Discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide as an efficacious inhaled muscarinic acetylcholine receptor antagonist for the treatment of COPD

Article abstract of DOI:10.1016/j.bmcl.2009.07.006

Design and syntheses of a novel series of muscarinic antagonists are reported. These efforts have culminated in the discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide (4a) as a potent and pan-active musca

Full text of DOI:10.1016/j.bmcl.2009.07.006

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4560–4562
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabi-
cyclo[3.2.1]octane bromide as an efficacious inhaled muscarinic acetylcholine
receptor antagonist for the treatment of COPD
*
Zehong Wan , Dramane I. Laine, Hongxing Yan, Chongjie Zhu, Katherine L. Widdowson, Peter T. Buckley,
Miriam Burman, James J. Foley, Henry M. Sarau, Dulcie B. Schmidt, Edward F. Webb,
Kristen E. Belmonte, Michael Palovich
Respiratory Centre of Excellence for Drug Discovery, Research and Development, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, PO Box 1539, 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:
Design and syntheses of a novel series of muscarinic antagonists are reported. These efforts have
culminated in the discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo-
[3.2.1]octane bromide (4a) as a potent and pan-active muscarinic antagonist as well as a functionally active
compound in a murine model of bronchoconstriction. The compound has also displayed pharmacokinetic
characteristics suitable for inhaled delivery.
Received 17 March 2009
Revised 26 June 2009
Accepted 2 July 2009
Available online 8 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Muscarinic
Antagonist
COPD
Tropane
Chronic obstructive pulmonary disease (COPD) is a progressive
respiratory disorder that causes significant deterioration of lung
function and chronic breathlessness.1 Six hundred million people
worldwide already live with COPD, but its prevalence is predicted
to rise to become the world’s third leading cause of death by 2020.2
Rather than a single pathologic condition, COPD is an umbrella
term encompassing chronic bronchitis, emphysema and small air-
ways disease.3 Bronchodilator drugs form a preferred choice of
COPD maintenance therapy with two principle classes of broncho-
dilation agent widely prescriped,4,5 b2-adrenoceptor agonists such
as SereventÒ (salmeterol xinafoate) and muscarinic acetylcholine
receptor (mAChR) antagonists such as SpirivaÒ (tiotropium bro-
mide).6,7 There are five sub-types (M1–M5) mAChRs playing a num-
ber of different pharmacological roles both centrally and
peripherally.8–10 As such, it would be beneficial to deliver an
anti-cholinergic to the lung that was not systemically active. Both
M2 and M3 mAChRs are located in smooth muscle and mucosal
glands and have been considered as a therapeutical targets for
COPD treatment.11–14 Herein, we disclose the design and syntheses
of a novel series of long-acting mAChR antagonists as exemplified
by (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoni-
abicyclo[3.2.1]octane bromide (4a).
Compounds described in this paper were synthesized from two
known intermediates (Schemes 1 and 2): 3-hydroxymethyltropane
(1) and 1,1-diphenyl-2-(3-tropanyl)ethanol (2).15,16 Starting from
1, iodination followed by alkylation of the resultant 3-iodomethyl-
tropane (3) with diphenylacetonitrile in the presence of NaH pro-
vided nitrile 4. Reduction of 4 with borane furnished the primary
amine 5, which was utilized to prepare a small array of compound
N
N
N
H
III
H
H
II
NH2
Ph
CN
Ph
R
4
5
1: R = CH2OH
3: R = CH2I
I
Ph
H
Ph
VI
V
IV
N
+
N
Br
N
H
H
R1
Ph
Ph
N
4a
CN
Ph
6a-f
7
H
Ph
Ph
Ph
Ph
Scheme 1. Preparation of muscarinic antagonists. Reagents and conditions: (I) I2,
PPh3, 77%; (II) Ph2CHCN, NaH, 93%; (III) BH3ÁTHF, 63%; (IV) (6a) ClSO2NCO, 38%; (6b)
PhCH2NCO 19%; (6c) CH3CH2NCO, 45%; (6d) Ac2O, 29%; (6e) PhSO2Cl, 54%; (6f)
MeSO2Cl, 28%; (V) Ph3CH, n-Buli, DMF, 17%; (VI) CH3Br.
* Corresponding author at present address: GlaxoSmithKline R&D China, Bldg 3,
898 Halei Road, Pudong, Shanghai 201203, China.
E-mail address: Zehong.2.Wan@gsk.com (Z. Wan).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.006

Z. Wan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4560–4562
4561
lene) linked with a tertiary amine (e.g., tropane), two key structural
features in well-documented muscarinic antagonists.18 The lipo-
philic part has been considered to drive interactions with the
receptors, while quaternization of the tertiary amine was utilized
to improve anti-muscarinic activities as well as to reduce side ef-
fects related to CNS penetration.19 We therefore started by search-
ing for appropriate replacements of the tertiary –OH that is directly
attached to the lipophilic unit in the lead with the purpose of
improving its potency against M3 receptors via modulating lipo-
philicity. Topological polar surface area (TPSA),20 a calculated prop-
erty that has been widely used to assess molecular interactions
with transport membranes, was later found to exhibit better corre-
lation with the lipophilicity of this chemical series than log D or
log P. For examples, insertion of an additional methylene group
does not change TPSA and the resultant primary alcohol 10 is as
potent as 2. Replacement with a tertiary nitrile (e.g., 4) slightly in-
creases TPSA value, but the resultant compound is ten fold more
potent as demonstrated later in the binding assays. Dramatic mod-
ifications of the polar surface area have been detrimental to po-
tency as evidenced by ureas 6a–c, amide 6d, sulfonamides 6e–f,
triphenylmethane 7, acid 8, and amide 9.
The compound inhibitory potency, IC50 of an EC80 concentration
of ACh, as well as a determination of any direct stimulation of the
receptors by the compounds was first determined using a standard
FLIPR protocol.22,23 Binding affinities of analogs with IC50 less than
10 nM in the FLIPR assays were measured in radioligand binding
assays. Compounds 2, 2a, 4, 4a, 10 and 10a all display high affini-
ties (Ki) with the three receptors. All compounds were devoid of
any agonist activities (data not shown). The antagonist potency
of all high potency compounds (IC50 <10 nM) was also further
quantified on FLIPR (pA2 determination) by measuring the ratio
of the ACh EC50 in presence and absence of a single concentration
N
N
N
H
H
H
H
I
III
2
8
OH
Ph
10
CO2H
Ph
OH
Ph
Ph
IV
Ph
IV
II
Ph
Ph
+
N
N
+
N
Br
Br
H
H
CONHBn
Ph
OH
Ph
2a
9
OH
Ph
10a
Ph
Ph
Scheme 2. Preparation of muscarinic antagonists. Reagents and conditions: (I)
HCO2H, H2SO4, 48%; (II) PhCH2NH2, EDC, HOBt, TEA, 30%; (III) LAH, THF, microwave,
100 °C, 71%; (IV) CH3Br.
including ureas 6a–c; amide 6d; and sulfonamides 6e–f. Alkylation
of 3 with triphenyl methane was studied under various conditions
furnishing compound 7 with a modest yield. This is very likely due
to the steric hindrance exhibited by the three phenyl groups. Acid 8
was prepared by mixing 2 with formic acid and concentrated sul-
furic acid in a freezer (À20 °C) for one week.17 Coupling with ben-
zyl amine afforded amide 9. Microwave assisted reduction of 8
with LiAlH4 furnished the primary alcohol 10. Quaternization of
the tertiary amine 2, 4, or 10 with CH3Br then gave rise to the cor-
responding quaternary salt 2a, 4a and 10a.
Screening of the GSK collection of compounds based on the
topography of the binding site of the muscarinic receptors M1–
M3, we uncovered the potent and pan-active antagonist 2 (Table 1).
This compound also shows high binding affinity for the three
receptors. It contains a bulky lipophilic unit (e.g., diphenyl methy-
Table 1
Evaluation of tropane series of muscarinic antagonists in vitro and in vivo
+
X
N
H
Ph
R
Ph
Compd
R
TPSAa
Xb
FLIPR (IC50, nM)c
M2
Binding affinity (pKi)d
FLIPR kinetics (pA2)e
In vivo
Inh.f
M1
M3
M1
M2
M3
M1
M2
M3
2
2a
4
–OH
–OH
–CN
–CN
23.5
—
27.0
—
—
Br
—
Br
—
—
—
—
—
—
—
—
—
—
Br
<10
<10
<10
<10
3829
1017
1276
2198
770
1597
105
277
<10
<10
<10
<10
5345
1273
1553
3478
458
2119
1195
240
<10
<10
<10
<10
6399
1338
1939
2895
1059
2732
206
9.3
8.8
10.0
10.3
—
—
—
—
—
—
—
—
—
9.0
8.9
8.2
8.4
9.3
9.8
—
—
—
—
—
—
—
—
—
7.9
8.4
8.7
8.5
9.8
10.2
—
—
—
—
—
—
—
—
—
8.6
8.6
9.1
9.6
10.8
11.4
—
—
—
—
—
—
—
—
—
8.9
8.8
8.4
9.3
9.5
10.3
—
—
—
—
—
—
—
—
—
8.2
8.3
8.6
9.3
10.2
10.7
—
—
—
—
—
—
—
—
—
8.4
8.5
<5%g
34%
11%
>95%
—
—
—
—
—
—
—
—
—
15%
23%
4a
6a
6b
6c
6d
6e
6f
7
8
9
10
10a
–CH2NHC(O)NH2
–CH2NHC(O)NHBn
–CH2NHC(O)NHEt
–CH2NHC(O)CH3
–CH2NHS(O2)Ph
–CH2NHS(O2)CH3
–Ph
–CO2H
–C(O)NHBn
–CH2OH
58.4
44.4
44.4
32.3
49.4
49.4
3.24
40.5
32.4
23.5
—
687
839
<10
<10
596
<10
<10
545
<10
<10
–CH2OH
a
TPSA values of tertiary amines were calculated with a GSK in-house program according to Ref. 20.
Compound is a tertiary amine unless the counter ion (X) is specified.
Functional assays were performed on CHO cells expressing cloned human M1–M3 receptors. Cells were stimulated with Ach. Calcium mobilization was measured using a
b
c
standard FLIPR protocol. Values are the mean of two or more independent assays.
d
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 two
or more independent assays.
e
The pA2 was determined by measuring the ratio of the ACh EC50 in presence and absence of compound.21
Screening was done with aerosolized methacholin induced bronchoconstriction in conscious mice. Compound was given by inhalation 24 h prior to the challenging at a
f
dose of 5
l
g per mouse.
g
Compound was administered at a dose of 50
lg per mouse.

4562
Z. Wan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4560–4562
40000
30000
20000
10000
0
- 0% inh
1.00
0.75
0.50
0.25
0.00
Vehicle
3.30E-10
1.00E-09
3.30E-09
1.00E-08
3.30E-08
0.5 µg/mouse
5.0 µg/mouse
pA2 = 10.7
- 100% inh
5
24
48
72
-11 -10 -9
-8
-7
-6
-5
-4
Time (hrs)
[Carbachol] (log M)
Figure 3. Duration of bronchoprotection for 4a.26
Figure 1. Functional antagonism of 4a in Ach-induced calcium mobilization (FLIPR)
employing CHO-M3.
Acknowledgment
of compound.21 All six compounds effectively antagonized the ago-
nist-induced responses resulting in a rightward shift of the acetyl-
choline concentration response curve in a manner similar to 4a at
the M3 receptor as shown in Figure 1. PA2 values are in good agree-
ment with the corresponding binding affinities (e.g., pKi). Schild
analysis21,24 of 4a against M3, Figure 1, would suggest competitive
antagonism.
Quaternary salts (e.g., 2a, 4a and 10a) were prepared to inves-
tigate its impact on potency. In general, their in vitro potency is
comparable to the amine. All quaternary salts, however, demon-
strate much greater magnitude inhibition than the corresponding
tertiary amines in response to aerosolized methacholine induced
bronchoconstriction in conscious mice when dosed by inhalation
Helpful discussions with Dr. James F. Callahan during the man-
uscript preparation are acknowledged.
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24 h prior to the challenging at the dose of 5 lg per mouse. Of
the compounds screened, 4a exhibited the greatest inhibition
and was thus selected for more of detailed studies in the same
murine model. This inhibition was found to be dose dependent
with ED50 of 0.01
was extended up to 48 h when given by inhalation at two different
doses of 0.5 g per mouse and 5.0 g per mouse (Fig. 3).
lg/mouse (Fig. 2) and the bronchoprotection
15. Zirkle, C. L.; Geissman, T. A.; Bloom, M.; Craig, P. N.; Gerns, F. R.; Indik, Z. K.;
Pavloff, A. M. J. Org. Chem. 1962, 27, 1269.
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l
l
Med. Chem. 1962, 5, 341.
The pharmacokinetics of 4a in the rat are characterized by low
drug absorption, high plasma clearance, high-to-moderate volume
of distribution at steady state, a short terminal half-life and low
oral bioavailability. These characteristics are considered desirable
for an inhaled drug candidate. These results, as well as detailed
SAR discussions of this novel series of antagonists, will be reported
in due course.
17. Takahashi, Y.; Yoneda, N.; Nagai, H. Chem. Lett. 1985, 11, 1733.
18. Miyachi, H.; Kiyota, H.; Segawa, M. Bioorg. Med. Chem. Lett. 1999, 9, 3003.
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Noguchi, K.; Ohtake, N. J. Med. Chem. 2006, 49, 5653.
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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; (b) Jin, J.;
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25. Balb/C mice (n = 4) were treated with the compound intra-nasally (in) and then
challenged after 5 h with 30 mg/ml methacholine (aerosolized, 2 min). The
magnitude of bronchoconstriction was measured as Penh over the next 5 min
using a standard Buxco plethysmography system. The data is expressed as a
ratio of the Penh achieved with methacholine after drug was given to that
achieved before drug was given. A ratio of 1.0 (dotted line) indicates that there
was no change in the Penh in response to aerosolized methacholine (i.e., 0%
inhibition), and a ratio of 0.0 means that there was no bronchoconstriction in
response to methacholine challenge (i.e., 100% inh.).
1.00
0.75
0.50
0.25
0.00
- 0% inh
ED50 = 0.01µg/mouse
- 100% inh
10
0.001
0.01
0.1
1
Dose of compound (µg, i.n.)
26. Same as Ref. 25 except the mice were treated with the compound for 0.25–48 h
before the challenging.
Figure 2. Dose–response of bronchoprotection for 4a.25