Design and synthesis of certain substituted cycloalkanecarboxamides structurally related to safinamide with anticonvulsant potential

Article abstract of DOI:10.1007/s11164-013-1488-2

A series of novel safinamide derivatives were synthesized and biologically evaluated for their anticonvulsant activity against maximal electroshock seizure assay and subcutaneous pentylenetetrazole (s.c. PTZ) screening test. Compound 13b is the most active derivative in s.c. PTZ screening test with an ED₅₀ value lower than that of safinamide by about tenfold. A molecular modeling study, including fitting to sodium channel blockers 3D-pharmacophore model and docking into a branched-chain aminotransferase enzyme active site were consistent with the in vivo results.

Full text of DOI:10.1007/s11164-013-1488-2

Res Chem Intermed
DOI 10.1007/s11164-013-1488-2
Design and synthesis of certain substituted
cycloalkanecarboxamides structurally related
to safinamide with anticonvulsant potential
•
Mohamed N. Aboul-Enein Aida A. El-Azzouny
•
•
Yousreya A. Maklad Mohamed A. Ismail
•
•
Nasser S. M. Ismail Rasha M. Hassan
Received: 17 June 2013 / Accepted: 12 November 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract A series of novel safinamide derivatives were synthesized and biologi-
cally evaluated for their anticonvulsant activity against maximal electroshock seizure
assay and subcutaneous pentylenetetrazole (s.c. PTZ) screening test. Compound 13b
is the most active derivative in s.c. PTZ screening test with an ED50 value lower than
that of safinamide by about tenfold. A molecular modeling study, including fitting to
sodium channel blockers 3D-pharmacophore model and docking into a branched-
chain aminotransferase enzyme active site were consistent with the in vivo results.
Keywords Epilepsy ꢀ Safinamide ꢀ Cycloalkanecarboxamides ꢀ
Anticonvulsants
Introduction
Epilepsy is a common chronic neurological disorder characterized by recurrent
unprovoked seizures [1]. These seizures are transient signs and/or symptoms of
Electronic supplementary material The online version of this article (doi:10.1007/s11164-013-1488-2)
contains supplementary material, which is available to authorized users.
M. N. Aboul-Enein (&) ꢀ A. A. El-Azzouny ꢀ R. M. Hassan
Medicinal and Pharmaceutical Chemistry Department (Medicinal Chemistry Group),
Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki,
Giza 12622, Egypt
e-mail: mnaboulenein@hotmail.com; mnaboulenein@yahoo.com
Y. A. Maklad
Medicinal and Pharmaceutical Chemistry Department (Pharmacology Group), Pharmaceutical and
Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt
M. A. Ismail ꢀ N. S. M. Ismail
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ain Shams, Abbassia,
Cairo 11566, Egypt
123

M. N. Aboul-Enein et al.
abnormal excessive or synchronous neuronal activity in the brain [2]. Despite the
availability of many new antiepileptic drugs (AEDs), estimates suggest that available
medication controls the seizures in only 50 % of patients or decreases the incidence in
only 75 % of patients [3, 4]. Moreover, these medications are associated with severe
side effects like ataxia, hepatotoxicity, and megaloblastic anemia [5, 6]. Accordingly,
the search for novel, more effective, and less toxic antiepileptic agents is essential.
Safinamide [(S)-(1)-2-(4-(3-fluorobenzyloxy) benzylamino) propanamide, methane-
sulfonate] (I) was reported as a promising antiepileptic agent acting via blocking of
voltage-sensitive sodium and calcium channels as well as glutamate release inhibition [7].
F
A
OH
S
H2
C
H2
C
H
N
O
O
O
C
H
C
O
NH2
Safinamide I
Through a literature survey, it was found that many geminally disubstituted
cyclohexane derivatives have proven to elicit anticonvulsant profile such as
Gabapentin (II). Aboul-Enein et al. [8] disclosed the synthesis and anticonvulsant
potential of N-benzyl-N-(1-((pyrrolidin-1-yl) methyl) cyclohexyl) benzenamine (III),
the N-benzyl-N-(1-((piperidin-1-yl) methyl) cyclohexyl) benzenamine (IV), and the
N-(4-methoxybenzyl)-N-(1-((piperidin-1-yl) methyl) cyclohexyl) benzenamine (V).
Further, certain indane derivatives such as 2,3 dihydro-1H-inden-1-amine (VI) [9] and
2-(2,3-dihydro-1H-inden-1-ylamino)acetamide (VII) [10] have been reported to
display anticonvulsant effect against maximal electroshock seizure (MES) assay.
R1
H2N
COOH
R2
N
N
Gabapentin II
R1
H
H
R2
III
IV
V
Pyrrolidino
Piperidino
Piperidino
OCH3
O
C
H2
C
NH2
H2N
NH
VI
VII
123

Design and synthesis of certain substituted cycloalkanecarboxamides
Based on the above considerations, it was found of interest to subject the
safinamide structure to certain molecular modifications directed to increase the
molecule lipophilicity, a parameter that would lead to enhancement of crossing
the blood–brain barrier, subsequently escalating the anticonvulsant potential [11],
by replacement moiety A of safinamide with the lipophilic cyclohexyl,
cycloheptyl, or 1-indanyl moieties in compounds 13a–e, 14a–e, and 15a–e,
respectively.
X
O
H2
C
H2
C
H
N
O
C
NH2
(CH2)n
13a-e (n=3)
14a-e (n=4)
X
O
C
H2
C
H2
C
H
N
O
NH2
15a-e
The molecular modeling study was carried out by the development of a
qualitative common feature pharmacophore model based on various sodium
channel blockers having anticonvulsant activity selected from the literature [12,
13]. The matching of the designed compounds with the generated pharmacophore
model was performed and these results will be compared with the anticonvulsant
potential of the these compounds against an MES test [14–17]. Another approach
for the molecular modeling study in this work is based on the structural
similarity between the designed compounds and gabapentin, where both the
synthesized compounds and gabapentin are 1,1-disubstituted cycloalkane deriv-
atives having amino and carbonyl moieties. Based on the previous concept,
docking studies of the designed compounds (13a–e, 14a–e, and 15a–e) at the
same binding site of gabapentin in the branched-chain aminotransferase enzyme
[18] will be performed. All the achieved data of the docking study of both
gabapentin and safinamide will be compared with the results of the in vivo
anticonvulsant activity in the subcutaneous pentylenetetrazole (s.c. PTZ) seizure
assay.
123

M. N. Aboul-Enein et al.
Experimental
Chemistry
All melting points were measured using Electrothermal Capillary melting point
apparatus and are uncorrected. Infrared (IR) spectra were recorded on a JASCO FT/
1
IR-6100 Spectrometer. H NMR and 13C NMR spectra were carried out on a Jeol
ECA 500 MHz spectrometer using TMS as internal standard, and chemical shift
values were recorded in ppm on d scale. Mass spectra (EI) were measured on a Jeol
JMS-AX500 Mass Spectrometer and ESI on a Finnigan LCQ Advantage MAX
Mass Spectrometer. Elemental analyses were carried out in the Microanalytical
Unit, National Research Centre, Egypt. TLC on silica gel plates (Merck, 60F 254)
were used for thin layer chromatography. Visualization was performed by
illumination with UV light source (254 nm). Column chromatography was carried
out on silica gel 60 (0.063–0.200 mm) obtained from Merck.
General procedure for synthesis of 4-(3-substituted benzyloxy) benzaldehydes
3a–e
A reaction mixture of 4-hydroxybenzaldehyde (2) (15 g, 0.12 mol), anhydrous
Na2CO3 (17 g, 0.16 mol) and the appropriate 3-substituted benzyl bromide (1a–
e) (0.136 mol) in 50 ml DMF was heated at 60 °C for 12 h. After cooling to room
temperature, the reaction mixture was poured onto 50 ml of water and extracted
with ethyl acetate (3 9 50 ml). The organic phase was washed with 10 % NaOH
(3 9 25 ml) then with distilled water (2 9 25 ml), dried over anhydrous Na2SO4,
and evaporated under reduced pressure to give 3a–e.
4-Benzyloxy benzaldehyde (3a)
M.p. 74–76 °C (Lit. [19]. 69 °C); IR spectra (KBr, cm-1) showed a band at 1689
(C=O) and absence of (OH) band at 3400; MS (EI) m/z (%): 213 (100) (M? ?1),
90.9 (32.38); yield 77 %.
4-(3-Fluoro benzyloxy) benzaldehyde (3b)
M.p. 42–44 °C (Lit. [20]. 43–45 °C); yield 79 %.
4-(3-Chloro benzyloxy) benzaldehyde (3c)
M.p. 52 °C (Lit. [20]. 53–55 °C); IR spectra (KBr, cm-1) showed a band at 1688
(C=O) and absence of (OH) band at 3400; yield 75 %.
4-(3-Methoxy benzyloxy) benzaldehyde (3d)
M.p. 60 °C (Lit. [21]. 60–62 °C); yield 72 %.
123

Design and synthesis of certain substituted cycloalkanecarboxamides
4-(3-Nitro benzyloxy) benzaldehyde (3e)
1
M.p. 80–82 °C H NMR (CDCl3): 5.25 (s, 2H, OCH2), 7.06 (d, J = 9.2, 2H, Har),
7.57 (t, J = 7.6, 1H, Har), 7.75 (d, J = 7.6, 1H, Har), 7.83 (d, J = 9.2, 2H, Har), 8.17
(d, J = 7.6, 1H, Har), 8.3 (s, 1H, Har), 9.87 (s, 1H, CHO); 13C-NMR (CDCl3): 68.92
(OCH2), 115.19 (2 CHar), 122.26 (CHar), 123.3 (CHar), 129.86 (CHar), 130.7 (CHar),
132.13 (2 CHar), 133.23 (Car), 138.27 (Car), 148.58 (Car), 163.07 (Car), 190.72
(C=O); yield 78 %.
General procedure for synthesis of 1- aminocycloalkanecarbonitriles 5a and 5b
To a cooled and stirred solution containing (13.7 g, 0.14 mol) of cyclohexanone
(4a) or (15.7 g, 0.14 mol) of cycloheptanone (5b), (7.8 g, 0.14 mol) of NH4Cl,
14.5 ml of methanol and 18.4 ml of water were added followed by the addition of a
solution of (9.5 g, 0.14 mol) KCN in 18.5 ml water. After the exothermic reaction
had subsided, the reaction mixture was stirred for 24 h at room temperature then
refluxed for 2 h.The organic layer was separated and the aqueous phase was
extracted with CH2Cl2 (3 9 50 ml). The combined organic layers were dried over
anhydrous Na2SO4 and rotavaped to afford 14 g (78 %) of 5a and 14.5 g (75 %) of
5b as crude yellow oils, which were subjected to acid base purification to achieve 5a
and 5b as pure colorless oils.
1-Aminocyclohexanecarbonitrile (5a)
M.p. of hydrochloride salt 188 °C (Lit. [22]. M.p. 183–185 °C); IR (KBr, cm-1):
3366–3297 (NH2) and 2222 (CN); yield 61 %.
1-Aminocycloheptanecarbonitrile (5b)
M.p. of hydrochloride salt 158 °C (Lit. [23]. M.p. 160–161 °C); yield 56 %.
General procedure for synthesis of 1-aminocycloalkanecarboxamides 6a and 6b
1-Aminocycloalkanecarbonitriles 5a (15.5 g, 0.125 mol) or 5b (17.2 g, 0.125 mol)
was added dropwise to 20 ml of cold conc. sulphuric acid and stirred at room
temperature for 48 h. The reaction mixture was poured over crushed ice and
rendered alkaline with 50 % NaOH, then extracted with ethyl acetate (3 9 50 ml),
dried over anhydrous Na2SO4, evaporated under reduced pressure, and crystallized
from petroleum–ether (40–60):ethyl acetate to give 6a and 6b as white crystals.
1-Aminocyclohexanecarboxamide (6a)
M.p. 98 °C (Lit. [24]. M.p. 105–106 °C); IR (KBr, cm-1): 3433–3395 (NH2) and 1669
1
(C=O, amide); MS (EI) m/z (%): 143 (28) (M? ?1), 99 (100); H NMR (CDCl3):
1.36–1.91 (m,10H, cyclohexane proton), 6.08, 7.49 (br s, 2H, exchangeable protons);
123

M. N. Aboul-Enein et al.
13C NMR (CDCl3): 21.27 (2CH2- cyclohexane); 25.29 (CH2- cyclohexane); 34.71
(2CH2–cyclohexane); 57.3 (C–cyclohexane); 181.68 (C=O,amide); yield 45 %.
1-Aminocycloheptanecarboxamide (6b)
1
M.p. 78–80 °C; IR (KBr, cm-1): 3431–3346 (NH2), 1675 (C=O, amide); H NMR
(CDCl3): 1.43–2.12 (m, 12H, cycloheptane proton), 5.93, 7.36 (br s, 2H,
exchangeable protons); 13C NMR : 22.91 (2CH2–cycloheptane); 29.67 (2CH2-
cycloheptane); 40 (2CH2–cycloheptane); 60.36 (C–cycloheptane); 181.68
(C=O,amide); yield 46 %.
Synthesis of spiro(imidazolidine-4,10-indane)2,5-dione (8)
A mixture of (2.64 g, 0.02 mol) 1-indanone (7), (9.1 g, 0.08 mol) (NH4)2CO3 and
(2.6 g, 0.04 mol) KCN in 50 ml 50 % ethanol was heated at 58–60 °C for 24 h.
After cooling, the solution was concentrated to approximately two-thirds of the
initial volume. The precipitate was filtered off, washed with cold water, and
crystallized from ethanol to give 8 as yellow crystals 2.8 g (69 %) yield, m.p
238–240 °C (Lit. [25]. M.p. 240 °C).
Synthesis of 1-amino-2,3-dihydro-1H-indene-1-carboxylic acid (9)
A mixture of 19.4 g (0.096 mol) of 8 and 53 g (0.17 mol) of Ba(OH)2 in 325 ml
water was refluxed for 72 h under vigorous stirring. After cooling, the precipitated
barium carbonate was filtered off. To the filtrate, 12 g (0.125 mol) of (NH4)2CO3
were added to precipitate all the barium cation as barium carbonate, which was
filtered off and washed several times with water. The combined aqueous filtrate and
washings were concentrated by distillation under normal pressure until the
precipitation of acid 9 started, then was allowed to cool. The precipitated acid 9
was filtered and crystallized from ethanol to give 11.5 g (68 %) yield of 9 as white
crystals, m.p. 245 °C (Lit. [26]. M.p. 247 °C).
Synthesis of 1-(tert-butoxycarbonylamino)-2,3-dihydro-1H-indene-1-carboxylic
acid (10)
To a solution of 40 ml 3.8 M NaOH was added (11.6 g, 0.066 mol) of 9 and 50 ml
tetrahydrofuran. The reaction mixture was cooled to 0–5 °C, and 14.5 g (0.066 mol)
of di-tert-butyl dicarbonate (BOC)2O was added portionwise. The reaction mixture
was stirred at room temperature for 15 h. The pH was adjusted to 3 with 2 M HCl,
extracted with CH2Cl2, dried over anhydrous Na2SO4, and evaporated under
reduced pressure to give 12.8 g (70 %) yield of 10 as white solid, m.p 164 °C; IR
(KBr, cm-1): 3317 (OH and NH), 1721 (C=O, acid), 1644 (C=O, amide); MS (EI)
1
m/z (%): 277(0.1) (M?), 232(60), 176 (100); H-NMR (DMSO-d6): 1.33(s, 9H,
OC(CH3)3), 2.14–2.89(m, 4H, aliphatic protons), 7.14–7.38(m, 4H, Har); 13C NMR
(DMSO-d6): 28.72 ((CH3)3); 30.67 (CH2–aliphatic); 39.53 (CH2–aliphatic); 69.08
(C–aliphatic); 78.72 (OC(CH3)3); 125 (CHar); 125.47(CHar); 126.71 (CHar); 128.89
123

Design and synthesis of certain substituted cycloalkanecarboxamides
(CHar); 142.77 (Car); 144.32 (Car); 155.9 (C=O, BOC), 174.39(C=O, acid); Anal.
Calcd for C15H19NO4 : C, 64.97; H, 6.91; N, 5.05. Found C, 65.07; H, 6.91; N, 5.16.
Synthesis of tert-Butyl 1-carbamoyl-2,3-dihydro-1H-inden-1-ylcarbamate (11)
To a solution of (14.4 g, 0.052 mol) of 10 in 200 ml CHCl3 was added slowly a
solution of N,N0-dicyclohexylcarbodiimide (DCC) (10.8 g, 0.052 mol) in 20 ml
CHCl3 at 10–15 °C. The reaction mixture was stirred at room temperature for 1 h.
To this mixture, NH4HCO3 (41.2 g, 0.522 mol) was added and stirred for 12 h. The
mixture was filtered, the residue was washed with CHCl3, and the filtrate was
evaporated under reduced pressure to give a white solid. This solid was purified by
filtration through column chromatography using silica gel as stationary phase and
ethyl acetate as mobile phase to give 9.5 g (66 %) of 11 as a white solid, m.p
152–154 °C; IR (KBr, cm-1): 3428, 3309 (NH2 and NH), 1688 (C=O, amide), 1652
(C=O, amide); MS (ESI) m/z : 276 (M?); 1H NMR (DMSO-d6): 1.34(s, 9H,
OC(CH3)3), 2.22–2.88(m, 4H, aliphatic protons), 7.16–7.39(m, 4H, Har); 13C NMR
(DMSO-d6): 28.63 ((CH3)3); 30.78 (CH2–aliphatic); 39.67 (CH2–aliphatic);
69.09(C–aliphatic); 78.87 (OC(CH3)3); 125.08 (CHar); 125.67(CHar); 126.98
(CHar); 128.98 (CHar); 142.89 (Car); 144.52 (Car); 155.87 (C=O, BOC),
175.22(C=O, acid); Anal. Calcd for C15H20N2O3: C, 65.20; H, 7.30; N, 10.14.
Found C, 65.60; H, 7.30; N, 10.30.
Synthesis of -1-amino-2,3-dihydro-1H-indene-1-carboxamide (12)
To a solution of (14.9 g,0.054 mol) 11 in 80 ml CH2Cl2 was passed hydrogen
chloride gas at 10 °C for 2 h. The hydrochloride salt was filtered off, basified with
10 % Na2CO3, extracted with CH2Cl2, dried over anhydrous Na2SO4, and rotavaped
to give 6.5 g (68 %) yield of 12 as a white solid, m.p 126–130 °C (Lit. [27]. m.p.
121–122 °C); IR (KBr, cm-1): 3353, 3289 (NH2), 1678 (C=O, amide); MS (EI) m/z
1
(%): 176 (0.12) (M?), 132 (100); H NMR (CDCl3): 1.98–2.02(m, 1H, aliphatic),
2.85–2.89 (m, 1H, aliphatic), 3.04–3.13 (m, 2H, aliphatic), 7.20–7.32(m, 4H,
aromatic); 13C NMR (CDCl3): 30.81 (CH2–aliphatic); 41.73 (CH2–aliphatic); 69.09
(C–aliphatic); 122.9 (CHar); 125.2 (CHar); 127.16 (CHar); 128.61 (CHar); 143.84
(Car); 146.08 (Car); 178.46 (C=O, amide); Anal. Calcd for C10H12N2O : C, 68.16; H,
6.86; N, 15.9. Found C, 68.3; H, 6.8; N, 16.1.
General procedure for synthesis of 1-[4-(3- substituted benzyloxy)benzylamino]
cycloalkanecarboxamides (13a–e, 14a–e) and 1-[4-(3- substituted
benzyloxy)benzylamino]-2,3-dihydro-1H-indene-1-carboxamides (15a–e)
To a solution of 0.0472 mol of the prepared amine (6a, 6b, or 12) in 75 ml DCM
0.046 mol of the appropriate aldehyde (3a–e) and 8 g (0.066 mol) of MgSO4 was
added. The reaction mixture was stirred at room temperature for 16 h, then filtered.
The solvent was removed under vacuum and the residue was reduced with NaBH4
(3.05 g, 0.08 mol) in 120 ml methanol at 0 °C for 8 h. The solvent was removed
under vacuum, the residue was dissolved in water, and extracted with ethyl acetate.
123

M. N. Aboul-Enein et al.
The organic layer was dried over anhydrous Na2SO4, evaporated under reduced
pressure to give 13a–e, 14a–e, and 15a–e as crude viscous oils. These oils were
purified by column chromatography using silica gel as stationary phase and
petroleum–ether (40–60):ethyl acetate (6:4) as mobile phase to achieve the desired
compounds in a pure form which were crystallized from mixture of petroleum–ether
(40–60):ethyl acetate to give the target compounds as pure crystals.
1-[4-(Benzyloxy)benzylamino]cyclohexanecarboxamide (13a)
White crystals; m.p. 126–128 °C; yield 40 %; IR (KBr, cm-1): 3355(NH), 1662
(C=O, amide); H NMR (CDCl3): 1.27–1.69 (m, 10H, cyclohexyl), 3.48 (s, 2H,
1
CH2–N), 4.99 (s, 2H, CH2–O), 6.87–6.89 (d, J = 8.4 Hz, 2H, Har.), 7.17–7.19 (d,
J = 8.4 Hz, 2H, Har.), 7.24–7.36 (m, 5H, Har.); 13C NMR (CDCl3): 21.43 (2CH2–
cyclohexane), 25.14 (CH2–cyclohexane), 31.79 (2CH2–cyclohexane), 46.46(CH2–
NH), 60.5( C–cyclohexane), 70.04 (CH2–O), 114.96, 127.41, 127.96, 128.57
(CHar.), 129.09, 136.93, 157.99 (Car.), 180.3 (C=O); MS (EI) m/z (%): 339(57.42)
(M??1), 294.2 (67.09), 197 (76.54), 90.9 (100); Anal. Calcd for C21H26N2O2 : C,
74.52; H, 7.74; N, 8.28. Found C, 74.16; H, 8.1; N, 8.03.
1-[4-(3-Fluorobenzyloxy)benzylamino] cyclohexanecarboxamide (13b)
White crystals; m.p. 96–98 °C; yield 45 %; IR (KBr, cm-1): 3412 (NH), 1661
1
(C=O, amide); H-NMR (CDCl3): 1.33–1.86 (m, 10H, cyclohexyl), 3.51 (s, 2H,
CH2–N), 5.02 (s, 2H, CH2–O), 6.92–7.36 (m, 8H, Har.); 13C NMR (CDCl3): 21.55
(2CH2–cyclohexane), 25.28 (CH2–cyclohexane), 31.95 (2CH2–cyclohexane), 46.56
(CH2–NH), 61.3(C–cyclohexane), 69.31 (CH2–O), 114.17 (d, JC-F = 21.45, CHar.),
114.79 (d, JC-F = 21.46, CHar.), 115.05 (2CHar.), 122.73 (CHar.), 129.26 (2CHar.),
130.18 (d, JC-F = 8.35, CHar.), 132.94 (Car), 139.71 (d, JC-F = 7.15, Car.), 157.78
(Car), 162.11 (d, JC-F = 244.4, Car.), 180.22 (C=O); MS (EI) m/z (%): 357.05(0.03)
(M??1), 312 (24.17), 230 (16.1), 215 (36.83), 109 (100); Anal. Calcd for
C21H25FN2O2 : C, 70.76; H, 7.07; N, 7.86. Found C, 70.9; H, 7.0; N, 7.9.
1-[4-(3-Chlorobenzyloxy)benzylamino] cyclohexanecarboxamide (13c)
White crystals; m.p. 122–124 °C; yield 42 %; IR (KBr, cm-1): 3412 (NH), 1661
(C=O, amide); H NMR (CDCl3): 1.32–1.89 (m, 10H, cyclohexyl), 3.54 (s, 2H,
1
CH2–N), 5.03 (s, 2H, CH2–O), 6.92–6.94 (d, J = 8.4 Hz, 2H, Har.), 7.25–7.26 (d,
J = 8.4 Hz, 2H, Har.), 7.30–7.53 (m, 4H, Har.); 13C NMR (CDCl3): 21.58 (2CH2–
cyclohexane), 25.35 (CH2–cyclohexane), 31.98 (2CH2–cyclohexane), 46.58 (CH2–
NH), 61.24(C–cyclohexane), 69.25 (CH2–O), 115.02 (2CHar.), 125.42 (CHar.),
127.46 (CHar.), 128.14 (CHar.), 129.32 (2CHar.), 129.97 (CHar.), 130.05 (Car.),
133.05 (Car.), 139.25 (Car.), 157.45 (Car.), 180.52 (C=O); MS (EI) m/z (%): 373.3
(7.24) (M??1), 371.3 (4.01) (M?-1), 330 (23.94), 328 (74.92), 248 (10.7), 246
(31.98), 233 (31.93), 231 (99.02), 127 (35.04), 125 (100); Anal. Calcd for
C20H24ClN2O2: C, 67.64; H, 6.76; N, 7.51. Found C, 67.80; H, 6.70; N, 7.50.
123

Design and synthesis of certain substituted cycloalkanecarboxamides
1-[4-(3-Methoxybenzyloxy)benzylamino] cyclohexanecarboxamide (13d)
White crystals; m.p. 118–120 °C; yield 39 %; IR (KBr, cm-1): 3454 (NH), 1678
(C=O, amide); H-NMR (CDCl3): 1.36–1.89 (m, 10H, cyclohexyl), 3.53 (s, 2H,
1
CH2–N), 3.81 (s, 3H, OCH3), 5.03 (s, 2H, CH2–O), 6.93–6.95(d, J = 8.4 Hz, 2H,
Har), 6.98–7.00 (m, 2H, Har.), 7.24–7.25 (d, J = 8.4 Hz, 2H, Har), 7.29–7.34 (m, 3H,
Har.); 13C NMR (CDCl3): 21.60 (2CH2–cyclohexane), 25.39 (CH2–cyclohexane),
32.01 (2CH2–cyclohexane), 46.61 (CH2–NH), 55.31 (OCH3), 61.23 (C–cyclohex-
ane), 69.98 (CH2-O), 113.01 (CHar.), 113.51 (CHar.), 115.05 (2CHar), 119.69 (CHar),
129.30 (2CHar.), 129.75 (CHar.), 132.79 (Car.), 138.76 (Car.), 158.03(Car.), 159.93
(Car.), 180.58 (C=O); MS (ESI) m/z : 366 (M?-2); Anal. Calcd for C22H28N2O3 : C,
71.71; H, 7.66; N, 7.60. Found C, 71.73; H, 8.00; N, 7.50.
1-[4-(3-Nitrobenzyloxy)benzylamino] cyclohexanecarboxamide (13e)
Yellow crystals; m.p. 130–132 °C; yield 42 %; IR (KBr, cm-1): 3408 (NH), 1690
(C=O, amide); H NMR (CDCl3): 1.32–1.89 (m, 10H, cyclohexyl), 3.55 (s, 2H,
1
CH2–N), 5.12 (s, 2H, CH2–O), 6.93–6.95(d, J = 8.4 Hz, 2H, Har), 7.26–7.27 (d,
J = 8.4 Hz, 2H, Har.), 7.56–8.28 (m, 4H, Har.); 13C NMR (CDCl3): 21.56 (2CH2–
cyclohexane), 25.27 (CH2–cyclohexane), 31.99 (2CH2–cyclohexane), 46.54 (CH2–
NH), 61.33 (C–cyclohexane), 68.83 (CH2–O), 115.06 (2CHar), 122.20 (CHar),
123.00 (CHar), 129.32 (2CHar.), 129.67 (CHar.), 133.15 (CHar.), 133.39 (Car.),
139.34 (Car.), 148.57 (Car.), 157.44 (Car.), 179.95 (C=O); MS (ESI) m/z : 383 (M?);
Anal. Calcd for C21H25N3O4 : C, 65.78; H, 6.57; N, 10.96. Found C, 66.08; H, 6.10;
N, 11.
1-[4-(Benzyloxy)benzylamino]cycloheptanecarboxamide (14a)
White crystals; m.p. 96–98 °C; yield 44 %; IR (KBr, cm-1): 3413 (NH), 1664
(C=O, amide); H NMR (CDCl3): 1.60–2.12 (m, 12H, cycloheptyl), 3.55 (s, 2H,
1
CH2–N), 5.05 (s, 2H, CH2–O), 6.93–6.95 (d, J = 8.4 Hz, 2H, Har.), 7.23–7.24 (d,
J = 8.4 Hz, 2H, Har.), 7.30–7.43 (m, 5H, Har.); 13C NMR (CDCl3): 22.63 (2CH2–
cycloheptane), 29.82 (2CH2–cycloheptane), 36.36 (2CH2–cycloheptane),
47.06(CH2–NH), 64.89(C–cycloheptane), 70.15(CH2–O), 115.05, 127.53, 128.07,
128.70, 129.17 (CHar.), 132.72, 137.08, 158.05 (Car.), 180.60(C=O); MS (ESI) m/z :
353 (M? ?1); Anal. Calcd for C22H28N2O2 : C, 74.97; H, 8.01; N, 7.95. Found C,
74.70; H, 8.00; N, 8.00.
1-[4-(3-Fluorobenzyloxy)benzylamino] cycloheptanecarboxamide (14b)
White crystals; m.p. 104–106 °C; yield 47 %; IR (KBr, cm-1): 3412 (NH), 1660
(C=O, amide); H NMR (CDCl3): 1.59–2.11 (m, 12H, cycloheptyl), 3.55 (s, 2H,
1
CH2–N), 5.04 (s, 2H, CH2–O), 6.91–7.35 (m, 8H, Har.); 13C NMR (CDCl3): 22.65
(2CH2–cycloheptane), 29.80 (2CH2–cycloheptane), 36.38 (2CH2–cycloheptane),
47.05 (CH2–NH), 64.90 (C–cycloheptane), 69.34 (CH2–O), 114.16 (d, JC-F
21.46, CHar.), 114.77 (d, JC-F = 21.46, CHar.), 115.05 (2CHar.), 122.74 (CHar.),
=
123

M. N. Aboul-Enein et al.
129.20 (2CHar.),130.16 (d, JC-F = 8.35, CHar.), 133.08 (Car), 139.71 (d, JC-F = 7.15,
Car.), 157.76 (Car), 162.12 (d, JC-F = 244.4, Car.), 180.64 (C=O); MS (EI) m/z (%):
371(0.13) (M??1), 326 (100), 230 (9.47), 215 (27.62); Anal. Calcd for
C22H27FN2O2 : C, 71.33; H, 7.35; N, 7.56. Found C, 71.00; H, 7.00; N, 7.40.
1-[4-(3-Chlorobenzyloxy)benzylamino] cycloheptanecarboxamide (14c)
White crystals; m.p. 98–100 °C; yield 38 %; IR (KBr, cm-1): 3407 (NH), 1666
1
(C=O, amide); H NMR (CDCl3): 1.48–2.10 (m, 12H, cycloheptyl), 3.55 (s, 2H,
CH2–N), 5.02 (s, 2H, CH2–O), 6.91–7.42 (m, 8H, Har.); 13C NMR (CDCl3): 22.63
(2CH2–cycloheptane), 29.83 (2CH2–cycloheptane), 36.36 (2CH2–cycloheptane),
47.04 (CH2–NH), 64.86(C–cycloheptane), 69.27 (CH2–O), 115.00 (2CHar.), 125.41
(CHar.), 127.47 (CHar.), 128.17 (CHar.), 129.23 (2CHar.), 129.97 (CHar.), 133.06
(Car.), 134.60 (Car.), 139.19(Car.), 157.70 (Car.), 180.76 (C=O); MS (EI) m/z (%):
386 (0.09) (M?), 342 (100), 246 (28.67), 231 (80.99), 125 (75.67); Anal. Calcd for
C22H27ClN2O2: C, 68.29; H, 7.03; N, 7.24. Found C, 68.30; H, 7.00; N, 7.30.
1-[4-(3-Methoxybenzyloxy)benzylamino] cycloheptanecarboxamide (14d)
White crystals; m.p. 98–100 °C; yield 40 %; IR (KBr, cm-1): 3408 (NH), 1690
1
(C=O, amide); H NMR (CDCl3): 1.58–2.10 (m, 12H, cyclohexyl), 3.54 (s, 2H,
CH2–N), 3.80 (s, 3H, OCH3), 5.03 (s, 2H, CH2–O), 6.92–7.28 (m, 8H, Har.); 13C
NMR (CDCl3): 22.62 (2CH2–cycloheptane), 29.80 (2CH2–cycloheptane), 36.34
(2CH2–cycloheptane), 47.06 (CH2–NH), 55.34 (OCH3), 64.87 (C–cyclohexane),
69.65 (CH2–O), 112.99 (CHar.), 113.52 (CHar.), 115.04 (2CHar), 119.68 (CHar),
127.82 (CHar), 129.19 (2CHar.), 129.75 (Car.), 138.70 (Car.), 158.00 (Car.), 159.92
(Car.), 180.58 (C=O); MS (EI) m/z (%): 381 (0.14) (M? ?1), 338 (16.84), 227
(14.35), 121 (100); Anal. Calcd for C23H30N2O3 : C, 72.22; H, 7.91; N, 7.32. Found
C, 72.60; H, 7.90; N, 7.40.
1-[4-(3-Nitrobenzyloxy)benzylamino] cycloheptanecarboxamide (14e)
Yellow crystals; m.p. 112–114 °C; yield 44 %; IR (KBr, cm-1): 3451 (NH), 1685
1
(C=O, amide); H NMR (CDCl3): 1.57–2.12 (m, 12H, cycloheptyl), 3.52 (s, 2H,
CH2–N), 5.10 (s, 2H, CH2–O), 6.92–8.29 (m, 8H, Har.); 13C NMR (CDCl3): 22.63
(2CH2–cycloheptane), 29.82 (2CH2–cycloheptane), 36.37 (2CH2–cycloheptane),
47.02 (CH2–NH), 64.88 (C–cycloheptane), 68.79 (CH2–O), 114.99 (2CHar), 122.19
(CHar), 122.99 (CHar), 129.31 (2CHar.), 129.68 (CHar.), 133.21 (CHar.), 133.46
(Car.), 139.35 (Car.), 148.52 (Car.), 157.37 (Car.), 179.95 (C=O); MS (ESI) m/z : 396
(M? -1); Anal. Calcd for C22H27N3O4 : C, 66.48; H, 6.85; N, 10.57. Found C,
66.10; H, 7.20; N, 10.50.
1-[4-(Benzyloxy)benzylamino]-2,3-dihydro-1H-indene-1-carboxamide (15a)
Colorless oil; m.p. of hydrochloride salt 202–204 °C; yield 40 %,; IR (KBr, cm-1):
3409 (NH), 1674 (C=O, amide); H NMR (CDCl3): 2.46–3.23 (m, 4H, aliphatic
1
123

Design and synthesis of certain substituted cycloalkanecarboxamides
protons), 3.80 (s, 2H, CH2–N), 5.09 (s, 2H, CH2–O), 6.97–7.72 (m, 13H, Har.); 13C
NMR (CDCl3): 33.12 (CH2–aliphatic), 39.49 (CH2–aliphatic), 47.29 (CH2–NH),
69.66 (C–aliphatic), 75.34 (CH2–O), 115.15 (2CHar), 115.63 (CHar), 125.82 (CHar),
126.05 (CHar), 127.45 (CHar.), 128.13 (2CHar.), 128.38 (CHar.), 128.98 (2CHar.),
130.72 (Car.), 132.54 (CHar.), 137.45 (Car.), 146.14 (Car.), 159.16 (Car.), 171.65
(C=O); MS (EI) m/z (%): 373 (0.05) (M??1), 328 (42), 197 (26.68), 91 (100);
Anal. Calcd for C24H25ClN2O2 : C, 70.49; H, 6.16; N, 6.85. Found C, 70.40; H,
6.10; N, 7.00.
1-[4-(3-Fluorobenzyloxy)benzylamino]-2,3-dihydro-1H-indene-1-carboxamide
(15b)
White crystals; m.p. 130–132 °C; yield 44 %; IR (KBr, cm-1): 3460 (NH), 1672
(C=O, amide); H NMR (CDCl3): 2.19–2.24 (m, 1H, aliphatic proton), 2.87–2.90
1
(m, 1H, aliphatic proton), 3.01–3.06 (m, 1H, aliphatic proton), 3.17–3.23 (m, 1H,
aliphatic proton), 3.56–3.58 (d, J = 12.2, 1H, CH–N), 3.67–3.69 (d, J = 12.2, 1H,
CH–N), 5.04 (s, 2H, CH2–O), 6.90–7.26 (m, 12H, Har.); 13C NMR (CDCl3): 31.33
(CH2–aliphatic), 35.73 (CH2–aliphatic), 48.02 (CH2–NH), 69.28 (C–aliphatic), 74.1
(CH2–O), 114.20 (d, JC-F = 22.6, CHar.), 114.82 (CHar.), 114.96 (2CHar.), 122.77
(CHar.), 124.04 (CHar.), 125.25 (CHar.), 126.64 (CHar.), 128.73 (CHar.), 129.34
(2CHar.), 130.20 (d, JC-F = 8.3, CHar.), 132.69 (Car), 139.69 (d, JC-F = 7.1, Car.),
143.77 (Car), 144.31 (Car), 157.75 (Car), 162.10 (d, JC-F = 244.4, Car.), 177.56
(C=O); MS (ESI) m/z : 390 (M?); Anal. Calcd for C24H23FN2O2 : C, 73.83; H, 5.94;
N, 7.17. Found C, 73.90; H, 5.90; N, 7.20.
1-[4-(3-Chlorobenzyloxy)benzylamino]-2,3-dihydro-1H-indene-1-carboxamide
(15c)
White crystals; m.p. 92–94 °C; yield 47 %; IR (KBr, cm-1): 3311 (NH), 1679
(C=O, amide); H NMR (CDCl3): 2.19–2.23 (m, 1H, aliphatic proton), 2.88–2.90
1
(m, 1H, aliphatic proton), 3.01–3.06 (m, 1H, aliphatic proton), 3.17–3.22 (m, 1H,
aliphatic proton), 3.56–3.59 (d, J = 12.2, 1H, CH–N), 3.67–3.69 (d, J = 12.2, 1H,
CH–N), 5.01 (s, 2H, CH2–O), 6.90–7.29 (m, 12H, Har.); 13C NMR (CDCl3): 31.34
(CH2–aliphatic), 35.73 (CH2–aliphatic), 48.04 (CH2–NH), 69.27 (C–aliphatic),
74.23 (CH2–O), 114.97 (2CHar.), 124.05 (CHar.), 125.25 (CHar.), 125.42 (CHar.),
126.65 (CHar.), 127.48 (CHar.), 128.18 (CHar.), 128.72 (CHar.), 129.36 (2CHar.),
129.97 (CHar.), 132.76 (Car), 134.60 (Car.), 139.21 (Car), 143.81 (Car), 144.31 (Car),
157.74 (Car), 177.60 (C=O); MS (ESI) m/z : 407 (M?); Anal. Calcd for
C24H24Cl2N2O2 : C, 65.02; H, 5.46; N, 6.32. Found C, 64.90; H, 5.30; N, 6.10.
1-[4-(3-Methoxybenzyloxy)benzylamino]-2,3-dihydro-1H-indene-1-carboxamide
(15d)
White crystals; m.p. 100–102 °C; yield 42 %; IR (KBr, cm-1): 3409 (NH), 1674
(C=O, amide); H NMR (CDCl3): 2.23–3.22 (m, 4H, aliphatic protons), 3.56–3.58
(d, J = 10, 1H, CH–N), 3.67–3.69 (d, J = 10, 1H, CH–N), 3.95 (s, 3H, OCH3), 5.02
1
123

M. N. Aboul-Enein et al.
(s, 2H, CH2–O), 6.87–7.38 (m, 12H, Har.); 13C-NMR (CDCl3): 31.35 (CH2-
aliphatic), 35.73 (CH2–aliphatic), 48.07 (CH2–NH), 55.36 (OCH3), 70.00 (C–
aliphatic), 74.21 (CH2–O), 112.99 (CHar.), 113.55 (CHar.), 114.99 (2CHar.), 119.71
(CHar.), 124.05 (CHar.), 125.25 (CHar.), 126.64 (CHar.), 128.72 (CHar.), 129.32
(2CHar.), 129.76 (CHar.), 132.44 (Car), 138.69 (Car.), 143.81 (Car), 144.31 (Car),
158.02 (Car), 159.92 (Car), 177.61 (C=O). MS (ESI) m/z : 401 (M? -1); Anal.
Calcd for C25H26N2O3 : C, 74.60; H, 6.51; N, 6.96. Found C, 74.70; H, 6.50; N,
7.00.
1-[4-(3-Nitrobenzyloxy)benzylamino]-2,3-dihydro-1H-indene-1-carboxamide (15e)
Colorless oil; m.p. of hydrochloride salt 200–202 °C; yield 41 %; IR (KBr, cm-1):
3420 (NH), 1677 (C=O, amide); H-NMR (CDCl3): 2.19–2.21 (m, 1H, aliphatic
1
proton), 2.86–2.88 (m, 1H, aliphatic proton), 3.03–3.04 (m, 1H, aliphatic proton),
3.16–3.18 (m, 1H, aliphatic proton), 3.55–3.58 (d, J = 15, 1H, CH–N), 3.65–3.68
(d, J = 15, 1H, CH–N), 5.07 (s, 2H, CH2–O), 6.91–8.28 (m, 12H, Har.); 13C NMR
(CDCl3): 31.34 (CH2–aliphatic), 35.70 (CH2–aliphatic), 48.00 (CH2–NH), 68.72
(C–aliphatic), 74.20 (CH2–O), 114.94 (2CHar.), 122.19 (CHar.), 122.98 (CHar.),
124.10 (CHar.), 125.24 (CHar.), 126.65 (CHar.), 128.72 (CHar.), 129.48 (2CHar.),
129.71 (CHar.), 133.11 (Car.), 133.28 (Car.), 139.36 (Car.), 143.81 (Car), 144.32 (Car),
148.47 (Car), 157.40 (Car), 177.82 (C=O); MS (EI) m/z (%): 418 (1.15) (M??1),
374 (76.83), 240 (99.27), 136 (100).
Biological evaluation
All the newly synthesized compounds, 13a–e, 14a–e, and 15a–e, were evaluated for
their initial anticonvulsant activity following the protocol given by the epilepsy
section of the National Institute of Neurological Disorders and Stroke (NINIDS)
using the standard protocol adopted by the Antiepileptic Drug Development (ADD)
program [28]. Those include the s.c. PTZ screen, which is used to identify
compounds that elevate seizure threshold and protect against generalized seizures of
the petit mal (absence) type, and the MES screen, which is indicative of the ability
of the test compounds to prevent seizure spread and their capability of protection
against generalized tonic–clonic seizures [17]. The selected compounds were
subjected to quantitative estimation (median effective dose against seizures, ED50)
and to the minimal motor impairment (neurotoxicity) test. The anticonvulsant
activity of the target compounds, 13a–e, 14a–e and 15a–e, was determined on adult
male Swiss albino mice weighing 18–25 g. Mice were purchased from the Animal
House Colony of the National Research Centre, Cairo, Egypt, and were housed
under standardized conditions (room temperature 23 2 °C; relative humidity
55 5 %; 12 h light/dark cycle). They were allowed free access to tap water and
standard mice chow throughout the whole experimental period. All animal
procedures were performed after permission the Ethics Committee of the National
Research Centre and in accordance with the recommendations for the proper care
and use of laboratory animals ‘‘Canadian Council on Animal Care Guidelines,
1984’’. After 7 days of adaptation to laboratory conditions, the animals were
123

Design and synthesis of certain substituted cycloalkanecarboxamides
randomly assigned to control, reference and tested experimental groups consisting
of 6–8 mice each. All the test compounds were suspended in 7 % Tween-80 saline
solutions.
Chemicals and drugs
Tween-80 (Sigma, St. Louis, MO, USA), Pentylenetetrazole (Sigma), Diphenylhy-
dantoin sodium (Nasr, Egypt), Phenobarbital (Memphis, Egypt), Gabapentin (Parke
Davis, USA) and Safinamide was synthesized according to the reported procedure
[29].
Maximal electroshock seizure (MES) assay [30]
In this test, the electroconvulsions were produced 30 min after intraperitoneal
injection of the tested compounds by a current (fixed current intensity of 25 mA,
0.2 s stimulus duration) delivered via ear clip electrodes by a Rodent Shocker
Generator (constant-current stimulator Type 221; Hugo Sachs Elektronik, Freiburg,
Germany). The criterion for the occurrence of seizure activity was the tonic hind
limb extension (i.e. the hind limbs of animals outstretched 180° to the plane of the
body axis).
Subcutaneous pentylenetetrazole (s.c. PTZ) screen
Aqueous solution of PTZ at a dose level of 85 mg/kg [31] was administered
subcutaneously in a loose fold of skin on the back of the mice neck. This dose
induced tonic-extensor convulsions in at least 97 % of controls. The test compounds
were administered intraperitoneally to mice 30 min before PTZ administration.
Mice were placed in single cages and observed for 30 min for the loss of righting
reflexes lasting at least 3 s after PTZ treatment [32], as the absence of loss of
righting reflexes was chosen as an index for protective effect.
Neurotoxicity
Grip strength [33]
In a preliminary experiment, the animals were tested for their normal reactivity. The
animals were exposed to a horizontal thin metallic wire suspended about 30 cm into
the air which they immediately grasped with the forepaws. The mouse was released
to hang on with its forelimbs. Normal animals are able to catch the thin wire with
the hind limbs and to climb up within 5 s. Only animals that fulfilled this criterion
were included in the experiment. Eight mice were used in the control and
experimental groups. The animals were tested directly after subcutaneous admin-
istration of the tested compounds and every 15 min for 2 h. Animals which were not
able to catch the thin wire with the hind limbs within 5 s or fell off from the wire
were considered to be impaired.
123

M. N. Aboul-Enein et al.
The wire mesh grasping test ‘‘the inverted screen test’’ [34]
Animals were tested for their normal reactivity. Each mouse was placed on the
center of a wire mesh (20–30 cm). After a few seconds, the mesh was turned 180°;
normal animals are able to climb the top of the wire mesh within 60 s after
inversion. Only animals that fulfilled this criterion were included in the experiment.
Eight mice were used in both the control and experimental groups. The animals
were tested directly after subcutaneous administration of the tested compounds and
every 15 min for 2 h. The animal that either fell off the inverted mesh or did not
climb the top of the screen within 60 s was considered to be impaired.
Molecular modeling
Pharmacophore model and molecular docking into the prepared enzyme were
performed using the Discovery Studio 2.5 software (Accelrys, San Diego, CA,
USA).
Generation of pharmacophore model for sodium channel blockers
The pharmacophore modeling method has been widely used in lead discovery and
optimization as a key tool of computer-aided drug design. A common feature
pharmacophore generation protocol was used to develop the pharmacophore model
for sodium channel blockers. The feature-based pharmacophore model was mapped
O
H2
C
H2
C
H
N
O
C
H
C NH2
CH2OH
H2
C
H2
C
H
N
I
O
C
H
C
O
NHCH3
F
F
VIII
O
H2 H2 H2
H2
C
H
C
C
C
O
N
C
H
C
NH2
O
H2 H2 H2 H2
H2
C
H
N
IX
C
C
C
C
O
C
H
C NH2
X
O
H2 H2 H2 H2
H2
C
H
N
H2C
C
C
C
C
O
C
H
C NH2
O
H2
C
H
XI
H3CO
N C C NH2
H
XII
O
C
H2
C
H2
H
O
C
N
C
OH
H
H2
C
N
XIII
O
H
NHCH3
O
F
O
XIV
Fig. 1 Structure of training set compounds
123

Design and synthesis of certain substituted cycloalkanecarboxamides
from a set of highly selective ligands (compounds I, VIII–XIV) (Fig. 1). The
selection of the training set used in generation of pharmacophore model for sodium
channel blockers was based on (1) using highly potent and selective ligands, (2) the
in vivo pharmacological screening for most of the selected training set was carried
out by the same method used for testing the in vivo activity for our compounds
using the MES seizure test, and (3) the results of in vivo testing for most of this
training set were consistent with their in vitro inhibitory activity [12]. The set of
conformational models of each structure of the lead compounds was performed and
used to generate the common feature hypotheses, by which ten hypotheses were
generated [35].
Molecular docking studies of branched-chain aminotransferase inhibitors
and binding conformation
All dock runs were conducted using Discovery Studio 2.5 to investigate the detailed
intermolecular interactions between the ligand and the target protein. An automated
docking study was carried out using the crystal structure of aminotransferase
Table 1 Anticonvulsant activity of 13a–e, 14a–e, and 15a–e against s.c. PTZ-induced seizures and
docking score
Compd. no.
Dosea
(mmol/kg)
Max. %
protection (%)
ED50 (confidence limits)
mg/kg
Docking
score
mmol/kg
Phenobarbital
Gabapentin
Safinamide
13a
0.107
0.22
100
100
83
13.2 (5.8–15.9)
ND
0.0568
ND
ND
-86
-93
-117
-125
-112
-87
-110
-82
-89
-107
-87
-95
-70
-73
-108
-69
-90
0.057
0.0148
0.007
0.0134
0.027
0.013
0.07
21 (16.66–26.47)
2.25 (1.66–3.05)
2 (1.49–2.67)
2 (1.38–3.13)
5 (3.95–8.51)
2.25 (1.56–3.34)
18.5 (12.01–28.52)
7 (4.63–10.75)
5 (2.97–8.71)
20 (14.89–26.87)
1.7 (0.985–2.94)
ND
0.052
0.0066
0.0056
0.0053
0.0135
0.0058
0.052
0.0188
0.0129
0.0522
0.0042
ND
100
100
100
83
13b
13c
13d
13e
100
83
14a
14b
0.04
83
14c
0.052
0.065
0.0062
0.049
0.038
0.049
0.049
0.022
100
83
14d
14e
83
15a
67
15b
67
ND
ND
15c
83
10 (6.06–18.68)
ND
0.0245
ND
15d
67
15e
83
6.5 (4.18–12.06)
0.0143
ND Not determined
a
The smallest dose which gives the best % inhibition
123

M. N. Aboul-Enein et al.
Fig. 2 The proposed binding mode of compounds gabapentin (A), 13a (B), 13b (C), and 13c (D) inside
the active site of aminotransferase resulting from docking, respectively. The most important amino acids
are shown together with their respective numbers. Compound 13b forms four hydrogen bonds: two with
Thr260 and with Tyr153 and Thr333
123

Design and synthesis of certain substituted cycloalkanecarboxamides
enzyme (2COJ) complexed with its ligand (Gabapentin), with a resolution of
˚
1.95 A. The prepared protein was used in the determination of the important amino
acids in the predicted binding pocket [18]. Interactive docking using the CDOCKER
protocol was carried out for all the conformers of each compound of the test set
(13a–e, 14a–e, and 15a–e) to the selected active site. The performance of the
docking method was evaluated by re-docking crystal ligand with 0.013 RMSD value
[36]. Each docked compound was assigned a score according to its binding mode
onto the binding site after energy minimization using the prepared ligand protocol.
The observed binding energies and the corresponding binding mode of interaction
taking place between the ligand (test compound) and the receptor (active site of the
protein) are listed in Table 1 and Fig. 2, and in the supplementary material.
Results and discussion
Chemistry
The synthesis of the target compounds 13a–e, 14a–e, and 15a–e and their
intermediates is illustrated in Schemes 1, 2, 3, and 4. 4-(3-substituted benzyloxy)
benzaldehydes (3a–e) [19–21] were prepared using the Williamson ether synthesis
by coupling 3-substituted benzyl bromides 1a–e with 4-hydroxybenzaldehyde (2) in
the presence of anhydrous Na2CO3 in DMF at 60 °C for 12 h.
Cycloalkanones 4a and 4b were allowed to react via Strecker synthesis with
NH4Cl and KCN in aqueous methanol to afford the carbonitrile derivatives 5a [22]
and 5b [23] which were subjected to hydrolysis using concentrated sulphuric acid at
room temperature for 2 days to obtain the respective carboxamides 6a [24] and 6b
[37]. Spiro (imidazolidine-4,10-indane)2,5-dione (8) [25] was synthesized from
1-indanone (7) in presence of potassium cyanide and ammonium carbonate
according to the Bucherer–Bergs reaction followed by alkaline hydrolysis using
barium hydroxide for 3 days to get 1-amino-2,3-dihydro-1H-indene-1-carboxylic
acid (9) [26]. The amino acid 9 was converted to the corresponding N-BOC
derivative 10 using di-tert-butyl dicarbonate (BOC)2O in the presence of sodium
CHO
CH2Br
CHO
i
+
X
X
H2
C
O
OH
1a-e
2
3a-e
Compounds 3a-e X: a=H, b=F, c=Cl, d=OCH3, e=NO2
Scheme 1 Synthesis of 4-(3-substituted benzyloxy) benzaldehydes 3a–e. Reagents and conditions:
(i) Na2CO3, DMF, 60 °C, 12 h
123

M. N. Aboul-Enein et al.
O
O
NC
NH2
H2N
C
NH2
ii
i
(CH2)n
4a,b
(CH2)n
(CH2)n
6a,b
5a,b
n
4,5,6
a
b
3
4
Scheme 2 Synthesis of 1-aminocycloalkanecarboxamides 6a and 6b. Reagents and conditions: (i) KCN,
NH4Cl, Methanol, H2O; (ii) Conc.H2SO4, r.t., 48 h
O
C
O
C
O
HN
O
HO
H
N
HO
O
BOC
NH
ii
NH2
i
iii
10
8
9
7
iv
O
C
O
H2N
H
N
H2N
C
BOC
NH2
v
12
11
Scheme 3 Synthesis of 1-amino-2,3-dihydro-1H-indene-1-carboxamide (12). Reagents and conditions:
(i) KCN, (NH4)2CO3, 50 % Ethanol, 55 °C, 24 h; (ii) Ba(OH)2, H2O, reflux, 72 h; (iii) (Boc)2O, 3.8 M
NaOH, THF, r.t., 15 h; (iv) (a) DCC, DCM, 15 °C, 1 h, (b) NH4 HCO3, r.t., 12 h; (v) HCl gas, DCM
hydroxide in THF at room temperature for 15 h. Coupling of N-BOC acid 10 with
NH4HCO3 using N,N’-dicyclohexylcarbodiimide (DCC) as condensing agent in
chloroform at room temperature yielded the corresponding primary amide 11 which
was purified by filtration through column chromatography. Subsequent N-
deprotection of 11 using dry HCl gas in dichloromethane afforded the respective
amine 12. The target compounds 13a–e, 14a–e, and 15a–e were prepared by
condensation of 6a, 6b, and 12, respectively, with the appropriate 4-(3-substituted
benzyloxy) benzaldehyde 3a–e in dichloromethane in presence of anhydrous
MgSO4 as drying agent. The formed intermediates were reduced without separation
to achieve the target secondary amines using NaBH4 in methanol. The spectral data
of the synthesized compounds in the present investigation were in accordance with
their assigned structures.
123

Design and synthesis of certain substituted cycloalkanecarboxamides
CHO
X
O
C
H2
O
O
C
H2N
H2N
C
NH2
NH2
3a-e
(CH2)n
6a,b
12
i
i
O
C
H2
C
O
C
H
N
H2
C
H
N
H2C
O
NH2
H2C
O
NH2
(CH2)n
X
X
15a-e
13a-e, 14a-e
Scheme 4 Synthesis of the target compounds 13a–e, 14a–e, and 15a–e. Reagents and conditions:
(i) (a) MgSO4, CH2Cl2, r.t., 16 h (b) NaBH4, CH3OH, 0 °C, 8 h
Biological evaluation
The anticonvulsant activity against s.c. PTZ assay and the MES assay of the target
compounds 1-[4-(3-substituted benzyloxy)benzylamino] cyclohexanecarboxamides
Table 2 Comparison of
Compd. no.
Fit value
Max. %
protection (%)
anticonvulsant activity of 13a–e,
14a–e, and 15a–e against the
MES test and fit values
Safinamide
Phenytoin
13a
3.612
–
100
67
17
50
50
33
33
33
17
33
33
33
33
33
50
50
50
compared to safinamide
1.756
1.839
1.997
1.998
1.998
1.804
1.993
1.998
1.999
1.929
2.582
2.591
2.781
2.666
2.709
13b
13c
13d
13e
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
123

M. N. Aboul-Enein et al.
series (13a–e), 1-[4-(3-substituted benzyloxy) benzylamino] cycloheptanecarboxa-
mides series (14a–e) and 1-[4-(3- substituted benzyloxy)benzylamino]-2,3-dihydro-
1H-indene-1-carboxamides series (15a–e) are given in Tables 1 and 2. The
anticonvulsant activity of the target compounds against MES induced seizures was
compared with that of safinamide at dose level equivalent to 0.062 mmol/kg at
which safinamide exhibited 100 % protection. The results revealed that all of the
compounds under investigation were less active than safinamide. The maximum
protection was only 50 % and it was obtained by 13b, 13c, 15c, 15d, and 15e. By
comparing the results of the anticonvulsant activity of the designed molecules
against the MES test with the fit values (Table 2) for these molecules obtained from
the pharmacophore hypothesis of sodium channel blockers, it was found that the
target compounds are less active than safinamide as anticonvulsants and as sodium
channel blockers. Moreover, 13a that has the lowest fit value (1.756) exhibited only
17 % protection against the MES test. Meanwhile, 15c which has the highest fit
value displayed 50 % protection against MES.
Concerning the s.c. PTZ screening test (Table 1), the different congeners of 1-(4-
(3-substituted benzyloxy) benzylamino) cyclohexanecarboxamides series (13a–e)
exhibited anticonvulsant potential greater than that of safinamide in wihch
compound 13a showed 100 % protection against s.c. PTZ-induced seizures at a
dose level of 0.0148 mmol/kg. Meanwhile, substitution of the benzyl moiety of 13a
with an electron-withdrawing group like fluorine, chlorine, and nitro enhance the
anticonvulsant activity against s.c. PTZ-induced seizures as displayed by com-
pounds 13b, 13c, and 13e which exhibited 100 % protection at a dose level of 0.007,
0.0134, and 0.013 mmol/kg, respectively. Substitution of benzyl moiety with an
electron-donating group as methoxy group evoked the least active compound in this
series (13d) which exhibited only 83 % protection at 0.027 mmol/kg but still more
active than safinamide which exhibited the same percentage protection (83 %) but at
higher dose level (0.057 mmol/kg).
Regarding the 1-(4-(3- substituted benzyloxy) benzyl amino) cycloheptanecarb-
oxamides series (14a–e), its anticonvulsant activity in general is weaker than that of
cyclohexanecarboxamide derivatives 13a–e. Also, in cycloheptanecarboxamides
series 14a–e, the compounds 14b, 14c, and 14e which were substituted with an
Table 3 Neurotoxicity of most
active compounds and
safinamide
Compound no.
Neurotoxicitya
Wire mesh
grasping test
Grip strength
13a
1/8
1/8
1/8
0/8
1/8
1/8
1/8
1/8
2/8
0/8
2/8
0/8
0/8
2/8
2/8
1/8
13b
13c
13e
14c
a
Number of animals exhibiting
14e
neurotoxicity/number of animals
tested at the dose level which
displayed the highest protection
in s.c. PTZ
15e
Safinamide
123

Design and synthesis of certain substituted cycloalkanecarboxamides
electron-withdrawing groups (F, Cl, NO2) (compounds) displayed better anticon-
vulsant activity than those substituted with electron-donating group (OCH3) (14d)
or unsubstituted one (14a).
In 1-(4-(3- substituted benzyloxy)benzylamino)-2,3-dihydro-1H-indene-1-car-
boxamides series (15a–e) only compounds 15c and 15e emerged as the most potent
members in this series, as they displayed 83 % protection at dose levels of 0.049 and
0.022 mmol/kg, respectively.
The most potent compounds in the s.c. PTZ screening test were subjected to
quantitative estimation (median effective dose against seizures, ED50) and to
neurotoxicity tests (Table 3). Compound 13b was the most promising candidate as
anticonvulsant in s.c. PTZ screen with 100 % protection at 0.007 mmol/kg with
ED50 value lower than that of safinamide by about 10-fold with minor neurotoxic
side effects.
It was observed that the anticonvulsant profile against s.c. PTZ screening test of
most of the tested compounds were consistent with their virtual docking scores at
the active site of branched-chain aminotransferase enzyme. Compounds 13a, 13b,
(A)
HBA1
HBD
HY
HBA2
(B)
HY
HBA2
HBD
HBA1
Fig. 3 Constraint distances and angles of sodium channel blocker hypothesis (A and B). The chemical
features colored light blue, green, and violet represent HY, HBA, and HBD, respectively. (Color figure
online)
123

M. N. Aboul-Enein et al.
Table 4 Constraint distances and angles between the features of generated sodium channel blocker
hypothesis
Dimentions
Features of sodium channel blocker hypothesis
˚
Constraint distance (A)
between features
HY–HBD, 12.153: HY–HBA1, 4.464: HY–HBA2, 16.211: HBD–HBA2,
5.580: HBD–HBA1, 8.588: HBA1–HBA2, 13.426.
Constraints angles (°)
between features
HY–HBA1–HBD, 15.11: HY–HBA1–HBA2, 121.47: HY–HBA2–HBD,
126.86: HBD–HBA1–HBA2, 15.93.
Fig. 4 Mapping of sodium channel blocker pharmacophore with 15c (A) and 15e (B), respectively
13c, 13e, and 14c gave higher docking scores than both gabapentin and safinamide
with 100 % protection against s.c. PTZ-induced seizures at lower doses than
gabapentin. On the other hand, compounds 15a, 15b, and 15d exhibited the least
docking scores and their maximum anticonvulsant protection of only 67 %.
Validation of the generated pharmacophore
Ten hypotheses were generated [38] and the one ranked number 8 was chosen as the
valid ideal hypothesis (Fig. 3) based on the following: (1) the hypothesis showed
full mapping of all its features without any steric clashes together with high fit
values with the training set (compounds VIII–XV, Fig. 1), (2) retrospectively, the
simulated fit values of test set compounds 13a–e, 14a–e, and 15a–e with hypothesis
8 were more consistent with the experimental results than the others [39], and (3)
the database search study for examining the affinity of such hypothesis with the
molecular structures of MiniMaybridge databases revealed that only 17 hits have
been retrieved from the databases (2,000 compounds). Such a low number of the
recognized database molecules by generated hypothesis may give an additional
advantage and selectivity to our hypothesis. Such an ideal hypothesis encompassed
four features, namely two hydrogen bonding acceptor (HBA), hydrogen bonding
donor (HBD), and hydrophobic features (HY). These pharmacophore features was
reported for sodium channel blocking activity by Unverferth et al. [40]. In this
article, we reported the constraint distances and the angles between the essential
features existed in the generated hypothesis, as shown in Fig. 3 and Table 4. The
structures of the test set were built using the Discovery studio software, and their
123

Design and synthesis of certain substituted cycloalkanecarboxamides
conformational models were generated in the energy range of 20 kcal/mol above the
estimated global energy minima. The fitting of each tested compound was
performed using best fit during the compare/fit process. Different mappings for all
the conformers of each compound of the test set to the hypothesis were visualized
and the fit values of the best-fitting conformers were shown in Table 2 and Fig. 4,
and in supplementary material.
Molecular docking studies of branched-chain aminotransferase inhibitors
The docking results suggested that, first, the Aryl group will increase the
hydrophobic binding interaction with the deep hydrophobic pocket created by
Gly288, Val200, Tyr193 and Ala334. And second, the hydrogen bonding
interactions have been found, between the crucial features of compounds with the
high docking scores and O–H group of Thr260, COO of Thr233, and Arg119.
Moreover, introduction of the indane ring system instead of cyclohexane or
cycloheptane will decrease the docking value.
Conclusion
The anticonvulsant potential of most of the target compounds against s.c. PTZ-
induced seizures and inactivity of these compounds againstthe MES test gives
indication that these compounds could decrease the threshold of seizures rather than
prevention of their spread. Moreover, these active compounds may be beneficial in
the treatment of petit mal seizures more than grand mal seizures.
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