Multivalent design of long-acting β2-adrenoceptor agonists incorporating biarylamines

Article abstract of DOI:10.1016/j.bmcl.2014.04.069

A series of potent β₂-adrenoceptor agonists incorporating a biarylamine secondary binding group was identified. The previously reported milveterol (5), identified by a multivalent approach and containing a typical β₂-agonist primary binding group linked via a phenethylamine linker to a hydrophilic secondary binding group, served as an initiation point. A more hydrophobic set of secondary binding groups was explored, prepared rapidly from a common intermediate by Buchwald-Hartwig amination. TD-5471 (25), a potent and selective full agonist of the human β₂- adrenoceptor, was identified as the most promising agent. It is potent, with slow onset in an in vitro guinea pig trachea model and shows a dose-dependent and long duration of action in an in vivo guinea pig model of bronchoprotection. TD-5471 is structurally differentiated from milveterol and its long duration of action is consistent with a correlation with hydrophobicity observed in other long-acting β₂-agonist discovery programs.

Full text of DOI:10.1016/j.bmcl.2014.04.069

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2625–2630
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multivalent design of long-acting b2-adrenoceptor agonists
incorporating biarylamines
⇑
John R. Jacobsen , James B. Aggen, Timothy J. Church, Uwe Klein, Juergen W. Pfeiffer,
Teresa M. Pulido-Rios, G. Roger Thomas, Cecile Yu, Edmund J. Moran
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:
A series of potent b2-adrenoceptor agonists incorporating a biarylamine secondary binding group was
identified. The previously reported milveterol (5), identified by a multivalent approach and containing
a typical b2-agonist primary binding group linked via a phenethylamine linker to a hydrophilic secondary
binding group, served as an initiation point. A more hydrophobic set of secondary binding groups was
explored, prepared rapidly from a common intermediate by Buchwald–Hartwig amination. TD-5471
(25), a potent and selective full agonist of the human b2-adrenoceptor, was identified as the most prom-
ising agent. It is potent, with slow onset in an in vitro guinea pig trachea model and shows a dose-depen-
dent and long duration of action in an in vivo guinea pig model of bronchoprotection. TD-5471 is
structurally differentiated from milveterol and its long duration of action is consistent with a correlation
with hydrophobicity observed in other long-acting b2-agonist discovery programs.
Received 26 March 2014
Revised 16 April 2014
Accepted 18 April 2014
Available online 26 April 2014
Keywords:
b2-Adrenoceptor agonist
Inhaled
LABA
Bronchodilator
Multivalent approach
Ó 2014 Elsevier Ltd. All rights reserved.
Inhaled b2-adrenoceptor agonists are highly effective bronchod-
ilators and are widely used in the treatment of both chronic
obstructive pulmonary disease (COPD) and asthma.1 Drugs with
short duration of action are useful for the as-needed relief of acute
bronchoconstriction, and long-acting b2-adrenoceptor agonists
(LABAs) are used for maintenance therapy in both asthma and
COPD. Salmeterol (1) and formoterol (2) (Fig. 1) are dosed twice-
daily for the treatment of asthma and COPD.2 Longer lasting
b2-adrenoceptor agonists have been sought to improve disease
control and patient compliance.3 Vilanterol4 (4) (in combination with
the inhaled corticosteroid fluticasone furoate) and indacaterol5 (3)
(as a single agent) have been approved in the US for the once-daily
treatment of COPD. Olodaterol6 and indacaterol (as a single agent
and in combination with the long-acting muscarinic antagonist
glycopyrronium)7 have been approved in the EU. Several additional
programs (LABA single agents,8–14 combinations,15 as well as
development programs for the treatment of asthma)16 are in
late-stage development or under regulatory review.
The optimized secondary binding group of milveterol is relatively
compact and hydrophilic. Herein we report on a second series of
compounds with considerably larger and more hydrophobic sec-
ondary binding groups. Following this approach, a second develop-
ment candidate was identified with markedly different
physicochemical properties relative to milveterol.
To further explore the potential of the milveterol scaffold to
afford novel, long-acting compounds, we prepared a number of
derivatives based on the 4-aminophenethylamine linking group.
We anticipated that a large number of derivatives could be rapidly
prepared using Pd-mediated coupling of a suitably protected aryl
bromide scaffold with a wide range of amines via Buchwald–Har-
twig amination.18,19 To facilitate the preparation of arrays by par-
allel synthesis, we selected intermediate 10, reasoning that the
acetonide and 4,40-dimethoxybenzyhydryl could be efficiently
removed under acidic conditions following coupling. The selection
of the racemic saligenin primary binding group for the initial sur-
vey was taken based on synthetic ease and on previous experience
that SAR generally translates well between primary binding
groups.
A wide variety of medicinal chemistry strategies have been
employed for the discovery of new LABAs.2 We previously reported
the discovery of the long-acting b2-adrenoceptor agonist milveter-
ol17 (5) (Fig. 2) following a multivalent approach to drug design.
Scaffold 10 was prepared in five steps from 5-bromo-2-
hydroxybenzyl alcohol (Scheme 1). Coupling to a variety of amines
was conducted as shown in Scheme 2, followed by deprotection to
afford compounds 12b–n. Compound 12a was prepared by direct
deprotection of 10 to afford the un-elaborated bromo derivative
as a comparator.
⇑
Corresponding author. Tel.: +1 650 808 6034; fax: +1 650 808 6120.
E-mail address: jjacobsen@theravance.com (J.R. Jacobsen).
http://dx.doi.org/10.1016/j.bmcl.2014.04.069
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2626
J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2625–2630
OH
O
considered an absolute requirement, b2/b1 selectivity inferior to
formoterol (2) was considered an unnecessary risk for off-target
pharmacology.
H
N
OH
OH
H
N
O
HO
O
2
HN
HO
HO
1
Following the synthetic approach outlined in Scheme 1, a fur-
ther series of analogs were prepared based on 12m and 12n. Data
are shown in Table 1. Analogs 12o–s further probed the SAR
around the relatively hydrophobic compound 12m. Compound
12s was selected for further profiling based on its favorable b2/b1
selectivity. Compounds 12t–z further probed the SAR around the
less hydrophobic compound 12n. Compound 12u demonstrated
enhanced potency in the b2 functional assay as well as encouraging
b2/b1 binding selectivity and was selected for further profiling.
To assess the efficacy of 12s and 12u in airway tissue, we
employed an electrically-stimulated superfused guinea pig tra-
cheal ring assay.21,22 In addition to providing a further measure
of potency, this model provides an assessment of onset of action
that has previously been shown to distinguish between the slow
onset of salmeterol (1) and the rapid onset of formoterol (2).23 Mil-
veterol (5) also shows high potency in this model and, like formo-
terol (2), a rapid onset of action (Table 2).
At this stage we chose to conduct a stereoselective synthesis of
(R)-12s, based on well-established stereochemical preference of
the b2-adrenoceptor.24 The benzyl-protected intermediate 17 was
prepared by coupling the doubly protected 4-bromophenethyl-
amine (14) with 4-methoxy-3-phenylaniline (15) under the condi-
tions employed in the earlier survey (Scheme 3) followed by
deprotection of the boc group. Reaction with chiral epoxide 18 fol-
lowed by deprotection afforded (R)-12s (Scheme 4).
Compound 12u was tested in the guinea pig trachea assay as a
racemic mixture and found to have modest potency approximately
10-fold less than salmeterol (1) (Table 2). The potency of (R)-12s
was 10-fold greater than that of salmeterol, and like salmeterol,
(R)-12s showed a slow onset of action. Either slow or fast onset
of action is entirely acceptable for a maintenance therapy in COPD
and asthma. We characterized the onset of action in tissue to assist
in the design and interpretation of subsequent in vivo efficacy
studies. In this case, the slow onset of (R)-12s was a biological dis-
tinction from the previously discovered clinical candidate milve-
terol (5), enhancing the value of this compound as a potential
backup to 5.
H
Cl
O
H
OH
H
N
N
O
Cl
HO
HO
HO
HN
4
3
O
Figure 1. Structures of the LABAs salmeterol (1), formoterol (2), indacaterol (3) and
vilanterol (4).
'Primary
Binding Group'
'Secondary
'Linker'
Binding Group'
OH
H
N
HO
N
H
HN
H
OH
5
O
Figure 2. Structure of milveterol (5) indicating the b2-adrenoceptor agonist
primary binding group, linker and secondary binding group.
The initial series of analogs is shown in Table 1 and is compared
to salmeterol (1), formoterol (2) and milveterol (5). Bromide 12a
was found to retain modest potency, albeit two orders of magni-
tude lower than that of 1, 2, and 5. A range of aryl-alkyl substitu-
ents including both straight-chain and cyclic linking groups
(compounds 12b–f) failed to improve potency relative to 5. Simi-
larly low potencies were obtained for compounds 12g–k in which
additional polar groups were introduced. In contrast, direct aryl
substitution to the nitrogen to afford diarylamines 12l–n provided
compounds with the greatest potency in this initial survey. In par-
ticular, compounds 12m and 12n have pEC50 values of 8.6 and 8.4,
respectively, and are structurally distinct from the milveterol ser-
ies. Selectivity for the b2- over the b1-adrenoceptor, measured as
the ratio of binding Ki’s, was significantly lower for compounds
12m and 12n relative to salmeterol (1) and milveterol (5). While
the extraordinary selectivity demonstrated by salmeterol was not
To further explore the SAR around compound (R)-12s, we pre-
pared compounds 22 and 25 containing established formanilide
O
O
Br
Br
e
Br
a
b
c
d
HO
HO
O
O
O
O
6
7
8
O
O
OMe
OMe
PG
N
PG
N
OH
O
PG =
*
O
Br
O
Br
9
10
O
O
Scheme 1. Reagents and conditions: (a) 2,2-dimethoxypropane, acetone, followed by ZnCl2; (b) n-BuLi, THF followed by N-methyl-N-methoxyacetamide; (c) NaHMDS, THF,
followed by TMSCl, followed by Br2; (d) 4-bromophenethylamine, Et3N, DCM, followed by bis(4-methoxyphenyl)methyl chloride; (e) NaBH4, MeOH, THF.
OH
PG
N
H
N
PG
N
OH
OH
*
*
b
*
a
HO
HO
R
O
R
O
Br
10
11b-n
12a-z
O
O
b
Scheme 2. Reagents and conditions: (a) HNR1R2, rac-BINAP, tris(dibenzylideneacetone)dipalladium(0), NaOtBu, toluene, 80 °C, 5 h; (b) 80% aq AcOH, 80 °C, 5 h. PG is as
defined in Scheme 1.

J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2625–2630
2627
Table 1
In vitro b2-agonist functional potency and binding selectivity of standards and derivatives of intermediate 1020
Compounds
R
b2 pEC50 SD (n)
b1 pKi SD (n)
b2 pKi SD (n)
b2/b1 Selectivitya
1
2
5
12a
Not applicable
Not applicable
Not applicable
Bromo
9.5 0.2 (345)
9.7 0.2 (648)
10.2 0.2 (28)
7.6 0.0 (2)
5.6 0.2 (462)
5.7 0.2 (359)
5.0 0.1 (22)
<5.0 (2)
8.6 0.2 (471)
7.5 0.2 (355)
8.5 0.1 (22)
4.5 0.0 (2)
1135
56
3027
ND
Cl
N
H
12b
5.8 0.2 (2)
<5.0 (2)
<5.0 (2)
ND
Cl
N
H
12c
12d
12e
6.8 0.0 (2)
6.4 0.2 (2)
6.3 0.0 (2)
<5.0 (2)
<5.0 (2)
ND
ND
4.8
N
<5.0 (2)
4.9 0.0 (2)
4.8 0.0 (2)
N
N
4.1 0.0 (2)
12f
5.8 0.0 (2)
5.9 0.1 (2)
3.9 0.0 (2)
3.9 0.0 (2)
3.7 0.0 (2)
4.4 0.0 (2)
0.6
3.4
3
N
12g
O
O
N
N
12h
12i
6.7 0.0 (2)
6.2 0.1 (2)
<5.0 (2)
<5.0 (2)
4.5 0.0 (2)
<5.0 (2)
ND
ND
O
N
N
N
N
12j
5.0 0.7 (2)
<5.0 (2)
<5.0 (2)
ND
N
O
12k
12l
6.0 0.1 (2)
7.7 0.0 (2)
<5.0 (2)
<5.0 (2)
ND
16
N
H
H
N
5.2 0.0 (2)
6.5 0.1 (2)
O
12m
12n
12o
12p
12q
12r
2-Naphthylamino
4-Methoxyphenylamino
1-Naphthylamino
5-Indanylamino
4-Biphenylamino
3-Biphenylamino
8.6 0.0 (2)
8.4 0.0 (2)
6.7 0.1 (2)
8.1 0.0 (2)
8.1 0.0 (2)
8.7 0.1 (4)
4.9 0.0 (2)
4.5 0.0 (2)
4.5 0.0 (2)
4.6 0.0 (2)
4.9 0.0 (2)
4.9 0.3 (3)
6.5 0.0 (2)
5.9 0.0 (2)
5.2 0.0 (2)
6.1 0.0 (2)
6.5 0.0 (2)
6.5 0.4 (3)
34
27
5.4
31
36
40
H
N
12s
8.9 0.4 (6)
4.7 0.1 (4)
7.2 0.3 (5)
319
O
12t
3-Methoxyphenylamino
4-Ethoxyphenylamino
4-Thiomethylphenylamino
4-Isopropoxyphenylamino
4-Hydroxyphenylamino
4-Fluorophenylamino
8.0 0.1 (2)
8.9 0.3 (6)
8.6 0.0 (2)
8.6 0.0 (2)
6.7 0.0 (2)
7.5 0.1 (2)
4.9 0.0 (2)
4.5 0.3 (5)
5.1 0.0 (2)
4.8 0.1 (2)
4.4 0.2 (2)
5.1 0.1 (2)
6.2 0.0 (2)
6.8 0.5 (5)
7.1 0.1 (2)
7.0 0.1 (2)
5.8 0.1 (2)
5.9 0.0 (2)
17
178
81
158
23
6.5
12u
12v
12w
12x
12y
H
N
12z
8.2 0.0 (2)
5.0 0.0 (2)
6.6 0.0 (2)
36
O
O
ND: not determined.
a
Selectivity is expressed as the ratio of Ki’s (b1 Ki/b2 Ki).
and carbostyril primary binding groups (Scheme 4). Both 22 and 25
have potencies within 10-fold of (R)-12s in the guinea pig tracheal
ring assay (Table 2). In the recombinant cellular assay (Table 3)
compounds 22 and 25 showed greater b2 potency than (R)-12s
and salmeterol (1). Selectivity for the human b2-adrenoceptor over
the b1- and b3-subtypes was assessed in functional assays in
recombinant cell lines and comparable to formoterol (2) for both
22 and 25. Potency was also assessed in the BEAS-2B cell line, a
transformed human airway epithelial line which endogenously
expresses the b2-adrenoceptor, allowing us to measure intrinsic
activity relative to isoproterenol (Table 4).25 Potencies of
formoterol (2) as well as compounds (R)-12s, 22 and 25 were all
Table 2
Potency and onset of action in electrically-stimulated superfused guinea pig trachea22
Compounds
pEC50 SEM (n)
Onseta
1
2
5
7.5 0.2 (8)
8.8 0.2 (5)
10.3 0.1 (17)
6.7 (2)
8.7 (2)
7.9 0.1 (3)
8.7 0.1 (5)
Slow
Fast
Fast
Slow
Slow
Slow
Slow
12u
(R)-12s
22
25
a
Onset is measured as time to achieve maximum effect at an EC80 concentration
where fast is 615 min and slow is >30 min.

2628
J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2625–2630
O
Bn
HN
Bn
N
H2N
Bn
N
O
O
e
a, b, c
15
H2N
Boc
Boc
N
H
N
H
d
Br
Br
13
14
16
17
Scheme 3. Reagents and conditions: (a) benzaldehyde, toluene, concentrate under vacuum at 45 °C; (b) NaBH4, THF, MeOH; (c) Et3N, Boc2O, MeOH; (d) 4-methoxy-3-
phenylaniline hydrochloride (15), rac-BINAP, tris(dibenzylideneacetone)dipalladium(0), NaOtBu, toluene, 95 °C, 6 h; (e) TFA, DCM.
OH
H
N
Bn
N
OH
O
O
b, c
O
17
HO
HO
N
H
BnO
MeO
BnO
MeO
N
H
a
(R)-12s
19
O
O
18
OH
H
H
N
Bn
N
OH
O
O
d
O
17
HO
N
BnO
BnO
N
H
a
HN
HN
H
H
22
HN
H
21
O
O
O
20
OH
H
N
Bn
N
OH
O
O
e
O
17
HO
N
BnO
BnO
N
H
a
HN
H
HN
23
HN
25
24
O
O
O
Scheme 4. Compounds (R)-12s, 22, and 25 were prepared from intermediate 17 by reaction with epoxides 18, 20, and 23, respectively. Reagents and conditions: (a) amine 17,
iPrOH, 78 °C, 16 h; (b) LiAlH4, THF, 0 °C; (c) 10% Pd/C, MeOH, THF, H2 (35 psi); (d) 10% Pd/C, MeOH, EtOH, H2 (1 atm); (e) 10% Pd/C, AcOH, H2 (40 psi).
Table 3
In vitro agonist potency and selectivity of agonists in recombinant cell lines expressing human b-adrenergic receptors20
Compounds
Agonist potency
Functional selectivitya
b2/b3
b2 pEC50 SD (n)
b1 pEC50 SD (n)
b3 pEC50 SD (n)
b2/b1
1
2
9.5 0.2 (345)
9.7 0.2 (648)
9.3 0.2 (4)
9.9 0.3 (8)
9.9 0.3 (44)
6.6 0.2 (162)
7.9 0.2 (615)
ND
8.0 0.4 (7)
8.1 0.3 (33)
6.0 0.2 (30)
7.6 0.3 (50)
ND
8.5 0.1 (3)
7.9 0.3 (22)
817
67
ND
75
2993
128
ND
26
100
(R)-12s
22
25
56
a
Selectivity is expressed as the ratio of EC50’s (b1 EC50/b2 EC50 and b3 EC50/b2 EC50), ND: not determined.
salmeterol (300 lg/mL) provides bronchoprotection lasting
Table 4
P24 h in this model, a 72-h time point was selected to provide
confidence that 25 would be suitable for once-daily dosing in
man. Compound 25-mediated bronchoprotection at 72 h was
dose-dependent and was significantly greater than that of salme-
In vitro agonist potency and intrinsic activity of agonists in a cell line endogenously
expressing the human b2-adrenergic receptor25
Compounds
b2 pEC50 SD (n)
IA (%) SD (n)
1
2
9.3 0.2 (359)
8.6 0.2 (186)
8.4 0.0 (4)
34 10 (361)
93 9 (186)
81 16 (4)
85 6 (7)
terol at nebulizer concentrations of P30 lg/mL (Fig. 3). Based on
its long duration in this model relative to an agent requiring
twice-daily administration in patients, compound 25 was selected
for further studies. The identification of a stable crystalline form is
critical to the development of an inhaled medicine.2,27 Crystalline
hydrochloride28 and 2,5-dichlorobenzenesulfonate29 salts of 25
have been identified, enabling further progression of this com-
pound to safety assessment and subsequently into clinical devel-
opment as a backup candidate (TD-5471) to milveterol (5).
The objective of this research was the identification of a long
acting b2-adrenoceptor agonist as a backup to milveterol (5). In
addition to a preclinical profile that provides confidence in efficacy
and duration of action, we sought both chemical and biological dif-
ferentiation from milveterol to maximize the potential to address
any deficiency that might arise with the lead compound. We
employed a multivalent approach to the identification of second-
ary binding groups that would impart these characteristics. The
secondary binding group in milveterol is relatively compact and
(R)-12s
22
9.1 0.3 (7)
25
9.4 0.4 (35)
83 9 (35)
IA: intrinsic activity.
approximately 10-fold lower in this cell line relative to the recom-
binant cell line used for primary screening. In contrast to the par-
tial agonist salmeterol (1), compounds (R)-12s, 22 and 25 were all
full agonists displaying intrinsic activities of >80%.
Compound 25 was prioritized for further evaluation over com-
pound 22 based on its superior potency in the guinea pig tissue
assay. In addition, the carbostyril b2-agonist primary binding group
of 25 provided structural differentiation relative to the lead candi-
date milveterol (5). In a guinea pig model of in vivo bronchoprotec-
tion,26 compound 25 showed excellent potency (measured 1.5 h
after nebulized dosing, data not shown). Because a high dose of

J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2625–2630
2629
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*
30
20
10
0
72 hours
*
0.1
1
10
100
β-Agonist (µg/ml)
Figure 3. Concentration-response for bronchoprotection measured 72-h after
dosing of 25 (closed circles; n = 10–12) and salmeterol (1, open circles; n = 8–
12).26 Data points are expressed as mean SEM. ⁄Significant difference (p < 0.05)
compared to water control (closed square; n = 3).
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Table 5
Calculated physicochemical properties of b2-adrenoceptor agonists
16. Loetvall, J.; Bateman, E. D.; Bleecker, E. R.; Busse, W. W.; Woodcock, A.; Follows,
R.; Lim, J.; Stone, S.; Jacques, L.; Haumann, B. Eur. Respir. J. 2012, 40, 570.
17. Jacobsen, J. R.; Choi, S. K.; Combs, J.; Fournier, E. J. L.; Klein, U.; Pfeiffer, J. W.;
Thomas, G. R.; Yu, C.; Moran, E. J. Bioorg. Med. Chem. Lett. 2012, 22, 1213.
18. Guram, A. S.; Rennels, R. A.; Buchwald, S. L. Angew. Chem., Int. Ed. Engl. 1995, 34,
1348.
tPSA (Å2)
MW
a
Compounds
cLogD7.4
5 (milveterol)
25 (TD-5471)
1 (salmeterol)
2 (formoterol)
3 (indacaterol)
4 (vilanterol)
0.91
3.8
3.0
0.36
2.7
3.0
114
103
82
91
82
436
522
416
344
392
486
19. Louie, J.; Hartwig, J. F. Tetrahedron Lett. 1995, 36, 3609.
20. For radioligand binding assays, cell membranes were incubated with
[3H]dihydroalprenolol in the presence of increasing concentrations of
unlabeled test compound (90 min at room temperature). Binding reactions
were stopped by rapid filtration over filter plates; bound radioactivity was
determined by liquid scintillation counting. Binding selectivities were
calculated from the ratio of the equilibrium dissociation constants Ki. In
order to determine the agonist potencies of test compounds, the ability of
compounds to stimulate cAMP accumulation in whole cells overexpressing
cloned human b2- or b1-adrenoceptor was determined. cAMP concentrations
91
a
clogD pH7.4 values were calculated using Pipeline Pilot.
hydrophilic. The current study led us to a hydrophobic aminophen-
ethylamine linker and hydrophobic biaryl secondary binding
group. It is interesting to note that a recently reported b2-agonist
clinical candidate PF-610355 also contains a biaryl secondary bind-
ing group, although it is linked via an amide linker.27 Our backup
candidate TD-5471 (25) has markedly different physicochemical
properties relative to our lead candidate milveterol (5) (Table 5).
Its higher calculated log D value and lower solubility are more sim-
ilar to those of salmeterol, vilanterol and indacaterol and is consis-
were measured by
PerkinElmer). Agonist selectivies were calculated from the ratio of the
agonist potencies EC50
a
homogeneous radioimmunoassay (Flashplate,
.
21. Coleman, R. A.; Nials, A. T.; Rabe, K. F.; Vardey, C. J.; Watson, N. Pulm.
Pharmacol. 1996, 9, 107.
22. To assess agonist potency and intrinsic activity of b2-adrenoceptor agonists in
guinea pig isolated tracheal preparations, male guinea pigs were pre-treated
with 6-hydroxy-dopamine (200 mg/kg) via intraperitoneal injection 24 h prior
to the study. On the day of the study, animals were euthanized by CO2
asphyxiation and the trachea was isolated and placed in an aerated high
potassium (30 mM) Krebs buffer (also containing 10
lM indomethacin, 1 lM
tent with
a trend observed by others correlating increased
choline, and 3 M guanethidine). Each tracheal ring (5 mm) was cut open,
l
sutured using 4.0 silk thread and positioned between two platinum electrodes
such that the circular smooth muscles were oriented vertically in the tissue
bath filled with normal potassium (4.7 mM) Krebs buffer at 37 °C. The
composition of the Krebs solution was 118 mM NaCl, 4.7 mM KCl, 2.52 mM
CaCl2, 1.64 mM MgSO4, 24.88 mM NaHCO3, 1.8 mM KH2PO4, 10 mM Glucose,
hydrophobicity with an extended duration of action in the
lung.5,10,27 The observation that milveterol provides potent and
long-lasting activity in our in vivo model suggests that drivers
other than hydrophobicity may be important for duration, as has
been proposed for a series of dibasic b2-adrenoceptor agonists.14,30
The possibility that the structural and physicochemical differences
between TD-5471 and milveterol result in different bases for dura-
tion supports the selection of TD-5471 as a backup to milveterol.
10 lM indomethacin, 1 lM choline, and 3 lM guanethidine. The preparations
were electric-field-stimulated with the following conditions: 9 V (just
maximal), 10 Hz, 1 ms pulse duration, 10 s train duration and 1 train every
100 s. An initial resting tension of 1 g was applied and a 1 h equilibration time
was allowed before test compounds were introduced. Concentration–response
relationships were determined by adding cumulative doses of b2-adrenoceptor
agonists to the bath. Onset of effect was determined as the time required to
achieve maximum effect at a sub-maximal dose.
References and notes
23. Nials, A. T.; Ball, D. I.; Butchers, P. R.; Coleman, R. A.; Humbles, A. A.; Johnson,
M.; Vardey, C. J. Eur. J. Pharmacol. 1994, 251, 127.
24. Patil, P. N.; Li, C.; Kumari, V.; Hieble, J. P. Chirality 2008, 20, 529.
25. In order to determine the agonist potency and intrinsic activity of test
compounds on the human b2-adrenoceptor, the ability of compounds to
1. Cazzola, M.; Calzetta, L.; Matera, M. G. Br. J. Pharmacol. 2011, 163, 4.
2. Jacobsen, J. R. Future Med. Chem. 2011, 3, 1607.
3. Toy, E. L.; Beaulieu, N. U.; McHale, J. M.; Welland, T. R.; Plauschinat, C. A.;
Swensen, A.; Duh, M. S. Respir. Med. 2011, 105, 435.
stimulate cAMP accumulation in
endogenously expressing the b2-adrenoceptor (BEAS-2B cells) was
determined. cAMP concentrations were measured by homogeneous
a human bronchial epithelial cell line
4. Procopiou, P. A.; Barrett, V. J.; Bevan, N. J.; Biggadike, K.; Box, P. C.; Butchers, P.
R.; Coe, D. M.; Conroy, R.; Emmons, A.; Ford, A. J.; Holmes, D. S.; Horsley, H.;
Kerr, F.; Li-Kwai-Cheung, A.-M.; Looker, B. E.; Mann, I. S.; McLay, I. M.;
Morrison, V. S.; Mutch, P. J.; Smith, C. E.; Tomlin, P. J. Med. Chem. 2010, 53, 4522.
5. Baur, F.; Beattie, D.; Beer, D.; Bentley, D.; Bradley, M.; Bruce, I.; Charlton, S. J.;
Cuenoud, B.; Ernst, R.; Fairhurst, R. A.; Faller, B.; Farr, D.; Keller, T.; Fozard, J. R.;
Fullerton, J.; Garman, S.; Hatto, J.; Hayden, C.; He, H.; Howes, C.; Janus, D.; Jiang,
Z.; Lewis, C.; Loeuillet-Ritzler, F.; Moser, H.; Reilly, J.; Steward, A.; Sykes, D.;
Tedaldi, L.; Trifilieff, A.; Tweed, M.; Watson, S.; Wissler, E.; Wyss, D. J. Med.
Chem. 2010, 53, 3675.
a
radioimmunoassay (Flashplate, PerkinElmer). The intrinsic activity IA of a
test compound is its maximal cAMP response expressed in % of the maximal
response obtained for isoproterenol.
26. Male guinea pigs were exposed for 10 min in a whole body exposure chamber
to nebulized aqueous solutions of test compound (1.0–100 lg/mL) delivered in
a mixture of gases (5% CO2, 21% O2, 74% N2). Post-treatment, pulmonary
resistance was measured in anesthetized, spontaneously-breathing animals

2630
using
J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2625–2630
a
head-out plethysmograph. Bronchoconstriction was induced by
28. Axt, S.; Stergiades, I. Patent U.S. 7,060,712, 2006.
increasing doses of intravenous acetylcholine (ACh) and the dose of ACh
needed to double the baseline resistance (PD2) was calculated.
29. Martin, W. P.; Smith, N.; Vernon, L. E. Patent Application U.S. 20090227547,
2009.
27. Glossop, P. A.; Lane, C. A. L.; Price, D. A.; Bunnage, M. E.; Lewthwaite, R. A.;
James, K.; Brown, A. D.; Yeadon, M.; Perros-Huguet, C.; Trevethick, M. A.;
Clarke, N. P.; Webster, R.; Jones, R. M.; Burrows, J. L.; Feeder, N.; Taylor, S. C. J.;
Spence, F. J. J. Med. Chem. 2010, 53, 6640.
30. Alcaraz, L.; Bailey, A.; Cadogan, E.; Connolly, S.; Jewell, R.; Jordan, S.; Kindon, N.;
Lister, A.; Lawson, M.; Mullen, A.; Dainty, I.; Nicholls, D.; Paine, S.; Pairaudeau,
G.; Stocks, M. J.; Thorne, P.; Young, A. Bioorg. Med. Chem. Lett. 2012, 22, 689.