Discovery of muscarinic acetylcholine receptor antagonist and beta 2 adrenoceptor agonist (MABA) dual pharmacology molecules

Article abstract of DOI:10.1016/j.bmcl.2011.01.043

We sought to design dual pharmacology bronchodilators targeting both the M₃ muscarinic acetylcholine and beta-2 adrenergic (β₂) receptors by applying our multivalent approach to drug discovery. Herein, we describe our initial discovery and the SAR of the first such compounds with matched potencies at both receptors.

Full text of DOI:10.1016/j.bmcl.2011.01.043

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1354–1358
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of muscarinic acetylcholine receptor antagonist and beta 2
adrenoceptor agonist (MABA) dual pharmacology molecules
⇑
Adam D. Hughes , Kay H. Chin, Sarah L. Dunham, Jeffrey R. Jasper, Kristin E. King,
Tae Weon Lee, Mathai Mammen, Jerri Martin, Tod Steinfeld
Theravance, Inc., 901 Gateway Blvd, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We sought to design dual pharmacology bronchodilators targeting both the M3 muscarinic acetylcholine
and beta-2 adrenergic (b2) receptors by applying our multivalent approach to drug discovery. Herein, we
describe our initial discovery and the SAR of the first such compounds with matched potencies at both
receptors.
Received 7 September 2010
Revised 11 January 2011
Accepted 12 January 2011
Available online 18 January 2011
Ó 2011 Published by Elsevier Ltd.
Keywords:
Multivalent approach
Dual pharmacology
Muscarinic acetylcholine receptor
antagonist
Beta 2 adrenoceptor agonist
MABA
LAMA
LABA
COPD
Bronchodilator
Inhaled
Chronic obstructive pulmonary disease (COPD) is the fifth lead-
ing cause of death worldwide and its prevalence is thought to be
greatly underestimated.1 COPD is most commonly associated with
cigarette smoking; however other risk factors include air pollu-
tants and occupational dust, vapors and fumes. This debilitating
disease is characterized by a progressive airflow limitation that is
not fully reversible. Treatment guidelines emphasize the use of
bronchodilators at all stages of the disease with a combination of
long-acting bronchodilators recommended for patients with
moderate to severe COPD.2 The addition of an inhaled corticoste-
roid (ICS) is recommended for severe and very severe COPD, but
recent studies indicate that a broader patient population may
benefit from treatment with an ICS in addition to a long-acting
bronchodilator.3
have shown that drugs from these two classes may be used effec-
tively in combination to provide enhanced efficacy.4 CombiventÒ
consists of a short acting muscarinic antagonist (SAMA) ipratropi-
um5 and a short acting beta agonist (SABA) albuterol6 formulated
in a single inhalation device.
Longer acting agents for both mechanisms are also prescribed
such as formoterol (1),7 salmeterol (2)8 (bid LABAs) and tiotropium
(3)9 (qd LAMA). New once daily LABAs such as indacaterol (4)10 and
GW64244411 and a twice daily LAMA, aclidinium (5),12 are in late
stage clinical trials. Combination studies with tiotropium and for-
moterol have confirmed that the complementary effects of the
two mechanisms can provide greater improvements in lung func-
tion compared to single agent bronchodilators.13
Due to the treatment successes of combination products such as
CombiventÒ, AdvairÒ3 (salmeterol and fluticasone propionate) and
SymbicortÒ14 (formoterol and budesonide), it is anticipated that a
coformulation of a LABA with a LAMA will soon be available.15
In this Letter, we describe an alternative approach to obtain
dual pharmacology through the design of compounds possessing
dual activity as muscarinic antagonists and b2 agonists (MABAs).
By providing both activities in a single molecule, there is the po-
tential to provide efficacy comparable to a combination product
The most frequently used bronchodilators for COPD are inhaled
beta-2 adrenergic receptor (b2) agonists and inhaled muscarinic
acetylcholine receptor antagonists. Short acting or ‘rescue’ com-
pounds with a 2–4 h duration of action, are recommended for
the relief of symptoms in patients with mild COPD. Clinical studies
⇑
Corresponding author.
E-mail address: ahughes@theravance.com (A.D. Hughes).
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.01.043

A. D. Hughes et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1354–1358
1355
Table 2
without the challenges of coformulation and clinical development
M3 potencies of n-alkyl primary amine derivatives of 8a
of a combination of two active agents. The MABA approach also of-
fers the potential for simplified pharmacokinetics and the opportu-
nity for a binary combination with an ICS to achieve ‘triple therapy’
in a single inhalation device.
Using our multivalent approach to drug design we investigated
the tolerability of a secondary binding site motif with an additional
pharmacology on both the muscarinic (M3) and b2 receptors. Our
prior experience with lead optimization for both muscarinic antag-
onists16 and b2 agonists17 indicated that distal secondary binding
sites are accessible on both receptors for single pharmacology
compounds.
Compound
n
hM3 KI (nM)
10a
10b
10c
10d
10e
10f
10g
10h
10i
2
3
4
5
6
7
8
9
50
67
84
98
19
0.53
1.8
0.75
1.6
2.5
2.6
10
11
12
10j
10k
Schemes 1–9 illustrate the preparation of the compounds de-
scribed in this Letter. We had previously investigated muscarinic
receptor antagonists for the treatment of overactive bladder and
had identified the biphenyl carbamate (8a) as a potent M3 receptor
antagonist with tolerance for piperidine N-substitution.18 Our
search for a secondary binding site began by evaluating different
alkyl chain lengths to probe for a lipophilic pocket while concom-
itantly assessing the tolerance of the piperidine nitrogen to steric
bulk, Table 1, Scheme 1.19
For alkyl chains of 6–9 atoms in length, a potency gain of
5–10-fold was observed at the M3 receptor relative to the unsubsti-
tuted piperidine 8a. Since both shorter and longer alkyl chains led
to reduced potency we concluded that compounds 9f–i were more
suited to accessing a secondary binding site on the muscarinic
receptor.
Scheme 2. Since all known efficacious b2 agonists contain a second-
ary amine as part of the epinephrine ligand mimic, this would be
vital for our MABA design.
Evaluation of a series of aminoalkyl substituents showed that
linkers of 7–12 carbon atoms between the two basic nitrogens
afforded low nanomolar potency at M3 (10f–k). Based on the com-
bined results from the alkyl and primary amine series, we focused
on 7- to 9-carbon linkers to elaborate the primary amine further to
build in b2 agonist activity.
Substituting the primary amine with selected b2 agonist frag-
ments (shown in Fig. 1) gratifyingly provided a further enhance-
ment of M3 receptor binding potency for all entries in Table 3,
To quickly assess the tolerance of a basic moiety at the second-
ary binding site, a primary amine was used as a probe, Table 2,
Table 1
M3 potencies of n-alkyl chain derivatives of 8a
Compound
n
hM3 KI20 (nM)
8a
9a
9b
9c
9d
9e
9f
9g
9h
9i
NA
0
1
2
3
4
5
6
7
46
22
55
77
17
14
4.8
5.1
7
8
9
10
9.5
25
48
9j
9k
Figure 1. Marketed respiratory medicines and those in late stage clinical
development.
Scheme 1. Reagents and conditions: (a) 70 °C, neat; (b) NH4HCO2, cat Pd/C, 40 °C, EtOH, 95% over 2 steps; (c) aldehyde, Na(OAc)3BH, 1:1 DCM–MeOH, 50–80%; (d) alkyl
halide, DIPEA, DMF, 45–75%.
Scheme 2. Reagents and conditions: (a) bromoalcohol, DIPEA, DMF, 50 °C, 49–82%; (b) DMSO, PyÁSO3, DIPEA, DCM, 0 °C, 60–90%; (c) dibenzylamine, Na(OAc)3BH, 1:1 DCM–
MeOH, 70–85%; (d) H2, cat Pd(OH)2, EtOH, 70–90%.

1356
A. D. Hughes et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1354–1358
Table 3
two compounds demonstrated matched, subnanomolar poten-
cies21 at both the M3 and b2 receptors (13c and 13d).
M3 potency, b2 potency and functional data (EC50) for biphenyl carbamate MABAs
containing various b2 pharmacophores
Maintaining the carbostyril b2 moiety, several other muscarinic
receptor binding motifs were investigated, Schemes 6–9. Table 4
illustrates that all of these compounds retained activity, but were
inferior to the biphenyl carbamate compounds described in Table
3. Intriguingly, not only was potency reduced at the M3 receptor,
but also at the b2 receptor, thus indicating the considerable influ-
ence of the secondary binding muscarinic motif on binding to the
b2 receptor.
It is interesting to note that although the marketed inhaled mus-
carinic antagonists ipratropium and tiotropium both contain quater-
nary ammonium groups, we found the incorporation of a quaternary
ammonium pharmacophore into our bifunctional MABA constructs
resulted in lower affinities for the M3 receptor (29 and 31).
We have discovered several compounds with matched in vitro
M3 and b2 potencies using a linked multivalent discovery approach.
22
Compound
n
hM3 KI (nM) HEK hb2 KI (nM) HEK hb2 EC50 (nM)
13a
13b
13c
13d
17
19
1
2
3
4
3
7
8
9
9
9
28
19
10
3.4
3.5
21
61
33
2.4
NA
61
1.3
0.74
0.32
0.01
0.14
0.14
1.1
0.32
0.36
2.6
14
NA NA
NA NA
NA 0.034
NA NA
0.19
0.33
NA
0.82
Schemes 3–5. The carbostyril and hydroxy formanilide b2 moieties
present in 13a–d and 17, respectively, offered superior b2 poten-
cies relative to the saligenin containing compound 19. Additionally
Scheme 3. Reagents and conditions: (a) 12,23 1,3-dibromopropane, NaHCO3, DMF, 50 °C, 55%; (b) H2, cat Pd(OH)2, EtOH; (c) Et3NÁ3HF, 1:9 DMF–DCM, 20% over 2 steps.
Scheme 4. Reagents and conditions: (a) Amine 1521 or 1624, Na(OAc)3BH, 1:1 DCM–MeOH, 60–75%; (b) Et3NÁ3HF, 1:1 DMF–DCM, 60–70%.
Scheme 5. Reagents and conditions: (a) 18,25 NaHCO3, NaI, THF, 80 °C, 60%; (b) Et3NÁ3HF, 1:9 DMF–DCM; (c) TFA, 1:1 THF–H2O, 30% over 2 steps.
Scheme 6. Reagents and conditions: (a) 10h or 11, Na(OAc)3BH, 20,24 DCE, 40–50%; (b) H2, cat Pd/C, MeOH, 10–28%.

A. D. Hughes et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1354–1358
1357
Scheme 7. Reagents and conditions: (a) 23,26 1-bromononan-9-ol, Et3N, MeCN, 50 °C, 62%; (b) DMSO, PyÁSO3, DIPEA, DCM, 0 °C, 90%; (c) benzylamine, Na(CN)BH3, MeOH,
46%; (d) 25,23 K2CO3, DMF, 45 °C; (e) NaBH4, EtOH, 50% over 2 steps; (f) H2, cat Pd/C, AcOH, 20%.
Scheme 8. Reagents and conditions: (a) 70 °C, neat, 95%; (b) 1,9-dibromononane, Et3N, MeCN, 50 °C, 52%; (c) NaH, di-tert-butyl iminodicarboxylate, DMF; (d) 1:3 TFA–DCM,
23% over 2 steps; (e) 20, Na(OAc)3BH, DCE, 38%; (f) H2, cat Pd/C, MeOH, 10%.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.043. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
Scheme 9. Reagents and conditions: (a) 30,27 1,9-dibromononane, DIPEA, DMF,
References and notes
50%; (b) 15, DMF, 50 °C; (c) Et3NÁ3HF, 1:9 DMF–DCM, 5% over 2 steps.
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26
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7.5
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A biphenyl carbamate was identified as the preferred muscarinic
moiety while a carbostyril was favored for b2 potency. Interestingly
potency at both receptors was affected when either group was
modified or changed, suggesting a secondary binding site is being
accessed in both cases.
Furthermore, the MABA compounds 13c and 13d, had subn-
anomolar M3 potencies and additionally possessed subnanomolar
b2 activity equivalent to LABAs such as formoterol (1), salmeterol
(2) and indacaterol (4).
The compounds in this article follow on from our first disclosure
of the MABA concept25 with publications from other organizations
now appearing in the literature.28 A related publication from Ther-
avance is now available suggesting that 13d exhibits a multivalent
bimodal orientation in the orthosteric and allosteric binding pock-
ets of the M3 and b2 receptors.29 Future publications will disclose
the in vivo activity of the lead compound 13d.
14. Lee, C.; Corren, J. Expert Rev. Respir. Med. 2008, 2, 551.
15. Clinical trials investigating the combination of a LAMA (NVA237) and LABA
(Indacaterol) are reported on the websites of Novartis and Sosei.
16. (a) Hughes, A. D.; Chin, K. H.; Hegde, S. S.; Husfeld, C. O.; Jasper, J. R.; Ji, Y.; King,
K. E.; Lee, T. W.; Li, L.; Mammen, M.; McNamara, A. J.; Mischki, T. K.; Obedencio,
G. P.; Pulido-Rios, T.; Steinfeld, T.; Sweazey, S. M.; Wilson, R. D. Abstracts of
Papers, 234th National Meeting of the American Chemical Society, Boston, MA,
Aug 19–23, 2007; American Chemical Society: Washington, DC; MEDI-023.; (b)
Steinfeld, T.; Mammen, M.; Smith, J. A.; Wilson, R. D.; Jasper, J. R. Mol.
Pharmacol. 2007, 72, 291.
Acknowledgments
17. Findings from Theravance’s LABA program will be reported at a later date.
18. (a) ‘Biphenyl compounds useful as muscarinic receptor antagonists’, Mammen,
M.; Ji, Y.-H.; Mu, Y.; Mischki, T.; Malathong, V. U.S. Patent 7,456,199, 2008.; (b)
The author would like to thank reviewers at Theravance and
GSK for their assistance in preparing this article.

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A. D. Hughes et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1354–1358
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19. All yields are unoptimized. Final step yields in each scheme refer to isolated
compounds after purification by reverse phase preparative HPLC.
20. Muscarinic receptor inhibition binding constants (KI) were measured using a
radioligand binding assay. Briefly, cell membranes expressing the human M3
muscarinic receptor were incubated for up to 6 h at 37 °C with 1 nM [3H]
N-methyl-scopolamine and various dilutions of test compounds in a buffer
consisting of 10 mM Hepes, 100 mM NaCl, 10 mM MgCl2, (pH 7.4 at 37 °C). KI
values were calculated from IC50 values according to Cheng, Y.; Prusoff, W. H.
Biochem. Pharmacol. 1973, 22, 3099. Muscarinic acetylcholine receptor
antagonist behavior has previously been reported in our patent publications
and confirmed in our in vivo models.
Boston, MA). Cells were stimulated with various agonist concentrations for
10 min at 37 °C, prior to measurement of bound [125I]cAMP. These data were
then fit to a 4-parameter logistic equation to determine agonist EC50 values.
23. Reference for compounds 12, 15, 25 (carbostyril building blocks): ‘Crystalline
form of a biphenyl compound’, Chao, R. S.; Rapta, M.; Colson, P.-J. U.S. Patent
7,521,558, 2009.
24. ‘Dialkylphenyl compounds having b2 adrenergic receptor agonist and
muscarinic receptor antagonist activity’, Colson, P.-J.; Hughes, A. D.; Husfeld,
C.; Mammen, M.; Rapta, M. U.S. Patent 7,524,965, 2007.
25. Saligenin and glyoxal reference: ‘Biphenyl derivatives’, Mammen, M.; Dunham,
S.; Hughes, A.; Lee, T. W.; Husfeld, C.; Stangeland, E. U.S. Patent 7,141,671,
2006.
26. ‘Substituted pyrrolidine and related compounds’, Mammen, M.; Hughes, A. D.;
Ji, Y.-H.; Li, L.; Zhang, W. U.S. Patent 7, 351, 718, 2008.
21. Beta 2-adrenoceptor inhibition binding constants (KI) were measured using a
radioligand binding assay. Briefly, cell membranes expressing the human beta
2-adrenoceptor were incubated for up to 4 h at 37 °C with 1 nM [3H]
27. ‘Azabicycloalkane compounds’, Mammen, M.; Hughes, A. D. U.S. Patent 7, 358,
244, 2008.
dihydroalprenolol and various dilutions of test compounds in
a
buffer
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Entwistle, D.; Fairman, D.; Feeder, N.; Fulton, C.; Hilton, L.; James, K.; Jones, R.
M.; Kenyon, A. S.; Marshall, S.; Newman, S. D.; Osborne, R.; Patel, S.; Selby, M.
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consisting of 75 mM Tris–HCl, 12.5 mM MgCl2, 1 mM EDTA (pH 7.4 at 37 °C).
KI values were calculated from IC50 values according to Cheng, Y.; Prusoff, W. H.
Biochem. Pharmacol. 1973, 22, 3099.
22. Beta 2-adrenoceptor agonist activities were assayed by measuring whole cell
cAMP levels using a homogeneous radioimmuno assay (Perkin–Elmer cAMP
Flashplate) according to the manufacturer’s instructions (Perkin–Elmer,
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