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

Article abstract of DOI:10.1016/j.bmc.2009.06.032

Earlier we have reported the effect of arecoline thiazolidinone and morpholino arecoline derivatives as muscarinic receptor 1 agonists in Alzheimer's presenile dementia models. To elucidate further our Structure-Activity Relationship (SAR) studies on the chemistry and muscarinic receptor 1 binding efficacy, a series of novel carboxamide derivatives of 2-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)morpholine molecule have been designed and synthesized as a new class of M1 receptor agonists with a low toxicity effect profile that enhances memory function in animal models of Alzheimer's presenile dementia and also modulates the APP secretion from rat brain cerebrocortical slices by activating M1 receptor in vitro. Results suggest that compound 9b having methyl group at the para position of the aryl group attached to the carboxamide of morpholino arecoline could emerge as a potent molecule having antidementia activity.

Full text of DOI:10.1016/j.bmc.2009.06.032

Bioorganic & Medicinal Chemistry 17 (2009) 5526–5534
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Muscarinic receptor 1 agonist activity of novel N-aryl carboxamide
substituted 3-morpholino arecoline derivatives in Alzheimer’s
presenile dementia models
Manish Malviya a, Y. C. Sunil Kumar a, R. B. Mythri b, C. Venkateshappa b, M. N. Subhash b, K. S. Rangappa a,
*
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India
b Department of Neurochemistry, National Institute of Mental Health and Neurosciences, Bangalore 560 029, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Earlier we have reported the effect of arecoline thiazolidinone and morpholino arecoline derivatives as
muscarinic receptor 1 agonists in Alzheimer’s presenile dementia models. To elucidate further our Struc-
ture–Activity Relationship (SAR) studies on the chemistry and muscarinic receptor 1 binding efficacy, a
series of novel carboxamide derivatives of 2-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)morpholine mol-
ecule have been designed and synthesized as a new class of M1 receptor agonists with a low toxicity
effect profile that enhances memory function in animal models of Alzheimer’s presenile dementia and
also modulates the APP secretion from rat brain cerebrocortical slices by activating M1 receptor in vitro.
Results suggest that compound 9b having methyl group at the para position of the aryl group attached to
the carboxamide of morpholino arecoline could emerge as a potent molecule having antidementia
activity.
Received 11 April 2009
Revised 14 June 2009
Accepted 16 June 2009
Available online 21 June 2009
Keywords:
Muscarinic agonist
Alzheimer’s disease
Amyloid precursor protein
Dementia
Ó 2009 Published by Elsevier Ltd.
1. Introduction
cursor protein (APP), a membrane associated glycoprotein
containing within its sequence the amyloid b-protein (Ab) which
is the major component of the senile plaques found in brains of
Alzheimer’s patients;11,12 reduces the Ab-peptide production;13–
Alzheimer’s disease (AD) is the most common neurodegenera-
tive disease that involves a gradual and progressive memory loss
and dementia.1 Neuropathologically, the disease is characterized
by the presence of extracellular deposits of amyloid b-peptide
(Ab) as fibril aggregates that form senile plaques, intracellular neu-
rofibrillary tangles and degeneration of presynaptic cholinergic
neurons that ascend from the basal forebrain to cortical and hippo-
campal areas.2,3 The M1 acetylcholine muscarinic receptor
(mAChR) is expressed in the cerebral cortex and hippocampus
and its major role is in cognitive processing including short-term
memory;4,5 however, muscarinic agonists were not effective in
AD treatment, inter alia, due to the lack of selectivity for the M1
mAChR, narrow safety margin, very low bioavailability and poor
tolerability.6,7
Activation of M1 mAChR by agonist modulates not only learning
and memory;4,8 but also protects cells from apoptotic effects in-
duced by H2O2 including DNA damage, oxidative stress and mito-
chondrial impairment in cortical neurons.9 M1 agonist also
blocks caspase activation in neurons deprived of serum;10 in-
creases the non-amyloidogenic processing of the b-amyloid pre-
15
reduces the tau hyperphosphorylation;16,17 increases inhibitory
postsynaptic currents,18,19 etc. However, the mechanism by which
mAChR activation protects neurons from insults is not well
understood.
In this study we have derivatized Arecoline molecule to develop
new class of M1 receptor selective agonists for the possible treat-
ment of AD dementia. Arecoline, an alkaloid obtained from the be-
tel nut (Areca catechu), the fruit of a palm tree, has been used
previously as centrally active muscarinic agents.20 The lack of M1
selectivity and efficacy due to dose limiting side effects associated
with M2 and M3 muscarinic receptors subtype stimulation has
produced disappointing results.20 Replacement of the ester func-
tionality of arecoline with either the 3-alkoxy-1,2,5 thiadiazole21
or the 3-alkyl-1,2,4-oxadiazole,22 has produced very potent musca-
rinic agonists. However, the systematic removal of a heteroatom in
the 3-methyl-1,2,4-oxadiazole giving oxazoles and furans caused a
decrease in affinity for the agonist binding site. The two isomers, 2-
methyl-1,2,4-oxadiazole and 5-methyl-1,2,4-oxadiazole,22 also had
shown lower affinities for M1 muscarinic receptor [see the struc-
ture of Arecoline and its above mentioned derivatives, Fig. 1].
Previously we have reported, muscarinic M1 subtype selectivity
for arecoline thiazolidinone23 and for 2-(1-methyl-1,2,5,6-tetrahy-
dropyridin-3-yl)morpholine.24,25 In a continued effort to discover
* Corresponding author. Tel.: +91 821 2412191, +91 821 2419661 (O); fax: +91
821 2412191.
E-mail addresses: rangappaks@gmail.com, rangappaks@chemistry.uni-mysore.
ac.in (K.S. Rangappa).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.06.032

M. Malviya et al. / Bioorg. Med. Chem. 17 (2009) 5526–5534
5527
Table 1
N
S
O
O
N
In vitro affinity of N-aryl carboxamide substituted 3-morpholino arecoline derivatives
9(a–h) towards M1 receptor of male Wistar rat cortex synaptosomal membrane
N
R
N
OMe
O
R
N
Compounds
Ki (
lM)
IC50 (lM)
N
N
CH3
CH
CH3
Arecoline
3
9a
9b
9c
9d
9e
9f
9g
9h
19 3.22
0.42 0.06
1.3 0.08
Nil
76 5.51
1.6 0.13
3-alkoxy-1,2,5-thiadiazole
3-alkyl-1,2,4-oxadiazole
R
4
1.50
Nil
Nil
618 20.35
142 11.14
215 13.50
460 18.18
N
N
O
N
R
Nil
O
N
126 13.83
48 7.32
65 6.12
88 11.25
N
CH3
N
CH3
Arecoline
2-methyl-1,2,4-oxadiazole
5-methyl-1,2,4-oxadiazole
Figure 1. Arecoline and arecoline derivatives.
less toxic arecoline class of muscarinic agonists and to further im-
prove their selectivity and potency, here, we are reporting our find-
ings of N-aryl carboxamide substituted 3-morpholino arecoline 9(
a–h) derivatives (Scheme 1) along with their in vitro muscarinic
receptor 1 binding studies by using [3H]QNB with male Wistar
rat brain synaptosomal membrane; estimation of second messen-
ger Inositol (1,4,5)-trisphosphate (IP3) formation; and modulation
of APP secretion in rat brain cerebrocortical slices in response to
M1 receptor activation by synthesized molecules, and also
in vivo evaluation of memory and learning in male Wistar rats
(passive avoidance tasks, plus and Y maze studies), as another po-
tent M1 receptor agonist for the symptomatic treatment of Alzhei-
mer’s dementia.
2. Results and discussion
Structure–Activity Relationship (SAR) can be drawn from the
in vitro radioligand affinity assay, which was carried out to find
out the affinity of the synthesized N-aryl carboxamide substituted
3-morpholino arecoline 9(a–h) derivatives for the cortical M1
receptor of rat brain (Scheme 1, Table 1 and Fig. 2). The most po-
Figure 2. Displacement graphs of four most potent compounds 9b, 9c, 9a and 9g.
The displacement studies were done with 0.2 nM [3H]QNB and different concen-
tration of N-aryl carboxamide substituted 3-morpholino arecoline derivatives 9(a–
h). The mean values of % bound are plotted against log of displacer concentration.
IC50 and Ki values are obtained from LIGAND–DRUG programme.
tent compound among all tested derivatives, 9b (Ki = 0.42 lM), is
one with methyl group at the para position of the aryl group at-
tached to the carboxamide of morpholino arecoline. Substitution
of chlorine group (9c), at para position also showed good affinity
tuted chlorine (9e) at 2 and 5 positions and also tertiary butyl
substituted derivative (9d) at the para position of the aryl group at-
tached to carboxamide of morpholino arecolines, showed no affin-
ity for the M1 receptor in vitro.
towards the M1 receptor in vitro (Ki = 1.3
lM). However, disubsti-
Ph
O
O
O
Br
N
OH
HBr, Br2
NaBH4
MeOH
K2CO3, DMF
CH3COOH
N-benzylaminoethanol
N
1
N
3
N
2
Ph
N
Boc
N
Ph
N
OH
(a) 10%Pd-C
OH
O
O
HCOONH4, MeOH, reflux
70% H2SO4
Reflux
(a) CH3I, acetone
(b) NaBH4, MeOH
N
4
N
5
N
6
(b) (Boc)2O, THF, K2CO3
R
C
N
Boc
N
H
N
O
TEA
O
R-CO-Cl,
O
O
HCl, MeOH
N
N
N
MDC
CH3
CH3
7
CH3
8
9
Scheme 1.

5528
M. Malviya et al. / Bioorg. Med. Chem. 17 (2009) 5526–5534
On the other hand substitution of nitro group (NO2) at the dif-
ferent positions of the aryl group attached to the carboxamide of
morpholino arecolines 9(f–h), reduced the affinity and potency of
the compounds for the M1 receptor. Among the derivatives having
electron withdrawing NO2 group at the aryl group, NO2 at meta po-
sition 9g (Ki = 48
compared it at para position 9h (Ki = 65
position 9f (Ki = 126 M) showed least affinity. Compound 9a hav-
l
M), showed considerable high affinity when
lM), while NO2 at ortho
l
ing phenyl group without any substitution on it, attached to the
carboxamide of morpholino arecoline showed moderate affinity
(Ki = 19 lM) to the muscarinic receptor 1 in vitro.
Muscarinic acetylcholine receptors (mAChR) play an important
role, among several events, in b-amyloid precursor protein (APP)
processing, as it was demonstrated in various cell lines transfected
with M1 and M3 receptors.26,27,15 Elevation of cytoplasmic calcium
and activation of protein kinase C, in these cells, play a role in mus-
carinic-mediated APP secretion,28 and it was also demonstrated
that phospholipase C (PLC), which activates phosphoinositide (PI)
hydrolysis, is involved in transduction signaling.29,30 Therefore,
we planned to measure the efficacy and potency of our synthesized
compounds 9a, 9b, 9c and 9g, which had shown high affinity for
the M1 receptor in radioligand binding studies, to elevate the IP3
formation in rat cerebrocortical slices and also to examine the
modulation of APP secretion by activating M1 receptor with these
compounds in rat brain cerebrocortical slices. Basal IP3 levels were
found to be 0.64 pmol/mg proteins, and it was significantly in-
creased by activating the M1 receptor with the compound 9b.
Compounds 9c and 9a could also elevate the IP3 formation when
it was compared with basal IP3 levels, but values were not signif-
icant (Table 2). The potency of these compounds to elevate the ba-
sal IP3 levels was compared with IP3 levels elevated by
acetylcholine iodide, which represents M1 receptor-linked PLC
activity (Fig. 3).
Figure 3. Inositol (1,4,5)-trisphosphate (IP3) levels were estimated using [3H]myo-
inositol, in rat brain cerebrocortical slices after stimulating the M1 receptor by
different concentration of N-aryl carboxamide substituted 3-morpholino arecoline
derivatives 9(a–h) and significance was compared with the basal IP3 level, and their
potency to elevate the IP3 formation were determined by comparing it with IP3
levels elevated by acetylcholine iodide as described in experimental section. This
figure represent scatchard graph of three most potent compounds (9b, 9c and 9a)
among all the tested derivatives 9(a–h). Data points are the means of three
experiments, each assayed in duplicate.
ulate the APP secretion by activating M1 receptor, as they already
had shown high affinity for M1 receptor in binding studies and also
have elevated IP3. The secretion of APP from the rat cerebrocortical
sliceswassignificantlyincreasedbymolecule9b (Fig. 4), whereas9a,
9c and 9g were not very effective in elevating the APP levels. The
inductionof APP released by 9b was inhibited by atropine, indicating
that the effect is mediated via the activation of mAChRs. These re-
sults show similarity to the neural cells that express M1 and M3
receptors which also respond to muscarinic activation by increasing
APP secretion.
The foresaid in vitro M1 receptor binding studies and subse-
quently IP3 formation studies and modulation of APP secretion
by activating M1 receptor, formed a basis for extending correlation
further to in vivo pharmacological studies to ascertain applicability
of the synthesized molecules 9(a–h) in scopolamine (muscarinic
antagonist) induced dementia models, using memory and learning
experiments (passive avoidance tasks, plus and Y maze studies), as
it has been demonstrated previously that cholinergic transmission
appears to be essential for learning and memory process.31,32 In
accordance with the degree of affinity and potency of synthesized
compounds 9(a–h) in vitro binding experiments, elicited almost
anticipated level of pharmacological actions in reversing scopol-
amine induced dementia in vivo (Table 3 and Fig. 5). Two synthe-
sized morpholino arecoline derivatives 9b and 9c are best among
all the derivatives 9(a–h) in reversing scopolamine-induced
dementia by making rats to commit less number of mistakes (No.
of mistakes done 11 and 15, respectively), when compared it with
the number of mistakes done by control rats (6 mistakes) and sco-
polamine-treated group (30 mistakes). Compound 9g also signifi-
cantly reversed the scopolamine induced memory loss. Other
compounds showed least or no activity against scopolamine in-
duced memory loss in rats (Table 3).
In vitro modulation of APP secretion in rat brain cerebrocortical
slices by activating M1 receptor with our synthesized M1 receptor
agonists were studied. Secreted APP from cerebrocortical slices ap-
peared as two protein bands with an apparent molecular weight of
105,000 (major band) and 82,000 (minor band). These APP bands
may correspond to secreted forms of APP751 and APP695.
11 Alterna-
tively, they may correspond to mature and immature (non-glycosyl-
ated) forms and activation of M1 receptor by our synthesized
molecules evoked APP release in rat brain slices. We used novel com-
pounds 9a, 9b, 9c and 9g, to study their efficacy and potency to mod-
Table 2
In vitro effect of N-aryl carboxamide substituted 3-morpholino arecoline derivatives
9(a–h) on the formation of Inositol (1,4,5)-trisphosphate (IP3) levels in the cerebro-
cortical slices of the rat brain
Agonists stimulation of M1 receptor
(100 V)
IP3 Concentration (pmol/mg of
protein)
l
Control (Basal)
9a-Stimulated
9b-Stimulated
9c-Stimulated
9d-Stimulated
9e-Stimulated
9f-Stimulated
9g-Stimulated
9h-Stimulated
Acetylcholine-stimulated
0.64 0.12
0.82 0.25
1.19 0.33*
0.94 0.31
0.63 0.17
0.64 0.30
0.65 0.17
0.80 0.21
0.64 0.20
1.60 0.09*
The efficacy of novel N-aryl carboxamide substituted 3-morpholino arecoline 9(a–
h) derivatives (100 mol/l) for the M1 receptor was measured by their potency to
elevate the basal IP3 levels, and it was compared with IP3 levels elevated by ace-
tylcholine iodide (20 mol/l), which represent M1 receptor-linked PLC activity. The
l
The plus maze experiments for synthesized derivatives 9(a–h)
measures the transfer latency (TL) in seconds taken to reach from
one extreme end of open arm to one of the closed arms in plus
maze. Difference in TL in seconds between 1st day and 2nd day
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 < 0.05.

M. Malviya et al. / Bioorg. Med. Chem. 17 (2009) 5526–5534
5529
TL2) were 18 s and 17 s, respectively, implying that these deriva-
tives failed to reverse acute memory and learning impairment in
male Wistar rats. TL for remaining compounds are given in Table
4. Similar results were obtained from Y maze experiment (data
not shown).
A
Cont.
10
50
100
500 1000 500+At (μM)
117 -
81 -
The finding of this study is somewhat similar to our previous
finding where arylthioureas are attached to the morpholino areco-
line.25 In both the studies, it is the position of the substitutes on the
aryl group which determines the affinity and potency of the mole-
cules for the M1 receptor. Substitution on the para position of the
aryl group attached to either amide or thiourea of morpholino
arecoline gives better affinity for the M1 receptor, when compared
it at meta or at ortho positions. Electron donating group (e.g.,
methyl, 9b) at the para position of aryl group attached to the car-
boxamide of morpholino arecoline has shown more affinity when
compared with the electron withdrawing group (e.g., chloro, 9c)
at the para position. In contrary to this, in N-arylthioureas substi-
tuted morpholino arecoline derivatives electron withdrawing
group (e.g., chloro) at the para position has shown better affinity
when compared it with electron donating group (e.g., methoxy)
at the para position.25 However when aryl group is replaced with
either alkyl group or with amino acids, it decreases the affinity
and potency of the molecules for the M1 receptor (ongoing work
in our laboratory).
β Actin
B
150
100
50
*
*
*
*
0
9b (μM) 10
50
100
500
1000 500+ At
Figure 4. (A) M1 mAChR activation by 9b induces APP secretion from rat
cerebrocortical slices. Protein extracts from rat brain slices stimulated with
different concentration of 9b, were prepared and electrophoresed on a PAGE gel.
Following western blotting, immunodetection with specific antibodies was carried
out to detect the levels of secreted APP on the blots. The last lane demonstrates the
3. Conclusion
In light of these findings, in vitro radioligand competitive bind-
ing assay for the M1 receptor; stimulation of IP3 formation by acti-
vating M1 receptor; modulation of APP secretion in rat brain
cerebrocortical slices and in vivo pharmacological experiments
for the synthesized compounds N-aryl carboxamide substituted
3-morpholino arecoline derivatives 9(a–h), to test the relation be-
tween affinity of these molecules to M1 receptor and their potency
to modulate the APP secretion and also their ability to reverse sco-
polamine-induced memory loss and learning impairment, for
ascertaining their applicability in Alzheimer’s dementia. We can
conclude that position of substituents on the aryl group attached
to the carboxamide of morpholino arecoline derivatives deter-
mines the affinity of the compounds for the M1 receptor. Substitu-
tion at the para position increases the affinity and potency of the
molecules for the M1 receptor. The compound 9a which dose not
having any substituted group on the aryl group is found to have
moderate affinity for the M1 receptor. But, when either methyl
(9b) or chlorine (9c) group is introduced at the para position, it in-
creases the affinity of the compounds several folds for the M1
receptor in vitro and showed greater potency in elevating the IP3
levels in rat cerebrocortical slices, and also have shown useful
blockade of 9b (500 lM) induced APP secretion by 10 lM atropine (At). (B) The
intensity of bands, specific for the secreted APP protein was measured densito-
metrically. The values were graphed as mean and standard error values of six
experiments each performed in duplicates for the 9b treated rat brain slices, and it
was compared to not treated control rat brain cerebrocortical slices in terms of fold
increase or decrease. *Indicates measurements significantly different to non-treated
control APP levels (*p < 0.05).
for scopolamine-treated group, and test compound along with sco-
polamine-treated groups were compared to evaluate learning (TL1)
and memory (TL2). Derivatives 9b and 9c significantly reversed
acute memory loss and learning impairment in male Wistar rats
(Table 4 and Fig. 6). Compound 9b produced lesser TL for 1st day
(TL1 = 33 s) compared to scopolamine-treated group (TL1 = 66 s),
but on 2nd day (TL2 = 13 s) TL was lesser than 1st day (9b) indicat-
ing how it is helpful in reversing learning impairment as compared
to rest of the derivatives. Overall difference between TL1 and TL2
were same for 9b, 9c (20 s), whereas for the control group it was
22 s. In contrary this, 9a and 9g molecules produced longer 1st
day TL (TL1 = 53 s and 64 s, respectively) and 2nd day TL
(TL2 = 35 s and 47 s, respectively) overall difference differs (TL1–
Table 3
Study of antiamnesic effect of N-aryl carboxamide substituted 3-morpholino arecoline derivatives 9(a–h) against scopolamine induced memory loss
S. No.
Experimental groups
Treatment (dose) mg/kg ip
Basal latency (s) of rat to reach shock free zone
Memory parameters
Latency (s) Mistakes
I
II
III
1
2
3
4
5
6
7
8
9
Control group*
Scopolamine treated group
Scop. + 9a
Saline (0.9%)
0.5
18
38
29
23
22
40
38
35
28
35
30
9
5
2
7
6
4
3
6
9
5
4
5
5
6
30
25
11
15
2
12
12
10
11
15
13
12
13
15
13
10
10
8
9
7
6
6
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
Scop. + 9b*
Scop. + 9c*
Scop. + 9d
Scop. + 9e
7
11
11
10
10
10
10
30 10
31
30
20
28
23
8
9
7
8
6
Scop. + 9f
Scop. + 9g*
10
11
Scop. + 9h
Scop. + Arecoline
Results are expressed as mean ( SEM), n = 10, *P < 0.05. (Scop. = Scopolamine).

5530
M. Malviya et al. / Bioorg. Med. Chem. 17 (2009) 5526–5534
Figure 5. Antiamnesic effect of N-aryl carboxamide substituted 3-morpholino arecoline derivatives 9(a–h) against scopolamine induced memory loss in male Wistar rats
(*p < 0.05), mean ( SEM).
Table 4
Acute studies of N-aryl carboxamide substituted 3-morpholino arecoline derivatives 9(a–h) on learning and memory against scopolamine-induced memory loss and learning
impairment on elevated plus maze in male Wistar rats
S. No.
Experimental groups
Treatment (dose) (mg/kg, ip)
First day transfer latency (TL1) in s
Second day transfer latency (TL2) in s
TL1–TL2
1
2
3
4
5
6
7
8
9
Control group*
Scopolamine treated group
Scop. + 9a
Saline (0.9%)
0.5
31
66
53
33
37
65
66
66
64
64
52
9
22 8.5
13 5.6
53
35
13
17
53
53
52
47
52
36
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
0.5 + 0.5
18
6
Scop. + 9b*
20 5.2
20 7.5
12 5.8
13 4.5
14 5.6
17 6.2
12 5.5
16 5.5
Scop. + 9c*
Scop. + 9d
Scop. + 9e
Scop. + 9f
Scop. + 9g
Scop. + 9h
Scop. + Arecoline
10
11
Results are expressed as mean ( SEM), n = 10, *P < 0.05 (Scop. = Scopolamine).
Figure 6. Effect of N-aryl carboxamide substituted 3-morpholino arecoline derivatives 9(a–h) on memory and learning against scopolamine induced memory loss and
learning impairment on elevated plus maze in male Wistar rats (*p < 0.05), mean ( SEM).

M. Malviya et al. / Bioorg. Med. Chem. 17 (2009) 5526–5534
5531
antidementia activity in vivo model tested. However, only 9b could
modulate the secretion of APP by activating M1 receptor in rat
cerebrocortical slices, which might shift the proteolytic processing
of APP towards the formation of non-amyloidogenic products and
might reduces the production of Ab. Consistent with our findings
WAL 2014, a predominantly M1 agonist, induced APP secretion
in cerebrocortical slices.33 However, when the aryl group was
disubstituted at 2 and 5 positions with chloro group (9e) or when
tert-butyl group (9d) is present at the para position, it showed nil
affinity for the M1 receptor. This may be due to steric hindrance.
Substitution of aryl group with nitro group once again decreased
the affinity of molecules for the M1 receptor. Among the molecules
substituted with nitro group, 9g with nitro group at meta position
showed good affinity, whereas nitro at ortho (9f) and at para (9h)
positions showed least affinity for M1 receptor in vitro and average
potency in reversing scopolamine induced memory loss in vivo. This
might be due to electron withdrawing nature of NO2 at ortho and at
para positions of the aryl group or substitutionat these two positions
might hinder the bindingof molecules at binding site of M1 receptor.
However, NO2 at meta position showed good affinity and potency
compared to NO2 at ortho and para positions, this may be due to less
electronwithdrawingnatureof NO2 at themetapositionorfitsbetter
at the binding site. These N-aryl carboxamide substituted 3-mor-
pholino arecoline 9(a–h) derivatives showed no visible cholinergic
toxicity at the dose tested (salivation, defecation, etc). One of the
promising new derivatives (9b) would emerge as the potent mole-
cule having antidementia activity.
(TMS) as an internal standard. Mass spectra were obtained on
LCMSD-Trap-XCT instrument. Elemental analysis were performed
on a variol EL, III Elementar C, H, N analyzer and values were within
the acceptable limits of the calculated values. The progress of the
reaction was monitored on pre coated silica gel plates (Merck) using
chloroform/methanol (9:1) as a solvent system. Spectral data (IR,
NMR and mass spectra) confirmed the structures of the synthesized
compounds. Elemental(C, H and N) analysisindicated thatthe calcu-
lated and observed values were within the acceptable limits ( 0.4%).
4.1.2. General procedure for the synthesis of compounds 9(a–h)
The intermediate compound 2-(1-methyl-1,2,5,6-tetrahydro-
pyridin-3-yl)morpholine (compound 8) was synthesized as sum-
marized in the Scheme 1 as per reported procedure (Kumar et al.,
2007). To a stirred solution of compound 8 in dichloromethane, tri-
ethylamine (5.0 equiv) was added and cooled to 0–5 °C, then acid
chloride (1.0 equiv) was added drop wise. The reaction mixture
was stirred for 4–5 h at room temperature (completion of reaction
was confirmed by TLC) then reaction mixture was washed with
water, followed by saturated NaCl solution and dried over sodium
sulfate. Dichloromethane was evaporated under reduced pressure
and the crude residue obtained was purified by column chroma-
tography using chloroform–methanol (9:1) as an eluent.
R=
CH3
9a =
9b =
9f =
9c =
9g =
CH3
Cl
NO2
C
CH3
9d =
9h =
CH3
Cl
9e =
4. Experimental
4.1. Chemistry
NO2
Cl
O2N
4.1.1. General
4.1.3. (2-(1,2,5,6-Tetrahydro-1-methylpyridin-3-yl)morpho-
lino)(phenyl)methanone (9a)
The synthetic route utilized for the preparation of the com-
pounds is shown in Scheme 1. The morpholino arecoline com-
pounds 9(a–h) were synthesized in total nine steps. Bromination
of 3-acetylpyridine 1 with Br2/HBr in glacial acetic acid gave the
HBr salt of bromoacetylpyridine 2. This was converted to amino
alcohol 3, by reaction with N-benzylaminoethanol in DMF in the
presence of K2CO3. The keto group of compound 3 was reduced
with NaBH4 in methanol to offer the dihydroxy compound 4. Treat-
ment of compound 4 with 70% H2SO4 under reflux conditions
caused dehydration to yield the cyclized product 5. The N-benzyl
group was removed by refluxing amine 5 in methanol in the pres-
ence of 10% Pd–C and ammonium formate. 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 hydroiodide salt by reaction
with methyl iodide in acetone. This on treatment 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 detail procedure for the synthesis of inter-
mediate compound 8 has been previously reported from our labo-
Compound 9a was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with benzoyl chloride (0.164 g, 0.0011 mol) and tri-
ethylamine (0.393 g, 0.0039 mol) in dichloromethane (3 ml). Yield:
91%; IR (Nujol, cmÀ1): 1670 (C@O), 1675 (–RC@CH–); 1H NMR
(CDCl3): d 7.62–7.51 (m, 5H), 5.76 (br s, 1H, –C@C–), 3.85–3.79
(m, 3H), 3.49–3.47 (m, 1H), 2.78–2.75 (m, 3H), 2.77–2.75 (m,
2H), 2.54–2.48 (m, 2H), 2.23 (s, 3H), 2.09 (m, 2H). MS m/z: 387.4
(M+). Anal. Calcd for C17H22N2O2: C, 71.30; H, 7.74; N, 9.78. Found:
C, 71.13; H, 7.98; N, 9.51.
4.1.4. (2-(1,2,5,6-Tetrahydro-1-methylpyridin-3-yl)-morpho-
lino)(p-tolyl)methanone (9b)
Compound 9b was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 4-methylbenzoyl chloride (0.18 g, 0.0011 mol)
and triethylamine (0.393 g, 0.0039 mol) in dichloromethane
(3 ml). Yield: 85%; IR (Nujol, cmÀ1): 1670 (C@O), 1675 (–RC@CH–
); 1H NMR (CDCl3): d 7.665–7.640 (d, 2H, J = 8.9 Hz), 7.360–7.338
(d, 2H, J = 8.80 Hz), 5.76 (br s, 1H, –C@C–), 3.85–3.72 (m, 3H),
3.50–3.45 (m, 1H), 2.91–2.89 (m, 3H), 2.79–2.76 (m, 2H), 2.52–
2.43 (m, 2H), 2.22 (s, 3H), 2.13 (s, 3H), 2.02 (m, 2H). MS m/z:
301.4 (M+); Anal. Calcd for C18H24N2O2 : C, 71.97; H, 8.05; N,
9.33. Found: C, 72.01; H, 8.03; N, 9.45.
ratory.24 Compound
8 on reacting with the respective acid
chlorides gave carboxamide compounds 9(a–h). 1H NMR spectra
of all compounds 9(a–h) showed a multiplet at 8–7 due to aro-
matic protons and 5.7–5.8 due to alkene of tetrahydro pyridine.
All the synthesized compounds were characterized by IR, 1H
NMR, mass spectroscopy and CHNS analysis.
4.1.5. (4-Chlorophenyl) (2-(1,2,5,6-tetrahydro-1-methylpyridin-
3-yl)-morpholino)methanone (9c)
All chemicals and reagents were obtained from Aldrich (USA),
Spectrochem Pvt. Ltd (India) and Rankem Pvt. Ltd (India) and were
used without further purification. The I.R. spectra were recorded
using Nujol on JASCO-FTTR, 41007 series. The 1H spectra were re-
corded on 400 MHz Bruker FT-NMR Spectrometer. The chemical
shifts were reported as parts per million (d ppm) tetramethyl silane
Compound 9c was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 4-chloro-benzoyl chloride (0.2 g, 0.0011 mol)
and triethylamine (0.393 g, 0.0039 mol) in dichloromethane
(3 ml). Yield: 90%; IR (Nujol, cmÀ1): 1675 (C@O), 1680 (–RC@CH–
); 1H NMR (CDCl3): d 7.91–7.88 (m, 2H), 7.50 (m, 2H), 5.76 (br s,

5532
M. Malviya et al. / Bioorg. Med. Chem. 17 (2009) 5526–5534
1H, –C@C–), 3.83–3.79 (m,3H), 3.49–3.46 (m, 1H), 2.78–2.75 (m,
3H), 2.76–2.73 (m, 2H), 2.54–2.48 (m, 2H), 2.23 (s, 3H), 2.09 (m,
2H). MS m/z: 321.8 (M+); Anal. Calcd for C17H21ClN2O2 : C, 63.64;
H, 6.60; N, 8.73. Found: C, 64.15; H, 6.39; N, 8.55.
2H), 2.24 (s, 3H), 2.07 (m, 2H). MS m/z: 332.3 (M+); Anal. Calcd for
C17H21N3O4: C, 61.62; H, 6.39; N, 12.68. Found: C, 61.3; H, 6.78; N,
12.11.
4.2. Biology
4.1.6. (4-tert-Butylphenyl)(2-(1,2,5,6-tetrahydro-1-methylpyri-
din-3-yl)-morpholino)methanone (9d)
4.2.1. Displacement study
Compound 9d was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 4-tert-butylbenzoyl chloride (0.230 g,
0.0011 mol) and triethylamine (0.393 g, 0.0039 mol) in dichloro-
methane (3 ml). Yield: 87%; IR (Nujol, cmÀ1): 1673 (C@O), 1678
(–RC@CH–); 1H NMR (CDCl3): d 7.895–7.873 (d, 2H, J = 8.77 Hz),
7.240–7.219 (d, 2H, J = 8.73 Hz), 5.76 (br s, 1H, –C@C–), 3.83–
3.76 (m, 3H), 3.49–3.44 (m, 1H), 2.89–2.85 (m, 3H), 2.77–2.75
(m, 2H), 2.54–2.48 (m, 2H), 2.24 (s, 3H), 2.08 (m, 2H), 1.02 (s,
9H). MS m/z: 343.5 (M+); Anal. Calcd for C21H30N2O2: C, 73.65, H,
8.83, N, 8.18. Found: C, 73.83; H, 8.99; N, 8.35.
The competitive inhibition study was done using N-aryl carbox-
amidesubstituted3-morpholino arecolinederivatives 9(a–h)tofind
out their affinity towards cortical M1 receptor. Male Wistar rat brain
cortex was 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.34 The tissue homogenate was centrifuged at a speed of
1000g for 10 min at 4 °C, to remove cellular debris. The supernatant
obtained was centrifuged at 32,000g for 20 min at 4 °C. Pellet ob-
tained was resuspended in 50 mmol/l phosphate assay buffer (pH
7.4) containing 1 mmol MgCl2. The protein concentration was esti-
mated by method described by Lowry et al.35
4.1.7. (2,4-Dichlorophenyl)(2-(1,2,5,6-tetrahydro-1-methylpyri-
din-3-yl)-morpholino)methanone (9e)
Compound 9e was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 2,5-dichlorobenzoyl chloride (0.244 g,
0.0011 mol) and triethylamine (0.393 g, 0.0039 mol) in dichloro-
methane (3 ml). Yield: 88%; IR (Nujol, cmÀ1): 1673 (C@O), 1680
(–RC@CH–); 1H NMR (CDCl3): d 7.896–7.889 (d, 1H, J = 2.0 Hz),
7.720–7.699 (d, 2H, J = 8.12 Hz), 5.78 (br s, 1H, –C@C–), 3.83–
3.72 (m, 3H), 3.49–3.44 (m, 1H), 2.78–2.76 (m, 3H), 2.79–2.76
(m, 2H), 2.54–2.43 (m, 2H), 2.22 (s, 3H), 2.02 (m, 2H). MS m/z:
356.3 (M+); Anal. Calcd for C17H20Cl2N2O2 : C, 57.47; H, 5.67; N,
7.89. Found: C, 57.62; H, 5.72; N, 7.67.
The affinity of various compounds towards M1 receptor were
estimated by using [3H]QNB (0.2 nM, specific activity 48 Ci/mmol,
Amersham, Little Chalfont, Bucks, UK) essentially following the
procedure described by Hyttel et al.36 and Yamamura and Sny-
der,37 with slight modification. In brief an aliquot of synaptosomal
membrane proteins (50
tration of compounds (0.1
(0.2 nM) and reaction volume was made up to 200
l
g) was incubated with different concen-
M–1 mM) as a displacer and [3H]QNB
l with assay
l
l
buffer and incubated for 2 h at 37 °C. The reaction for all displace-
ment assay was stopped by adding ice-cold assay buffer and reac-
tion mixtures were rapidly filtered through GF/B filters under
vacuum. The filters were transferred to vials containing scintilla-
tion fluid, (5 ml) and allowed to equilibrate overnight. Radioactiv-
ity was measured in a liquid scintillation counter (Tris-Carb
2100TR, Packard, US) at 65% efficiency. The data from displacement
were analyzed and IC50 and Ki values are obtained from LIGAND–DRUG
programme.38 The mean values of % bound are plotted against log
of displacer concentration.
4.1.8. (2-(1,2,5,6-Tetrahydro-1-methylpyridin-3-yl)morpho-
lino)(2-nitrophenyl)methanone (9f)
Compound 9f was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 2-nitro benzoyl chloride (0.217 g, 0.0011 mol)
and triethylamine (0.393 g, 0.0039 mol) in dichloromethane
(3 ml). Yield: 90%; IR (Nujol, cmÀ1): 1670 (C@O), 1675 (–RC@CH–);
1H NMR (CDCl3): d 8.364–8.343 (d, 1H, J = 8.8 Hz), 8.125–8.102 (d,
1H, J = 8.4 Hz), 7.87–7.85 (m, 2H), 5.78 (br s, 1H,–C@C–), 3.83–3.72
(m, 3H), 3.49–3.44 (m, 1H), 2.79–2.76 (m, 2H), 2.78–2.76 (m, 3H),
2.54–2.43 (m, 2H), 2.19 (s, 3H), 2.02 (m, 2H). MS m/z: 332.4 (M+);
Anal. Calcd for C17H21N3O4: C, 61.62; H, 6.39; N, 12.68. Found: C,
61.46; H, 6.45; N, 12.55.
4.2.2. IP3 levels estimation
Inositol (1,4,5)-trisphosphate (IP3) levels were estimated using
[3H]myoinositol (s.a. 16.0 Ci/mmol, Amersham) in rat cerebral cor-
tex slices, at basal level and also after stimulation by different con-
centrations of synthesized N-aryl carboxamide substituted 3-
4.1.9. (2-(1,2,5,6-Tetrahydro-1-methylpyridin-3-yl)morpho-
lino)(3-nitrophenyl)methanone (9g)
morpholino arecoline derivatives 9(a–h) (1 lM–1 mM), to find
out their efficacy and potency to elevate the IP3 formation, by com-
paring it with basal IP3 levels and IP3 formation elevated by acetyl-
choline iodide, following the procedure described by Kendall and
Naharoski;39 Van Rooijen et al.,40 and Gonzales et al.,41 with slight
Compound 9g was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 3-nitro benzoyl chloride (0.217 g, 0.0011 mol)
and triethylamine (0.393 g, 0.0039 mol) in dichloromethane
(3 ml). Yield: 85%; IR (Nujol, cmÀ1): 1670 (C@O), 1675 (–RC@CH–
); 1H NMR (CDCl3): d 8.850–8.845 (d, 1H, J = 1.2 Hz), 8.30–8.22
(m, 2H), 7.23 (m, 1H), 5.78 (br s, 1H, –C@C–), 3.85–3.81 (m, 3H),
3.49–3.44 (m, 1H), 2.79–2.76 (m, 2H), 2.78–2.76 (m, 3H), 2.55–
2.45 (m, 2H), 2.22 (s, 3H), 2.02 (m, 2H). MS m/z: 332.3 (M+); Anal.
Calcd for C17H21N3O4: C, 61.62; H, 6.39; N, 12.68. Found: C, 62.05;
H, 5.99; N, 12.25.
modification. In brief: Cross chopped (300 Â 300
lm) rat brain
cerebrocortical slices (0.5 g) were prepared and incubated in oxy-
genated Kreb’s Ringer bicarbonate (KRB) buffer (pH 7.4) with
0.5 l
Ci [3H]myoinositol for 1 h at 37 °C. Tissues were washed with
the same buffer and incubated with LiCl (10 mM). Assay was ter-
minated by the addition of 10% trichloracetic acid (TCA). Samples
were kept on ice for 20 min and homogenized and sedimented
by centrifugation at 3000g. Supernatant was washed 4–5 times
with water saturated diethyl ether and neutralized with NaHCO3
(5 mM). Dowex-1-chloride (AG-X8: 200-400 mesh size) resin was
used for separation of [3H]IP3 form the reaction mixture by an-
ion-exchange chromatography. The bound [3H] IP3 was eluted
from the column using 0.8 M formate and 0.1 M formic acid. The
radioactivity of [3H]IP3 formed was measured in a liquid scintilla-
tion counter at 65% efficiency. The levels of IP3 were expressed as
picomoles of [3H]IP3 formed per milligram of protein.
4.1.10. (2-(1,2,5,6-Tetrahydro-1-methylpyridin-3-yl)morpho-
lino)(4-nitrophenyl)methanone (9h)
Compound 9h was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 4-nitro benzoyl chloride (0.217 g, 0.0011 mol)
and triethylamine (0.393 g, 0.0039 mol) in dichloromethane
(3 ml). Yield: 95%; IR (Nujol, cmÀ1): 1670 (C@O), 1675 (–RC@CH–);
1H NMR (CDCl3): d 8.324–8.299 (d, 2H, J = 9.2 Hz), 7.931–7.908 (d,
2H, J = 9.16 Hz), 5.76 (br s, 1H, –C@C–), 3.83–3.76 (m, 3H), 3.49–
3.44 (m, 1H), 2.77–2.75 (m, 3H), 2.79–2.76 (m, 2H), 2.54–2.48 (m,

M. Malviya et al. / Bioorg. Med. Chem. 17 (2009) 5526–5534
5533
4.2.3. Modulation of APP Secretion in rat brain cerebrocortical
slices
4.2.7. Dose–response curve
Different doses (0.05–2 mg/kg of the body wt.) of the deriva-
tives were selected to find out optimum dose (found to be
0.5 mg/kg) for in vivo studies.
Rat cerebral cortex was dissected out and minced in two per-
pendicular directions by Mcllwain tissue chopper (300 Â 300
lm)
and rinsed three times with oxygenated (95% O2/5% CO2) Krebs
Ringer Bicarbonate buffer (pH 7.4) consisting of (120 mM NaCl;
4.65 mM KCl; 1.2 mM MgSO4; 1.2 mM KH PO4; 11.2 mM glucose;
25 mM NaHCO3; 2.5 mM CaCl2). Washed slices were equilibrated
for 50 min at 37 °C under oxygen atmosphere. The buffer was then
5. Data analysis
The data from the displacement assay was analyzed using ‘LI-
GAND–DRUG’ software programme38 to obtain the IC50 and Ki values
changed to a fresh Krebs buffer containing 50
albumin and a cocktail of protease inhibitors (0.1 mM phenylmeth-
ylsulfonyl fluoride, 5 g/ml leupeptin, 5 g/ml pepstatin and 5
lg/ml bovine serum
(both are expressed in
lmol). All the data are expressed as
mean SD. The statistical analysis was done by using student’s t-
test. Differences were considered to be significant at P < 0.05. All
analysis were performed with the ‘JANDEL-SCIENTIFIC-SIGMA STAT’ soft-
ware, version 2.0 for windows.
l
l
units/ml aprotinin. Aliquots of the slices were transferred to Ep-
pendorff tubes (50 mg slices/0.5 ml) and stimulated for 1 h at
37 °C with the tested compounds while oxygenating and gently
stirring the slices every few minute. At the end of the incubation
period, the medium was collected for secreted APP determinations.
Protein content of the collected medium was measured by
Acknowledgements
Manish Malviya is grateful to Dr. Srinivas Bharath M.M., Depart-
ment of Neurochemistry, NIMHANS, Bangalore, India, for kindly
providing him the lab facilities to carry out radioligand binding
experiments. Manish Malviya is also grateful to the Indian Council
of Medical Research (Ref. No. 45/51/2007/Pha/BMS) for providing
him the Senior Research 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 Pro-
ject 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.
method described by Lowery et al. (1953), and loaded (50 lg/lane)
on 10% SDS–PAGE (BioRad). The gel was run at 35 mA, 100 W for
2 h. Proteins were then transferred on to a nitrocellulose mem-
brane (Millipore) using a semidry transfer unit (BioRad). Mem-
brane was blocked in 5% non-fat milk powder in PBS-T (0.1%
Tween 20 in PBS). The membrane was incubated with primary
monoclonal antibody 22C11 (anti-Alzheimer precursor protein
A4; Chemicon/Millipore) of the appropriate concentration for 2 h.
The membrane was then washed 3 times for 10 min each on a
rocker with Phosphate Buffer Saline (PBS), incubated with an
appropriate horse radish peroxidase conjugated secondary anti-
body for 1 h and then sequentially washed 2 Â 2 min, 1 Â 15 min
and finally 3 Â 5 min with PBS-T. For APP detection, the Renais-
sance Chemiluminescence Reagent (PerkinElmer Life Sciences)
was employed followed by exposure to an autoradiography film
(Hyperfilm-ECL; Amersham). Signal intensities were quantified
using SCION IMAGE software.
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4.2.5. Elevated plus maze
The elevated plus maze experiments were done to elucidate the
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against scopolamine induced memory loss in rats by following
the procedure described by Itoh et al.44 and also by Sharma and
Kulkarni45 with slight modification. In brief: This was employed
for the measurement of transfer latency (TL). The male Wistar rats
(weighing 200–250 g, n = 10) were selected, grouped and were
administered scopolamine and test compound along with scopol-
amine to respective animal groups. Thirty minutes later they were
placed individually at the end of one arm facing away from the
central platform and the time they take 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. Control
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was measured in the similar way on the same animals. The resul-
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Rats (6 per group), which had fasted 16 h, were treated orally
with various doses of the compounds and observed for 1 week
after treatment, deaths were recorded daily. None of the rats died
within one week after administration under test dose.

5534
M. Malviya et al. / Bioorg. Med. Chem. 17 (2009) 5526–5534
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