Efficient conversion of a nonselective norepinephrin reuptake inhibitor into a dual muscarinic antagonist-β2-agonist for the treatment of chronic obstructive pulmonary disease

Article abstract of DOI:10.1021/jm2007535

Following interrogation of a wide-ligand profile database, a nonselective norepinephrin reuptake inhibitor was converted into a novel muscarinic antagonist using two medicinal chemistry transformations (M3/NRI selectivity of >1000). Conjugation to a β₂ agonist motif furnished a molecule with balanced dual pharmacology, as demonstrated in a guinea pig trachea tissue model of bronchoconstriction. This approach provides new starting points for the treatment of chronic obstructive pulmonary disease and illustrates the potential for building selectivity into GPCR modulators that possess intrinsic promiscuity or reverse selectivity.

Full text of DOI:10.1021/jm2007535

BRIEF ARTICLE
pubs.acs.org/jmc
Efficient Conversion of a Nonselective Norepinephrin Reuptake
Inhibitor into a Dual Muscarinic Antagonistꢀβ2-Agonist for the
Treatment of Chronic Obstructive Pulmonary Disease
Rachel Osborne,† Nick Clarke,# Paul Glossop,† Amy Kenyon,† Hao Liu,† Sheena Patel,# Susan Summerhill,#
and Lyn H. Jones*,†
†WorldWide Medicinal Chemistry and #Allergy and Respiratory Biology, Pfizer R&D, Ramsgate Road, Sandwich, Kent, CT13 9NJ, U.K.
S Supporting Information
b
ABSTRACT: Following interrogation of a wide-ligand profile database, a nonselective norepinephrin reuptake inhibitor was
converted into a novel muscarinic antagonist using two medicinal chemistry transformations (M3/NRI selectivity of >1000).
Conjugation to a β2 agonist motif furnished a molecule with balanced dual pharmacology, as demonstrated in a guinea pig trachea
tissue model of bronchoconstriction. This approach provides new starting points for the treatment of chronic obstructive pulmonary
disease and illustrates the potential for building selectivity into GPCR modulators that possess intrinsic promiscuity or reverse
selectivity.
’ INTRODUCTION
trivial), and our efforts were therefore focused on the muscarinic
antagonist motif. The caveat to this approach is that it is parti-
cularly difficult to design selective G-protein-coupled receptor
(GPCR) modulators, and the antimuscarinic field is extremely
well studied, thus making the creation of completely novel and
selective scaffolds challenging.
Rather than modification of existing selective antimuscarinic
templates, our strategy involved searching the Pfizer Bioprint
(Cerep SA)5 wide-ligand profile (WLP) database for molecules
that possessed muscarinic antagonism but in a template not
necessarily classed or claimed in the literature as specifically
“antimuscarinic”. Any “hits’” would likely have been designed for
a different purpose, i.e., where the primary and desired pharma-
cology results from targeting an alternative protein. M3 SARs
would then be harnessed to efficiently enhance antimuscarinic
activity while switching selectivity, ultimately yielding novel
muscarinic antagonists and dual pharmacology MABAs.
Chronic obstructive pulmonary disease (COPD) will become
the third largest cause of death by 2030 and is characterized by
chronic bronchitis and emphysema, mainly caused by cigarette
smoking.1 Inhaled adrenergic (β2) agonists and muscarinic (M3)
antagonists are used as bronchodilators to treat the symptoms of
the disease. More recently, long acting muscarinic antagonists
(LAMAs) and long acting β2 agonists (LABAs) have been
developed to enable once-daily dosing regimens.2
Triple therapy for COPD, via the synergistic combination of a
LABA, LAMA, and an inhaled corticosteroid (ICS), would
significantly enhance the treatment of the disease,3 but the com-
bination of three drugs into a dry powder inhalation device is
extremely challenging. As a result, we and others have recently
reported the concept of dual pharmacology muscarinic antag-
onistsꢀβ2-agonists (MABAs) that should facilitate the combina-
tion with an ICS in a single device to enable the triple therapy
paradigm.4
We have reported conjugation of a known muscarinic antago-
nist tolterodine 1 with the known quinolinone β2 agonist trigger
to create MABAs such as 2 that demonstrate balanced potency
and pharmacological duration, incorporating the principles of
“inhalation by design”.4c Theravance published a similar strategy
using the known biarylcarbamoylpiperidine antimuscarinic motif
to furnish dual pharmacology molecules such as 3.4d Although a
mix-and-match approach to novel MABA creation is possible
using known β2 and M3 fragments, the available intellectual pro-
perty space, and the opportunity to construct something truly
innovative, soon becomes limited. Therefore, a need exists to
create novel single pharmacology agents to broaden the palette
that can enable dual pharmacology MABAs and subsequently the
triple therapeutic approach.
’ RESULTS
Following a search of our wide-ligand profile database for
muscarinic antagonists, we discovered indazole 4 (Scheme 1), a
known norepinephrin reuptake inhibitor (NRI, assessed through
the inhibition of the norepinephrin transporter, NET)6 that pos-
sessed moderate M3 activity (Table 1). We hoped that even-
tually, the presence of the piperidine moiety would provide an
opportunity for conjugation to a β2 pharmacophore through the
nitrogen atom in a manner similar to that described previously by
us and Theravance (e.g., MABA 3). Not surprisingly, this rather
lipophilic, basic compound is a promiscuous binder of many
proteins7 (see WLP in the Supporting Information).
It is noticeable that 4 lacks a hydrogen bonding group which
is present in other selective antimuscarinics, such as the phenol
We felt that the subtle structureꢀactivity relationships (SARs)
that trigger β2 agonism may significantly hinder the creation of
novel headgroup templates (avoiding β1 activity is also far from
Received: June 11, 2011
Published: August 25, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/jm2007535 J. Med. Chem. 2011, 54, 6998–7002
|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 1a
Figure 2. Molecular overlay of tolterodine 1 (blue, from small molecule
crystal structure)9 and energy minimized indazole 4 (pink). Arrows
indicate design focus.
a Reagents and conditions: (a) tBuOK, THF, reflux, then HCl in
dioxane.
Scheme 2a
Table 1. Antimuscarinic M3 Potency, Human Liver Micro-
some (HLM) Metabolic Stability and Lipophilicity (log D) for
Compounds 1, 4, 5, 11a
1
4
5
11
M3 Ki (nM)c
NET Ki (nM)d
log D
3.6
49
10
2.5
15% at 10 μM
6.8
1.4
<8
ndb
0.6
<8
2900
0.3
1.8
<8
HLM
<8
((μL/min)/mg)
a All data are geometric mean values with 95% CI, n = 4. b nd = not
determined. c Binding assay, human cloned M3 receptor using [3H]N-
methylscopolamine. d Binding assay, human cloned NET receptor
using [3H]nisoxetine.
a Reagents and conditions: (a) Ac2O, toluene; (b) 2-oxocyclohexane-
carboxylic acid methyl ester, PCl3, MeCN; (c) PPh3, DIAD, THF;
(d) HCl, dioxane; (e) tBuOK, dioxane, reflux, then HCl in dioxane;
(f) 9-bromononanol, NaHCO3, MeCN; (g) (i) SO3 pyridine, DMSO,
3
NEt3; (ii) 14, EtOH, NaB(OAc)3H, CH2Cl2; (iii) NEt3 3HF, THF.
3
5-fold. Interestingly, this is the first report of a molecule contain-
ing the N-(o-hydroxyphenyl)pyrazole substructure.
Additionally, M3 SAR suggests that one of the benzene rings
usually present in aminergic GPCR inhibitors can be saturated to
a cyclic aliphatic system, resulting in an improvement in affinity.11
There is limited precedence that ring B in tolterodine can be
saturated in this manner,12 and therefore, guided by the overlay
in Figure 2, the benzene ring of the indazole was “reduced”.
Acetylation of commercially available hydrazine 6 provided 7
that was cyclized with the requisite keto ester yielding hydroxy-
pyrazole 8 (Scheme 2). Mitsunobu alkylation to 9 and subse-
quent deprotection provided 10 that was converted to the phenol
11 using the conditions described above.
Figure 1. Structures of tolterodine 1 and MABAs 2 and 3.
ꢀOH present in tolterodine (Figure 1; see also the M3 dock
with azoniabicyclo[2.2.2]octane muscarinic antagonists by Lainꢀe
et al. suggesting a key hydrogen bond between the ligand-OH and
Ser7:46).8 A molecular overlay of the small molecule crystal struc-
ture of tolterodine9 with an energy minimized structure of 410
suggested that replacing one of the fluorine atoms with a
hydroxyl group may improve affinity (Figure 2). In fact, treat-
ment of 4 with potassium tert-butoxide and acidic workup
provided the more potent analogue 5 (Table 1). In a single syn-
thetic transformation, the M3 antimuscarinic potency was improved
This transformation resulted in a further improvement in
potency such that the two-step medicinal chemistry conversion
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Table 2. Pharmacology, Metabolism, and Membrane Flux of
MABA 13a
compound to recover by 50% of the inhibition induced. As
expected, 13 is a potent bronchodilator in this model with
impressive duration of action (Table 2). To delineate the M3
drive, the β2 antagonist propranolol was added to the EFS model
to block the β2 effect. MABA 13 retained M3 potency as pre-
dicted from the in vitro experiment, but surprisingly, the DoA
was significantly longer than that predicted by the offset assay.
These results are significant, since generally there are good
correlations between M3 offset and pharmacological duration
in the GPT assay (suggesting that an alternative mechanism is at
play and further work is warranted to explain this observation).
The relatively high clearance in human liver microsomes com-
bined with poor membrane permeability (assessed as flux across
the human colon carcinoma CACO-2 monolayer, Table 2) pre-
dicts that MABA 13 would have poor oral bioavailability, a
necessary attribute for inhaled molecules where the risks of sys-
temically driven side effects from the swallowed component can
be ameliorated in this way.4
M3 Kib
β2 EC50
β1 EC50
M3 offsetd
β2 wash-offe
2.2 nM
c
14 nM
>10 μM
<16 min
1.5-fold
28 nM
f
GPT EFS EC50
GPT EFS DoAf
>16 h
g
GPT (+propranolol) EC50
27 nM
GPT (+propranolol) DoAg
>16 h
log D
2.4
cLogP
5.4
HLM
25 (μL/min)/mg
<1 ꢁ 10ꢀ6 cm/s
CACO-2 flux
a All data are geometric mean values with 95% CI, n = 4. b Potency of
compounds at the human cloned M3 receptor using [3H]NMS. c Ability
to stimulate intracellular cAMP production in CHO-hβ2 cells. d Offset
kinetics from human cloned M3 receptor using [3H]NMS (dilution
method). e Fold-difference in β2 potency following receptor wash.
f GPT = guinea pig trachea. EFS = electric field stimulation. Potency
and duration for β2 and M3 components of bronchodilation. Submax-
imal concentration used for the duration is that which gives 70%
inhibition of the EFS response. g Potency and duration for M3 compo-
nent of bronchodilation.
’ CONCLUSIONS
Introduction of hydrogen bonding potential and benzene ring
saturation of a Bioprint hit resulted in the successful conversion
of a nonselective NRI into a selective muscarinic antagonist.
Subsequent conjugation to a quinolinone β2 agonist motif effi-
ciently furnished a MABA with balanced potency and pharma-
cological duration. Further exploration of this series includes a
detailed in vivo pharmacological analysis and optimization of the
synthetic chemistry to furnish additional molecules from this
class. This research expands the opportunities for triple ther-
apeutic approaches for the treatment of COPD. Additionally, this
article provides a proof of concept that hits from a wide-ligand
profile database can be converted into novel and selective GPCR
modulators.
of Bioprint hit 4 to pyrazole 11 improved M3 potency 20-fold,
even though the molecule was 10-fold more polar (Table 1). As
we predicted, the selectivity of 11 was significantly improved over
starting point 4 and possessed considerably less activity as an
NRI (>1000-fold selective over NET; see Table 1). Compound
11 also possessed excellent metabolic stability as assessed in human
liver microsomes (HLM), suggesting that molecules from this
class would be interesting to pursue as oral anticholinergic leads.
Conjugation of the selective antimuscarinic 11 to the quino-
linone β2 agonist headgroup is continued in Scheme 2. Installa-
tion of the C9-linker (previously proven optimal because of
possible multivalent effects in the resultant MABA)13 yielded 12,
and oxidation of the alcohol followed by reductive amination
with the known amine 144e and desilylation furnished MABA 13.
The pharmacological profile of 13 is shown in Table 2. MABA
13 possessed impressive primary biochemical pharmacology
against M3 and β2. The kinetics of pharmacological duration
were assessed for β2 agonism through a simple receptor wash-off
assay as previously described,14 and the small shift in potency
following wash is indicative of long duration. Kinetics for the M3
component were assessed using a dilution-offset methodology
whereby the offset was inferred from the on rate of coadminis-
tered 3H-NMS (N-methylscopolamine) and is expressed as the
’ EXPERIMENTAL SECTION
General Methods. Unless otherwise stated, all reactions were
carried out under a nitrogen atmosphere, using commercially available
anhydrous solvents. Thin-layer chromatography was performed on
glass-backed precoated Merck silica gel (60 F254) plates, and FCC
(flash column chromatography) was carried out using 40ꢀ63 μm silica
gel. NMR spectra were carried out on a Varian Mercury 400 spectro-
meter in the solvents specified. Mass and LCMS spectra were recorded
on a Waters Micromass ZQ using electrospray chemical ionization
(ESCI). Purities for all compounds were determined to be g95% by
LCMS. Other abbreviations are used in conjunction with standard
chemical practice.
3-Fluoro-2-[3-(piperidin-4-yloxy)indazol-1-yl]phenol (5).
4 (615 mg, 1.68 mmol) was dissolved in THF (30 mL). Potassium
tert-butoxide (754 mg, 6.72 mmol) was added and the mixture heated to
reflux for 16 h. HCl (aq, 4 M in dioxane, 20 mL) was added carefully and
the solution refluxed for 2 h. The solvents were removed under reduced
pressure and the mixture partitioned between EtOAc and saturated
NaHCO3 (aq). The organic layer was washed with water, then brine,
separated, dried (MgSO4), filtered, and concentrated. The mixture
was triturated with diethyl ether, washed with cold methanol to yield
5: 550 mg (75%).
3
time taken to reach 50% of total H-NMS for solvent treated
membranes.15 The fast offset from the M3 receptor may be
indicative of short pharmacological duration. We have previously
suggested that cLogP > 5.5 for MABAs such as 2 is required for
long offset kinetics.4c
We then assessed the potency of 13 in the guinea pig trachea
(GPT) model using electric field stimulation (EFS) to release
endogenous acetylcholine.14 The drive for bronchoconstriction
in this model operates through both β2 and M3 mechanisms, and
therefore, values in this assay reflect combined bronchodilatory
effects. A “duration of action” (DoA) was defined as the time
taken for the muscle tone at a submaximal concentration of the
Acetic Acid N0-(2,6-Difluorophenyl)hydrazide (7). 6 (5.0 g,
23 mmol) was partitioned between saturated NaHCO3 (aq) and EtOAc.
The organics were separated, dried (MgSO4), filtered, and concentrated.
The residue was dissolved in toluene (25 mL). Acetic anhydride
(2.40 mL, 25.3 mmol) was added. After 1 h, the solvent was removed
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dx.doi.org/10.1021/jm2007535 |J. Med. Chem. 2011, 54, 6998–7002

Journal of Medicinal Chemistry
BRIEF ARTICLE
under reduced pressure and the residue triturated with diethyl ether to
yield 7: 2.90 g (68%).
’ ASSOCIATED CONTENT
S
Supporting Information. Spectroscopic details for 5,
b
1-(2,6-Difluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-3-ol (8).
7 (2.90 g, 15.6 mmol) and 2-oxocyclohexanecarboxylic acid methyl
ester (2.43 g, 15.6 mmol) were dissolved in acetonitrile (10 mL). PCl3
(1.63 mL, 18.7 mmol) was added carefully. The mixture was heated to
50 °C for 40 h, then cooled to rt, poured on ice, extracted with EtOAc,
washed with water, then brine, separated, dried (MgSO4), filtered,
and concentrated. The residue was triturated with MeOH to provide
8: 2.0 g (51%).
7ꢀ12; selectivity data for 1, 4, and 11. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +44 1304 644256. E-mail: lyn.jones@pfizer.com.
4-[1-(2,6-Difluorophenyl)-4,5,6,7-tetrahydro-1H-indazol-
3-yloxy]piperidine-1-carboxylic Acid tert-Butyl Ester (9). 8
(5.0 g, 20 mmol) and 4-hydroxypiperidine-1-carboxylic acid tert-butyl
ester (4.0 g, 20 mmol) were dissolved in THF (100 mL). PPh3 (5.8 g,
22 mmol) and then DIAD (4.4 g, 22 mmol) were added. After 16 h, the
mixture was diluted with EtOAc, washed with water, then brine, sep-
arated, dried (MgSO4), filtered, and concentrated. The residue was puri-
fied by FCC (heptanes/EtOAc, 70/30) to provide 9: 8.7 g (76%).
1-(2,6-Difluorophenyl)-3-(piperidin-4-yloxy)-4,5,6,7-tetra-
hydro-1H-indazole Hydrochloride (10). 9 (6.6 g, 15.2 mmol) was
treated with HCl (4 M in dioxane, 20 mL, 80 mmol). After 1 h the sol-
vent was removed under reduced pressure and the residue triturated
with CH2Cl2 to provide 10: 5.0 g (99%).
3-Fluoro-2-[3-(piperidin-4-yloxy)-4,5,6,7-tetrahydroinda-
zol-1-yl]phenol (11). 10 (5.0 g, 15 mmol) was dissolved in dioxane
(80 mL). Potassium tert-butoxide (6.8 g, 60 mmol) was added and the
mixture heated to reflux for 16 h. HCl (aq, 4 M in dioxane, 100 mL) was
added carefully and the solution refluxed for 2 h. The solvents were re-
moved under reduced pressure and the mixture partitioned between
EtOAc and saturated NaHCO3 (aq). The organic layer was washed with
water, then brine, separated, dried (MgSO4), filtered, and concentrated.
The mixture was triturated with diethyl ether, washed with cold methanol to
yield 11: 4.5 g (90%).
’ ACKNOWLEDGMENT
We thank Maria Stanley, Michele Coghlan, Matthew Strawbridge,
Malyn Asuncion, Mike Trevethick, Jessica Watson, Emilio Stewart,
and Richard Mold for pharmacological screening and David Fairman
and Rhys Jones for metabolic screening.
’ ABBREVIATIONS USED
CHO, Chinese hamster ovary; COPD, chronic obstructive pul-
monary disease; DIAD, diisopropyl azodicarboxylate; DMSO,
dimethylsulfoxide; DoA, duration of action; EFS, electric field
stimulationn; ESCI, electrospray chemical ionization; FCC, flash
column chromatography; GPCR, G-protein-coupled receptor; GPT,
guinea pig trachea; HLM, human liver microsome; LABA, long
acting β-2 agonist; LAMA, long-acting muscarinic antagonist;
LCMS, liquid chromatographyꢀmass spectrometry; MABA,
muscarinic antagonistꢀβ2-agonist; nd, not determined; NET,
norepinephrin transporter; NMR, nuclear magnetic resonance;
NMS, N-methylscopolamine; NRI, norepinephrin reuptake in-
hibitor; rt, room temperature; SAR, structureꢀactivity relation-
ship; THF, tetrahydrofuran; WLP, wide-ligand profile
3-Fluoro-2-{3-[1-(9-hydroxynonyl)piperidin-4-yloxy]-4,5,
6,7-tetrahydroindazol-1-yl}phenol (12). 11 (250 mg, 0.75 mmol),
9-bromononanol (168 mg, 0.75 mmol), and NaHCO3 (190 mg, 2.26 mmol)
were added to acetonitrile (20 mL) and heated at relux for 16 h. The sol-
vents were removed under reduced pressure, and the mixture was parti-
tioned between EtOAc and water. The organic layer was separated, dried
(MgSO4), filtered, and concentrated to provide 12: 320 mg (90%).
5-[2-(9-{4-[1-(2-Fluoro-6-hydroxyphenyl)-4,5,6,7-tetrahydro-
1H-indazol-3-yloxy]piperidin-1-yl}nonylamino)-1-hydroxy-
ethyl]-8-hydroxy-1H-quinolin-2-one (13). 12 (160 mg, 0.34 mmol)
was dissolved in DMSO (5 mL). Then NEt3 (470 μL, 3.38 mmol) and
sulfur trioxideꢀpyridine complex (215 mg, 1.35 mmol) were added. The
mixture was stirred for 16 h. The mixture was partitioned between EtOAc
and water. The organic layer was washed with brine, separated, dried
(MgSO4), filtered, and concentrated. The crude aldehyde was dissolved in
CH2Cl2 (9mL) andEtOH(1mL), andtheamine144e wasadded(106mg,
0.32 mmol). NaB(OAc)3H (201 mg, 0.95 mmol) was added and the mix-
ture stirred for 16 h. The solvents were removed under reduced pressure,
and the residue was partitioned between EtOAc and water. The organic
layer was separated, dried (MgSO4), filtered, and concentrated. The crude
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