Design, synthesis, evaluation and molecular modeling studies of some novel N-substituted piperidine-3-carboxylic acid derivatives as potential anticonvulsants

Article abstract of DOI:10.1007/s00044-018-2141-9

Novel Schiff bases of 1-(2-Aminoethyl)piperidine-3-carboxylic acid were synthesized, characterized and screened for anticonvulsant activity. Compounds were evaluated for in vitro blood–brain barrier (BBB) permeability by parallel artificial membrane permeability BBB assay (PAMPA-BBB). Compounds 5d, 5f, 5j, 5l, 5m, 5n, 5w, 5x and 5y elicited considerable in vitro permeability across BBB and further screened for in vivo anticonvulsant activity by sc-PTZ and DMCM-induced seizure models. The outcome of the in vivo models suggested that 5d, 5w, and 5y were most potent amongst the synthesized compounds. The neurotoxicity evaluation of 5d, 5w, and 5y by rotarod indicates no impairment of muscle coordination in comparison to standard diazepam. The MTT assay revealed that the test compounds (5d, 5w, and 5y) were not found to alter the cell viability considerably. In silico molecular docking and dynamics simulations were carried out on the homology modeled protein of human GABA transporter 1 (GAT1), which exhibited complementary interactions of compound 5w within the active binding pocket.

Full text of DOI:10.1007/s00044-018-2141-9

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2141-9
ORIGINAL RESEARCH
Design, synthesis, evaluation and molecular modeling studies of
some novel N-substituted piperidine-3-carboxylic acid derivatives as
potential anticonvulsants
Ankit Seth1 Piyoosh A. Sharma1 Avanish Tripathi1 Priyanka K. Choubey1 Pavan Srivastava1
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Prabhash Nath Tripathi1 Sushant Kumar Shrivastava
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Received: 25 September 2017 / Accepted: 20 January 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Novel Schiff bases of 1-(2-Aminoethyl)piperidine-3-carboxylic acid were synthesized, characterized and screened for
anticonvulsant activity. Compounds were evaluated for in vitro blood–brain barrier (BBB) permeability by parallel artificial
membrane permeability BBB assay (PAMPA-BBB). Compounds 5d, 5f, 5j, 5l, 5m, 5n, 5w, 5x and 5y elicited considerable
in vitro permeability across BBB and further screened for in vivo anticonvulsant activity by sc-PTZ and DMCM-induced
seizure models. The outcome of the in vivo models suggested that 5d, 5w, and 5y were most potent amongst the synthesized
compounds. The neurotoxicity evaluation of 5d, 5w, and 5y by rotarod indicates no impairment of muscle coordination in
comparison to standard diazepam. The MTT assay revealed that the test compounds (5d, 5w, and 5y) were not found to alter
the cell viability considerably. In silico molecular docking and dynamics simulations were carried out on the homology
modeled protein of human GABA transporter 1 (GAT1), which exhibited complementary interactions of compound 5w
within the active binding pocket.
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Keywords Schiff bases Anti-convulsant Piperidine-3-carboxylic acid PAMPA-BBB Rota rod test MTT assay
Introduction
Moreover, the poor tolerability and reported side effects of
the antiepileptic drugs have affected the quality of life of the
epilepsy patients (El-Helby et al. 2017). Thus, to address
these massive challenges, development of novel anti-
epileptic drugs with improved efficacy, considerable toler-
ability, and lower toxicity is a paramount necessity.
Epilepsy is a complex set of neurological disorders (Villalba
et al. 2016), which is mainly characterized by unpredictable
and frequent disturbances of normal brain function in the
form of convulsive seizure episodes and/or loss of con-
sciousness (Siddiqui et al. 2017). Despite the availability of
numerous antiepileptic agents in the drug market, nearly
20–30% of the 70 million epilepsy patients worldwide have
insufficient control over seizures and are resistant to the
currently available pharmacotherapy (Ghareb et al. 2017).
GABA is the main inhibitory neurotransmitter in the
brain of mammals that plays a considerable role in the
pathogenesis of epilepsy (Holmes 1995). Low brain GABA
concentration and diminution in GABA-ergic neuro-
transmission have been observed in a range of epileptic
syndromes (Petroff et al. 1996). The transport of GABA
from synaptic cleft to the glial cells as well as presynaptic
neurons is mediated by GABA transporters (GATs), which
results in the termination of GABA-ergic neurotransmis-
sion. GATs are the member of sodium symporters, which
belong to solute carrier 6 (SLC6) transporter gene family in
humans that mediates neurotransmitter transport (Chen et al.
2004). Elevation in GABA concentration within the
synaptic cleft is proved to be advantageous in the man-
agement of epilepsy. One such strategy for the up-
regulation of GABA would be the blockade of specific
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2141-9) contains supplementary
material, which is available to authorized users.
* Sushant Kumar Shrivastava
skshrivastava.phe@iitbhu.ac.in
1
Pharmaceutical Chemistry Research Laboratory, Department of
Pharmaceutical Engineering & Technology, Indian Institute of
Technology (Banaras Hindu University), Varanasi 221005, India

Medicinal Chemistry Research
subtypes of GATs, which are responsible for neuronal and
glial uptake of GABA from the synaptic cleft (Quandt et al.
2013). Inhibitors of GABA uptake are of utmost sig-
nificance as regulators of neurotransmitter levels that
increase the duration of inhibitory postsynaptic potentials to
elicit antiepileptic activity (Thompson and Gahwiler 1992;
Suzdak and Jansen 1995).
in vivo anticonvulsant activity. Despite the synthesis of
these diverse derivatives, BBB permeation remains the key
bottleneck for brain drug delivery. The single marketed
derivative of the piperidine-3-carboxylic acid, i.e., tiagabine
is a GAT-1 selective GABA uptake inhibitor with con-
siderable antiepileptic potential. Reported GABA uptake
inhibition triggered by tiagabine has been attributed to the
presence of GABA mimetic moiety in the form of
piperidine-3-carboxylic acid and a lipophilic di-aromatic
region connected by a linker (Jurik et al. 2013). Tiagabine is
effectively used as adjunctive therapy for the management
of complex partial seizures in epilepsy patients (Prescott
1997). However, its long-term use is associated with
adverse effects like asthenia, tremor, concentration diffi-
culties, lethargy, nervousness, and depression (Ortinski and
Meador 2004; Sveinbjornsdottir et al. 1994; Kälviäinen
2001). Therefore, scope to develop novel compounds with
improved BBB permeation and lesser side effects is still a
challenge for medicinal chemists in the discovery of new
drugs to treat epilepsy.
Schiff bases are reported to be attractive scaffolds in the
exploration of new chemical entities as antiepileptics, owing
to their lipophilic nature and reported applications in the
design and synthesis of derivatives with marked anti-
epileptic activity (Pandeya and Rajput 2012; Swathi and
Sarangapani 2015; Bhat and Al-Omar 2011; Kulkarni et al.
2017; Aly et al. 2010). These are the condensation products
of primary amines with aldehydes or ketones. In the current
work, Schiff bases have been synthesized by the reaction of
1-(2-aminoethyl)piperidine-3-carboxylic acid with corre-
sponding aromatic aldehydes under acidic condition to
obtain novel compounds with improved lipophilicity as
compared to the parent scaffold. The synthesized com-
pounds have been evaluated for their ability to permeate the
blood–brain barrier (BBB) by an in vitro parallel artificial
membrane permeability BBB assay (PAMPA-BBB). The
potential leads exhibiting in vitro BBB permeability were
further evaluated for anticonvulsant activity in sc-Pentyle-
netetrazole (PTZ) (Subcutaneous pentylenetetrazol) and
DMCM (Methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-car-
boxylate) induced seizure models. Neurological side effects
of antiepileptic drugs have been reported in several litera-
tures. Therefore, the effects of the leads on motor coordi-
nation were evaluated by rota-rod test on rodents. Also, the
effects of the most active compounds on cell viability was
determined in neuroblastoma cell line (SH-SY5Y) by 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. The leads were then subjected to molecular
docking on a homology modeled protein to identify and
compare the complementary interactions of the compounds
with the amino acid residues of the active pocket. The
binding modes of the most active compound were further
probed using molecular dynamics simulation at the GAT1
The discovery of piperidine-3-carboxylic acid (nipecotic
acid) as a high-affinity substrate for GABA transporter
remarkably added new prospects of targeting GABA uptake
systems. It has been reported to exhibit significant in vitro
activity as an inhibitor of uptake of GABA into neuronal
and glial cells (Krogsgaard-Larsen 1980). However, it is
unable to cross BBB owing to its zwitterionic and polar
nature (Andersen et al. 2001a). The synthetic versatility of
piperidine-3-carboxylic acid led to the discovery of lipo-
philic derivatives which have been explored as useful
inhibitors of GABA uptake. Initially, N-(4,4-diphenyl-3-
butenyl)-nipecotic acid (SK&F 89976 A) (Yunger et al.
1984; Löscher 1985; Ali et al. 1985), and NO-328 (tiaga-
bine) (Nielsen et al. 1991; Braestrup et al. 1990), 1-(2-(bis
(4-(trifluoromethyl)phenyl)methoxy)ethyl)-1,2,5,6-tetra-
hydropyridine-3-carboxylic acid, (Cl966) (Bjorge et al.
1990; Taylor et al. 1990) (S)-1-(2-(tris(4-methoxyphenyl)
methoxy)ethyl)piperidine-3-carboxylic acid (SNAP-5114)
(Dhar et al. 1994) (Fig. 1) and N-(mono)-(diaryl methoxy)
alkyl or N-(diaryl methoxy)alkyl derivatives (Falch and
Krogsgaard-Larsen 1989) have demonstrated considerable
in vitro GABA uptake inhibition thereby translating into
effective anticonvulsants in some in vivo models. The
findings have led to further exploration of the piperidine-3-
carboxylic acid, resulting in the discovery of N-(benzhydryl
ethyl ether) derivatives (Pavia et al. 1992), N-alkylene, alkyl
or cycloalkylene derivatives (Andersen et al. 2001a), N-
tricyclic derivatives (Andersen et al. 2001b), as in vitro
inhibitors of GABA uptake with some leads demonstrating
Fig. 1 Derivatives of piperidine-3-carboxylic acid as GABA uptake
inhibitors

Medicinal Chemistry Research
active site. A mechanistic hypothesis based on the binding
mode of the ligand was also proposed.
N) analyses and results obtained were within 0.4% of the
theoretical values.
Designing consideration
General procedure for the synthesis of ethyl 1-(2-
aminoethyl) piperidine-3-carboxylate (3)
In vivo modulation of specific GAT isoform to selectively
block neuronal or glial GABA uptake represents a hercu-
lean challenge with currently available pharmacological
tools. This is due to the inability of the compounds to cross
highly selective BBB. This problem can be circumvented
by developing lipophilic analogs of the existing scaffolds
with proven in vitro GABA uptake inhibitory potential. One
such scaffold is piperidine-3-carboxylic acid, which has
gained attention owing to its synthetic versatility (Krogs-
gaard‐Larsen and Johnston 1975). Tiagabine, a new gen-
eration antiepileptic drug and a well-known GABA uptake
inhibitor is a marketed derivative of piperidine-3-carboxylic
acid. Considering tiagabine as a benchmark and taking
cognizance of structure-activity relationship of the reported
N-substituted piperidine-3-carboxylic acid derivatives, an
attempt was made to synthesize a series of novel Schiff
bases of 1-(2-aminoethyl) piperidine-3-carboxylic acid.
Presence of an ethylene bridge in title compounds provides
the necessary flexibility to the overall structure. Thus, the
objective of this study was to design and synthesize some
novel nipecotic acid derivatives with increased lipophilicity
and permeability across BBB.
Compound 3 was synthesized according to the reported
procedure with slight modification (Murali Dhar et al.
1999). A mixture of ethyl nepicotate (7.76 mmol), K2C03
(19.5 mmol) and KI (3.9 mmol) was dissolved in 1,4-diox-
ane with gentle heating. 2-Bromo ethylamine hydrobromide
(16 mmol) was then added, and the reaction mixture was
refluxed for 30 h. After completion, the reaction mixture
was cooled to room temperature, and dioxane was evapo-
rated under vacuum. The obtained residue was treated with
ice-cold 3 N NaOH and further extracted with EtOAc (4 ×
120 mL). The extracted organic layer was dried over
(Na2SO4) and concentrated. The crude product was further
purified by column chromatography on silica gel using
DCM/methanol (9.5:0.5) as the mobile phase to afford the
compound 3 as viscous brown oil.
FT-IR (KBr) νmax 3366 (Asymmetric N–H), 3298
1
(Symmetric N–H), 2868 (C–H), 1748 (C=O). H NMR
(500 MHz, CDCl3) δ = 4.21 (2H, q, J = 6.0 Hz, OCH2CH3),
3.25 (1H, dd, J = 12.4, 7.8 Hz, piperidine H-2a), 2.96–2.90
(1H, m, piperidine H-3), 2.88–2.65 (4H, m, H-1′, H-2′,
C=NCH2, NCH2), 2.56 (1H, t, J = 4.9 Hz, piperidine H-
6a), 2.52–2.41 (1H, m, piperidine H-4b), 2.38–2.26 (1H, m,
piperidine H-6b, 2b), 1.91–1.64 (3H, m, piperidine H-4a,
5), 1.49 (1H, s, NH2), 1.42 (3H, t, J = 6.0 Hz, OCH2CH3).
13C NMR (125 MHz, CDCl3) δ = 173.9 (C=O, COOC2H5),
61.7 (CH2, OCH2CH3), 60.8 (CH2, C-1′), 56.8 (CH2,
piperidine C-2), 53.6 (CH2, piperidine C-6), 44.6 (CH,
piperidine C-3), 39.3 (CH2, C-2′), 24.9 (CH2, piperidine C-
4), 22.6 (CH2, piperidine C-5), 14.7(CH3, OCH2CH3). Anal.
calcd. for C10H20N2O2: C, 59.97; H, 10.07; N, 13.99.
Found: C, 59.82; H, 10.04; N, 13.95.
Materials and methods
Chemistry
Analytical grade chemicals and solvents procured from
Sigma-Aldrich (India), Merck (Germany) and SD fine
Chemicals (India) were used for the synthesis. To determine
the melting points, open capillary tubes were used on a
Stuart Melting Point apparatus (SMP10). Thin layer chro-
matography was performed on a pre-coated Merck silica gel
60F254 aluminum sheets (Merck, Germany) to observe the
progress of the reaction. TLC visualization was accom-
plished by using UV cabinet (254 nm) or iodine vapors.
Experimental Log P values of the synthesized compounds
were determined by shake flask method using Shimadzu
UV/Visible spectrophotometer. FT-IR spectrophotometer
(Shimadzu 8400S) was used to obtain the infrared spectrum
of the intermediates and final compounds; analysis of the oil
samples was done by their application in the form of films
of fixed thickness while the solid samples were analyzed as
General procedure for the synthesis of 1-(2-aminoethyl)
piperidine-3-carboxylic acid (4)
The synthesized compound 3 was hydrolyzed following
the reported protocol with slight modification (Andersen
et al. 2001a). The corresponding compound 2 (1.0 mmol)
and 4 N NaOH solution (3.0 mmol) were taken in etha-
nol (3 mL). The reaction mixture was stirred at room tem-
perature (3–6 h). Completion of the reaction was
monitored by TLC using DCM/methanol (9.5:0.5). The
reaction mixture was kept on an ice bath followed by the
workup with 1 N HCl and then dried under vacuum to yield
the compound 4.
1
KBr pellets. H-NMR spectra (500 MHz) and 13C-NMR
(125 MHz) was recorded using Bruker Advance spectro-
photometer using TMS as an internal standard. Exeter CE-
440 Elemental Analyzer was used for elemental (C, H, and
FT-IR (KBr) νmax 3363 (Asymmetric N–H), 3304
(Symmetric N–H), 3212 (O–H), 2860 (C–H), 1724

Medicinal Chemistry Research
(C=O).1H NMR (500 MHz, CDCl3) δ = 11.46 (1H, s,
COOH), 3.01 (1H, dd, J = 12.4, 7.6Hz, piperidine H-2a),
2.83–2.68 (5H, m, H-1′, H-2′, piperidine H-3, C=NCH2,
NCH2), 2.56 (1H, t, J = 4.9 Hz, piperidine H-6a), 2.53–2.41
(1H, m, piperidine H-2b), 2.32–2.23 (1H, m, piperidine H-
6b), 2.11 (1H, dd, J = 14.0, 6.1 Hz, piperidine H-4a),
1.77–1.59 (3H, m, piperidine H-4b, 5), 1.45 (2H, s, NH2).
13C NMR (125 MHz, CDCl3) δ = 182.9 (C=O, COOH),
67.4 (CH2, C-1′), 62.0 (CH2, piperidine C-2), 60.2 (CH2,
piperidine C-6), 47.5 (CH, piperidine C-3), 45.9 (CH2, C-
2′), 31.7 (CH2, piperidine C-4), 29.2 (CH2, piperidine C-5).
Anal. calcd. for C8H16N2O2: C, 55.79; H, 9.36; N, 16.27.
Found: C, 55.61; H, 9.34; N, 16.31.
H-3′, imine CH=N), 7.44–7.22 (m, 4H, phenyl H-2- H-5,
Ar–H), 3.58 (2H, t, J = 7.1 Hz, H-2′, C=NCH2), 3.18 (1H,
dd, J = 12.3, 7.7 Hz, piperidine H-2a), 2.71 (2H, t, J = 7.1
Hz, H-1′, NCH2), 2.68–2.57 (2H, m, piperidine H-2b, 6a),
2.51–2.47 (1H, m, piperidine H-6b), 2.41–2.28 (1H, m,
piperidine H-3), 2.08–2.04 (1H, m, piperidine H-4a),
1.76–1.59 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 179.4 (C=O, COOH), 161.9 (imine CH,
C-3′), 136.4 (CH, Ar–C, phenyl C-6), 134.2 (C, Ar–C,
phenyl C-1), 132.4 (CH, Ar–C, phenyl C-4), 130.7 (CH,
Ar–C, phenyl C-5), 128.5 (CH, Ar–C, phenyl C-2), 124.3
(CH, Ar–C, phenyl C-3), 60.1 (CH2, C-1′), 57.8 (CH2, C-
2′), 56.1 (CH2, piperidine C-2), 55.7 (CH2, piperidine C-6),
48.2 (CH, piperidine C-3), 24.6 (CH2, piperidine C-4), 22.1
(CH2, piperidine C-5). Anal. calcd. for C15H19ClN2O2: C,
61.12; H, 6.50; N, 9.50. Found: C, 61.03; H, 6.49; N, 9.52.
General procedure for the synthesis of compounds (5a–5y)
Compound 4 (1 mmol) was dissolved in absolute ethanol
(20 mL) with gentle heating, followed by addition of 2–3
drops of glacial acetic acid. Corresponding aromatic alde-
hydes (1.2 mmol) were added, and the reaction mixture was
refluxed till the completion of reaction (3–5 h) as monitored
by TLC using DCM/methanol (9.5:0.5) as the mobile phase.
The obtained precipitate was filtered, dried and purified by
column chromatography on silica gel with few drops of
triethylamine using DCM/methanol as the mobile phase to
yield the target compounds (5a–5y).
1-(2-((4-chlorobenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5c) Yield: 133 mg, 45.24%. FT-IR (KBr)
νmax 3238 (O–H), 3041 (Ar C–H), 2878 (C–H), 1712
(C=O), 1614 (C=N), 1582 (Ar C=C) 812 (C-Cl).1H NMR
(500 MHz, CDCl3) δ = 11.41 (s, 1H, COOH), 8.28 (s, 1H,
H-3′, imine CH=N), 7.51 (2H, d, J = 7.5 Hz, Ar–H, phenyl
H-2, 6), 7.34 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-3, 5), 3.66
(2H, t, J = 4.9 Hz, H-2′, C=NCH2), 2.98 (1H, dd, J = 12.4,
7.8 Hz, piperidine H-2a), 2.76 (2H, t, J = 4.9 Hz, H-1′,
NCH2), 2.66–2.51 (2H, m, piperidine H-2b, 6a), 2.47–2.31
(1H, m, piperidine H-6b), 2.27–2.16 (1H, m, piperidine H-
3), 2.09–1.95 (1H, m, piperidine H-4a), 1.68–1.47 (3H, m,
piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3) δ =
177.1 (C=O, COOH), 160.9 (imine CH, C-3′), 137.7 (C,
Ar–C, phenyl C-4), 135.1 (CH, Ar–C, phenyl C-1), 129.7
(2 × CH, Ar–C, phenyl C-6, 2), 128.9 (2 × CH, Ar–C,
phenyl C-5, 3), 60.1 (CH2, C-1′), 58.8 (CH2, C-2′), 57.5
(CH2, piperidine C-2), 56.7 (CH2, piperidine C-6), 55.1
(CH, piperidine C-3), 25.4 (CH2, piperidine C-4), 21.4
(CH2, piperidine C-5). Anal. calcd. for C15H19ClN2O2: C,
61.12; H, 6.50; N, 9.50. Found: C, 61.29; H, 6.52; N, 9.47.
1-(2-(benzylideneamino)ethyl)piperidine-3-carboxylic acid
(5a) Yield: 125 mg, 48.07%. FT-IR (KBr) νmax 3220
(O–H), 3042 (Ar C–H), 2880 (C–H), 1712 (C=O), 1616
1
(C=N), 1586 (Ar C=C). H NMR (500 MHz, CDCl3) δ =
11.32 (s, 1H, COOH), 8.42 (s, 1H, H-3′, imine CH=N),
7.48–7.27 (m, 5H, phenyl H-2- H-6, Ar–H), 3.67 (2H, t, J
= 7.4 Hz, H-2′, C=NCH2), 3.04 (1H, dd, J = 12.5, 7.7 Hz,
piperidine H-2a), 2.84 (2H, t, J = 7.2 Hz, H-1′, NCH2),
2.78–2.67 (2H, m, piperidine H-2b, 6a), 2.63 (1H, m,
piperidine H-6b), 2.32 (1H, m, piperidine H-3), 2.11 (1H,
m, piperidine H-4a), 1.77–1.64 (3H, m, piperidine H-5, 4b).
13C NMR (125 MHz, CDCl3) δ = 181.2 (C=O, COOH),
163.3 (imine CH, C-3′), 135.7 (C, Ar–C, phenyl C-1),
132.1 (CH, Ar–C, phenyl C-4), 128.8 (2 × CH, Ar–C,
phenyl C-3, 5), 125.5 (2 × CH, Ar–C, phenyl C-2, 6), 63.1
(CH2, C-1′), 59.8 (CH2, C-2′), 59.5 (CH2, piperidine C-2),
56.3 (CH2, piperidine C-6), 49.1 (CH, piperidine C-3), 24.4
(CH2, piperidine C-4), 20.4 (CH2, piperidine C-5). Anal.
calcd. for C15H20N2O2: C, 69.20; H, 7.74; N, 10.76. Found:
C, 69.39; H, 7.76; N, 10.74.
1-(2-((2,3-dichlorobenzylidene)amino)ethyl)piperidine-3-
carboxylic acid (5d) Yield: 168 mg, 51.22%. FT-IR (KBr)
νmax 3233 (O–H), 3041 (Ar C–H), 2876 (C–H), 1725
1
(C=O), 1617 (C=N), 1590 (Ar C=C) 797, 788 (C–Cl). H
NMR (500 MHz, CDCl3) δ = 11.39 (s, 1H, COOH), 8.77 (s,
1H, H-3′, imine CH=N), 7.58–7.20 (m, 3H, phenyl H-2- H-
4, Ar–H), 4.00 (2H, t, J = 4.8 Hz, H-2′, C=NCH2), 3.23
(1H, dd, J = 12.3, 7.7 Hz, piperidine H-2a), 3.05–2.96 (2H,
m, H-1′, NCH2), 2.86–2.79 (2H, m, piperidine H-2b, 6a),
2.65 (1H, dd, J = 12.3, 7.7 Hz, piperidine H-6b), 2.52–2.47
(1H, m, piperidine H-3), 2.37–2.31 (1H, m, piperidine H-
4a), 1.92–1.83 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 173.68 (C=O, COOH), 160.7 (imine
CH, C-3′), 132.4 (CH, Ar–C, phenyl C-1), 131.5 (C, Ar–C,
1-(2-((2-chlorobenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5b) Yield: 153 mg, 52.04%. FT-IR (KBr)
νmax 3236 (O–H), 3042 (Ar C–H), 2889 (C–H), 1716
(C=O), 1612 (C=N), 1585 (Ar C=C) 798 (C-Cl). 1H NMR
(500 MHz, CDCl3) δ = 11.42 (s, 1H, COOH), 8.32 (s, 1H,

Medicinal Chemistry Research
phenyl C-6), 131.1 (CH, Ar–C, phenyl C-4), 130.9 (CH,
Ar–C, phenyl C-5), 125.5 (CH, Ar–C, phenyl C-3), 123.8
(CH, Ar–C, phenyl C-2), 56.8 (CH2, C-1′), 52.7 (CH2, C-
2′), 52.6 (CH2, piperidine C-2), 51.0 (CH2, piperidine C-6),
38.2 (CH, piperidine C-3), 22.2 (CH2, piperidine C-4), 19.9
(CH2, piperidine C-5). Anal. calcd. for C15H18Cl2N2O2: C,
54.72; H, 5.51; N, 8.51. Found: C, 54.89; H, 5.52; N, 8.50.
1-(2-((3-fluorobenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5g) Yield: 144 mg, 51.79%. FT-IR (KBr)
νmax 3229 (O–H), 3049 (Ar C–H), 2890 (C–H), 1721
(C=O), 1625 (C=N), 1594 (Ar C=C) 1278 (C–F). 1H
NMR (500 MHz, CDCl3) δ = 11.51 (s, 1H, COOH), 8.30 (s,
1H, H-3′, imine CH=N), 7.39–7.03 (m, 4H, phenyl H-2, 3,
4, 6, Ar–H), 3.70 (2H, t, J = 5.0 Hz, H-2′, C=NCH2), 2.99
(1H, dd, J = 12.4, 7.6 Hz, piperidine H-2a), 2.77 (2H, t, J =
5.0 Hz, H-1′, NCH2), 2.68–2.54 (2H, m, piperidine H-2b,
6a), 2.46–2.29 (1H, m, piperidine H-6b), 2.23–2.19 (1H, m,
piperidine H-3), 2.04–1.97 (1H, m, piperidine H-4a),
1.71–1.50 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 177.4 (C=O, COOH), 164.3 (d, JC,F =
259.4 Hz, C, Ar–C, phenyl C-5), 162.1 (d, JC,F = 4.5 Hz,
imine CH, C-3′), 139.8 (d, JC,F = 7.1 Hz, C, Ar–C, phenyl
C-1), 129.5 (d, JC,F = 7.1 Hz, C, Ar–C, phenyl C-3), 124.8
(d, JC,F = 4.1 Hz, C, Ar–C, phenyl C-2), 118.4 (d, JC,F =
26.2 Hz, C, Ar–C, phenyl C-4), 115.5 (d, JC,F = 27.3 Hz, C,
Ar–C, phenyl C-6), 59.4 (CH2, C-1′), 55.5 (CH2, C-2′),
55.1 (CH2, piperidine C-2), 53.4 (CH2, piperidine C-6),
40.7 (CH, piperidine C-3), 25.9 (CH2, piperidine C-4), 22.9
(CH2, piperidine C-5). Anal. calcd. for C15H19FN2O2: C,
64.73; H, 6.88; N, 10.07. Found: C, 64.94; H, 6.89; N,
10.03.
1-(2-((2,4-dichlorobenzylidene)amino)ethyl)piperidine-3-
carboxylic acid (5e) Yield: 148 mg, 45.12%. FT-IR (KBr)
νmax 3230 (O–H), 3040 (Ar C–H), 2880 (C–H), 1718
1
(C=O), 1619 (C=N), 1591 (Ar C=C) 826, 798 (C–Cl). H
NMR (500 MHz, CDCl3) δ = 11.51 (s, 1H, COOH), 8.31 (s,
1H, H-3′, imine CH=N), 7.41–7.21 (m, 3H, phenyl H-2, 3,
4, Ar–H), 3.54 (2H, t, J = 7.41 Hz, H-2′, C=NCH2), 3.18
(1H, dd, J = 12.3, 7.7 Hz, piperidine H-2a), 2.68 (2H, t, J =
7.1 Hz, H-1′, NCH2), 2.66–2.55 (2H, m, piperidine H-2b,
6a), 2.49 (1H, dd, J = 12.3, 7.7 Hz, piperidine H-6b),
2.40–2.0 (1H, m, piperidine H-3), 2.13–2.02 (1H, m,
piperidine H-4a), 1.78–1.59 (3H, m, piperidine H-5, 4b).
13C NMR (125 MHz, CDCl3) δ = 178.4 (C=O, COOH),
161.7 (imine CH, C-3′), 137.8 (C, Ar–C, phenyl C-4),
135.5 (C, Ar–C, phenyl C-6), 132.1 (C, Ar–C, phenyl C-1),
130.9 (C, Ar–C, phenyl C-5), 130.3 (C, Ar–C, phenyl C-2),
128.8 (C, Ar–C, phenyl C-3), 59.5 (CH2, C-1′), 55.5 (CH2,
C-2′), 55.3 (CH2, piperidine C-2), 53.7 (CH2, piperidine C-
6), 40.99 (CH, piperidine C-3), 24.2 (CH2, piperidine C-4),
21.3 (CH2, piperidine C-5). Anal. calcd. for
C15H18Cl2N2O2: C, 54.72; H, 5.51; N, 8.51. Found: C,
54.85; H, 5.53; N, 8.53.
1-(2-((4-fluorobenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5h) Yield: 122 mg, 43.88%. FT-IR (KBr)
νmax 3228 (O–H), 3039 (Ar C–H), 2887 (C–H), 1729
(C=O), 1620 (C=N), 1598 (Ar C=C) 1318 (C–F). 1H
NMR (500 MHz, CDCl3) δ = 11.18 (s, 1H, COOH), 8.27 (s,
1H, H-3′, imine CH=N), 7.64 (2H, d, J = 7.5 Hz, Ar–H,
phenyl H-2, 6), 7.06 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-3,
5), 3.59 (2H, t, J = 5.3 Hz, H-2′, C=NCH2), 3.05 (1H, dd,
J = 12.3, 7.7 Hz, piperidine H-2a), 2.90 (2H, t, J = 5.3 Hz,
H-1′, NCH2), 2.83–2.69 (2H, m, piperidine H-2b, 6a),
2.68–2.49 (1H, m, piperidine H-6b), 2.33–2.17 (1H, m,
piperidine H-3), 2.16–1.91(1H, m, piperidine H-4a),
1.86–1.42 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 176.4 (C=O, COOH), 163.3 (d, JC,F =
248.8 Hz, C, Ar–C, phenyl C-4), 162.6 (imine CH, C-3′),
133.4 (d, JC,F = 3.8 Hz, C, Ar–C, phenyl C-1), 131.7 (d, JC,
F = 8.1 Hz, 2 × CH, Ar–C, phenyl C-6, 2), 115.6 (d, JC,F =
24.8 Hz, 2 × CH, Ar–C, phenyl C-5, 3), 60.1 (CH2, C-1′),
55.8 (CH2, C-2′), 55.2 (CH2, piperidine C-2), 53.7 (CH2,
piperidine C-6), 40.9 (CH, piperidine C-3), 25.9 (CH2,
piperidine C-4), 22.6 (CH2, piperidine C-5). Anal. calcd. for
C15H19FN2O2: C, 64.73; H, 6.88; N, 10.07. Found: C,
64.89; H, 6.86; N, 10.09.
1-(2-((2,3,6-trichlorobenzylidene)amino)ethyl)piperidine-3-
carboxylic acid (5f) Yield: 172 mg, 47.51%. FT-IR (KBr)
νmax 3226 (O–H), 3066 (Ar C–H), 2871 (C–H), 1727
(C=O), 1611 (C=N), 1586 (Ar C=C) 811, 787, 778 (C-Cl).
1H NMR (500 MHz, CDCl3) δ = 11.21 (s, 1H, COOH),
8.40 (s, 1H, H-3′, imine CH=N), 7.23–7.15 (m, 2H, phenyl
H-3, 4, Ar–H), 3.50 (2H, t, J = 5.1 Hz, H-2′, C=NCH2),
2.97 (1H, dd, J = 12.3, 7.7 Hz, piperidine H-2a), 2.33 (2H,
t, J = 7.1 Hz, H-1′, NCH2), 2.66–2.55 (2H, m, piperidine H-
2b, 6a), 2.49 (1H, dd, J = 12.3, 7.7 Hz, piperidine H-6b),
2.35–2.30 (1H, m, piperidine H-3), 2.14–1.95 (1H, m,
piperidine H-4a), 1.77–1.47 (3H, m, piperidine H-5, 4b).
13C NMR (125 MHz, CDCl3) δ = 174.4 (C=O, COOH),
159.7 (imine CH, C-3′), 136.8 (C, Ar–C, phenyl C-1),
134.5 (C, Ar–C, phenyl C-6), 132.8 (C, Ar–C, phenyl C-4),
131.9 (C, Ar–C, phenyl C-2), 131.3 (C, Ar–C, phenyl C-5),
129.9 (C, Ar–C, phenyl C-3), 59.1 (CH2, C-1′), 57.5 (CH2,
C-2′), 56.2 (CH2, piperidine C-2), 54.1 (CH2, piperidine C-
6), 40.4 (CH, piperidine C-3), 25.2 (CH2, piperidine C-4),
21.9 (CH2, piperidine C-5). Anal. calcd. for
C15H17Cl3N2O2: C, 49.54; H, 4.71; N, 7.70. Found: C,
49.40; H, 4.70; N, 7.72.
1-(2-((2,4-difluorobenzylidene)amino)ethyl)piperidine-3-
carboxylic acid (5i) Yield: 153 mg, 51.69%. FT-IR (KBr)
νmax 3242 (O–H), 3036 (Ar C–H), 2872 (C–H), 1716
(C=O), 1612 (C=N), 1588 (Ar C=C) 1319, 1292 (C–F).

Medicinal Chemistry Research
1H NMR (500 MHz, CDCl3) δ = 11.21 (s, 1H, COOH),
8.29 (s, 1H, H-3′, imine CH=N), 7.53–6.80 (m, 3H, phenyl
H-2, 3, 4, Ar–H), 3.53 (2H, t, J = 5.3 Hz, H-2′, C=NCH2),
3.06–2.81 (1H, m, piperidine H-2a), 2.74 (2H, t, J = 7.1 Hz,
H-1′, NCH2), 2.64–2.57 (2H, m, piperidine H-2b, 6a),
2.34–2.29 (1H, m, piperidine H-6b), 2.21–2.08 (1H, m,
piperidine H-3), 2.05–1.99 (1H, m, piperidine H-4a),
1.72–1.61 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 175.8 (C=O, COOH), 165.3 (dd, JC,F =
190.1, 12.7 Hz, Ar–C–F, phenyl C-4), 163.4 (dd, JC,F =
188.3, 12.8 Hz, Ar–C–F, phenyl C-6), 160.6 (d, JC,F = 6.7,
imine CH, C-3′), 133.7 (dd, JC,F = 11.1, 3.5 Hz, CH, Ar–C,
phenyl C-2), 121.7 (dd, JC,F = 8.3, 3.4 Hz, CH, Ar–C,
phenyl C-1), 113.6 (dd, JC,F = 22.0, 3.6 Hz, CH, Ar–C,
phenyl C-3), 103.7 (t, JC,F = 24.9 Hz, CH, Ar–C, phenyl C-
5), 60.2 (CH2, C-1′), 55.7 (CH2, C-2′), 54.8 (CH2, piper-
idine C-2), 53.8 (CH2, piperidine C-6), 41.3 (CH, piperidine
C-3), 24.9 (CH2, piperidine C-4), 22.1 (CH2, piperidine C-
5). Anal. calcd. for C15H18F2N2O2: C, 60.80; H, 6.12; N,
9.45. Found: C, 60.66; H, 6.11; N, 9.47.
6.2 Hz, H-1′, NCH2), 2.74–2.70 (2H, m, piperidine H-2b,
6a), 2.52–2.48 (1H, m, piperidine H-6b), 2.34–2.29 (1H, m,
piperidine H-3), 2.24–2.09 (1H, m, piperidine H-4a),
1.84–1.62 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 176.5 (C=O, COOH), 162.9 (imine CH,
C-3′), 138.1 (CH, Ar–C, phenyl C-1), 134.3 (C, Ar–C,
phenyl C-4), 131.4 (CH, Ar–C, phenyl C-6), 130.6 (CH,
Ar–C, phenyl C-3), 126.5 (CH, Ar–C, phenyl C-2), 121.8
(CH, Ar–C, phenyl C-5), 69.2 (CH2, C-1′), 57.9 (CH2, C-
2′), 56.2 (CH2, piperidine C-2), 55.4 (CH2, piperidine C-6),
47.3 (CH, piperidine C-3), 24.4 (CH2, piperidine C-4), 21.9
(CH2, piperidine C-5). Anal. calcd. for C15H19BrN2O2: C,
53.11; H, 5.65; N, 8.26. Found: C, 53.30; H, 5.65; N, 8.24.
1-(2-((4-bromobenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5l) Yield: 143 mg, 42.31%. FT-IR (KBr) νmax
3258 (O–H), 3053 (Ar C–H), 2898 (C–H), 1724 (C=O),
1
1619 (C=N), 1602 (Ar C=C), 639 (C–Br). H NMR (500
MHz, CDCl3) δ = 11.42 (s, 1H, COOH), 8.26 (s, 1H, H-3′,
imine CH=N), 7.49 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-2,
6), 7.45 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-3, 5), 3.57 (2H,
t, J = 5.3 Hz, H-2′, C=NCH2), 3.05 (1H, dd, J = 12.5, 7.7
Hz, piperidine H-2a), 2.90 (2H, t, J = 5.3 Hz, H-1′, NCH2),
2.66–2.50 (2H, m, piperidine H-2b, 6a), 2.37 (1H, dd, J =
12.5, 7.7 Hz, piperidine H-6b), 2.26–2.21 (1H, m, piper-
idine H-3), 2.15–1.96 (1H, m, piperidine H-4a), 1.74–1.54
(3H, m, piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3)
δ = 177.1 (C=O, COOH), 160.9 (imine CH, C-3′), 137.7
(C, Ar–C, phenyl C-4), 135.1 (CH, Ar–C, phenyl C-1),
129.7 (2 × CH, Ar–C, phenyl C-6, 2), 128.9 (2 × C, Ar–C,
phenyl C-5, 3), 60.1 (CH2, C-1′), 58.8 (CH2, C-2′), 57.5
(CH2, piperidine C-2), 56.7 (CH2, piperidine C-6), 55.1
(CH, piperidine C-3), 25.4 (CH2, piperidine C-4), 21.4
(CH2, piperidine C-5). Anal. calcd. for C15H19BrN2O2: C,
53.11; H, 5.65; N, 8.26. Found: C, 53.02; H, 5.64; N, 8.27.
1-(2-((3,4,5-trifluorobenzylidene)amino)ethyl)piperidine-3-
carboxylic acid (5j) Yield: 168 mg, 53.50%. FT-IR (KBr)
νmax 3238 (O–H), 3038 (Ar C–H), 2868 (C–H), 1710
(C=O), 1616 (C=N), 1588 (Ar C=C) 1306, 1292, 1286
(C–F). 1H NMR (500 MHz, CDCl3) δ = 11.24 (s, 1H,
COOH), 8.29 (s, 1H, H-3′, imine CH=N), 7.10 (2H, dd, J
= 8.0, 4.9 Hz, phenyl H-3, 4, Ar–H), 3.83 (2H, t, J = 4.7
Hz, H-2′, C=NCH2), 3.47 (1H, dd, J = 12.4, 7.9 Hz,
piperidine H-2a), 2.90 (2H, t, J = 4.7 Hz, H-1′, NCH2),
2.77–2.54 (2H, m, piperidine H-2b, 6a), 2.26 (1H, dd, J =
12.3, 7.7 Hz, piperidine H-6b), 2.18–2.11 (1H, m, piper-
idine H-3), 2.14–2.08 (1H, m, piperidine H-4a), 1.73–1.37
(3H, m, piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3)
δ = 174.4 (C=O, COOH), 161.4 (t, JC,F = 4.1 Hz, C, imine
CH, C-3′), 149.8 (ddd, JC,F = 223.8, 24.8, 7.2 Hz, C, Ar–C,
phenyl C-5, 3), 140.5 (dt, JC,F = 49.8, 28.2 Hz, C, Ar–C,
phenyl C-4), 135.8 (td, JC,F = 5.9, 3.9 Hz, C, Ar–C, phenyl
C-1), 111.9 (ddd, JC,F = 27.9, 7.2, 4.1 Hz, C, Ar–C, phenyl
C-6, 2), 59.2 (CH2, C-1′), 57.8 (CH2, C-2′), 56.4 (CH2,
piperidine C-2), 54.6 (CH2, piperidine C-6), 40.1 (CH,
piperidine C-3), 25.8 (CH2, piperidine C-4), 21.4 (CH2,
piperidine C-5). Anal. calcd. for C15H17F3N2O2: C, 57.32;
H, 5.45; N, 8.91. Found: C, 57.21; H, 5.47; N, 8.89.
1-(2-((2,6-dibromobenzylidene)amino)ethyl)piperidine-3-
carboxylic acid (5m) Yield: 196 mg, 47.12%. FT-IR (KBr)
νmax 3255 (O–H), 3059 (Ar C–H), 2865 (C–H), 1718
1
(C=O), 1622 (C=N), 1598 (Ar C=C), 616 (C–Br). H
NMR (500 MHz, CDCl3) δ = 11.51 (s, 1H, COOH), 8.38 (s,
1H, H-3′, imine CH=N), 7.44–7.07 (m, 3H, phenyl H-2, 3,
4, Ar–H), 3.51 (2H, t, J = 5.1 Hz, H-2′, C=NCH2), 3.05
(1H, dd, J = 12.3, 7.7 Hz, piperidine H-2a), 2.96 (2H, t, J =
5.0 Hz, H-1′, NCH2), 2.80–2.55 (2H, m, piperidine H-2b,
6a), 2.34 (1H, dd, J = 12.4, 7.8 Hz, piperidine H-6b),
2.31–2.25 (1H, m, piperidine H-3), 2.10–2.06 (1H, m,
piperidine H-4a), 1.80–1.51 (3H, m, piperidine H-5, 4b).
13C NMR (125 MHz, CDCl3) δ = 173.4 (C=O, COOH),
156.7 (imine CH, C-3′), 137.9 (C, Ar–C, phenyl C-1),
132.9 (2 × C, Ar–C, phenyl C-5, 3), 131.3 (C, Ar–C, phenyl
C-4), 124.9 (2 × C, Ar–C, phenyl C-2, 6), 59.7 (CH2, C-1′),
55.9 (CH2, C-2′), 55.6 (CH2, piperidine C-2), 54.7 (CH2,
1-(2-((3-bromobenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5k) Yield: 166 mg, 49.11%. FT-IR (KBr)
νmax 3242 (O–H), 3028 (Ar C–H), 2868 (C–H), 1719
(C=O), 1622 (C=N), 1588 (Ar C=C), 628 (C-Br). 1H
NMR (500 MHz, CDCl3) δ = 11.52 (s, 1H, COOH), 8.28 (s,
1H, H-3′, imine CH=N), 7.90–7.19 (m, 4H, phenyl H-2- H-
5, Ar–H), 3.59 (2H, t, J = 6.2 Hz, H-2′, C=NCH2), 3.10
(1H, dd, J = 12.2, 7.7 Hz, piperidine H-2a), 2.81 (2H, t, J =

Medicinal Chemistry Research
piperidine C-6), 40.1 (CH, piperidine C-3), 24.8 (CH2,
piperidine C-4), 22.6 (CH2, piperidine C-5). Anal. calcd. for
C15H18Br2N2O2: C, 43.09; H, 4.34; N, 6.70. Found: C,
43.22; H, 4.35; N, 6.68.
νmax 3368 (sharp, O–H), 3238 (broad, O–H), 3039 (Ar
C–H), 2869 (C–H), 1717 (C=O), 1616 (C=N), 1588 (Ar
C=C). 1H NMR (500 MHz, CDCl3) δ = 11.37 (s, 1H,
COOH), 9.02 (1H, s, Phenyl OH), 8.23 (s, 1H, H-3′, imine
CH=N), 7.45–6.78 (m, 4H, phenyl H-2- H-5, Ar–H), 3.62
(2H, t, J = 6.8 Hz, H-2′, C=NCH2), 3.22 (1H, dd, J = 12.3,
7.6 Hz, piperidine H-2a), 2.81 (2H, t, J = 6.8 Hz, H-1′,
NCH2), 2.75–2.61 (2H, m, piperidine H-2b, 6a), 2.66–2.54
(1H, m, piperidine H-6b), 2.39–2.32 (1H, m, piperidine H-
3), 2.13–2.09 (1H, m, piperidine H-4a), 1.75–1.65 (3H, m,
piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3) δ =
176.4 (C=O, COOH), 164.1 (imine CH, C-3′), 160.8 (CH,
Ar–C, phenyl C-6), 133.1 (C, Ar–C, phenyl C-3), 129.3
(CH, Ar–C, phenyl C46), 121.7 (CH, Ar–C, phenyl C-2),
120.3 (CH, Ar–C, phenyl C-1), 117.9 (CH, Ar–C, phenyl
C-5), 62.2 (CH2, C-1′), 59.4 (CH2, C-2′), 57.7 (CH2,
piperidine C-2), 55.7 (CH2, piperidine C-6), 40.9 (CH,
piperidine C-3), 24.4 (CH2, piperidine C-4), 22.1 (CH2,
piperidine C-5). Anal. calcd. for C15H20N2O3: C, 65.20; H,
7.30; N, 10.14. Found: C, 65.01; H, 7.28; N, 10.12.
1-(2-((4-nitrobenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5n) Yield: 162 mg, 53.11%. FT-IR (KBr)
νmax 3233 (O–H), 3062 (Ar C–H), 2879 (C–H), 1706
1
(C=O), 1612 (C=N), 1596 (Ar C=C), 1562 (N=O). H
NMR (500 MHz, CDCl3) δ = 11.28 (s, 1H, COOH), 8.56 (s,
1H, H-3′, imine CH=N), 8.28 (2H, d, J = 7.5 Hz, Ar–H,
phenyl H-2, 6), 8.11 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-3,
5), 3.66 (2H, t, J = 5.3 Hz, H-2′, C=NCH2), 2.95 (1H, dd,
J = 12.4, 7.9 Hz, piperidine H-2a), 2.71 (2H, t, J = 6.2 Hz,
H-1′, NCH2), 2.64–2.47 (2H, m, piperidine H-2b, 6a), 2.39
(1H, dd, J = 12.5, 7.7 Hz, piperidine H-6b), 2.34–2.18 (1H,
m, piperidine H-3), 2.14–1.92 (1H, m, piperidine H-4a),
1.87–1.32 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 176.3 (C=O, COOH), 161.2 (imine CH,
C-3′), 151.1 (C, Ar–C, phenyl C-4), 140.5 (CH, Ar–C,
phenyl C-1), 130.7 (2 × CH, Ar–C, phenyl C-6, 2), 124.6
(2 × C, Ar–C, phenyl C-5, 3), 61.3 (CH2, C-1′), 57.2 (CH2,
C-2′), 55.5 (CH2, piperidine C-2), 54.7 (CH2, piperidine C-
6), 41.1 (CH, piperidine C-3), 26.4 (CH2, piperidine C-4),
21.8 (CH2, piperidine C-5). Anal. calcd. for C15H19N3O4: C,
59.01; H, 6.27; N, 13.76. Found: C, 59.21; H, 6.25; N,
13.80.
1-(2-((4-hydroxybenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5q) Yield: 146 mg, 52.89%. FT-IR (KBr)
νmax 3376 (sharp, O–H), 3239 (broad, O–H), 3068 (Ar
C–H), 2862 (C–H), 1724 (C=O), 1628 (C=N), 1592 (Ar
C=C). 1H NMR (500 MHz, CDCl3) δ = 11.28 (s, 1H,
COOH), 8.90 (1H, s, Phenyl OH), 8.25 (s, 1H, H-3′, imine
CH=N), 7.40 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-2, 6),
6.81 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-3, 5), 3.69 (2H, t,
J = 5.0 Hz, H-2′, C=NCH2), 2.98 (1H, dd, J = 12.3, 7.7
Hz, piperidine H-2a), 2.77 (2H, t, J = 5.0 Hz, H-1′, NCH2),
2.49–2.27 (2H, m, piperidine H-2b, 6a), 2.29 (1H, dd, J =
12.5, 7.7 Hz, piperidine H-6b), 2.10–1.93 (1H, m, piper-
idine H-3), 1.68–1.47 (1H, m, piperidine H-4a), 1.46–1.32
(3H, m, piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3)
δ = 176.4 (C=O, COOH), 161.6 (imine CH, C-3′), 159.5
(C, Ar–C, phenyl C-4), 131.3 (2 × CH, Ar–C, phenyl C-6,
2), 127.5 (CH, Ar–C, phenyl C-1), 116.6 (2 × C, Ar–C,
phenyl C-5, 3), 60.5 (CH2, C-1′), 57.4 (CH2, C-2′), 55.7
(CH2, piperidine C-2), 52.9 (CH2, piperidine C-6), 40.8
(CH, piperidine C-3), 23.4 (CH2, piperidine C-4), 21.5
(CH2, piperidine C-5). Anal. calcd. for C15H20N2O3: C,
65.20; H, 7.30; N, 10.14. Found: C, 65.42; H, 7.31; N,
10.17.
1-(2-((2-nitrobenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5o) Yield: 178 mg, 58.36%. FT-IR (KBr)
νmax 3242 (O–H), 3055 (Ar C–H), 2869 (C–H), 1721
1
(C=O), 1619 (C=N), 1585 (Ar C=C), 1545 (N=O). H
NMR (500 MHz, CDCl3) δ = 11.42 (s, 1H, COOH), 8.54 (s,
1H, H-3′, imine CH=N), 8.09–7.39 (m, 4H, phenyl H-2- H-
5, Ar–H), 3.65 (2H, t, J = 6.2 Hz, H-2′, C=NCH2), 2.98
(1H, dd, J = 12.2, 7.7 Hz, piperidine H-2a), 2.79 (2H, t, J =
6.2 Hz, H-1′, NCH2), 2.68–2.63 (2H, m, piperidine H-2b,
6a), 2.57–2.41 (1H, m, piperidine H-6b), 2.37–2.24 (1H, m,
piperidine H-3), 2.26–2.11 (1H, m, piperidine H-4a),
1.74–1.51 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 172.9 (C=O, COOH), 160.1 (imine CH,
C-3′), 148.2 (CH, Ar–C, phenyl C-6), 133.7 (C, Ar–C,
phenyl C-3), 132.6 (CH, Ar–C, phenyl C46), 131.7 (CH,
Ar–C, phenyl C-2), 128.3 (CH, Ar–C, phenyl C-1), 124.9
(CH, Ar–C, phenyl C-5), 67.2 (CH2, C-1′), 58.4 (CH2, C-
2′), 56.9 (CH2, piperidine C-2), 55.2 (CH2, piperidine C-6),
40.3 (CH, piperidine C-3), 24.1 (CH2, piperidine C-4), 22.5
(CH2, piperidine C-5). Anal. calcd. for C15H19N3O4: C,
59.01; H, 6.27; N, 13.76. Found: C, 59.18; H, 6.28; N,
13.73.
1-(2-((3-methoxybenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5r) Yield: 128 mg, 44.14 FT-IR (KBr) νmax
3237 (O–H), 3049 (Ar C–H), 2885 (C–H), 1723 (C=O),
1618 (C=N), 1590 (Ar C=C). 1H NMR (500 MHz, CDCl3)
δ = 11.21 (s, 1H, COOH), 8.27 (s, 1H, H-3′, imine CH=N),
7.34-6.87 (m, 4H, phenyl H-2,4,5,6, Ar–H), 3.81 (3H, s,
OCH3), 3.72 (2H, t, J = 5.4 Hz, H-2′, C=NCH2), 2.98 (1H,
dd, J = 12.3, 7.7 Hz, piperidine H-2a), 2.84 (2H, t, J = 5.4
1-(2-((2-hydroxybenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5p) Yield: 161 mg, 58.33%. FT-IR (KBr)

Medicinal Chemistry Research
Hz, H-1′, NCH2), 2.74–2.70 (2H, m, piperidine H-2b, 6a),
2.67–2.48 (1H, m, piperidine H-6b), 2.45–2.28 (1H, m,
piperidine H-3), 2.20–2.08 (1H, m, piperidine H-4a),
1.79–1.56 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 175.4 (C=O, COOH), 162.7 (imine CH,
C-3′), 160.1 (CH, Ar–C, phenyl C-1), 139.3 (C, Ar–C,
phenyl C-4), 129.3 (CH, Ar–C, phenyl C-6), 122.6 (CH,
Ar–C, phenyl C-3), 117.5 (CH, Ar–C, phenyl C-2), 112.1
(CH, Ar–C, phenyl C-5), 59.2 (CH2, C-1′), 56.8 (CH3,
phenyl OCH3), 55.9 (CH2, C-2′), 55.2 (CH2, piperidine C-
2), 53.5 (CH2, piperidine C-6), 40.8 (CH, piperidine C-3),
24.1 (CH2, piperidine C-4), 22.3 (CH2, piperidine C-5).
Anal. calcd. for C16H22N2O3: C, 66.18; H, 7.64; N, 9.65.
Found: C, 66.40; H, 7.64; N, 9.63.
(CH, Ar–C, phenyl C-1), 124.6 (CH, Ar–C, phenyl C-2),
113.4 (CH, Ar–C, phenyl C-3), 112.8 (C, Ar–C, phenyl C-
6), 60.7 (CH2, C-1′), 57.8 (CH2, C-2′), 56.7 (2 × CH3,
phenyl OCH3), 55.7 (CH2, piperidine C-2), 52.5 (CH2,
piperidine C-6), 40.7 (CH, piperidine C-3), 24.2 (CH2,
piperidine C-4), 22.1 (CH2, piperidine C-5). Anal. calcd. for
C17H24N2O4: C, 63.73; H, 7.55; N, 8.74. Found: C, 63.51;
H, 7.53; N, 8.76.
1-(2-((3,4,5-trimethoxybenzylidene)amino)ethyl)piperidine-
3-carboxylic acid (5u) Yield: 175 mg, 50.00%. FT-IR
(KBr) νmax 3219 (O–H), 3040 (Ar C–H), 2873 (C–H),
1711 (C=O), 1620 (C=N), 1581 (Ar C=C). 1H NMR (500
MHz, CDCl3) δ = 11.39 (s, 1H, COOH), 8.24 (s, 1H, H-3′,
imine CH=N), 6.87 (2H, s, phenyl H-2, 6, Ar–H), 3.83 (6H,
s, 2 × OCH3), 3.79 (2H, t, J = 4.9 Hz, H-2′, C=NCH2), 3.68
(3H, s, OCH3), 3.51 (1H, dd, J = 12.5, 7.9 Hz, piperidine H-
2a), 2.84 (2H, t, J = 4.9 Hz, H-1′, NCH2), 2.69–2.53 (2H,
m, piperidine H-2b, 6a), 2.28 (1H, dd, J = 12.3, 7.7 Hz,
piperidine H-6b), 2.25–2.09 (1H, m, piperidine H-3),
1.76–1.58 (1H, m, piperidine H-4a), 1.52–1.39 (3H, m,
piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3) δ =
177.4 (C=O, COOH), 161.6 (imine CH, C-3′), 154.3 (2 ×
C, Ar–C, phenyl C-5, 3), 140.6 (C, Ar–C, phenyl C-4),
135.7 (C, Ar–C, phenyl C-1), 108.9 (2 × C, Ar–C, phenyl
C-6, 2), 59.8 (CH2, C-1′), 56.4 (3 × CH3, phenyl OCH3),
57.4 (CH2, C-2′), 56.7 (CH2, piperidine C-2), 54.2 (CH2,
piperidine C-6), 40.8 (CH, piperidine C-3), 25.2 (CH2,
piperidine C-4), 21.1 (CH2, piperidine C-5). Anal. calcd. for
C18H26N2O5: C, 61.70; H, 7.48; N, 7.99. Found: C, 61.85;
H, 7.46; N, 7.80.
1-(2-((4-methoxybenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5s) Yield: 122 mg, 42.06%. FT-IR (KBr)
νmax 3229 (O–H), 3040 (Ar C–H), 2875 (C–H), 1716
(C=O), 1622 (C=N), 1590 (Ar C=C). 1H NMR (500 MHz,
CDCl3) δ = 11.36 (s, 1H, COOH), 8.24 (s, 1H, H-3′, imine
CH=N), 7.53 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-2, 6),
6.93 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-3, 5), 3.83 (3H, s,
OCH3), 3.68 (2H, t, J = 4.9 Hz, H-2′, C=NCH2), 2.99 (1H,
dd, J = 12.5, 7.7 Hz, piperidine H-2a), 2.76 (2H, t, J = 4.9
Hz, H-1′, NCH2), 2.64–2.52 (2H, m, piperidine H-2b, 6a),
2.40 (1H, dd, J = 12.5, 7.7 Hz, piperidine H-6b), 2.24–2.15
(1H, m, piperidine H-3), 2.05–1.93 (1H, m, piperidine H-
4a), 1.76–1.46 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 175.4 (C=O, COOH), 162.3 (C, Ar–C,
phenyl C-4), 161.3 (imine CH, C-3′), 131.7 (2 × CH, Ar–C,
phenyl C-6, 2), 128.4 (CH, Ar–C, phenyl C-1), 114.5 (2 ×
C, Ar–C, phenyl C-5, 3), 60.2 (CH2, C-1′), 57.8 (CH2, C-
2′), 56.9 (CH3, phenyl OCH3), 55.2 (CH2, piperidine C-2),
52.1 (CH2, piperidine C-6), 40.9 (CH, piperidine C-3), 24.4
(CH2, piperidine C-4), 22.8 (CH2, piperidine C-5). Anal.
calcd. for C16H22N2O3: C, 66.18; H, 7.64; N, 9.65. Found:
C, 66.29; H, 7.66; N, 9.68.
1-(2-((4-methylbenzylidene)amino)ethyl)piperidine-3-car-
boxylic acid (5v) Yield: 123 mg, 44.89%. FT-IR (KBr)
νmax 3229 (O–H), 3049 (Ar C–H), 2872 (C–H), 1718
(C=O), 1622 (C=N), 1591 (Ar C=C). 1H NMR (500 MHz,
CDCl3) δ = 11.47 (s, 1H, COOH), 8.18 (s, 1H, H-3′, imine
CH=N), 7.51 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-2, 6),
7.19 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-3, 5), 3.69 (2H, t,
J = 4.9 Hz, H-2′, C=NCH2), 2.99 (1H, dd, J = 12.3, 7.7
Hz, piperidine H-2a), 2.76 (2H, t, J = 4.9 Hz, H-1′, NCH2),
2.70––2.52 (2H, m, piperidine H-2b, 6a), 2.42 (1H, dd, J =
12.3, 7.7 Hz, piperidine H-6b), 2.36 (3H, s, CH3), 2.22–2.17
(1H, m, piperidine H-3), 2.03–1.96 (1H, m, piperidine H-
4a), 1.67–1.49 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 174.4 (C=O, COOH), 161.24 (imine
CH, C-3′), 142.5 (C, Ar–C, phenyl C-4), 132.4 (CH, Ar–C,
phenyl C-1), 129.5 (2 × CH, Ar–C, phenyl C-3, 5), 128.4
(2 × C, Ar–C, phenyl C-6, 2), 59.6 (CH2, C-1′), 55.7 (CH2,
C-2′), 55.5 (CH2, piperidine C-2), 53.78 (CH2, piperidine
C-6), 41.1 (CH, piperidine C-3), 24.9 (CH2, piperidine C-4),
23.6 (CH2, piperidine C-5), 20.8 (CH3, phenyl CH3). Anal.
1-(2-((3,4-dimethoxybenzylidene)amino)ethyl)piperidine-3-
carboxylic acid (5t) Yield: 157 mg, 49.66%. FT-IR (KBr)
νmax 3228 (O–H), 3034 (Ar C–H), 2865 (C–H), 1716
(C=O), 1618 (C=N), 1585 (Ar C=C). 1H NMR (500 MHz,
CDCl3) δ = 11.37 (s, 1H, COOH), 8.19 (s, 1H, H-3′, imine
CH=N), 7.26-6.89 (3H, phenyl H-2, 3, 6), 3.83 (3H, s,
OCH3), 3.81 (3H, s, OCH3), 3.71 (2H, t, J = 4.9 Hz, H-2′,
C=NCH2), 2.97 (1H, dd, J = 12.5, 7.7 Hz, piperidine H-
2a), 2.74 (2H, t, J = 4.9 Hz, H-1′, NCH2), 2.63–2.54 (2H,
m, piperidine H-2b, 6a), 2.38 (1H, dd, J = 12.5, 7.7 Hz,
piperidine H-6b), 2.25–2.16 (1H, m, piperidine H-3),
2.02–1.95 (1H, m, piperidine H-4a), 1.66–1.46 (3H, m,
piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3) δ =
175.7 (C=O, COOH), 162.5 (imine CH, C-3′), 152.7 (C,
Ar–C, phenyl C-4), 149.5 (CH, Ar–C, phenyl C-5), 131.4

Medicinal Chemistry Research
calcd. for C16H22N2O2: C, 70.04; H, 8.08; N, 10.21. Found:
69.79; H, 8.10; N, 10.18.
(KBr) νmax 3243 (O–H), 3068 (Ar C–H), 2878 (C–H), 1719
(C=O), 1616 (C=N), 1588 (Ar C=C), 1346 (C-F). 1H
NMR (500 MHz, CDCl3) δ = 11.33 (s, 1H, COOH), 8.46 (s,
1H, H-3′, imine CH=N), 7.89–7.25 (m, 4H, phenyl H-
2,4,5,6, Ar–H), 3.77 (2H, t, J = 5.1 Hz, H-2′, C=NCH2),
3.06 (1H, dd, J = 12.5, 7.7 Hz, piperidine H-2a), 2.87 (1H,
t, J = 5.0 Hz, H-1a′, NCH2), 2.81–2.65 (3H, m, H-1b′,
NCH2, piperidine H-2b, 6a), 2.53–2.29 (2H, m, piperidine
H-3, 6b), 2.23–2.07 (1H, m, piperidine H-4a), 1.88–1.64
(3H, m, piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3)
δ = 176.4 (C=O, COOH), 162.1(imine CH, C-3′), 137.0
(CH, Ar–C, phenyl C-1), 129.75 (C, Ar–C, phenyl C-2),
129.73 (CH, Ar–C, phenyl C-5), 129.6 (CH, Ar–C, phenyl
C-4), 129.0 (q, JC,F = 33.2 Hz, CH, Ar–C, phenyl C-3),
124.1 (CH, Ar–C, phenyl C-6), 123.6 (q, JC,F = 272.5 Hz,
CF3), 59.5 (CH2, C-1′), 55.4 (CH2, C-2′), 55.3 (CH2,
piperidine C-2), 53.7 (CH2, piperidine C-6), 40.9 (CH,
piperidine C-3), 24.9 (CH2, piperidine C-4), 22.6 (CH2,
piperidine C-5). Anal. calcd. for C16H19F3N2O2: C, 58.53;
H, 5.83; N, 8.53. Found: C, 58.69; H, 5.81; N, 8.51.
1-(2-((naphthalen-2-ylmethylene)amino)ethyl)piperidine-3-
carboxylic acid (5w) Yield: 149 mg, 48.06%. FT-IR (KBr)
νmax 3239 (O–H), 3039 (Ar C–H), 2881 (C–H), 1710
(C=O), 1618 (C=N), 1588 (Ar C=C). 1H NMR (500 MHz,
CDCl3) δ = 11.6 (s, 1H, COOH), 8.10 (s, 1H, H-3′, imine
CH=N), 8.08–7.26 (7H, m, Ar–H, napthyl H), 3.89 (2H, t,
J = 4.8 Hz, H-2′, C=NCH2), 3.08 (1H, dd, J = 12.3, 7.7
Hz, piperidine H-2a), 2.81 (2H, m, H-1′, NCH2), 2.74–2.57
(2H, m, piperidine H-2b, 6a), 2.47 (1H, dd, J = 12.5, 7.7
Hz, piperidine H-6b), 2.38–2.30 (1H, m, piperidine H-3),
2.25–2.12 (1H, m, piperidine H-4a), 1.87–1.60 (3H, m,
piperidine H-5, 4b). 13C NMR (125 MHz, CDCl3) δ =
176.4 (C=O, COOH), 162.4 (imine CH, C-3′), 134.9 (C,
Ar–C, phenyl C-4), 133.8 (C, Ar–C, phenyl C-9), 133.5
(CH, Ar–C, phenyl C-1), 129.7 (CH, Ar–C, phenyl C-8),
129.3 (CH, Ar–C, phenyl C-3), 129.0 (CH, Ar–C, phenyl
C-10), 128.4 (CH, Ar–C, phenyl C-5), 126.98 (C, Ar–C,
phenyl C-2), 126.92 (C, Ar–C, phenyl C-6), 126.4 (C,
Ar–C, phenyl C-7), 59.5 (CH2, C-1′), 55.4 (CH2, C-2′),
55.3 (CH2, piperidine C-2), 53.7 (CH2, piperidine C-6),
40.9 (CH2, piperidine C-3), 24.9 (CH2, piperidine C-4),
22.6 (CH2, piperidine C-5). Anal. calcd. for C19H22N2O2: C,
73.52; H, 7.14; N, 9.03. Found: C, 73.77; H, 7.13; N, 9.05.
Determination of partition coefficient (log P)
Log P value of all the synthesized compounds (5a–5y) was
determined experimentally by shake flask method in n-
octanol and buffer (pH 7.4) as per the reported procedure
(Ghadimi et al. 2008). A calibration curve was plotted using
different concentrations of the compound in water using
methanol as co-solvent. The known quantity compounds
were dissolved separately in n-octanol and shaken with the
buffer on a mechanical shaker for 30 min. To accomplish
complete phase separation, the mixture was centrifuged for
20 min, and the n-octanol phase was separated. The absor-
bance of the buffer phase was then measured, and the Log P
was calculated by correlating the absorbance with the
concentration in the standard plot.
1-(2-((4-(trifluoromethoxy)benzylidene)amino)ethyl)piperi-
dine-3-carboxylic acid (5x) Yield: 187 mg, 54.36%. FT-IR
(KBr) νmax 3237 (O–H), 3046 (Ar C–H), 2881 (C–H), 1712
1
(C=O), 1619 (C=N), 1579 (Ar C=C), 1356 (C–F). H
NMR (500 MHz, CDCl3) δ = 11.55 (s, 1H, COOH), 8.27 (s,
1H, H-3′, imine CH=N), 7.55 (2H, d, J = 7.5 Hz, Ar–H,
phenyl H-2, 6), 6.97 (2H, d, J = 7.5 Hz, Ar–H, phenyl H-3,
5), 3.60 (2H, t, J = 5.3 Hz, H-2′, C=NCH2), 3.05 (1H, dd,
J = 12.3, 7.7 Hz, piperidine H-2a), 2.89 (2H, t, J = 5.3 Hz,
H-1′, NCH2), 2.77–2.67 (2H, m, piperidine H-2b, 6a), 2.66
(1H, dd, J = 12.5, 7.7 Hz, piperidine H-6b), 2.28–2.20 (1H,
m, piperidine H-3), 2.11–1.98 (1H, m, piperidine H-4a),
1.79–1.57 (3H, m, piperidine H-5, 4b). 13C NMR (125
MHz, CDCl3) δ = 173.4 (C=O, COOH), 161.8 (imine CH,
C-3′), 152.6 (C, Ar–C, phenyl C-4), 134.8 (CH, Ar–C,
phenyl C-1), 129.4 (2 × CH, Ar–C, phenyl C-6, 2), 121.8 (q,
JC,F = 325.1, 193.1 Hz, C, OCF3), 121.2 (2 × C, Ar–C,
phenyl C-5, 3), 60.1 (CH2, C-1′), 57.5 (CH2, C-2′), 56.4
(CH3, phenyl OCH3), 55.8 (CH2, piperidine C-2), 52.6
(CH2, piperidine C-6), 40.4 (CH, piperidine C-3), 23.4
(CH2, piperidine C-4), 21.5 (CH2, piperidine C-5). Anal.
calcd. for C16H19F3N2O3: C, 55.81; H, 5.56; N, 8.14.
Found: C, 55.63; H, 5.57; N, 8.11.
Pharmacology
In vitro PAMPA-BBB assay
PAMPA-BBB assay was performed to predict the in vivo
penetration of the synthesized compounds (5a–5y) across
BBB by the reported method (Di et al. 2003). The porcine
polar brain lipid (PBL) was obtained from the Avanti Polar
Lipids. Dodecane was purchased from the Avra Synthesis
Pvt. Ltd. Acceptor microplates with PVDF membrane pore
size 0.45 μm and donor microplates were procured from the
Merck Millipore. The test compounds were dissolved in
dimethylsulfoxide (DMSO) to form the primary stock
solutions (5 mg/mL). An aliquot of 10 µL from each pri-
mary stock solution was diluted 200 times using a buffer of
pH 7.5 to form the corresponding secondary stock solutions
1-(2-((3-(trifluoromethyl)benzylidene)amino)ethyl)piperi-
dine-3-carboxylic acid (5y) Yield: 176 mg, 53.66%. FT-IR

Medicinal Chemistry Research
(25 µg/mL). The donor wells were filled with 200 µL of the
secondary stock solution. The coating of the porous filter
disk at the bottom of each well of the acceptor plate was
accomplished using a solution of PBL in an inert organic
solvent (4 µL of 20 mg/mL PBL in dodecane). 200 µL of
buffer (pH 7.4) was then filled into the acceptor plate. The
acceptor plate was then carefully stacked over the donor
plate to create the sandwich and incubated for 18 h to allow
the diffusion of the test compound from the donor well to
the acceptor well via lipid membrane. The drug con-
centration in the acceptor, donor, and reference well was
then determined by UV spectroscopy (96 well UV micro-
plate reader). The samples were scanned in triplicate for at
least five different wavelengths. The validation of the model
was done by using nine commercial drugs with known BBB
permeability which were purchased from Sigma-Aldrich
(India). A linear correlation was established by using the
experimentally obtained permeability [Pe (Exp)] and refer-
ence permeability [Pe (Ref)] (Di et al. 2003).
from Sigma Aldrich and administered s.c. at the dose of
100 mg/kg. Test compounds and a standard drug (tiagabine)
were administered i.p. 1 h before PTZ challenge at equi-
molar dose relative to 10 mg/kg tiagabine. Control group
animals received physiological saline (0.9%) containing
2.5% tween 80. Latency to first seizure and frequency of
seizures were noted up after PTZ administration.
DMCM induced seizures in mice
Compounds eliciting considerable anticonvulsant activity in
the sc-PTZ model were further screened using the standard
DMCM induced seizure model (Andersen et al. 2001a).
This test measures the efficiency of drugs to delay the onset
of tonic and clonic seizures caused by DMCM. Test com-
pounds (5d, 5l, 5w, 5x and 5y) were injected i.p. at the dose
of 10 mg/kg equivalent to standard tiagabine. DMCM was
injected at the dose of 15 mg/kg, i.p. The latency to the first
convulsion was noted.
Animals
Rota-rod performance test in mice
In vivo experiments were performed on Swiss albino mice
(20 5 g) of either sex, which were procured from the
Central Animal House, Institute of Medical Sciences,
Banaras Hindu University, Varanasi. The animals were
housed in polypropylene cages at a controlled temperature
of 25 1 °C and 45–55% relative humidity, under 12:12 h
light/dark cycle. The rodents had free access to commercial
food pellets and water ad libitum unless stated otherwise.
Rodents were allowed to acclimatize with the laboratory
environment before experiments for at least 1 week. Peri-
odical measurement of the body weight of animals was
done, and the animals were given identification marks
cryptically encoding the group as well as dose level. Prin-
ciples of laboratory animal care guidelines (NIH publication
number 85–23, revised 1985) were followed. All the
experimental procedures and protocols have been approved
by the Institutional Animal Ethical Committee, Institute of
Medical Sciences, Banaras Hindu University, Varanasi
(Letter No. Dean/12–13/CAEC/17).
The test is used to observe any motor incoordination caused
by the drugs. The test involves measurement of fall off time
of mice on a rotating rod (10 rpm). The test was conducted
in two sessions- before drug treatment and 1 h after drug
treatment. Only those animals that stay more than 3 min on
rotating rods were selected for the test. Each mouse was
placed on rotating rod and “fall-off” time was noted.
Immediately after the test animals were treated with the
respective drug, i.e., test, standard, and the vehicle in case
of control. One hour after the drug treatment, animals were
again placed on rotating rod and “fall-off” time was noted.
MTT assay for the measurement of cell viability
The effect of the most active compounds (5d, 5w, and 5y)
on neuroblastoma cell line SH-SY5Y (procured from
National Center for Cell Science, Pune) was evaluated by
MTT assay (Regulska et al. 2010; Shidore et al. 2016).
Initially, 96-well plates were seeded with SH-SY5Y cells at
a density of 1 × 105/well in 100 μL of the medium, followed
by 24 h of incubation at 37 °C in 5% CO2. Different con-
centrations of the test compounds (ranging from 1 µM to
160 µM) were then incubated with the cells for further 24 h.
After the exposure of cells to the different concentrations of
the test drugs, 20 μL of 5 mg/mL MTT reagent was added to
each well and incubated for 3 h (at 37 °C, 5% CO2). After
incubation, the formation of a purple colored precipitate
was observed under a microscope and solubilized by adding
1 mL of DMSO. Cell viability was measured by determin-
ing absorbance at 570 nm in a microplate-reader (Synergy
HTX, BioTek, Germany). The outcomes were expressed as
sc-PTZ induced seizures in mice
The mice model of sc-PTZ induced seizures as described by
Kowalczyk et al. 2014; was used to screen the compounds
5d, 5f, 5j, 5l, 5m, 5n, 5w, 5x and 5y. These compounds
exhibited better permeability in PAMPA-BBB assay
(Kowalczyk et al. 2014). Clonus of the whole body lasting
more than 3 s followed by loss of righting reflex is an
indicator of seizures in this test. Delay in the onset of sei-
zures and reduced frequency by test and the standard drug is
considered as anti-epileptic activity. The PTZ was procured

Medicinal Chemistry Research
a growth percentage in each well relative to the control cells
incubated in the absence of test compounds. Percentage cell
viability was measured by using the following formula:
on Kernel Linux operating environment. The potential
ligands 5d, 5w, and 5y were selected and prepared using
LigPrep module. The minimum energy conformers of
selected ligands were generated using OPLS2005 force
field. Homology modeled protein structure (PDB Code:
4XP4) of GAT1 GABA transporter (GAT) was refined and
corrected using Protein Preparation Wizard module. The
structure of the protein was further optimized using PropKa
method at default pH value 7.0, and restrained minimization
was performed for heavy atoms to RMSD 0.30 Å. Receptor
Grid was generated surrounding the active binding pocket
of the protein. The grid box of 10 Å was created by sup-
plying the x, y and z coordinates (x = 29 Å, y = 27 Å and z
= 22 Å) surrounding the active pocket in which the tiaga-
bine binds. The prepared grid and docking simulation pro-
tocols of Glide (Grid-Based Ligand Docking with
Energetics) extra precision (XP) mode were validated by
docking the tiagabine. During the docking simulations, the
protein was kept rigid, and ligands were kept flexible. All
other parameters of Glide module were maintained at their
default values. The docking results were studied using the
Glide XP visualizer module to gain insights of the inter-
actions of ligands with the amino acid residues. The results
of score and interactions were analyzed in comparison to
tiagabine.
Percentage Cell Viability ¼Absorption of sample=
absorption of control  100
Statistical analysis
The experimental results are expressed as the mean S.D
(n = 6) followed by a one-way analysis of variance.
Tukey’s multiple comparisons test were applied for deter-
mining the statistical significance between different groups.
InStat Graph Pad Software (San Diego, CA, USA) was used
for all statistical analyses and a p-value <0.05 was con-
sidered significant.
Computational studies
Homology modeling of GAT1
Homology modeling was performed as per the method of
Zaman and coworkers (Singh et al. 2017). It was carried out
in three principle steps. The first step involves the alignment
of the amino acid sequence of Drosophila dopamine trans-
porter (dDAT) with that of the target sequence (GAT-1).
The second step includes model building from the align-
ment, followed by validation.
Molecular dynamics simulations
From UniProt website (http://www.uniprot.org/uniprot/
P30531) the complete sequence of human sodium-
dependent and chloride-dependent GABA transporter 1
(GAT1) (UniProtKB: P30531) was downloaded. To find the
suitable template psi-BLAST was performed against Protein
Data Bank (PDB). A template of Drosophila melanogaster
dopamine transporter (dDAT) (PDB ID: 4XP4_A) (Wang
et al. 2015) was chosen as the template for building multiple
homology models. The sequences of the template
(4XP4_A) GAT1 were aligned using multiple sequence
alignment programs (Clustal W). The generated models
were then subjected to loop refinement using the Prime
module implemented in the Schrödinger suite. During
model preparation, the Na+ and Cl- ions were retained. The
quality of the models was assessed by using Ramachandran
plot. The best model was validated by docking tiagabine (a
known inhibitor) which matches with the previous studies
and mutagenesis data.
To confirm the stability of binding mode interactions for the
most potent compound 5w, its docked complex was further
utilized for molecular dynamics simulation run using the
Desmond module of Schrödinger Maestro 10.5.014 pro-
gram with an OPLS-AA force field in an explicit solvent
with the TIP3P water model. The docked complex of 5w
with GABA GAT1 transporter protein (PDB Code: 4XP4)
was soaked adequately in 14,594 TIP3P water molecules,
and the system was neutralized by adding 4Na+ ions to
balance the overall charge of the system. The generated
system with water molecules consisted of total 52,414
atoms. The system was further minimized to maximum
20,000 steps. The recording interval energy was kept at 1.2
ps, and the trajectory was set at 9.6. At constant number of
atoms (N), pressure (P), and temperature (T) (NVT) mole-
cular dynamics was performed for the first 100 ps, during
which the temperature of the system was raised from 0–300
K. For further simulations, the system was maintained at
constant temperature (300 K) and pressure (1.0132 bar) till
the complete cycle of 50 ns simulation run.
In silico docking simulations
In silico docking simulation protocols were performed using
Schrödinger Glide module in Schrödinger Suite 10.5.014
MM Share Version 3.3.014 Release 2016-1 with work-
station 4× Intel(R) Xeon(R) CPU E5-1607 v3 @ 3.10 GHz
In silico ADME studies
QikProp module of Schrödinger Maestro 10.5.014 Release
2016-1 was used to predict the “drug likeliness” of the most

Medicinal Chemistry Research
Table 1 Chemical structures and physicochemical properties of the
synthesized compounds (5a–5y)
Comp. Ar–group
Rfa Log Pb Melting point (oC)
5a
5b
5c
5d
5e
5f
Phenyl
0.46 2.36
0.37 2.62
0.37 2.60
0.30 3.16
0.28 3.31
0.26 3.48
0.43 2.42
0.42 2.46
0.41 2.51
0.34 2.90
0.29 3.28
0.31 3.20
0.27 3.47
0.25 3.41
0.26 3.37
0.52 2.12
0.56 2.08
0.49 2.27
0.48 2.29
0.51 2.18
0.54 2.10
0.35 2.82
0.28 3.42
192–194
162–164
167–169
181–183
172–174
187–189
158–160
166–168
177–179
186–188
155–157
158–160
148–150
212–214
222–224
198–200
213–215
204–206
209–211
224–226
217–219
177–179
228–230
163–165
181–183
Scheme 1 Synthesis of compounds 5a–5y; Reagents and conditions: a
K2CO3, KI, 1,4-dioxane, reflux, 30 h; b (i) 3 N NaOH, EtOH, RT, (ii)
1 N HCl; c Corresponding aromatic aldehydes, glacial acetic acid (2–3
drops), EtOH, reflux, 3–6 h
2-Chlorophenyl
4-Chlorophenyl
2,3-Dichlorophenyl
2,4-Dichlorophenyl
2,3,6-Trichlorophenyl
3-Fluorophenyl
active compounds 5d, 5w, and 5y. Before being subjected
to QikProp analysis, the best-fit ligands were neutralized.
Several principal descriptors were estimated.
5g
5h
5i
4-Fluorophenyl
2,4-Difluorophenyl
3,4,5-Trifluorophenyl
3-Bromophenyl
4-Bromophenyl
2,6-Dibromophenyl
4-Nitrophenyl
5j
Results and discussion
5k
5l
Chemistry
5m
5n
5o
5p
5q
5r
5s
The synthesis of the target compounds 5a–5y was per-
formed according to the reaction sequence outlined in
Scheme 1. At the outset, Ethyl 1-(2-aminoethyl)piperidine-
3-carboxylate (3) was synthesized by piperidine-3-
carboxylic acid ethyl ester (1) and 2-bromoethylamine
hydrobromide (2) by nucleophilic substitution (SN2) reac-
tion. Compound 3 which is an ester derivative of the parent
cyclic amine (nipecotic acid) was used for protecting car-
boxyl group in the reaction. The ethyl ester group of
compound 3 was hydrolyzed in alcoholic alkaline solution
to generate compound 4. Compounds 5a–5y were synthe-
sized by the nucleophilic addition of the amino group of
compound 4 to the carbonyl group of corresponding aro-
matic aldehydes forming an unstable aminomethanol
intermediate followed by dehydration in an acidic envir-
onment to generate an imine (Scheme 1).
2-Nitrophenyl
2-Hydroxyphenyl
4-Hydroxyphenyl
3-Methoxyphenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
4-Methylphenyl
Napthyl
5t
5u
5v
5w
5x
5y
4-Trifluoromethoxyphenyl 0.24 3.78
3-Trifluoromethylphenyl 0.30 3.27
aRf values are determined using DCM/methanol (9.5:0.5) as mobile
phase
bLog P values of all the compounds were determined experimentally
using shake flask method
The structures of the synthesized compounds were
ascertained by FT-IR, H NMR, 13C NMR and elemental
1
analysis. The amino group in compound 3 was confirmed
by the presence of asymmetric N–H stretching around 3366
cm−1, respectively. A singlet varying from δH11.6–11.18
ppm in H NMR belonged to piperidine-CO–OH, and the
1
cm−1 and symmetric N–H stretching around 3298 cm−1
,
singlet appeared at δH9.02–8.10 ppm revealed the presence
of N = CH– methaneimine protons. The chemical shifts
from 181.2–172.9 and 164.3–156.7 ppm in 13C NMR con-
firmed the existence of C=O and C=N, respectively.
Results of the elemental analysis were found within 0.4%
of the theoretical values and were well within the limit. The
respectively. The characteristic broad O–H stretching peak
observed at around 3236 cm−1 in compound 4 depicted the
presence of H-bonded O–H group. The absorption bands in
the compounds 5a–5y showed characteristic skeletal fre-
quencies for C=O and C=N at 1729–1706 and 1628–1612

Medicinal Chemistry Research
Table 2 Effect of drugs on s.c. administered PTZ-induced seizures
partition co–efficient was determined using the octanol/
water “shake-flask” method. The Rf values, melting point
and Log P values of the compounds 5a–5y are presented in
Table 1.
Comp.
Latency of seizures (s)a Frequency of seizures
(numbers)a
Control
5d
554.16 18.84
1036.50 20.56*
555.33 12.82
564.16 25.46
843.16 21.94*
560.83 19.45
565.16 46.82
1181.66 19.16*
792.33 19.59*
1119.83 21.84*
4.33 0.81
1.66 0.81*
3.66 0.81
3.83 0.75
1.83 0.75*
4.33 0.81
4.16 0.75
1.16 0.40*
2.16 0.75*
1.33 0.51*
1.16 0.40*
Pharmacology
5f
5j
PAMPA-BBB assay
5l
5m
5n
Determination of brain permeability is very crucial for
antiepileptic drugs to reach the target site and elicit its
effect. PAMPA-BBB is a technique to observe the BBB
permeation of drug molecules. In the current work, the
permeability of the synthesized compounds was evaluated
by PAMPA-BBB as per the reported procedure of Di et al.
(Di et al. 2003) This system is a prototype of BBB that
measures the effective permeability (Pe, cm/s) of an artifi-
cial lipid membrane and thereby predicts the rate of trans-
cellular passive diffusion of drugs across the BBB. A plot of
experimentally obtained permeability [Pe(Exp)] versus per-
meability reported in the literature [Pe(Ref)] provided a good
5w
5x
5y
Tiagabine 1276.33 17.50*
Control: Physiological saline (0.9%) containing 2.5% tween 80;
Tiagabine: 10 mg/kg, i.p.; all the test compounds were administered
intraperitoneally at an equimolar dose relative to 10 mg/kg tiagabine
*p < 0.05 compared to control
aValues are expressed as the Mean SD (n = 6)
linear correlation
Pe(Exp) = 1.308 P
e(Ref)–0.8394 (R2 =
Table 3 Effect of compounds on DMCM induced seizures
0.9317). Using this equation, we have calculated the cut-off
limits for determining the BBB permeability of the test
compounds. The values of Pe(Ref) were taken from the limits
established by Di et al. The findings suggested that the
compounds 5d, 5f, 5j, 5l, 5m, 5n, 5w, 5x, and 5y exhibited
considerable permeability across BBB. 5w was more
permeable (Pe = 8.93) than the standard tiagabine (Pe =
7.96) (See Supplementary Table S1)
Comp.
Latency of seizures
(s)a
Control
222.33 6.02
5d
373.33 8.35*
272.33 11.62*
417.83 9.17*
267.66 13.93*
411.50 16.67*
438.66 10.46*
5l
5w
5x
5y
PTZ-induced convulsions in mice
Tiagabine
Control: Physiological saline (0.9%) containing 2.5% tween 80;
Tiagabine: 10 mg/kg, i.p.; all the test compounds were administered
intraperitoneally at an equimolar dose relative to 10 mg/kg Tiagabine
aValues are expressed as the Mean SD (n = 6)
Manipulation of GABA metabolism, synaptic uptake
mechanism and its receptor complex along with neuronal
ion channels has been a central theme for research to dis-
cover safe and effective novel drugs for the treatment of
epilepsy. Tiagabine is a recent entrant in the category of
anti-epileptic drug that has a distinct mechanism of reuptake
inhibition of GABA at the synapse. Tiagabine augments the
level and neuro-inhibitory activity of GABA by interfering
the function of GATs specifically GAT1.
*p < 0.05 compared to control
DMCM (Methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-
carboxylate)-induced seizures in mice
Subcutaneous injection of PTZ is validated and most
commonly used rodent model of epilepsy. In this test
inhibitory potential of test drugs to suppress or delay the
seizures induced by PTZ is measured. It was observed that
the test compounds 5d, 5l, 5w, 5x and 5y significantly
delayed the onset of seizures and its frequency. However,
compounds 5n, 5f, 5m, and 5j failed to exhibit anti- seizure
activity in this model (Table 2). Tiagabine also significantly
delayed the onset of seizures and frequency of seizures. 5d,
5w, and 5y were most potent amongst synthesized
compounds.
Only those compounds that exhibited significant anti-
epileptic activity in the sc-PTZ model were selected for
further screening in DMCM induced seizure test. DMCM is
a potent convulsant agent having dual effect-augmenting
excitatory amino acid and attenuating GABA inhibitory
function. DMCM has been identified to possess specific
benzodiazepine binding sites (Petersen 1983). The outcome
of the model was similar to that of sc-PTZ induced seizure
model. All the test compounds (5d, 5l, 5w, 5x and 5y) and
standard drug significantly delayed the onset of convulsion
(Table 3).

Medicinal Chemistry Research
Rota-rod performance test in mice
dehydrogenases to reduce MTT to form the insoluble for-
mazan is interpreted as the measure of cell viability. The
formazan upon solubilization can be measured spectro-
photometrically, which is directly proportional to the viable
cell number (Lim et al. 2015). The results of the experiment
reveal that the MTT reduction was not effected significantly
by test compounds (5d, 5w, and 5y), thus corresponds to the
insignificant cell death in the concentrations of the test
compounds ranging from 1 µM to 80 µM (Table 5).
Drugs acting on CNS do have potential to cause motor
incoordination. In order to assess the putative motor
incoordination effect of drugs, rota rod test is widely used.
In this test rodents are placed on a rotating rod and fall off
time is measured before and after the drug treatment. A
significant decrease in the fall off time indicates the motor
incoordination effect of the drug. In this test, all the test
compounds were found to be devoid of any adverse effect
on muscle coordination (Table 4). Standard drug diazepam
showed a significant reduction in fall off time.
Computational studies
Homology modeling
Cell viability and neurotoxicity
For model building, human GAT1 protein has been selec-
ted. Structures are accessible for dDAT, which is one of the
closest transporter protein related to human GAT1.
Homology model of both the occluded and the open-to-out
conformations was constructed taking dDAT structure as a
template (See Supplementary Fig. S7). After the alignment,
46% sequence identity and 67% similarity of the 4XP4
template with GAT1 sequence has been obtained. In the
model generation, a known disulfide bridge between C164
and C173 located in the extracellulAr–loop 2 of GAT1 was
included (Jurik et al. 2015). A total of 10 models were
prepared using the knowledge-based method implemented
in the Schrödinger Suite 2016 (Kim and Cho 2016). The
best model thus generated was then subjected to loop
refinement. In the Ramachandran plot, all the amino acids
except Phe174 and Ser178 were present in the favored/
allowed regions, which enabled to use the best model for
the docking study (See Supplementary Fig. S8). These two
residues are not the part of the binding site. The models
were exhaustively tested and verified through comparison to
functional and mutational data reported in the literature. The
homology model was evaluated through computations of
molecular interactions fields and sequence identities. Tem-
plate in an open-to-out conformation was used for allowing
access to bulky synthesized molecules. A validated
homology model was finally obtained followed by the
identification of putative spots for ligand (Tiagabine)
Some antiepileptic drugs and their metabolites have been
reported to possess neurotoxicity (Araújo et al. 2004;
Ambrósio et al. 2000; Liu et al. 2015; Gao and Chuang
1992; Gao et al. 1995; Nonaka et al. 1998). Ideally, anti-
epileptic drugs should prevent the seizures without produ-
cing neuronal toxicity. Therefore, the therapeutic suitability
of the most active compounds (5d, 5w, and 5y) and their
effects on cell viability was determined in neuroblastoma
cell line (SH-SY5Y). The ability of intracellular
Table 4 Effect of compounds on rota-rod performance test in mice
Comp.
Fall off time before
treatment (s)a
Fall off time after
treatment (s)a
Control
5d
318.33 11.37
323.50 6.41
331.83 15.06
325.16 16.64
326.66 12.95
328.16 5.56
333.33 8.23
331.83 15.35
322.66 5.98
152.66 12.12a
5w
5y
Tiagabine 320.83 13.07
Diazepam 334.83 15.86
Control: Physiological saline (0.9%) containing 2.5% tween 80;
Tiagabine: 10 mg/kg, i.p.; Diazepam: 4 mg/kg, i.p.
All the test compounds were administered intraperitoneally at an
equimolar dose relative to 10mg/kg tiagabine
*p < 0.05 compared to control
aValues are expressed as the Mean SD (n = 6)
Table 5 Cell Viability of the test compounds at different concentrations in neuroblastoma cell line (SH-SY5Y)
Comp.
Percentage cell viabilitya
1 μm
10 μm
20 μm
40 μm
80 μm
5d
5w
5y
99.99 0.054
99.74 0.127
99.60 0.117
99.95 0.101
99.46 0.220
99.40 0.216
99.91 0.115
96.50 0.380
95.81 0.378
98.63 0.059
92.22 0.467
91.48 0.470
89.25 0.116
83.52 0.304
84.92 0.384
Values are expressed as the percentage cell viability SD of at least five independent experiments
aPercentage cell viability of SH-SY5Y cells incubated with increasing concentration of test compounds

Medicinal Chemistry Research
selectivity. Tiagabine was added as a co-crystallized ligand
in the model which was further utilized for molecular
docking and dynamics.
Tyr139, Tyr140, Ile143, Gln291, Phe294, Ser295 and Na
atom.
The docking conformations of the compounds 5d, 5w,
and 5y reveal salt bridge formation between oxygen (O)
atom of carboxyl group and sodium (Na611). Another
oxygen (O) atom present in carboxyl group of compound 5y
shown to have additional metal coordination interaction
with sodium (Na611). The O atom of the carboxyl group in
all the docked ligands interacted through a network of
hydrogen bonding with backbone atom of Gly65. Com-
pounds 5d and 5w involved in the hydrogen bonding with
side chain hydroxyl groups of Tyr140. Additionally, the NH
group of all the ligands was also involved in hydrogen
bonding interactions with Phe294 similar to tiagabine. The
NH group of compound 5y was additionally involved in
π–cationic interaction with Tyr60. The charged interactions
with Arg69 and Asp451 were also responsible for stabiliz-
ing the aromatic rings of the ligands. The detailed interac-
tion results of tiagabine, 5d, 5w, and 5y with active site
amino acid residues are summarized in Table 6. Overall,
these interactions of all the docked ligands with the mod-
eled protein of GAT1 GABA transporter showed com-
plementary binding with active site amino acids residues as
shown in Figs. 3, 4 and 5.
In silico docking study
In silico docking studies were performed using the Schrö-
dinger Maestro program to gain insight into the possible
mode of protein–ligand interactions using a generated and
validated model of GAT1 GABA transporter (PDB Code:
4XP4). The validation of the prepared grid and docking
protocols was performed by generating a minimum energy
conformer of tiagabine, and it’s docking on a prepared grid.
The results demonstrate that tiagabine occupied the same
active site within the binding pocket leading to its com-
plementary interaction with the amino acid residues within
the active site (Petrera et al. 2016; Skovstrup et al. 2010;
Jurik et al. 2013) (Fig. 2).
The binding affinity of the active compounds 5d, 5w and
5y were carried out using GAT1 GABA transporter mod-
eled protein. The molecular docking studies yielded the best
possible conformation for all the ligands 5d (Glide Score:
−3.9); 5w (GLIDE Score: −6.2) and 5y (GLIDE Score:
−7.3) occupying the similar binding pocket as that of tia-
gabine (GLIDE Score: −4.6) (See Supplementary Fig. S9,
S10 and S11). In the present study, the active binding
pocket was selected on the basis of the outcomes of Petrera
et al. The active pocket was present around the amino acid
residues Tyr60, Ala61, Gly63, Gly65, Trp68, Arg69,
Molecular dynamics simulations
The dynamics simulation runs of the generated minimized
complex of 5w with GABA GAT1 transporter protein of
Fig. 2 3D Ribbon structure representation of docking conformation of
the tiagabine in the hydrophobic pocket of modeled protein structure
of GAT1 GABA transporter (PDB Code: 4XP4). Structure of tiagabine
is shown as ball and stick model; light brown color surface is showing
hydrophobic pocket; blue and red surface is charged surface of the
protein; green dotted line is showing H-bonding interactions between
active site amino acid residues and ligand

Medicinal Chemistry Research
Table 6 Details of protein–ligand interactions of tiagabine, 5d, 5w, and 5y
Comp.
Parameters
Glide
score
Interacting residuesa
H-bonding
Salt
bridge
Metal
coordination
π–π
cation
Hydrophobic
Polar
Charged
Tiagabine −4.6
Gly65, Tyr140,
Phe294
Na611
Na611
None
None
None
None
Tyr60, Ala61, Gly63, Trp68, Tyr139,
Ile143, Gly297, Phe447
Asn66, Gln291,
Ser295, Ser396
Arg69,
Asp451
5d
5w
5y
−3.9
−6.2
−7.3
Gly65, Tyr140,
Phe294
Tyr60, Ala61, Ile62, Gly63, Leu64,
Trp68, Leu136, Tyr139, Gly297,
Leu300, Leu460
Asn66, Ser295, Ser396 Arg69,
Asp451
Gly65, Tyr140,
Phe294
Na611
Na611
None
None
Tyr60, Ala61, Gly63, Leu64, Leu136,
Tyr139, Tyr296, Gly297, Leu300,
Ala455, Leu460
Asn66, Ser295,
Ser396, Ser456
Asp451
Gly65, Phe294
Na611
Tyr60
Ala61, ile62, Gly63, Leu64, Leu136,
Tyr139, Tyr140, Tyr296, Gly297,
Leu300, Ala455, Leu460
Asn66, Ser396, Ser456 Asp451
aAll the interactions of protein–ligand were observed within the distance of 4 Å
Fig. 3 3D Ribbon structure representation of docking conformation of
the compound 5d in the hydrophobic pocket of modeled protein
structure of GAT1 GABA transporter (PDB Code: 4XP4). Structure of
compound 5d is shown as ball and stick model; light brown color
surface showing hydrophobic pocket; the blue and red surface is
charged surface of the protein; green dotted lines are H-bonding, and
the red dotted line is charged interaction between active site amino
acid residues and ligand
4XP4 was performed for 50 ns to predict the stability of
binding mode interactions. The overall stability of the sys-
tem was evaluated by RMSD (root mean square deviation)
and RMSF (root mean square fluctuation) calculations. The
results of the RMSD values confirmed that all frames of the
complex were in trajectory throughout the simulation with
average fluctuation in the range of 1–3 Å (See Supple-
mentary, Fig. S12).
The graphical representation of binding interactions
of compound 5w showed the active site interactions
throughout the simulation run (Fig. 6). The results demon-
strated that compound 5w efficiently interacted with active
site residues Ala61, Gly65, Asn66, Arg69, Tyr140 and
Phe294 through H-bonds. Besides, it also interacted with
Phe294 through hydrophobic π-stacking. The carboxylate O
atom also involved in salt bridge formation with Na611
atom.
The interaction fraction with individual amino acid
residues was also calculated and represented in a stacked
bar chart (Fig. 7). The interaction fraction of a percentage of
total contact maintained throughout the run. For example,
0.8 suggests that interaction was maintained 80% of the
total simulation run.

Medicinal Chemistry Research
Fig. 4 3D Ribbon structure representation of docking conformation of
the compound 5w in the hydrophobic pocket of modeled protein
structure of GAT1 GABA transporter (PDB Code: 4XP4). Structure of
compound 5w is shown as ball and stick model; light brown color
surface showing hydrophobic pocket; the blue and red surface is
charged surface of the protein; green dotted lines are H-bonding and
orange dotted lines are Van der Waals interactions between active site
amino acid residues and ligand
Fig. 5 3D Ribbon structure representation of docking conformation of
the compound 5y in the hydrophobic pocket of modeled protein
structure of GAT1 GABA transporter (PDB Code: 4XP4). Structure of
compound 5y is shown as ball and stick model; light brown color
surface showing hydrophobic pocket; the blue and red surface is
charged surface of the protein; green dotted lines are H-bonding and
orange dotted lines are Van der Waals interactions between active site
amino acid residues and ligand
In silico prediction of “drug likeliness”
to be active for CNS and might be permeable across BBB.
(Das et al. 2014) These results are comparable with the
outcome of PAMPA-BBB assay and experimental log P
values. However, the experimental log P values differ with
that of the predicted values. The predicted PSA (polar
surface area) values were found to be in the range of
58.378–59.856, which revealed that the selected
The results of some principle descriptors for the prediction
of in silico “drug likeliness” of the most active compounds
(5d, 5w, and 5y) are mentioned in Table 7. The predicted
values for QPlogBB and CNS activity predicted by QikProp
method indicated that the selected compounds were found

Medicinal Chemistry Research
compounds showed lower polar surface area. Lower PSA is
a key requisite for the compounds designed for CNS dis-
orders.(Meena et al. 2015) The absence of reactive func-
tional groups that causes decomposition, reactivity, or
toxicity problems in vivo was predicted by the outcome of
“rtvFG” value, which was found to be 0 for all the tested
compounds. The test compounds demonstrated drug like-
liness as per the Lipinski’s rule of five (mol_MW < 500,
QPlogPo/w < 5, donorHB ≤ 5, accptHB ≤ 10). QPlogKHSA
values for the tested compounds fall within the limit, indi-
cating considerable binding of the compounds with plasma
proteins. Overall the predicted parameters revealed that the
compounds 4a, 4b, and 4i fulfill drug-like characteristics.
(Banerjee et al. 2016)
suggested that compounds 5d, 5w, 5y were comparatively
more active than 5d and 5l, while the compound 5n was
found inactive. Compounds 5d, 5w and 5y exhibited
desirable physicochemical and drug-like properties. Based
on the outcome of the in silico studies, we can reasonably
infer that the potential derivatives inhibit GAT1 in a manner
similar to tiagabine which may explain their underlying
mechanism of action. Also the compounds 5d, 5w and 5y
were found to be devoid of neurotoxicity as indicated by the
results of MTT assay and rota rod test. Moreover the most
active compound 5w can be further quantified by in vitro
GAT1 inhibitory/binding assay to provide a mechanistic
pathway for anticonvulsant activity. All together the effi-
cacy and safety of the potential leads justifies the rationale
behind the study and provide a valuable insight towards the
Conclusion
In summary, we have successfully synthesized a series of
novel N-substituted nipecotic acid derivatives following a
synthetic approach of Schiff base formation through an
ethylene bridge linker. Determination of brain permeability
is very crucial for antiepileptic drugs to reach to the target
site and elicit pharmacological activity. Amongst the syn-
thesized compounds some potential leads have been iden-
tified with the ability to permeate the BBB by an in vitro
PAMPA- BBB assay, mitigating the rationale of design.
The permeability of the compounds 5d, 5f, 5j, 5l, 5m, 5n,
5w, 5x, and 5y were comparable to tiagabine. The findings
of in vivo PTZ, and DMCM induced rodent model epilepsy
Fig. 7 Stacked bar charts of protein interactions with ligand 5w as
monitored throughout the MD simulation
Fig. 6 The detailed atomic interactions of ligand 5w with the key active amino acid residues with GABA GAT1 transporter protein of 4XP4

Medicinal Chemistry Research
Table 7 In silico prediction of drug like properties of the active compounds 5d, 5w, 5y
Comp. Mol_MW
QPlogBB
CNS
QPlog Po/w PSA
QP log KHSA Lipinski’s rule
donorHB
accptHB
#rtvFG
(130–725)
(−3–1.2)
(−2– + 2) (−2–6.8)
(7-200) (−1.5–1.5)
of five (Max. 4) (0–6)
(2–20)
(0–2)
5d
5w
5y
310.39
329.22
328.33
−0.267
0.059
0
1
0
1.579
1.47
59.856 0.053
58.378 −0.106
59.195 −0.041
0
0
0
1
1
1
5.5
5.5
5.5
0
0
0
−0.005
1.625
Mol_MW molecular weight, QPlogBB predicted brain/blood partition coefficient, CNS predicted central nervous system activity, QPlogPo/w
predicted octanol/water partition coefficient, PSA polar surface area, QPlogKHSA prediction of binding to human serum albumin, Rule of five no.
of violations of Lipinski’s rule of five, donorHB No. of hydrogen bonds that would be donated by the solute to water molecules in an aqueous
solution, accptHB No. of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution, #rtvFG number of
reactive functional group
Bjorge S, Black A, Bockbrader H, Chang T, Gregor VE, Lobbestael
development and optimization more promising compounds
SJ, Nugiel D, Pavia MR, Radulovic L, Woolf T (1990) Synthesis
with superior anticonvulsant effects.
and metabolic profile of Cl‐966: a potent, orally‐active inhibitor
of GABA uptake. Drug Dev Res 21:189–193
Braestrup C, Nielsen EB, Sonnewald U, Knutsen LJ, Andersen KE,
Jansen JA, Frederiksen K, Andersen PH, Mortensen A, Suzdak
PD (1990) R)‐N‐[4, 4‐Bis (3‐Methyl‐2‐Thienyl) but‐3‐en‐1‐yl]
nipecotic acid binds with high affinity to the brain γ‐aminobutyric
acid uptake carrier. J Neurochem 54:639–647
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Chen N-H, Reith ME, Quick MW (2004) Synaptic uptake and beyond:
the sodium-and chloride-dependent neurotransmitter transporter
References
family SLC6. Pflug Arch 447:519–531
̈
Das N, Garabadu D, Banerjee AG, Krishnamurthy S, Shrivastava SK
(2014) Synthesis and pharmacological evaluation of some N3-
aryl/heteroaryl-substituted 2-(2-chlorostyryl)-6, 7-dimethoxy-
quinazolin-4 (3H)-ones as potential anticonvulsant agents. Med
Chem Res 23:4167–4176
Dhar TM, Borden LA, Tyagarajan S, Smith KE, Branchek TA,
Weinshank RL, Gluchowski C (1994) Design, synthesis and
evaluation of substituted triarylnipecotic acid derivatives as
GABA uptake inhibitors: identification of a ligand with moderate
affinity and selectivity for the cloned human GABA transporter
GAT-3. J Med Chem 37:2334–2342
Ali FE, Bondinell WE, Dandridge PA, Frazee JS, Garvey E, Girard
GR, Kaiser C, Ku TW, Lafferty JJ, Moonsammy GI (1985)
Orally active and potent inhibitors of gamma-aminobutyric acid
uptake. J Med Chem 28:653–660
Aly MM, Mohamed YA, El-Bayouki KA, Basyouni WM, Abbas SY
(2010) Synthesis of some new 4 (3H)-quinazolinone-2-carbox-
aldehyde thiosemicarbazones and their metal complexes and a
study on their anticonvulsant, analgesic, cytotoxic and anti-
microbial activities–Part-1. Eur J Med Chem 45:3365–3373
Ambrósio AF, Silva AP, Araújo I, Malva JO, Soares-da-Silva Pc,
Carvalho AP, Carvalho CM (2000) Neurotoxic/neuroprotective
profile of carbamazepine, oxcarbazepine and two new putative
antiepileptic drugs, BIA 2-093 and BIA 2-024. Eur J Pharmacol
406:191–201
Di L, Kerns EH, Fan K, McConnell OJ, Carter GT (2003) High
throughput artificial membrane permeability assay for
blood–brain barrier. Eur J Med Chem 38:223–232
El-Helby AGA, Ayyad RR, Sakr HM, Abdelrahim AS, El-Adl K,
Sherbiny FS, Eissa IH, Khalifa MM (2017) Design, synthesis,
molecular modeling and biological evaluation of novel 2, 3-
dihydrophthalazine-1, 4-dione derivatives as potential antic-
onvulsant agents. J Mol Struct 1130:333–351
Andersen KE, Lau J, Lundt BF, Petersen H, Huusfeldt PO, Suzdak
PD, Swedberg MD (2001a) Synthesis of novel GABA uptake
inhibitors. Part 6: preparation and evaluation of N-Ω asymme-
trically substituted nipecotic acid derivatives. Bioorg Med Chem
9:2773–2785
Falch E, Krogsgaard-Larsen P (1989) GABA uptake inhibitors con-
taining mono-and diarylmethoxyalkyl N-substituents. Drug Des
Deliv 4:205–215
Gao X-M, Chuang D-M (1992) Carbamazepine-induced neurotoxicity
and its prevention by NMDA in cultured cerebellar granule cells.
Neurosci Lett 135:159–162
Gao X-M, Margolis RL, Leeds P, Hough C, Post RM, Chuang D-M
(1995) Carbamazepine induction of apoptosis in cultured cere-
bellar neurons: effects ofN-methyl-d-aspartate, aurintricarboxylic
acid and cycloheximide. Brain Res 703:63–71
Ghadimi S, Latif Mousavi S, Javani Z (2008) Synthesis, lipophilicity
study and in vitro evaluation of some rodenticides as acet-
ylcholinesterase reversible inhibitors. J Enzyme Inhib Med Chem
23:213–217
Andersen KE, Sørensen JL, Lau J, Lundt BF, Petersen H, Huusfeldt
PO, Suzdak PD, Swedberg MD (2001b) Synthesis of novel γ-
aminobutyric acid (GABA) uptake inhibitors. 5. 1 Preparation
and structure− activity studies of tricyclic analogues of known
GABA uptake inhibitors. J Med Chem 44:2152–2163
Araújo IM, Ambrósio AF, Leal EC, Verdasca MJ, Malva JO, Soares‐
da‐Silva P, Carvalho AP, Carvalho CM (2004) Neurotoxicity
induced by antiepileptic drugs in cultured hippocampal neurons: a
comparative study between carbamazepine, oxcarbazepine, and
two new putative antiepileptic drugs, BIA 2‐024 and BIA 2‐093.
Epilepsia 45:1498–1505
Banerjee AG, Das N, Shengule SA, Sharma PA, Srivastava RS,
Shrivastava SK (2016) Design, synthesis, evaluation and mole-
cular modelling studies of some novel 5, 6-diphenyl-1, 2, 4-
triazin-3 (2H)-ones bearing five-member heterocyclic moieties as
potential COX-2 inhibitors: a hybrid pharmacophore approach.
Bioorg Chem 69:102–120
Ghareb N, Daim MMA, El-Sayed NM, Elgawish MS (2017) Synth-
esis, molecular modelling, and preliminary anticonvulsant activ-
ity evaluation of novel naphthalen-2-yl acetate and 1, 6-dithia-4,
9-diazaspiro [4.4] nonane-3, 8-dione derivatives. Bioorg Chem
71:110–119
Bhat MA, Al-Omar MA (2011) Synthesis, characterization and in vivo
anticonvulsant and neurotoxicity screening of Schiff bases of
phthalimide. Acta Pol Pharm 68:375–380

Medicinal Chemistry Research
Holmes GL (1995) Role of glutamate and GABA in the pathophy-
siology of epilepsy. Dev Disabil Res Rev 1:208–219
Jurik A, Reicherstorfer R, Zdrazil B, Ecker GF (2013) Classification of
high‐activity tiagabine analogs by binary QSAR modeling. Mol
Inform 32:415–419
Jurik A, Zdrazil B, Holy M, Stockner T, Sitte HH, Ecker GF (2015) A
binding mode hypothesis of tiagabine confirms liothyronine
effect on γ-aminobutyric acid transporter 1 (GAT1). J Med Chem
58:2149–2158
Petersen EN (1983) DMCM: a potent convulsive benzodiazepine
receptor ligand. Eur J Pharmacol 94:117–124
Petrera M, Wein T, Allmendinger L, Sindelar M, Pabel J, Höfner G,
Wanner KT (2016) Development of highly potent GAT1 inhi-
bitors: synthesis of nipecotic acid derivatives by Suzuki–Miyaura
cross‐coupling reactions. ChemMedChem 11:519–538
Petroff OA, Rothman DL, Behar KL, Mattson RH (1996) Low brain
GABA level is associated with poor seizure control. Ann Neurol
40:908–911
Kälviäinen R (2001) Long‐term safety of Tiagabine. Epilepsia
42:46–48
Prescott L (1997) Tiagabine offers effective seizure control. Inpharma
Wkly 1085:15–16
Kim M, Cho AE (2016) Incorporating QM and solvation into docking
for applications to GPCR targets. Phys Chem Chem Phys
18:28281–28289
Kowalczyk P, Sałat K, Höfner GC, Mucha M, Rapacz A, Podkowa A,
Filipek B, Wanner KT, Kulig K (2014) Synthesis, biological
evaluation and structure–activity relationship of new GABA
uptake inhibitors, derivatives of 4-aminobutanamides. Eur J Med
Chem 83:256–273
Quandt G, Höfner G, Wanner KT (2013) Synthesis and evaluation of
N-substituted nipecotic acid derivatives with an unsymmetrical
bis-aromatic residue attached to a vinyl ether spacer as potential
GABA uptake inhibitors. Bioorg Med Chem 21:3363–3378
Regulska M, Pomierny B, Basta-Kaim A, Starek A, Filip M, Lasoń W,
Budziszewska B (2010) Effects of ethylene glycol ethers on cell
viability in the human neuroblastoma SH-SY5Y cell line. Phar-
macol Rep 62:1243–1249
Krogsgaard-Larsen P (1980) Inhibitors of the GABA uptake systems.
Mol Cell Biochem 31:105–121
Krogsgaard‐Larsen P, Johnston G (1975) Inhibition of GABA uptake
in rat brain slices by nipecotic acid, various isoxazoles and related
compounds. J Neurochem 25:797–802
Shidore M, Machhi J, Shingala K, Murumkar P, Sharma MK, Agrawal
N, Tripathi A, Parikh Z, Pillai P, Yadav MR (2016)
Benzylpiperidine-linked Diarylthiazoles as potential anti-Alzhei-
mer’s agents: synthesis and biological evaluation. J Med Chem
59:5823–5846
Kulkarni A, Wankhede S, Dhawale N, Yadav P, Deore V, Gonjari I
(2017) Synthesis, characterization and biological behavior of
some Schiff’s and Mannich base derivatives of Lamotrigine. Arab
J Chem 10:S184–S189
Lim S-W, Loh H-S, Ting K-N, Bradshaw TD, Allaudin ZN (2015)
Reduction of MTT to purple formazan by vitamin E isomers in
the absence of cells. Trop Life Sci Res 26:111
Liu Y, Wang X-y, Li D, Yang L, Huang S-p (2015) Short-term use of
antiepileptic drugs is neurotoxic to the immature brain. Neural
Regen Res 10:599
Löscher W (1985) Anticonvulsant action in the epileptic gerbil of
novel inhibitors of GABA uptake. Eur J Pharmacol 110:103–108
Meena P, Nemaysh V, Khatri M, Manral A, Luthra PM, Tiwari M
(2015) Synthesis, biological evaluation and molecular docking
study of novel piperidine and piperazine derivatives as multi-
targeted agents to treat Alzheimer’s disease. Bioorg Med Chem
23:1135–1148
Murali Dhar T, Nagarathnam D, Marzabadi MR, Lagu B, Wong WC,
Chiu G, Tyagarajan S, Miao SW, Zhang F, Sun W (1999) Design
and synthesis of novel α1a adrenoceptor-selective antagonists. 2.
Approaches to eliminate opioid agonist metabolites via mod-
ification of linker and 4-methoxycarbonyl-4-phenylpiperidine
moiety. J Med Chem 42:4778–4793
Nielsen EB, Suzdak PD, Andersen KE, Knutsen LJ, Sonnewald U,
Braestrup C (1991) Characterization of tiagabine (NO-328), a
new potent and selective GABA uptake inhibitor. Eur J Phar-
macol 196:257–266
Nonaka S, Katsube N, Chuang D-M (1998) Lithium protects rat cer-
ebellar granule cells against apoptosis induced by antic-
onvulsants, phenytoin and carbamazepine. J Pharmacol Exp Ther
286:539–547
Ortinski P, Meador KJ (2004) Cognitive side effects of antiepileptic
drugs. Epilepsy Behav 5:60–65
Siddiqui N, Alam MS, Sahu M, Naim MJ, Yar MS, Alam O (2017)
Design, synthesis, anticonvulsant evaluation and docking study
of 2-[(6-substituted benzo [d] thiazol-2-ylcarbamoyl) methyl]-1-
(4-substituted phenyl) isothioureas. Bioorg Chem 71:230–243
Singh RB, Singh GK, Chaturvedi K, Kumar D, Singh SK, Zaman MK
(2017) Design, synthesis, characterization, and molecular mod-
eling studies of novel oxadiazole derivatives of nipecotic acid as
potential anticonvulsant and antidepressant agents. Med Chem
Res 27:137–152
Skovstrup S, Taboureau O, Bräuner‐Osborne H, Jørgensen FS (2010)
Homology modelling of the GABA transporter and analysis of
tiagabine binding. ChemMedChem 5:986–1000
Suzdak PD, Jansen JA (1995) A review of the preclinical pharma-
cology of tiagabine: a potent and selective anticonvulsant GABA
uptake inhibitor. Epilepsia 36:612–626
Sveinbjornsdottir S, Sander J, Patsalos P, Upton D, Thompson P,
Duncan J (1994) Neuropsychological effects of tiagabine, a
potential new antiepileptic drug. Seizure 3:29–35
Swathi K, Sarangapani M (2015) Synthesis and antiepileptic activity
of Schiff’s bases of dialkylamino alkoxy isatin derivatives.
GeNeDis 2014. Springer International Publishing, Swizerland
Taylor CP, Vartanian MG, Schwarz RD, Rock DM, Callahan MJ,
Davis MD (1990) Pharmacology of Cl‐966: A potent GABA
uptake inhibitor, in vitro and in experimental animals. Drug Dev
Res 21:195–215
Thompson SM, Gahwiler B (1992) Effects of the GABA uptake
inhibitor tiagabine on inhibitory synaptic potentials in rat hip-
pocampal slice cultures. J Neurophysiol 67:1698–1701
Villalba ML, Enrique AV, Higgs J, Castaño RA, Goicoechea S,
Taborda FD, Gavernet L, Lick ID, Marder M, Blanch LEB
(2016) Novel sulfamides and sulfamates derived from amino
esters: synthetic studies and anticonvulsant activity. Eur J Phar-
macol 774:55–63
Pandeya SN, Rajput N (2012) Synthesis and anticonvulsant activity of
various Mannich and Schiff bases of 1,5-benzodiazepines. Int J
Med Chem 2012:1–10
Wang KH, Penmatsa A, Gouaux E (2015) Neurotransmitter and
psychostimulant recognition by the dopamine transporter. Nature
521:322–327
Pavia MR, Lobbestael SJ, Nugiel D, Mayhugh DR, Gregor VE, Taylor
CP, Schwarz RD, Brahce L, Vartanian MG (1992) Structure-
activity studies on benzhydrol-containing nipecotic acid and
guvacine derivatives as potent, orally-active inhibitors of GABA
uptake. J Med Chem 35:4238–4248
Yunger L, Fowler P, Zarevics P, Setler P (1984) Novel inhibitors of
gamma-aminobutyric acid (GABA) uptake: anticonvulsant
actions in rats and mice. J Pharmacol Exp Ther 228:109–115