Design, synthesis and structure-activity relationship of N-substituted tropane muscarinic acetylcholine receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.02.015

A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24 displayed much slower reversibility at mAChRs than

Full text of DOI:10.1016/j.bmcl.2012.02.015

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3366–3369
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and structure–activity relationship of N-substituted
tropane muscarinic acetylcholine receptor antagonists
Dramane I. Lainé , Hongxing Yan, Haibo Xie, Roderick S. Davis, Jeremy Dufour,
Katherine L. Widdowson, Michael R. Palovich, Zehong Wan, James J. Foley,
Dulcie B. Schmidt, Gerald E. Hunsberger, Miriam Burman, Alicia M. Bacon,
Edward F. Webb, Mark A. Luttmann, Michael Salmon à, Henry M. Sarau,
⇑
Sandra T. Umbrecht, Philip S. Landis, Brian J. Peck, Jakob Busch-Petersen
GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine
receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24
displayed much slower reversibility at mAChRs than the methyl-substituted parent molecule darotropi-
um. In addition, it was shown that this characteristic appeared to translate into enhanced which duration
of action in a mouse model of bronchonstriction.
Received 15 December 2011
Revised 3 February 2012
Accepted 6 February 2012
Available online 15 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Inhaled
Muscarinic antagonist
COPD
Tropane
Quaternary ammonium salts
Muscarinic acetylcholine receptors (mAChRs) belong to the
superfamily of G-protein coupled 7-transmembrane (TM) recep-
tors. Five subtypes of mAChRs, termed M1–M5, have been identi-
fied to-date.1,2 The mAChRs are widely distributed in mammalian
organs where they mediate many of the vital functions.1–3 In the
lungs, mAChRs have been localized to smooth muscle in the tra-
chea and bronchi, the submucosal glands, and the parasympathetic
ganglia.4 The three subtypes of mAChRs which are known to exert
their physiological effect in the lungs through the action of the na-
tive ligand, acetylcholine (Ach), are the M1, M2 and M3 receptors.4
The M3 mAChRs, are located on the airway smooth muscle and also
on the pulmonary submucosal glands where they mediate muscle
contraction and mucus secretion, respectively.5,6 The M2 mAChRs,
which make up the majority of the cholinergic receptor population
on airway smooth muscle, are also located on postganglionic para-
sympathetic nerves,7 where their role is autoinhibitory, to provide
tightly regulated control of acetylcholine release. M1 mAChRs are
found in the pulmonary parasympathetic ganglia where they func-
tion to facilitate neurotransmission.8
mAChR dysfunction in the lungs has been noted in a variety of
different pathophysiological states.9 In particular, in asthma and
chronic obstructive pulmonary disease (COPD), inflammatory
conditions lead to loss of inhibitory M2 mAChR autoreceptor func-
tion on parasympathetic nerves supplying the pulmonary smooth
muscle, causing increased acetylcholine release following vagal
nerve stimulation.10 This mAChR dysfunction results in airway
hyperreactivity and hyperresponsiveness mediated by increased
stimulation of M3 mAChRs. Thus, mAChR antagonists are useful
therapeutics in mAChR-mediated disease states and particularly
COPD. Inhaled anticholinergic agents approved for the treatment
of COPD include ipratropium bromide, oxitropium bromide and
more recently tiotropium bromide.11 Ipratropium and oxitropium
have relatively short durations of action (4–8 h) whereas tiotropi-
um has a duration of action of over 24 h, making it suitable for
once-daily treatment.12 As part of a general strategy to develop
new respiratory products, our goal was to discover novel long-
acting muscarinic antagonists. The investigation of several series
of muscarinic antagonists developed within our laboratory has
previously been disclosed.13–18 Darotropium bromide 1 (Fig. 1),
in particular, has been reported as a very potent mAChR antagonist
with long duration of action in a mice.13,14 However, further work
⇑
Corresponding author. Tel.: +1 610 270 7831; fax: +1 610 270 4451.
E-mail address: jakob.2.busch-petersen@gsk.com (J. Busch-Petersen).
Present address: Hoffmann-La Roche Inc. 340 Kingsland Street, Nutley, NJ 07110,
USA.
à
Present address: Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston,
MA 021115, USA.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.015

D. I. Lainé et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3366–3369
3367
Table 1
Br
Tropane N-substitution-initial SAR
N+
X
H
CN
R
N+
1
Ph
N
Ph
Figure 1. Darotropium bromide.
was carried out in order to enhance the understanding of the inter-
play between molecular structure, potency, receptor kinetics and
duration of action. In this letter we report the structural–activity
relationships (SAR) at the M3 receptor of a N-substituted series of
analogs of 1.
a
b
Entry
Stereochemistry
R
X
M3 IC50
M3 pA2
1
3
4
5
6
7
8
9
10
11
12
13
14
15
20
21
22
23
–Me
Br
Br
Br
Br
Br
I
Br
Br
Br
Br
Br
Br
Br
Br
<10
<10
<10
<10
<10
<10
<10
<10
89.6
318
109
85.2
<10
64
10.7
10.3
10.3
10.3
9.8
8.9
9.3
9.8
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
Mixed
N-Endo
N-Exo
N-Endo
N-Exo
–CH2–cPr
–Et
–Pr
–(CH2)2OH
Two complementary methods of preparation of the differen-
tially substituted quaternary salts are shown in Scheme 1. The pre-
viously described nitrile 213 was treated with an alkyl halide (other
than a methylating agent) in the presence of a base to give quater-
nary salts 3–15 as a mixture of two isomers. One of the compounds
formed possessed the pending chain on the opposite side as the
C-3 substituent (referred to as N-Exo) whereas the other isomer
had the chain on the same side of the C-3 substituent (referred
to as N-Endo). Although synthetically convenient, this approach
did not prove to be very selective and variable ratio of both isomers
were obtained. Subsequent SAR studies, however, showed that the
N-Endo derivatives were the more potent of the two isomers. Con-
sequently, a more appropriate route to the preparation to these
isomers was designed. Treatment of the known alcohol 16 with
iodine,19 followed by reaction with diphenylacetonitrile and a base
afforded the nitrile derivative 5. Removal of the benzyl group
–CH2CH@CH2
–(CH2)2OMe
–(CH2)3CN
–CH2–cHex
–CH2–Ph
–(CH2)2cHex
–(CH2)4CH@CH2
–(CH2)2O(CH2)2OMe
–(CH2)11CH3
9.2
–CH2–cPr
<10
26.3
<10
32
10.6
–CH2–cPr
–(CH2)4CH@CH2
–(CH2)4CH@CH2
>11
a
Functional assays were performed on CHO cells expressing cloned human M3
receptors. Cells were stimulated with ACh. Calcium mobilization was measured
using the standard FLIPR protocol.22 Values are in nM and are a mean of two or
more independent assays.
b
The pA2 value was determined by measuring the ratio of the ACh EC50 in the
presence and absence of compound.21
either by hydrogenation or via
a
quaternization/dealkylation
Bn
N
N
H
H
CN
Ph
OH
Ph
16
2
b, c
a
R
R
Bn
R
N+
N+
N+
X
X
N
X
f
H
CN
H
H
H
+
CN
CN
Ph Ph
N-Exo
CN
Ph
Ph
Ph
17
Ph
3-15
Ph Ph
N-Endo
20, 22, 24-32
21, 23
e
d
R
N
H
N
a
H
H
CN
Ph
CN
Ph
Ph
18
Ph
19
Scheme 1. Preparation of mixed and stereospecific quaternary ammonium salts. Reagents and conditions: (a) RX, K2CO3; (b) I2, PS-PPh3, DCM; (c) PhCH(CN)Ph, NaH, DMF; (d)
ClCO2CHClCH3, mw, 140 °C or H2, 10% Pd(OH)2; (e) MeX, K2CO3; (f) separation by chromatography.

3368
D. I. Lainé et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3366–3369
Table 2
of 1 was replaced with a variety of substituents was quickly syn-
M3 activities of N-Endo derivatives
thesized. At that point, and chiefly for practical reasons, the com-
pounds were tested as mixtures of diasterosiomers of unknown
composition (Table 1). Generally, it was observed that the intro-
duction of relatively small groups, such as ethyl, propyl or cyclo-
propylmethyl gave compounds with slightly decreased potency
compared to 1 (3–5). Elongation of the chain by one of two atoms
further decreased the compound potencies by about half a log unit
(6–9). The activity of compound containing cyclohexyl or phenyl
rings at the terminal chain position was also markedly reduced
(10–12). Finally, we found out that the introduction of chains
containing 6 or more carbon atoms did not increase compound
activity either (13–15). Although, this first round of SAR was some-
what disappointing, we decided to proceed with the separation of
the isomeric mixture of the most potent compound, 3, in the hope
that one of the diasteroisomers could be differentiated with re-
gards to potency. Indeed this proved to be the case as the isomer
20 with the larger group pointing towards the same side as the
biphenylcyano moiety (N-Endo) was found to be almost equipotent
to 1. Positioning the substituent in the opposite direction (N-Exo),
however, led to a sharp lost of potency (21). Similar results were
obtained for the alkene derivative 13. In that case, the N-Endo
derivative, 22, was found to be much more potent than 13, sug-
gesting that this isomer was only present as a minor component
of the original mixture. As with the cyclopropyl derivatives, the
N-Exo isomer 23 was markedly less potent than the N-Endo isomer.
These results clearly indicated the importance of the stereochem-
istry at the tropane nitrogen and suggested that the testing of
the compounds as diasterosiomeric mixtures was not the most
appropriate way to proceed.
X
R
N+
N
a
b
Entry
R
X
M3 IC50
M3 pA2
20
24
25
26
27
28
29
30
31
32
–CH2–cPr
Br
Br
I
Br
Br
Br
Br
Br
Br
Br
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
10.6
>11
>11
>11
>11
>11
>11
>11
10.4
10.1
–(CH2)4CH@CH2
–(CH2)2OCH2Ph
–(CH2)3Ph
–(CH2)3OPh
–(CH2)3CH3
–(CH2)5CH3
–(CH2)2OPh
–(CH2)3OCH2Ph
–(CH2)7CH3
a
Functional assays were performed on CHO cells expressing cloned human M3
receptors. Cells were stimulated with ACh. Calcium mobilization was measured
using the standard FLIPR protocol.22 Values are in nM and are a mean of two or
more independent assays.
b
The pA2 value was determined by measuring the ratio of the ACh EC50 in the
presence and absence of compound.21
two-steps process, led to the secondary amine 17.20 The larger sub-
stituent was attached onto 17 by treatment with the appropriate
alkyl halide to give the tertiary amine 18. Subsequent methylation
of 18 delivered a mixture of the two diastereoisomeric salts which
could be separated by chromatography to give the N-Endo deriva-
tives (24–32) as the major products and the N-Exo derivatives as
the minor products. Using this approach, we found that the selec-
tivity was generally high for the desired N-Endo derivative (80–
90%). This selectivity probably originated from the preferential
approach of the electrophile from the least hindered face.
The compound inhibitory potency (IC50 of an EC80 concentration
of Ach) at cloned human M3 receptors was first determined by cal-
cium mobilization assays. When a compound IC50 was lower than
10 nM, antagonist potency was further quantified by a pA2.21
In order to probe the initial SAR for potency around the N-
substitution, a series of derivatives where one of the methyl groups
The next step of our investigation was to further explore the
SAR of the N-Endo derivatives. The compounds were prepared
using the chemical route via the secondary amine 18, as previously
described. As shown in Table 2, a wide variety of groups were tol-
erated at that position. It appears that the receptor pocket accom-
modating these substituents can tolerate linear alkyl chains of up
to 7 carbon atoms. In fact, the majority of the compounds were
so potent that their pA2’s could not be accurately determined using
this assay and they are simply listed as >11.
To further assess the pharmacological profile of the series the
N-Endo derivatives, one exemplar, 24 was chosen for additional
evaluation and comparison with the previously reported profile
Table 3
Comparison 24 versus 1
Properties
1
24
Binding affinitiesa
M1 (nM)
M2 (nM)
M3 (nM)
0.07 0.01
0.17 0.01
0.06 0.02
10.0 0.2
0.12 0.02
0.10 0.02
0.16 0.02
NCc
>600
0.02
>96 (0.05)
125 14
NQ
b
In vitro tissue studies
Mouse in vivo studies
Rat PK properties
pA2
Off t50 (min)d
85
5
ED50
(l
g/mouse)e
0.01
f
Hours duration (dose (
Cl (ml/min/kg)g
F (%)h
l
g/mouse))
ꢀ48 (0.5)
74 21
NQi
a
Radioligand binding assays were conducted using CHO cell membrane preparations in SPA format versus 0.5 nM [3H]-N-methyl scopolamine. Values are the mean of
three independent assays.
b
Potency against carbachol-induced contraction in human bronchus.
NC = non competitive.
Reversal time for 50% of carbachol-induced contraction to return to maximum relaxation in human bronchus after incubation at [10 nM] antagonist (The human
c
d
biological samples were sourced ethically and their research use was in accord with the terms of the informed consents).
e
Potency in conscious mice against MCh-induced bronchoconstriction (n = 1).
Duration of bronchoprotection in conscious mice against methacholine-induced bronchoconstriction. Time to 50% loss of maximum protection.
Cl – systemic plasma clearance. iv doses: 1 mg/kg.
F – percent bioavailability. po doses: 2 mg/kg.
f
g
h
i
NQ = no quantifiable plasma levels following oral administration. (All studies were conducted after review by the GSK Institutional Animal Care and Use Committee and
in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals.).

D. I. Lainé et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3366–3369
3369
and low bioavailability; two properties considered desirable for a
drug delivered by inhalation.
In conclusion, a novel series of N-Endo tropane derivatives was
characterized as potent M3 mAChR antagonists. Kinetic washout
studies at the human M3 cloned receptor and in human bronchus
showed that analog 24 displayed slower reversibility at the mACh-
Rs than the parent molecule 1. This profile translated in a much
longer in vivo duration of action for 24 compared to 1. Taken to-
gether, these results suggest that the introduction of alkyl chains
on the tropane nitrogen of 1 has created a novel class of mAChR
antagonists with a better likelihood to achieve prolonged duration
of bronchodilation in humans. Further studies of these compounds
will be reported elsewhere.
Figure 2. Concentration-dependent shifts in ACh concentration response curve
after 90 min incubation with 24 and reversibility following a 180 min wash-out at
the human M3 receptor.
References and notes
1. Caulfield, M. P.; Birdsall, N. J. M. Pharmacol. Rev. 1998, 50, 279.
2. Eglen, R. M. Prog. Med. Chem. 2005, 43, 105.
3. Caulfield, M. P. Pharmacol. Ther. 1993, 58, 319.
4. Lee, A. M.; Jacoby, D. B.; Fryer, A. D. Cur. Opin. Pharmacol. 2001, 1, 223.
5. Barnes, P. J. Thorax 1989, 44, 161.
6. Gater, P. J.; Alabastar, V. J.; Piper, I. Pulm. Pharmacol. 1989, 2, 87.
7. Faulkner, D.; Fryer, A. D.; MacLagan, J. Br. J. Pharmacol. 1986, 88, 181.
8. Lammers, J.-W. J.; Minette, P.; McCusker, M.; Barnes, P. J. Am. Rev. Respir. Dis.
1989, 139, 446.
9. Hay, D. W. P. Curr. Opin. Chem. Biol. 2000, 4, 412.
10. Fryer, A. D.; Adamko, D. J.; Yost, B. L.; Jacoby, D. B. Life Sci. 1999, 64, 449.
11. Gross, J. N. Eur. J. Pharmacol. 2006, 533, 36.
12. Busch-Petersen, J.; Laine, D. I. Future Med. Chem. 2011, 3, 1623.
13. Wan, Z.; Laine, D. I.; Yan, H.; Zhu, C.; Widdowson, K. L.; Buckley, P. T.; Burman,
M.; Foley, J. J.; Sarau, H. M.; Schmidt, D. B.; Webb, E. F.; Belmonte, K. E.;
Palovich, M. R. Bioorg. Med. Chem. Lett. 2009, 19, 4560.
14. Laine, D. I.; Wan, Z.; Yan, H.; Zhu, C.; Xie, H.; Fu, W.; Busch-Petersen, J.; Neipp,
C.; Davis, R.; Widdowson, K. L.; Blaney, F. E.; Foley, J.; Bacon, A. M.; Webb, E. F.;
Luttmann, M. A.; Burman, M. A.; Burman, M.; Sarau, H. M.; Salmon, M.;
Palovich, M. R.; Belmonte, K. E. J. Med. Chem. 2009, 52, 5241.
15. Laine, D. I.; Xie, H.; Buffet, N.; Foley, J. J.; Buckey, P. T.; Webb, E. F.; Widdowson,
K. L.; Palovich, M. R.; Belmonte, K. E. Bioorg. Med. Chem. Lett. 2007, 17, 6066.
16. Jin, J.; Wang, Y.; Shi, D.; Wang, F.; Davis, R. S.; Jin, Q.; Fu, W.; Foley, J. J.; Webb, E.
F.; Dehaas, C. J.; Berlanga, M.; Burman, M.; Sarau, H. M.; Morrow, D. M.; Rao, P.;
Kallal, L. A.; Moore, M. L.; Rivero, R. A.; Palovich, M.; Salmon, M.; Belmonte, K.
E.; Busch-Petersen, J. J. Med. Chem. 2008, 51, 4866.
17. Jin, J.; Budzik, B.; Wang, Y.; Shi, D.; Wang, F.; Xie, H.; Wan, Z.; Zhu, C.; Foley, J. J.;
Webb, E. F.; Berlanga, M.; Burman, M.; Sarau, H. M.; Morrow, D. M.; Moore, M.
L.; Rivero, R. A.; Palovich, M.; Salmon, M.; Belmonte, K. E.; Laine, D. I. J. Med.
Chem. 2008, 51, 5915.
18. Laine, D. I.; McCleland, B.; Thomas, S.; Neipp, C.; Underwood, B.; Dufour, J.;
Widdowson, K. L.; Palovich, M. R.; Blaney, F. E.; Foley, J. J.; Webb, E. F.;
Luttmann, M. A.; Burman, M.; Belmonte, K.; Salmon, M. J. Med. Chem. 2009, 52,
2493.
of 1 (Table 3).14 The selection was made on the basis of the ability
of 24 to form a stable crystalline form with a high melting point
(mp >200 °C), which is considered a desirable property for a dry
powder inhaled drug as it may help prevent polymorphic transfor-
mation or chemical degradation during manufacturing processes
such as micronisation or spray-drying.23 In radioligand binding
studies, 24 was found to be comparably potent to 1 and competed
with [3H]-N-methylscopolamine binding to M3 mAChR (Ki of
0.10 nM). No clear selectivity for M3 over the two other receptor
subtypes was observed for both molecules. Studies to evaluate
the reversibility of 24 at the M3 receptor were also conducted in
the FLIPR assay using a 30 min incubation interval followed by a
180 min washout period. As shown in Figure 2, following the
washout, acetylcholine concentration–response curves did not
fully reverse to levels obtained in the absence of drug at any con-
centration of the compound tested. Similar findings were obtained
at endogenously expressed M3 mAChRs. Thus, in human bronchus
kinetic studies, 24 was found to have a very long off rate (off
t50 >600 min), which was considerably longer than 1 (85 min).
Taken together, these data indicate that antagonist blockade re-
mained after washout, suggesting that 24 is very slowly reversible
at the M3 receptor. To examine the impact of the compound kinet-
ics on in vivo duration of action, 24 was further characterized in a
mouse plethysmography model. Both compounds exhibited
similar potency when dosed intranasally with ED50’s of 0.02 and
19. Laine, D. I.; Palovich, M. R.; Preston, A. G.; Cooper, Anthony W.J.
WO2005067537.
0.01
lg per animal for 24 and 1, respectively. However, when
dosed at the ED80 (0.05
lg/mouse), 24 displayed considerable long-
20. Wan, Z.; Yan, H.; Palovich, M. R.; Laine, D. I. WO2005046586.
21. Arunlakshana, O.; Schild, H. O. Br. J. Pharmacol. 1959, 14, 48–58.
22. Sarau, H. M.; Ames, R. S.; Chamber, J.; Ellis, C.; Elshourbagy, N.; Foley, J. J.;
Schmidt, D. B.; Muccitelli, R. M.; Jenkins, O.; Murdock, P. R.; Herrity, N. C.;
Halsey, W.; Sathe, G.; Muir, A. I.; Nuthulaganti, P.; Dytko, G. M.; Buckley, P. T.;
Wilson, S.; Bergsma, D. J.; Hay, D. W. P. Mol. Pharmacol. 1999, 56, 657.
23. Tong, H. H. Y.; Chow, A. H. L. KONA 2006, 24, 27.
er duration of bronchoprotection (>96 h) than 1 (ꢀ48 h) even
though the latter was given at a dose which exceeded the ED90
(0.5 lg/mouse). This is most likely due to the slower target off-rate
of 24. From a pharmacokinetic viewpoint, both compounds are
characterized in the art as having very high systemic clearance