Synthesis, molecular modeling studies, and anticonvulsant evaluation of novel 1-((2-hydroxyethyl)(aryl)amino)-N-substituted cycloalkanecarboxamides and their acetate esters

Article abstract of DOI:10.1002/ardp.201800269

A series of 1-((2-hydroxyethyl)(aryl)amino)-N-substituted cycloalkanecarboxamides IXa–l and their acetate esters Xa–l were designed and synthesized as new anticovulsant agents. The evaluation of the anticonvulsant effect was performed in vivo by subcutaneous pentylenetetrazole (scPTZ) and maximal electroshock (MES) tests in mice. Further, neurotoxicity, hepatotoxicity, and acute toxicity were determined. All the new candidates displayed 100% anticonvulsant activity in the scPTZ screen in the dose range of 0.0057–0.283 mmol/kg. The most potent compounds in the scPTZ screen were Xh (ED₅₀ = 0.0012 mmol/kg), Xd (ED₅₀ = 0.002 mmol/kg), Xf (ED₅₀ = 0.004 mmol/kg), IXj (ED₅₀ = 0.0047 mmol/kg), Xl (ED₅₀ = 0.0076 mmol/kg), and Xi (ED₅₀ = 0.008 mmol/kg). They exhibited higher fold activity in the anticonvulsant potential than the gold standards, phenobarbital and ethosuximide. Compound Xf was active in both scPTZ and MES screens. It showed ED₅₀ of 0.016 mmol/kg in MES screen. In the neurotoxicity screens, none of the test compounds displayed any minimal motor impairment at the maximum administered dose. The 3D pharmacophore model using Biova 1 Discovery Studio 2016 programs exhibited high fit value. The anticonvulsant evaluation results were compatible with the molecular modeling study.

Full text of DOI:10.1002/ardp.201800269

Received: 8 September 2018
Revised: 16 October 2018
Accepted: 17 October 2018
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DOI: 10.1002/ardp.201800269
FULL PAPER
Synthesis, molecular modeling studies, and anticonvulsant
evaluation of novel 1-((2-hydroxyethyl)(aryl)-
amino)-N-substituted cycloalkanecarboxamides and their
acetate esters
Mohamed N. Aboul-Enein1
Aida A. El-Azzouny1
Kamilia M. Amin2
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Mona E. Aboutabl3
Mai I. Abo-Elmagd1
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1 Department of Medicinal and
Pharmaceutical Chemistry, Medicinal
Chemistry Group, Pharmaceutical and Drug
Industries Research Division, National
Research Centre (ID: 60014618), Dokki, Giza,
Egypt
Abstract
A series of 1-((2-hydroxyethyl)(aryl)amino)-N-substituted cycloalkanecarboxamides
IXa–l and their acetate esters Xa–l were designed and synthesized as new
anticovulsant agents. The evaluation of the anticonvulsant effect was performed
in vivo by subcutaneous pentylenetetrazole (scPTZ) and maximal electroshock (MES)
tests in mice. Further, neurotoxicity, hepatotoxicity, and acute toxicity were
determined. All the new candidates displayed 100% anticonvulsant activity in the
scPTZ screen in the dose range of 0.0057–0.283 mmol/kg. The most potent
compounds in the scPTZ screen were Xh (ED50 = 0.0012 mmol/kg), Xd
(ED50 = 0.002 mmol/kg), Xf (ED50 = 0.004 mmol/kg), IXj (ED50 = 0.0047 mmol/kg), Xl
(ED50 = 0.0076 mmol/kg), and Xi (ED50 = 0.008 mmol/kg). They exhibited higher fold
activity in the anticonvulsant potential than the gold standards, phenobarbital and
ethosuximide. Compound Xf was active in both scPTZ and MES screens. It showed
ED50 of 0.016 mmol/kg in MES screen. In the neurotoxicity screens, none of the test
compounds displayed any minimal motor impairment at the maximum administered
dose. The 3D pharmacophore model using Biova 1 Discovery Studio 2016 programs
exhibited high fit value. The anticonvulsant evaluation results were compatible with
the molecular modeling study.
2 Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Cairo University,
Cairo, Egypt
3 Department of Medicinal and
Pharmaceutical Chemistry, Pharmacology
Group, Pharmaceutical and Drug Industries
Research Division, National Research Centre
(ID: 60014618), Dokki, Giza, Egypt
Correspondence
Prof. Dr. Mohamed N. Aboul-Enein,
Department of Medicinal and Pharmaceutical
Chemistry, Medicinal Chemistry Group,
Pharmaceutical and Drug Industries Research
Division, National Research Centre (ID:
60014618), 33 El Bohouth St., P.O. 12622,
Dokki, Giza, Egypt.
Email: mnaboulenein@yahoo.com
Funding information
National Research Centre (ID: 60014618),
Dokki, Giza, Egypt, Grant number: 11010311
K E Y W O R D S
1-[(2-hydroxyethyl)(aryl)amino]-N-substituted cycloalkanecarboxamides, acetate esters,
anticonvulsants, epilepsy, molecular modeling studies
Epilepsy affects about 70 million people worldwide.[1,2] Actually,
epilepsy is considered as the third most frequent neurological disease
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1
INTRODUCTION
Epilepsy is a common neurologic affection characterized predomi-
nantly by periodic and sudden occurrence of seizures that are caused
by abnormal excessive or synchronous cerebral neurons discharge.
in the elderly after cerebrovascular diseases and dementia.[3]
Presently, the chronic administration of antiepileptic drugs (AEDs) is
the main treatment for epilepsy. Despite the great marketed
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Arch Pharm Chem Life Sci. 2018;e1800269.
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© 2018 Deutsche Pharmazeutische Gesellschaft
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carbamoyl)cycloalkyl)(aryl)amino)ethyl acetates (Xa–l) were prepared
to increase the lipophilicity and BBB penetration consequently the
anticonvulsant profile. Noteworthy, the structural framework of
compounds IXa–l and Xa–l comprise the following pharmacophoric
moieties (Figure 3): (A) 1,1-disubstituted cycloalkanes; (B) selected
substituents present in the molecule of the active diazaspiroalka-
nediones; (C) presence of the ethanol amine entity; (D) the acetate
fragment, thus aiming to reach new candidates exhibiting pro-
nounced anticonvulsant potential. Also, molecular modeling studies
and anticonvulsant evaluation of the novel target compounds were
performed. The anticonvulsant activity was assessed via MES and
scPTZ methods in addition to neuro- and hepatotoxicity. The
structure of the designed compounds was confirmed by elemental
analysis, IR, 13C NMR, 1H NMR, and MS sperctral data as well as X-
ray crystallographic analysis of IXg. Additionally, the target com-
pounds contain the essential pharmacophoric features that are
proposed to be necessary for a molecule to display anticonvulsant
activity.[30]
FIGURE 1 Structures of gabapentin and certain anticonvulsants
bearing the 1,1-disubstituted cycloalkane moiety
therapeutic arsenal of old and new AEDs, about 30% of patients are
not seizure-free.[4] Moreover, many AEDs are effective toward only
60% of patients besides inducing some undesirable chronic side-
effects, which seriously cause devastating outcomes on patient's
quality and style of life.[5] Therefore, there is an enormous demand for
the development of novel AEDs with more escalating efficacy and
fewer side effects.[6]
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
Synthesis of the target compounds IXa–l and Xa–l is illustrated in
Scheme 1. The cycloalkanone II was subjected to Strecker synthesis
using either toluidine or anisidine in the presence of potassium cyanide
and glacial acetic acid to give the respective nitriles IIIa–d.[31] Partial
hydrolysis of III using sulfuric acid at room temperature yielded the
respective amide derivatives IVa–d.[32] The cyano methyl group was
introduced using paraformaldehyde and potassium cyanide in glacial
acetic acid to afford compounds Va–d[33] which were subjected to
hydrolysis using sodium hydroxide in ethanol to furnish the
corresponding acids VIa–d.[33] Cyclization was accomplished using
4 N hydrochloric acid and ethylenediamine in dioxane to obtain the
diketopiperazine derivatives VIIa–d. Subsequent alkylation using
either benzyl chloride or phenethylbromide in the presence of
potassium carbonate and catalytic amount of tetrabutylammoniun
bromide gave compounds VIIIa–f.[33] The new intermediates IIIf, Ve,f,
VIe,f, VIIe,f, and VIIIi–l possessing the cycloheptane nucleus were
synthesized as their previously mentioned congeners. The target
compounds IXa–l were reached through reductive cleavage of the
cyclic imide ring of VIIIa–l using sodium borohydride in ethanol. Their
structures were confirmed through elemental analysis, IR, 13C NMR,
1,1-Disubstituted cycloalkanes were reported to display a broad
spectrum of biological activities[7–16] among which the anticonvulsant
one[17–24] and the antiepileptic one such as gabapentin[25,26] as
depicted in Figure 1.
Moreover, several investigations have reported that ethanol-
amine-O-sulfate (EOS) was active against audiogenic seizures by
irreversibly inhibiting the GABA-transaminase (4-aminobutyrate-2-
oxoglutarate) which is the major catabolic enzyme for GABA in
mammalian brain, consequently raises the brain GABA concentra-
tion.[27] Also, the ethanolamine moiety is proven to improve and
enhance drug penetration to the central nervous system as in the
prodrug ester of dexibuprofen which is used in the management of
neurodegenerative disorders (Figure 2).[28]
Besides, it was previously disclosed the anticonvulsant potential of
certain diazaspiroalkanediones 1 (Figure 3).[29] Accordingly, it was
found of interest to design and synthesize a set of novel 1-((2-
hydroxyethyl)(aryl)amino)-N-substituted
cycloalkanecarboxamides
(IXa–l). Also, the acetate esters of the latter, namely, 2-((1-substituted
FIGURE 2 Structures of ethanolamine-O-sulfate (EOS) and dexibuprofen prodrug

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FIGURE 3 Pharmacophoric structural fragments of title compounds. (A) 1,1-Disubstituted cycloalkanes (purple), (B) selected substituent
present in the active diazaspiroalkanediones (green), (C) the ethanol amine entity (blue), (D) the acetate fragment (red)
SCHEME 1 Synthesis of compounds IXa–l and Xa–l. Reagents and conditions: (i) KCN, glacial acetic acid, 24 h, r.t.; (ii) conc. H2SO4, 48 h,
r.t.; (iii) glacial acetic acid, paraformaldehyde, KCN, 37% formaldehyde; (iv) 50% aqueous ethanol, NaOH, 24 h, reflux; (v) dioxane, 4 N HCl,
ethylenediamine, 24 h, reflux; (vi) ClCH2C6H5 or BrCH2CH2C6H5, acetone, K2CO3, tetrabutylammoniumbromide, 7 h, reflux; (vii) NaBH4,
ethanol, 24 h, r.t.; (viii) acetic anhydride, dichloromethane, triethylamine, DMAP, 24 h, r.t.

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1H NMR, and MS spectral data as well as X-ray crystallographic
analysis of IXg as a representative example that showed the cleavage
of the cyclic imide (Figure 4). The IR showed broad band at 3500–3000
for the OH group, 1H NMR exhibited exchangeable NH peak, in
addition to the absence of one carbonyl cyclic imide peak in 13C NMR.
The acetate esters Xa–l were achieved using acetic anhydride,
triethylamine and catalytic amount of 4-dimethylaminopyridine and
were confirmed by the presence of both amidic and ester carbonyl
peaks in IR and 13C NMR.
hydroxyethylamino derivatives IXa–l and their acetates Xa–l was
evaluated using the scPTZ and MES tests and was expressed as %
protection besides their neurotoxicity are presented in Tables 2 and 3.
It was reported that PTZ test represents a valid model for generalized
myoclonic seizures. By contrast, the MES test is a predictor of possible
therapeutic efficacy against generalized tonic-clonic seizures.[35] The
obtained data showed that all the compounds were effective in scPTZ
test while most of them were effective against MES test except
compounds IXj, Xj, and Xk.
All the scPTZ screening tests of the hydroxyethylamino deriva-
tives IXa–l were more potent than the reference drugs (phenobarbital
and ethosuximide) as they showed the same antiseizure profile (100%
protection) at lower doses on molecular bases except compound IXa
was less potent than phenobarbital. Compounds IXa–l dispalyed 100%
protection against scPTZ-induced seizures at dose levels of 0.0253–
0.283 mmol/kg ≡ 10–100 mg/kg as compared with phenobarbital
(0.13 mmol/kg ≡ 30 mg/kg) and ethosuximide (1.06 mmol/kg ≡
150 mg/kg) where compound IXj possessing the cycloheptane
scaffold was the most active congener. It exhibited 11.9- and 196.8-
fold more potent anticonvulsant effect than phenobarbital and
ethosuximide, respectively. Concerning the MES test of IXa–l, the
dose which displayed 100% anticonvulsant activity in the scPTZ
screening or higher doses have been selected. In this screening test,
compounds IXa, IXe–g, IXi, IXk, and IXl gave protection in half or more
of the tested mice after 0.5 h post-administration. Meanwhile
compounds IXb, IXd, and IXh exhibited 33% protection. On the other
hand, only compound IXj did not exert protection against electro-
induced seizures in MES test. In the neurotoxicity screen, all the target
compounds did not show any minimal motor impairment in the
maximum dose administered (Table 2).
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2.2 X-ray structure determination
The study of the title structure IXg was undertaken to establish its
three-dimensional structure. Single crystals of IXg (CCDC#: 1854009)
compatible for X-ray diffraction were achieved by slow evaporation of
ethanol from ethanolic solution of the compound to give cubic
colorless crystals with the following crystallographic parameters:
a = 10.2710(4) Å, b = 19.4956(9) Å, c =10.5602(6) Å, α = 90.00°,
β = 102.771(3)°, γ = 90.00°, V = 2062.3(2) Å3, (Z) = 4, goodness-of-fit
on F2 = 1.146 (Table 1). The crystal structure of IXg is monoclinic with a
space group of P21/n. All diagrams and calculations were performed
using maXus. The X-ray crystal structure of IXg is shown in Figure 4.
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2.3 Anticonvulsant activity
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2.3.1 Phase I anticonvulsant evaluation
The initial pharmacological evaluation (phase 1 screening) was adopted
using the standard procedure of Antiepileptic Drug Development
(ADD) program of the National Institute of Neurological Disorders and
Stroke (NINIDS), Epilepsy section.[34] This protocol consists of using
the “gold standards” screens, namely subcutaneous pentylenetetra-
zole (scPTZ) screen and the maximal electroshock seizure (MES)
screen. The anticonvulsant activity of the newly synthesized
Regarding the scPTZ screening of the amino ethyl acetate
derivatives Xa–l, all the compounds were more potent than the
reference drugs, except Xa and Xk. Moreover, all the compounds
evoked 100% protection against scPTZ-induced seizures at dose levels
FIGURE 4 ORTEP diagram of IXg with thermal ellipsoids drawn at 50% probability

ABOUL-ENEIN ET AL.
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TABLE 1 Crystal data and structure refinement for IXg
post-administration. Moreover, compounds Xc–e exhibited 33%
protection while compounds Xj–k displayed no activity in MES test.
Therefore, it is suggested that the compounds that demonstrated
activity in MES test could have a mode of action through inhibiting the
voltage gated sodium channels.
CCDC number
Empirical formula
Molecular weight
Temperature
1854009
23H30N2O3
C
382.5
298 K
Furthermore, the generalized myoclonic seizures induced subcuta-
neously by PTZ resemble generalized myoclonic seizures in human.[37]
Previous studies have reported that scPTZ-induced seizures were
prevented by drugs that enhance GABAA receptor-mediated inhibitory
transmission such as phenobarbital[38] and also by drugs that inhibit
T-type Ca2+ currents such as ethosuximide.[36,37,39] Accordingly, the
findings of this study suggest that compounds IXa–l and Xa–l possibly
exhibited their anticonvulsant potential through inhibiting or attenuat-
ing the PTZ-induced seizures in mice by enhancing the GABA-ergic
neurotransmission and/or inhibiting Ca2+ channel.
Wavelength
0.71073
Crystal system
Space group
Monoclinic
P21/n
Cell dimensions
a = 10.2710 (4) Å
b = 19.4956 (9) Å
c = 10.5602 (6) Å
α = 90.00°
β = 102.771 (3)°
γ = 90.00°
In the neurotoxicity screen, all the test compounds did not show
any minimal motor impairment in the maximum dose administered.
Volume
2062.26 (17) Å3
4
Z
Density
1.232 Mg/m3
0.200 × 0.3400.480 × mm
0.081 mm
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2.3.2 Phase II anticonvulsant evaluation
Crystal size
In this study, we found that the acetate derivatives Xa–l were more
active than their corresponding alcohol ones IXa–l. The promising
results of phase I anticonvulsant activity encouraged us to undergo
phase II anticonvulsant evaluation. The protective index (as a ratio of
TD50 and ED50) values for the compounds Xa–l was calculated. The
most active compounds namely Xd, Xf, Xh, Xi, and Xl with the lowest
ED50 showed protective index ranging from >5.68 to >2.89 using the
dose that demonstrated 100% protection in the scPTZ test and
showed no neurotoxicity. Also, for compound Xf, the PI was calculated
according to the dose that showed 100% protection and devoid of
neurotoxicity in the MES test. Compound Xf demonstrated similar PI
index in both scPTZ and MES test.
Absorption coefficient
F000
824
Theta range for data collection
Index range
2.916–31.988°
−14 ≤ h ≤ 14, 0 ≤ k ≤ 27, 0 ≤ l ≤ 15
Multi scan
Absorption correction
Max. and min. transmission
Refinement method
No. of parameters
No. of restraints
Goodness-of-fit on F2
R (reflections)
0.98 and 0.61
Full matrix least square on F2
253
0
1.146
0.048 (2079)
0.15 (2079)
0.432
Additionally, among the active acetate derivatives, we selected
the most active compounds namely Xd, Xf, Xh, Xi, and Xl to investigate
the possible hepatotoxic effect. Hepatotoxicity is considered as one of
the most serious side effects of antiepileptic drugs. The serum enzyme
activity assay was carried out for the most active compounds Xd, Xf,
Xh, Xi, and Xl. Elevated liver enzymes are considered as specific
markers of liver dysfunction. The data of liver enzymes analysis
including: ALT, AST, and ALP, as well as albumin and total protein were
represented as mean SEM in Table 4. The nominated compounds did
not exhibit any significant change in serum enzymes level value as well
as albumin and total protein as compared to control. This result
demonstrated that there are no signs of hepatotoxicity. Moreover, all
animals were devoid of acute toxicity signs at the tested dose range
during the observation period.
wR2 (reflections)
Data completeness
of 0.0057–0.2 mmol/kg ≡ 2.5–80 mg/kg as compared with the
reference standards (Table 3). The most active congeners were
compounds Xh, Xd, Xf, Xl, and Xi. They possessed more potent
activities which reached 46.7-, 28-, 14-, 7.3-, and 7-fold than
phenobarbital, and 770.8-, 462.5-, 231.3-, 121.7-, and 115.6-fold
than ethosuximide, respectively.
MES test is considered a unique and effective screen test in
detecting drugs among which is phenytoin that blocks human
generalized tonic–clonic seizures and whose mechanism of action is
reported to be via blocking the sodium channels. Interestingly,
phenytoin acts via this mechanism of action and is reported to be
active in MES test.[36] In this study, compound Xf displayed 100%
activity in MES test at dose level 0.094 mmol/kg with
ED50 = 0.0166 mmol/kg which is 2.3-fold more potent than the
reference drug phenytoin. Meanwhile, compounds Xa,b, Xg–i, and
Xl showed protection in half or more of the tested mice after 0.5 h
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2.3.3 Structure–activity relationship (SAR)
As a result of the in vivo studies, the anticonvulsant effect of the
synthesized compounds IXa–l and Xa–l in the scPTZ test is mostly
attributed to the electron donating properties of −CH3 or −OCH3
groups at the four position of the anilino ring, the benzyl or phenethyl

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TABLE
2
Anticonvulsant and neurotoxicity evaluation of compounds IXa–l as well as the reference drugs against subcutaneous
pentylenetertrazole and electro-induced seizures in mice, as well as their fit values
scPTZ
MES
Compound
no.
Dosea mg/kg
(mmol/kg)
Max. %
Dose mg/kg
(mmol/kg)
Max. %
ED50 mg/kg (confidence limits)c
ED50 mmol/kg (confidence limits)c
Fit
protection
protection
Neurotoxicityb
value
IXa
100 (0.283)
20 (0.054)
40 (0.1)
100
100
100
100
100
100
100
100
100
100
100
100
100
100
–
100 (0.283)
50
0/6
35.14 (44.39–26.42)
0.099 (0.125–0.074)
3.723 (4.122–3.367)
0.01 (0.011–0.009)
7.462 (8.277–6.734)
0.02 (0.022–0.018)
8.958 (11.45–6.872)
0.023 (0.029–0.017)
4.482 (5.588–3.346)
0.012 (0.015–0.009)
12.37 (12.4–12.33)
0.032 (0.0325–0.0324)
14.47 (17.15–12.05)
0.037 (0.044–0.031)
8.953 (12.21–6.334)
0.022 (0.03–0.015)
3.319 (3.622–3.035)
0.0087 (0.009–0.0079)
1.862 (2.061–1.683)
0.0047 (0.005–0.0042)
7.446 (8.243–6.734)
0.018 (0.02–0.0169)
16.14 (20.39–12.26)
0.039 (0.049–0.0298)
13.2 (15.90–6.80)
2.990
2.995
2.993
2.978
2.998
2.996
2.981
2.988
2.995
2.988
2.980
2.980
1.980
–
IXb
20 (0.054)
40 (0.1)
–
0/6
0/6
0/6
33.3
16.7
IXc
40 (0.1)
IXd
40 (0.1)
40 (0.1)
–
0/6
0/6
0/6
80 (0.2)
33.3
50
IXe
20 (0.054)
80 (0.21)
100 (0.26)
40 (0.1)
20 (0.054)
IXf
80 (0.21)
50
50
0/6
0/6
1/6
IXg
100 (0.26)
IXh
40 (0.1)
–
0/6
0/6
0/6
100 (0.252)
20 (0.052)
33.3
50
IXi
20 (0.052)
10 (0.0253)
40 (0.1)
IXj
10 (0.0253)
–
0/6
IXk
40 (0.1)
33.3
66.7
66.7
0/6
0/6
0/6
120 (0.3)
40 (0.097)
IXl
40 (0.097)
30 (0.13)
150 (1.06)
–
Phenobarbital
Ethosuximide
Phenytoin
–
–
nd
nd
nd
0.056 (0.068–0.029)
130.55 (177.87–98.78)
0.925 (1.260–0.699)
9.50 (10.96–7.24)
–
–
45 (0.16)
100
–
0.0376 (0.0434–0.0287)
– Indicates absence of activity. nd: not determined.
aThe minimal dose which exhibited the maximum anticonvulsant activity in scPTZ test.
bRotarod test: number of mice exhibiting neurotoxicity (falling of the rotarod)/number of mice tested. Control group demonstrated 0/6 in the neurotoxicity
test and were devoid of anticonvulsant activity.
cED50: median effective dose in scPTZ test.
carboxamide side chain, in addition to the cycloalkyl ring size scaffold
(cyclopentyl, cyclohexyl, or cycloheptyl), Tables 2 and 3, respectively.
In the hydroxyethyl amino derivatives linked to cyclopentane ring
IXa–d, it was found that IXb was the most active congener. It contains
the cyclopentyl ring linked to both electron donating −CH3 group at
the four position of the anilino moiety, and phenethyl carboxamide
side chain. In IXc and IXd when −OCH3 as electron releasing group
replaced the −CH3 at the four position of the anilino moiety in the
presence of either benzyl or phenethyl carboxamide side chains,
respectively, nearly equipotent anticonvulsant activity was observed.
The least anticonvulsant activity was observed with both 4-methyl
anilino and benzyl carboxamide substituent in compound IXa. In the
cyclohexane series IXe–h, the most active congeners were IXe
having the 4-methyl anilino and the benzyl carboxamide side chain,

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then IXh having the 4-methoxyanilino and the phenethyl carboxamide
side chain. By increasing the cycloalkane ring size as in the
cycloheptane series IXi–l, compound IXj was the most active congener
where the cycloheptane ring is linked to both the 4-methyl anilino and
the phenethyl carboxamide moieties. The different congeners of IXa–l
exhibited anticonvulsant potential in the following decreasing order:
IXj > IXi > IXb = IXe > IXk > IXc = IXd = IXh > IXf > IXg = IXl > IXa.
Regarding the scPTZ screening of the amino ethyl acetate
derivatives, it was found that compound Xd was the most acive
congener among Xa–d where the cyclopentane ring was attached to
the phenethyl carboxamide side chain and the anilino group having at
the 4-position the electron releasing OCH3 group. Moreover, Xd
showed the highest fit value among Xa–l in the 3D pharmacophore
model. Meanwhile, replacement of the methoxy group with the
electron donor −CH3 group decreased the anticonvulsant activity as in
Xb and Xc regardless whether the side chain was benzyl or phenethyl.
In compounds Xe–h bearing the cyclohexane scaffold, the most active
congeners were Xh then Xf where the cyclohexyl ring was linked to the
phenethyl carboxamide side chain and either electron donor 4-OCH3
or 4-CH3 anilino groups, respectively. Also, the anticonvulsant effect
was reduced in Xe and Xg on linking the cyclohexyl scaffold with the
benzyl carboxamide side chain and with either the 4-CH3 or 4-OCH3
anilino groups, respectively. Increasing the size of the cycloalkane ring
to cycloheptane (Xi–l) produced two of the most active congeners Xl
having 4-OCH3 anilino and phenethyl carboxamide moieties, then Xi
possessing 4-CH3 anilino and benzyl carboxamide moieties, where
compound Xl possessed higher fit value than Xi in the 3D
pharmacophore model. Surprisingly, compounds possessing the
cycloheptane nucleus showed less anticonvulsant potentiality than
their congeners in the cyclopentane and cyclohexane series where
compounds Xj–l were less potent than their analogues in the
cyclopentyl and cyclohexyl series, Xb–d and Xf–h, respectively. One
explanation of this reduced activity might be due to the low solubility
of the more lipophilic analogs in the aqueous plasma where they easily
bind to plasma proteins which may lead to lower drug
concentrations available for diffusion into the CNS.[40] Hence,
delivering a drug via the circulatory system for treatment of CNS
diseases requires a balance between BBB permeability and plasma
solubility. The different congeners of the acetate series showed
anticonvulsant potential in the following decreasing order:
Xh > Xd > Xf > Xl = Xi > Xb = Xg = Xe > Xc = Xj > Xk > Xa. The results
of their fit values were consistent with the anticonvulsant activity in
the scPTZ test.
Generally, the acetate derivatives Xa–l showed better anticonvul-
sant activity than the precursor hydroxyethylamino derivatives IXa–l.
Also, the occurrence of electron donating group such as −OCH3 or
−CH3 at position 4 of the anilino moiety improves the anticonvulsant
activity. The phenethyl carboxamide side chain as a substituent of the
cycloalkyl ring is preferable than the benzyl carboxamide one. The
presence of both 4-OCH3 anilino and phenethyl carboxamide
fragments in one molecule augment the activity. The cyclopentyl
and cyclohexyl ring size may be preferable than the cycloheptyl one in
the acetate derivatives.

ABOUL-ENEIN ET AL.
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|
TABLE 4 Determination of liver enzyme, albumin and total protein with the most active compounds Xd, Xf, Xh, Xi, and Xl
Compound no.
ALT (U/L)
AST (U/L)
ALP (U/L)
Albumin (g/dL)
3.14 0.34
3.41 0.07
3.28 0.14
3.35 0.12
3.85 0.07
3.50 0.07
Total protein (g/dL)
7.30 0.06
Control
Xd
23.00 2.51
20.18 3.15
18.46 2.93
29.42 0.95
22.95 4.46
20.65 0.38
70.00 7.76
58.67 2.25
57.00 4.58
74.33 10.38
85.78 7.15
81.67 4.25
12.00 0.64
13.95 1.25
10.76 2.86
11.06 0.48
12.35 0.74
10.35 1.61
7.78 0.33
Xf
8.21 0.64
Xh
7.71 0.31
Xi
8.11 0.08
Xl
9.00 0.38
Data are represented as mean SEM and were analyzed by ANOVA followed by Student Newman Keul test, n = 8. ALT, alanine aminotransferase; AST,
aspartate aminotransferase; ALP, alkaline phosphatase.
|
2.4 Molecular modeling study
possessed more potent activities which reached 46.7-, 28-, 14-,
11.9-, 7.3-, and 7-fold than phenobarbital, and 770.8-, 462.5-, 231.3-,
196.8-, 121.7-, and 115.6-fold than ethosuximide, respectively. It is
noteworthy to mention that compound Xf was active in both scPTZ
and MES screens. It showed ED50 of 0.016 mmol/kg in MES screens
which is 2.3-fold more potent than phenytoin. In the neurotoxicity
screen, none of the test compounds evoked any minimal motor
impairment at the maximum administered dose. Furthermore, the
achieved molecular simulation fitting to the anticonvulsant 3D-
pharmacophore model exhibited high-fit values. The determined
fitting scores were correlated with the scPTZ experimental results.
The molecular modeling study was accomplished aiming to analyze and
investigate the anticonvulsant activity of compounds IXa–l and Xa–l.
This was done by running the common feature pharmacophore model
protocol in Discovery Studio 2016Biova 1 software. The essential
pharmacophoric elements of antiepileptic drugs and numerous
anticonvulsant compounds include the occurrence of ring aromatic
(RA), hydrogen bond acceptor (HBA), and hydrophobic moiety (HY).
The used training sets containing the essential features to generate the
pharmacophore model were demonstrated in Figure 5. In addition, the
generated hypothetical pharmacophore model was presented in
Figure 6, which might be responsible for interface with the receptor
active site. Distances and angles between the pharmacophoric
features of the training sets (A, HBA, HY) are shown in Table 5.[41–44]
The validation of the generated pharmacophore was based on full
mapping of all essential features and high fit values with the used
training sets (compounds 2–11, Figure 7). Validating retrospectively
the latter simulated fit values of test set compounds IXa–l and Xa–l
with the given hypothesis were more consistent with the experimental
values than other hypotheses. The fit values of test compounds IXa–l
and Xa–l are shown in Tables 2 and 3. Mapping of anticonvulsants
pharmacophoric features of the most potent acetate ester candidates
according to their ED50 values, Xh, Xd, Xf, Xi, and Xl, and phenobarbital
is illustrated in Figure 8.
|
3
CONCLUSION
The anticonvulsant potential of certain 1-((2-hydroxyethyl)(aryl)
amino)-N-substituted cycloalkanecarboxamides IXa–l and 2-((1-
substituted carbamoyl)cycloalkyl)(aryl)amino)ethyl acetates Xa–l was
investigated. All the compounds displayed 100% anticonvulsant
activity in the scPTZ screen at a dose range from 0.0057 to
0.283 mmol/kg. It could be concluded that the acetate esters were
more potent than their starting alcohols due to expected higher
lipophilicity. The most potent compounds in the scPTZ screen
were Xh (ED50 = 0.0012 mmol/kg), Xd (ED50 = 0.002 mmol/kg),
Xf (ED50 = 0.004 mmol/kg), IXj (ED50 = 0.0047 mmol/kg), Xl
(ED50 = 0.0076 mmol/kg), and Xi (ED50 = 0.008 mmol/kg). They
FIGURE 5 Representative examples of anticonvulsants (training
set) with their ED50 values in scPTZ test. 2: ED50 = 0.49 mmol/kg[42]
3: ED50 = 0.0056 mmol/kg[44]; 4: ED50 = 0.45 mmol/kg[1]; 5:
ED50 = 0.011 mmol/kg[29]; 6: ED50 = 0.017 mmol/kg[29]; 7:
ED50 = 0.19 mmol/kg[1]; 8: ED50 = 0.012 mmol/kg[29]; 9:
ED50 = 0.071 mmol/kg[29]; 10: ED50 = 0.15 mmol/kg[41]; 11:
ED50 = 0.019 mmol/kg[29]
;

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|
(for oils) in KBr discs or as KBr pellets (for solids) with Jasco IR
and FTIR-6100 spectrometer and values are represented in
.
cm−1 1H NMR and 13C NMR spectra were carried out on Jeol
ECA 500 MHz or Bruker Avance 300 spectrometers using TMS as
internal standard. Chemical shift values are recorded in ppm δ
scale. The 1H NMR data were represented as follows: chemical
shifts, multiplicity (s. singlet, br.s. broad singlet, d. doublet, t. triplet,
and m. multiplet), number of protons, and type of protons.
13C NMR data are represented as chemical shifts and type of
carbon atoms.
The X-ray was performed using maXus (Bruker Nonius, Delft &
MacScience, Japan) at the X-ray Unit, National Research Centre.
Purification was performed using column chromatography and
solvent system petroleum ether (40–60)/ethyl acetate (7:3) for
compounds VIIIa–f and dichloromethane/ethyl acetate (9:1) for
compounds IXa–l and Xa–l. Also, aluminum oxide 60G F254 neutral
plates for TLC from Merck were used for thin layer chromatography.
Visualization was performed by illumination with UV-light source
(254 nm).
FIGURE 6 Pharmacophore model of anticonvulsants generated
by Discovery Studio 2016 Biova 1 software. Pharmacophore
features are colored: green for hydrogen bond acceptor (HBA),
orange for aromatic region (RA), and light blue for hydrophobic
region (HY)
Compounds IIIa–e, IVa–f, Va–d, VIa–d, VIIa–d, VIIIa–f were
prepared according to adopted procedures.[17,29,31,32] Please also see
the Supporting Information for further details.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
Interestingly, these compounds could be considered as promising
antiepileptic candidates.
|
4.1.2 Synthesis of 1-[(4-methoxyphenyl)amino]-
|
cycloheptanecarbonitrile (IIIf)
4
EXPERIMENTAL
Compound IIIf was synthesized according to the reported
procedure.[31] Yellow solid, m.p. 58°C, yield 73.68%. IR (KBr, cm−1):
2221 (nitrile group); MS (EI) m/z (%): C15H20N2O, 244 (M+, 39), 122
(100).
|
4.1 Chemistry
|
4.1.1 General
Mass spectra were run on Jeol JMS-AX500 mass spectrometer.
Elemental analysis were carried out in Microanalytical Units at
National Research Centre and the results were within 0.4% of the
theoretical value. Infrared (IR) spectra were recorded as thin film
|
4.1.3 Synthesis of 1-[(aryl)(cyanomethyl)amino]-
cycloheptanecarboxamides Ve,f
Compounds Ve,f were synthesized according to the reported
procedure.[33]
1-[(Cyanomethyl)(4-methylphenyl)amino]-
TABLE 5 The constraint distances and angles between the features
of the generated anticonvulsant pharmacophore model
cycloheptanecarboxamide (Ve)
Buff solid, m.p. 84°C, yield 82%. IR (KBr, cm−1): 2220 (nitrile group),
1671 (amide carbonyl group); MS (EI) m/z (%): C17H23N3O, 285
(M+, 19), 57 (100); 1H NMR (CDCl3) δ ppm 1.46–2.2 (m, 12H, 6 x CH2,
cycloheptyl), 2.27 (s, 3H, CH3), 3.9 (s, 2H, CH2-CN), 6 (s, 2H, NH2), 6.5–
7 (m, 4H, Har.); 13C NMR (CDCl3) δ ppm 20.51 (CH3), 22.92, 29.9, 36.2
(6 x CH2, cycloheptyl), 41 (CH2-CN), 63.68 (Cq), 116 (CN), 116.09,
Features of the given
Dimensions
anticonvulsant model
Constraint distances (Å)
between feature
HBA-RA: 5.53
HBA-HY: 8.55
RA-HY: 10.02

129.68 (CHar.), 128.03, 142.26 (2 x Car.), 180.34 (C O).
Constraint angles between
features
HBA-HY-RA: 32.38
1-[(Cyanomethyl)(4-methoxyphenyl)amino]-
cycloheptanecarboxamide (Vf)
HBA-RA vector: 64.73
HY-HBA vector: 106.73
HY-RA vector: 87.59
Brown solid, m.p. 84–85°C, yield 95%.MS (EI) m/z (%):
C
17H23N3O2, 301 (M+, 10), 218 (100); 1H NMR (CDCl3) δ ppm

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FIGURE 7 (A and B) Constraint distances and angels for pharmacophore model of the training set (2–11), test set (IXa–l and Xa–l) and
phenobarbital. The chemical features colored green, orange, and light blue represent hydrogen bonding acceptor (HBA), ring aromatic (RA),
and hydrophobic features (HY), respectively
|
1.2–2 (m, 12H, 6 x CH2, cycloheptyl), 3.7 (s, 3H, OCH3), 4.1 (s, 2H,
CH2-CN), 6.3 (s, 2H, NH2), 6.6–7 (m, 4H, Har.); 13C NMR (CDCl3) δ
ppm 22.9, 29.9, 34.2 (6 x CH2, cycloheptyl), 41.4 (CH2-CN), 55.4
(OCH3), 64.6 (Cq), 116 (CN), 114.59, 128.6 (CHar.), 141.2, 157.8 (2
4.1.4 Synthesis of [(aryl)(1-carbamoylcycloheptyl)-
amino]acetic acids VIe,f
Compounds VIe,f were synthesized according to the reported
procedure.[33]

x Car.), 180 (C O).
FIGURE 8 Mapping of anticonvulsants pharmacophore fragments of the most active test compounds Xh, Xd, Xf, Xl, Xi, and phenobarbital

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[(1-Carbamoylcycloheptyl)(4-methylphenyl)amino]acetic acid
(VIe)
4-Benzyl-1-(4-methylphenyl)-1,4-diazaspiro(5.6)dodecane-3,5-
dione (VIIIi)
Yellow viscous oil; yield (67%). IR (KBr, cm−1): 1725, 1675 (C O, imide

Yellow solid, m.p. 80–82°C, yield 70%. MS (EI) m/z (%): C17H24N2O3,
304 (M+, 21), 203 (100); 1H NMR (CDCl3) δ ppm 1.52–2.2 (m, 12H, 6 x
CH2, cycloheptyl), 2.29 (s, 3H, CH3), 4.1 (s, 2H, CH2-COOH), 6.7–7 (m,
4H, Har.), 7.08 (s, 2H, NH2), 8.6 (s, H, OH); 13C NMR (CDCl3) δ ppm
20.52 (CH3), 23.87, 30.3, 35.93 (6 x CH2, cycloheptyl), 50.06 (CH2-
COOH), 69.4 (Cq), 119.8, 129 (CHar.), 129.9, 143.24 (2 x Car.), 177.61,
carbonyls); MS (EI) m/z (%): C24H28N2O2, 376.33 (M+, 93.62), 91 (100);
1H NMR (CDCl3) δ ppm 1.27–2.02 (m, 12H, 6 x CH2, cycloheptyl), 2.26

(s, 3H, CH3), 4.08 (s, 2H, O C-CH2-N), 5.04 (s, 2H, CH2-C6H6), 6.7 (d,
2H, J = 8.6 Hz, Har.), 6.9 (d, 2H, J = 8.6 Hz, Har.), 7.3–7.4 (m, 5H, Har.);
13C NMR (CDCl3) δ ppm 22.36, 29.83, 34.8 (6 x CH2, cycloheptyl),


183.64 (2 x C O).
32.05 (CH3), 42.7 (CH2-C6H5), 55.04 (O C-CH2-NH2), 64.9 (Cq),
127.11, 127.6, 128.5, 129.2, 129.8 (CHar.), 135.7, 137.4, 145.8 (3 x

[(1-Carbamoylcycloheptyl)(4-methoxyphenyl)amino]acetic acid
(VIf)
Car.), 171.07, 176.6 (2 x C O).
Brown viscous oil, yield 84%. IR (KBr, cm−1): 3431 (COOH), 1658 (C O,

1-(4-Methylphenyl)-4-(2-phenethyl)-1,4-diazaspiro(5.6)-
dodecane-3,5-dione (VIIIj)
Yellow viscous oil, yield 61%. IR (KBr, cm−1): 1728, 1677 (C O,
imide carbonyls); MS (EI) m/z (%): C25H30N2O2, 390.05 (M+, 26.69),
271 (91.52), 91 (100); 1H NMR (CDCl3) δ ppm 1.494–2.026 (m, 12H,
6 x CH2, cycloheptyl), 2.285 (s, 3H, CH3), 2.88 (t, J = 10 Hz, 2H, CH2-

amide), 1720 (C O, carboxylic acid); MS (EI) m/z (%): C17H24N2O4, 320
(M+, 6), 218 (100); 1H NMR (CDCl3) δ ppm 1.46–2.04 (m, 12H, 6 x CH2,
cycloheptyl), 3.73 (s, 3H, OCH3), 3.9 (s, 2H, CH2-COOH), 6.5–6.8 (m, 4H,
Har.), 7.06 (s, 2H, NH2), 8.3 (s, H, OH); 13CNMR(CDCl3)δppm 23.78, 29.7,
35.7 (6 x CH2, cycloheptyl), 52 (CH2-COOH), 55.5 (OCH3), 70.18 (Cq),


114.1, 125 (CHar.), 139.15, 155.81 (2 x Car.), 176.5, 182.8 (2 x C O).

C6H5), 4.03 (s, 2H, O C-CH2-N), 4.1 (t, J = 10 Hz, 2H, CH2-CH2-
C6H5), 6.8 (d, 2H, J = 8 Hz, Har.), 7.02 (d, 2H, J = 8 Hz, Har.), 7.203–
7.296 (m, 5H, Har.); 13C NMR (CDCl3) δ ppm 20.89 (CH3) 22.39,
29.63, 34.63 (6 x CH2, cycloheptyl), 33.98 (CH2-C6H5), 40.55 (CH2-
|
4.1.5 Synthesis of 1-aryl-1,4-diazaspiro[5.5]-
dodecane-3,5-diones VIIe,f

CH2-C6H6), 54.84 (O C-CH2-N), 64.83 (Cq), 126.53, 126.94,
Compounds VIIe,f were synthesized according to the reported
procedure.[33]
128.47, 129.07, 129.88 (CHar.), 135.68, 138.4, 145.94 (3 x Car.),

170.87, 176.6 (2 x C O).
1-(4-Methylphenyl)-1,4-diazaspiro(5,6)dodecane-3,5-dione
(VIIe)
4-Benzyl-1-(4-methoxyphenyl)-1,4-diazaspiro[5.6]dodecane-
3,5-dione (VIIIk)
White solid, m.p. 120°C, yield 60%. MS (EI) m/z (%): C17H22N2O2, 286
(M+, 40), 105 (100); 1H NMR (CDCl3) δ ppm 1.462–2.024 (m, 12H, 6 x
Yellow viscous oil, yield 80%. IR (KBr, cm−1): 1720, 1673 (C O, imide

carbonyl); MS (EI) m/z (%): 392.25 (M+, 100), 91 (100); 1H NMR (CDCl3)

CH2, cycloheptyl), 2.213 (s, 3H, CH3), 3.92 (s, 2H, O C-CH2-N), 6.893
δ ppm 1.53–2.033 (m, 12H, 6 x CH2, cycloheptyl), 3.7 (s, 3H, OCH3),
(d, 2H, J = 10 Hz, Har.), 6.998 (d, 2H, J = 10 Hz, Har.), 8.85 (s, 1H, NH);
13C NMR (CDCl3) δ ppm 20.9 (CH3) 22.31, 29.55, 34.11 (6 x CH2,

4.04 (s, 2H, O C-CH2-N), 5.02 (s, 2H, CH2-C6H5), 6.6 (d, 2H,
J = 8.6 Hz, Har.), 6.77 (d, 2H, J = 8.6 Hz, Har.), 7.3–7.39 (m, 5H, Har.);
13C NMR (CDCl3) δ ppm 22.28, 29.63, 34.7 (6 x CH2, cycloheptyl), 42.6
(CH2-C6H5), 54.9 (OC-CH2-N), 55.42 (OCH3), 65.25 (Cq), 114.307,
127.66, 128.44, 128.5, 129.2 (CHar.), 136.95, 141.2, 157.6 (3 x Car.),

cycloheptyl), 54.43 (O C-CH2-N), 64.64 (Cq), 126.95, 129.95 (CHar.),

135.85, 145.8 (2 x Car.), 172.25, 177.44 (2 x C O).
1-(4-Methoxyphenyl)-1,4-diazaspiro(5,6)dodecane-3,5-dione
(VIIf)

171.1, 176.5 (2 x C O).
Buff solid, m.p. 92°C, yield 60%. IR (KBr, cm−1): 3426 (NH) and 1675, 1641
(C O, imide carbonyl); MS (EI) m/z (%):C17H22N2O3, 302 (M+, 100);
1HNMR(CDCl3)δppm 1.23–2.05 (m, 12H, 6 x CH2, cycloheptyl), 3.7 (s, 3H,
1-(4-Methoxyphenyl)-4-(2-phenylethyl)-1,4-diazaspiro[5.6]-
dodecane-3,5-dione (VIIIl)

Yellow viscous oil, yield 60%. IR (KBr, cm−1): 1722, 1673 (C O, imide
carbonyl); MS (EI) m/z (%): C25H30N2O3, 406.26 (M+, 100); 1H NMR


OCH3), 3.97 (s, 2H, O C-CH2-N), 6.78 (d, 2H, J = 8.6 Hz, Har.), 7 (d, 2H,
J = 8.6 Hz,Har.), 8.3 (s, 1H, NH); 13CNMR(CDCl3)δppm 22.29, 29.73, 34.12
(6 x CH2, cycloheptyl), 54.41 (OC-CH2-NH2), 55.49 (OCH3), 65.06 (Cq),
(CDCl3) δ pp m 1.4–1.97 (m, 12H, 6 x CH2, cycloheptyl), 2.89 (t,


114.51, 128.37 (CHar.), 141.16, 157.78 (2 x Car.), 171.96, 177.15 (2 x C O).
J = 7.6 Hz, 2H, CH2-C6H5), 3.75 (s, 3H, OCH3), 4 (s, 2H, O C-CH2-N),
4.08 (t, J = 7.6 Hz, 2H, CH2-CH2-C6H5), 6.7 (d, 2H, J = 9.6 Hz, Har.), 6.9
(d, 2H, J = 9.6 Hz, Har.), 7.22–7.29 (m, 5H, Har.); 13C NMR (CDCl3) δ
ppm 22.37, 29.82, 34.62 (6 x CH2, cycloheptyl), 34 (CH2-C6H5), 40.5
|
4.1.6 Synthesis of 1-aryl-4-substituted-1,4-
diazaspiro[5.5]-undecane-3,5-diones VIIIi–l

(CH2-CH2-C6H6), 54.6 (O C-CH2-N), 55.4 (OCH3), 65.1 (Cq), 114.4,
Compounds VIIIi–l were synthesized according to the reported
126.6, 128.3, 128.5, 129.1 (CHar.), 138.4, 141.2, 157.6 (3 x Car.),
procedure.[29]
171.07, 176.64 (2 x C O).


ABOUL-ENEIN ET AL.
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|
4.1.7 General procedure for the synthesis of 1-((2-
(CH2-C6H5), 52.67 (CH2-CH2-OH), 55.49 (OCH3), 60.034 (CH2-OH),
76.92 (Cq), 113.97, 127.29, 127.87, 128.67, 129.02 (CHar.), 138.4,
hydroxyethyl)(aryl)amino)-N-substituted cycloalkane
carboxamides IXa–l

139.26, 156.91 (3 x Car.), 177.71 (C O). Anal. calcd. for C22H28N2O3:
C, 71.71; H, 7.66; N, 7.6. Found: C, 71.45; H, 7.7; N, 7.3.
Sodium borohydride (0.02 mol) was added portionwise during
5–10 min to the solution of the appropriate substituted diazaspir-
oalkanedione (VIIIa–l) (0.01 mol) in ethanol (30 mL) at room tempera-
ture. The mixture was stirred for 24 h, then the solvent was removed in
vacuum. Water (150 mL) was added and the mixture extracted with
dichloromethane (3 × 50 mL). The extracts were dried with anhydrous
sodium sulfate. Evaporation of the solvent gave the title compounds.
1-[(2-Hydroxyethyl)(4-methoxyphenyl)amino]-N-(2-
phenylethyl)cyclopentane carboxamide (IXd)
Yellow viscous oil, yield 89%. IR (KBr, cm−1): 3422 (OH), 3282 (NH) and
+

1639 (C O, amide); MS (EI) m/z (%): 383 (M +1, 33), 234 (100);
1H NMR (CDCl3) δ ppm 1.63–2.07 (m, 8H, 4 x CH2, cyclopentyl), 2.81
(t, J = 6.7 Hz, 2H, CH2-C6H5), 3.1 (t, J = 5.7 Hz, 2H, CH2-CH2-C6H5), 3
(t, J = 4.75 2H, CH2-CH2-OH), 3.3 (q, J = 5.7 Hz, 2H, CH2-OH), 3.7 (s,
3H, OCH3), 6.77 (d, 2H, J = 8.6 Hz, Har.), 7.03 (d, 2H, J = 8.6 Hz, Har.),
7.23–7.3 (m, 5H, Har.), 8.2 (s, 1H, NH, D2O exchangeable), OH (spread
over the chart); 13C NMR (CDCl3) δ ppm 24.96, 34.7 (4 x CH2,
cyclopentyl), 43.56 (CH2-C6H5), 48 (CH2-CH2-C6H5), 52.67 (CH2-
CH2-OH), 55.49 (OCH3), 60.034 (CH2-OH), 76.92 (Cq), 113.97,
127.29, 127.87, 128.67, 129.7 (CHar.), 138.4, 139.26, 156.91 (3 x
N-Benzyl-1-[(2-hydroxyethyl)(4-methylphenyl)amino]-
cyclopentane carboxamide (IXa)
Brown solid, m.p. 115–117°C, yield 80%. IR (KBr, cm−1): 3432 (OH),
+

3305 (NH) and 1644 (C O, amide); MS (EI) m/z (%): 353 (M +1, 21), 91
(100), 218 (29); 1H NMR (CDCl3) δ ppm 1.68–2.1 (m, 8H, 4 x CH2,
cyclopentyl), 2.28 (s, 3H, CH3), 3.2 (t, J = 4.5 Hz, 2H, CH2-CH2-OH), 3.4
(q, J = 5 Hz, 2H, CH2-OH), 4.4 (d,J = 5.7 Hz, 2H, CH2-C6H5), 6.9 (d, 2H,
J = 8.4 Hz, Har.), 7.02 (d, 2H, J = 8.4 Hz, Har.), 7.25–7.28 (m, 5H, Har.),
8.2 (s, 1H, NH, D2O exchangeable), OH (spread over the chart);
13C NMR (CDCl3) δ ppm 20.83 (CH3), 24.75, 35.11 (4 x CH2,
cyclopentyl), 43.59 (CH2-C6H5), 51.2 (CH2-CH2-OH), 60.1 (CH2-OH),
76.93 (Cq), 125.07, 127.21, 127.77, 128.61, 129.42 (CHar.), 133.01,
139.17, 143.03 (3 x Car.), 177.77 (CO). Anal. calcd. for C22H28N2O2: C,
74.97; H, 8.01; N, 7.95. Found: C, 74.5; H, 7.88; N, 7.7.

Car.), 177.71 (C O). Anal. calcd. for C23H30N2O3: C, 72.22; H, 7.91; N,
7.32. Found: C, 72.5; H, 7.8; N, 7.2.
N-Benzyl-1-[(2-hydroxyethyl)(4-methylphenyl)amino]-
cyclohexanecarboxamide (IXe)
Yellow solid, m.p. 140°C, yield 90%. IR (KBr, cm−1): 3429 (OH), 3313
+

(NH) and 1631 (C O, amide); MS (EI) m/z (%): 367 (M +1, 21), 232
(100); 1H NMR (CDCl3) δ ppm 1.45–1.95 (m, 10H, 5 x CH2,
cyclohexyl), 2.3 (s, 3H, CH3), 3.17 (t, J = 5.7 Hz, 2H, CH2-CH2-OH),
3.26 (q, J = 6.5 Hz, 2H, CH2-OH), 4.4 (d, J = 5.7 Hz, 2H, CH2-C6H5),
6.94 (d, 2H, J = 8.4 Hz, Har.), 7.07 (d, 2H, J = 8.4 Hz, Har.), 7.23–7.35
(m, 5H, Har.), 7.43 (s, 1H, NH, D2O exchangeable), OH (spread over
the chart); 13C NMR (CDCl3) δ ppm 20.86, 22.7, 25.44 (5 x CH2,
cyclohexyl), 33.04 (CH3), 35.56 (CH2-C6H5), 50.25 (CH2-CH2-OH),
59.8 (CH2-OH), 66.18 (Cq), 126.37, 127.72, 128.52, 128.91, 129.14
1-[(2-Hydroxyethyl)(4-methylphenyl)amino]-N-(2-phenylethyl)-
cyclopentane carboxamide (IXb)
Yellow viscous oil, yield 86%. IR (KBr, cm−1): 3432 (OH), 3298 (NH) and
+

1642 (C O, amide); MS (EI) m/z (%): 376 (M +1, 21), 218 (100);
1H NMR (CDCl3) δ ppm 1.64–2.04 (m, 8H, 4 x CH2, cyclopentyl), 2.27
(s, 3H, CH3), 2.79 (t, J = 6.7 Hz, 2H, CH2-C6H5), 3.1 (t, J = 6.7 Hz, 2H,
CH2-CH2-C6H5), 3.3 (t, J = 4.8 Hz, 2H, CH2-CH2-OH), 3.5 (q, J = 6.7 Hz,
2H, CH2-OH), 6.9 (d, 2H, J = 8.6 Hz, Har.), 7.03 (d, 2H, J = 8.6 Hz, Har.),
7.13–7.27 (m, 5H, Har.), 7.91 (s, 1H, NH, D2O exchangeable), OH
(spread over the chart); 13C NMR (CDCl3) δ ppm 20.86 (CH3), 24.76,
35.17 (4 x CH2, cyclopentyl), 35.64 (CH2-C6H5), 40.76 (CH2-CH2-
C6H5), 50.68 (CH2-CH2-OH), 60.04 (CH2-OH), 76.95 (Cq), 124.42,
128.51, 129.06, 129.13, 129.35 (CHar.), 133, 139.53, 143.03 (3 x Car.),

(CHar.), 134.15, 139.46, 142.29 (3 x Car.), 176.54 (C O). Anal. calcd.
for C23H30N2O2: C, 75.37; H, 8.25; N, 7.64. Found: C, 75.8; H, 8.01;
N, 7.21.
1-[(2-Hydroxyethyl)(4-methylphenyl)amino]-N-(2-phenylethyl)-
cyclohexanecarboxamide (IXf)
Yellow viscous oil, yield 82%. IR (KBr, cm−1): 3432 (OH), 3282 (NH)
+

177.8 (C O). Anal. calcd. for C23H30N2O2: C, 75.37; H, 8.25; N, 7.64.

and 1639 (C O, amide); MS (EI) m/z (%): 381 (M +1, 21), 232 (100),
91 (76); 1H NMR (CDCl3) δ ppm 1.45–1.95 (m, 10H, 5 x CH2,
cyclohexyl), 2.32 (s, 3H, CH3), 2.87 (t, J = 10 Hz, 2H, CH2-C6H5), 3.27
(t, J = 10 Hz, 2H, CH2-CH2-C6H5), 3.33 (t, J = 5 Hz, 2H, CH2-CH2-
OH), 3.55 (q, J = 10 Hz, 2H, CH2-OH), 6.94 (d, 2H, J = 10 Hz, Har.),
7.07 (d, 2H, J = 10 Hz, Har.), 7.23–7.35 (m, 5H, Har.), 7.43 (s, 1H, NH,
D2O exchangeable), OH (spread over the chart); 13C NMR (CDCl3) δ
ppm 20.86, 22.7, 25.44 (5 x CH2, cyclohexyl), 33.04 (CH3), 35.56
(CH2-C6H5), 40.47 (CH2-CH2-C6H5), 50.25 (CH2-CH2-OH), 59.8
(CH2-OH), 66.18 (Cq), 126.37, 127.72, 128.52, 128.91, 129.14
Found: C, 74.9; H, 7.8; N, 7.3.
N-Benzyl-1-[(2-hydroxyethyl)(4-methoxyphenyl)amino]-
cyclopentane carboxamide (IXc)
White solid, m.p. 106–108°C, yield 80%. IR (KBr, cm−1): 3461 (OH),
+

3308 (NH) and 1644 (C O, amide); MS (EI) m/z (%): 369 (M +1, 27),
234 (100); 1H NMR (CDCl3) δ ppm 1.64–2.07 (m, 8H, 4 x CH2,
cyclopentyl), 3.07 (t, J = 4.75, 2H, CH2-CH2-OH), 3.3 (q, J = 5.7 Hz, 2H,
CH2-OH), 3.7 (s, 3H, OCH3), 4.43 (d, J = 5.75, 2H, CH2-C6H5), 6.7 (d,
2H, J = 8.6 Hz, Har.), 7.03 (d, 2H, J = 8.6 Hz, Har.), 7.23–7.3 (m, 5H, Har.),
8.2 (s, 1H, NH, D2O exchangeable), OH (spread over the chart);
13C NMR (CDCl3) δ ppm 24.96, 34.7 (4 x CH2, cyclopentyl), 43.56

(CHar.), 134.15, 139.46, 142.29 (3 x Car.), 176.54 (C O). Anal. calcd.
for C24H32N2O2: C, 75.75; H, 8.48; N, 7.36. Found: C, 75.5; H, 8.8;
N, 7.3.

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N-Benzyl-1-[(2-hydroxyethyl)(4-methoxyphenyl)amino]-
J = 6.5 Hz, 2H, CH2-OH), 6.99 (d, 2H, J = 5 Hz, Har.), 7.03 (d, 2H,
J = 5 Hz, Har.), 7.2–7.29 (m, 5H, Har.), 7.86 (s, 1H, NH,
D2O exchangeable), OH (spread over the chart); 13C NMR (CDCl3) δ
ppm 20.93 (CH3), 23.83, 30.8, 35.18 (6 x CH2, cycloheptyl), 35.53
(CH2-C6H5), 40.65 (CH2-CH2-C6H5), 51.65 (CH2-CH2-OH), 59.78
(CH2-OH), 70.38 (Cq), 126.34, 127.73, 128.33, 128.94, 129.32 (CHar.),
cyclohexanecarboxamide (IXg)
White solid, m.p. 100°C, yield 88%. IR (KBr, cm−1): 3432 (OH), 3282
+

(NH) and 1636 (C O, amide); MS (EI) m/z (%): 382 (M +1, 10), 248
(100), 91 (55); 1H NMR (CDCl3) δ ppm 1.42–2.1 (m, 10H, 5 x CH2,
cyclohexyl), 3.1 (t, J = 6.5 Hz, 2H, CH2-CH2-OH), 3.28 (q, J = 6.5 Hz,
2H, CH2-OH), 3.75 (s, 3H, OCH3), 4.4 (d, J = 6 Hz 2H, CH2-C6H5), 6.74
(d, 2H, J = 11 Hz, Har.), 7.02 (d, 2H, J = 11 Hz, Har.), 7.29–7.313 (m, 5H,
Har.), 7.6 (s, 1H, NH, D2O exchangeable), OH (spread over the chart);
13C NMR (CDCl3) δ ppm 22.89, 25.47, 33.09 (5 x CH2, cyclohexyl),
43.41 (CH2-C6H5), 51.02 (CH2-CH2-OH), 55.36 (OCH3), 59.8 (CH2-
OH), 66.35 (Cq), 113.7, 127.28, 127.95, 128.62, 129.8 (CHar.), 137.66,

134.81, 139.6, 142.83 (3 x Car.), 178.17 (C O). Anal. calcd. for
C25H34N2O2: C, 76.1; H, 8.69; N, 7.1. Found: C, 75.84; H, 8.9; N, 6.92.
N-Benzyl-1-[(2-hydroxyethyl)(4-methoxyphenyl)amino]-
cycloheptanecarboxamide (IXk)
White solid, m.p. 73–76°C, yield 80%. IR (KBr, cm−1): 3389 (OH), 3286
+


139.04, 157.05 (3 x Car.), 176.23 (C O). Anal. calcd. for C23H30N2O3:
(NH) and 1641 (C O, amide); MS (EI) m/z (%): 397 (M +1, 21), 263
C, 72.22; H, 7.91; N, 7.32. Found: C, 71.78; H, 8.01; N, 7.21.
(100); 1H NMR (CDCl3) δ ppm 1.26–2.12 (m, 12H, 6 x CH2,
cycloheptyl), 2.9 (t, J = 5.7 Hz, 2H, CH2-CH2-OH), 3.2 (q, J = 4.8 Hz,
2H, CH2-OH), 3.7 (s, 3H, OCH3), 4.4 (d, J = 6.7 Hz, 2H, CH2-C6H5), 6.8
(d, 2H, J = 8.6 Hz, Har.), 7.1 (d, 2H, J = 8.6 Hz, Har.), 7.2–7.3 (m, 5H, Har.),
8 (s, 1H, NH, D2O exchangeable), OH (spread over the chart); 13C NMR
(CDCl3) δ ppm 23.82, 30.58, 34.86 (6 x CH2, cycloheptyl), 43 (CH2-
C6H5), 52.06 (CH2-CH2-OH), 55.4 (OCH3), 59.8 (CH2-OH), 70.18 (Cq),
113.6, 128.4, 128.6, 129.01, 129.1 (CHar.), 138.2., 139.7, 157.5 (3 x
1-[(2-Hydroxyethyl)(4-methoxyphenyl)amino]-N-(2-
phenylethyl)-cyclohexanecarboxamide (IXh)
Yellow viscous oil, yield 80%. IR (KBr, cm−1): 3419 (OH), 3282 (NH) and
+

1631 (C O, amide); MS (EI) m/z (%): 397 (M +1, 21), 248 (100);
1H NMR (CDCl3) δ ppm 1.23–1.859 (m, 10H, 5 x CH2, cyclohexyl), 2.84
(t, J = 6.7 Hz, 2H, CH2-C6H5), 3.13 (t, J = 6.5 Hz, 2H, CH2-CH2-C6H5),
3.23 (t, J = 6.7 Hz, 2H, CH2-CH2-OH), 3.52 (q, J = 6.7 Hz, 2H, CH2-OH),
3.77 (s, 3H, OCH3), 6.77 (d, 2H, J = 8.4 Hz, Har.), 6.96 (d, 2H, J = 8.4 Hz,
Har.), 7.202–7.293 (m, 5H, Har.), 7.4 (s, 1H, NH, D2O exchangeable),
OH (spread over the chart); 13C NMR (CDCl3) δ ppm 22.87, 25.51,
33.15 (5 x CH2, cyclohexyl), 35.59 (CH2-C6H5), 40.5 (CH2-CH2-C6H5),
50.84 (CH2-CH2-OH), 55.46 (OCH3), 59.83 (CH2-OH), 66.29 (Cq),
114.37, 128.62, 128.52, 128.95, 129.78 (CHar.), 137.7, 139.49, 157.1

Car.), 178.17 (C O). Anal. calcd. for C24H32N2O3: C, 72.7; H, 8.13; N,
7.06. Found: C, 72.49; H, 7.98; N, 6.9.
1-[(2-Hydroxyethyl)(4-methoxyphenyl)amino]-N-(2-
phenylethyl)-cycloheptanecarboxamide (IXl)
Colourless viscous oil, yield 85%. IR (KBr, cm−1): 3440 (OH), 3282 (NH)
+

and 1636 (NH-C O, amide); MS (EI) m/z (%): 411 (M +1, 25), 98 (100);
1H NMR (CDCl3) δ ppm 1.4–2.01 (m, 12H, 6 x CH2, cycloheptyl), 2.86
(t, J = 6.7 Hz, 2H, CH2-C6H5), 2.91 (t, J = 6.5 Hz, 2H, CH2-CH2-C6H5),
3.17 (t, J = 6.7 Hz, 2H, CH2-CH2-OH), 3.51 (q, J = 6.7 Hz, 2H, CH2-OH),
3.77 (s, 3H, OCH3), 6.77 (d, 2H, J = 8.6 Hz, Har.), 7.04 (d, 2H, J = 8.6 Hz,
Har.), 7.22–7.3 (m, 5H, Har.), 7.8 (s, 1H, NH, D2O exchangeable), OH
(spread over the chart); 13C NMR (CDCl3) δ ppm 23.91, 31.03, 35.15 (6
x CH2, cycloheptyl), 35.56 (CH2-C6H5), 40.71 (CH2-CH2-C6H5), 52.21
(CH2-CH2-OH), 55.45 (OCH3), 59.81 (CH2-OH), 70 (Cq), 113.88,
128.58, 128.64, 129.04, 129.09 (CHar.), 138.17, 139.73, 157.43 (3 x

(3 x Car.), 176.28 (C O). Anal. calcd. for C24H32N2O3: C, 72.7; H, 8.13;
N, 7.06. Found: C, 72.94; H, 8.2; N, 7.21.
N-Benzyl-1-[(2-hydroxyethyl)(4-methylphenyl)amino]-
cycloheptanecarboxamide (IXi)
Yellow solid, m.p. 108°C, yield 80%. IR (KBr, cm−1): 3389 (OH), 3286
+

(NH) and 1641 (C O, amide); MS (EI) m/z (%): 381 (M +1, 21), 98
(100), 246 (77); 1H NMR (DMSO) δ ppm 1.31–2.01 (m, 12H, 6 x CH2,
cycloheptyl), 2.22 (s, 3H, CH3), 3 (t, J = 4.5 Hz, 2H, CH2-CH2-OH), 3.2
(q, J = 5 Hz, 2H, CH2-OH), 4.2 (d, J = 5.75 Hz, 2H, CH2-C6H5), 7.04 (d,
2H, J = 8.4 Hz, Har.), 7.11 (d, 2H, J = 8.4 Hz, Har.), 7.2–7.25 (m, 5H, Har.),
8.6 (s, 1H, NH, D2O exchangeable), OH (spread over the chart);
13C NMR (CDCl3) δ ppm 20.93 (CH3), 23.82, 30.58, 34.86 (6 x CH2,
cycloheptyl), 42.77 (CH2-C6H5), 52.06 (CH2-CH2-OH), 59.13 (CH2-
OH), 70.18 (Cq), 127.08, 127.48, 127.89, 128.7, 129.41 (CHar.),

Car.), 178.14 (C O). Anal. calcd. for C25H34N2O3: C, 73.14; H, 8.35; N,
6.82. Found: C, 73.48; H, 8.19; N, 6.68.
|
4.1.8 General procedure for the synthesis of 2-((1-
substituted carbamoyl)cycloalkyl)(aryl)amino)ethyl
acetates Xa–l

133.65, 140.46, 144 (3 x Car.), 177.44 (C O). Anal. calcd. for
C
24H32N2O2: C, 75.75; H, 8.48; N, 7.36. Found: C, 75.9; H, 8.52; N, 7.5.
A solution of the hydroxyethyl amino derivative (0.071 mol), triethyl-
amine (194.6 mmol, 19.7 g), dimethylaminopyridine (1.22 mmol, 0.15 g),
acetic anhydride (142.03 mmol, 14.5 g), and dichloromethane (2.34 mol,
150 mL) was stirred overnight at about 20–25°C. To the resulting
solution water was added followed by addition of HCl till pH of the
resulting solution was 1–2. The solvent was distilled out completely.
Ethyl acetate was added to the resulting residue, washed with water,
dried using anhydrous Na2SO4 and the organic layer was evaporated
under reduced pressure to give the target compounds Xa–l.
1-[(2-Hydroxyethyl)(4-methylphenyl)amino]-N-(2-phenylethyl)-
cycloheptanecarboxamide (IXj)
White solid, m.p. 78–80°C, yield 90%. IR (KBr, cm−1): 3432 (OH), 3319
+

(NH) and 1645 (C O, amide); MS (EI) m/z (%): 395.2 (M +1, 21), 98
(100); 1H NMR (CDCl3) δ ppm 1.4–2.01 (m, 12H, 6 x CH2, cycloheptyl),
2.29 (s, 3H, CH3), 2.84 (t, J = 5 Hz, 2H, CH2-C6H5), 3 (t, J = 5.5 Hz, 2H,
CH2-CH2-C6H5), 3.22 (t, J = 5.5 Hz, 2H, CH2-CH2-OH), 3.5 (q,

ABOUL-ENEIN ET AL.
15 of 19
|
Purified by column chromatography using a mixture of methylene
chloride/ethyl acetate (7:3) as a mobile phase.
2-{(4-Methoxyphenyl)[1-((2-phenylethyl)carbamoyl)cyclopentyl]-
amino}ethyl acetate (Xd)
Yellow viscous oil, yield 78.4%. IR (KBr, cm−1): 3350 (NH), 1735 (C O,

2-{[1-(Benzylcarbamoyl)cyclopentyl](4-methylphenyl)amino}-
ethyl acetate (Xa)
ester carbonyl), 1652 (CO, amide carbonyl); MS (EI) m/z (%): 425.29
(M++1, 38.5), 276.14 (100); 1H NMR (CDCl3) δ ppm 1.3–2.04 (m, 8H, 4
Yellow solid, m.p. 80–82°C, yield 88%. IR (KBr, cm−1): 3350 (NH), 1735
x CH2, cyclopentyl), 1.84 (s, 3H, O C-CH3), 2.81 (t, J = 6.7 Hz, 2H,



(C O, ester carbonyl), 1652 (C O, amide carbonyl); MS (EI) m/z (%):
395.3 (M++1, 38.5), 260 (100); 1H NMR (CDCl3) δ ppm 1.33–2.08 (m,
CH2-C6H5), 3.06 (t, J = 5.75 Hz, 2H,CH2-CH2-O), 3.53 (q, J = 6.7 Hz,
2H, CH2-CH2-C6H5), 3.72 (t, J = 5.75 Hz, 2H, CH2-OH), 3.76 (s, 3H,
OCH3), 6.77 (d, 2H, J = 8.4 Hz, Har.), 6.92 (d, 2H, J = 8.4 Hz, Har.), 7.17–
7.28 (m, 5H, Har.), 7.7 (s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3)

8H, 4 x CH2, cyclopentyl), 1.7 (s, 3H, O C-CH3), 2.26 (s, 3H, CH3), 3.2
(t, J = 5.75 Hz, 2H, CH2-CH2-O), 3.82 (t, J = 5.75 Hz, 2H, CH2-O), 4.4 (d,
J = 5.75 Hz, 2H, CH2-C6H5), 6.85 (d, 2H, J = 8.4 Hz, Har.), 7.04 (d, 2H,
J = 8.4 Hz, Har.), 7.13–7.27 (m, 5H, Har.), 7.5 (s, 1H, NH,
D2O exchangeable); 13C NMR (CDCl3) δ ppm 20.84 (CH3), 20.8

δ ppm 20.82 (O C-CH3), 25.25, 34.38 (4 x CH2, cyclopentyl), 35.93
(CH2-C6H5), 40.81 (CH2-CH2-C6H5), 49.55 (CH2-CH2-O), 55.46
(OCH3), 62.51 (CH2-O), 76.96 (Cq), 114.06, 126.53, 128.66, 128.69,


(O C-CH3), 25.19, 34.78 (4 x CH2, cyclopentyl), 42.3 (CH2-C6H5),
128.93 (CHar.), 138.09, 139.26, 157.28 (3 x Car.), 170.7 (O C-CH3),

48.68 (CH2-CH2-O), 62.64 (CH2-O), 76.94 (Cq), 125.41, 126.53,
128.64, 128.92, 129.54 (CHar.), 133.87, 139.27, 143 (3 x Car.), 170.67
177.68 (C O). Anal. calcd. for C25H32N2O4: C, 70.73; H, 7.6; N, 6.6.
Found: C, 70.47; H, 7.52; N, 6.57.


(O C-CH3), 177.35 (C O). Anal. calcd. for C24H30N2O3: C, 73.07; H,
7.67; N, 7.1. Found: C, 73.46; H, 7.78; N, 7.21.
2-{[1-(Benzylcarbamoyl)cyclohexyl)(4-methylphenyl)amino}ethyl
acetate (Xe)
2-{(4-Methylphenyl)[1-((2-phenylethyl)carbamoyl)cyclopentyl]-
amino}ethyl acetate (Xb)
Yellow viscous oil, yield 88%. IR (KBr, cm−1): 3389 (NH), 1738 (CO,
White solid, m.p. 76–78°C, yield 90%. IR (KBr, cm−1): 3344 (NH),


1733 (C O, ester carbonyl), 1648 (C O, amide carbonyl); MS (EI)
m/z (%): 409.5 (M++1, 21), 98 (100); 1H NMR (CDCl3) δ ppm 1.33–


ester carbonyl), 1652 (C O, amide carbonyl); MS (EI) m/z (%): 409
(M++1, 21), 260 (100); 1H NMR (CDCl3) δ ppm 1.35–2.06 (m, 8H, 4 x
1.97 (m, 10H, 5 x CH2, cyclohexyl), 1.75 (s, 3H, O C-CH3), 2.3 (s,
3H, CH3), 3.2 (t, J = 5.75 Hz, 2H, CH2-CH2-O), 3.7 (t, J = 5.75 Hz,
2H, CH2-O), 4.48 (d, J = 5.75 Hz, 2H, CH2-C6H5), 6.9 (d, 2H,
J = 8.6 Hz, Har.), 7 (d, 2H, J = 8.6 Hz, Har.), 7.25–7.33 (m, 5H, Har.),
7.4 (s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3) δ ppm 20.66

CH2, cyclopentyl), 1.86 (s, 3H, O C-CH3), 2.28 (s, 3H, CH3), 2.79 (t,
J = 5.75 Hz, 2H, CH2-C6H5), 3.1 (t, J = 5.75 Hz, 2H, CH2-CH2-O), 3.52
(q, J = 5.75 Hz, 2H, CH2-CH2-C6H5), 3.82 (t, J = 5.75 Hz, 2H, CH2-O),
6.85 (d, 2H, J = 8.4 Hz, Har.), 7.04 (d, 2H, J = 8.4 Hz, Har.), 7.13–7.27 (m,
5H, Har.), 7.5 (s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3) δ ppm

(CH3), 20.97 (CH3-C O), 23.04, 25.48, 32.95 (5 x CH2, cyclohexyl),
43.39 (CH2-C6H5), 47.93 (CH2-CH2-O), 62.81 (CH2-O), 66.83 (Cq),

20.84 (CH3), 20.8 (O C-CH3), 25.19, 34.78 (4 x CH2, cyclopentyl),
127.43, 127.71, 128.76, 128.87, 129.37 (CHar.), 135.4, 139.04,


35.94 (CH2-C6H5), 40.82 (CH2-CH2-C6H5), 48.68 (CH2-CH2-O), 62.64
(CH2-O), 76.94 (Cq), 125.41, 126.53, 128.64, 128.92, 129.54 (CHar.),
142.24 (3 x Car.), 170.76 (O C-CH3), 176.41 (NH-C O). Anal.
calcd. for C25H32N2O3: C, 73.5; H, 7.9; N, 6.86. Found: C, 73.82; H,
7.79; N, 6.82.


133.87, 139.27, 143 (3 x Car.), 170.67 (O C-CH3), 177.35 (C O).
Anal. calcd. for C25H32N2O3: C, 73.5; H, 7.9; N, 6.86. Found: C, 73.75;
H, 7.79; N, 6.605.
2-{(4-Methylphenyl)[1-((2-phenylethyl)carbamoyl)cyclohexyl]-
amino}ethyl acetate (Xf)
2-{[1-(Benzylcarbamoyl)cyclopentyl](4-methoxyphenyl)amino}-
ethyl acetate (Xc)
White solid, m.p. 45°C, yield 90%. IR (KBr, cm−1): 3344 (NH),


1735 (C O, ester carbonyl), 1646 (C O, amide carbonyl); MS
Yellow viscous oil, yield 66%. IR (KBr, cm−1): 3372 (NH), 1738 (C O,
(EI) m/z (%): 423 (M++1, 25.3), 274 (100); 1H NMR (CDCl3) δ ppm



ester carbonyl), 1662 (C O, amide carbonyl); MS (EI) m/z (%): 411.32
(M++1, 66), 276.05 (100); 1H NMR (CDCl3) δ ppm 1.35–2.08 (m, 8H, 4 x
1.47–1.89 (m, 10H, 5 x CH2, cyclohexyl), 1.8 (s, 3H, O C-CH3),
2.3 (s, 3H, CH3), 2.8 (t, J = 6.7 Hz, 2H, CH2-C6H5), 3.2 (t, J = 6.7 Hz,
2H, CH2-CH2-O), 3.5 (q, J = 6.7 Hz, 2H, CH2-CH2-C6H5), 3.7 (t,
J = 6.7 Hz, 2H, CH2-O), 6.9 (d, 2H, J = 8.6 Hz, Har.), 7 (d, 2H,

CH2, cyclopentyl), 1.73 (s, 3H, O C-CH3), 3.16 (t, J = 6.7 Hz, 2H, CH2-
CH2-O), 3.75 (s, OCH3), 3.8 (t, J = 6.7 Hz, 2H, CH2-O), 4.4 (d,
J = 5.75 Hz, 2H, CH2-C6H5), 6.8 (d, 2H, J = 8.4 Hz, Har.), 7.02 (d, 2H,
J = 8.6 Hz,
H
ar.), 7.16–7.25 (m, 5H, Har.), 7.31 (s, 1H, NH,
ppm 20.86 (CH3),
J = 8.4 Hz, Har.), 7.23–7.3 (m, 5H, Har.), 7.9 (s, 1H, NH,
13
D2O exchangeable); 13C NMR (CDCl3)
δ


D2O exchangeable); C NMR (CDCl3) δ ppm 20.5 (O C-CH3),
20.98 (CH3-C O), 22.96, 25.46, 32.78 (5 x CH2, cyclohexyl),
36.01 (CH2-1C6H5), 40.54 (CH2-CH2-C6H5), 47.83 (CH2-CH2-O),
62.83 (CH2-O), 66.67 (Cq), 126.56, 127.72, 128.68, 128.85,
25.21, 34.37 (4 x CH2, cyclopentyl), 43.59 (CH2-C6H5), 49.78 (CH2-
CH2-O), 55.46 (OCH3), 62.47 (CH2-O), 76.94 (Cq), 114.11, 127.41,
127.67, 128.75, 128.81 (CHar.), 138.1, 139.04, 157.36 (3 x Car.),

129.33 (CHar.), 135.42, 139.25, 142.35 (3 x Car.), 170.67 (O C-


170.76 (OC-CH3), 177.54 (C O). Anal. calcd. for C24H30N2O4: C,
CH3), 176.36 (NH-C O). Anal. calcd. for C26H34N2O3: C, 73.9; H,
70.22; H, 7.37; N, 6.82. Found: C, 70.06; H, 7.5; N, 6.74.
8.11; N, 6.63. Found: C, 73.65; H, 8.04; N, 6.58.

16 of 19
ABOUL-ENEIN ET AL.
|
2-{[1-(Benzylcarbamoyl)cyclohexyl](4-methoxyphenyl)amino}-
ethyl acetate (Xg)
437 (M++1, 3), 288 (100); 1H NMR (CDCl3) δ ppm 1.4–2.07 (m, 12H, 6 x

CH2, cycloheptyl), 1.85 (s, 3H, O C-CH3), 2.29 (s, 3H, CH3), 2.83 (t,
Yellow viscous oil, yield 80%. IR (KBr, cm−1): 3384 (NH), 1734 (C O,
J = 6.7 Hz, 2H, CH2-C6H5), 3.06 (t, J = 5.75 Hz, 2H,CH2-CH2-O), 3.53
(q, J = 6.65 Hz, 2H, CH2-CH2-C6H5), 3.67 (t, J = 5.75 Hz, 2H, CH2-O),
6.98 (d, 2H, J = 8.4 Hz, Har.), 7.05 (d, 2H, J = 8.4 Hz, Har.), 7.2–7.28 (m,
5H, Har.), 7.5 (s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3) δ ppm


ester carbonyl), 1648 (C O, amide carbonyl); MS (EI) m/z (%): 425
(M++1, 11), 290 (100); 1H NMR (CDCl3) δ ppm 1.2–2 (m, 10H, 5 x CH2,

cyclohexyl), 1.5 (s, 1H, NH, D2O exchangeable), 1.87 (s, 3H, O C-

CH3), 3.2 (t, J = 5.75 Hz, 2H, CH2-CH2-O), 3.7 (t, J = 5.75 Hz, 2H, CH2-
O), 3.81 (s, 3H, OCH3), 4.5 (d, J = 7.5 Hz, 2H, CH2-C6H5), 6.8 (d, 2H,
J = 11 Hz, Har.), 7 (d, 2H, J = 11 Hz, Har.), 7.29–7.238 (m, 5H, Har.);
13C NMR (CDCl3) δ ppm 20.7, 25.51, 32.92 (5 x CH2, cyclohexyl), 23.06
20.84 (CH3), 21.02 (O C-CH3), 23.93, 30.92, 34.91 (6 x CH2,
cycloheptyl), 36.06 (CH2-C6H5), 40.78 (CH2-CH2-C6H5), 48.85 (CH2-
CH2-O), 62.44 (CH2-O), 70.1 (Cq), 126.55, 128.65, 128.71, 128.9,

129.52 (CHar.), 135.53, 139.36, 142.63 (3 x Car.), 170.61 (O C-CH3),


(CH3-C O), 43.38 (CH2-C6H5), 48.22 (CH2-CH2-O), 55.44 (OCH3),
177.63 (NH-C O). Anal. calcd. for C27H36N2O3: C, 74.28; H, 8.31; N,
62.79 (CH2-O), 66.84 (Cq), 113.86, 127.44, 127.73, 128.77, 130.37
6.42. Found: C, 73.8; H, 7.94; N, 6.22.

(CHar.), 137.58, 139.03, 157.64 (3 x Car.), 170.78 (O C-CH3), 176.3

(NH-C O). Anal. calcd. for C25H32N2O4: C, 70.73; H, 7.6; N, 6.6.
2-{[1-(Benzylcarbamoyl)cycloheptyl](4-methoxyphenyl)amino}-
ethyl acetate (Xk)
Found: C, 70.49; H, 7.73; N, 6.58.
White solid, m.p. 80°C, yield 90%. IR (KBr, cm−1): 3388 (NH), 1725


2-{(4-Methoxyphenyl)[1-((2-phenylethyl)carbamoyl)cyclohexyl]-
amino}ethyl acetate (Xh)
(C O, ester carbonyl), 1673 (C O, amide carbonyl); MS (EI) m/z (%):
439 (M++1, 3.17), 304 (100); 1H NMR (CDCl3) δ ppm 1.4–2.16 (m, 12H,
Light brown solid, m.p. 76–78°C, yield 90%. IR (KBr, cm−1): 3335 (NH),
6 x CH2, cycloheptyl), 1.75 (s, 3H, O C-CH3), 3.1 (t, J = 5.7 Hz, 2H,



1732 (C O, ester carbonyl), 1647 (C O, amide carbonyl); MS (EI) m/z
(%): 439.3 (M++1, 15), 290 (100); 1H NMR (CDCl3) δ ppm 0.9 (s, 1H,
NH, D2O exchangeable), 1.2–1.83 (m, 10H, 5 x CH2, cyclohexyl), 1.87
CH2-CH2-O), 3.7 (t, J = 5.7 Hz, 2H, CH2-O), 3.77 (s, 3H, OCH3), 4.4 (d,
J = 6.7 Hz, 2H, CH2-C6H5), 6.78 (d, 2H, J = 8.6 Hz, Har.), 7 (d, 2H,
J = 8.6 Hz, Har.), 7.24–7.31 (m, 5H, Har.), 7.84 (s, 1H, NH,
D2O exchangeable); 13C NMR (CDCl3) δ ppm 20.61 (OC-CH3),
23.95, 30.98, 34.93 (6 x CH2, cycloheptyl), 43.51 (CH2-C6H5), 49.27
(CH2-CH2-O), 55.44 (OCH3), 62.34 (CH2-O), 71.05 (Cq), 114.04,
127.4, 127.62, 128.75, 130.21 (CHar.), 137.82, 139.14, 157.83 (3 x

C-CH3), 2.8 (t, J = 6.7 Hz, 2H, CH2-C6H5), 3.16 (t,
(s, 3H,
O
J = 5.75 Hz, 2H, CH2-CH2-O), 3.5 (q, J = 5.75 Hz, 2H, CH2-CH2-
C6H5), 3.7 (t, J = 5.75 Hz, 2H, CH2-O), 3.8 (s, 3H, OCH3), 6.7 (d, 2H,
J = 8.6 Hz, Har.), 6.9 (d, 2H, J = 8.6 Hz, Har.), 7.2–7.31 (m, 5H, Har.);
13C NMR (CDCl3) δ ppm 20.88, 25.49, 32.9 (5 x CH2, cyclohexyl), 22.99
(CH3-CO), 36 (CH2-C6H5), 40.5 (CH2-CH2-C6H5), 48.14 (CH2-CH2-O),
55.44 (OCH3), 62.82 (CH2-O), 66.69 (Cq), 113.84, 126.57, 128.69,


Car.), 170.69 (O C-CH3), 177.66 (NH-C O). Anal. calcd. for
C26H34N2O4: C, 71.21; H, 7.81; N, 6.39. Found: C, 71.56; H, 7.93;
N, 6.16.

128.86, 130.34 (CHar.), 137.69, 139.25, 157.6 (3 x Car.), 170.7 (O C-

CH3), 176.26 (NH-C O). Anal. calcd. for C26H34N2O4: C, 71.21; H,
2-{(4-(Methoxyphenyl)[(1-((2-phenylethyl)carbamoyl)-
cycloheptyl]amino}ethyl acetate (Xl)
7.81; N, 6.39. Found: C, 70.9; H, 8.21; N, 5.923.
White solid, m.p. 90°C, yield 90%. IR (KBr, cm−1): 3334 (NH), 1734


2-{[1-(Benzylcarbamoyl)cycloheptyl)(4-methylphenyl)amino}-
ethyl acetate (Xi)
(C O, ester carbonyl), 1643 (C O, amide carbonyl); MS (EI) m/z (%):
453 (M++1, 0.39), 304 (100); 1H NMR (CDCl3) δ ppm 1.23–2.05 (m,
White solid, m.p. 88°C, yield 78%. IR (KBr, cm−1): 3338 (NH), 1732
12H, 6 x CH2, cycloheptyl), 1.85 (s, 3H, O C-CH3), 2.8 (t, J = 6.7 Hz,



(C O, ester carbonyl), 1647 (C O, amide carbonyl); MS (EI) m/z (%):
2H, CH2-C6H5), 3 (t, J = 5.75 Hz, 2H, CH2-CH2-O), 3.5 (q, J = 6.7 Hz,
2H, CH2-CH2-C6H5), 3.6 (t, J = 5.75 Hz, 2H, CH2-O), 3.77 (s, 3H,
OCH3), 6.7 (d, 2H, J = 8.6 Hz, Har.), 7 (d, 2H, J = 8.6 Hz, Har.), 7.2–7.31
(m, 5H, Har.), 7.5 (s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3) δ
+
1

423 (5) (M +1), 288 (100); H NMR (CDCl3) δ ppm 1.72 (s, 3H, O C-
CH3), 1.42–2.15 (m, 12H, 6 x CH2, cycloheptyl), 2.3 (s, 3H, CH3),
3.1 (t, J = 6.7 Hz, 2H, CH2-CH2-O), 3.72 (t, J = 6.7 Hz, 2H, CH2-O), 4.4
(d, J = 5.75 Hz, 2H, CH2-C6H5), 6.98 (d, 2H, J = 8.4 Hz, Har.), 7.05 (d, 2H,
J = 8.4 Hz, Har.), 7.2–7.3 (m, 5H, Har.), 7.8 (s, 1H, NH,
D2O exchangeable); 13C NMR (CDCl3) δ ppm 20.6 (CH3), 21.02

ppm 20.83 (O C-CH3), 23.92, 30.94, 34.85 (6 x CH2, cycloheptyl),
36.06 (CH2-C6H5), 40.75 (CH2-CH2-C6H5), 49.16 (CH2-CH2-O), 55.43
(OCH3), 62.39 (CH2-O), 70.87 (Cq), 114, 126.56, 128.65, 128.89,


(O C-CH3), 23.97, 30.95, 34.99 (6 x CH2, cycloheptyl), 43.53 (CH2-
130.18 (CHar.), 137.91, 139.35, 157.77 (3 x Car.), 170.59 (O C-CH3),

C6H5), 48.97 (CH2-CH2-O), 62.4 (CH2-O), 71.01 (Cq), 126.55, 128.65,
128.71, 128.9, 129.52 (CHar.), 135.77, 139.12, 142.55 (3 x Car.),
177.6 (NH-C O). Anal. calcd. for C27H36N2O4: C, 71.65; H, 8.02; N,
6.19. Found: C, 71.25; H, 8.42; N, 5.915.


170.72 (O C-CH3), 177.7 (NH-C O). Anal. calcd. for C26H34N2O3: C,
73.9; H, 8.11; N, 6.63. Found: C, 73.6; H, 8.01; N, 6.42.
|
4.2 Anticonvulsant activity
2-{(4-Methylphenyl)[1-((2-phenylethyl)carbamoyl)cycloheptyl]-
amino}ethyl acetate (Xj)
|
4.2.1 Animals and chemicals
White solid, m.p. 110°C, yield 88.8%. IR (KBr, cm−1): 3331 (NH), 1734
The anticonvulsant activity of the compounds under investigation


(C O, ester carbonyl), 1641 (C O, amide carbonyl); MS (EI) m/z (%):
IXa–l and Xa–l was tested on adult male albino mice weighing 19–25 g.

ABOUL-ENEIN ET AL.
17 of 19
|
|
All experimental procedures involving animals were performed
according to the guidelines of the ethical committee of the National
Research Centre for Experimental Animal Use. The mice were obtained
from Animals House Colony of the National Research Centre, Cairo,
Egypt and were housed in polypropylene cages under the standard
conditions of light (12 h light/dark cycle) and temperature (23 2°C).
The mice were allowed free access to water and standard chow and
were allowed to acclimatize to laboratory conditions before the start of
the experiment.
4.2.4 Neurotoxicity screen
Neurotoxicity is determined using rotarod test which is well-established
test for the detection of minimal neurological deficit. The mice were
trained to maintain equilibrium on a rotating 1-inch-diameter knurled
plastic rod at a speed of 10 rpm for at least 1 min in each of three trials
using a rotarod device (UGO Basile, 47600, Varese, Italy). Only animals
that fulfill this criterion were included into the experiment. The selected
trained mice were classified into control groupandexperimentalgroups.
The animalsinthe experimentalgroupsweregiventhereferencedrug or
one of the test compounds via i.p. route at dose which exerted 100%
protection in PTZ test; meanwhile, the control group received the
vehicle. Thirty minutes after the administration of the test compound or
vehicle, the mice were placed again on the rotating rod and the
neurotoxicity was indicated by the inability of the animal to maintain
equilibrium on the rod for at least 1 min.[53]
The animals were randomly assigned to control, reference and
tested experimental groups, each consisting of six mice. Each mouse
was used only once and all tests were performed between 09:00 a.m.
and 04:00 p.m. All the tested compounds were suspended in 7%
Tween 80 as a vehicle. All chemicals were purchased from Sigma–
Aldrich (St. Louis, MO, USA).
4.2.2 scPTZ-induced seizures test[45]
|
|
4.2.5 Serum liver enzyme activity
scPTZ test was performed to access the production of threshold or
minimal (clonic) seizures. PTZ, prepared as an aqueous solution, was
administered subcutaneously in the loose fold of the skin on the back
of the mice neck at a dose of 85 mg/kg (0.61 mmol/kg)[46] which is
known to produce clonic seizures that last for a period of at least 5 s in
97% (CD97) of the animals tested. Six mice were used in both the
control and the experimental groups. The control experiments were
performed using the solvent alone. The other groups, each of which
received individually the reference drugs, ethosuximide (150 mg/
kg ≡ 1.06 mmol/kg)[47] or phenobarbital[48] (30 mg/kg ≡ 0.13 mmol/
kg) or one of the test compounds in graded doses, IXa–l and Xa–l
(2.5–100 mg/kg, i.p.).
Mice were divided into six groups (n = 6). Group 1 served as control
group and received the vehicle (7% aqueous suspension of Tween 80)
only; Groups 2–6 received compound Xd, Xf, Xh, Xi, and Xl at doses (5,
10, 2.5, 10, and 10 mg/kg, i.p.), the doses that caused 100% protection
in the scPTZ test, dissolved in vehicle. The treatments were continued
out for a period of 7 days. Twenty-four hours after the last
administration of the compounds, the animals were anaesthetized.
The blood samples were collected by cardiac puncture followed by
centrifugation at 3000 rpm for 10 min for the separation of sera. The
serum samples obtained were used for the analyses of the liver
enzymes AST, ALT, and ALP, as well as total protein and albumin were
determined using commercially available kits (Biodiagnostic, Egypt).
The mice were observed for 30 min after PTZ administration for
the occurrence of seizures. A threshold convulsion is defined as one
episode of clonic convulsions which persist for at least 5 s. Failure to
observe such threshold seizure indicates the ability of the test
compound to abolish PTZ induced seizure threshold and is considered
as protection.[49,50]
|
4.2.6 Acute toxicity test
Various doses of the test compounds ranging from 10 to 300 mg/kg
dissolved in 7% tween 80 were given via i.p. to different groups of
healthy adult mice.[54] After the administration of the compounds, mice
were observed continuously for the first 2 h for any gross behavioral
changes and deaths, and occasionally for 4 h, then intermittently for the
next 24 h and for the onset of any delayed effects. The behavior of the
animal and any other toxic symptoms were also observed for 72 h and
then the animals were kept on observation for 14 days.[55]
4.2.3 MES test[51]
|
MES is a model used to identify generalized tonic-clonic seizures.
Animals were randomly assigned to groups each of six mice. The first
group served as the control group. The second group received
phenytoin (45 mg/kg) as a reference drug and the other groups of mice
received the test compounds individually by i.p. with the dose, which
induces 100% protection in pentylenetetrazole test, or higher. Thirty
minutes later, an electric current of fixed current intensity of 25 mA
and 0.2 s stimulus duration was produced via Rodent Shocker
generator (constant-current stimulator Type 221, Hugo Sachs
Elektronik, Freiburg, Germany) and delivered via ear-clip electrode
producing electroconvulsions in mice. The ability of the test compound
to overcome the tonic hind limb extension (i.e., 120° outstretching of
the mouse hind limb to the plane of the body axis) indicates its
capability to inhibit MES-induced seizure spread.[52]
|
4.3 Molecular modeling studies
The hypothesis was generated using common feature pharmacophore
model protocol in Discovery Studio 2016 Biova 1 software. The test
sets (lead compounds) 2–13 (Figure 5) which were reported to have
anticonvulsant activity were used to generate the anticonvulsant
pharmacophore. A set of conformational models of each structure of
the lead compounds was performed and used to generate the common
feature hypotheses, where ten hypotheses were generated (Support-
ing Information).

18 of 19
ABOUL-ENEIN ET AL.
|
|
4.4 Single X-ray crystallography
[22] M. N. Aboul-Enein, A. El-Azzouny, F. Ragab, M. S. Abdel-Maksoud,
Y. Maklad, Egypt Pharmaceut. J. 2014, 13, 1.
Single crystals of suitable dimensions were chosen for X-ray diffraction
studies. The X-ray intensity data were collected at a temperature of
298 K. All diagrams and calculations were performed using maXus.[56]
The structure of title compound was refined using SHELXL-97.
[23] M. N. Aboul-Enein, A. A. El-Azzouny, O. A. Saleh, K. M. Amin, Y. A.
Maklad, R. M. Hassan, Arch. Pharm. 2015, 348, 575.
[24] M. N. Aboul-Enein, A. A. El-Azzouny, Y. A. Maklad, M. A. Ismail, N. S.
Ismail, R. M. Hassan, Res. Chem. Intermediat. 2015, 41, 3767.
[25] K. Fink, D. J. Dooley, W. P. Meder, N. Suman-Chauhan, S. Duffy,
H. Clusmann, M. Göthert, Neuropharmacology 2002, 42, 229.
[26] N. O. Dalby, E. B. Nielsen, Epilepsy Res. 1997, 28, 63.
[27] R. W. Horton, G. M. Anlezark, M. C. B. Sawaya, B. S. Meldrum, Eur. J.
Pharmacol. 1977, 41, 387.
ACKNOWLEDGMENTS
The authors would like to thank the National Research Centre (ID:
60014618), Dokki, Giza, Egypt, for the support of this research. The
authors thank Dr. Walaa Hamada Abd-Allah for her assistance in
performing the molecular modeling study.
[28] X. Zhang, X. Liu, T. Gong, X. Sun, Z. Zhang, Acta Pharmacol. Sin. 2012,
33, 279.
[29] M. N. Aboul-Enein, A. A. El-Azzouny, M. I. Attia, Y. A. Maklad,
M. E. Aboutabl, F. Ragab, W. H. El-Hamid, Int. J. Mol. Sci. 2014, 15,
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CONFLICT OF INTEREST
[30] K. Unverferth, J. Engel, N. Höfgen, A. Rostock, R. Günther, H. Lankau,
M. Menzer, A. Rolfs, J. Liebscher, B. Müller, J. Med. Chem. 1998,
41, 63.
The authors have declared no conflict of interest.
[31] H. Henecka, P. Kurz, E. Müller, Methoden der Organischen Chemie. 4
Auflage, Stuttgart 1952, 3, 427.
ORCID
[32] P. R. Desai, D. R. Desai, J. S. Saharia, B. R. Saharia, J. Indian Chem. Soc.
1963, 40, 525.
Mohamed N. Aboul-Enein
http://orcid.org/0000-0001-8749-0556
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How to cite this article: Aboul-Enein MN, El-Azzouny AA,
Amin KM, Aboutabl ME, Abo-Elmagd MI. Synthesis,
molecular modeling studies, and anticonvulsant evaluation of
novel 1-((2-hydroxyethyl)(aryl)amino)-N-substituted
cycloalkanecarboxamides and their acetate esters. Arch
Pharm Chem Life Sci. 2018;1–19.
SUPPORTING INFORMATION
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Supporting Information section at the end of the article.
https://doi.org/10.1002/ardp.201800269