Lead optimization studies towards the discovery of novel carbamates as potent AChE inhibitors for the potential treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2012.09.005

The optimization of our previous lead compound 1 (AChE IC₅₀ = 3.31 μM) through synthesis and pharmacology of a series of novel carbamates is reported. The synthesized compounds were evaluated against mouse brain AChE enzyme using the colorimetric method described by Ellman et al. The three compounds 6a (IC₅₀ = 2.57 μM), 6b (IC₅₀ = 0.70 μM) and 6i (IC₅₀ = 2.56 μM) exhibited potent in vitro AChE inhibitory activities comparable to the drug rivastigmine (IC₅₀ = 1.11 μM). Among them, the compound 6b has been selected as possible optimized lead for further neuropharmacological studies. In addition, the AChE-carbamate Michaelis complexes of these potent compounds including rivastigmine and ganstigmine have been modeled using covalent docking protocol of GOLD and important direct/indirect interactions contributing to stabilization of the AChE-carbamate Michaelis complexes have been investigated.

Full text of DOI:10.1016/j.bmc.2012.09.005

Bioorganic & Medicinal Chemistry 20 (2012) 6313–6320
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lead optimization studies towards the discovery of novel carbamates as potent
AChE inhibitors for the potential treatment of Alzheimer’s disease q
Kuldeep K. Roy a, Santoshkumar Tota b, Tusha Tripathi a, Subhash Chander a, Chandishwar Nath c,
Anil K. Saxena a,
⇑
a Division of Medicinal and Process Chemistry, CSIR Central Drug Research Institute, Lucknow 226 001, India
b Division of Pharmacology, CSIR Central Drug Research Institute, Lucknow 226 001, India
c Division of Toxicology, CSIR Central Drug Research Institute, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The optimization of our previous lead compound 1 (AChE IC50 = 3.31 lM) through synthesis and pharma-
cology of a series of novel carbamates is reported. The synthesized compounds were evaluated against
mouse brain AChE enzyme using the colorimetric method described by Ellman et al. The three com-
Received 3 August 2012
Revised 3 September 2012
Accepted 5 September 2012
Available online 12 September 2012
pounds 6a (IC50 = 2.57
inhibitory activities comparable to the drug rivastigmine (IC50 = 1.11
l
M), 6b (IC50 = 0.70
l
M) and 6i (IC50 = 2.56
l
M) exhibited potent in vitro AChE
M). Among them, the compound
l
6b has been selected as possible optimized lead for further neuropharmacological studies. In addition,
the AChE–carbamate Michaelis complexes of these potent compounds including rivastigmine and gan-
stigmine have been modeled using covalent docking protocol of GOLD and important direct/indirect
interactions contributing to stabilization of the AChE–carbamate Michaelis complexes have been
investigated.
Keywords:
Alzheimer’s disease
AChE
Synthesis
Dementia
Ó 2012 Elsevier Ltd. All rights reserved.
Carbamates
1. Introduction
caused by the peripheral activity of these drugs on cholinesterase
enzyme. As the average age is increasing all over the world, and so
Alzheimer’s disease (AD) is the most common form of irrevers-
ible neurological disorder of elderly patients that affects more than
37 million people worldwide.1 It is clinically characterized by
progressive cognitive impairments or decline defined by a loss of
memory and learning ability, together with a reduced ability to per-
form daily routine activities and a diverse array of neuropsychiatric
symptoms such as apathy, verbal and physical agitation, irritability,
anxiety, depression, delusions and hallucinations.2 Characteristic
neuropathologic findings include selective neuronal and synaptic
losses, extracellular neuritic plaques containing the b-amyloid pep-
tide and neurofibrillary tangles (NFTs) composed of hyperphospho-
the AD (66% in the developing countries), there is an urgent need
for novel therapeutics, which could act as anti-Alzheimer agents
with low or no side effects associated with the known commercial
drugs for the treatment of the AD. Specifically, there is a need for
new cholinesterase inhibitors with wider therapeutic window, to
be useful as potential anti-Alzheimer agents without interacting
with peripheral cholinesterase enzymes. Extensive efforts by
different researchers in the span of 20 years have led to discovery
of a number of potent AChE inhibitors with structural diversity, such
as xanthostigmine,10 physostigmine,11 phenserine,12 huperzine-A,13
bis-tacrine,14 bis-huperzin-B,15 quilostigmine,16 eptastigmine,13 Ro-
46-5934,17a P10358,17b CHF2819,18 memoquin,19 lipocrine,19 and hup-
rine-X.20
Till date, despite of extensive efforts in the area of neurobiology
proposing different hypotheses for AD, the cholinergic hypothesis
has been the most productive leading to the four clinically effective
drugs (tacrine, donepezil, rivastigmine, galanthamine) for treat-
ment of AD. Interestingly, the development of allosteric agonists
and modulators of the muscarinic acetylcholine receptor subtype
1 (mAChR1 or M1) is being envisaged as a new path in medicinal
chemistry for getting new promising AD therapeutics.21 The
ongoing effort to develop more therapeutically efficacious AChE
inhibitors is currently driven by the remarkable progress made
during the last 20 years in elucidating the structural and functional
rylated forms of the tau (s
) protein.3 Existing treatments for mild to
moderate AD include the use of acetylcholinesterase (AChE) inhibi-
tors such as tacrine,4 donepezil,5 rivastigmine,6 galanthamine,7 and
the use of N-methyl-D
-aspartate (NMDA) antagonist memantine.8
However, these drugs are unable to slow or prevent AD progression,
rather can provide symptomatic benefits only and suffer from major
drawback of loss of therapeutic potential with time.9 Thus, increas-
ing daily doses in such circumstances increases the side effects until
the pause of the treatment. The major side effects are specifically
q
The CDRI communication number allotted to this manuscript is 8318.
⇑
Corresponding author. Tel.: +91 (522) 2624273; fax: +91 (522) 2623938.
E-mail address: anilsak@gmail.com (A.K. Saxena).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.005

6314
K. K. Roy et al. / Bioorg. Med. Chem. 20 (2012) 6313–6320
properties of the AChE enzyme using X-ray crystallography, com-
putational, and site-direct mutagenesis techniques. Presently,
there is an availability of ample information on the functional sub-
sites in the active site gorge of AChE involved in the reactivity of
the natural substrate (ACh), synthetic covalent and non-covalent
inhibitors. The active site gorge of the AChE can be classified into
two sites: (i) the catalytic site (CAS) located at the bottom of the
gorge catalyzing the hydrolysis of the substrate, and (ii) the periph-
eral anionic site (PAS) formed by W279 and Y79 present at the
mouth of the gorge, which serves as a relay station for the sub-
strate entry into the active site. The CAS, in turn, comprises anionic
subsite, acyl pocket, and oxyanion hole. While the W84 and F330
previously described23 by us. Briefly, the ring reduction of
the 6-methoxyquinoline (2) using in situ produced Raney Ni at the
room temperature (rt) afforded the first intermediate compound,
6-methoxy-1,2,3,4-tetrahydroquinoline (3), which upon reaction
with benzyl or 2,4-dichlorobenzyl chloride in the presence of potas-
sium carbonate (K2CO3) and potassium iodide (KI) in anhydrous N,
N-dimethylformamide (DMF) afforded the intermediate com-
pounds 4a or 4b respectively. The O-demethylation of the second
intermediate (4a or 4b) was achieved using molar solution of boron
tribromide (BBr3) to afford the final hydroxy-intermediates (5a and
5b). The reaction of the final hydroxyl intermediates (5a or 5b) with
substituted or unsubstituted alkyl/aryl isocyanates in the nonpolar
solvent using the inorganic base afforded title compounds.
contribute to the anionic subsite of the CAS, making
p–cation
interactions with the quaternary group of the substrate, the
W233, F288 and F290 form the acyl pocket, which determines
the specificity of the substrate ACh. The two residues Y121 and
F330 make important contributions to the narrow bottleneck in
the midway of the active site gorge.22 A number of these residues
have been experimentally established to be involved in the interac-
2.2. Lead optimization strategies
Considering the fact of the high aromatic content of the active
site gorge and the dominating hydrophobic interactions of AChE
with its inhibitors, we targeted the site-1 and site-2 of the lead
compound 123 (Fig. 1) for site-specific modification/optimization
in order to explore the suitable substituent(s) at these two sites
to identify novel and more potent AChE inhibitors. In view of this,
we replaced the benzyl group attached to the nitrogen of 1,2,3,
4-tetrahydroquinoline (site-1) with comparatively more hydro-
phobic 2,4-dichlorobenzyl group. We attempted major modifica-
tions at the site-2 of the lead compound 1, where both
substituted aromatic and long-chain (C6–C8) alkyl groups were
incorporated in novel derivatives of the lead 1 in view of the
important contribution of the hydrophobicity at this site as previ-
ously reported by us based on the systematic three-dimensional
quantitative structure–activity relationship (3D-QSAR) studies
carried out on diverse carbamate-class of AChE inhibitors.24,25
tions with the known anticholinesterase agents via both cation–
and
stacking interactions.22
In the recent past, we have reported the discovery of novel orally
potent lead compound 1 (AChE IC50 = 3.31 M) through the state-
p
p–p
l
of-the-art computer-aided drug design techniques including phar-
macophore modeling, virtual screening and docking studies.23 In or-
der to further improve the AChE inhibitory potential, we embarked
on further optimization of the identified lead 1 considering the fact
that the accommodation of covalent and non-covalent ligands by
the AChE is dominated by the hydrophobic interactions.24,25 It has
been evidenced by the X-ray crystallographic studies that the high
aromatic content of deeply buried (ꢀ20 Å) cylindrical active-site
gorge, lined by 14 conserved aromatic amino acid residues, is in-
volved in the strong hydrophobic interactions with AChE inhibitors
via both cation–p and p–p stacking interactions. In the present
study, we describe the further optimization of the lead compound
based on above mentioned hypothesis through the synthesis and
pharmacological evaluation of a novel series of 16 carbamates.
Among these compounds, the three compounds exhibited promis-
ing in vitro AChE inhibitory activities comparable to the existing
drug rivastigmine used in the treatment of AD.
Site 2
Cl
H
N
O
O
N
Site 1
2. Results and discussions
2.1. Chemistry
1
AChE IC50 = 3.31 µM
Figure 1. The structure and AChE inhibitory activity of the lead compound 1
previously reported from our research laboratory. Sites 1 and 2 are the two sites
targeted for the optimization studies presented here.
The steps for the synthesis of intermediates and title compounds
(6a–m, 7a,b) are outlined in Scheme 1, which are essentially same as
O
O
O
ii
i
N
H
N
N
R1
4a: R1=2,4-dichlorobenzyl
2
3
4b
: R1=benzyl
H
N
HO
O
O
R2
N
iv
N
R1
iii
R1
5a: R1=2,4-dichlorobenzyl
5b: R1=benzyl
6a-m, 7a, 7b
Scheme 1. Synthesis of carbamates (6a–m, 7a,b). Reagents and conditions: (i) NickelÀaluminum alloy, ethanol, 10% NaOH in water (w/v), 5À6 h; (ii) R1-Cl, dry DMF, baked
K2CO3, KI, rt, 3À5 h; (iii) 1 M BBr3 in DCM, 3À6 h; (iv) R2NCO, Et3N, THF or DCM.

K. K. Roy et al. / Bioorg. Med. Chem. 20 (2012) 6313–6320
6315
Figure 2. An outline of the synthesized title compounds (6a–m) based on the site-specific modifications of the lead compound 1.
Figure 2 outlines the site-specific modifications carried out at the
tally determined AChE inhibitory activities (IC50
,
lM) of the com-
two sites of the lead compound 1 as envisaged.
pounds (6a–m, 7a,b) along with rivastigmine and tacrine.
2.4. Structure–activity relationship (SAR) Interpretation
2.3. Determination of AChE inhibitory activity in vitro
The SAR studies on the synthesized and screened compounds
(Table 1) against AChE enzyme suggested that the substitution at
the ortho (o) position of the phenyl ring attached to the carbamyl
nitrogen as the most preferred position for the potential inhibition
of the AChE enzyme. Among the substituted phenyl groups at-
tached to carbamyl nitrogen, the order of preference was: ortho
(o) > meta (m) > para (p) positions for the effective AChE inhibition.
These findings corroborated well with our earlier studies23–25 ex-
cept the compounds 6b containing m-tolyl at site-2 and 2,4-dichlo-
robenzyl groups at site-1. This compound 6b demonstrated the
The AChE inhibitory activity of the synthesized novel deriva-
tives (6a–m, 7a,b) of the lead compound 1 was determined using
the Ellman26 method. The AChE enzyme was purified from red
blood cells obtained after the perfusion of the brain of anesthetized
adult Swiss albino mice (20–25 g). The kinetic profile of the AChE
enzyme activity was studied spectrophotometrically (Shimadzu,
USA) and the specific AChE inhibitory activity was calculated on
the basis of percent (%) decrease in AChE activity from control val-
ues, that is AChE activity without incubation with any standard or
test drug. In this assay, tacrine and rivastigmine were used as the
two standard AChE inhibitors. Table 1 summarizes the experimen-
highest AChE inhibitory activity (AChE IC50 = 0.70
about three fold higher than that of the compound 6a containing
o-tolyl group at site-2 (AChE IC50 = 2.57 M), and was comparable
to the drug rivastigmine (AChE IC50 = 1.11 M).
lM) which was
l
l
Table 1
Among the compounds with halogen (F, Cl and Br) groups
substituted at ortho, meta and para positions of the phenyl ring
at site-2, the fluorophenyl derivatives (6i and 6j) exhibited better
AChE inhibitory activity than the chlorophenyl (6d–f) and bromo-
phenyl (6g) derivatives. The compound 6i containing 2-fluoro-
phenyl group at site-2 also exhibited potent AChE inhibitory
Inhibition of AChE by rivastigmine, tacrine and synthesized new compounds (6a–m,
7a,b)
H
N
H
N
O
O
R
R
O
O
activity (AChE IC50 = 2.56 lM) which was comparable to the com-
pound 6a. The compound 6h containing 2-methoxyphenyl group
N
N
at site-2 exhibited moderate AChE inhibitory activity with AChE
IC50 of 13.80
groups at the site-2, the compound 6k containing hexyl chain
exhibited moderate AChE inhibition (AChE IC50 of 14.08 M), but
was better than the compounds 6l (IC50 = 105.13 M) and 6m
(IC50 >10,000 M) possessing heptyl and octyl chains respectively
lM. Among the compounds with long (C6–C8) alkyl
Cl
6a-mCl
7a-b
l
Title
R
AChE IC50 S.E.M. (lM)
l
l
1
2-Chlorophenyl
2-Methylphenyl
3-Methylphenyl
4-Methylphenyl
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
3-Bromophenyl
2-Methoxyphenyl
2-Fluorophenyl
4-Fluorophenyl
Hexyl
3.31 0.25
2.57 0.20
0.70 0.05
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
7a
7b
at the site-2 (Table 1).
Moreover, it is noteworthy that the introduction of a more
hydrophobic group 2,4-dichlorobenzyl at site-1 resulted in the
increase in AChE inhibitory activity as the compounds 6a and 6b
showed better activity than the corresponding compounds (7a:
2-methylphenyl; 7b: 3-methylphenyl) possessing benzyl in place
of 2,4-dichlorobenzyl group except the 2-chlorophenyl derivatives
(6d) which was less active than the corresponding compound 1.
850.60 58.90
11.87 2.80
210.70 50.10
3476.50 68.70
140 17.10
13.80 0.23
2.56 0.28
89.37 9.60
14.08 1.70
105.13 22.48
>10,000
Heptyl
Octyl
2-Methylphenyl
3-Methylphenyl
2.5. Computational modeling of TcAChE–carbamate Michaelis
complexes
14.30 0.23
60.20 13.40
Among the two X-ray structures of TcAChE (PDB-IDs: 1gqr27
and 2bag28) available in RCSB protein data bank (www.rcsb.org),
the bound ligands (rivastigmine and ganstigmine respectively)
Rivastigmine
Tacrine
1.11 0.05
0.13 0.002

6316
K. K. Roy et al. / Bioorg. Med. Chem. 20 (2012) 6313–6320
Figure 3. Tetrahedral Michaelis complexes of: (A) TcAChE–rivastigmine. (B) TcAChE–ganstigmine. Ligands are shown in amber-colored stick. (C) The superposed view of the
TcAChE–rivastigmine (amber colored) and TcAChE–ganstigmine (green colored) complexes depicting the differences in their orientation and the induced conformations of the
active site residues after running 100 ps MDS. (D) TcAChE–compound 6a; (E) TcAChE–compound 6b; (F) TcAChE–compound 6c. The protein is represented as cartoon form.
are present in broken state, with the carbamyl group being cova-
lently bound to the O atom of the catalytic site residue Ser200,
rivastigmine is a second-order reaction, while the decarbamylation
reaction is a first-order reaction.
c
and the NAP being non-carbamate part of rivastigmine, is located
near the anionic subsite characterized by the presence of Trp84
and Phe330 residues,27 while the geneseroline being non-carba-
mate part of ganstigmine, is not retained in the catalytic pocket
of AChE due to an unknown reason.28 The ‘back door’ opening
hypothesis has provided an indirect evidence for the release of
the leaving group of ganstigmine, physostigmine, and MF268.29–
In order to gain an insight into the formation of the tetrahedral
intermediate and to elucidate the specific direct/indirect interac-
tions contributing to their stabilization, molecular models of
TcAChE–carbamate Michaelis complexes for rivastigmine, ganstig-
mine, and novel discovered compounds (6a, 6b, and 6c) were gener-
ated using the covalent docking protocol available in GOLD docking
program, considering the X-ray crystal structure of TcAChE–gan-
stigmine complex (PDB-ID: 2bag). The small scale (100 ps) molecu-
lar dynamics simulations (MDS) using MacroModel33 suite were
performed to relax the initial molecular models to afford stable
TcAChE–ligand Michaelis complexes.
Figure 3 depicts the modeled tetrahedral intermediates of the
rivastigmine, ganstigmine, and new compounds (6a, 6b and 6c)
with TcAChE. Analyses of these transition states revealed that
rivastigmine and ganstigmine, although shared the same binding
site, but attained distinct orientation with respect to the active site
architecture of TcAChE enzyme. In case of TcAChE–rivastigmine
tetrahedral intermediate (Fig. 3A), the positively ionized nitrogen
31
However, its relevance as far as the mechanism of substrate
hydrolysis is concerned, remains yet to be established.
Since the rates of formation of the covalent adducts depend pri-
marily on the stabilities of the corresponding AChE–carbamate
Michaelis complexes (tetrahedral intermediates), it is imperative
to elucidate the specific direct/indirect interactions contributing
to stabilization of these complexes. Over the decades, some re-
search groups have reported a number of favorable tetrahedral
intermediate of the different covalent (reversible/irreversible)
AChE inhibitors in order to explain the rates (kinetics) of the for-
mation of the covalent adducts, and different key interactions play-
ing roles in the stabilization of the AChE–substrate tetrahedral
intermediate.32 It is widely accepted that the carbamylation
reaction or covalent adduct formation takes place in two steps:
(i) formation of AChE–carbamate Michaelis complexes, (ii) release
of the leaving group to result in the carbamylated AChE. A number
of kinetic studies32 on human AChE and BuChE have indicated that
the inhibition of AChE by majority of carbamates including
exhibited cation–p interaction with the cation binding subsite-
Trp84, and the alkyl groups (methyl and ethyl) attached to the
carbamyl nitrogen were occupied the acyl pocket formed by
Phe290, Phe292 and Tyr121. In case of TcAChE-ganstigmine
Michaelis complex (Fig. 3B), the 2-ethylphenyl group of ganstigmine
was projected toward the cation binding subsite-Trp84 exhibiting
aromatic
p–p interactions and the o-ethyl group exhibited

K. K. Roy et al. / Bioorg. Med. Chem. 20 (2012) 6313–6320
6317
hydrophobic interaction with the two aromatic residues Phe330
3. Conclusion
and Phe331. The carbonyl oxygen was accommodated in the oxyan-
ion hole through the direct tridentate H-bonds with the amidic NH
of the three residues, namely Gly118, Gly119, and Ala201. The par-
ticular arrangement of H-bond donors (amidic NH groups of Gly118,
Gly119, and Ala201 forming the oxyanion hole) may increase the
stability of the TcAChE–carbamate Michaelis complex by favorably
accommodating the negatively charged carbonyl oxygen, and thus
facilitating the nucleophilic addition by the catalytic residue
Ser200, being located in its close proximity, through polarization
of the C@O bond of the covalent substrate. Such direct H-bond inter-
actions with the amidic NH groups of the residues forming oxyanion
hole are not specifically required for accommodation of non-cova-
lent inhibitors.32c
The tricyclic geneseroline group of ganstigmine was extended
towardsthenarrowbottleneckandaccommodatedinthehydropho-
bic pocket formed by Tyr121, Phe288, Phe290, and Phe331
(Fig. 3B). However, as aforementioned, this tricyclic group is not re-
tained in the X-ray co-crystal structure of TcAChE–ganstigmine
(PDB-ID: 2bag) due to unknown reason. This may be mainly due to
its relatively more bulkiness as compared to the leaving group NAP
of the rivastigmine. It is noteworthy to note herein that there were
apparently some steric perturbations between the tricyclic geneser-
oline groups and the residues present in the bottleneck region.
In order to explore the conformational landscape of the residues,
of the whole AChE–carbamate Michaelis complex, and to confirm
the chances of steric perturbations mentioned above, small scale
(100 ps) molecular dynamics simulation (MDS) studies were exper-
imented on the TcAChE–carbamate Michaelis complexes. As ex-
pected, there was angular shift of the two aromatic residues
Phe331 and Tyr121 forming the bottleneck that in turn led into
the widening of the gorge opening by about 2 Å as reflected from
the superposed view of the TcAChE–rivastigmine and TcAChE–gan-
stigmine Michaelis complexes (Fig. 3C). Such incidence was not ob-
served in case of TcAChE–rivastigmine after running MDS and the
residues in the bottleneck region were quite stable and did not suf-
fer any perturbation(s) due to the small size of the leaving group
(NAP) present in the rivastigmine. Our observations are in strong
agreement with the reported insights based on the previous molec-
ular modeling and dynamics simulation studies.29,32,34
The interaction patterns of the two new potent carbamates (6a
and 6b) were very similar to that of the ganstigmine (Fig. 3D and
E). Examination of these Michaelis complexes revealed that the o-
tolyl and m-tolyl groups in compounds 6a and 6b respectively
was located in the cation binding subsite-Trp84, exhibiting hydro-
phobic interactions with Trp84, Phe330 and Phe331. However, the
compound 6c, which is an structural analogue of the compounds
6a and 6b, exhibited steric perturbation (distance 62.5 Å) with
the residue Trp84, which was unfavorable towards the stability of
the resulting complex and thus, for potential inhibition of the AChE
( Fig. 3F). The favorable interaction with this aromatic residue
(Trp84) has previously been reported32 to be highly crucial for the
stabilization of Michaelis complex and hence, for carbamylation
process of AChE. The unfavorable steric perturbation of p-tolyl
group of the compound 6c with Trp84 may be suggested to be one
of the potential reasons behind its poor AChE inhibitory activity
Considering the evidence from the X-ray crystallographic and
computational studies, that hydrophobic interaction plays key role
in the accommodation of ligands within the active-site gorge of
AChE, we have embarked on the optimization of the previously dis-
covered lead compound 1 (AChE IC50 = 3.31 lM). For this, we have
synthesized and evaluated a series of novel compounds which has
led to the discovery of the three novel promising compounds, 6a
(IC50 = 2.57
almost comparable in vitro AChE inhibitory activities as compared
to the drug rivastigmine (IC50 = 1.11 M). Among these three com-
lM), 6b (IC50 = 0.70 lM) and 6i (IC50 = 2.56 lM) with
l
pounds, the compound 6b has been identified as possible opti-
mized lead for further detailed in vivo neuropharmacological
studies. Furthermore, the AChE–carbamate Michaelis complexes
of these potent compounds including rivastigmine and ganstig-
mine have been modeled using covalent docking method imple-
mented in GOLD and important structural factors governing the
complex stability have been investigated.
4. Experimental section
4.1. Chemistry
General: Reagents were purchased from common commercial
suppliers and were used without further purification. Solvents
were purified and dried by standard procedures, when necessary.
Chromatographic separations of the synthesized intermediates
and title compounds were performed on silica gel (Merck:
100–200 mesh). Thin-layer chromatography was used to monitor
the reactions. Melting points (uncorrected) were determined with
Büchi 510 apparatus. Characterization of the synthesized com-
pounds was accomplished in the sophisticated analytical instru-
ment facility (SAIF) department of CDRI, Lucknow, India. The IR
spectroscopy was carried out using Perkin–Elmer 881 spectropho-
tometer and the values are expressed as vmax cmÀ1. Mass spectra
(MS) were recorded on a Jeol (Japan) SX 102/DA-6000 Mass Spec-
trometer. 1H NMR and 13C NMR spectra were recorded on Bruker
Spectrospin spectrometer at 300 and 200 MHz respectively. The
chemical shifts are reported in d scale (ppm) and are relative to tet-
ramethyl silane (TMS) as internal standard. The coupling constants
J are given Hertz and spin multiplicities are expressed s (singlet), d
(doublet), t (triplet), dd (double doublet), and m (multiplet).
4.1.1. General procedure for synthesis of title compounds (6a–
m, 7a,b)
A mixture of 1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquino-
lin-6-ol (5, 1 mM) and triethylamine (1.2 mM) in dry tetrahydrofu-
ran (THF) was stirred for half an hour under nitrogen atmosphere
at rt. To the stirred reaction mixture, desired isocyanate (1.2 mM)
was added at once and then the reaction mixture was further stir-
red for 24–72 h under N2 atmosphere at the rt. The duration of the
reaction was variable depending upon the nature of isocyanate
used. The reaction mixture was concentrated under vacuum and
then distilled water (15 mL) was added followed by the extraction
with ether (3 Â 15 mL). The ether layer was dried over sodium sul-
fate and concentrated under vacuum to afford the crude product,
which was finally chromatographed to give the corresponding final
product in good yield.
(IC50 = 850.60 lM) compared to its other two structural analogues
(6a and 6b). However, unlike ganstigmine, the leaving group
1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol, common
in these new compounds (6a–c) did not suffer from any steric per-
turbations with the residues forming the bottleneck as assured
using small scale MDS (100 ps). This leaving group exhibited hydro-
phobic interactions with the surrounding residues, namely Trp279,
Leu282, Ile287, and Tyr334 (Fig. 3D–F). The chloro groups attached
to 2nd and 3rd positions of the phenyl ring are in hydrophobic con-
tacts with the two aliphatic residues Leu282 and Ile287.
4.1.1.1. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
o-tolylcarbamate (6a).
This compound was synthesized using
the general carbamoylation method by taking a solution of 1-(2,4-
dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM) and tri-
ethylamine (1.2 mM) in dry THF, and o-tolyl isocyanate (1.2 mM).
% Yield: 81%; mp 105 °C, 1H NMR (CDCl3, 300 MHz). d ppm 7.92 (br

6318
K. K. Roy et al. / Bioorg. Med. Chem. 20 (2012) 6313–6320
s, 1H), 7.49 (s, 1H), 7.26–7.24 (d, J = 7.71 Hz, 2H), 7.21–7.19 (d,
J = 5.94 Hz, 2H), 7.09–7.04 (t, J = 7.30 Hz, 1H), 6.88–6.87 (d,
J = 2.21 Hz, 1H), 6.81–6.77 (m, 1H), 6.71–6.68 (d, J = 8.88 Hz, 1H),
6.27–6.25 (d, J = 8.82 Hz, 1H), 4.47 (s, 2H), 3.42–3.38 (t, J = 5.55 Hz,
2H), 2.89–2.85 (t, J = 6.18 Hz, 2H), 2.33 (s, 3H), 2.11–2.03 (m, 2H).
13C NMR (CDCl3, 200 MHz). d ppm 155.12, 143.32, 140.19, 135.47,
134.68, 133.88, 132.51, 131.74, 130.26, 129.12, 127.52, 126.67,
125.58, 122.49, 120.45, 119.37, 114.44, 110.59, 59.83, 50.12, 27.34,
22.11, 20.84. FTIR (KBr): cmÀ1 3405, 2945, 2819, 2366, 1713, 1598,
1441, 1351, 1246, 1157, 972, 771, 692. ESMS: m/z 442 (M+1)+; HRMS
calcd for C24H22Cl2N2O2 (M+1)+ 441.1058; found 441.1153.
4.1.1.5.
1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
This compound was synthe-
3-chlorophenylcarbamate (6e).
sized using the general carbamoylation method by taking a solution
of 1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM)
and triethylamine (1.2 mM) in dry THF, and 3-chlorophenyl isocya-
nate (1.2 mM). % Yield: 80%; mp 125 °C, 1H NMR (CDCl3 + CD3OD,
300 MHz). d ppm 7.60 (s, 1H), 7.45–7.36 (m, 3H), 7.33–7.31 (m,
3H), 7.29–7.26 (m, 2H), 7.06–7.02 (m, 1H), 6.89–6.86 (m, 1H), 4.61
(s, 2H), 3.54–3.50 (t, J = 5.610 Hz, 2H), 2.98–2.93 (t, J = 6.375 Hz,
2H), 2.22–2.18 (m, 2H). 13C NMR (CDCl3, CD3OD, 200 MHz). d ppm
153.06, 142.61, 140.64, 139.40, 134.20, 133.94, 133.18, 132.71,
129.56, 129.01, 128.30, 126.86, 122.86, 121.78, 119.64, 118.41,
116.44, 110.66, 59.95, 53.11, 49.82, 27.77, 21.97. FTIR (KBr): cmÀ1
3677, 3287, 2931, 2365, 2334, 1714, 1615, 1593, 1478, 1299,
1231, 1195, 1055, 757. ESMS: m/z: 461 (M+); HRMS calcd for
4.1.1.2. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
m-tolylcarbamate (6b).
This compound was synthesized
using the general carbamoylation method by taking a solution of
1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM)
and triethylamine (1.2 mM) in dry THF, and m-tolyl isocyanate
(1.2 mM). % Yield: 81%; 1H NMR (CDCl3, 300 MHz). d ppm 8.17
(br s, 1H), 7.40–7.34 (m, 3H), 7.28–7.23 (m, 1H), 7.16 (s, 1H),
7.02–6.97 (m, 1H), 6.85–6.84 (d, J = 2.35 Hz, 1H), 6.78–6.74 (d,
J = 9.98 Hz, 2H), 6.24–6.21 (d, J = 8.78 Hz, 1H), 4.44 (s, 2H), 3.38–
3.34 (t, J = 5.535 Hz, 2H), 2.86–2.83 (t, J = 6.105 Hz, 2H), 2.30 (s,
3H), 2.09–2.02 (m, 2H). 13C NMR (CDCl3, 200 MHz). d ppm
154.82, 143.44, 141.20, 138.72, 136.00, 135.45, 134.81, 133.91,
131.23, 130.12, 128.25, 126.76, 125.30, 124.48, 123.15, 121.56,
120.44, 119.53, 118.49, 110.51, 60.12, 49.99, 27.52, 22.83, 21.24,
20.76. FTIR (Neat): cmÀ1 3410, 2948, 2823, 2387, 1714, 1598,
1348, 1254, 1157, 975, 771. ESMS: m/z: 442 (M+1)+; HRMS calcd
for C24H22Cl2N2O2 (M+1)+ 441.1058; found 441.1156.
C
23H19Cl3N2O2 (M+) 461.0512; found 461.0573.
4.1.1.6. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl 4-
chlorophenylcarbamate (6f). This compound was synthesized
using the general carbamoylation method by taking a solution of 1-
(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM) and
triethylamine (1.2 mM) in dry THF, and 4-chlorophenyl isocyanate
(1.2 mM).
%
Yield: 82%; mp 158 °C, 1H NMR NMR (CD3OD,
300 MHz). d ppm 7.89 (br s, 1H), 7.47–7.46 (d, J = 4.62 Hz, 1H),
7.43–7.40 (m, 2H), 7.37 (s, 1H), 7.27–7.24 (m, 2H), 7.18–7.17 (d,
J = 1.5 Hz, 2H), 6.83–6.82 (d, J = 2.76 Hz, 1H), 6.25–6.22 (d,
J = 8.82 Hz, 1H), 4.47 (s, 2H), 3.42–3.38 (t, J = 5.640 Hz, 2H), 2.87–
2.83 (t, J = 6.225 Hz, 2H), 2.09–2.02 (m, 2H). 13C NMR (CDCl3, CD3OD,
200 MHz). d ppm 153.21, 142.43, 140.58, 136.76, 134.52, 133.86,
133.04, 132.57, 128.83, 128.27, 127.64, 126.68, 122.72, 121.64,
119.89, 119.50, 110.50, 59.65, 52.91, 49.67, 48.92, 27.60, 21.82. FTIR
(KBr): cmÀ1 3332, 3106, 3033, 2929, 2844, 2367, 1713, 1601, 1542,
1507, 1349, 1220, 1195, 1012, 826, 691, 560. ESMS: m/z 461 (M+);
HRMS calcd for C23H19Cl3N2O2 (M+) 461.0512; found 461.0533.
4.1.1.3. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
p-tolylcarbamate (6c).
This compound was synthesized
using the general carbamoylation method by taking a solution of
1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM)
and triethylamine (1.2 mM) in dry THF, and p-tolyl isocyanate
(1.2 mM). % Yield: 78%; mp 130 °C, 1H NMR (CDCl3, 300 MHz). d
ppm 7.40–7.38 (d, J = 6.33 Hz, 1H), 7.31–7.28 (d, J = 8.18 Hz, 1H),
7.26–7.25 (d, J = 4.95 Hz, 2H), 7.22–7.15 (m, 2H), 7.12–7.09 (m,
2H), 6.83–6.82 (d, J = 2.64 Hz, 1H), 6.76–6.69 (m, 1H), 6.23–6.16
(m, 1H), 4.44 (s, 2H), 3.38–3.34 (t, J = 5.565 Hz, 2H), 2.85–2.81 (t,
J = 6.375 Hz, 2H), 2.30 (s, 3H), 2.05–1.99 (m, 2H). 13C NMR (CDCl3,
200 MHz). d ppm 155.96, 143.37, 142.93, 135.07, 134.25, 133.93,
133.52, 133.05, 129.58, 128.62, 128.41, 127.23, 123.09, 122.19,
121.53, 120.72, 120.05, 119.81, 118.81, 111.05, 50.17, 28.14,
22.33, 21.20. FTIR (KBr): cmÀ1 3403, 2965, 2839, 2366, 1713,
1600, 1496, 1348, 1239, 1157, 998, 762, 686. ESMS: m/z 442
(M+1)+; HRMS calcd for C24H22Cl2N2O2 (M+1)+ 441.1058; found
441.1154.
4.1.1.7. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl 3-
bromophenylcarbamate(6g).
Thiscompoundwassynthesized
using the general carbamoylation method by taking a solution of 1-
(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM) and
triethylamine (1.2 mM) in dry THF, and 3-bromophenyl isocyanate
(1.2 mM). % Yield: 80%; mp: 132 °C; 1H NMR (CDCl3, 300 MHz). d
ppm 7.59 (s, 1H), 7.44–7.35 (m, 3H), 7.33–7.31 (m, 3H), 7.27–7.24
(d, J = 2.58 Hz, 1H), 7.01–6.98 (m, 1H), 6.85–6.81 (m, 1H), 4.52 (s,
2H), 3.53–3.50 (t, J = 5.613 Hz, 2H), 2.97–2.93 (t, J = 6.372 Hz, 2H),
2.25–2.14 (m, 2H). 13C NMR (CDCl3, 200 MHz). d ppm 153.16,
142.71, 140.73, 139.49, 134.29, 134.04, 133.28, 132.81, 129.65,
129.11, 128.39, 126.96, 122.96, 121.88, 119.74, 118.51, 116.54,
110.75, 58.98, 53.65, 49.92, 27.87, 22.07. FTIR (KBr) cmÀ1 3316,
3039, 2936, 2844, 2368, 1712, 1605, 1505, 1359, 1021, 828, 771,
666. ESMS: m/z 505 (M+1)+; HRMS calcd for C23H19BrCl2N2O2
(M+1)+ 505.0007; found 505.0023.
4.1.1.4. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl 2-
chlorophenylcarbamate (6d).
Thiscompoundwas synthesized
using the general carbamoylation method bytaking a solution of 1-(2,
4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM) and tri-
ethylamine (1.2 mM) in dry THF, and 2-chlorophenyl isocyanate
(1.2 mM). % Yield: 81%; mp 222 °C, 1H NMR (CDCl3, 300 MHz). d
ppm 8.17 (br s, 1H), 7.40–7.34 (m, 3H), 7.28–7.23 (m, 2H), 7.16 (s,
1H), 7.02–6.97 (m, 1H), 6.85–6.84 (d, J = 2.28 Hz, 1H), 6.78–6.74 (d,
J = 2.61 Hz, 1H), 6.24–6.21 (d, J = 8.82 Hz, 1H), 4.44 (s, 2H), 3.38–
3.34 (t, J = 5.535 Hz, 2H), 2.86–2.83 (t, J = 6.105 Hz, 2H), 2.09–2.02
(m, 2H). 13C NMR (CDCl3, CD3OD 200 MHz). d ppm 153.14, 140.58,
134.20, 134.00, 133.32, 132.88, 129.19, 128.98, 128.40, 127.57,
127.20, 127.03, 124.07, 123.77, 122.42, 121.84, 119.74, 110.84,
60.19, 53.28, 49.96, 27.93, 22.09. FTIR (KBr): cmÀ1 3677, 3291, 2928,
2364, 2340, 1713, 1591, 1476, 1386, 1294, 1232, 1197, 1054, 752.
ESMS: m/z 461 (M+); HRMS calcd for C23H19Cl3N2O2 (M+) 461.0512;
found 461.0578.
4.1.1.8. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl 2-
methoxyphenylcarbamate (6h).
This compound was synthe-
sized using the general carbamoylation method by taking a solution
of 1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM)
and triethylamine (1.2 mM) in dry THF, and 2-methoxyphenyl isocy-
anate (1.2 mM). % Yield:81%; mp158 °C; 1H NMR (CDCl3, 300 MHz). d
ppm 8.15 (br s, 1H), 7.42–7.34 (m, 3H), 7.27–7.21 (m, 1H), 7.17 (s, 1H),
7.00–6.95 (m, 2H), 6.87–6.75 (m, 2H), 6.22–6.19 (d, J = 8.84 Hz, 1H),
4.44 (s, 2H), 4.32 (s, 3H), 3.38–3.33 (t, J = 5.537 Hz, 2H), 2.86–2.82 (t,
J = 6.111 Hz, 2H), 2.07–2.01 (m, 2H). 13C NMR (CDCl3, 200 MHz). d
ppm 153.21, 141.08, 134.32, 134.11, 133.33, 133.00, 129.22, 128.52,
127.64, 127.27, 124.13, 124.03, 122.93, 122.34, 121.88, 120.31,
119.44, 110.64, 60.04, 58.65, 53.35, 49.98, 27.87, 22.04. FTIR (KBr):
cmÀ1 3676, 3290, 2928, 2364, 2340, 1712, 1647, 1591, 1476, 1386,

K. K. Roy et al. / Bioorg. Med. Chem. 20 (2012) 6313–6320
6319
1294, 1232, 1197, 1054, 752. ESMS: m/z: 456 (M+); HRMS calcd for
24H22Cl2N2O3 (M+) 456.1007; found 456.0995.
4.48 (s, 2H), 3.35–3.31 (t, J = 5.626 Hz, 2H), 3.28–3.19 (m, 2H),
2.83–2.80 (t, J = 6.236 Hz, 2H), 2.05–1.97 (m, 2H), 1.73–1.61 (m,
2H), 1.56–1.47 (m, 3H), 0.93–0.83 (m, 8H). 13C NMR (CDCl3,
200 MHz). d ppm 155.46, 142.54, 141.44, 134.25, 133.36, 132.91,
129.24, 128.57, 127.12, 122.89, 122.13, 119.94, 110.93, 50.08,
41.18, 31.71, 29.77, 29.14, 28.03, 26.67, 23.94, 22.56, 22.28,
14.01. FTIR (Neat): cmÀ1 3450, 3015, 2930, 2859, 2363, 2340,
1713, 1616, 1507, 1462, 1355, 1219, 1173, 1026, 769, 670. ESMS:
m/z 449 (M+); HRMS calcd for C24H30Cl2N2O2 (M+) 449.1684; found
449.1712.
C
4.1.1.9. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
2-fluorophenylcarbamate (6i). This compound was synthe-
sized using the general carbamoylation method by taking a solu-
tion of 1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5,
1 mM) and triethylamine (1.2 mM) in dry THF, and 2-fluorophenyl
isocyanate (1.2 mM). % Yield: 80%; mp 133 °C, 1H NMR (CDCl3,
300 MHz). d ppm 8.17 (br s, 1H), 7.40–7.34 (m, 3H), 7.28–7.23
(m, 1H), 7.16 (s, 1H), 7.02–6.97 (m, 2H), 6.85–6.74 (m, 2H), 6.24–
6.21 (d, J = 8.83 Hz, 1H), 4.44 (s, 2H), 3.38–3.34 (t, J = 5.535 Hz,
2H), 2.86–2.83 (t, J = 6.105 Hz, 2H), 2.09–2.02 (m, 2H). 13C NMR
(CDCl3, 200 MHz). d ppm 153.21, 142.16, 139.82, 133.44, 133.24,
132.56, 132.12, 128.43, 128.22, 127.64, 126.44, 123.31, 123.00,
122.27, 121.66, 119.51, 118.98, 110.08, 58.67, 52.24, 49.20, 27.17,
21.71. FTIR (KBr): cmÀ1 3653, 3303, 2826, 2370, 2341, 1715,
1598, 1495, 1456, 1354, 1258, 1202, 1106, 1060, 847, 755, 654.
ESMS: m/z 445 (M+); HRMS calcd for C23H19Cl2FN2O2 (M+)
445.0808; found 445.0873.
4.1.1.13. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
n-octylcarbamate (6m).
This compound was synthesized
using the general carbamoylation method by taking a solution of
1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM)
and triethylamine (1.2 mM) in dry THF, and n-octyl isocyanate
(1.2 mM). % Yield: 83%; 1H NMR (CDCl3, 300 MHz). d ppm 7.49 (s,
1H), 7.23 (s, 1H), 7.17–7.16 (d, J = 1.16 Hz, 2H), 6.80–6.79 (d,
J = 2.52 Hz, 1H), 6.71–6.68 (m, 1H), 6.22–6.19 (d, J = 8.84 Hz, 1H),
4.45 (s, 2H), 3.37–3.32 (t, J = 5.626 Hz, 2H), 3.29–3.21 (m, 2H),
2.85–2.81 (t, J = 6.238 Hz, 2H), 2.02–1.94 (m, 2H), 1.71–1.64 (m,
2H), 1.55–1.48 (m, 3H), 0.91–0.83 (m, 10H). 13C NMR (CDCl3,
200 MHz). d ppm 155.55, 142.63, 141.53, 134.33, 133.45, 132.99,
129.33, 128.66, 127.20, 122.98, 122.22, 120.03, 111.02, 50.16,
41.27, 31.80, 29.85, 29.22, 28.11, 26.76, 24.83, 24.03, 22.65,
22.36, 14.10. FTIR (Neat): cmÀ1 3455, 3022, 2931, 2862, 2345,
1712, 1617, 1508, 1463, 1362, 1178, 1022, 771, 669, 585. ESMS:
m/z 464 (M+1)+; HRMS calcd for C25H32Cl2N2O2 (M+) 463.1841;
found 463.1858.
4.1.1.10. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
4-fluorophenylcarbamate (6j).
This compound was synthe-
sized using the general carbamoylation method by taking a solution
of 1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM)
and triethylamine (1.2 mM) in dry THF, and 4-fluorophenyl isocya-
nate (1.2 mM). % Yield: 82%; mp 156 °C, 1H NMR (CDCl3, 300 MHz). d
ppm 8.09 (br s, 1H), 7.49–7.44 (d, 1H), 7.41–7.38 (m, 2H), 7.35 (s,
1H), 7.28–7.23 (m, 2H), 7.19–7.17 (d, J = 4.59 Hz, 2H), 6.83–6.81
(d, J = 6.34 Hz, 1H), 6.25–6.22 (d, J = 8.79 Hz, 1H), 4.47 (s, 2H),
3.41–3.37 (t, J = 5.638 Hz, 2H), 2.87–2.82 (t, J = 6.232 Hz, 2H),
2.09–2.02 (m, 2H). 13C NMR (CDCl3, 200 MHz). d ppm 153.29,
142.51, 140.66, 136.84, 135.19, 133.94, 133.46, 133.12, 132.65,
128.91, 128.35, 127.72, 126.76, 122.80, 121.72, 119.97, 119.58,
110.58, 60.08, 53.56, 49.75, 27.68, 21.90. FTIR (KBr): cmÀ1 3156,
3082, 2928, 2856, 2365, 1887, 1712, 1552, 1504, 1448, 1407,
1352, 1313, 1226, 969, 691. ESMS: m/z 445 (M+); HRMS calcd for
4.1.1.14. 1-Benzyl-1,2,3,4-tetrahydroquinolin-6-yl o-tolylcarba-
mate (7a).
This compound was synthesized using the general
carbamoylation method by taking a solution of 1-benzyl-1,2,3,4-tet-
rahydroquinolin-6-ol (1 mM)22 and triethylamine (1.2 mM) in dry
THF, and o-tolyl isocyanate (1.2 mM). % Yield: 76%; mp 132 °C, 1H
NMR (CDCl3, 300 MHz). d ppm 7.49–7.32 (m, 4H), 7.24–7.19 (m,
5H), 7.05–7.01 (m, 2H), 6.80–6.78 (d, J = 2.18 Hz, 1H), 4.45 (s, 2H),
3.42–3.37 (t, J = 5.68 Hz, 2H), 2.87–2.83 (t, J = 6.12 Hz, 2H), 2.38 (s,
3H), 2.08–2.03 (m, 2H). 13C NMR (CDCl3, 200 MHz). d ppm 153.92,
143.22, 140.19, 135.37, 134.82, 133.48, 132.11, 131.24, 130.56,
129.72, 126.97, 125.38, 122.19, 120.51, 119.37, 118.25, 114.82,
109.92, 60.13, 52.65, 48.98, 27.43, 22.12, 20.78. FTIR (KBr): cmÀ1
3405, 2951, 2822, 1713, 1598, 1440, 1245, 1155, 775. ESMS: m/z
373 (M+1)+; HRMS calcd for C24H24N2O2 (M+1)+ 373.1432; found
373.1434.
C
23H19Cl2FN2O2 (M+) 445.0808; found 445.0874.
4.1.1.11. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
n-hexylcarbamate (6k). This compound was synthesized
using the general carbamoylation method by taking a solution of
1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM)
and triethylamine (1.2 mM) in dry THF, and n-hexyl isocyanate
(1.2 mM). % Yield: 84%; 1H NMR (CDCl3, 300 MHz). d ppm 7.40 (s,
1H), 7.26 (s, 1H), 7.16–7.15 (d, J = 1.17 Hz, 2H), 6.78–6.77 (d,
J = 2.7 Hz, 1H), 6.70–6.66 (m, 1H), 6.21–6.18 (d, J = 8.82 Hz, 1H),
4.43 (s, 2H), 3.37–3.33 (t, J = 5.625 Hz, 2H), 3.26–3.20 (m, 2H),
2.84–2.80 (t, J = 6.240 Hz, 2H), 2.06–1.98 (m, 2H), 1.72–1.65 (m,
2H), 1.56–1.50 (m, 3H), 0.92–0.86 (m, 6H). 13C NMR (CDCl3,
200 MHz). d ppm 155.49, 142.57, 141.47, 134.27, 133.39, 132.93,
129.27, 128.60, 127.14, 122.92, 119.97, 110.96, 50.10, 41.21,
31.74, 29.79, 29.16, 28.05, 26.70, 23.97, 22.59, 22.30, 14.04. FTIR
(Neat): cmÀ1 3454, 3025, 2925, 2862, 2350, 1715, 1616, 1509,
1462, 1362, 1222, 1178, 1022, 765, 668, 578. ESMS: m/z 435
(M+1)+; HRMS calcd for C23H28Cl2N2O2 (M+1)+ 435.1528; found
435.1542.
4.1.1.15. 1-Benzyl-1,2,3,4-tetrahydroquinolin-6-yl m-tolylcarba-
mate (7b).
This compound was synthesized using the general
carbamoylation method by taking a solution of 1-benzyl-1,2,3,4-tet-
rahydroquinolin-6-ol (1 mM)22 and triethylamine (1.2 mM) in dry
THF, and m-tolyl isocyanate (1.2 mM). % Yield: 61%; 1H NMR (CDCl3,
300 MHz). d ppm 7.68 (s, 1H), 7.28–7.23 (m, 8H), 6.83–6.79 (m, 2H),
6.46 (d, J = 8.62 Hz, 1H), 4.44 (s, 2H), 3.38–3.34 (t, J = 5.535 Hz, 2H),
2.86–2.83 (t, J = 6.11 Hz, 2H), 2.32 (s, 3H), 2.07–2.01 (m, 2H). 13C
NMR (CDCl3, 200 MHz). d ppm 153.82, 143.46, 141.05, 138.75,
136.00, 135.43, 134.28, 133.39, 131.23, 128.24, 126.75, 125.73,
124.41, 123.12, 120.44, 119.53, 118.42, 109.91, 59.96, 52.45, 48.89,
27.27, 22.34, 21.05. FTIR (Neat): cmÀ1 3405, 2951, 2822, 1711,
1598, 1440, 1245, 1155, 975, 771. ESMS: m/z: 373 (M+1)+; HRMS
calcd for C24H24N2O2 (M+1)+ 373.1432; found 373.1443.
4.1.1.12. 1-(2,4-Dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-yl
n-heptylcarbamate (6l).
This compound was synthesized
using the general carbamoylation method by taking a solution of
1-(2,4-dichlorobenzyl)-1,2,3,4-tetrahydroquinolin-6-ol (5, 1 mM)
and triethylamine (1.2 mM) in dry THF, and n-heptyl isocyanate
(1.2 mM). % Yield: 84%; 1H NMR (CDCl3, 300 MHz). d ppm 7.43 (s,
1H), 7.28 (s, 1H), 7.15–7.14 (d, J = 1.23 Hz, 2H), 6.80–6.79 (d,
J = 2.67 Hz, 1H), 6.72–6.68 (m, 1H), 6.23–6.20 (d, J = 8.85 Hz, 1H),
4.1.2. Biological methods
4.1.2.1. Preparation of AChE enzyme.
mice (20–25 g) were perfused under mild ether anesthesia through
heart with ice cooled normal saline (0.9% NaCl) to remove blood-
born cholinesterase from the brain. After perfusion the whole brain
The adult Swiss albino

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K. K. Roy et al. / Bioorg. Med. Chem. 20 (2012) 6313–6320
was taken out. A 10% (w/v) homogenate of brain was prepared first
by homogenizing in an Ultra-Turrax T25 homogenizer at a speed of
9500 rpm thrice giving intervals for few seconds in between the
runs, with sodium phosphate buffer (0.03 M, pH 7). The brain
characterization of compounds. The technical assistance of Messrs
A. S. Kushwaha, Z. Ali and D. Vishwakarma is also acknowledged.
References and notes
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Acknowledgments
The authors (K.K.R. and S.T.) are thankful to the Indian Council of
Medical Research, New Delhi and Council of Scientific and Industrial
Research, New Delhi for the financial assistance in the form of a fel-
lowship, and to Sophisticated Analytical Instrument Facility (SAIF)
department of Central Drug Research Institute, Lucknow for the
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