4-FC6H4

>100

>100

2

4b

2-ClC6H4

29.52±0.05

46.10±0.23

3

4c

4-ClC6H4

>100

>100

4

4d

2,4-(Cl)2C 6H3

36.21±0.86

>100

5

4e

4-BrC6H4

>100

>100

6

4f

3-MeC6H4

35.60±0.34

82.06±0.74

7

4g

3-MeOC6H4

>100

>100

8

4h

4-MeOC6H4

>100

>100

9

4i

3,4-(MeO)2C 6H3

>100

>100

10

4j

3-NO2C6H4

42.990.26

>100

11

Rivastigmine

11.07±0.01

7.72±0.02

Inhibitor concentration (mean ± SD of three experiments) required for 50% inactivation of AChE and BChE.

was carried using DS Views Pro (Accelyrs, Inc). Preliminary results by visual examination of the docked compound 4b showed that the best poses of this compound docked almost similarly and the best pose of the compound 4b was further analyzed. 3. CHEMISTRY To prepare our desired anticholinesterase compounds 4, we followed the synthetic route as depicted in Scheme 1. It was started from ethyl 5-arylisoxazole-3-carboxylate 1 which were synthesized through the reaction of different ethyl 4aryl-2,4-dioxobutanoates and hydroxylamine hydrochloride in the presence of sodium methoxide in refluxing ethanol [33]. Hydrolysis of ester group in the presence of potassium hydroxide (KOH) in refluxing MeOH afforded the related carboxylic acid 2. Finally, reaction of compound 2 and tryptamine 3 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and hydroxybenzotriazole (HOBt) in dry CH3CN gave the corresponding indoleisoxazole hybrids 4. 4. BIOLOGICAL ACTIVITY The evaluation of the anti-acetylcholinesterase (AChE) activity of compounds 4 was performed using Ellman’s method [27] and compared with rivastigmine as the reference drug (IC50 = 11.07 µM). All results are depicted in Table 1. It was revealed that compounds 4a, 4c, 4e, 4g, 4h, and 4i showed no activity. Compounds 4b, 4d, 4f, and 4j showed good antiAChE activity and among them compound 4b was the most potent derivative (IC50 = 29.52 µM). It seems that the presence of chlorine at 2-position of aryl group connected to isoxazole ring induced more anti-AChE activity. However,

increasing the number of chlorine at 2- and 4- positions decreased the inhibitory activity (compound 4d, IC50 = 36.21 µM). Compound 4f having methyl group at 3-position of aryl group connected isoxazole ring showed inhibitory activity with IC50 = 35.64 µM which almost as potent as compound 4d. Introduction of strong electron withdrawing group (NO2) into 3-position of aryl group (4j) led to lower activity (IC50 = 42.99 µM) comparing with compounds 4b, 4d, and 4f. Our results revealed that hybridization of indole and isoxazole scaffolds did not lead to highly active inhibitors as compared to our previous report on isoxazole-1,2,3-triazole hybrids [16], indicating the fact that 1,2,3-triazole ring induced better anti-AChE activity. Anti-butyrylcholinesterase assay showed that all compounds except compounds 4b (IC50 = 42.10 µM) and 4f (IC50 = 82.06 µM) were inactive toward BChE. 5. DOCKING STUDY Docking study was conducted to determine the binding mode of the most active compound 4b in the active site of AChE (Fig. 1) and BChE (Fig. 2). As can be seen in Fig. 1, indole moiety plays vital role in ligand recognition via π-π stacking through Phe330 and Phe331 amino acid residues. Oxygen of carbonyl group and chlorine formed hydrogen bond with Tyr130 and Tyr121, respectively. However, the isoxazole ring was oriented towards Gly117 via hydrogen bonding. In the case of BChE (Fig. 2), oxygen of carbonyl formed hydrogen bond with Ser198. Isoxazole ring aligned with Trp82 through π-π T-shaped link. Also, chlorine attached to the phenyl ring as well as isoxazole of oxazole displayed H-

716 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 6

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connected to isoxazole ring showed the best cholinesterase inhibitory activity. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS This research was supported by grant from the Research Council of Tehran University of Medical Sciences with Project No. 91-03-33-19357. This paper is dedicated to the memory of our unique teacher in Chemistry and Medicinal Chemistry, Professor Abbas Shafiee. REFERENCES [1] [2]

Fig. (1). The binding mode of compound 4b in the active site of AChE.

[3]

[4] [5]

[6]

[7] [8] [9]

[10] [11]

[12]

Fig. (2). The binding mode of compound 4b in the active site of BChE.

bond with Tyr128. Phenyl ring was aligned toward Trp82 and Leu125 via π-lone pair and π-sigma interactions. CONCLUSION In conclusion, novel indole-isoxazole hybrids were synthesized and evaluated for their anti-AChE and antiBChE activities. Among the synthesized compounds, N-(2(1H-Indol-3-yl)ethyl)-5-(2-chlorophenyl)isoxazole-3-carboxamide (4b) possessing chlorine at 2-position of aryl group

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