Effect of novel N-aryl sulfonamide substituted 3-morpholino arecoline derivatives as muscarinic receptor 1 agonists in Alzheimer's dementia models

Article abstract of DOI:10.1016/j.bmc.2008.03.019

A series of novel, potent, and selective muscarinic receptor 1 agonists (M1 receptor agonists) that employ a key N-substituted morpholine Arecoline moiety has been synthesized as part of research effort for the therapy of Alzheimer's diseases. The ester group of arecoline (which is reported as muscarinic agonist) has been replaced by N-substituted morpholine ring. The structure-activity relationship reveals that the electron donating 4-substituted sulfonyl derivatives (9a, 9b, 9c, and 9e) on the nitrogen atom of the morpholine ring increases the affinity of M1 receptor binding 50- to 80-fold greater than the corresponding arecoline. Other derivatives also showed considerable M1 receptor binding affinity.

Full text of DOI:10.1016/j.bmc.2008.03.019

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5157–5163
Effect of novel N-aryl sulfonamide substituted 3-morpholino
arecoline derivatives as muscarinic receptor 1 agonists
in Alzheimer’s dementia models
Y. C. Sunil Kumar,a Manish Malviya,a J. N. Narendra Sharath Chandra,a
C. T. Sadashiva,a C. S. Ananda Kumar,a S. B. Benaka Prasad,a D. S. Prasanna,a
M. N. Subhashb and K. S. Rangappaa,*
aDepartment of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
bDepartment of Neurochemistry, National Institute of Mental Health and Neurosciences, Bangalore 560029, India
Received 18 January 2008; revised 4 March 2008; accepted 5 March 2008
Available online 8 March 2008
Abstract—A series of novel, potent, and selective muscarinic receptor 1 agonists (M1 receptor agonists) that employ a key N-substi-
tuted morpholine Arecoline moiety has been synthesized as part of research effort for the therapy of Alzheimer’s diseases. The ester
group of arecoline (which is reported as muscarinic agonist) has been replaced by N-substituted morpholine ring. The structure–
activity relationship reveals that the electron donating 4-substituted sulfonyl derivatives (9a, 9b, 9c, and 9e) on the nitrogen atom
of the morpholine ring increases the affinity of M1 receptor binding 50- to 80-fold greater than the corresponding arecoline. Other
derivatives also showed considerable M1 receptor binding affinity.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
with some muscarinic agonists arecoline,3 oxotremorine,
RS 86,4 pilocarpine,5 and bathenechol showed results
that ranked from modest improvement to lack of bene-
ficial effects.6 It is thus, important to understand the
drawbacks of the tested muscarinic agonists in order
to design better drugs.
Senile dementia of the Alzheimer type (SDAT) is an age-
related neurodegenerative disorder, characterized by a
progressive deterioration of cognitive function with
impairments in faculty to use language, visuospatial
ability, and memory. In recent years, the total number
of individuals suffering from Alzheimer’s Diseases
(AD) is rapidly increasing as the percentage of elderly
population is increasing due to the improved health care
and quality of life. Although abnormalities in many neu-
rotransmitter systems have been documented in AD, the
most consistent deficit involves the cholinergic system.1
The cholinergic hypothesis of aging and of dementia
suggests that the loss of central forebrain cholinergic
neurons contributes to the decline in cognitive abilities
associated with AD.2 The presynaptic cholinergic defi-
cits in AD indicate that a cholinergic replacement ther-
apy might be beneficial in alleviating some of the
cognitive dysfunctions in this disorder. The clinical trials
Most of the potent muscarinic agonists evaluated in AD
patients show adverse central and peripheral side effects
and are neither non-selective nor M2 > M1 selective.
They activate the inhibitory M2 autoreceptors resulting
in decreased acetylcholine (ACh) release.6 M1-type
mAChRs are predominant in cerebral cortex and hippo-
campus and exhibited important roles in cognitive pro-
cesses relevant to AD.6 Hence the following probes for
mAChRs were suggested as a rational treatment strat-
egy in AD; (a) M1 agonists6,7 (b) M2 antagonists6; (c)
mixed M1 agonist and M2 antagonist in the same
compound.8
N-methyl tetrahydropyridine embodying the muscarinic
pharmacophore in a framework of semirigid template is
one such selective M1 agonist and at the same time is
flexible enough to bring about conformational change
in M1 receptor during its activation. The arecoline based
Keywords: Alzheimer’s diseases; M1 agonists; Morpholino arecolines;
Displacement assay; Rat brain; In vitro; In vivo.
*
Corresponding author. Tel./fax: +91 821 2412191; e-mail addresses:
rangappaks@gmail.com; rangappaks@chemistry.uni-mysore.ac.in
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.019

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Y. C. Sunil Kumar et al. / Bioorg. Med. Chem. 16 (2008) 5157–5163
muscarinic agonists have been found to overcome the
limited oral activity and short duration of action of clas-
sical agonists and they are relatively specific in their ac-
tion between central and peripheral effects. Arecoline
oximes and/or oxadiazoles,9 arecoline thiadiazoles, arec-
oline oxazoles, arecoline amides10 are few arecolines
with the above advantages. Many arecoline bioisosters
classes of M1 receptor agonists are still in clinical trials
and Alvameline, Milameline, sabcomeline, xanomeline,
Ly 593093, and YM 796 have been discontinued during
different phases of clinical trials. Most of the derivatives
were disappointing in clinical trials due to their low effi-
cacy and high cholinergic side effects (salivation, gastric
secretion, nausea and vomiting).
2. Chemistry
The synthetic route utilized for the preparation of the
compounds is shown in Scheme 1. The morpholino arec-
oline compounds 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 bromoacetyl-
pyridine 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 dihy-
droxy compound 4. Treatment of compound 4 with 70%
H2SO4 under reflux conditions caused dehydration to
yield the cyclized product 5. The N-benzyl group was re-
moved by refluxing amine 5 in methanol in the presence
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 com-
pound 6. This was converted to the corresponding
methylamine hydroiodide salt by reaction with methyl
iodide in acetone. This on treatment with sodium boro-
hydride in methanol gave the reduced product 7. Final-
ly, the Boc group was removed using methanolic HCl to
yield the free amine as HCl salt 8. The detail procedure
for the synthesis of compound 8 has been previously re-
ported.14 This on reacting with respective sulfonyl chlo-
The effect of muscarinic agonist arecoline was investi-
gated first on human volunteers and found to improve
the performance in serial learning.11 In patients with
dementia of the Alzheimer’s type12 reported a significant
enhancement of performance in a picture recognition
test after the infusion of 4 mg arecoline administration
compared to placebo treatment in same subjects but
the effects were not statistically significant.13 This low
efficacy of arecoline can be explained by its poor tolera-
bility paired with a short biological half-life. Many
structurally modified arecoline based muscarinic ago-
nists have been tested mainly to overcome these limita-
tions. Both affinity and efficacy were significantly
enhanced by tetrahydropyridine analogues which pro-
vide semirigid template and have good affinity for the
M1 receptor.14 Several five and six membered heterocy-
clic ring attached arecoline basic nuclei have been ex-
plored as M1 receptor agonists in AD research. The
lack of M1 selectivity and efficacy due to dose limiting
side effects associated with M2 and M3 muscarinic
receptor subtype stimulation have produced disappoint-
ing results.12 Replacement of the ester functionality of
arecoline with either the 3-alkyl-1,2,4-oxadiazole or the
3-alkyl-1,2,4-thiadiazole has produced very potent mus-
carinic agonists.15 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 also
had lower affinities for muscarinic receptors.16
1
rides gave sulfonamides 9(a–h). H 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 character-
ized by IR, H NMR, Mass spectroscopy, and CHNS
analysis.
1
3. Result and discussion
In the present study, we have synthesized N-aryl sulfo-
nyl morpholino arecoline derivatives 9(a–h), which were
subjected to in vitro competitive muscarinic receptor 1
studies by using radiological [3H] Qunclidinyl Benzillate
(QNB), in male Wistar rat brain synaptosomal mem-
brane. Also we extended the findings of in vitro studies
(Table 1) to in vivo pharmacological experiment involv-
ing evaluation of learning and memory in male Wistar
rats (Rodent memory evaluation, plus and Y maze stud-
ies) to ascertain their applicability in dementia model.
In our previous study we have reported arecoline
thiazolidinones as muscarinic receptor 1 agonists.17
In continued efforts to discover less toxic arecoline
class of muscarinic agonists and to further improve
their selectivity and potency here we are reporting
our finding of N-Aryl sulfonamide substituted 3-mor-
pholino arecoline derivatives as another potent M1
receptor agonists for the symptomatic treatment of
Alzheimer’s dementia. The C-functionalized morphol-
ines are found in a variety of natural and biologically
active compounds.18 Herein, we have described the
synthesis of N-aryl sulfonyl morpholino arecolines
9(a–h) along with their in vitro muscarinic binding
assay by using [3H] Qunclidinyl Benzillate (QNB),
with male Wistar rat brain synaptosomal membrane
and in vivo evaluation of memory and learning in
male Wistar rats.
Structure–activity relation (SAR) was drawn from the
in vitro affinity assay for N-arylsulfonyl-3-morpholino
arecoline derivatives 9(a–h). It reveals that affinity and
potency of all molecules 9(a–h) were dependent on the
position and the substituents of the sulfonylated aro-
matic ring (Scheme 1, Table 1 and Fig. 1). The most po-
tent compound was 9c (Ki = 0.26 lM) with electron
donating chlorine group at the para position. The disub-
stituted chlorine compound 9d at ortho and meta posi-
tions reduces the affinity of the M1 receptor
(Ki = 35 lM). Electron donating groups at para position
such as tertiary butyl 9b (Ki = 4 lM) and methyl group
9a (Ki = 14 lM) also exhibited considerably high affinity
and potency for the M1 receptor. On the other hand,
substitution of electron withdrawing group such as
NO2 on the aromatic ring 9(f–h), reduced the affinity

Y. C. Sunil Kumar et al. / Bioorg. Med. Chem. 16 (2008) 5157–5163
5159
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
(b) NaBH4, MeOH
N
6
N
5
N
4
Reflux
R
O
S
N
O
Boc
N
H
R
N
O
S
O
, TEA
O
Cl
O
HCl, MeOH
O
N
MDC
N
N
CH3
7
CH3
9
CH3
8
R=
CH3
CH3
9a =
9e =
C
CH3
9b =
9c =
9d =
9f =
CH3
NO2
O2N
Cl
9g =
9h =
Cl
NO2
Cl
Scheme 1.
Table 1. In vitro affinity and potency of morpholino arecoline
derivatives 9(a–h) towards M1 receptor of male Wistar rat cortex
synaptosomal membrane
considerable high affinity when compared to ortho posi-
tion 9f (Ki= 96 lM) and para position 9h (Ki = 64 lM).
This is may be due to the strong electron withdrawing
affinity of NO2, which is in order of ortho > para > me-
ta. Compound 9e having no substituent showed inter-
mediate affinity with Ki value of 27 lM.
Compounds
Ki (lM)
IC50 (lM)
9a
9b
14 2.26
4
61 1.23
19 1.27
0.82
9c
9d
0.26 .04
35 4.56
27 3.22
96 1.98
62 3.28
64 5.36
86 1.11
0.92 .11
115 9.73
90 2.95
The foresaid in vitro M1 receptor binding studies
formed a basis for extending correlation further to
in vivo pharmacological studies to ascertain applicabil-
ity of the N-aryl sulfonamide morpholino arecoline
derivatives 9(a–h) in scopolamine induced dementia
models (male Wistar rats) using memory and learning
experiments (passive avoidance, plus and Y maze tasks).
In accordance with the degree of affinity and potency of
synthesized morpholino arecoline derivatives 9(a–h)
in vitro binding experiments elicited almost anticipated
9e
9f
9g
314 11.87
201 7.12
208 8.56
469 2.18
9h
Arecoline
and potency of the compounds for the M1 receptor.
Among the derivative having electron withdrawing
groups, NO2 at meta position 9g (Ki = 62 lM), showed

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Y. C. Sunil Kumar et al. / Bioorg. Med. Chem. 16 (2008) 5157–5163
Effect of 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
30
Control
Scopolamine
25
20
15
10
5
9a
9b
9c
9d
9e
9f
9g
9h
0
Figure 1. Displacement graphs of three most potent compounds 9c, 9b
and 9a. The displacement studies were done with 0.2 nM [3H] QNB
and different concentrations of N-aryl sulfonamide 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.
Different groups of experimental rats
Figure 3. Effect of morpholino arecoline derivatives 9(a–h) on memory
and learning.
seconds for 2nd day and 1st day for scopolamine trea-
ted group and test compound along with scopolamine
treated groups are compared to evaluate learning
(TL1) and memory (TL2). Derivatives 9c, 9b, 9a,
and 9e reversed acute memory loss and learning
impairment in a better way than other synthesized
derivatives. Compound 9c produced lesser TL for 1st
day (TL1 = 32 s) compared to scopolamine treated
group (TL1 = 67 s.), but on 2nd day (TL2 = 08 s) TL
was even lesser than 1st day indicating helpful in
reversing learning impairment as compared to rest of
the derivatives. Overall difference between TL2 and
TL1 was lesser for 9c among the other synthesized
compounds. In contrary to this, compound 9f pro-
duced longer 1st day TL (TL1 = 35 s.) and 2nd day
TL (TL2 = 12 s) overall difference differs (TL2–
TL2 = 16 s), implying that 9f is less significant than
other derivatives, to reverse acute memory and learn-
ing impairing in male Wistar rats. TL for remaining
of the compounds is given in Figure 3.
level of pharmacological actions in reversing scopol-
amine induced dementia.
The in vivo passive avoidance results (Fig. 2) for com-
pounds 9(a–h) using rodent memory evaluator in male
Wistar rats show reversed scopolamine-induced demen-
tia by making rats to commit less number of mistakes.
Compounds 9c, 9b, and 9a (No. of mistakes done 9, 9
and 10, respectively) were highly potent when compared
with the number of mistakes done by control rats (8 mis-
takes) and scopolamine treated group (33 mistakes).
Compounds 9e, 9d, and 9g were also significantly re-
versed the scopolamine induced memory loss. Com-
pound 9f was the least potent.
The in vivo plus maze experiment for synthesized
morpholino arecoline derivatives 9(a–h) in male Wis-
tar rats, measures the transfer latency (TL) in seconds
to reach from one extreme ends of open arm to one
of the closed arms in plus maze. Difference in TL in
Effect of morpholino arecoline 9(a-h) on spatial
Antiamnesic effect of morpholino arecolines 9(a-h) against
scopolamine induced memory loss in male wistar rats
working memory using Y maze task in male wistar rats
70
35
Control
Control
60
Scopolamine
30
25
20
15
10
5
Scopolamine
9a
9a
9b
9c
9d
9e
9f
50
9b
40
30
20
10
0
9c
9d
9e
9f
9g
9h
9g
9h
0
Different groups of experimental rats
Different groups of experimental rats
Figure 2. Antidementia activity of morpholino arecoline derivatives
9(a–h).
Figure 4. Effect of morpholino arecoline derivatives 9(a–h) on acute
spatial memory.

Y. C. Sunil Kumar et al. / Bioorg. Med. Chem. 16 (2008) 5157–5163
5161
The N-aryl sulfonamide morpholino arecoline com-
pounds 9(a–h) were also subjected to in vivo Y maze
study using male Wistar rats. Rats were allowed to ex-
plore three arm Y maze in which rat move in sequence,
noting the percentage animal does by not entering into
already entered arm before completing the sequence.
The alteration behavior in terms of percentage of alter-
ation behavior was noted for test compounds in the
presence of scopolamine and was compared with scopol-
amine administered animal group. The percentage of
alteration behavior for 9c, 9a, and 9b (64%, 62%, and
61%, respectively) indicating that these derivatives sig-
nificantly reverse scopolamine induced spatial working
memory loss (in terms of alteration behavior). Com-
pound 9e and 9d (60% and 59%, respectively) also mod-
erately reverse scopolamine induced spatial working
memory loss. The percentage of alteration behavior
for the rest of the compounds is shown in Figure 4.
(d ppm) tetramethyl silane (TMS) as an internal stan-
dard. Mass spectra were obtained on LCMSD-Trap-
XCT instrument. Elemental analysis was performed on
a variol EL, III Elementar C, H, N analyzer and values
were within the acceptable limits of the calculated val-
ues. 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)
analysis indicated that the calculated and observed val-
ues were within the acceptable limits ( 0.4%).
5.1.2. General procedure for the synthesis of compounds
9(a–h). The intermediate compound 2-(1-methyl-1,2,5,6-
tetrahydropyridin-3-yl)morpholine 8 was synthesized as
summarized in Scheme 1 as per reported procedure.19
To a stirred solution of compound 8 in dichlorometh-
ane, triethylamine (5.0 equiv) was added and cooled to
0–5 °C, then sulfonyl chloride (1.0 equiv) was added
dropwise. The reaction mixture was stirred for 4–5 h
at room temperature (completion of reaction was con-
firmed by TLC) then the reaction mixture was washed
with water, followed by saturated NaCl solution and
dried over sodium sulfate. Dichloromethane was evapo-
rated under reduced pressure and the crude residue ob-
tained was purified by column chromatography using
chloroform: methanol (9:1) as an eluent.
4. Conclusion
In the current study, in vitro competitive M1 receptor
displacement assay was done using male Wistar rat
brain synaptosomal membrane and in vivo pharmaco-
logical experiments for compounds 9(a–h) to testify the
reversal of scopolamine induced memory loss and learn-
ing impairment to ascertain their applicability in demen-
tia. The derivatives with electron donating group at para
position of the sulfanamide (Scheme 1) such as Cl (9c),
C(CH3)3 (9b), CH3(9a) showed considerable high affin-
ity and potency for the M1 receptor in vitro and useful
antidementia activity in vivo model tested. This finding
can be attributed due to the delocalization of electrons
from electron donating group towards the oxygen of sul-
fonyl group through benzene ring, which influences the
affinity of the compound for the receptor. On the other
hand, Cl at ortho and meta positions (9d) showed less
affinity and potency compared to other electron donat-
ing group at para positions. Because Cl at meta position
act as electron withdrawing rather donating group. This
is further evidenced by the decrease in the affinity and
potency of the derivatives having electron withdrawing
group (NO2) at ortho or para positions of the benzene
ring. However, NO2 at meta position showed good affin-
ity and potency compared to NO2 at ortho and para po-
sition, this is due to the less electron withdrawing nature
of NO2 at the meta position. All derivatives 9(a–h)
showed no visible cholinergic toxicity (salivation, defe-
cation, etc.) at the dose tested.
5.1.3. 4-(4-Methyl-benzenesulfonyl)-2-(1-methyl-1,2,5,6-
tetrahydro-pyridin-3-yl)-morpholine (9a). Compound 9a
was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 4-methyl benzene sulfonyl chloride
(0.15 g, 0.00078 mol) and triethylamine (0.393 g,
0.0039 mol) in dichloromethane (3 ml). Yield: 80%; IR
(Nujol, cmÀ1): 1365, 1162 (O@S@O), 1675 (–
1
RC@CH–); H NMR (CDCl3): d 7.663–7.639 (d, 2H,
J = 8.9 Hz), 7.363–7.341 (d, 2H, J = 8.80 Hz), 5.76 (bs,
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:
337.45 (M+); Anal. Calcd for C17H24N2O3S; C: 60.69,
H: 7.19, N: 8.32, S: 9.52. Found: C: 60.79, H: 7.39, N:
8.22, S: 9.23.
5.1.4.
4-(4-tert-Butyl-benzenesulfonyl)-2-(1-methyl-
1,2,5,6-tetrahydro-pyridin-3-yl)-morpholine (9b). Com-
pound 9b was obtained by reaction of compound 8
(0.2 g, 0.00078 mol) with 4-tributyl-benzene sulfonyl
chloride (0.183 g, 0.00078 mol) and triethylamine
(0.393 g, 0.0039 mol) in dichloromethane (3 ml). Yield:
85%; IR (Nujol, cmÀ1): 1360, 1160 (O@S@O), 1678 (–
1
5. Experimental procedure
5.1. Chemistry
RC@CH–); H NMR (CDCl3): d 7.892–7.870 (d, 2H,
J = 8.77 Hz), 7.243–7.221 (d, 2H, J = 8.73 Hz), 5.76
(bs, 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:
379.5 (M+); Anal. Calcd for C20H30N2O3S; C: 63.46,
H: 7.99, N: 7.40, S: 8.47. Found: C: 64.56, H: 8.19, N:
7.48, S: 8.66.
5.1.1. General. 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
5.1.5. 4-(2-Chloro-benzenesulfonyl)-2-(1-methyl-1,2,5,6-
tetrahydro-pyridin-3-yl)-morpholine (9c). Compound 9c

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Y. C. Sunil Kumar et al. / Bioorg. Med. Chem. 16 (2008) 5157–5163
was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 2-chloro-benzenesulfonyl chloride
(0.166 g, 0.00078 mol) and triethylamine (0.393 g,
0.0039 mol) in dichloromethane (3 ml). Yield: 86%; IR
(Nujol, cmÀ1): 1368, 1162 (O@S@O), 1680 (–
RC@CH–); 1H NMR (CDCl3): d 7.89–7.87 (m, 2H),
7.52 (m, 2H), 5.76 (bs, 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: 357.5 (M+); Anal. Calcd for C16H21ClN2O3S;
C: 53.85, H: 5.93, N: 7.84, S: 8.99. Found: C: 54.15,
H: 5.89, N: 7.92, S: 8.59.
(0.174 g, 0.00078 mol) and triethylamine (0.393 g,
0.0039 mol) in dichloromethane (3 ml). Yield: 82%; IR
(Nujol, cmÀ1): 1360, 1158 (O@S@O), 1675 (–
1
RC@CH–); H NMR (CDCl3): d 8.847–8.844 (d, 1H,
J = 1.2 Hz), 8.29–8.22 (m, 2H), 7.25 (m, 1H), 5.78 (bs,
1 H, –C@C–), 3.85–3.81 (m, 3 H), 3.49–3.44 (m, 1 H),
2.79–2.76 (m, 2 H), 2.78–2.76 (m, 3 H), 2.55–2.45 (m,
2 H), 2.22 (s, 3 H), 2.02 (m, 2 H). MS m/z: 368.32
(M+); Anal. Calcd for C16H21N3O5S; C: 52.3, H: 5.76,
N: 11.4, S:8.73. Found: C: 52.29, H: 5.66, N: 10.95, S:
8.99.
5.1.10. 4-(4-Nitro-benzenesulfonyl)-2-(1-methyl-1,2,5,6-
tetrahydro-pyridin-3-yl)-morpholine (9h). Compound 9h
was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 4-nitrobenzenesulfonyl chloride
(0.174 g, 0.00078 mol) and triethylamine (0.393 g,
0.0039 mol) in dichloromethane (3 ml). Yield: 93%; IR
(Nujol, cmÀ1): 1369, 1158 (O@S@O), 1675 (–
5.1.6.
4-(2,5-Dichloro-benzenesulfonyl)-2-(1-methyl-
1,2,5,6-tetrahydro-pyridin-3-yl)-morpholine (9d). Com-
pound 9d was obtained by reaction of compound 8
(0.2 g, 0.00078 mol) with 2,5-dichloro-benzenesulfonyl
chloride (0.193 g, 0.00078 mol) and triethylamine
(0.393 g, 0.0039 mol) in dichloromethane (3 ml). Yield:
90%; IR (Nujol, cmÀ1): 1368, 1160 (O@S@O), 1680 (–
1
RC@CH–); H NMR (CDCl3): d 8.321–8.298 (d, 2H,
1
RC@CH–); H NMR (CDCl3): d 7.892–7.887 (d, 1H,
J = 9.2 Hz), 7.931–7.908 (d, 2H, J = 9.16 Hz), 5.76 (bs,
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,
2H), 2.24 (s, 3H), 2.07 (m, 2H). MS m/z: 368.2 (M+);
Anal. Calcd for C16H21N3O5S; C: 52.3, H: 5.76, N:
11.4, S:8.73. Found: C: 52.23, H: 5.86, N: 10.94, S: 8.97.
J = 2.0 Hz), 7.720–7.699 (d, 2H, J = 8.12 Hz), 5.78 (bs,
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: 392.1 (M+);
Anal. Calcd for C16H20Cl2N2O3S; C: 49.13, H: 5.16,
N: 7.15, S: 8.23. Found: C: 49.33, H: 5.36, N: 7.25, S:
8.33.
5.2. Biology
5.1.7. 4-Benzenesulfonyl-2-(1-methyl-1,2,5,6-tetrahydro-
pyridin-3-yl)-morpholine (9e). Compound 9e was ob-
tained by reaction of compound 8 (0.2 g, 0.00078 mol)
with benzene sulfonyl chloride (0.138 g, 0.00078 mol)
and triethylamine (0.393 g, 0.0039 mol) in dichlorometh-
ane (3 ml). Yield: 88%; IR (Nujol, cmÀ1): 1366, 1168
(O@S@O), 1675 (–RC@CH–); 1H NMR (CDCl3): d
7.59–7.51 (m, 5H), 5.76 (bs, 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: 323.7 (M+); Anal. Calcd for
C16H22N2O3S; C: 59.6, H: 6.88, N: 8.69, S: 9.99. Found:
C: 59.56, H: 7.12, N: 8.99, S: 10.19.
5.2.1. Displacement study. The competitive inhibition
study was done using N-aryl sulfonamide morpholino
arecoline derivatives 9(a–h) to find 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, fol-
lowing the procedure described by Creese and Snyder.20
The tissue homogenate was centrifuged at a speed of
1000g for 10 min at 4 degrC, to remove cellular debris.
The supernatant obtained was centrifuged at 15,000g
for 20 min at 4 °C. Pellet obtained 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.21
5.1.8. 4-(2-Nitrobenzenesulfonyl)-2-(1-methyl-1,2,5,6-tet-
rahydro-pyridin-3-yl)-morpholine (9f). Compound 9g was
obtained by reaction of compound
8
(0.2 g,
0.00078 mol) with 2-nitrobenzenesulfonyl chloride
(0.174 g, 0.00078 mol) and triethylamine (0.393 g,
0.0039 mol) in dichloromethane (3 ml). Yield: 90%; IR
(Nujol, cmÀ1): 1358, 1162 (O@S@O), 1675 (–
The affinity of various compounds towards M1 receptor
was estimated by using [3H]QNB (0.2 nM, specific activ-
ity 48 Ci/mmol, Amersham, Little Chalfont, Bucks, UK)
essentially following the procedure described by Hyttel
et al.22; Yamamura and Snyder23 with slight modifica-
tion. In brief an aliquot of synaptosomal membrane
proteins (50 lg) was incubated with different concentra-
tions of compounds (0.1–200 lM) as a displacer and
[3H]QNB (0.2 nM) and reaction volume was made up
to 200 ll with assay buffer and incubated for 2 h at
37 °C. The reaction for all displacement assay was
stopped by adding ice-cold assay buffer and the reaction
mixtures were rapidly filtered through GF/B filters un-
der vacuum. The filters were transferred to vials contain-
ing scintillation fluid, (5 ml) and allowed to equilibrate
overnight. Radioactivity was measured in a liquid scin-
tillation counter (Tris-Carb 2100TR, Packard, US) at
1
RC@CH–); H NMR (CDCl3): d 8.123–8.102 (d, 1H,
J = 8.4 Hz), 8.363–8.341 (d, 1H, J = 8.8 Hz), 7.89–7.87
(m, 2H), 5.78 (bs, 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: 368.3 (M+); Anal. Calcd for C16H21N3O5S;
C: 52.3, H: 5.76, N: 11.4, S: 8.73. Found: C: 51.83, H:
5.86, N: 10.94, S: 8.97.
5.1.9. 4-(3-Nitrobenzenesulfonyl)-2-(1-methyl-1,2,5,6-tet-
rahydro-pyridin-3-yl)-morpholine (9g). Compound 9g
was obtained by reaction of compound 8 (0.2 g,
0.00078 mol) with 3-nitrobenzenesulfonyl chloride

Y. C. Sunil Kumar et al. / Bioorg. Med. Chem. 16 (2008) 5157–5163
5163
65% efficiency. The data from displacement were ana-
Acknowledgments
lysed and IC50 and Ki values are obtained from Li-
gand–Drug programme.24 The mean values of %
bound are plotted against log of displacer concentration.
The authors are grateful to the Department of Science
and Technology (DST), New Delhi, for financial sup-
port 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) pro-
grammes are greatly acknowledged.
5.2.2. Antiamnesic activity. It was carried out for synthe-
sized N-aryl morpholino arecoline derivatives 9(a–h)
against scopolamine induced memory loss using passive
avoidance step down task paradigm in male Wistar rats
weighing 200–250 g (n = 8) according to the method de-
scribed by Sharma and Kulkarni.25,26
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and were administered scopolamine and test compound
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of one arm facing away from the central platform and
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enclosed arms (TL) was measured. On the 1st day male
Wistar rats were allowed to explore the plus maze for
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ine and on 2nd day TL was measured in the similar way
on the same animals. The resultant data were subjected
to statistical analysis.26
9. Saunders, J.; Freedman, S. B. Trends Pharmacol. Sci.
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memory through the spontaneous alteration behavior in
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administered with test compound along with scopol-
amine. 30 min later they were allowed to explore in Y-
maze. The ability of the rats to alternate in the Y-maze re-
quires the rat to know which arm they have already vis-
ited.27 The series of arm entry, including possible
returns into the same arm, are recorded visually. Alter-
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the three arms, on overlapping triplet sets. The percentage
ofalteration is calculated as the ratiosofactual alterations
to possible alterations, defined as the total number of arm
entries minus two, and multiplied by 100.
13. Tariot, P. N.; Cohen, R. M.; Welkowitz, J. A.; Sunder-
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one week after administration under test dose.
19. Sunil Kumar, Y. C.; Sadashiva, M. P.; Rangappa, K. S.
Tetrahedron Lett. 2007, 48, 4565.
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6. Data analysis
The data from the displacement assay were analyzed
using ‘Ligand–Drug’ software program24 to obtain the
IC50 and Ki values (both are expressed in lMol). All
the data are expressed as means SD. The statistical
analysis was done by using student’s t-test. Differences
were considered to be significant at P < 0.05. All analy-
ses were performed with the ‘Jandel-Scientific-Sigma
stat’ software, version 2.0 for windows.
24. McPherson, G. A. Comput. Programs Biomed. 1983, 17,
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25. Sharma, A. C.; Kulkarni, S. K. Methods Find. Exp. Clin.
Pharmacol. 1990, 12, 175.
26. Sharma, A. C.; Kulkarni, S. K. Methods Find. Exp. Clin.
Pharmacol. 1991, 13, 155.
27. Sarter, M.; Bodewitz, G.; Stephens, D. N. Psychopharma-
cology (Berl.) 1988, 94, 491.