Discovery of (R)-1-(3-((2-Chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl)amino)methyl)-5-methoxyphenyl)amino)-3-oxopropyl)piperidin-4-yl [1,1′-biphenyl]-2-ylcarbamate (TD-5959, GSK961081, Batefenterol): First-in-Class dual pharmacology multivalent muscarinic antagonist and β2 agonist (MABA) for the treatment of chronic obstructive pulmonary disease (COPD)

Article abstract of DOI:10.1021/jm501915g

Through application of our multivalent approach to drug discovery we previously reported the first discovery of dual pharmacology MABA bronchodilators, exemplified by 1. Herein we describe the subsequent lead optimization of both muscarinic antagonist and β₂ agonist activities, through modification of the linker motif, to achieve 24 h duration of action in a guinea pig bronchoprotection model. Concomitantly we targeted high lung selectivities, low systemic exposures and identified crystalline forms suitable for inhalation devices. This article culminates with the discovery of our first clinical candidate 12f (TD-5959, GSK961081, batefenterol). In a phase 2b trial, batefenterol produced statistical and clinically significant differences compared to placebo and numerically greater improvements in the primary end point of trough FEV1 compared to salmeterol after 4 weeks of dosing in patients with moderate to severe chronic obstructive pulmonary disease (COPD).

Full text of DOI:10.1021/jm501915g

Drug Annotation
pubs.acs.org/jmc
Discovery of (R)‑1-(3-((2-Chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-
1,2-dihydroquinolin-5-yl)ethyl)amino)methyl)-5-
methoxyphenyl)amino)-3-oxopropyl)piperidin-4-yl [1,1′-Biphenyl]-2-
ylcarbamate (TD-5959, GSK961081, Batefenterol): First-in-Class Dual
Pharmacology Multivalent Muscarinic Antagonist and β2 Agonist
(MABA) for the Treatment of Chronic Obstructive Pulmonary Disease
(COPD)
Adam D. Hughes,*,† Yan Chen,† Sharath S. Hegde,‡ Jeffrey R. Jasper,∥ Sarah Jaw-Tsai,§ Tae-Weon Lee,∥
Alexander McNamara,‡ M. Teresa Pulido-Rios,‡ Tod Steinfeld,∥ and Mathai Mammen†
†
‡
§
∥
Departments of Medicinal Chemistry, Pharmacology, Drug Metabolism and Pharmacokinetics, and Molecular and Cellular
Biology, Theravance Biopharma, Inc., 901 Gateway Boulevard, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Through application of our multivalent approach to drug discovery we previously reported the first discovery of
dual pharmacology MABA bronchodilators, exemplified by 1. Herein we describe the subsequent lead optimization of both
muscarinic antagonist and β2 agonist activities, through modification of the linker motif, to achieve 24 h duration of action in a
guinea pig bronchoprotection model. Concomitantly we targeted high lung selectivities, low systemic exposures and identified
crystalline forms suitable for inhalation devices. This article culminates with the discovery of our first clinical candidate 12f (TD-
5959, GSK961081, batefenterol). In a phase 2b trial, batefenterol produced statistical and clinically significant differences
compared to placebo and numerically greater improvements in the primary end point of trough FEV1 compared to salmeterol
after 4 weeks of dosing in patients with moderate to severe chronic obstructive pulmonary disease (COPD).
(LABA) are more desirable because of their 24 h duration of
action controlling disease symptoms and improved patient
compliance owing to their once-daily dosing regimen (Chart
1).2 Binary combinations of these agents, such as QVA149
(glycopyrronium plus indacaterol)3 and ANORO (umeclidi-
nium bromide and vilanterol),4 show even greater improve-
ments in lung function, while “triple therapy”, through the
addition of an inhaled corticosteroid (ICS), is recommended in
COPD patients at high risk for exacerbations. Historically
inhaler devices have been limited to binary combinations
though several companies are now applying their specific
technologies to enable the formulation of three components.5
INTRODUCTION
■
Chronic obstructive pulmonary disease (COPD) is now
recognized as a global health problem and is projected to be
the third leading cause of death worldwide by 2020. The
pulmonary component of this debilitating disease is charac-
terized by persistent and progressive airflow limitation. A
combination of small airways disease (obstructive bronchiolitis)
and parenchymal destruction (emphysema) are responsible for
the airflow limitation component of COPD, with relative
contributions varying across the patient population.1 Broncho-
dilators are recommended at all stages of COPD, with
muscarinic acetylcholine receptor antagonists (MAs) and β2
adrenergic receptor agonists (BAs) being the most frequently
used. For more advanced stages of the disease, long acting (LA)
bronchodilators such as tiotropium (LAMA) and indacaterol
Received: December 10, 2014
© XXXX American Chemical Society
A
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Drug Annotation
simultaneously and achieve the appropriate balance of
activities.9
Chart 1. Once-Daily Long-Acting Bronchodilator Reference
Standards
In our first reported MABA drug discovery efforts, we
identified an optimal linker length of nine atoms between the
amine atoms of the biphenyl carbamate muscarinic and
carbostyril pharmacophores, culminating in the early lead
compound 1 (THRX-198321) (Chart 2).10 Further elaboration
of this nonylalkyl chain to introduce additional functionalities
such as secondary and tertiary amides, carbo- and heterocycles,
while maintaining the overall nine atom linker length, proved to
be well tolerated in vitro and in vivo.11 While a napadisylate
crystalline form12 of 1 was identified, all attempts to crystallize
the amide-linked MABAs 2 (THRX-109156) and 3 (THRX-
494732) (Chart 2) failed despite their promising biological
profiles. In contrast, arylamide linked MABAs 4 and 5 (Chart
3) showed promising crystalline properties but lacked 24 h
duration of action as β2 agonists in vivo. Using these
compounds as a foundation for achieving suitable crystalline
forms, in this article we describe the optimization of their
biological properties toward the discovery of the clinical
candidate 12f (TD-5959).
However, the regulatory path forward becomes even more
complex with three, rather than two, agents. One possible
solution is to combine two of the pharmacologies in a single
molecule. Preclinical data support the hypothesis that
muscarinic (M2, M3) and β2 receptors in the lung act through
complementary pathways6 such that the resulting MABA would
offer a desirable solution to achieving “triple therapy” in a single
device, concomitantly matching the pharmacokinetics and
pharmacodynamics of two of the three components.
This project presented two significant drug discovery
challenges: (1) designing compounds suitable for inhaled
delivery with low systemic exposures; (2) designing dual
pharmacology compounds such that both mechanisms of action
demonstrate matched duration of action in vivo. To ensure
minimal systemic exposure, an inhaled drug requires a low dose
and poor oral bioavailability, since up to 90% of the dose may
be swallowed.7 Antedrug approaches, such that the drug is
rapidly metabolized upon reaching the bloodstream, further
control for low systemic exposures.8 Current inhalation devices
require highly crystalline material, and therefore, early screening
for suitable crystalline forms in the discovery effort is critical.
Dual pharmacology molecules are attractive because of the
prospect of greater efficacy over single pharmacology agents
and simplified pharmacokinetics over fixed dose combinations,
but the challenges in early drug discovery are much greater
because of the need to optimize both pharmacologies
CHEMISTRY
■
The synthetic route for compounds 4 and 12a−j (Scheme 1)
began with the 1,4-addition of our biphenyl carbamate
muscarinic orthostere 7, formed in one step from commercially
available starting materials, to the appropriately aryl-substituted
acrylamide 6. Lithium borohydride mediated reduction of the
aryl ester afforded the alcohol, which was then subsequently
oxidized to the aldehyde 10. Reductive amination with
protected carbostyrilamine orthostere 1113 and subsequent
removal of the silyl protecting group gave the MABA
compounds 4, 12a−j.
Reverse amides 5 and 19a−c were synthesized from amine
building blocks 13 and 16 (Scheme 2). Primary amine 13 was
prepared by alkylation of 7 with BOC-protected aminoethyl
bromide, followed by BOC removal under standard conditions.
Secondary amine 16 was accessed from amino alcohol 14, after
Cbz protection and subsequent oxidation; this material was
further used in a reductive amination with 7, and after
hydrogenation of the Cbz group, 16 was isolated. Amines 13
and 16 were then individually coupled with commercially
Chart 2. Previously Described Theravance MABAs
B
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Drug Annotation
Chart 3. Unsubstituted Arylamide Linked MABAs
a
Scheme 1. Synthesis of Compounds 4, 12a−j
a
Reagents: (a) DIPEA, 0 °C, DCM, 67%; (b) (i) 70 °C, 12 h; (ii) 50 °C EtOH, 6 M HCl; (iii) NH4HCO2, Pd/C (10 wt %), 40 °C, 12 h, 100%; (c)
6, 50 °C, THF/MeOH (9:1), 70−80%; (d) LiBH4, 0 °C, THF/MeOH (9:1) 20−80%; (e) DMSO, DIPEA, Py·SO3, −20 to 0 °C, DCM; (f) (i) 11,
Na(OAc)3BH, DCM/MeOH (1:1); (ii) Et3N·3HF, DCM, 25−40% over three steps.
available benzoic acid 17 and oxidized to give 18. The desired
MABA compounds 5, 19a−c were isolated upon subjection of
18 to reductive amination with the carbostyrilamine 11 and silyl
group removal.
The meta-substituted anilides 30a−c were prepared in four
steps from commercially available amino esters 25. The esters
were converted to the acrylamides 26 and used in a 1,4-addition
with the muscarinic orthostere 7. The resulting esters 27 were
reduced to the alcohol and nosylated to give intermediate 28.
The nosylate activation of the alcohols was the most viable
route for the synthesis of these compounds, since the
corresponding aldehyde was unstable because of its propensity
to polymerize. Alkylation of the benzyl protected carbostyril
building block 2912 with 28 followed by benzyl and silyl
protecting group removal gave the MABA compounds 30a−c
(Scheme 4).
N-Methyl compounds 24a−c were accessed through two
different pathways depending on the availability of the
substituted aryl ring (Scheme 3). Commercially available
anilines were methylated by way of formylation and then
reduced to give methylanilines 21. The acrylamide was then
formed to facilitate a 1,4-addition of 7 to give intermediate 22.
Aryl iodide (path A) was converted to benzaldehyde 23 using a
palladium-mediated reaction with acetic formic anhydride. Aryl
esters (path B) were converted to aldehyde 23, as described
previously, through reduction to the alcohol and then
oxidation. Reductive amination and silyl group removal
completed the syntheses of MABAs 24a−c.
RESULTS AND DISCUSSION
■
In a study of physical properties of inhaled drugs, high
molecular weight and high total polar surface area (tPSA) were
C
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Drug Annotation
a
Scheme 2. General Syntheses of Compounds 5, 19a−c
a
Reagents: (a) (i) 2-tert-butoxycarbonylaminoethyl bromide, DIPEA, MeCN, 50 °C; (ii) TFA/DCM (1:3), 70% over two steps; (b) (i) benzyl
chloroformate, aq Na2CO3, THF; (ii) DMSO, DIPEA, Py·SO3, −20 to 0 °C, DCM, 95% over two steps; (c) (i) 7, Na(OAc)3BH, MeOH; (ii) Pd/C
(10 wt %), H2(g), MeOH, 44% over two steps; (d) (i) HATU, DIPEA, DMF; (ii) DMSO, DIPEA, Py·SO3, −20 to 0 °C, DCM, 50% over two steps;
(e) (i) 11, Na(OAc)3BH, DCM/MeOH (1:1); (ii) Et3N·3HF, DCM, 6−20% over two steps.
a
Scheme 3. Synthesis of Compounds 24a−c
a
Reagents: (a) (i) HCO2H, Ac2O; (ii) BH3·Me2S, THF, 80−90%; (b) (i) acryloyl chloride, NaHCO3, DCM; (ii) 7, DCM/EtOH (1:1), 90−95%;
(c) AcOCHO, Et3SiH, DIPEA, Pd2dba3, dppe, MeCN, 78%; (d) (i) LiAlH4, THF; (ii) DMSO, DIPEA, Py·SO3, −20 to 0 °C, DCM, 69−82%; (e)
(i) 11, Na(OAc)3BH, MeOH/DCM (1:1); (ii) Et3N·3HF, DMF/DCM (1:9), 26−52% over two steps.
identified as the key drivers for achieving long acting lung
retained compounds.14 More recently, increases in basicity have
been proposed to extend pharmacokinetic duration and favor
lung, rather than systemic, exposure.7 Long residence time on
the receptor is also a precedented design feature for achieving
long duration of action, with tiotropium often believed to be
the best example to date in the inhaled drug space.15 However,
while some elegant postrationalization to understand the
D
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Drug Annotation
a
Scheme 4. General Syntheses of Compounds 30a−c
a
Reagents: (a) acryloyl chloride, NaHCO3, DCM, 95%; (b) 7, DCM/iPrOH (1:1), 60 °C, 90%; (c) (i) LiAlH4, THF, 0 °C; (ii) DABCO, DCM,
nosyl chloride; (d) (i) NaHCO3, MeCN, 43% over three steps; (ii) Pd(OH)2, H2(g), AcOH, MeOH; (iii) Et3N·3HF, DMF/DCM (1:9), 59% over
two steps.
features contributing to long residence times may be possible, it
still remains challenging to prospectively design for slow off-
rate kinetics.16 Additionally, tiotropium has recently been
shown to have much shorter off-rate kinetics under
physiological versus nonphysiological conditions (46 and 462
min, respectively) than previously reported, suggesting that the
molecule’s physical properties play a significant role in its
duration of action.17
the behavior of established once-daily compounds such as
tiotropium and indacaterol. The 2-substituted compounds 12a,
12b, and 12g improved BA activity in vivo relative to 4 but
failed to meet our BA criterion at 24 h (Table 5). However, the
3-methoxy compounds 12d and 12f both displayed significant
BA bronchoprotection and pleasingly also retained MA activity.
Our model also enabled us to assess the two activities working
in union as MABAs. Greater bronchoprotection was observed
than either MA or BA alone, demonstrating the cooperation
between the M3 and β2 pathways.
A more limited investigation of aryl ring substitution effects,
due to the availability of commercial starting materials, was
performed on the reverse amide 5 (Table 2). In this case,
reduction in M3 potency was observed for both 3-methoxy 19a
and 2,5-dimethyl 19b derivatives. The unsubstituted N-methyl
analog 19c recovered M3 potency; however, this compound
failed to produce crystalline forms in our early screens. Our
general methodology for screening for crystalline compounds is
described in detail in the Supporting Information; however, this
was usually conducted in three stages: broad counterion
screening in different solvent systems using evaporative
crystallizations, identification of optimal crystallization con-
ditions (e.g., vapor diffusion, antisolvent additions), and finally
scale up and characterization of promising salt forms. Over the
course of the MABA program the selection of the initial
screen’s counterions was adapted as we learned more about the
general properties of these molecules.
Compounds 4 and 5 possessed good physical property
starting points for designing compounds with long duration of
action given their high molecular weight (676 Da) and tPSA
(156 Å2). Given the proximity of the linker aryl group to the
carbostyril β2 orthostere, we decided to investigate the effect of
modulating the ring electronics on the BA in vivo properties. A
survey of electron donating and withdrawing groups and
combinations thereof revealed some interesting trends in vitro
(Table 1). The addition of chlorine in both the 2- and 3-
positions (12a and 12c, respectively) retained potency at both
targets, while the 2-methoxy compound 12b exhibited reduced
M3 potency, compared to its 3-substituted counterpart 12d. In
contrast methyl substitution at the 2-position 12g was preferred
for high M3 potency. Given these small structural changes are
somewhat distal to the biphenyl carbamate orthostere, it is
quite remarkable what effects are bestowed on the M3 potency
profiles, highlighting the contributions these linker moieties are
making toward in vitro activity on both targets. Retaining the
optimal 3-methoxy group and adding the chlorine or methyl
groups to either ortho position (to the anilide) also gave
compounds 12e, 12f, 12i, and 12j worthy of further evaluation.
Compounds 12a, 12b, 12d, 12f, and 12g were progressed
into our in vivo bronchoprotection model18 after showing early
signs of crystallinity and offering a good representation of the
substituents surveyed. Our criteria for this assay were to achieve
40% or greater bronchoprotection at 24 h on both MA and BA
activities. From our earlier MABA work, this threshold was
considered a robust, reproducible response and consistent with
Methylation of a selection of anilides from Table 1 offered
mixed effects on M3 and β2 activities in vitro (Table 3). While
M3 potency increased for the 2-chloro, 5-methoxy derivative
24a, the 3-methoxy 24b and 2-methyl 24c compounds
displayed weaker M3 activity and marked reductions in β2
activity. In vivo, 24a demonstrated 24 h duration of action as a
MA at 30 μg/mL; however, the lack of crystalline forms halted
any further characterization of this compound.
E
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Drug Annotation
a
Table 1. M3, β2 Potency, β1 Selectivity, tPSA, and MW for Compounds 12a−j
a
M3 Ki was measured at a time point where equilibrium was reached (either 1 or 6 h; see Supporting Information). Log sel. β1/β2 EC50 is described
in the Experimental Section.
F
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Table 2. M3, β2 Potency, β1 Selectivity for Compounds 19a−c
Table 3. M3, β2 Potency, β1 Selectivity for Compounds 24a−c
We also investigated meta-substituted anilides that retained
the nine-atom linker length between the orthosteric nitrogen
atoms (Table 4). This phenylethyl substituted β2 orthostere
motif is present in marketed LABAs such as arformoterol19 and
indacaterol. In contrast to the benzylamides described earlier
(Tables 1−3), the most interesting compound from this small
set was the unsubstituted 30a, which offered the best in vitro
profile when compared to the methoxy-substituted compounds
30b and 30c. In vivo, robust MA, BA, and MABA efficacies
were demonstrated at 24 h, warranting further characterization
of this compound (Table 5).
Having identified 12d, 12f, and 30a as early in vitro leads
with 24 h MA and BA duration of action in vivo, we conducted
further characterization to assess onset of bronchoprotection
and lung selectivity. All of these compounds demonstrated fast
onset of action with significant and maximal MA and BA
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Table 4. M3, β2 Potency, β1 Selectivity for Compounds 30a−c
a
Table 5. Guinea Pig Einthoven Model of Bronchoprotection Data at 24 h for Compounds 1, 4, 5, 12a, 12b, 12d, 12f, 12g, 30a
24 h, % bronchoprotection
MA
BA
MABA
compd
dose, μg/mL
tiotropium
10
75
7
6
n = 6
NA
43
NA
NA
indacaterol
100
100
100
100
100
100
100
100
100
100
NA
72
3
8
n = 6
n = 6
n = 6
n = 4
n = 3
n = 6
n = 8
n = 3
n = 6
n = 6
1
n = 6
n = 5
10
4
98 0.3
NT
−2
25 13
33 20
3
12a
12b
12d
12f
5
22 16
n = 3
n = 6
n = 8
n = 3
n = 6
n = 5
81
48
74
92
3
9
3
3
72
47
20
20
46
4
5
1
7
6
77
4
n = 8
12g
30a
96
72
1
4
n = 6
n = 6
51 14
a
The TFA salt form was dosed in all of the initial experiments. The edisylate salt form of 12f did not alter the efficacy or dose concentration
delivered to the animals when compared with the TFA salt form.
Table 6. Guinea Pig Einthoven Model of Bronchoprotection and Pilocarpine Induced Salivation Data at 1.5 h for Compounds
1, 4, 12d, 12f, 30a
1.5 h, % bronchoprotection
MA
BA
MABA
1.5 h, % inh of pilocarpine
% inh
compd
dose, μg/mL
dose, μg/mL
tiotropium
10
90
2
1
n = 6
n = 6
NA
74
63
47
70
73
80
NA
NA
NT
NT
NT
92
100
NA
99 0.3
n = 6
indacaterol
100
100
100
100
100
100
NA
94
6
5
5
5
4
7
n = 6
n = 6
n = 6
n = 6
n = 8
n = 6
1
NT
4
NT
94
300
450
500
500
82
5
n = 6
n = 6
n = 5
n = 6
12d
12f
30a
1
5
6
n = 6
n = 8
n = 6
−3 12
75
3
n = 8
n = 6
4
12
5
77
93
2
23
activities at the earliest time point measured (1.5 h) (Table 6).
Interestingly, both compounds (1 and 4) that lacked BA
duration at 24 h showed substantial BA bronchoprotection at
this early time point. One explanation for this observation
might be that tachyphylaxis is occurring via the β-arrestin
pathway; however, the biased agonism nature of these
molecules has not been characterized.20 The lung retention
and selectivity of our MABA compounds were measured by
evaluating the sialagogue response to pilocarpine in guinea pigs
after pretreatment with various doses of the test compound.21
Dry mouth is the most common side effect of tiotropium.22 At
1.5 h postdose, 12d, 12f, and 30a all displayed significantly
reduced effects when compared with tiotropium and the
unsubstituted arylamide 4 in inhibiting sialagogue responses to
pilocarpine. While a crystalline edisylate salt form of 12f had
been discovered,23 no crystal forms of 12d or 30a had been
H
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Table 7. In Vitro and in Vivo Pharmacokinetics for 12f in Rats
in vitro data
iv
compd
CACO-2 Papp × 10−6 (cm/s)
HLM T1/2 (min)
rat in vivo PK, dose (mg/kg)
0.25
CL (L h−1 kg−1
4.52
)
Vdss (L/kg)
0.32
T1/2 (h)
po, F (%)
12f
<0.4
11.3
0.18
<0.45
Table 8. In Vitro Major Metabolites of 12f and Their Activities
compd
hM3 pKI
HEK hβ2 pEC50
log β1/β2 EC50
12f
31
32
8.7
7.9
5.0
9.6
<5
8.3
2.6
NA
3.2
a
a
CHO hβ2 pEC50. Potency differences between HEK and CHO cell expressions were negligible.
identified despite significant efforts. Therefore, because of the
structural similarity between 12f and 12d, 12f was prioritized
for further characterization.
in rats following inhalation of 12f. At 600 nM, 12f resulted in
<20% inhibition of CYP2C9, 2C19, 2D6, and 3A4 activities,
while a 25% inhibition on CYP1A2 activity was observed. On
the basis of the low inhibitory effect observed for 12f at 600 nM
(i.e., 2000-fold above the β2 EC50 and 460-fold above M3 Ki)
and anticipated low plasma concentration, the risk for 12f to
cause pharmacokinetic drug−drug interactions in humans by
inhibition of the major drug-metabolizing enzymes was
predicted to be very low.
12f exhibited an ideal pharmacokinetic profile for an inhaled
agent coupled with an excellent in vitro and in vivo biological
profile and was subsequently evaluated in safety assessment
studies. There was no evidence of inhibition of hERG
potassium channel currents by 12f at a concentration of 150
nM (approximately 100 times its binding affinity toward M3
and approximately 500 times the EC50 at β2). No evidence of
mutagenicity was noted in an exploratory (non-GLP) Ames
assay at up to 5000 μg/plate in the presence or absence of a
metabolic activation system. In a 7-day inhalation study in the
rat dosing up to 333 μg kg−1 day−1, there was no evidence of
respiratory irritation and systemic exposures did not increase.
Pharmacology studies of 12f in the guinea pig have shown that
the therapeutic index for antisialagogue activity is approximately
800-fold, 55-fold greater than that of tiotropium. Systemic β2
effects were evaluated using hypotensive end points in guinea
pigs.26 Consistent with the very low systemic exposures of 12f,
its lung selectivty is 78-fold, 14-fold greater lung selectivity than
that of salmeterol in this assay.
These preclinical data supported nomination of 12f as a
development candidate. In collaboration with GSK, the
compound was progressed into clinical trials. Proof of concept
was demonstrated in a phase 2a trial (NCT00478738)
compared to placebo and twice-daily salmeterol plus tiotropium
in 50 patients with moderate to severe COPD.27 A phase 2b
trial (NCT01319019) evaluated dose response, dose interval,
efficacy, and safety in 436 moderate to severe COPD patients
over a 4-week treatment period, with salmeterol (50 μg twice-
daily) included as an active comparator. All doses were well
tolerated and produced statistical and clinically significant
differences compared to placebo and numerically greater
improvements in the primary end point, the change from
In kinetic radioligand dissociation studies at the human M3
receptor, 12f was found to have a short dissociation half-life (3
min); half-lives for other MABAs such as 4 were not tested.
Therefore, the MA duration of action in vivo cannot be
attributed to receptor residence time for 12f. We believe the
lung retention is facilitated by the possibility of rebinding as
proposed for LABAs,24 as well as our own cooperative binding
hypothesis,25 and the aforementioned basicity and physical
properties such as high molecular weight and tPSA. Excellent
topical retention was displayed by 12f after inhalation dosing in
guinea pig, with high lung to plasma ratios from the initial
postdose out to 21 days and a pharmacokinetic half-life of 4
days, consistent with the long duration of action in our efficacy
models. In guinea pig isolated trachea expressing native
muscarinic M3 and β2, 12f produced smooth muscle relaxation
through a dual mechanism involving competitive antagonism of
the M3 receptor (EC50 = 50 nM) and agonism of the β2
receptor (EC50 = 25 nM).26 The combined effect on both
muscarinic receptors and β2 receptors was more potent than
either function working alone (EC50 = 10 nM).
The in vitro pharmacokinetic properties of 12f met our
criteria for low systemic exposures through rapid CYP-
mediated metabolism and negligible permeability (Table 7).
Metabolic lability of 12f was observed across rat, dog, guinea
pig, and human liver microsomes. No human specific
metabolites were observed for 12f in human liver microsomes.
The metabolites generated were less potent than the parent at
either respective M3 or β2 target. The metabolite 31, exhibiting
7-fold lower M3 potency than 12f, was not detected in vivo. A
second major metabolite 32 was 14-fold less potent and more
β2 selective than the parent molecule (Table 8). In vivo levels
for this metabolite were <4% in dog and guinea pig studies
(0.01 and 0.5 mg/kg, respectively, iv). In vivo pharmacokinetic
studies of 12f in rats were consistent with our in vitro findings;
the compound exhibited a rapid rate of clearance and short
half-life. Consequently the oral bioavailability was very low
because of the combination of high clearance and poor
permeability. Very low systemic exposure was also observed
I
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Drug Annotation
Figure 1. Comparison of molecular interactions of 4 and 12f modeled using the β2 crystal structure: (1A) 12f modeled using the 3P0G crystal
structure; (1B) H-bonding event of the aryl methoxy group with TYR316; (1C) lack of a H-bond for 4 in this region.
baseline in trough forced expiratory volume in 1 s on day 29,
compared to salmeterol. Batefenterol was generally well
tolerated at all doses tested (100, 400, 800 μg once-daily;
100, 200, 400 μg twice-daily) with the optimal dose found to be
400 μg, either once-daily or as 200 μg twice-daily.28
properties of the molecule that are likely key drivers for
duration of action at both targets. This article serves to
highlight the significant challenge, but substantial benefit, of
designing compounds exhibiting balanced dual pharmacology
profiles in vitro and in vivo and the need for early screening and
identification of suitable crystalline forms that are compatible
with inhalation devices due to the high attrition rate and lack of
predictability.
CONCLUSION
■
We have described the application of our multivalent approach
to drug discovery to identify 12f as the first MABA compound
to enter clinical trials and demonstrate proof of concept and
efficacy in COPD patients. Further confirmatory trials are
warranted, but this compound has the potential to improve
lung function in COPD patients as well as represent a simpler
development strategy toward achieving “triple therapy” in one
inhalation device. Preclinically, we were able to optimize
duration of action in the lung, retaining MA duration at 24 h
from our scaffold start point 4 and build in BA duration by
virtue of modulating the electronics and substitution of the
linker aryl ring. Using the recently described β2 crystal
structure29 as our start point, 12f was modeled using MOE
(Figure 1).30 Comparing the interactions with the unsub-
stituted 4, we retrospectively (since the β2 crystal structure was
not available at the time of our drug discovery efforts) speculate
that the improved duration of action at β2 may be partly
attributable to the proximal nature of this substituted benzyl
group to the β2 orthosteric site, making a key hydrogen bond
with Tyr316 through the methoxy oxygen and adopting a
conformation that does not induce the arrestin pathway and
associated tachyphylaxis.31 Another explanation, given 24 h MA
bronchoprotection is achieved without the need for a long
residence time, can be derived from the overall physical
EXPERIMENTAL SECTION
■
Chemistry. Unless otherwise indicated, all reactions were
performed under a nitrogen atmosphere, using commercially available
anhydrous solvents and starting materials. Progress of reaction
mixtures was monitored by analytical high performance liquid
chromatography (HPLC) and mass spectrometry, the details of
which are given below. Thin layer chromatography was performed on
Merck 0.25 mm Kieselgel 60 F254 plates, and compounds were
visualized under UV light and/or by staining with aqueous KMnO4
solution. Reaction mixtures were worked up as described specifically in
each reaction and routinely purified by preparative HPLC. Mass
spectrometric identification of compounds was performed using
standard electrospray ionization methods (ESMS) with a PerkinElmer
SCIEX API 150 EX. High resolution mass spectrometry was
performed using standard electrospray ionization methods (ESMS)
with an Applied Biosystems/MDS SCIEX QSTAR. Analytical reverse
phase HPLC−MS analysis was conducted on a 1100 series HPLC
system from Agilent Technologies (Santa Clara, CA), consisting of a
G1312A binary pump, G1313A autosampler, G1315A PDA detector,
G1316A column compartment, and G1322A degasser and solvent tray.
The system is connected to an AB SCIEX 150EX mass spectrometer
containing an electrospray ionization (ESI) source and operated with
Analyst software. The mobile phases are LC−MS grade water (A) and
MeCN (B) from Honeywell Burdick & Jackson, both containing 0.1%
J
DOI: 10.1021/jm501915g
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Drug Annotation
formic acid. For HPLC, the 2.1 mm × 50 mm analytical column used
is a BONUS RP C14 column from Agilent, each with a particle size of
3.5 μm. The flow rate is 0.5 mL/min (4 min methods). Reverse phase
(RP) preparative HPLC was performed using LC−MS grade water
(A) and MeCN (B) from Honeywell Burdick & Jackson, both
containing 0.1% TFA, a flow rate of 20 mL/min, and 21.2 mm × 150
mm and 21.2 mm × 250 mm preparative columns; Zorbax Bonus RP
C14 from Agilent and Luna C18 from Phenomenex (Torrance, CA). A
semipreparative autopurification LC−MS system from Waters
Corporation (Milford, MA), consisting of a 2767 sample manager
providing open bed fraction collection, a 2998 PDA coupled with a
3100 series mass spectrometer containing an ESI source, and full
software control provided by MassLynx software, was used for high
throughput compound purification. The LC−MS instrument contains
high-pressure 2545 solvent pumps and a dual-column fluidics
organizer. Method gradients were determined based on analytical
scouting runs, and fractions were collected by MS triggering on the (M
+ H)+ ion or UV at a specified wavelength (typically 214 nm).
Collected fractions were dried on a GeneVac HT 24 evaporator
(Genevac LTD, Gardiner, NY), weighed on a Bohdan automated
robotic workstation by Mettler-Toledo AutoChem (Columbia MD),
and analyzed by gradient analytical LC−MS to determine the purity
(UV area percent at 214 nm). Characterization of reaction products
was routinely performed by 1H and 13C NMR spectrometry in
deuterated solvents specified in the examples, acquired under standard
parameters using a Varian Gemini 2000 instrument (400 or 300 MHz)
or Bruker UltraShieldplus NMR spectrometer (600 MHz). Com-
pounds submitted for biological evaluation were isolated in ≥95%
purity (assessed by analytical HPLC or LC−MS) unless otherwise
stated. Organic solutions were dried over anhydrous Na2SO4 unless
otherwise stated.
(Scheme 1) General Procedure for the Preparation of the
Aryl Ester 8. To the acrylamide (5 mmol) were added biphenyl-2-
ylcarbamic acid piperidin-4-yl ester (1.33 g, 4.5 mmol) and a mixture
of THF (22.5 mL) and MeOH (2.5 mL). This mixture was heated at
50 °C with stirring for 16 h, and then the solvent was removed in
vacuo. The residue was chromatographed (silica gel; EtOAc) to give
the title compound, e.g., R = 2-Cl, 5-MeO (2.0 g; Rf = 0.4, 78%), as an
off-white foam (95% purity). MS m/z 566.4 (M + H, expected 565.20
1
for C30H32ClN3O6). H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H),
8.15 (s, 1H), 7.78 (s, 1H), 7.45−7.26 (m, 8H), 4.57−4.44 (m, 1H),
3.79 (s, 3H), 3.77 (s, 3H), 2.76 (s, 2H), 2.61 (s, 4H), 2.24 (s, 2H),
1.78 (d, J = 8.9 Hz, 2H), 1.52 (d, J = 9.8 Hz, 2H).
(Scheme 1) General Procedure for the Preparation of
Alcohol 9 and Aldehyde 10. To a solution of the ester 8 (0.82 g,
1.45 mmol) in a mixture of THF (4.5 mL) and MeOH (0.5 mL) at 0
°C was added lithium borohydride (32 mg, 1.45 mmol). The reaction
mixture was allowed to warm to room temperature and was stirred
overnight at this temperature. The reaction mixture was quenched by
the addition of 1 N HCl at 0 °C. The solvent was removed in vacuo,
and the residue was dissolved in MeCN (2 mL). This solution was
purified by RP-HPLC (gradient, 2−50% MeCN in water with 0.05%
TFA). The appropriate fractions were collected, combined, and
lyophilized to give the title compound as a trifluoroacetate salt (98%
purity by HPLC). This salt was treated with isopropyl acetate (10 mL)
and 1 N NaOH (10 mL), and the organic layer was collected, dried,
filtered and the solvent was removed under reduced pressure to give
the alcohol 9 (161 mg, 21%) as a white foam, e.g., R = 2-Cl, 5-MeO:
MS m/z 538.4 (M + H, expected 537.20 for C29H32ClN3O5). 1H
NMR (600 MHz, DMSO-d6) δ 8.70 (s, 1H), 7.76 (s, 1H), 7.50−7.23
(m, 9H), 4.49 (br s, 1H), 4.44 (d, J = 5.7 Hz, 2H), 3.73 (s, 3H), 2.74
(br s, 2H), 2.60 (s, 2H), 2.54 (s, 2H), 2.17 (br s, 2H), 1.77 (br s, 2H),
1.51 (br s, 2H). To a stirred solution of the resulting alcohol (161 mg,
0.3 mmol) in DCM (3 mL) were added DMSO (213 μL, 3.0 mmol)
and DIPEA (261 μL, 1.5 mmol). This mixture was cooled to −20 °C,
and sulfur trioxide pyridine complex (238 mg, 1.5 mmol) was added
slowly. After 30 min, the reaction mixture was quenched by adding
water (3 mL). The layers were separated and the organic layer was
dried, filtered and the solvent was removed under reduced pressure to
give the aldehyde 10 as a light yellow solid (98%), e.g., R = 2-Cl, 5-
MeO: MS m/z 536.3 (M + H, expected 535.19 for C29H30ClN3O5).
1H NMR (600 MHz, DMSO-d6) δ 10.20 (s, 1H), 8.85 (d, J = 15.0 Hz,
Chiral purity was determined on a Beckman P/ACE MDQ capillary
electrophoresis system employing a 50 μm, 60 cm fused silica capillary
and 25 μM triethylammonium phosphate/10% w/v highly sulfated γ-
cyclodextrin (Beckman) buffer system.
General procedures contain representative reagent quantities and
product yields for the purpose of illustration.
Biphenyl-2-ylcarbamic Acid Piperidin-4-yl Ester 7. Biphenyl
2-isocyanate (97.5 g, 521 mmol) and 4-hydroxy-1-benzylpiperidine
(105 g, 549 mmol) were heated together at 70 °C for 12 h. The
reaction mixture was then cooled to 50 °C, and EtOH (1 L) was
added, followed by slow addition of 6 M HCl (191 mL). The reaction
mixture was then cooled to room temperature, and ammonium
formate (98.5 g, 1.56 mol) was added and nitrogen gas bubbled
through the solution for 20 min. Palladium (10 wt % (dry basis) on
activated carbon) (20 g) was then added. The reaction mixture was
heated at 40 °C for 12 h and then filtered through a Celite pad.
Solvent was removed under reduced pressure and 1 M HCl (40 mL)
added to the crude residue. 10 N NaOH was added to adjust the pH
to 12. The aqueous layer was extracted with EtOAc (2 × 150 mL) and
dried (MgSO4) and solvent removed under reduced pressure to give
the title compound 7 (155 g, 100%). HPLC (10−70) tR = 2.52 (99%
purity). LRMS m/z: found, 297.3 [M + H]+; C18H20N2O2 requires
1H), 7.95 (d, J = 11.1 Hz, 1H), 7.72 (s, 1H), 7.55−7.22 (m, 8H), 4.70
(m, 1H), 3.89 (s, 3H), 3.41 (m, 4H), 3.00 (m, 4H), 2.16−1.88 (m,
2H), 1.88−1.60 (m, 2H).
(Scheme 1) General Reductive Amination and tert-Butyldi-
methylsilyl Group Removal Procedure for the Preparation of
Compounds 4, 12a−j. To a stirred solution of the aldehyde 10 (0.3
mmol) in a mixture of DCM (0.5 mL) and MeOH (0.5 mL) was
added 5-[(R)-2-amino-1-(tert-butyldimethylsilanyloxy)ethyl]-8-hy-
droxy-1H-quinolin-2-one·acetate 11 (124.1 mg, 0.3 mmol), and the
resulting mixture was stirred at room temperature for 1.5 h. Sodium
triacetoxyborohydride (190.7 mg, 0.9 mmol) was added, and the
solution was stirred at room temperature for 15 h. The reaction was
quenched by adding water (about 0.2 mL) and the mixture was
concentrated under reduced pressure, e.g., R = 2-Cl, 5-MeO: MS m/z
854.5 (M + H, expected 853.36 for C46H56ClN5O7Si). 1H NMR (600
MHz, DMSO-d6) δ 8.18 (s, 1H), 7.68−7.24 (m, 10H), 7.07 (s, 1H),
6.97 (s, 1H), 6.56 (m, 2H), 5.47 (br s, 1H), 4.69 (m, 1H), 4.38−4.02
(m, 2H), 3.77 (s, 3H), 3.36 (m, 6H), 3.03 (m, 4H), 2.20−1.46 (m,
4H), 0.81 (s, 9H), 0.06 (s, 3H), −0.23 (s, 3H). DCM (1.0 mL) was
added followed by triethylamine trihydrofluoride (245 μL, 1.5 mmol),
and this mixture was stirred at room temperature for 45 h before
removing the solvent under reduced pressure. The crude mixture was
purified by RP-HPLC (2−50% MeCN/water with 0.05% TFA) to
afford the title compound 4 or 12a−j (100 mg, 34%) as an off-white
solid, e.g., R = 2-Cl, 5-MeO: (R)-1-(3-((2-chloro-4-(((2-hydroxy-2-(8-
hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl)amino)methyl)-5-
methoxyphenyl)amino)-3-oxopropyl)piperidin-4-yl [1,1′-biphenyl]-2-
ylcarbamate (12f). LCMS found: 740; C40H42ClN5O7 requires 740.2
1
297.2. H NMR (400 MHz, CD3OD) δ 7.56 (d, J = 7.6 Hz, 1H),
7.47−7.21 (m, 8H), 4.66 (m, 1H), 3.00−2.90 (m, 2H), 2.63 (ddd, J =
12.7, 9.2, 3.3 Hz, 2H), 1.87−1.77 (m, 2H), 1.49 (dtd, J = 12.7, 8.9, 3.8
Hz, 2H).
(Scheme 1) General Procedure for the Preparation of the
Acrylamides 6. To a stirred solution of the methyl 4-aminobenzoate
(5 mmol) and triethylamine (2.1 mL, 15 mmol) in DCM (10 mL) at 0
°C was added acryloyl chloride (812 μL, 10 mmol) dropwise. After 2 h
at 0 °C, MeOH (2 mL) was added and the reaction mixture allowed to
warm to room temperature before removing the solvent under
reduced pressure. DCM (30 mL) and water (30 mL) were added and
organics separated, dried, and solvent was removed under reduced
pressure. The crude acrylamide 6 was used directly in the subsequent
step. 1H NMR (400 MHz, CD3OD) δ 8.05 (s, 1H), 7.86 (s, 1H), 6.64
(dd, J = 16.9, 10.2 Hz, 1H), 6.45 (dd, J = 16.9, 1.6 Hz, 1H), 5.86 (dd, J
= 10.2, 1.6 Hz, 1H), 3.90 (d, J = 1.8 Hz, 3H), 3.86 (s, 3H).
1
(98% purity). H NMR (300 MHz, CD3OD) δ 8.07 (d, J = 9.8 Hz,
K
DOI: 10.1021/jm501915g
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Drug Annotation
1H), 7.76 (s, 1H), 7.60−7.15 (m, 11H), 7.00 (d, J = 8.5 Hz, 1H), 6.56
(d, J = 10.7 Hz, 1H), 5.36 (dd, J = 8.5, 3.5 Hz, 1H), 4.79 (m, 1H),
4.38−4.05 (m, 2H), 3.80 (s, 3H), 3.56 (m, 4H), 3.22−2.89 (m, 6H),
2.35−1.59 (m, 4H).
Compound Name (Synthetic Route). (R)-1-(9-((2-Hydroxy-2-
(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl)amino)nonyl)piperidin-
4-yl [1,1′-Biphenyl]-2-ylcarbamate (1) (Reference 10). LCMS found:
641; C38H48N4O5 requires 640.81. 1H NMR (400 MHz, DMSO-d6) δ
9.79 (s, 1H), 8.79 (m, 2H), 8.60 (s, 1H), 8.20 (d, J = 9.9 Hz, 1H),
7.48−7.27 (m, 9H), 7.15 (d, J = 8.2 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H),
6.58 (d, J = 10.0 Hz, 1H), 6.16 (s, 1H), 5.35 (d, J = 9.3 Hz, 1H), 4.72−
4.52 (m, 1H), 3.39 (m, 3H), 3.14−2.76 (m, 9H), 1.95 (m, 2H), 1.72
(m, 6H), 1.28 (m, 10H).
(R)-1-(3-((5-((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-
yl)ethyl)amino)pentyl)amino)-3-oxopropyl)piperidin-4-yl [1,1′-Biphenyl]-
2-ylcarbamate (2) (Reference 32). HRMS found: 655.337; C37H45N5O6
requires 656.344. 1H NMR (400 MHz, CD3OD) δ 8.35 (d, J = 9.9 Hz,
1H), 7.49 (d, J = 7.1 Hz, 1H), 7.42−7.18 (m, 9H), 7.00 (d, J = 8.2 Hz,
1H), 6.62 (d, J = 9.8 Hz, 1H), 5.41 (dd, J = 8.3, 5.0 Hz, 1H), 4.75 (br
s, 1H), 3.28−2.99 (m, 12H), 2.64 (t, J = 7.0 Hz, 2H), 2.08−1.91 (m,
2H), 1.90−1.65 (m, 4H), 1.53 (p, J = 7.1 Hz, 2H), 1.40 (q, J = 8.1 Hz,
2H).
1-(3-(((1R,4r)-4-((((R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-
quinolin-5-yl)ethyl)amino)methyl)cyclohexyl)amino)-3-oxopropyl)-
piperidin-4-yl [1,1′-Biphenyl]-2-ylcarbamate (3) (Reference 32). LCMS
found: 683 C39H47N5O6 requires 681.82. 1H NMR (400 MHz,
CD3OD) δ 8.39 (d, J = 9.9 Hz, 1H), 7.55 (s, 1H), 7.50−7.25 (m, 9H),
7.04 (d, J = 8.2 Hz, 1H), 6.69 (d, J = 9.9 Hz, 1H), 5.44 (t, J = 6.9 Hz,
1H), 4.91 (m, 1H), 3.74−3.54 (m, 2H), 3.52−3.35 (m, 3H), 3.25 (d, J
= 6.9 Hz, 2H), 3.19−3.03 (m, 2H), 3.00 (dt, J = 7.9, 3.6 Hz, 2H), 2.71
(s, 2H), 2.17 (s, 1H), 2.09−1.86 (m, 6H), 1.79 (dt, J = 7.3, 3.9 Hz,
2H), 1.26 (dq, J = 51.2, 12.5 Hz, 4H).
7.18 (m, 9H), 7.03 (d, J = 8.2 Hz, 1H), 6.61 (d, J = 9.8 Hz, 1H), 5.45
(dd, J = 9.3, 3.3 Hz, 1H), 4.78 (m, 1H), 4.50−4.35 (m, 2H), 3.74−
3.37 (m, 4H), 3.35−3.21 (m, 2H), 3.20−3.05 (m, 2H), 2.96 (s, 2H),
2.27−1.62 (m, 4H).
(R)-1-(3-((4-(((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-
yl)ethyl)amino)methyl)-3-methoxyphenyl)amino)-3-oxopropyl)piperidin-
4-yl [1,1′-Biphenyl]-2-ylcarbamate (12d) (Scheme 1). LCMS found:
1
706; C40H43N5O7 requires 705.8. H NMR (300 MHz, CD3OD) δ
8.03 (d, J = 9.9 Hz, 1H), 7.53 (s, 1H), 7.51−7.22 (m, 11H), 7.07 (dd, J
= 19.9, 8.1 Hz, 2H), 6.55 (d, J = 9.8 Hz, 1H), 5.38 (dd, J = 8.3, 3.3 Hz,
1H), 4.85−4.67 (m, 1H), 4.34−4.13 (m, 2H), 3.81 (s, 3H), 3.52 (s,
2H), 3.35−3.06 (m, 6H), 2.97 (s, 2H), 2.36−1.69 (m, 4H).
(R)-1-(3-((2-Chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-
quinolin-5-yl)ethyl)amino)methyl)-3-methoxyphenyl)amino)-3-
oxopropyl)piperidin-4-yl [1,1′-Biphenyl]-2-ylcarbamate (12e) (Scheme
1
1). LCMS found: 740; C40H42ClN5O7 requires 740.2. H NMR (300
MHz, CD3OD) δ 8.16 (d, J = 9.9 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H),
7.53 (s, 1H), 7.48−7.17 (m, 10H), 7.03 (d, J = 8.2 Hz, 1H), 6.61 (d, J
= 9.8 Hz, 1H), 5.42 (dd, J = 8.8, 3.8 Hz, 1H), 4.86−4.73 (m, 1H), 4.36
(s, 2H), 3.94 (s, 3H), 3.52 (t, J = 6.3 Hz, 4H), 3.29−2.93 (m, 6H),
2.31−1.61 (m, 4H).
(R)-1-(3-((4-(((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-
yl)ethyl)amino)methyl)-2-methylphenyl)amino)-3-oxopropyl)piperidin-4-
yl [1,1′-Biphenyl]-2-ylcarbamate (12g) (Scheme 1). LCMS found: 690;
1
C40H43N5O6 requires 689.8. H NMR (400 MHz, CD3OD) δ 8.26−
8.03 (m, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.47−7.22 (m, 12H), 7.04 (d, J
= 8.2 Hz, 1H), 6.63 (d, J = 9.9 Hz, 1H), 5.48−5.30 (m, 1H), 4.85−
4.74 (m, 1H), 4.33−4.14 (m, 2H), 3.67 (m, 2H), 3.52 (d, J = 6.7 Hz,
2H), 3.29−2.93 (m, 6H), 2.31 (s, 3H), 2.04 (m, 4H).
(R)-1-(3-((4-(((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-
yl)ethyl)amino)methyl)-3-methylphenyl)amino)-3-oxopropyl)piperidin-4-
yl [1,1′-Biphenyl]-2-ylcarbamate (12h) (Scheme 1). LCMS found: 690;
1
C40H43N5O6 requires 689.80. H NMR (400 MHz, CD3OD) δ 8.21
(R)-1-(3-((4-(((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-
yl)ethyl)amino)methyl)phenyl)amino)-3-oxopropyl)piperidin-4-yl [1,1′-
Biphenyl]-2-ylcarbamate (4) (Scheme 1). HRMS found: 676.306;
C39H41N5O6 requires 676.313. 1H NMR (400 MHz, CD3OD) δ
8.12 (d, J = 9.9 Hz, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.54 (s, 1H), 7.48−
7.22 (m, 11H), 7.02 (d, J = 8.2 Hz, 1H), 6.59 (d, J = 9.8 Hz, 1H), 5.38
(dd, J = 9.1, 3.6 Hz, 1H), 4.96−4.88 (m, 1H), 4.88−4.83 (m, 11H),
4.31−4.17 (m, 2H), 3.64 (s, 1H), 3.59−3.43 (m, 3H), 3.27−3.01 (m,
4H), 2.95 (s, 2H), 2.28−1.72 (m, 5H).
(d, J = 9.9 Hz, 1H), 7.54 (d, J = 6.9 Hz, 2H), 7.49−7.22 (m, 11H),
7.05 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 9.8 Hz, 1H), 5.44 (dd, J = 9.3, 3.5
Hz, 1H), 4.97−4.86 (m, 1H), 4.39−4.27 (m, 2H), 3.71 (m, 1H),
3.60−3.43 (m, 5H), 3.31−3.03 (m, 2H), 2.98 (br s, 2H), 2.42 (s, 3H),
2.30−1.69 (m, 4H).
(R)-1-(3-((4-(((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-
yl)ethyl)amino)methyl)-3-methoxy-2-methylphenyl)amino)-3-oxopropyl)-
piperidin-4-yl [1,1′-Biphenyl]-2-ylcarbamate (12i) (Scheme 1). LCMS
found: 720; C41H45N5O7 requires 719.8. 1H NMR (400 MHz,
CD3OD) δ 8.09 (d, J = 9.8 Hz, 1H), 7.55 (m, 1H), 7.48−7.19 (m,
11H), 7.03 (d, J = 8.2 Hz, 1H), 6.59 (d, J = 9.8 Hz, 1H), 5.40 (dd, J =
8.9, 3.5 Hz, 1H), 4.85 (m, 1H), 4.45−4.20 (m, 2H), 3.80 (s, 3H), 3.66
(m, 2H), 3.58−3.41 (m, 2H), 3.28−3.07 (m, 4H), 3.02 (d, J = 6.1 Hz,
2H), 2.21 (m, 7H).
(R)-1-(3-((4-(((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-
yl)ethyl)amino)methyl)-5-methoxy-2-methylphenyl)amino)-3-oxopropyl)-
piperidin-4-yl [1,1′-Biphenyl]-2-ylcarbamate (12j) (Scheme 1). LCMS
found: 720; C41H45N5O7 requires 719.8. 1H NMR (400 MHz,
CD3OD) δ 8.04 (d, J = 9.8 Hz, 1H), 7.55 (s, 1H), 7.49−7.23 (m,
10H), 7.18 (s, 1H), 7.12−6.97 (m, 1H), 6.58 (d, J = 9.8 Hz, 1H), 5.38
(dd, J = 8.7, 3.4 Hz, 1H), 4.92 (m, 1H), 4.33−4.14 (m, 2H), 3.81 (s,
3H), 3.65 (m, 2H), 3.61−3.44 (m, 2H), 3.28−3.08 (m, 4H), 3.03 (m,
2H), 2.22 (s, 3H), 2.17−1.66 (m, 4H).
(R)-5-(2-Amino-1-((tert-butyldimethylsilyl)oxy)ethyl)-8-hydroxyquino-
lin-2(1H)-one·acetate (11) (Reference 13). LCMS found: 335;
1
C17H26N2O3Si requires 334.5. H NMR (400 MHz, CD3OD) δ 8.39
(d, J = 9.9 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H),
6.69 (d, J = 9.9 Hz, 1H), 5.35 (dd, J = 7.7, 4.4 Hz, 1H), 3.26−3.09 (m,
2H), 1.94 (s, 3H), 0.90 (s, 9H), 0.12 (s, 3H), −0.14 (s, 3H).
(R)-1-(3-((2-Chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-
quinolin-5-yl)ethyl)amino)methyl)phenyl)amino)-3-oxopropyl)piperidin-
4-yl [1,1′-Biphenyl]-2-ylcarbamate (12a) (Scheme 1). LCMS found: 710
C39H40ClN5O6 requires 710.22. 1H NMR (400 MHz, CD3OD) δ 8.17
(d, J = 9.9 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 2.0 Hz, 1H),
7.55 (d, J = 5.6 Hz, 1H), 7.48−7.21 (m, 10H), 7.03 (d, J = 8.2 Hz,
1H), 6.62 (d, J = 9.8 Hz, 1H), 5.39 (dd, J = 9.0, 3.8 Hz, 1H), 4.96−
4.86 (m, 1H), 4.34−4.20 (m, 2H), 3.65 (s, 1H), 3.58−3.43 (m, 4H),
3.31−3.10 (m, 3H), 3.06 (d, J = 5.1 Hz, 2H), 2.03 (s, 4H).
(R)-1-(3-((4-(((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-
yl)ethyl)amino)methyl)-2-methoxyphenyl)amino)-3-oxopropyl)piperidin-
4-yl [1,1′-Biphenyl]-2-ylcarbamate (12b) (Scheme 1). LCMS found:
ASSOCIATED CONTENT
■
1
S
* Supporting Information
706; C40H43N5O7 requires 705.80. H NMR (400 MHz, CD3OD) δ
8.11 (dd, J = 17.3, 9.3 Hz, 1H), 7.84 (s, 1H), 7.55 (s, 1H), 7.50−7.19
(m, 8H), 7.18−6.82 (m, 4H), 6.57 (d, J = 9.8 Hz, 1H), 5.39 (dd, J =
8.8, 3.3 Hz, 1H), 4.90 (s, 1H), 4.23 (m, 2H), 4.01−3.77 (m, 4H), 3.68
(s, 1H), 3.50 (m, 3H), 3.44−3.33 (m, 1H), 3.29−2.88 (m, 4H), 2.28−
1.66 (m, 4H).
(R)-1-(3-((3-Chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-
quinolin-5-yl)ethyl)amino)methyl)phenyl)amino)-3-oxopropyl)piperidin-
4-yl [1,1′-Biphenyl]-2-ylcarbamate (12c) (Scheme 1). LCMS found:
710; C39H40ClN5O6 requires 710.22. 1H NMR (400 MHz, CD3OD) δ
8.23 (d, J = 9.9 Hz, 1H), 7.96 (s, 1H), 7.52 (d, J = 10.9 Hz, 3H), 7.47−
Biology experimental details for cell culture and membrane
preparation, in vitro potency and selectivity at recombinant
human β1- and β2-adrenoceptors expressed in HEK and CHO
cells, in vitro potency at recombinant human M3 receptors
expressed in CHO cells, human M3 cold kinetics and ligand
dissociation, in vivo bronchoprotection studies, antisialagogue
activity, in vitro permeability in CACO-2 cells, metabolic
stability in human liver microsomes, pharmacokinetics and
bioavailability of 12f in rats after intravenous or oral
L
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Drug Annotation
(7) Cooper, A. E.; Ferguson, D.; Grime, K. Optimisation of DMPK
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crystalline compounds, additional chemistry experimental
details and analytical data for the MABA compounds described
and key common intermediates. This material is available free
of charge via the Internet at http://pubs.acs.org.
(8) 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. Synthesis and structure−activity relationships of
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 650-808-3737. E-mail: ahughes@theravance.com.
Author Contributions
The manuscript was written by A.D.H. All authors have given
approval to the final version of the manuscript.
(9) Morphy, R.; Rankovic, Z. Designed multiple ligands. An
emerging drug discovery paradigm. J. Med. Chem. 2005, 48 (21),
6523−6543.
(10) Hughes, A. D.; Chin, K. H.; Dunham, S. L.; Jasper, J. R.; King,
K. E.; Lee, T. W.; Mammen, M.; Martin, J.; Steinfeld, T. Discovery of
muscarinic acetylcholine receptor antagonist and beta 2 adrenoceptor
agonist (MABA) dual pharmacology molecules. Bioorg. Med. Chem.
Lett. 2011, 21, 1354−1358.
(11) Hughes, A. D. Discovery of muscarinic acetylcholine receptor
antagonist and beta-2 adrenoceptor agonist (MABA) dual pharmacol-
ogy molecules. In RDD Europe 2013; Dalby, R. N., Byron, P. R., Peart,
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Commonwealth University: Richmond, VA, 2013; Vol, 1, pp 47−58.
(12) Chao, R.; Rapta, M.; Colson, P.-J.; Lee, J. Crystalline form of a
biphenyl compound. 20050182092, February 11, 2005; Theravance,
Inc., USA.
(13) Chao, R.; Rapta, M.; Colson, P.-J. Crystalline form of a biphenyl
compound. U.S. Patent 7,521,558, 2009.
(14) Ritchie, T. J.; Luscombe, C. N.; Macdonald, S. J. F. Analysis of
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Erik Fenster, Jeff Haganah, Ryan Hudson,
John Jacobsen, Uwe Klein, Dan Marquess, and Donna A. A.
Wilton for their reviews of this manuscript. Erik Fenster is
thanked for his syntheses of compounds for characterization.
Kevin DeMent, Kanaka Hettiarachchi, Uwe Klein, Alex
McNamara, Venkat Thalladi, and Qifeng Xue are thanked for
their assistance with the Supporting Information. Erin Bradley
is thanked for her computational chemistry modeling work and
1
Jayaprakasam Bolleddula for gathering 600 MHz H NMR
spectra. The authors thank all the talented scientists that
synthesized and evaluated biological properties of molecules
decsribed herein. GSK is thanked for its review of this
manuscript.
(15) Mundy, C.; Kirkpatrick, P. Tiotropium bromide. Nat. Rev. Drug
Discovery 2004, 3, 643−644.
ABBREVIATIONS USED
■
(16) Tautermann, C. S.; Kiechle, T.; Seeliger, D.; Diehl, S.; Wex, E.;
Banholzer, R.; Gantner, F.; Pieper, M. P.; Casarosa, P. Molecular basis
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the muscarinic M3 receptor. J. Med. Chem. 2013, 56, 8746−8756.
(17) Sykes, D. A.; Dowling, M. R.; Leighton-Davies, J.; Kent, T. C.;
Fawcett, L.; Renard, E.; Trifilieff, A.; Charlton, S. J. The influence of
receptor kinetics on the onset and duration of action and the
therapeutic index of NVA237 and tiotropium. J. Pharmacol. Exp. Ther.
2012, 343 (2), 520−528.
COPD, chronic obstructive pulmonary disease; MA, muscarinic
antagonist; BA, β2 agonist; MABA, muscarinic antagonist β2
agonist; CHO, Chinese hamster ovary; ACh, acetylcholine;
DPI, dry powder inhaler; LABA, long-acting β2 agonist; LAMA,
long-acting muscarinic antagonist; HEK, human embryonic
kidney; GPCR, G-protein-coupled receptor
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