Muscarinic receptor 1 agonist activity of novel N-arylthioureas substituted 3-morpholino arecoline derivatives in Alzheimer's presenile dementia models

Article abstract of DOI:10.1016/j.bmc.2008.06.053

As part of our continuing effort aimed at the development of selective, efficacious and centrally active M1 muscarinic agonists for the treatment of Alzheimer's presenile dementia, a series of N-arylthioureas substituted 3-morpholino arecoline derivatives 9(a-j) were synthesized by using N-benzyl amino ethanol coupling with α-bromo acetyl pyridine followed by reduction and cyclization to develop a new class of M1 receptor agonists. Subsequently the synthesized compounds were subjected to in vitro radioligand M1 receptor affinity studies, IP3 formation studies and also to in vivo pharmacological evaluation of memory and learning in male Wistar rats. Derivatives with chloro (9f) and methoxy (9c) groups on the para position of the benzene ring attached to the nitrogen of thiourea showed several fold high affinity for the M1 receptor (in vitro) among all the synthesized molecules 9(a-j), and also significantly elevated IP3 levels and as well elicited beneficial effects in vivo in memory and learning models in rats (rodent memory evaluation, plus and Y maze studies).

Full text of DOI:10.1016/j.bmc.2008.06.053

Bioorganic & Medicinal Chemistry 16 (2008) 7095–7101
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Muscarinic receptor 1 agonist activity of novel N-arylthioureas substituted
3-morpholino arecoline derivatives in Alzheimer’s presenile dementia models
Manish Malviya a, Y. C. Sunil Kumar a, D. Asha a, J. N. Narendra Sharath Chandra a, M. N. Subhash b,
K. S. Rangappa a,
*
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
b Department of Neurochemistry, National Institute of Mental Health and Neurosciences, Bangalore 560029, India
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of our continuing effort aimed at the development of selective, efficacious and centrally active M1
muscarinic agonists for the treatment of Alzheimer’s presenile dementia, a series of N-arylthioureas
substituted 3-morpholino arecoline derivatives 9(a–j) were synthesized by using N-benzyl amino ethanol
Received 12 June 2008
Revised 26 June 2008
Accepted 27 June 2008
Available online 2 July 2008
coupling with a-bromo acetyl pyridine followed by reduction and cyclization to develop a new class of
M1 receptor agonists. Subsequently the synthesized compounds were subjected to in vitro radioligand
M1 receptor affinity studies, IP3 formation studies and also to in vivo pharmacological evaluation of
memory and learning in male Wistar rats. Derivatives with chloro (9f) and methoxy (9c) groups on
the para position of the benzene ring attached to the nitrogen of thiourea showed several fold high affin-
ity for the M1 receptor (in vitro) among all the synthesized molecules 9(a–j), and also significantly ele-
vated IP3 levels and as well elicited beneficial effects in vivo in memory and learning models in rats
(rodent memory evaluation, plus and Y maze studies).
Keywords:
Alzheimer’s disease
Dementia
M1 receptor
Agonist
Inositol (1,4,5)-trisphosphate
Displacement assay
Rat brain
Ó 2008 Elsevier Ltd. All rights reserved.
In vitro
In vivo
1. Introduction
Arecoline, an alkaloid obtained from the betel nut (Areca cate-
chu), the fruit of a palm tree, has been used previously as centrally
9
Neurochemical examination of the brain material from Alzhei-
mer’s patients has demonstrated the loss of presynaptic marker
enzyme choline acetyltransferase and the muscarinic receptors of
the M2 subtype, which are mainly responsible in causing deficits
in central cholinergic transmission in Alzheimer’s patients.1–3 The
postsynaptic muscarinic receptors, which primarily are of the M1
subtype, to a large extent, seem to survive the loss of cholinergic
nerve endings.4 These findings have led to attempts at restoring
cholinergic function by means of cholinomimetic drugs such as
muscarinic agonists, acetylcholinesterase inhibitors and acetylcho-
line releasing agents, the hypothesis being that enhancement of
cholinergic neurotransmission would alleviate the symptoms of
the diseases, particularly the deficits in cognition and memory.4
Four types of muscarinic receptors are known, named M1 to
M4,5 and five subtypes of muscarinic receptors have been cloned
and designated m1–m5.6,7 Identifying M1 selective muscarinic
agonists, which are capable of crossing the blood–brain barrier is
the subject of active research for pharmacological application.8
active muscarinic agents. The lack of M1 selectivity and efficacy
due to dose limiting side effects associated with M2 and M3 mus-
carinic receptor subtype stimulation has produced disappointing
results.9 Replacement of the ester functionality of arecoline with
either the 3-alkoxy-1,2,5 thiadiazole8 or the 3-alkyl-1,2,4-oxadiaz-
ole10 has produced very potent muscarinic agonists. However, the
systematic removal of a heteroatom in the 3-methyl-1,2,4-oxadi-
azole giving oxazoles and furans caused a decrease in affinity for
the agonist binding site. The two isomers, 2-methyl-1,2,4-oxadiaz-
ole and 5-methyl-1,2,4-oxadiazole also had lower affinities for
muscarinic receptors;10 see the structure of Arecoline and its deriv-
atives (Fig. 1). No muscarinic M1 subtype selectivity has been
reported for 2-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)morpho-
line. C-Functionalized morpholines are found in various naturally
occurring products as well as in drugs.11 Since compound 2-(1-
methyl-1,2,5,6-tetrahydropyridin-3-yl) morpholine is a conforma-
tionally restricted arecoline analogue, we were encouraged to pur-
sue this compound.
In our previous studies, we have reported arecoline thiazolidi-
nones12 and N-arylsulphonamide substituted 3-morpholino areco-
line derivatives13 as muscarinic receptor 1 agonist. In a continued
effort to discover less toxic arecoline class of muscarinic agonist
* Corresponding author. Tel.: +91 821 2419661; fax: +91 821 2412191.
E-mail addresses: rangappaks@gmail.com, rangappaks@chemistry.uni-mysor-
e.ac.in (K.S. Rangappa).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.053

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M. Malviya et al. / Bioorg. Med. Chem. 16 (2008) 7095–7101
and Fig. 2), which is in the order of 9f > 9c > 9h > 9g. The most po-
tent compound among all tested derivatives, 9f (Ki = 0.31 M), is
N
S
O
O
N
l
N
R
one with chlorine, at the para position of the aryl group attached
to the nitrogen of thiourea. Substitution of methoxy group (9c),
at para position of the aryl group also showed good affinity to-
wards the M1 receptor in vitro. However, when chloro and meth-
oxy groups are substituted at meta position (9e and 9b,
respectively) they showed moderate affinity, and when they are
substituted at ortho position (9d and 9a, respectively) they showed
least or no affinity, respectively, for the M1 receptor in vitro. In
similar fashion, introduction of fluoro group at para position (9h)
also showed good affinity for the M1 receptor, whereas when
introduced at ortho position (9g) it decreases the affinity of com-
pound for the receptor in vitro. However, disubstituted chlorine
(9i) at ortho and para positions showed only moderate affinity
for the receptor. Compound 9j with benzene (without any substi-
tution on it) ring substituted on the nitrogen of thiourea showed
average affinity towards the M1 receptor in vitro.
N
OMe
O
R
N
N
N
CH3
CH3
CH3
3-alkoxy-1,2,5-thiadiazole
Arecoline
3-alkyl-1,2,4-oxadiazole
R
N
N
O
N
R
O
N
N
CH3
N
CH3
5-methyl-1,2,4-oxadiazol
2-methyl-1,2,4-oxadiazole
e
Figure 1. Arecoline and arecoline derivatives.
and to further improve their selectivity and potency, here, we are
reporting our findings of N-arylthioureas substituted 3-morpho-
lino arecoline derivatives [9(a–j), scheme 1] along with their
in vitro muscarinic binding studies by using [3H]QNB with male
Wistar rat brain synaptosomal membrane, IP3 levels estimation
stimulated by the synthesized molecules 9(a–j) and also in vivo
evaluation of memory and learning in male Wistar rats, as another
potent M1 receptor agonist for the symptomatic treatment of Alz-
heimer’s dementia.
H3CO
Cl
9a =
9b =
9c =
9d =
9h =
F
9e =
Cl
OCH3
9i =
9j =
Cl
9f =
9g =
Cl
F
OCH3
Cl
2. Results and discussion
Structure–activity relationship (SAR) can be drawn from the
in vitro affinity assay for the synthesized N-arylthioureas substi-
tuted 3-morpholino arecoline 9(a–j) derivatives (Scheme 1). Four
derivatives among all synthesized compounds 9(a–j) showed
greater affinity and potency towards the M1 receptor (Table 1
Agonist stimulation of the M1 receptor results in a rapid elevation
of IP3 levels.14 Therefore, we planned to measure the efficacy and
Ph
O
O
O
Br
N
HBr, Br2
OH
K2CO3, DMF
NaBH4
MeOH
CH3COOH
N-benzylaminoethanol
N
1
N
2
N
3
Ph
N
Boc
N
Ph
OH
(a) 10%Pd-C
N
O
O
OH
70% H2SO4
HCOONH4, MeOH, reflux
(b) (Boc)2O, THF, K2CO3
(a) CH3I, acetone
N
6
N
5
N
4
Reflux
(b) NaBH4, MeOH
R
HN
S
Boc
N
H
N
C
N
O
O
O
S
C
N R
HCl, MeOH
TEA,
N
MDC
N
N
CH3
7
CH3
CH3
8
9
Scheme 1.

M. Malviya et al. / Bioorg. Med. Chem. 16 (2008) 7095–7101
7097
Table 1
Table 2
In vitro affinity of N-arylthioureas substituted 3-morpholino arecoline derivatives
9(a–j) towards M1 receptor of male Wistar rat cortex synaptosomal membrane
In vitro effect of N-arylthioureas substituted 3-morpholino arecoline derivatives 9(a–
j) on the formation of Inositol (1,4,5)-trisphosphate (IP3) levels in the cerebral cortex
slices of the rat brain
Compounds
Ki (lM)
IC50 (lM)
Rat-group
IP3 Conc. (pM)
Stimulation (%)
9a
9b
9c
9d
9e
9f
9g
9h
98 12.36
41 9.08
362 17.47
120 4.21
15 2.73
515 28.69
236 11.87
1.2 0.25
67 10.56
31 5.12
Control (Basal)
9a-Stimulated
9b*-Stimulated
9c*-Stimulated
9d-Stimulated
9e-Stimulated
9f*-Stimulated
9g*-Stimulated
9h*-Stimulated
9i-Stimulated
0.61 0.05
0.61 0.02
0.65 0.06
1.09 0.05
0.61 0.06
0.63 0.05
1.32 0.06
0.79 0.03
0.95 0.03
0.71 0.04
0.62 0.04
1.64 0.06
—
4
0.36
100
106
178
100
103
216
129
155
116
101
268
214 21.92
85 6.98
0.31 0.12
15 3.36
7
1.28
9i
9j
31 4.90
49 5.36
86 8.39
112 08.16
153 8.59
469 17.54
Arecoline
9j-Stimulated
Acetylcholine*-Stimulated
The efficacy of novel N-arylthioureas substituted 3-morpholino arecoline 9(a–j)
derivatives (100 mol/l) for the M1 receptor was measured by its potency to ele-
vate the basal IP3 levels, and it was compared with IP3 levels elevated by acetyl-
choline iodide (20 mol/l), which represent M1 receptor linked PLC activity. The
l
l
values are mean and standard deviation of three experiments, each assayed in
duplicate. IP3 formation was expressed as picomoles of [3H]IP3 formed per milli-
gram of tissue. n = 6, *P = <0.001.
Figure 2. Displacement graphs of three potent compounds 9f, 9c and 9h. The
displacement studies were done with 0.2 nM [3H]QNB and different concentrations
of N-arylthioureas substituted 3-morpholino arecolines 9(a–j). The mean values of%
bound are plotted against log of displacer concentration. IC50 and Ki values are
obtained from Ligand–Drug programme.
potency of synthesized compounds 9(a–j) to elevate the IP3 forma-
tion in rat cerebral cortex slices. Basal IP3 levels was found to be
0.61 pmol/mg protein, and it was significantly increased by all the
four derivatives (9f, 9c, 9h and 9g), which were showing high affin-
ity for M1 receptor in binding studies. The potency of these com-
pounds to elevate the basal IP3 levels was compared with that of
IP3 levels elevated by acetylcholine iodide, which represents M1
receptor-linked PLC activity (Table 2 and Fig. 3).
It has been demonstrated that the M1 receptor subtypes pre-
dominate in the cerebral cortex and hippocampus areas15,16 in
which cholinergic transmission appears to be essential for learning
and memory. The foresaid in vitro M1 receptor binding studies and
subsequently IP3 formation studies formed a basis for extending
correlation further to in vivo pharmacological studies to ascertain
applicability of the synthesized N-arylthioureas substituted 3-mor-
pholino arecoline 9(a–j) derivatives in scopolamine (muscarinic
antagonist)-induced dementia models using memory and learning
experiments (passive avoidance tasks, plus and Y maze studies). In
accordance with the degree of affinity and potency of synthesized
molecules 9(a–j) in vitro binding experiments elicited almost
anticipated level of pharmacological actions in reversing scopol-
amine-induced dementia (Fig. 4). Three synthesized morpholino
arecoline derivatives 9f, 9c and 9h were best among all 9(a–h) syn-
thesized compounds in reversing scopolamine-induced dementia
by making rats to commit less number of mistakes (Number of
mistakes done 10, 10 and 11, respectively) when compared with
Figure 3. The scatchard graph of four potent compounds (9f, 9c, 9h and 9g) among
all the tested derivatives 9(a–j). Inositol (1,4,5)-trisphosphate (IP3) levels were
estimated using [3H]myoinositol, in rat cerebral cortex slices after the stimulation
by different concentrations of N-arylthioureas substituted 3-morpholino arecoline
derivatives 9(a–j), and significance was compared with the basal IP3 levels, and
their potency to elevate the IP3 formation was determined by comparing it with IP3
levels elevated by acetylcholine iodide as described in Section 4. Data points are the
means of three experiments, each assayed in duplicate.
the number of mistakes done by control rats (8 mistakes) and sco-
polamine-treated group (33 mistakes). Compounds 9g, 9b and 9i
also significantly reversed the scopolamine-induced memory loss.
Compounds 9d, 9a and 9e were the least potent compounds
among all tested derivatives.
The in vivo plus maze for synthesized derivatives 9(a–j) in male
Wistar rats measures the transfer latency (TL) in seconds taken to
reach from one extreme ends of open arm also to one of the closed
arms in a plus maze. Difference in TL in seconds between 1st day
and 2nd day for scopolamine-treated group and test compound
along with scopolamine-treated groups was compared to evaluate
learning (TL1) and memory (TL2). Derivatives 9f, 9c and 9h signif-
icantly reversed acute memory loss and learning impairment in

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M. Malviya et al. / Bioorg. Med. Chem. 16 (2008) 7095–7101
45
40
35
30
25
20
15
10
5
Control
Scopolamine
Scopo.+ 9a
Scopo.+ 9b
Scopo.+ 9c
Scopo.+ 9d
Scopo.+ 9e
Scopo.+ 9f
Scopo.+ 9g
Scopo.+ 9h
Scopo.+ 9i
Scopo.+ 9j
0
Different groups of experimental rats
Figure 4. Antiamnesic effect of N-arylthioureas substituted 3-morpholino arecolines 9(a–j) against scopolamine-induced memory loss in male Wistar rats. Mean ( SEM).
35
Control
Scopolamine
30
25
Scopo.+ 9a
Scopo.+ 9b
Scopo.+ 9c
20
15
10
5
Scopo.+ 9d
Scopo.+ 9e
Scopo.+ 9f
Scopo.+ 9g
Scopo.+ 9h
Scopo.+ 9i
Scopo.+ 9j
0
Different groups of experimental rats
Figure 5. Effect of N-arylthioureas substituted 3-morpholino arecoline derivatives 9(a–j) on memory and learning against scopolamine-induced memory loss and learning
impairment on elevated plus maze in male Wistar rats. Mean ( SEM).
male Wistar rats (Fig. 5). 9f produced lesser TL for 1st day
(TL1 = 34 s) compared to scopolamine-treated group (TL1 = 68 s),
but on 2nd day (TL2 = 9 s) TL was lesser than 1st day (9f) indicating
how it is helpful in reversing learning impairment as compared to
rest of the derivatives. Overall difference between TL2 and TL1 was
same for 9f, 9c and control groups (25 s). In contrary to this, com-
pound 9d produced longer 1st day TL (TL1 = 60 s) and 2nd day TL
(TL2 = 43 s) overall difference differs (TL1–TL2 = 17 s), implying
that the derivative fails to reverse acute memory and learning
impairing in male Wistar rats. TL for the remaining compounds is
given in Figure 5.
The synthesized derivatives were also subjected to in vivo Y
maze studies using male Wistar rats. Male Wistar rats were al-
lowed to explore three-arm Y maze in which rats move in se-
quence. The percentage of alteration behaviour an animal does
by not entering into the already entered arm before completing
the sequence was noted. The alteration behaviour in terms of per-
centage of alteration behaviour was noted for test compounds in
presence of scopolamine and was compared with scopolamine
administered animal group (Fig. 6). The percentage of alteration
behaviour for 9f, 9h, 9c and 9g (64, 61, 60 and 58%) indicates that
derivatives significantly reverse scopolamine-induced spatial
working memory loss (in terms of alteration behaviour). For the
rest of the derivatives percentage of alteration behaviour is shown
in Figure 6.
3. Conclusion
In light of these findings, in vitro competitive M1 receptor affin-
ity assay; stimulation of IP3 formation studies and in vivo pharma-
cological experiments for the synthesized compounds 9(a–j), to
test the relation between affinity of these molecules to M1 receptor
and their ability to reverse scopolamine-induced memory loss and
learning impairment, have ascertained their applicability in
dementia. We can conclude that position of substitutions on the
benzene ring attached to the nitrogen of thioureas determines
the affinity of the compound for the M1 receptor. The compound
9j, which is not having any substitute group on the benzene at-
tached to the nitrogen of thiourea, is found to have moderate affin-
ity for the M1 receptor. But when chloro (9f) and methoxy (9c) are
present at the para position of the benzene, it increases the affinity
of the compounds several folds for the M1 receptor in vitro, and
also showed greater potency in elevating the IP3 levels in rat cere-
bral slices and useful antidementia activity in the in vivo model
tested. However, when the same groups are present at meta (9e
and 9b, respectively) or at ortho position (9d and 9a, respectively),
it decreases the affinity of compounds for the M1 receptor. In the
similar fashion, when fluoro group is present at para position
(9h) it increases the affinity of compound for the receptor but,
when present at ortho position (9g) it decreases the affinity of
compound for the M1 receptor. Decrease in the affinity of the com-

M. Malviya et al. / Bioorg. Med. Chem. 16 (2008) 7095–7101
7099
80
70
60
50
40
30
20
10
0
Control
Scopolamine
Scopo.+ 9a
Scopo.+ 9b
Scopo.+ 9c
Scopo.+ 9d
Scopo.+ 9e
Scopo.+ 9f
Scopo.+ 9g
Scopo.+ 9h
Scopo.+ 9i
Scopo.+ 9j
Different groups of experimental rats
Figure 6. Effect of N-aryl thioureas substituted 3-morpholino arecoline derivatives 9(a–j) on spatial working memory using Y maze task in male Wistar rats. Mean ( SEM).
pounds having substitute at ortho position may be due to the hin-
drance in binding to the active site of M1 receptor. Compound 9i
having disubstituted chlorine at ortho and para positions showed
moderate affinity for the M1 receptor in vitro and average potency
in reversing scopolamine-induced memory loss in vivo. These N-
aryl morpholino arecoline derivatives 9(a–j) showed no visible
cholinergic toxicity at the dose tested (salivation, defecation,
etc.). One of the promising new derivatives would emerge as the
potent molecule having antidementia activity.
performed using Merck 7734 silica gel (60–120 mesh) and Merck
made TLC plates. All chemicals and reagents were obtained from
Aldrich (USA), Spectrochem Pvt. Ltd. (India) and Rankem Pvt. Ltd.
(India), and were used without further purification.
4.1.1. General procedure for the synthesis of N-arylthioureas
substituted 3-morpholino arecoline derivatives 9(a–j)
The intermediate compound 2-(1-methyl-1,2,5,6-tetrahydro-
pyridin-3-yl)morpholine (compound 8, scheme 1) was synthesized
by eight step reactions according to the previously described pro-
cedure.17 To the stirred solution of compound 8 (1 equiv) in dichlo-
romethane, triethylamine (5 equiv) was added and cooled to 0 °C,
respective isothiocyanates (1 equiv) were added at cold condition
and stirred at room temperature for 4–5 h (completion of reaction
was confirmed by TLC). Reaction mixture was quenched using
water and extracted using dichloromethane. The combined dichlo-
romethane layer was washed with brine solution and dried over
anhydrous sodium sulfate. Dichloromethane was evaporated un-
der reduced pressure, and the crude product obtained was purified
by silica gel (60–120 mesh) column. The compounds 9(a–j) were
eluted at 8% to 10% methanol in chloroform.
4. Experimental procedure
4.1. Chemistry
The N-arylthioureas substituted 3-morpholino arecoline deriva-
tives 9(a–j) were synthesized in nine steps as shown in the scheme
1. Bromination of 3-acetylpyridine 1 with Br2/HBr in glacial acetic
acid gave the HBr salt of bromoacetylpyridine 2. This was con-
verted to amino alcohol 3, by reaction with N-benzylaminoethanol
in DMF in the presence of K2CO3. The keto group of compound 3
was reduced using NaBH4 in methanol to obtain the dihydroxy
compound 4. Treatment of compound 4 with 70% H2SO4 under re-
flux conditions caused dehydration to yield the cyclized product 5.
The N-benzyl group was removed by refluxing amine 5 in metha-
nol in the presence of 10% Pd–C and ammonium formate, and
the resulting free amine was treated with Boc-anhydride in THF
in the presence of K2CO3 to yield the Boc-protected compound 6.
This was converted to the corresponding methylamine hydroio-
dide salt by reaction with methyl iodide in acetone. This on treat-
ment with sodium borohydride in methanol gave the reduced
product 7. Finally, the Boc group was removed using methanolic
HCl to yield the free amine as HCl salt 8. The detailed procedure
for the synthesis of compound 8 has been reported in our previous
paper.17 This on reacting with respective isothiocyanates gave N-
arylthioureas substituted 3-morpholino arecoline derivatives
9(a–j). 1H NMR spectra of all compounds 9(a–j) showed a multiplet
at 9–7 due to aromatic protons and 5.7–5.8 due to double bond of
tetrahydro pyridine. All the synthesized compounds were charac-
terized by IR, 1H NMR, Mass spectroscopy and CHNS analysis.
Infrared (IR) spectra were recorded using Nujol on JASCO-FTIR,
4100 series. Nuclear magnetic resonance (1H NMR) spectra were
recorded on Shimadzu AMX 400-Bruker, 400MHz spectrometer
using DMSO as a solvent and TMS as internal standard (chemical
shift in d ppm). Spin multiplets are given as s (singlet), d (doublet),
t (triplet) and m (multiplet). Mass and purity were recorded on a
LC-MSD-Trap-XCT. Elemental (CHNS) analyses were obtained on
Vario EL III Elementar. Silica gel column chromatography was
4.1.2. Synthesis of 2-(1,2,5,6-tetrahydro-1-methylpyridin-3yl)-
N–(2-methoxyphenyl)morpholine-4-carbothioamide (9a)
The compound (9a) was obtained by the reaction of compound
8 (0.25 g, 0.00098 mol) with 2-methoxy phenyl isothiocyanate
(0.162 g, 0.00098 mol) and triethylamine (0.394 g, 0.0039 mol) in
dichloromethane (3 ml). Yield: 82%; IR (Nujol, cmÀ1): 3201(–NH–),
1389 (–CS–N–), 1675 (–RC@CH–). 1H NMR (CDCl3): d 9.23(s, 1H),
7.16–7.14 (m, 1H), 6.88–6.86 (m, 2H), 6.784–6.763 (d, 1H, J = 8.4
Hz), 5.76 (br s, 1H, –C@CH–), 3.85–3.74 (m, 3H), 3.73 (s, 3H),
3.52–3.48 (m, 1H), 3.31–3.26 (m, 3H), 2.80–2.76 (m, 2H), 2.52–
2.48 (m, 2H), 2.21 (s, 3H), 2.02 (m, 2H). MS (m/z): 348.53 (M+).
4.1.3. Synthesis of 2-(1,2,5,6-tetrahydro-1-methylpyridin-3yl)-
N–(3-methoxyphenyl)morpholine-4-carbothioamide (9b)
The compound (9b) was obtained by the reaction of compound
8 (0.25 g, 0.00098 mol) with 3-methoxy phenyl isothiocyanate
(0.162 g, 0.00098 mol) and triethylamine (0.394 g, 0.0039 mol) in
dichloromethane (3 ml). Yield: 83%; IR (Nujol, cmÀ1): 3206(–NH–),
1398 (–CS–N–), 1676 (–RC@CH–). 1H NMR (CDCl3): d 9.23 (s, 1H),
7.19–7.16 (m, 1H), 6.89–6.84 (m, 2H), 6.677–6.657 (dd, 1H,
J = 1.6 and 1.6 Hz), 5.74 (br s, 1H, –C@CH–), 3.84–3.73 (m, 3H),
3.70 (s, 3H), 3.53–3.49 (m, 1H), 3.33–3.28 (m, 3H), 2.81–2.76 (m,
2H), 2.52–2.47 (m, 2H), 2.23 (s, 3H), 2.01 (m, 2H). MS (m/z):
348.51 (M+).

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M. Malviya et al. / Bioorg. Med. Chem. 16 (2008) 7095–7101
4.1.4. Synthesis of 2-(1,2,5,6-tetrahydro-1-methylpyridin-3yl)-
N–(4-methoxyphenyl)morpholine-4-carbothioamide (9c)
The compound (9c) was obtained by the reaction of compound
8 (0.25 g, 0.00098 mol) with 4-methoxy phenyl isothiocyanate
(0.162 g, 0.00098 mol) and triethylamine (0.394 g, 0.0039 mol) in
dichloromethane (3 ml). Yield: 86%; IR (Nujol, cmÀ1): 3204
(–NH–), 1386 (–CS–N–), 1678 (–RC@CH–). 1H NMR (CDCl3): d 9.20
(s, 1H), 7.141–7.119 (d, 2H, J = 8.8 Hz), 6.863–6.842 (d, 2H, J = 8.4
Hz), 5.78 (bs, 1H, –C@CH–), 3.85–3.74 (m, 3H), 3.74 (s, 3H), 3.52–
3.47 (m, 1H), 3.33–3.28 (m, 3H), 2.84–2.79 (m, 2H), 2.53–2.48 (m,
2H), 2.21 (s, 3H), 2.00 (m, 2H). MS (m/z): 348.51 (M+).
(–NH–), 1394 (–CS–N–), 1676 (–RC@CH–). 1H NMR (CDCl3): d
9.02 (s, 1H), 7.419–7.404 (d, 2H, J = 7.20 Hz), 7.072–7.041 (d, 2H,
J = 6.02 Hz), 5.74 (bs, 1H, –C@CH–), 3.85–3.75 (m, 3H), 3.50–3.48
(m, 1H), 3.31–3.23 (m, 3H), 2.80–2.74 (m, 2H), 2.54–2.46 (m,
2H), 2.24 (s, 3H), 2.05 (m, 2H). MS (m/z): 336.48 (M+).
4.1.10. Synthesis of N-(2,4-dichlorophenyl)-2-(1,2,5,6-tetra-
hydro-1-methylpyridin-3-yl)morpholine-4-carbothioamide (9i)
The compound (9i) was obtained by the reaction of compound 8
(0.25 g, 0.00098 mol) with 2,4-dichloro phenyl isothiocyanate
(0.20 g, 0.00098 mol) and triethylamine (0.394 g, 0.0039 mol) in
dichloromethane (3 ml). Yield: 87%; IR (Nujol, cmÀ1): 3211 (–NH–
), 1382 (–CS–N–), 1676 (–RC@CH–). 1H NMR (CDCl3): d 9.06 (s, 1H),
7.596–7.591 (d, 1H, J = 2.0 Hz), 7.368–7.347 (d, 1H, J = 8.4 Hz),
7.513–7.492 (d, 1H, J = 8.4 Hz), 5.70 (bs, 1H, –C@CH–), 3.83–3.78
(m, 3H), 3.54–3.50 (m, 1H), 3.32–3.24 (m, 3H), 2.80–2.74 (m, 2H),
2.54–2.46 (m, 2H), 2.25 (s, 3H), 2.08 (m, 2H). MS (m/z): 387.36 (M+).
4.1.5. Synthesis of N-(2-chlorophenyl)-2-(1,2,5,6-tetrahydro-1-
methylpyridin-3-yl)morpholine-4-carbothioamide (9d)
The compound (9d) was obtained by the reaction of compound
8
(0.25 g, 0.00098 mol) with 2-chloro phenyl isothiocyanate
(0.166 g, 0.00098 mol) and triethylamine (0.394 g, 0.0039 mol) in
dichloromethane (3 ml).Yield: 88%; IR (Nujol, cmÀ1): 3221 (–NH–),
1389 (–CS–N–), 1678 (–RC@CH–). 1H NMR (CDCl3): d 9.17 (s, 1H),
7.473–7.454 (d, 1H, J = 7.6 Hz), 7.25–7.19 (m, 3H), 5.73 (bs, 1H,
–C@CH–), 3.80–3.75 (m, 3H), 3.53–3.50 (m, 1H), 3.30–3.23 (m,
3H), 2.80–2.72 (m, 2H), 2.54–2.46 (m, 2H), 2.26 (s, 3H), 2.05 (m,
2H). MS (m/z): 352.90 (M+).
4.1.11. Synthesis of 2-(1,2,5,6-tetrahydro-1-methylpyridin-3yl)-
N-phenylmorpholine-4-carbothioamide (9j)
The compound (9j) was obtained by the reaction of compound 8
(0.2 g, 0.00098 mol) with phenylisothiocyanate (0.132 g, 0.00098
mol) and triethylamine (0.394 g, 0.0039 mol) in dichloromethane
(3 ml). Yield: 89%; IR (Nujol, cmÀ1): 3212 (–NH–), 1389 (–CS–N–),
1674 (–RC@CH–). 1H NMR (CDCl3): d 9.20 (s, 1H), 7.13–7.06 (m,
2H), 6.95–6.90 (m, 3H), 5.76 (b, 1H, –C@CH–), 3.85–3.76 (m, 3H),
3.53–3.49 (m, 1H), 3.32–3.28 (m, 3H), 2.79–2.78 (m, 2H), 2.53–
2.48 (m, 2H), 2.20 (s, 3H), 2.04 (m, 2H), 1.24 (m, 3H). MS (m/z):
318.48 (M+).
4.1.6. Synthesis of N-(3-chlorophenyl)-2-(1,2,5,6-tetrahydro-1-
methylpyridin-3-yl)morpholine-4-carbothioamide (9e)
The compound (9e) was obtained by the reaction of compound 8
(0.25 g, 0.00098 mol) with 3-chloro phenyl isothiocyanate (0.166 g,
0.00098 mol) and triethylamine (0.394 g, 0.0039 mol) in dichloro-
methane (3 ml). Yield: 92%; IR (Nujol, cmÀ1): 3224(–NH–), 1408
(–CS–N–), 1677 (–RC@CH–). 1H NMR (CDCl3): d 9.36 (s, 1H),
7.395–7.390 (d, 1H, J = 2.06 Hz), 7.27–7.20 (m, 2H), 7.133–7.116
(d, 1H, J = 7.8 Hz), 5.73 (bs, 1H, –C@CH–), 3.84–3.78 (m, 3H),
3.50–3.48 (m, 1H), 3.33–3.27 (m, 3H), 2.80–2.75 (m, 2H), 2.53–
2.48 (m, 2H), 2.24 (s, 3H), 2.04 (m, 2H). MS (m/z): 352.90 (M+).
4.2. Biology
4.2.1. Displacement study
The competitive affinity assay was done using various synthe-
sized N-arylthioureas substituted 3-morpholino arecoline deriva-
tives 9(a–j) to find out their affinity towards cortical M1
receptor. Male Wistar rat brain cortex was taken out and used
for synaptosomal membrane preparation. Crude membrane pellet
was obtained from brain tissue, homogenized in 20 volumes of
Tris–HCl buffer (50 mmol/L, pH 7.4) containing 0.32 mol/L sucrose,
following the procedure described by Creese and Snyder.18 The tis-
sue homogenate was centrifuged at a speed of 1000g for 10 min at
4 °C, to remove cellular debris. The supernatant obtained was cen-
trifuged at 32,000g for 20 min at 4 °C. Pellet obtained was resus-
pended in 50 mmol/L phosphate assay buffer (pH 7.4) containing
1 mmol MgCl2. The protein concentration was estimated by meth-
od described by Lowry et al.19
4.1.7. Synthesis of N-(4-chlorophenyl)-2-(1,2,5,6-tetrahydro-1-
methylpyridin-3-yl)morpholine-4-carbothioamide (9f)
The compound (9f) was obtained by the reaction of compound 8
(0.25 g, 0.00098 mol) with 4-chloro phenyl isothiocyanate (0.166 g,
0.00098 mol) and triethylamine (0.394 g, 0.0039 mol) in dichloro-
methane (3 ml). Yield: 92%; IR (Nujol, cmÀ1): 3221 (–NH–), 1383
(–CS–N–), 1669 (–RC@CH–). 1H NMR (CDCl3): d 9.04 (s, 1H),
7.351–7.330 (d, 2H, J = 8.4 Hz), 7.234–7.215 (d, 2H, J = 7.6 Hz),
5.75 (bs, 1H, –C@CH–), 3.84–3.79 (m, 3H), 3.53–3.48 (m, 1H),
3.33–3.28 (m, 3H), 2.81–2.75 (m, 2H), 2.55–2.48 (m, 2H), 2.28 (s,
3H), 2.05 (m, 2H). MS (m/z): 352.90 (M+).
The affinity of various compounds towards M1 receptor was
estimated by using [3H]QNB (0.2 nM, specific activity 48 Ci/mmol,
Amersham, Little Chalfont, Bucks, UK) essentially following the
4.1.8. Synthesis of N-(2-fluorophenyl)-2-(1,2,5,6-tetrahydro-1-
methylpyridin-3-yl)morpholine-4-carbothioamide (9g)
The compound (9g) was obtained by the reaction of compound
procedure described by Hyttel et al.20; Yamamura and Snyder21
,
8
(0.25 g, 0.00098 mol) with 2-fluoro phenyl isothiocyanate
with slight modification. In brief, an aliquot of synaptosomal mem-
brane proteins (50 g) was incubated with different concentra-
tions of compounds (0.1–500
M) as a displacer and [3H]QNB
(0.2 nM), and reaction volume was made up to 200 l with assay
buffer in 96-well plates and incubated for 2 h at 37 °C. The reac-
tion for all displacement assays was stopped by adding ice-cold as-
say buffer, and reaction mixtures were rapidly filtered through GF/
B filters under vacuum. The filters were transferred to vials and
added 5 ml of scintillation fluid and allowed to equilibrate over-
night. Radioactivity was measured in a liquid scintillation counter
(Tris-Carb 2100TR, Packard, US) at 65% efficiency.
(0.150 g, 0.00098 mol) and triethylamine (0.394 g, 0.0039 mol)
in dichloromethane (3 ml). Yield: 90%; IR (Nujol, cmÀ1): 3232
(–NH–), 1392 (–CS–N–), 1675 (–RC@CH–). 1H NMR (CDCl3): d
9.13 (s, 1H), 7.464–7.442 (d, 1H, J = 8.8 Hz), 7.31–7.29 (m, 2H),
7.18–7.16 (m, 1H), 5.73 (bs, 1H, –C@CH–), 3.85–3.75 (m, 3H),
3.53–3.48 (m, 1H), 3.33–3.27 (m, 3H), 2.81–2.76 (m, 2H), 2.54–
2.49 (m, 2H), 2.24 (s, 3H), 2.04 (m, 2H). MS (m/z): 336.48 (M+).
l
l
l
4.1.9. Synthesis of N-(4-fluorophenyl)-2-(1,2,5,6-tetrahydro-1-
methylpyridin-3-yl)morpholine-4-carbothioamide (9h)
The compound (9h) was obtained by the reaction of compound
The data from displacement were analyzed, and IC50 and Ki values
22
8
(0.25 g, 0.00098 mol) with 4-fluoro phenyl isothiocyanate
are obtained from Ligand–Drug programme (McPherson).
The
(0.150 g, 0.00098 mol) and triethylamine (0.394 g, 0.0039 mol) in
mean values of % bound are plotted against log of displacer
concentration.
dichloromethane (3 ml). Yield: 94%; IR (Nujol, cmÀ1): 3243

M. Malviya et al. / Bioorg. Med. Chem. 16 (2008) 7095–7101
7101
4.2.2. IP3 levels estimation
4.2.8. Data analysis
Inositol (1,4,5)-trisphosphate (IP3) levels were estimated using
[3H]myoinositol (s.a. 16.5 Ci/mmol, Amersham) in rat cerebral cor-
tex slices, at basal level and also after stimulation by different con-
centrations of synthesized N-arylthioureas substituted 3-
morpholino arecoline 9(a–j) derivatives (1–500 lmol/l), to find
out their efficacy and potency to elevate the IP3 formation, by com-
The data from the radioligand displacement assay were ana-
lyzed using ‘LIGAND–DRUG’ software programme (McPherson)22]
to obtain the IC50 and Ki values (both are expressed in lmol/l). All
the data are expressed as means SD. The statistical analysis was
done by using one-way analysis of variance (ANOVA). Differences
were considered to be significant at P < 0.05. All analyses were per-
formed with the ‘Jandel-Scientific-Sigma stat’ software, version 2.0
for windows.
paring it with basal IP3 levels and IP3 formation elevated by acetyl-
choline iodide (1–100
by Kendall and Naharoski.
lmol/l), following the procedure described
23
The radioactivity of [3H]IP3 formed
was measured in a liquid scintillation counter at 65% efficiency.
The levels of IP3 were expressed as picomoles of [3H]IP3 formed
per milligram of protein.
Acknowledgements
Manish Malviya is grateful to the Indian Council of Medical Re-
search (Ref. No. 45/51/2007/Pha/BMS) for providing the Senior Re-
search Fellowship (IRIS Cell No. 2007-04110). The authors are also
grateful to the Department of Science and Technology (DST), New
Delhi, for financial support under the project SR/SO/HS-58/2003.
The CHNS, IR and other data obtained from the instruments
granted under DST-FIST and UGC-SAP (phase I) programmes are
greatly acknowledged.
4.2.3. Antiamnesic activity
It was carried out for synthesized N-arylthioureas substituted
3-morpholino arecoline derivatives 9(a–j) against scopolamine-in-
duced memory loss using passive avoidance step down task para-
digm in male Wistar rats weighing 200–250 g (n = 8) according to
the method described by Sharma and Kulkarni.24,25
4.2.4. Elevated plus maze
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(TL). The male Wistar rats (weighing 200–250 g) were placed indi-
vidually at the end of one arm facing away from the central plat-
form, and the time they took to move from open arm to either of
enclosed arms (TL) was measured. On the 1st day, male Wistar rats
were allowed to explore the plus maze for 90 s with scopolamine,
and scopolamine with test compounds treatment to respective ani-
mal groups, 30 min before the plus maze exploration. Control
group animals were treated with saline (0.9%). Second day TL
was measured in the similar way on the same animals. The resul-
tant data were subjected to statistical analysis. P < 0.05 was con-
sidered statistically significant.25
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This task was used to measure the spatial memory through the
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respective animal groups. The ability to alternate requires that the
rats know which arm they have already visited. 26 The series of arm
entry, including possible returns into the same arm, is recorded
visually. Alteration is defined as the number of successive entries
into the three arms, on overlapping triplet sets. The percentage
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4.2.7. Dose–response curve
Different doses (0.05–0.2 mg/kg) of the derivatives were selected
to find optimum dose (found to be 0.1 mg/kg) for in vivo studies.