Chemistry

All melting points were recoded using “Electrothermal Capillary apparatus” and all were uncorrected. IR spectra were determined as KBr pellets, values are characterized in cm−1. Mass spectral data were recoded using electron impact ionization technique at 70 eV. “1H NMR (500 MHz) and 13C NMR” (100 MHz) bands were achieved on “Jeol ECA 500 MHz spectrometer” and chemical shifts were presented as ppm. “Elemental microanalyses were done on Microanalytical Units in Cairo University and the National Research Center”. All final compounds exhibited purity above 95% as detected by HPLC. Analysis was carried out using Agilent 1100 series apparatus. Detection method was using diode array (DAD) ultraviolet (UV) detector was used at 254 nm. Column used was C18 column Zorbax ODS (4.6 ×150 mm i.d.,5 mm) and eluent was water: acetonitrile (30:70), flow rate = 1 mL/min, temperature 25 °C.

Preparation of 1a-c, 2a-c, 3b and 4b was achieved as per to stated methods [25, 47, 48].

Synthesis of 1- (2-chloro – N - arylacetamido) cyclolkane-1-carboxamides 3a and 3c

Chloroacetyl chloride (1.317 g, 0.0116 mol) was added to 0.0097 mol of 2a or 2c in chloroform (30 mL). The mixture was stirred at rt for 24 h followed by adding aqueous solution 10% NaOH (2 ×30 mL) and separation of the 3a-c.

1-(2-Chloro- N- phenylacetamido) cyclopentane-1-carboxamide (3a)

Pale yellow oil, yield 89.2%, 1H NMR (CDCl3) 1.64–1.81 (m, 6H, 3CH2, cyclopentyl), 2.20–2.40 (m, 2H, CH2, cyclopentyl), 3.64 (s, 2H, CH2-Cl), 6.99 (s, 2H, NH2), 7.26 (s, NH2, 2H), 7.35–7.51 (m, 5H, Har.); IR (KBr, cm−1) 3454, 3361 (NH2), 1732, 1662 (2 C = O); “13C NMR” (CDCl3) δ ppm 23.95, 35.32 (2CH2, cyclopentyl), 45.32 (CH2-Cl), 66.60 (Cq), 129.41, 129.76, 128.81, 137.78 (aromatic carbons), 168.2 (CO-CH2), 172.45 (CO-NH2); MS (EI) m/z (%): 280.75 (M+, 9.5%). Anal. calcd. for C14H17ClN2O2: C, 59.89; H, 6.10; N, 9.98. Found: C, 59.85; H, 6.14; N, 9.94.

1-(2-Chloro-N-(p-tolyl)acetamido)cyclohexane-1-carboxamide (3c)

Pale yellow oil, yield 84%, 1H NMR (CDCl3) 1.72–1.80 (m, 10H, 5CH2, cyclohexyl), 2.43 (s, 3H, CH3), 3.80 (s, 2H, CH2-Cl), 7.28 (s, 2H, NH2), 7.45 (d, 2H, J = 4 Hz, Har.), 7.47 (d, 2H, J = 4, Har.); “13C NMR” (CDCl3) δ ppm 222.41 (CH3), 22.69, 24.77, 38.68 (3CH2, cyclohexyl), 42.99 (CO-CH2), 62.69 (Cq), 129.83, 129.86, 129.88, 138.28 (arenes carbons), 176.1, 173.03 (2 C = O); IR (KBr, cm−1) 3458, 3392 (NH2), 1743, 1680 (2 C = O); MS (EI) m/z (%): 308.81 (M+, 6.2%), Anal. calcd. for C16H21ClN2O2:: C, 62.23; H, 6.85; N, 9.07. Found: C, 62.90; H, 6.95; N, 9.13.

Synthesis of 1-(2-hydrazineyl-N-arylacetamido)cycloalkane-1-carboxamides 4a and 4c

A.02 mol of hydrazine hydrate 99% was added to 0.0105 mol of 3a or 3c in absolute ethanol. For 8 hours, the reaction mixture was heated to a simmer. In order to get 4a and 4c, the produced precipitate was filtered out and refined by crystallization from ethanol.

1-(2-Hydrazineyl-N- phenylacetamido) cyclopentane -1-carboxamide (4a)

Yellowish white solid, m.p 108–110 °C, yield 85%, IR (KBr, cm−1) 3446, 3309, 3282, 3198 (NH2, NH), 1687, 1656 (C = O). “1HNMR” (CDCl3) 1.44–1.72 (m, 2H, CH2, cyclopentyl), 1.80–1.83 (m, 2H, CH2, cyclopentyl), 1.91–2.36 (m, 4H, 2CH2, cyclopentyl), 4.37 (s, 2H, CH2-CO), 7.12 (m, 2H, Har.), 7.21 (br.s, 1H, NH), 7.39–7.46 (m, 3H, Har.). (EI) m/z (%): 276.34 (M+, 15%), Anal. calcd. for C14H20N4O2: C, 60.85; H, 7.30; N, 20.28. Found: C, 60.90; H, 7.35; N, 20.35.

1-(2-Hydrazineyl-N-(p-tolyl)acetamido)cyclohexane-1-carboxamide (4c)

Yellowish brown oil, yield 83%, IR (KBr, cm−1) 3311, 3186, 3055 (NH2, NH), 1682, 1602 (C = O). “1HNMR” (CDCl3) 1.68–1.90 (m, 10H, 5CH2, cyclohexyl), 2.30 (s, 3H, CH3), 3.92 (br.s, 1H, NH), 4.35 (s, 2H, CO─CH2), 5.06 (br.s, 1H, NH), 7.28 (s, 2H, NH2), 7.36–7.46 (m, 4H, Har.). (EI) m/z (%): 304.39 (M+, 12%), Anal. calcd. for C16H24N4O2: C, 63.13; H, 7.95; N, 18.41. Found: C, 62.15; H, 7.00; N, 18.45.

Synthesis of 2-(2-((1-carbamoylcycloalkyl)(aralkyl)amino)-2-oxoethyl)-N-alkyl/aralkylhydrazine-1-carboxamides 5a-d, 6a-d and 7a-d

A 0.01 mol of the suitable isocyante was added to 0.01 mol of hydrazine 4a-c in dichloroethane and the mixture was for 3 h. The mixture was filtered off and the residue was washed with diethyl ether to get 5a-d, 6a-d and 7a-d.

2-(2-((1-Carbamoylcyclopentyl)(phenyl)amino)-2-oxoethyl)-N-propylhydrazine-1-carboxamide (5a)

Yield 84%, “1HNMR” (CDCl3d6, δ, ppm): 0.93 (t, 3H, J = 7.5 Hz, CH3-CH2), 1.46–1.50 (m, 2H, CH2-CH3), 1.60–1.67 (m, 4H, 2CH2-cyclopentyl), 1.90–1.93 (m, 2H, CH2-cyclopentyl), 2.46–2.51 (m, 2H, CH2-cyclopentyl), 3.72 (s, 2H, CH2-CH2-CH3), 3.82–3.86 (m, 2H, CH2-NH), 5.91 (br.s, 1H, NH), 7.14–7.16 (m, 2H, Har.), 7.28–7.45 (m, 3H, Har.), 7.95 (s, 2H, NH2). 13CNMR (CDCl3d6, δ, ppm): 11.38 (CH3), 22.75 (CH2-CH3),, 23.20, 34.18 (CH2-cyclopentyl), 42.17 (CH2-CH2-CH3), 55.33 (CH2-CO) 63.22 (Cq), 128.76, 129.48, 130.14, 137.06 (aromatic carbons), 157.54, 166.98, 170.65 (3C = O), IR (KBr, cm−1) 3240, 3120, 3096, 2950 (3NH, NH2), 1701, 1664 (C = O). MS (EI) m/z (%): 361.45 (M+, 10%), Anal. calcd. for C18H27N5O3: C, 59.81; H, 7.53; N, 19.39. Found: C, 59.50; H, 7.33; N, 19.50.

2-(2-((1-Carbamoylcyclopentyl)(phenyl)amino)-2-oxoethyl)-N-isopropylhydrazine-1-carboxamide (5b)

Yellowish brown oil, yield 88%,; “1HNMR” (CDCl3d6, δ, ppm): 1.07 (d, 3H, J = 6.5 Hz, CH3-CH), 1.11–1.15 (m, 2H, CH2-cyclopentyl), 1.52–1.97 (m, 2H, 2CH2-cyclopentyl), 2.33–2.42 (m, 2H, CH2-cyclopentyl), 3.82 (s, 2H, CH2-NH), 4.38 (s, 1H, (CH3)2), 5.60 (br.s, 1H, NH), 7.02–7.14 (m, 2H, Har.), 7.23 (s, 2H, NH2), 7.40–7.44 (m, 3H, Har.). 13CNMR (CDCl3d6, δ, ppm): 23.30 (2CH3), 25.37, 37.22 (CH2-cyclopentyl), 41.63 (CH-CH3), 45.0 (CH2-NH), 70.8 (Cq), 128.70, 129.39, 129.51, 137.59 (aromatic carbons), 165.97, 170.08, 171.67 (3C = O), IR (KBr, cm−1) 3321, 3232, 3086, 2974 (3NH, NH2), 1666 (C = O), MS (EI) m/z (%): 361.45 (M+, 10%), Anal. calcd. for C18H27N5O3: C, 59.81; H, 7.53; N, 19.38. Found: C, 59.50; H, 7.32; N, 19.50.

N-Butyl-2-(2-((1-carbamoylcyclopentyl)(phenyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (5c)

Yield 89%, “1HNMR” (CDCl3d6, δ, ppm): 0.83(t, 3H, J = 7.65 Hz, CH3), 1.22–1.46 (m, 8H, 4CH2-cyclopentyl), 1.62–2.05 (m, 4H, (CH2)2-CH3), 2.99 (t, 2H, J = 6.7 Hz, CH2-(CH2)2-CH3), 3.40 (s, 2H,CH2-CO), 4.29 (br.s, 1H, NH), 7.01–7.15 (m, 2H, Har.), 7.10 (s, 2H, NH2), 7.11–7.40 (m, 5H, Har.), 8.29 (br.s, 1H, NH). 13CNMR (CDCl3d6, δ, ppm): 13.82 (CH3-CH2), 20.09 (CH3-CH2), 24.81, 25.00 (2CH2-cyclopentyl), 32.06 (CH3-CH2-CH2), 40.50 (CH2-(CH2)2-CH3), 55.08 (CH2-CO), 67.91 (Cq), 115.23, 116.56, 129.26, 129.37 (aromatic carbons), 158.89, 175.99, 192.75 (3 C = O); IR (KBr, cm−1) 3458, 3358, 3302 (3NH, NH2), 16604 (C = O); MS (EI) m/z (%): 375.47 (M+, 10%), Anal. calcd. for C19H29N5O3: C, 60.78; H, 7.79; N, 18.65. Found: C, 60.75; H, 7.77; N, 18.62.

N-Benzyl-2-(2-((1-carbamoylcyclopentyl)(phenyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (5d)

Pale yellow oil, yield 89%, “1HNMR” (CDCl3d6, δ, ppm): 1.40–1.71 (m, 8H, CH2-cyclopentyl), 2.13–2.16 (m, 3H, CH2-cyclohexyl), 4.42 (s, 2H, CH2-CO), 4.46 (br.s, 1H, NH), 7.05 (br.s, 1H, NH), 7.12 (br.s, 1H, NH), 7.24 (s, 2H, NH2), 7.26–7.36 (m, 7H, Har.), 7.39–7.42 (m, 3H, Har.). 13CNMR (CDCl3d6, δ, ppm): 22.71, 25.4, 32.41 (2CH2-cyclopentyl), 44.23 (CH2-C6H5), 57.4 (CH2-CO), 68.51 (Cq), 119.72, 121.9, 122.7, 126.8, 127.5, 128.4, 134.59, 136.31 (aromatic carbons), 157.78, 164.7, 178.0 (C = O), IR (KBr, cm−1) 3369, 3288, 3030, 2937 (3NH, NH2), 1703, 1691, 1650 (3C = O);MS (EI) m/z (%): 409.21 (M+, 18%), Anal. calcd. for C22H27N5O3: C, 64.53; H, 6.65; N, 17.10. Found: C, 64.50; H, 6.68; N, 17.15.

2-(2-((1-Carbamoylcyclohexyl)(phenyl)amino)-2-oxoethyl)- N- propyl hydrazine -1 -carboxamide (6a)

White solid, m.p. 200 °C, yield 85%, “1HNMR” (CDCl3d6, δ, ppm): 0.83 (t, 3H, J = 8 Hz, CH3), 1.38–1.81 (m, 12H, 5CH2-cyclohexyl, 2H, CH2-CH3), 2.99 (t, 2H, J = 8 Hz, CH2-CH2-CH3), 3.37 (s, 2H, CH2-CO), 4.31 (br.s, 1H, NH), 7.14 (s, 2H, NH2), 7.41–7.45 (m, 10H, Har.), 8.364.31 (br.s, 1H, NH). 13CNMR (CDCl3d6, δ, ppm): 11.76 (CH3), 21.08 (CH2-CH3), 22.70, 23.68, 33.97 (3CH2-cyclohexyl), 41.50 (CH2-CH2-CH3), 55.54 (CH2-CO), 62.45 (Cq), 127.93, 129.09, 130.51, 137.45 (aromatic carbons), 157.85, 166.58, 168.76 (3C = O), IR (KBr, cm−1) 3371, 3300, 3223 (3NH, NH2), 1701, 1687, 1678 (C = O); MS (EI) m/z (%): 375.47 (M+, 22%). Anal. calcd. for C19H29N5O3: C, 60.78; H, 7.79; N, 18.65. Found: C, 60.75; H, 7.76; N, 18.62.

2-(2-((1-Carbamoylcyclohexyl)(phenyl)amino)-2-oxoethyl)-N-isopropylhydrazine-1-carboxamide (6b)

Yellowish white oil, yield 86%, “1HNMR” (CDCl3d6, δ, ppm): 1.20 (d, 3H, J = 6.5 Hz, CH3), 1.84 (s, 4H, 2CH2-cyclohexyl), 1.96 (s, 6H, 3 CH2-cyclohexyl), 3.64 (s, 2H, CH2-CO), 3.96–4.03 (m, 1H, CH-CH3)2, 4.39 (br.s, 1H, NH), 7.29 (s, 2H, NH2), 7.39–7.48 (m, 5H, Har.), 13CNMR(CDCl3d6, δ, ppm): 22.90 (CH3), 23.26, 24.51, 30.91 (3CH2-cyclopentyl), 41.63 (CH-(CH3)2), 51.93 (CH2-CO), 64.32 (Cq), 129.73, 129.99, 130.02, 137.02 (aromatic carbons), 163.94, 166.73, 175.60 (3C = O), IR (KBr, cm−1) 3429, 3421, 3196, 2981 (3NH, NH2), 1747, 1708, 1685 (C = O);MS (EI) m/z (%): 375.47 (M+, 15%), Anal. calcd. for C19H29N5O3: C, 60.78; H, 7.79; N, 18.65. Found: C, 60.78; H, 7.81; N, 18.67.

N-Butyl-2-(2-((1-carbamoylcyclohexyl)(phenyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (6c)

Yield 87%, “1HNMR” (CDCl3d6, δ, ppm): 0.86 (t, 3H, J = 4 Hz, CH3), 1.27–1.40 (m, 10H, 5CH2-cyclohexyl), 1.48–1.81 (m, 4H, (CH2)2-CH3), 3.01 (t, 2H, J = 8 Hz, CH2-(CH2)2-CH3), 3.35 (s, 2H,CH2-CO), 4.29 (br.s, 1H, NH), 7.03–7.16 (m, 2H, Har.), 7.27 (s, 2H, NH2), 7.40–7.48 (m, 3H, Har.), 8.26 (br.s, 1H, NH). 13CNMR (CDCl3d6, δ, ppm): 13.73 (CH3-CH2), 20.08 (CH3-CH2), 22.72, 24.43, 32.06 (3 CH2-cyclohexyl), 34.17 (CH3-CH2-CH2), 42.89 (CH2-(CH2)2-CH3), 58.28 (CH2-CO), 63.22 (Cq), 129.42, 130.16, 137.12, 149.12 (aromatic carbons), 157.67, 167.19, 170.49 (3C = O), IR (KBr, cm−1) 3369, 3300, 3226, 2956 (3NH, NH2), 1703, 1691, 1619 (C = O); MS (EI) m/z (%): 389.59 (M+, 15%), Anal. calcd. for C19H29N5O3: C, 60.78; H, 7.79; N, 18.65. Found: C, 60.75; H, 7.76; N, 18.62.

N-Benzyl-2-(2-((1-carbamoylcyclohexyl)(phenyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (6d)

White solid, m.p. 210–212 °C, 84.5%, “1HNMR”(CDCl3d6, δ, ppm): 1.40–1.55 (m, 5H, CH2-cyclohexyl), 1.72–1.82 (m, 2H, CH2-cyclohexyl), 2.13–2.16 (m, 3H, CH2-cyclohexyl), 4.40 (s, 2H, CH2-CO), 4.48 (br.s, 1H, NH), 7.09 (br.s, 1H, NH), 7.11 (br.s, 1H, NH), 7.24 (s, 2H, NH2), 7.26–7.34 (m, 7H, Har.), 7.39–7.45 (m, 3H, Har.). 13CNMR (CDCl3d6, δ, ppm): 22.71, 25.4, 32.41 (3CH2-cyclohexyl), 44.23 (CH2-C6H5), 57.4 (CH2-CO), 68.55 (Cq), 119.70, 121.7, 122.7, 126.6, 127.6, 128.3, 134.59, 136.31 (aromatic carbons), 156.79, 164.7, 178.0 (3C = O), IR (KBr, cm−1) 3369, 3288, 3203 (3NH, NH2), 1705, 1687, 1672 (C = O);MS (EI) m/z (%): 423.52 (M+, 5%), Anal. calcd. for C23H29N5O3: C, 65.23; H, 6.90; N, 16.54. Found: C, 65.25; H, 6.93; N, 16.55.

2-(2-((1-Carbamoylcyclohexyl)(p-tolyl)amino)-2-oxoethyl)-N-propylhydrazine-1-carboxamide (7a)

Pale yellow oil, 87%, “1HNMR” (CDCl3d6, δ, ppm): 0.95 (t, 3H, J = 4 Hz, CH3-CH2), 1.64–1.79 (m, 10H, 5CH2-cyclohexyl), 1.80 (s, 3H, CH3), 1.93–1.96 (m, 2H, CH2-CH2-CH3), 2.32 (s, 3H, CH3), 2.70 (br.s, 1H, NH), 3.73 (br.s, 1H, NH), 3.84 (t, 3H, J = 8, CH2-CH2-CH3), 4.38 (s, 2H, CH2-CO), 2.70 (s, 1H, NH), 7.22 (d, 2H, J = 4 Hz, Har.), 7.24 (d, 2H, J = 4 Hz, Har.), 7.28 (s, 2H, NH2). 13CNMR (CDCl3d6, δ, ppm): 11.19 (CH3-CH2), 22.45, 23.34, 37.21 (3CH2, cyclohexyl), 23.41 (CH2-CH3), 44.43 (CH2-CH2-CH3), 53.94 (CH2-CO), 70.83 (Cq), 129.08, 129.22, 137.58, 140.49 (aromatic carbons), 149.06, 166.00, 171.69 (3C = O), IR (KBr, cm−1) 3290, 3215, 3196 (3NH, NH2), 1662 (C = O); MS (EI) m/z (%): 389.50 (M+, 10%), Anal. calcd. for C20H31N5O3: C, 61.67; H, 8.02; N, 17.98. Found: C, 61.65; H, 8.05; N, 17.97.

2-(2-((1-Carbamoylcyclohexyl)(p-tolyl)amino)-2-oxoethyl)-Nisopropylhydrazine-1-carboxamide (7b)

Pale yellow oil, 90%, “1HNMR” (CDCl3d6, δ, ppm): 1.08–1.13 (m, 4H, 2CH2-cyclohexyl), 1.15 (d, 2H, J = 8 Hz, CH3-CH), 1.17–1.27 (m, 6H, 3CH2-cyclohexyl), 1.83 (s, 3H, CH3), 2.77 (br.s, 1H, NH), 3.75 (s, 2H, CH2-CO), 4.40 (s, 1H, CH-CH3)2, 4.67 (br.s, 1H, NH), 6.70 (br.s, 1H, NH), 7.15 (d, 2H, J = 8 Hz, Har.), 7.25 (d, 2H, J = 8 Hz, Har.), 7.28 (s, 2H, NH2). 13CNMR (CDCl3d6, δ, ppm): 23.77 (CH3), 23.97 (CH-(CH3)2), 24.21, 29.69, 37.22 (3CH2-cyclohexyl), 54.15 (CH2-CO), 70.84 (Cq), 129.20, 129.39, 129.46, 137.60 (aromatic carbons), 165.96, 170.08, 178.78 (3C = O), IR (KBr, cm−1) 3313, 3234, 3084 (3NH, NH2), 1689, 1680, 1656 (C = O); MS (EI) m/z (%): 389.50 (M+, 15%), Anal. calcd. for C20H31N5O3: C, 61.67; H, 8.02; N, 17.98. Found: C, 61.65; H, 8.03; N, 17.95.

N-Butyl-2-(2-((1-carbamoylcyclohexyl)(p-tolyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (7c)

Pale yellow, 89%, “1HNMR” (CDCl3d6, δ, ppm): 0.87 (t, 3H, J = 7.5 Hz, CH3-CH2), 1.15 (t, 4H, J = 7 Hz, CH2, CH2-CH3), 1.95–1.1.98 (m, 10H, 5CH2-cyclohexyl), 3.62 (s, 2H,CH2-CO), 3.79 (t, 2H, J = 7.5 Hz, CH2-CH2-CH2-CH3), 4.31 (br.s, 1H, NH), 7.07 (d, 2H, J = 5.8 Hz, Har.), 7.16 (d, 2H, J = 5.8 Hz, Har.), 7.20 (s, 2H, NH2), 7.98 (br.s, 1H, NH), 8.91 (br.s, 1H, NH), 13CNMR (CDCl3d6, δ, ppm): 13.7 (CH3-CH2), 18.2 (CH3-CH2), 20.8 (CH3), 22.4, 25.3 (2CH2-cyclohexyl), 31.8 (CH2-cyclohexyl), 36.81 (CH3-CH2-CH2), 42.8 (CH2-(CH2)2-CH3), 58.2 (CH2-CO), 71.0 (Cq), 129.0, 129.2, 137.5, 140.4 (aromatic carbons), 159.1, 166.7, 180.2 (3 C = O), IR (KBr, cm−1) 3330, 3214, 3050, 2937 (3NH, NH2), 1730, 1688, 1672 (C = O); MS (EI) m/z (%): 403.53 (M+, 7%), Anal. calcd. for C21H33N5O3: C, 62.51; H, 8.24; N, 17.36. Found: C, 62.54; H, 8.25; N, 17.35.

N-Benzyl-2-(2-((1-carbamoylcyclohexyl)(p-tolyl)amino)-2-oxoethyl)hydrazine-1-carboxamide (7d)

Oil, 85%, “1HNMR” (CDCl3d6, δ, ppm): 1.68–2.00 (m, 10H, 5CH2-cyclohexyl), 2.34 (s, 3H, CH3), 4.28 (s, 2H, CH2-CO), 5.23 (s, 2H, CH2-C6H5), 7.10 (s, 1H, NH), 7.22–7.29 (m, 9H, Har.), 8.99 (br.s, 1H, NH). 13CNMR (CDCl3d6, δ, ppm): 21.02 (CH3), 22.21, 24.25, 30.21 (3CH2-cyclohexyl), 43.86 (CH2-C6H5), 52.43 (CH2-CO), 70.55 (Cq), 127.15, 127.34, 128.49, 129.69, 129.91 131.21, 134.21, 137.52 (aromatic carbons), 158.21, 166.82, 180.10 (3C = O), IR (KBr, cm−1) 3400, 3332, 3294, (3NH, NH2), 1693, 1662 (3C = O); MS (EI) m/z (%): 437.54 (M+, 30%), Anal. calcd. for C24H31N5O3: C, 65.88; H, 7.14; N, 16.01. Found: C, 65.89; H, 7.15; N, 16.03.

Synthesis of substituted (2,4,5-trioxoimidazolidin-1-yl)amino-N-phenylacetamido)cycloalkane-1-carboxamides 8a-d, 9a-d and 10a-d

To a 0.001 mol of 5a-d, 6a-d or 7a-d in dry benzene a 0.002 mol of oxalyl chloride was supplemented. The mix was heated for 3 h then benzene was allowed to evaporate. The residual substance was subjected to column chromatography using mixture of CHCl2 and CH3OH as mobile phase to obtain 8a-d, 9a-d and 10a-d.

1-(N- Phenyl-2-((2,4,5 - trioxo-3-propyl imidazolidin-1-yl)amino)acetamido) cyclopentane-1-carboxamide (8a)

White solid, m.p. 110–112 °C, yield 80%, HPLC: purity 98.7%, Rt = 3.98 min. “1HNMR” (CDCl3d6, δ, ppm): 0.91–1.01 (m, 3H, CH3-CH2), 1.61–1.91 (m, 10H, 4CH2-cyclopentyl, CH2-CH3), 3.63 (t, 2H, J = 8, CH2-CH2-CH3), 3.73 (s, 2H, CH2-CO), 4.59 (br.s, 1H, NH), 7.22 (s, 2H, NH2), 7.38–7.54 (m, 5H, Har.). 13CNMR (CDCl3d6, δ, ppm): 11.13 (CH3), 20.94 (CH2-CH3), 23.92, 36.14 (2 CH2-cyclopentyl), 41.67 (CH2-CH2-CH3), 44.47 (CH2-CO), 71.09 (Cq), 115.56, 128.35, 130.10, 135.70 (aromatic carbons), 149.09, 152.55, 154.54 (3C = O, imidazolidine), 166.5, 169.30 (2C = O); IR (KBr, cm−1) 3365, 3251 (NH, NH2), 1755, 1689, 1631 (C = O);MS (EI) m/z (%): 415.45 (M+, 4%), Anal. calcd. for C20H25N5O5: C, 57.82; H, 6.07; N, 16.86. Found: C, 57.81; H, 6.15; N, 16.75.

1-(2-((3 – Isopropyl - 2,4,5-trioxo imidazolidin-1-yl)amino) – N -phenylacetamido)cyclopentane-1-carboxamide (8b)

Yellowish brown oil, yield 75%, HPLC: purity 99.4%, Rt = 4.30 min. “1HNMR” (CDCl3d6, δ, ppm): 1.07–1.11 (m, 2H, CH2-cyclopentyl), 1.14 (d, 6H, J = 6.5, (CH3)2), 1.22–1.26 (m, 2H, CH2-cyclopentyl), 1.39–1.50 (m, 4H, 2CH2-cyclopentyl) 2.07 (br.s, 1H, NH), 3.71 (s, 2H, CH2-CO), 4.51 (m, 1H, CH-(CH3)2), 7.26 (s, 2H, NH2), 7.36–7.45 (m, 5H, Har.). 13C NMR (CDCl3, δ, ppm): 20.43 (CH3), 23.00, 37.00 (2CH2-cyclopentyl), 42.47 (CH2-CO), 51.16 (CH-(CH3)2), 63.85 (Cq), 129.65, 129.77, 130.12, 137.57 (aromatic carbons), 148.78, 151.81, 159.06 (3 C = O, imidazolidine), 165.06, 177.20 (2 C = O), IR (KBr, cm−1) 3304, 3234, 3219 (NH, NH2), 1662, 1610, 1593 (C = O); MS (EI) m/z (%): 415.45 (M+, 25%), Anal. calcd. for C20H25N5O5: C, 57.82; H, 6.07; N, 16.86. Found: C, 57.72; H, 6.15; N, 16.69.

1-(2-((3-Butyl-2,4,5-trioxoimidazolidin-1-yl)amino)-N-phenylacetamido)cyclopentane-1-carboxamide (8c)

Yellowish brown oil, yield 80 %, HPLC: purity 96.5%, Rt = 3.39 min. “1HNMR” (CDCl3d6, δ, ppm): 0.87 (t, 3H, J = 8 Hz, CH3), 1.13–1.18 (m, 2H, CH3-CH2), 1.23–1.27 (m, 2H, CH2-CH2-CH3), 1.29–1.68 (m, 8H, 4CH2-cyclopentyl), 3.50 (s, 2H, CH2-CO), 3.80 (t, 2H, J = 8 Hz, CH2-CH2-CH2-CH3), 4.50 (br.s, 1H, NH), 7.22 (br.s, 2H, NH2), 7.27–7.38 (m, 5H, Har.). 13CNMR (CDCl3d6, δ, ppm): 13.70 (CH3), 18.18 (CH2-CH3), 24.14, 36.64 (2 CH2-cyclopentyl), 29.38 (CH2-CH2- CH3), 42.88 (CH2-CH2- CH2-CH3), 52.98 (CH2-CO), 72.38 (Cq), 119.31, 128.65, 129.02 (3CHar.), 137.38 (Car.), 148.45, 155.07, 159.22 (3C = O, imidazolidine), 163.07, 177.28 (2C = O). IR (KBr, cm−1) 3444, 3419, 3360 (NH, NH2), 1755, 1747, 1734 (C = O); MS (EI) m/z (%): 429.48 (M+, 20%), Anal. calcd. for C21H27N5O5: C, 58.73; H, 6.34; N, 16.31. Found: C, 58.80; H, 6.31; N, 16.35.

1-(2- ((3 -Benzyl- 2,4,5- trioxo imidazolidin-1-yl) amino)- N- phenylacetamido) cyclopentane-1-carboxamide (8d)

Pale yellow viscous oil, yield 71%, HPLC: purity 98%, Rt = 7.18 min. “1HNMR” (CDCl3d6, δ, ppm): 1.14–1.27 (m, 4H, 2CH2-cyclopentyl), 1.94–2.08 (m, 4H, 2CH2-cyclopentyl), 4.71 (s, 2H, CH2-C6H5), 4. 76 (br.s, 1H, NH), 7.14 (s, 2H, NH2), 7.16–7.29 (m, 10H, Har.), 13CNMR (CDCl3d6, δ, ppm): 25.01, 35.55 (2 CH2-cyclopentyl), 42.93 (CH2-C6H5), 43.95 (CH2-CO), 67.90 (Cq), 128.96, 128.98, 129.01 129.07, 129.08, 129.12, 136.90, 137.25 (aromatic carbons), 148.56, 151.68, 153.43 (3C = O, imidazolidine), 166.92, 172.75 (2C = O). IR (KBr, cm−1) 34195, 3273, 3186, 3089 (NH, NH2), 1759, 1720, 1680, 1665, 1631 (5 C = O); MS (EI) m/z (%): 463.39 (M+, 10%), Anal. calcd. for C24H25N5O5: C, 62.19; H, 5.44; N, 15.11. Found: C, 62.21; H, 5.45; N, 15.15.

1-(N- Phenyl-2-((2,4,5 - trioxo- 3-propyl imidazolidin-1-yl)amino) acetamido) cyclohexane-1-carboxamide (9a)

White solid, m.p. 120 °C, yield 75%, HPLC: purity 97.6%, Rt = 5.05 min. “1HNMR” (CDCl3d6, δ, ppm): 1.10–1.23 (m, 3H, CH3-CH2), 1.41–1.91 (m, 12H, 5CH2-cyclohexyl, CH2-CH3), 3.61 (t, 2H, J = 8, CH2-CH2-CH3), 3.75 (s, 2H, CH2-CO), 4.59 (br.s, 1H, NH), 7.22 (s, 2H, NH2), 7.38–7.54 (m, 5H, Har.). 13CNMR (CDCl3d6, δ, ppm): 11.14 (CH3), 20.84 (CH2-CH3), 23.92, 36.14 (2 CH2-cyclopentyl), 41.61 (CH2-CH2-CH3), 44.47 (CH2-CO), 69.12 (Cq), 115.56, 128.35, 130.10 (3CHar.), 135.91 (Car.), 149.09, 152.55, 154.54 (3C = O, imidazolidine), 166.2, 169.30 (2C = O), IR (KBr, cm−1) 3313, 3294, 3142 (NH, NH2), 1690, 1685, 1630 (C = O); MS (EI) m/z (%): 429.48 (M+, 25%), Anal. calcd. for C21H27N5O5: C, 58.73; H, 6.34; N, 16.31. Found: C, 58.71; H, 6.38; N, 16.29.

1-(2- ((3-Isopropyl -2,4,5 – trioxo imidazolidin-1-yl) amino) -N-phenyl acetamido) cyclohexane-1-carboxamide (9b)

White solid, m.p. 160 °C, yield 80%, HPLC: purity 99.4%, Rt = 4.27 min. “1HNMR” (CDCl3d6, δ, ppm): 1.11 (d, 6H, J = 4 Hz, (CH3)2), 1.38–1.61 (m, 10H, 5CH2-cyclohexyll), 3.65 (s, 2H, CH2-CO), 4.33–4.50 (m, 1H, CH-(CH3)2), 4.93 (br.s, 1H, NH), 7.14 (s, 2H, NH2), 7.27 (s, 2H, Har.), 7.36–7.48 (m, 3H, Har.); 13C NMR (CDCl3, δ, ppm): 20.68 CH3, 22.49, 25.85, 29.68 (3 CH2-cyclohexyl), 46.79 (CH2-CO), 53.57 (CH-(CH3)2), 66.06 (Cq), 129.61, 129.75, 129.87, 134.78 (aromatic carbons), 148.23, 156.44, 157.21 (3C = O, imidazolidine), 164.89, 166.19 (2 C = O), IR (KBr, cm−1) 3209, 3184, (NH, NH2), 1762, 1728, 1697 (C = O); MS (EI) m/z (%): 429.48 (M+, 10%), Anal. calcd. for C21H27N5O5: C, 58.73; H, 6.34; N, 16.31. Found: C, 58.75; H, 6.34; N, 16.30.

1-(2-((3-Butyl-2,4,5-trioxo imidazolidin-1-yl) amino)- N-phenyl acetamido) cyclohexane-1-carboxamide (9c)

White solid, m.p. 170 °C, yield 75%, HPLC: purity 97.6%, Rt = 6.59 min. “1HNMR” (CDCl3d6, δ, ppm): 0.96 (t, 3H, J = 4 Hz, CH3), 1.25 36–1.41 (m, 2H, CH2- CH3), 1.61–1.63 (m, 10H, 5CH2-cyclohexyl), 3.66 (s, 2H, CH2-CO), 3.73 (t, 2H, CH2-(CH2)2 -CH3), 4.54 (br.s, 1H, NH), 7.18 (s, 2H, Har.), 7.26 (br.s, 2H, NH2), 7.27–7.46 (m, 3H, Har.). 13CNMR (CDCl3d6, δ, ppm): 13.73 (CH3), 19.89 (CH2-CH3), 20.04, 22.51, 29.82 (3CH2-cyclohexyl), 29.96 (CH2-CH2- CH3), 42.90 (CH2-CH2- CH2-CH3), 53.42 (CH2-CO), 63.23 (Cq), 129.61, 129.82, 130.08, 136.56, 137.38 (aromatic carbons.), 148.78, 151.81, 153.71 (3C = O, imidazolidine), 165.06, 167.69 (2 C = O). MS (EI) m/z (%): 443.50 (M+, 10%), IR (KBr, cm−1) 3392, 3385, 3275 (NH, NH2), 1795, 1759, 1685 (C = O); Anal. calcd. for C22H29N5O5: C, 59.58; H, 6.59; N, 15.79. Found: C, 59.55; H, 6.62; N, 15.83.

1-(2-((3-Benzyl-2,4,5- trioxo imidazolidin-1-yl) amino)-N-phenyl acetamido) cyclohexane-1-carboxamide (9d)

White solid, m.p. 175 °C, yield 90%, HPLC: purity 97.9%, Rt = 8.25 min. “1HNMR” (CDCl3d6, δ, ppm): 1.25 (s, 4H, 2CH2-cyclohexyl), 1.46–1.72 (m, 6H, 3 CH2-cyclohexyl), 4.84 (s, 2H, CH2-CO), 4.87 (s, 2H, CH2-C6H5),7.25 (s, 2H, NH2), 7.35–7.37 (m, 10H, Har.) 7.50 (br.s, 1H, NH). 13CNMR (CDCl3d6, δ, ppm): 22.89, 34.52, 34.58 (3CH2-cyclohexyl), 43.52 (CH2-C6H5), 44.03 (CH2-CO), 64.40 (Cq), 128.79, 128.97, 129.01, 129.03, 129.12, 129.18, 133.82, 134.99 (aromatic carbons), 149.94, 151.59, 153.31 (3C = O, imidazolidine), 164.89, 166.19 (2 C = O), MS (EI) m/z (%): 477.52 (M + , 54), IR (KBr, cm−1) 3419, 3273, 3186 (NH, NH2), 1759, 1680, 1625 (C = O); Anal. calcd. for C25H27N5O5: C, 62.88; H, 5.70; N, 14.67. Found: C, 62.86; H,5.68; N, 14.63.

1-(N-(p-Tolyl)-2-((2,4,5-trioxo-3-propylimidazolidin-1-yl)amino)acetamido)cyclohexane-1-carboxamide (10a)

White solid, m.p. 110–112 °C, yield 75%, HPLC: purity 97%, Rt = 3.81 min. “1HNMR” (CDCl3d6, δ, ppm): 0.92 (t, 3H, J = 4 Hz, CH2-CH3), 1.56–1.59 (m, 2H, CH2-CH3), 1.66–1.73 (m, 10H, 5CH2-cyclohexyl), 1.79 (s, 3H, CH3), 3.85 (t, 2H, J = 8, CH2-CH2-CH3), 3.73 (s, 2H, CH2-CO), 4.53 (br.s, 1H, NH), 7.28 (s, 2H, NH2), 7.36 (d, 2H, J = 8, Har.), 7.46 (d, 2H, J = 8 Hz, Har.). 13CNMR (CDCl3d6, δ, ppm): 11.10 (CH3-CH2), 21.14 (CH3-C6H5), 23.94, 24.17, 36.97 (3CH2-cyclohexyl), 42.39 (CH2-CH2-CH3), 44.46 (CH2-CO), 72.38 (Cq), 128.75, 129.56, 129.73, 140.71 (aromatic carbons), 149.0, 152.55, 154.54 (3C = O, imidazolidine), 172.28, 177.53 (2 C = O). MS (EI) m/z (%): 443.50 (M+, 50%), IR (KBr, cm−1) 3290, 3165 (NH, NH2), 1685, 1662 (C = O); Anal. calcd. for C22H29N5O5: C, 59.58; H, 6.59; N, 15.79. Found: C, 59.54; H, 6.57; N, 15.78.

1-(2- ((3-Isopropyl -2,4,5 –trioxo imidazolidin-1-yl) amino)-N- (p-tolyl) acetamido) cyclohexane-1-carboxamide (10b)

White solid, m.p. 150–152 °C, yield 83%, HPLC: purity 99%, Rt = 5.94 min. “1HNMR” (CDCl3d6, δ, ppm): 1.14 (d, 6H, J = 6.5 Hz, (CH3)2), 1.43–1.54 (m, 10H, 5CH2-cyclohexyl), 1.77 (s, 3H, C6H5-CH3), 3.87 (s, 2H, CH2-CO), 4.41–4.57 (m, 1H, CH-(CH3)2), 7.17 (d, 2H, J = 6.5 Hz, Har.), 7.27 (s, 2H, NH2), 7.44 (d, 2H, J = 6.5 Hz, Har.); “13C NMR” (CDCl3, δ, ppm): 20.42 (CH-(CH3)2), 21.15 (CH3-C6H5), 22.32, 25.16, 29.72 (3 CH2-cyclohexyl), 46.17 (CH2-CO), 53.52 (CH-(CH3)2), 67.12 (Cq), 129.41, 129.65, 129.81, 135.73 (aromatic carbons), 148.21, 154.42, 157.13 (3C = O, imidazolidine), 164.42, 167.16 (2 C = O), MS (EI) m/z (%): 443.50 (M+, 50%), IR (KBr, cm−1) 3155, 3091 (NH, NH2), 1745, 1708, 1680 (C = O); Anal. calcd. for C22H29N5O5: C, 59.58; H, 6.59; N, 15.79. Found: C, 59.57; H, 6.55; N, 15.77.

1-(2-((3-Butyl -2,4,5-trioxo imidazolidin-1-yl) amino)-N-(p-tolyl) acetamido) cyclohexane-1-carboxamide (10c)

White solid, m.p. 120 °C, yield 75%, HPLC: purity 99%, Rt = 6.07 min. “1HNMR” (CDCl3d6, δ, ppm): 0.86 (t, 3H, J = 4.4 Hz, CH2-CH3), 1.24–1.30 (m, 2H, CH2-CH2-CH3), 1.24–1.30 (m, 2H, CH2-CH2-CH3), 1.47–1.69 (m, 10H, 5CH2-cyclohexyl), 1.97 (s, 3H, C6H4-CH3), 3.58 (s, 2H, CH2-CO), 3.80 (t, 2H, J = 7.4 Hz, CH2-(CH2)2 -CH3), 4.49 (br.s, 1H, NH), 7.20 (br.s, 2H, NH2), 7.27 (d, 2H, J = 8.4 Hz, Har.), 7.31 (d, 2H, J = 8.4 Hz, Har.). MS (EI) m/z (%): 457.53 (M+, 15%), IR (KBr, cm−1) 3385, 3311 (NH, NH2), 1755, 1687 (C = O); Anal. calcd. for C23H31N5O5: C, 60.38; H, 6.83; N, 15.31. Found: C, 60.36; H, 6.81; N, 15.30.

1-(2-((3-Benzyl - 2,4,5- trioxoimidazolidin-1-yl)amino)-N-(p-tolyl)acetamido)cyclohexane-1-carboxamide (10d)

White solid, m.p. 160 °C, yield 75%, HPLC: purity 95.9%, Rt = 6.60 min. “1HNMR” (CDCl3d6, δ, ppm): 1.19–1.37 (m, 10H, 5CH2-cyclohexyl), 1.74 (CH3-C6H4), 3.66 (s, 2H, CH2-CO), 4.77 (s, 2H, CH2-C6H5), 4.85 (br.s, 1H, NH), 5.29 (s, 2H, NH2), 7.17–7.42 (m, 10H, Har.). 13CNMR (CDCl3d6, δ, ppm): 20.7 (CH3), 22.4, 25.1, 36.9 (2 CH2-cyclohexyl), 44.6 (CH2-C6H5), 53.1 (CH2-CO), 70.9 (Cq), 129.06, 129.54, 129.64, 129.68, 129.77, 133.81, 137.05, 136.57 (aromatic carbons), 150.41, 154.44, 159.31 (3C = O, imidazolidine), 166.20, 177.51 (2C = O). MS (EI) m/z (%): 491.55 (M+, 30%), IR (KBr, cm−1) 3307 (NH, NH2), 1747, 1714, 1645 (C = O); Anal. calcd. for C26H29N5O5: C, 63.53; H, 5.95; N, 14.25. Found: C, 63.50; H, 5.61; N, 14.27.

Biological EvaluationAnimals

The anticonvulsant effect of the new hydrids 8a-d, 9a-d as well as 10a-d was carried on Male mature white mice range in weight from “20–25 g” that obtained from “European Reef Animal House Colony (Egypt)”. They were retained in the ambient condition for humidity, light/dark cycle and temperature. The mice were fed a regular diet and had free access to water. Prior to the start of the pharmacological experiments, mice were acclimated to laboratory conditions. Every part of the experimental procedures involving animals were conducted in agreement with the Ethics Committee of the Misr University for Science & Technology (Approval number MC-7). All of the compounds under investigation were suspended in a solution of 1 percent of Tween 80.

Calculation of the median lethal dose (LD50) and acute toxicity test

The mice were distributed randomly into groups (6 mice per group) Preliminary investigations were performed to detect the minimum dose that causes death of all mice (LD100). If the LD100 is not achieved, the chemical has a good safety profile, and the LD50 will be reported as greater than 0.5 g/kg. Animals were observed during the first 2 hours after receiving the maximal i.p. dosage of these chemicals for any signs of abnormal behavior or fatalities, and then once an hour for the next 4 hours. The monitoring was kept up each 2 h till the 12th h then at 24 h to inspect any late changes. The behaviors and any other toxic manifestations of the mice were monitored every day for three days and the animals were maintained under monitoring for two weeks.

Stage I: Preliminary evaluation of anticonvulsant effect Subcutaneous pentylenetetrazole (scPTZ)-induced seizures method

A group of 6 animals serves as negative control and the test compounds or the standard antiepileptic drug were given i.p. to groups of 6 mice. Subsequently one hour of intraperitoneal injection of the substances, pentylenetetrazole (PTZ) was administrated 85 mg/kg subcutaneous injection, in the fatty fold of skin near the base of the neck [49, 50]. This dosage has been reported to produce clonic seizures that continues for at least five seconds in 97% of the mice under investigations.

Each mouse is put in a separate plastic cage for monitoring that persists for half an hour. The occurrence of tonic-clonic convulsions long-lasting for more than five seconds was detected. Lacking of a single five seconds episode of clonic convulsions through the period of monitoring (30 min) is considered as the end point in this experiment [51]. The results were expressed as percent protection.

Maximal electroshock seizure (MES) test

The experiment was carried out following the reported method described by White et al. and Krall et al. [52, 53]. Thirty min after the intraperitoneal injection of the compounds, in order to cause convulsions in mice, an electrical current was applied. Ugo Basil, ECT Unit, 57800 adjusted to a fixed 25 mA current intensity and 0.2 s stimulus duration, and at 50 Hz” via auricle electrodes. The fact that the test chemical was able to prevent tonic 180-degree hind limb flexion raises the possibility that it might slow the spread of MES-induced convulsions [54].

Neurotoxicity

Employing the motor coordination apparatus, neuronal toxicity of examined compounds was assessed for every signs of changes in motion coordination [51]. Before beginning the experiment, the mice were trained to keep their equilibrium on “a rotating 2.5 cm diameter of plastic rod (Ugo-Basile Accele. ROTA-ROD for mice, UGO Basile, 7650, Italy) at a fixed speed of 10 rpm for one minute”. Mice which fulfilling this prerequisite criteria and effectively maintain their balance on the rod for three times were selected to enter the experiment. Each test substance was injected, and then the experiment was conducted 30 minutes later. The mice were put back on the rotating rod, and their motor abilities were monitored for up to 60 seconds. The inability to keep the balance on the rod during one minute is considered as a sign of motor impairment and neurotoxicity [55].

Stage II: Determination of ED50

ED50 is defined as “the dose of the compound which protecting half of the mice in the group against scPTZ or MES seizures”. To determine the ED50, the chemical was administered into every groups of mice (n = 6) at varying dosages, resulting in a spectrum of seizure protection from 16 to 83%. The ED50 and its associated confidence interval might be deduced from the provided data plot.

Quantitative determination of GABA level in whole brain

Animals were divided into three groups and each group contains 6 mice. The first group was negative control group and was injected using a Tween 80 water suspension at concentration of 1%.The mice in the second and third groups were administered compounds 8b or 10b, correspondingly of 100 mg/ kg, intraperitoneally. The compounds were given for 7 days. One day after the final dose of the examined substance, the mice were sedated followed by brains dissection and were laid on ice and stored at a temperature of −80 degrees Celsius. Consequently, brains were crushed using “a homogenizer (Medical instruments, MPW-120, Poland), with ice-cooled saline (0.9% NaCl) to prepare 20% w/v homogenate. The homogenate was then centrifuged at 4000 rpm for 5 min at 4 °C using a cooling centrifuge to remove cell debris (Laborzentrifugen, 2k15, Sigma, Germany) then the supernatant was used for determination of brain’s γ- aminobutyric acid (GABA) content” following the previously reported method [56].

Hepatotoxicity

Animals were divided into seven groups (n = 6). The first group was the control group and was injected with 1% Tween 80 suspension; Groups (2–7) were administered substances 8b, 9a-d and 10b, correspondingly of 100 mg/ kg, intraperitoneally suspended in 1% Tween 80. The compounds were given for seven days. One day afterward the final dosing of the examined substances, the mice were sedated followed by collection of blood samples from the heart perforation then centrifugation at 4000 rpm for ten min to separate the serum. Analyses of the liver serum enzymes, total protein in addition of albumin were measured using commercially available kits “Biodiagnostic, Egypt”.

Statistical analysis

Before proceeding in the statistical analysis, data values were checked for normality using the Shapiro test and for heteroscedasticity using the Brown-Forsythe test. The data are presented as means ± S.E. Data were processed by one-way ANOVA followed by the Tukey–Kramer Post hoc test. GraphPad Prism software (version 8; GraphPad Software, Inc., San Diego, CA, USA) was employed to perform the statistical analysis and to establish the graphical representation.

In-Silico studiesPharmacophore generation

The hypothesis was created through common feature pharmacophore model protocol in Discovery studio software 4.1 (Accelrys, Inc, San Diego, USA). Training set 1–10 was used to generate the anticonvulsant pharmacophore; they were stated to have antiepileptic action. There are different conformational models were constructed for of each structure of the test sets to generate the common feature hypotheses, whereas ten hypotheses were generated.

Docking study

Docking study was performed using Discovery studio software 4.1 (Accelrys, Inc, San Diego, USA). The crystal structure of GABA-AT (PDB: 1OHW) [57] was downloaded from RCSB protein data bank (http://www.rscb.org/pdb). In general, the docking workflow was listed as: (1) Protein was prepared using the “Protein Preparation protocol”. All water molecules were deleted from the structure of the complex. Hydrogen atoms and charges were added during a brief relaxation. After optimizing the hydrogen bond network, the crystal structure was minimized. (2) The ligand was prepared with ligand preparation protocol, including adding hydrogen atoms, then energy minimization. (3) The center of the grid box, grid position and spacing was defined with the intrinsic ligand. The top-ranking poses were determined. Ten of the most stable docking poses for each ligand were obtained and arranged according to their docking scores (-CDOCKER energy). The ideal pose which fulfills the reported key binding features was selected.

Physicochemical parameters

All physicochemical descriptors and pharmacokinetic properties were calculated using swissADME a fee online web tool