Muscarinic M2 antagonists: Anthranilamide derivatives with exceptional selectivity and in vivo activity

Article abstract of DOI:10.1016/j.bmc.2003.11.005

Anthranilamide analogues such as 23 are potent and highly selective muscarinic M2 antagonists that also show good oral bioavailability and in vivo activity.

Full text of DOI:10.1016/j.bmc.2003.11.005

Bioorganic & Medicinal Chemistry 12 (2004) 319–326
Muscarinic M2 antagonists: anthranilamide derivatives with
exceptional selectivity and in vivo activity
John W. Clader,* William Billard, Herbert Binch, III, Lian-Yong Chen, Gordon Crosby, Jr.,
Ruth A. Duffy, Jennifer Ford, Joseph A. Kozlowski, Jean E. Lachowicz, Shengjian Li,
Charles Liu, Stuart W. McCombie, Susan Vice, Gowei Zhou and William J. Greenlee
Schering-Plough Research Institute 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
Received 12 September 2003; revised 30 October 2003; accepted 10 November 2003
Abstract—Anthranilamide analogues such as 23 are potent and highly selective muscarinic M2 antagonists that also show good oral
bioavailability and in vivo activity.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
that piperazines such as 1 and 2 show improved selec-
Alzheimer’s disease (AD) is a neurodegenerative disease
characterized by a profound cognitive impairment
which progresses eventually to an inability to function
independently and ultimately to death. One of the con-
sistent findings in brains of AD patients is loss of choli-
nergic markers, including levels of acetylcholine (ACh).
The cholinergic approach to treatment of AD involves
counteracting this loss in cholinergic activity by phar-
macologic intervention to increase cholinergic transmis-
sion.1 While use of acetylcholinesterase inhibitors
remains the most common approach to cholinergic
therapy, an alternative approach involves inhibition of
central presynaptic M2 receptors, thus causing an
increase in acetylcholine release. Successful implemen-
tation of this approach requires high selectivity for the
M2 receptor over the other four muscarinic receptor
subtypes, especially the M1 receptor that mediates the
cognitive effects of acetylcholine.2 Additionally, com-
pounds need to have appropriate bioavailability and
CNS penetration.
tivity versus earlier compounds. In particular, com-
pounds similar to 1 show >100ꢁ selectivity for the M2
receptor. Key pharmacophore elements required to
achieve this level of selectivity are the (R)-methylpiper-
azine moiety and the 2-substitituted benzamide. Unfor-
tunately, the overall pharmacokinetic profile of this
class of compounds is poor, resulting in low plasma
levels after oral administration and low efficacy in sev-
eral animal models predictive of cognitive activity. In
this Letter, we describe our efforts in a piperidine series
related to 1 (Fig. 1), which has yielded a compound
suitable for clinical evaluation.18,19
2. Results and discussion
Several measures were used to evaluate progress in this
program. In addition to determining activity at each of
the five cloned human muscarinic receptors, in vivo
activity was evaluated by measuring release of acetyl-
choline in conscious rats using microdialysis as pre-
viously described.20 Activity was measured as the
maximum percent increase in acetylcholine release over
baseline up to three h after oral administration of drug.
Although failure at several points (e.g., absorption,
first-pass metabolism, brain penetration, and receptor
occupancy) could result in lack of activity, we found
this to be a particularly useful screen for identifying
compounds with the best potential for showing activity
in more detailed and time-intensive animal models of
Previous reports from these laboratories have described
a series of piperidine and piperazine derivatives which
are potent muscarinic M2 antagonists with promising
selectivity versus other muscarinic receptor sub-
types.3ꢀ17 Some of these reports3,4 have demonstrated
* Corresponding author. Tel.:+1-908-7403478; fax:+1-908-2592160;
e-mail: john.clader@spcorp.com
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.11.005

320
J. W. Clader et al. / Bioorg. Med. Chem. 12 (2004) 319–326
cognition. In particular, compounds with good M2
selectivity which also induced a sustained acetylcholine
release of at least 150% over baseline generally showed
good oral activity in the juvenile rat passive-avoidance
response (PAR) model.21 Activity in rat microdialysis
generally paralleled plasma levels in an abbreviated rat
pharmacokinetic screen which has previously been
described.22
gave 6. This was treated with n-butyllithium followed by
treatment with arylsulfonyl fluoride 7 to give dia-
rylsulfone 8. The Boc group was removed and the
resulting piperidine was coupled with a carboxylic acid
under standard conditions.
In an alternative synthesis (Scheme 2), methylidene
piperidine 10 was treated with 9-BBN and the resulting
borane was coupled with bromoarylsulfone 11 under
palladium catalysis to give 12.23 The Boc group was
removed, and the core was elaborated to target com-
pounds using the same procedures as outlined in
Scheme 1.
Among the variety of core structures available for this
study, we focused on core 9 (Scheme 1) because it was
achiral and gave us maximal flexibility for evaluating
changes on either terminus of the core structure, that is,
the left-hand aromatic substituent R1 and the piperidine
substituent R2. From a survey of a variety of sub-
stituents on the piperidine nitrogen, including aryl and
alkyl amides, sulfonamides, carbamates, and ureas, we
focused primarily on a series aryl amides because they
consistently provided the highest potency and selectivity
for the M2 receptor. The synthesis of the core structure
and subsequent derivatization was carried out as shown
in Schemes 1 and 2. Piperidine 3 was protected as its
trifluoroacetamide and brominated with dibromodi-
methylhydantoin (DBDMH) to give primarily the 4-
bromo derivative 4 after base-induced deprotection.
Reductive alkylation of 4 with N-Boc piperidinone 5
Initial results in the piperidine series indicated that 2-
substituted benzamides provided potent muscarinic M2
antagonists with very good selectivity versus the other
muscarinic receptor subtypes (Table 1). Avariety of
substituents R1 on the arylsulfone moiety were exam-
ined, but in general the structure–activity relationship
trends are reflected by results where R1 is either 4-
methoxy or 3-chloro. Thus, toluamides 13 and 15
showed very good selectivity for the M2 receptor which
was further improved by addition of a chloro group at
the 3-position (e.g., 14, 16). That this effect was pri-
marily steric was suggested by the fact that the 2-chloro-
Figure 1. Piperazine M2 antagonists.
Scheme 1.

J. W. Clader et al. / Bioorg. Med. Chem. 12 (2004) 319–326
321
Scheme 2.
Table 1. 2-Substituted benzamides
with additional substitution at the 3-position of the
anthranilamide, although potency and selectivity
remained high except for 3-fluoro derivative 26. How-
ever, the anthranilamides consistently caused a greater
release of acetylcholine than other benzamides, with
several compounds showing a sustained release of at
least 150% of baseline. Based largely on its superior
activity in microdialysis and correspondingly higher rat
plasma levels, compound 23 was selected for more
detailed evaluation.
No. R1
R2
Ki (nM) M1/ M3/ M4/ M5/
AcCh
Figure 2 shows the complete time course of acetylcholine
release for compound 23 over a range of doses, demon-
strating that 23 causes a robust and long-lasting increase
in acetylcholine release. Consistent with these results, 23
improved cognition in the juvenile rat PAR model, with
a maximally effective dose of 0.1 mg/kg po. Further
details of these studies will be reported elsewhere.
M2a M2 M2 M2 M2 release (%)b
13 4-CH3O 2-CH3
14 4-CH3O 2-CH3, 3-Cl
2.53
1.29
2.7
1.52
1.2
144 99 55 24
651 774 223 100
230 172 14 48
536 542 155 37
461 795 107 39
70 ND ND ND
115
164
ND
125
153
ND
ND
137
150
15
16
17
3-Cl
2-CH3
3-Cl 2-CH3, 3-Cl
3-Cl 2-Cl, 3-CH3
18 4-CH3O
19 4-CH3O 2-CH3O
20 4-CH3O
21 3-Cl
2-OH
17.1
4.9
2.2
0.2
141 62
414 495 42 89
2926 2722 148 186
7
20
2-NH2
2-NH2
3. Conclusions
ND, not detected.
a Activity at the m2 receptor expressed as a Ki (nM). Selectivity versus
the other receptors expressed as the ratio of the Ki (nM) at each
receptor versus the Ki (nM) at the m2 receptor. All Ki values are the
mean of at least two determinations.
We have evaluated a series of benzamide derivatives for
their potency and selectivity for the M2 receptor versus
other muscarinic receptors. Compounds have also been
evaluated for their ability to increase acetylcholine
release after oral administration. Simple toluamide
derivatives show excellent potency and selectivity but
cause only a modest increase in acetylcholine release in
most cases. By contrast, anthranilamides gave both
excellent in vitro activity and caused a robust increase
in acetylcholine release. Compound 23 is also active in
animal models of cognition. Based on these results,
compound 23 was selected for clinical evaluation as a
potential new treatment for Alzheimer’s disease.
b Acetylcholine release expressed as the maximum percent increase
over predosing baseline.
3-methyl derivative 17 gave nearly identical potency and
selectivity compared to 16. Unfortunately, as was the
case with analogous piperazines, none of these com-
pounds showed exceptional in vivo activity as measured
by the percent increase in acetylcholine release. Believ-
ing that these compounds might in fact be too lipo-
philic, we also examined several more polar benzamides.
While initial results with 2-hydroxy and 2-methoxy
derivatives 18 and 19 were not promising, we were
pleasantly surprised to find that anthranilamides 20 and
21 gave very promising potency and selectivity.
4. Experimental
4.1. Biology: in vitro assay using cloned human muscari-
nic receptors
Based on these promising results, we evaluated the
anthranilamide class in more detail (Table 2). Unlike
earlier benzamides, anthranilamides did not show sig-
nificant improvement in either potency or selectivity
Inhibition of muscarinic receptors was measured by dis-
placement of tritiated QNB (l-quinuclidinyl benzilate) at

322
J. W. Clader et al. / Bioorg. Med. Chem. 12 (2004) 319–326
Table 2. Anthranilamides
No.
R1
R3
Ki (nM)
M2a
M1/
M2
M3/
M2
M4/
M2
M5/
M2
AcCh
release (%)
Rat AUC0ꢀ6 h
(h ng/ml)a
20
21
22
23
24
25
26
27
28
29
30
4-CH3O
3-Cl
4-CH3O
3-Cl
4-CH3O
3-Cl
4-CH3O
3-Cl
H
2.2
414
2926
456
734
426
1297
165
528
259
350
229
495
2722
481
787
262
1650
174
365
285
194
265
42
148
67
69
88
80
62
68
86
54
20
89
186
140
95
165
65
112
82
89
27
137
150
130
180
138
145
ND
162
ND
128
150
3484
4587
2918
7800
3349
4903
ND
5324
ND
2692
3750
H
3-CH3
3-CH3
3-Cl
3-Cl
3-F
3-F
4-F
4-F
5-F
0.22
2.37
0.89
1.43
0.67
5.7
1.71
3.5
2.63
2.88
4-CH3O
3-Cl
3-Cl
24
ND, not detected.
a AUC0ꢀ6 h after 10 mg/kg oral dose in an abbreviated rat PK protocol. See ref 22.
maximum percent increase in acetylcholine versus the
pre-dosing baseline.
4.2. Pharmacokinetics
Rat AUC values were determined using an abbreviated
protocol as described in ref 22. All compounds were
dosed orally at 30 mg/kg in 20% HPbCD. AUCs are
expressed at hꢁng per mL.
4.3. Chemistry
All melting points were taken on a Thomas-Hoover
melting point apparatus and are uncorrected. Proton
NMR spectra were determined with a Gemini 300 MHz
instrument using Me4Si as an internal standard. Cou-
pling constants are given in hertz.
Figure 2. Effect of 23 on ACh release from striatum of conscious rat
following po administration. * Significant stimulation over baseline
(p=0.05, Duncan’s multiple range statistic).
the five cloned human receptors. For radioligand bind-
ing analysis, each muscarinic receptor subtype was sta-
bly expressed in CHO-K1 cells. Clonal cell lines were
selected which expressed receptors at levels between 1
and 9 pmol/mg protein. The Kd of QNB at each recep-
tor subtype was determined by saturation binding using
5–2500 pM [3H] QNB in 10 mM potassium phosphate
buffer, pH 7.4. Protein concentrations were adjusted for
each assay to achieve between 700 and 1500 cpm specific
binding. Competition binding experiments were per-
formed using 180 pM [3H] QNB. All binding experi-
ments were performed in the presence of 1% DMSO
and 0.4% methylcellulose. Nonspecific binding was
determined by 0.5 mM atropine. After equilibrium was
reached (120-min incubation at room temperature),
bound and free radioactivity were separated by fil-
tration using Whatman GF-C filters. Ki was expressed
as mean of duplicate value (SEM<15%). All determi-
nations were performed at least twice. Acetylcholine
release in conscious rats was measured via microdialysis
as previously described. Activity is expressed as the
4.3.1. Synthesis of intermediate 4-(4-bromo-benzyl)-
[1,40]bipiperidinyl-10-carboxylic acid tert-butyl ester (6).
Asolution of 4-benzylpiperidine 3 (202 g, 1.15 mol) in
dichloromethane (1.5 L) was treated with 216 mL (1.53
mol) of trifluoroacetic anhydride added drop-wise over
30 min. The mixture was allowed to stir an additional 90
min at room temperature, then cooled to 0 ꢂC in an ice
bath. Methanesulfonic acid (306 mL) was added in
portions followed by dibromodimethylhydantoin (171
g, 0.6 mol) also added in portions, and the resulting
mixture was stirred overnight while coming to room
temperature. After again cooling in an ice bath, the
mixture was quenched by addition of saturated Na2SO3
(1.6 L) added over 30 min. The aqueous layer was
separated and washed with dichloromethane (2ꢁ2 L).
The combined organic layers were dried over magne-
sium sulfate and concentrated. The residue was purified
by silica gel chromatography, eluting with hexanes (16
L), 5% ethyl acetate–hexane (16 L), and 10% ethyl
acetate–hexane to yield 105 g of brominated compound,
which was used directly in the next step. Asolution of

J. W. Clader et al. / Bioorg. Med. Chem. 12 (2004) 319–326
323
the brominated compound in 1.7 L methanol was trea-
ted with 90 g potassium carbonate in 300 mL water. The
mixture was stirred for 3 h, then concentrated under
vacuum. The residue was treated with 2 N NaOH (2 L)
and extracted with dichloromethane (2ꢁ2 L). The com-
bined organic layers were dried over magnesium sulfate
and evaporated to give 76 g of compound 4 as an oil
which partially crystallized.
2.95 (m, 2H), 3.08–3.20 (m, 2H), 7.21–7.30 (m, 3H),
7.40–7.55 (m, 2H), 7.81 (d, 2H, J=8 Hz), 7.90 (s, 1).
4.3.5. 4-[4-(4-Methoxy-benzenesulfonyl)-benzyl]-[1,40]bi-
piperidinyl-10-carboxylic acid tert-butyl ester (8b). This
was prepared in a similar manner from 6 and 4-meth-
oxybenzenesulfonyl fluoride. 1H NMR (300 MHz,
CDCl3) d 1.1–1.40 (m, 3H), 1.40–1.50 (m, 2H), 1.50–
1.68 (m, 2H), 1.70–2.00 (m, 2H), 2.00–2.20 (m, 4H),
2.30–2.45 (m, 1H), 2.50–2.65 (m, 4H), 2.80–2.95 (m,
2H), 3.08–3.20 (m, 2H), 3.78 (s, 3H), 6.92 (d, 2H, J=8
Hz), 7.20 (d, 2H, J=8 Hz).
Asuspension of compound 4 in 1 L dichloromethane
was treated with 1-tert-butoxycarbonyl-4-piperidone 5
(64 g, 0.32 mol), glacial acetic acid (38 mL), and sodium
triacetoxyborohydride (192.12 g, 0.9 mol). The mixture
was allowed to stir overnight at room temperature, then
poured into 2 N NaOH (2 L). After stirring an addi-
tional 30 min, the layers were separated, and the aqu-
eous layer was extracted with ethyl acetate (2ꢁ2 L). The
combined organic layers were dried over magnesium
sulfate and evaporated, and the residue was purified by
flash chromatography, eluting with ethyl acetate (40 L)
to give 30.2 g of compound 6, as well as an additional
54.4 g of ꢃ50% pure compound 6. 1H NMR (300 MHz,
CDCl3) d 1.1–1.8 (m, 7H), 1.40 (s, 9H), 2.05–2.40 (m,
2H), 2.40–2.70 (m, 7H), 2.80 (m, 2H), 4.15 (m, 2H), 7.18
(d, 2H, J=9 Hz), 7.41 (d, 2H, J=9 Hz).
4.3.6. Alternate synthesis of intermediate 8a. To a
cooled (0 ꢂC) mixture of 12a17 (4.74 g; 10.5 mmol),
CH2Cl2 (35 mL) and H2O (0.19 mL) was added, drop-
wise, trifluoroacetic acid (7 mL). The cooling bath was
removed and the mixture was stirred for 30 min. Tri-
fluoroacetic acid (1.0 mL) and H2O (0.18 mL) were
added. The stirring was continued for 2 h, the volatile
materials were removed in vacuo, CH2Cl2 (20 mL) and
10% NaOH (2 mL) was added, and the resulting mix-
ture was stirred for 3 min. The CH2Cl2 layer was
removed, the aqueous layer was extracted with CH2Cl2
(3ꢁ5 mL), the organic extracts were dried over Na2SO4,
filtered and evaporated to give the free amine as a white
foam (3.10 g) in 88% yield.
4.3.2. Synthesis of 3-chlorophenylsulfonyl fluoride (7a).
To a mixture of 3-chlorosulfonyl chloride (12 g, 0.12
mol) in 185 mL acetone was added drop-wise a solution
of 17.4 g (0.24 mol) KF in 150 mL water. The mixture
was stirred at room temperature overnight, then con-
centrated to one-half volume under vacuum. The resi-
due was diluted with 100 mL ethyl acetate and extracted
3ꢁ30 mL ethyl acetate. The combined extracts were
dried over magnesium sulfate. Toluene (5 mL) was
added and the solution evaporated to give 16.64 g
To a solution of the above amine (1.69 g), N-tert-butoxy
piperidone 5 (4.80 g), CH2Cl2 (12 mL) and acetic acid
(0.28 mL) was added NaB(OAc)3H (1.42 g) in four
portions over 15 min. The resulting solution was stirred
for 4 h when acetic acid (0.14 mL) and NaB(OAc)3H
(1.42 g) were added. After stirring at room temperature
for 16 h, the reaction was diluted with CH2Cl2 (50 mL)
and made basic with 2 N NaOH (15 mL). The CH2Cl2
layer was removed and the aqueous layer was extracted
with CH2Cl2 (2ꢁ15 mL). The organic extracts were
combined, washed with water, brine and dried over
MgSO4, then filtered and evaporated to give a crude
solid which was purified by silica gel chromatography
(320 g silica; 1:1 hexanes–EtOAc then 76:19:5 EtOAc–
hexanes: Et3N as eluant) to give the product, 4, as a
waxy solid (2.27 g) in 88% yield.
1
(72%) of a clear liquid. H NMR (300 MHz, CDCl3) d
7.58 (t, 1H, J=8 Hz), 7.75 (d, 1H, J=8 Hz), 7.96 (d,
1H, J=8 Hz), 7.99 (s, 1H).
4.3.3. 4-Methoxyphenysulfonyl fluoride (7b). This was
prepared in a similar manner from 4-methoxy-
phenylsulfonyl chloride.
4.3.4. Synthesis of intermediate 4-[4-(3-chloro-benzene-
sulfonyl) - benzyl] - [1,40]bipiperidinyl - 10 - carboxylic acid
tert-butyl ester (8a). Asolution of compound 6 (8.8 g,
0.02 mol) in 35 mL dry THF at ꢀ78 ꢂC was treated with
8.05 mL (0.02 mol) of 2.5 M n-butyllithium in hexanes
followed by a solution of 3-chlorobenzenesulfonyl fluo-
ride (3.92 grams, 0.02 mol) in 20 mL dry THF. The
mixture was stirred at ꢀ78 ꢂC for 2 h then allowed to
come to room temperature overnight. The reaction was
quenched with water, concentrated under vacuum, and
partitioned between ethyl acetate and 10% sodium car-
bonate. The organic layer was washed with water, dried
over magnesium sulfate, and evaporated. The residue
was purified over silica gel, eluting with 5% methanol–
ethyl acetate, and the purified residue was crystallized
from ethyl acetate to give 3.03 g of 8a. 1H NMR
(300 MHz, CDCl3) d 1.20–1.53 (m, 2H), 1.35–1.55 (m,
3H), 1.55–1.68 (m, 2H), 1.70–1.90 (m, 2H), 2.00–2.20
(m, 4H), 2.30–2.45 (m, 1H), 2.50–2.65 (m, 4H), 2.80–
4.4. General synthesis of amides 9
Compound 8 (2.6 mmol) was dissolved in 10 mL of di-
chloromethane was treated with 2 mL trifluoroacetic
acid and 0.046 mL water for 30 min. The mixture was
concentrated then partitioned between dichloromethane
(20 mL) and 10% sodium hydroxide (2 mL). The aqu-
eous layer was extracted with dichloromethane (3ꢁ5
mL), and the combined organic layers were dried over
magnesium sulfate and evaporated to give the free
amine.
EDCI (0.032 g, 0.21 mmol) was added to a mixture of
the free amine (0.14 mmol), dimethylformamide (2 mL),
hydroxobenzotriazole (0.028 g, 0.21 mmol), diisopropy-
lethylamine (0.1 mL), and the appropriate benzoic acid
(0.21 mmol). The resulting solution was stirred at room
temperature overnight then partitioned between 10 mL

324
J. W. Clader et al. / Bioorg. Med. Chem. 12 (2004) 319–326
ethyl acetate and 1 mL 2 N sodium hydroxide. The
aqueous layer was extracted with ethyl acetate (3ꢁ4
mL), and the combined organic layers were dried over
sodium sulfate and evaporated. The residue was purified
by preparative silica gel thin-layer chromatography to
give the desired compound as its free base.
4.4.7. 10-(2-Methoxybenzoyl)-4-[4-[(4-methoxyphenyl)sul-
fonyl]phenyl]methyl]-1,40-bipiperidine (19). 1H NMR
(300 MHz, CDCl3) d 1.1–1.35 (m, 3H), 1.4–1.75 (m,
6H), 1.8–1.9 (m, 1H), 2.0–2.25 (m, 2H), 2.4–2.6 (m, 2H),
2.6–2.8 (m, 1H), 2.8–2.9 (m, 4H), 3.4–3.55 (m, 1H), 3.78
(s, 3H), 3.8 (s, 3H), 6.8–6.95 (m, 4H), 7.1–7.32 (m, 4H),
7.77 (d, 2H, J=8 Hz), 7.85 (d, 2H, J=8 Hz); HRMS
calcd for C32H39N2O5S 563.2579, found 563.2589.
The free base was dissolved in 2 mL ethyl acetate and
treated with 50 mL of 4 M HCl in dioxane at 0 ꢂC. The
salt was precipitated by addition of ether, centrifuged,
washed with ether, and dried under vacuum to give the
HCl salt.
4.4.8. 10-(2-Aminobenzoyl)-4-[4-[(4-methoxyphenyl)sulfo-
nyl]phenyl]methyl]-1,40-bipiperidine (20). 1H NMR
(300 MHz, CDCl3) d 1.15–1.35 (m, 2H), 1.35–1.7 (m,
4H), 1.7–1.9 (m, 3H), 2.0–2.2 (m, 3H), 2.4–2.6 (m, 4H),
2.7–3.0 (m, 4H), 3.8 (s, 3H), 4.25 (br s, 2H), 6.66 (m,
2H), 6.93 (d, 2H, J=7 Hz), 7.02 (d, 1H, J=7 Hz), 7.13
(dd, 1H, J=7.7 Hz), 7.22 (m, 2H), 7.79 (d, 2H, J=7
Hz), 7.85 (d, 2H, J=7 Hz); HRMS calcd for
C31H38N3O4S 548.2683, found 548.2700.
Using the above methods, the following compounds
were prepared.
4.4.1. 4-[4-[(4-Methoxyphenyl)sulfonyl]phenyl]methyl]-10-
(2-methylbenzoyl)-1,40-bipiperidine (13). HRMS calcd
for C32H38N2O4S 547.2631, found 547.2630.
4.4.9. 10-(2-Aminobenzoyl)-4-[4-[(3-chlorophenyl)sulfo-
nyl]phenyl]methyl]-1,40-bipiperidine (21). 1H NMR
(300 MHz, CDCl3) d 1.20–1.70 (m, 9H), 1.75–1.95 (m,
2H), 2.05–2.20 (m, 2H), 2.45–2.65 (m, 3H), 2.75–3.00
(m, 4H), 4.25 (br s, 2H), 6.65–6.72 (m, 2H), 7.05 (d, 1H,
J=8 Hz), 7.15 (dd, 1H, J=8.8 Hz), 7.20–7.30 (m, 2H),
7.4 (dd, 1H, J=8.8 Hz), 7.5 (d, 1H, J=8 Hz), 7.78–7.81
(m, 3H), 7.9 (s, 1H); HRMS calcd for C30H35ClN3O3S
552.2087, found 552.2088.
4.4.2. 10-(3-Chloro-2-methylbenzoyl)-4-[4-[(4-methoxy-
phenyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (14). 1H
NMR (300 MHz, CDCl3) d 1.1–1.2 (m, 11H), 2.25, 2.75
(two s, 3H), 2.4–2.6 (m, 3H), 2.6–2.8 (m, 1H), 2.8–2.95
(m, 3H), 3.35–3.48 (m,1H), 4.7–4.85 (m, 1H), 3.8 (s,
3H), 6.91 (d, 2H, J=8 Hz), 6.9–7.15 (m, 2H), 7.19 (d,
2H, J=8 Hz), 7.3 (d, 1H, J=8 Hz), 7.6 (d, 2H, J=8
Hz), 7.72 (d, 2H, J=8 Hz); HRMS calcd for
C32H37ClN2O4S 581.2241, found 581.2224.
4.4.10. 10-(2-Amino-3-methylbenzoyl)-4-[4-[(4-methoxy-
phenyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (22). Mp
150 ꢂC (dec), 1H NMR (300 MHz, CDCl3) d 1.2–2.1 (m,
11H), 2.15 (m, 3H), 2.4–2.6 (m, 3H), 2.6–2.8 (m, 6H),
3.8 (s, 3H), 4.2 (br s, 2H), 6.6 (m, 1H), 6.9 (m, 3H), 7.05
(m, 1H), 7.2 (m, 2H), 7.78 (d, 2H, J=7 Hz), 7.82 (d, 2H,
J=7 Hz); HRMS calcd for C32H39N3O4S 562.2739,
found 562.2734.
4.4.3. 10-(2-Methylbenzoyl)-4-[4-[(3-chlorophenyl)sulfo-
nyl]phenyl]methyl]-1,40-bipiperidine (15). 1H NMR
(300 MHz, CDCl3) d 1.2–1.8 (m, 6H), 1.8–2.2 (m, 4H),
2.25 (m, 3H), 2.50 (m, 4H), 2.70 (m, 2H), 2.90 (m, 4H),
7.0–7.3 (m, 6H), 7.3–7.55 (m, 2H), 7.80 (m, 3H), 7.90 (s,
1H), HRMS calcd for C31H35ClN2O3S 551.2135, found
551.2140.
4.4.4. 10-(3-Chloro-2-methylbenzoyl)-4-[4-[(3-chlorophe-
nyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (16). 1H
NMR (300 MHz, CDCl3) d 1.1–1.2 (m,11H), 2.22,2.30
(two s, 3H), 2.4–2.6 (m, 3H), 2.65–2.8 (m, 1H), 2.8–3.0
(m, 3H), 3.40–3.50 (m, 1H), 4.8 (m, 1H), 6.95–7.2 (m,
2H), 7.25 (d, 2H, J=8 Hz), 7.3–7.5 (m, 3H), 7.77–7.82
(m, 3H), 7.9 (s, 1H);. HRMS calcd for C31H34Cl2N2O3S
585.1745, found 585.1744.
4.4.11. 10-(2-Amino-3-methylbenzoyl)-4-[4-[(3-chlorophe-
nyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (23). 1H
NMR (300 MHz, CDCl3) d 1.30–1.90 (m, 11H), 2.05–
215 (m, 2H), 2.18 (s, 3H), 2.47 (m, 1H), 2.58 (d, 2H,
J=7 Hz), 2.80–2.95 (m, 4H), 4.28 (m, 2H), 6.66 (dd, 1H,
J=7.7 Hz), 6.92 (d, 1H, J=7 Hz), 7.05 (d, 1H, J=7
Hz), 7.28 (m, 2H), 7.43 (dd, 1H, J=7.7 Hz), 7.52 (m,
1H), 7.82 (m, 3H), 7.92 (s, 1H). Anal:
.
.
(C31H36ClN3O3S 2HCl H2O) C,H,N,S,Cl.
4.4.5. 10-(2-Chloro-3-methylbenzoyl)-4-[4-[(3-chlorophe-
nyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (17). 1H
NMR (300 MHz, CDCl3) d 1.1–2.1 (m, 11H), 2.31 (s,
4H), 2.4–2.6 (m, 4H), 2.6–3.1 (m, 3H), 3.38–3.42 (m,
1H), 6.95–7.3 (m, 5H), 7.35–7.5 (m, 2H), 7.71–7.8 (m,
3H), 7.9 (s, 1H); HRMS calcd for C31H34Cl2N2O3S
585.1745, found 585.1747.
4.4.12. 10-(2-Amino-3-chlorobenzoyl)-4-[4-[(4-methoxy-
phenyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (24). Mp
130 ꢂC (dec), H NMR (300 MHz, CDCl3) d 1.20–1.70
1
(m, 7H), 1.80–1.90 (m, 2H), 2.08 (m, 2H), 2.40–2.60 (m,
3H), 2.70–3.00 (m, 6H), 3.8 (s, 3H), 4.7 (s, 2H), 6.66 (dd,
1H, J=7.7 Hz), 6.92 (m, 3H), 7.18–7.25 (m, 3H), 7.78
(dd, 2H, J=7 Hz), 7.85 (dd, 2H, J=7 Hz); HRMS calcd
for C31H36ClN3O4S 582.2193, found 582.2204.
4.4.6. 10-(2-Hydroxybenzoyl)-4-[4-[(4-methoxyphenyl)sul-
fonyl]phenyl]methyl]-1,40-bipiperidine (18). 1H NMR
(300 MHz, CDCl3) d 1.1–1.3 (m, 3H), 1.4–1.7 (m, 5H),
1.8–1.9 (m, 2H), 2.0–2.15 (m, 2H), 2.45–2.55 (m, 2H),
2.8–3.0 (m, 4H), 3.81 (s, 3H), 4.3–4.45 (m, 2H), 6.9–7.0
(m, 3H), 7.15–7.35 (m, 5H), 7.77 (d, 2H, J=8 Hz), 7.85
(d, 2H, J=8 Hz); HRMS calcd for C31H37N2O5S
549.2423, found 549.2408.
4.4.13. 10-(2-Amino-3-chlorobenzoyl)-4-[4-[(3-chlorophe-
nyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (25). Mp
133 ꢂC (dec), H NMR (300 MHz, CDCl3) d 1.20–1.70
1
(m, 8H), 1.80–1.90 (m, 2H), 2.00–2.15 (m, 2H), 2.40–2.60
(m, 3H), 2.70–3.00 (m, 5H), 4.66 (s, 2H), 6.55–6.65 (m,
1H), 6.90–6.98 (m, 1H), 7.20–7.30 (m, 3H), 7.36–7.55 (m,

J. W. Clader et al. / Bioorg. Med. Chem. 12 (2004) 319–326
325
2H), 7.80–7.85 (m, 3H), 7.9 (s, 1H); HRMS calcd for
C30H33Cl2N3O3S 586.1698, found 586.1698.
D.; Vice, S.; Zhao, H.; Zhou, G.; Billard, W.; Binch, H.;
Crosby, R.; Duffy, R.; Lachowicz, J.; Coffin, V.; Watkins,
R.; Ruperto, V.; Strader, C.; Taylor, L.; Cox, K. Farmaco
2001, 56, 247.
4.4.14. 10-(2-Amino-3-fluorobenzoyl)-4-[4-[(4-methoxy-
2. Clader, J. W. Curr. Opin. Drug Discov. Develop. 1999, 2, 311.
3. McCombie, Stuart W.; Lin, Sue-Ing; Tagat, Jayaram R.;
Nazareno, D.; Vice, S.; Ford, J.; Asberom, T.; Leone, D.;
Kozlowski, Joseph A.; Zhou, G.; Ruperto, Vilma B.;
Duffy, Ruth A.; Lachowicz, Jean E. Bioorg. Med. Chem.
Lett. 2002, 12, 795.
4. Kozlowski, Joseph A.; Zhou, Guowei; Tagat, Jayaram R.;
Lin, Sue-Ing; McCombie, Stuart W.; Ruperto, Vilma B.;
Duffy, Ruth A.; McQuade, Robert A.; Crosby, Gordon;
Taylor, Lisa A.; Billard, W.; Binch, H.; Lachowicz, Jean
E. Bioorg. Med. Chem. Lett. 2002, 12, 791.
5. Wang, Y. C.; Samuel, Hu Z.; McKittrick, Brian A.;
Greenlee, W.; Ruperto, V.; Duffy, Ruth A.; Lachowicz,
Jean E. Bioorg. Med. Chem. Lett. 2002, 12, 1087.
6. Wang, Y.; Chackalamannil, S.; Chang, W.; Greenlee, W.;
Ruperto, V.; Duffy, R. A.; McQuade, R.; Lachowicz, J. E.
Bioorg. Med. Chem. Lett. 2001, 11, 891.
7. Boyle, C. D.; Chackalamannil, S.; Clader, J. W.; Green-
lee, W. J.; Josien, H. B.; Kaminski, J. J.; Kozlowski, J. A.;
McCombie, S. W.; Nazareno, D. V.; Tagat, J. R.; Wang,
Y.; Zhou, G.; Billard, W.; Crosby, G.; Cohen-Williams,
M.; Coffin, V. L.; Cox, K. A.; Grotz, D. E.; Duffy, R. A.;
Ruperto, V.; Lachowicz, J. E. Bioorg. Med. Chem. Lett.
2001, 11, 2311.
8. Boyle, C. D.; Chackalamannil, S.; Chen, L.-Y.; Dugar, S.;
Pushpavanam, P.; Billard, W.; Binch, H.; Crosby, G.;
Cohen-Williams, M.; Coffin, V. L.; Duffy, R. A.;
Ruperto, V.; Lachowicz, J. E. Bioorg. Med. Chem. Lett.
2000, 10, 2727.
9. Wang, Y.; Chackalamannil, S.; Hu, Z.; Clader, J. W.;
Greenlee, W.; Billard, W.; Binch, H.; Crosby, G.;
Ruperto, V.; Duffy, R. A.; McQuade, R.; Lachowicz, J. E.
Bioorg. Med. Chem. Lett. 2000, 10, 2247.
10. Kozlowski, J. A.; Lowe, D. B.; Guzik, H. S.; Zhou, G.;
Ruperto, V. B.; Duffy, R. A.; McQuade, R.; Crosby, G.;
Taylor, L. A.; Billard, W.; Binch, H.; Lachowicz, J. E.
Bioorg. Med. Chem. Lett. 2000, 10, 2255.
11. Billard, W.; Binch, H.; Bratzler, K.; Chen, L. Y.; Crosby,
G., Jr; Duffy, R. A.; Dugar, S.; Lachowicz, J.; McQuade,
R.; Pushpavanam, P.; Ruperto, V. B.; Taylor, L. A.; Cla-
der, J. W. Bioorg. Med. Chem. Lett. 2000, 10, 2209.
12. Wang, Y.; Chackalamannil, S.; Chang, W.; Greenlee, W.;
Ruperto, V.; Duffy, R. A.; McQuade, R.; Lachowicz, J. E.
Bioorg. Med. Chem. Lett. 2001, 11, 891.
13. Kozlowski, Joseph A.; Zhou, Guowei; Tagat, Jayaram R.;
Lin, Sue-Ing; McCombie, Stuart W.; Ruperto, Vilma B.;
Duffy, Ruth A.; McQuade, Robert A.; Crosby, Gordon;
Taylor, Lisa A.; Billard, W.; Binch, H.; Lachowicz, Jean
E. Bioorg. Med. Chem. Lett. 2002, 12, 791.
14. McCombie, Stuart W.; Lin, Sue-Ing; Tagat, Jayaram R.;
Nazareno, D.; Vice, S.; Ford, J.; Asberom, T.; Leone, D.;
Kozlowski, Joseph A.; Zhou, G.; Ruperto, Vilma B.;
Duffy, Ruth A.; Lachowicz, Jean E. Bioorg. Med. Chem.
Lett. 2002, 12, 795.
15. Wang, Y.; Chackalamannil, S.; Hu, Z.; McKittrick, Brian
A.; Greenlee, W.; Ruperto, V.; Duffy, Ruth A.; Lacho-
wicz, Jean E. Bioorg. Med. Chem. Lett. 2002, 12, 1087.
16. Boyle, Craig D.; Vice, S. F.; Campion, J.; Chacka-
lamannil, S.; Lankin, C. M.; McCombie, S. W.; Billard,
W.; Binch, H.; Crosby, G.; Williams, M.-C.; Coffin, V. L.;
Cox, K. A.; Grotz, D. E.; Duffy, R. A.; Ruperto, V.;
Lachowicz, J. E. Bioorg. Med. Chem. Lett. 2002, 12, 3479.
17. Wang, Y.; Chackalamannil, S.; Hu, Z.; Greenlee, W. J.;
Clader, J.; Boyle, C. D.; Kaminski, J. J.; Billard, W.;
Binch, H., III; Crosby, G.; Ruperto, V.; Duffy, R. A.;
phenyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (26). Mp
ꢂ
1
136 C (dec), H NMR (300 MHz, CDCl3) d 1.20–1.70
(m, 7H), 1.80–1.95 (m, 2H), 2.05–2.20 (m, 2H), 2.45–
2.55 (m, 3H), 2.75–3.00 (m, 6H), 3.81 (s, 3H), 4.35 (s,
2H), 6.55–6.65 (m, 1H), 6.80–6.85 (m, 1H), 6.90–7.00
(m, 3H), 7.18–7.25 (m, 2H), 7.75 (d, 2H, J=8 Hz), 7.85
(d, 2H, J=8 Hz); HRMS calcd for C31H36FN3O4S
566.2489, found 566.249.
4.4.15. 10-(2-Amino-3-fluorobenzoyl)-4-[4-[(3-chlorophe-
nyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (27). Mp
ꢂ
1
138 C (dec), H NMR (300 MHz, CDCl3) d 1.20–1.70
(m, 11H), 1.80–2.20 (m, 3H), 2.40–2.60 (m, 3H), 2.70–
3.10 (m, 3H), 4.35 (s, 2H), 6.60–6.68 (m, 1H), 6.85 (d,
1H, J=8 Hz), 6.95 (dd, 1H, J=8.8 Hz), 7.25 (d, 2H,
J=8 Hz), 7.48 (d, 1H, J=8 Hz), 7.55 (d, 1H, J=8 Hz),
7.81–7.88 (m, 3H), 7.9 (s, 1H); HRMS calcd for
C30H33ClFN3O3S 570.1993, found 570.199.
4.4.16. 10-(2-Amino-4-fluorobenzoyl)-4-[4-[(4-methoxy-
phenyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (28). 1H
NMR (300 MHz, CDCl3) d 1.20–1.65 (m, 8H), 1.80–
1.90 (m, 2H), 2.00–2.20 (m, 2H), 2.40–2.60 (m, 3H),
2.70–3.00 (m, 5H), 3.8 (s, 3H), 4.45 (s, 2H), 6.30–6.40
(m, 2H), 6.91 (d, 2H, J=8 Hz), 6.90–7.05 (m, 1H), 7.15–
7.25 (m, 2H), 7.78 (d, 2H, J=8 Hz), 7.82 (d, 2H, J=8
Hz); HRMS calcd for C31H36FN3O4S 566.2489, found
566.2499.
4.4.17. 10-(2-Amino-4-fluorobenzoyl)-4-[4-[(3-chlorophe-
nyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (29). 1H
NMR (300 MHz, CDCl3) d 1.20–1.70 (m, 8H), 1.75–
1.90 (m, 2H), 2.00–2.20 (m, 2H), 2.40–2.60 (m,3H),
2.70–3.00 (m, 5H), 4.5 (s, 2H), 6.30–6.40 (m, 2H), 6.95–
7.05 (m, 1H), 7.25 (d, 2H, J=8 Hz), 7.40–7.55 (m, 2H),
7.75–7.82 (m, 3H), 7.9 (m, 1H); HRMS calcd for
C30H33ClFN3O3S 570.1993, found 570.1997.
4.4.18. 10-(2-Amino-5-fluorobenzoyl)-4-[4-[(3-chlorophe-
nyl)sulfonyl]phenyl]methyl]-1,40-bipiperidine (30). 1H
NMR (300 MHz, CDCl3) d 1.10–1.70 (m, 8H), 1.70–
1.95 (m, 2H), 2.00–2.20 (m, 2H), 2.40–2.60 (m, 3H),
2.70–3.00 (m, 5H), 4.5 (s, 2H), 6.60–6.70 (m, 1H), 6.70–
6.80 (m, 1H), 6.80–6.90 (m, 1H), 7.25 (d, 2H, J=8 Hz),
7.40–7.60 (m, 2H), 7.77–7.88 (m, 3H), 7.9 (m, 1H);
HRMS calcd for C30H33ClFN3O3S 570.1993, found
570.1997.
Acknowledgements
We are grateful for the help and support of Drs. A. K.
Ganguly and Catherine D. Strader throughout all pha-
ses of this project.
References and notes
1. Greenlee, W.; Clader, J.; Asberom, T.; McCombie, S.;
Ford, J.; Guzik, H.; Kozlowski, J.; Li, S.; Liu, C.; Lowe,

326
J. W. Clader et al. / Bioorg. Med. Chem. 12 (2004) 319–326
Cohen-Williams, M.; Coffin, V. L.; Cox, K. A.; Grotz,
D. E.; Lachowicz, J. E. J. Med. Chem. 2002, 45, 5415.
21. Smith, R. D. K.; Michael, K.; Cohen-Williams, Mary;
Coffin, V. L. Brain Res. 1996, 707, 13.
18. Apreliminary account of this work was presented at the
221st meeting of the American Chemical Society, 2001.
19. For other approaches towards improving the the phar-
macokinetic profiles of piperidine muscarinic M2 antago-
nists, see refs 7–9, 12, 15.
22. Korfmacher, W. A.; Cox, K. A.; Ng Kwokei, J.; Veals, J.;
Hsieh, Y.; Wainhaus, S.; Broske, L.; Prelusky, D.;
Nomeir, A.; White, R. E. Rapid Commun. Mass Spectrom.
2001, 15 (5), 335.
23. Vice, S.; Bara, T.; Bauer, A.; Evans, C.; Anderson, F. J. J.;
Hubert McCombie, S.; Miller, M.; Nazareno, D.; Palani,
A.; Tagat, J. J. Org. Chem. 2001, 66, 2487.
20. Billard, W.; Binch, H., III; Crosby, G.; McQuade, R. D.
J. Pharmacol. Exp. Ther. 1995, 273, 273.