Inhalation by design: Novel ultra-long-acting β2- adrenoreceptor agonists for inhaled once-daily treatment of asthma and chronic obstructive pulmonary disease that utilize a sulfonamide agonist headgroup

Article abstract of DOI:10.1021/jm1005989

A novel series of potent and selective sulfonamide derived β₂-adrenoreceptor agonists are described that exhibit potential as inhaled ultra-long-acting bronchodilators for the treatment of asthma and chronic obstructive pulmonary disease. Analogues from this series mediate very long-lasting smooth muscle relaxation in guinea pig tracheal strips. The sulfonamide agonist headgroup confers high levels of intrinsic crystallinity that could relate to the acidic sulfonamide motif supporting a zwitterionic form in the solid state. Optimization of pharmacokinetic properties was achieved through targeted introduction of a phenolic moiety to support rapid phase II clearance, thereby minimizing systemic exposure following inhalation and reducing systemically mediated adverse events. Compound 38 (PF-610355) is identified as a clinical candidate from this series, with in vivo duration of action studies confirming its potential for once-daily use in humans. Compound 38 is currently in advanced phase II clinical studies.

Full text of DOI:10.1021/jm1005989

6640 J. Med. Chem. 2010, 53, 6640–6652
DOI: 10.1021/jm1005989
Inhalation by Design: Novel Ultra-Long-Acting β2-Adrenoreceptor Agonists for Inhaled
Once-Daily Treatment of Asthma and Chronic Obstructive Pulmonary Disease That Utilize
a Sulfonamide Agonist Headgroup
Paul A. Glossop,*,† Charlotte A. L. Lane,*,† David A. Price,†,# Mark E. Bunnage,† Russell A. Lewthwaite,† Kim James,†
Alan D. Brown,† Michael Yeadon,‡ Christelle Perros-Huguet,‡,§ Michael A. Trevethick,‡ Nicholas P. Clarke,‡,
Robert Webster,§ Rhys M. Jones,§ Jane L. Burrows,^ Neil Feeder,^ Stefan C. J. Taylor,^ and Fiona J. Spence
†Department of Worldwide Medicinal Chemistry, ‡Allergy and Respiratory Research Unit, §Department of Pharmacokinetics,
Dynamics, and Metabolism, Department of Drug Safety, and ^Department of Pharmaceutical Sciences, Pfizer Global Research and
Development, Sandwich Laboratories, Ramsgate Road, Kent CT13 9NJ, U.K. # Present address: Pfizer Global Research and Development,
Eastern Point Road, Groton, Connecticut 06340.
Received May 17, 2010
A novel series of potent and selective sulfonamide derived β2-adrenoreceptor agonists are described that
exhibit potential as inhaled ultra-long-acting bronchodilators for the treatment of asthma and chronic
obstructive pulmonary disease. Analogues from this series mediate very long-lasting smooth muscle
relaxation in guinea pig tracheal strips. The sulfonamide agonist headgroup confers high levels of
intrinsic crystallinity that could relate to the acidic sulfonamide motif supporting a zwitterionic form in
the solid state. Optimization of pharmacokinetic properties was achieved through targeted introduction
of a phenolic moiety to support rapid phase II clearance, thereby minimizing systemic exposure
following inhalation and reducing systemically mediated adverse events. Compound 38 (PF-610355) is
identified as a clinical candidate from this series, with in vivo duration of action studies confirming its
potential for once-daily use in humans. Compound 38 is currently in advanced phase II clinical studies.
Chart 1
Introduction
Respiratory diseases such as asthma and chronic obstruc-
tive pulmonary disease (COPDa) are highly prevalent diseases
that affect millions of people worldwide. Long acting
β2-adrenoreceptor agonists are a highly precedented drug
class used for the treatment of asthma and COPD. There
are currently two marketed long-acting β2-adrenoreceptor
agonists (LABAs), salmeterol and formoterol (Chart 1),
which exhibit sufficient duration of action to support twice-
daily dosing regimens following inhaled delivery. However,
neither of these agents are approved for once-daily dosing.
A major area of current focus in the respiratory field is to
identify novel, ultra-long-acting β2-adrenoreceptor agonists
that are suitable for use as once-a-day inhaled agents, either
as stand-alone therapies or for use in combination products
with other bronchodilator and/or anti-inflammatory agents.
The improved patient convenience and compliance associated
with such once-a-day treatments, especially when delivered in
convenient dry powder inhaler devices, is expected to support
much more effective therapy for both asthma and COPD.
A number of reports from our laboratories and others have
described various chemical series that offer promise as inhaled
once-daily β2-adrenoreceptor agonists.1-9 Indacaterol (QAB-
149) is the most advanced agent (Chart 2), having been
recently approved as its maleate salt in the European Union
(2009) and launched (2010) asthe Onbrez Breezhaler for once-
daily treatment of COPD.3 Advanced agents from other
efforts include milveterol (GSK-159797),4 vilanterol (GSK-
642444),5 and olodaterol (BI-1744-CL)6 that have largely
been progressed to phase II trials in asthma and phase III
trials in COPD (Chart 2). We have previously reported the
design and profile of two novel series of β2-adrenoreceptor
agonists exemplified by compounds 1, 2, and 3 (Chart 3).7-9
In addition, 1 and 3 have been disclosed as compounds that
were selected as development candidates.10 Both compounds
*To whom correspondence should be addressed. For P.A.G.: phone,
þ44 1304 649188; fax, þ441304651821; e-mail, paul.glossop@pfizer.
com. For C.A.L.L.: phone, þ441304648231; fax, þ441304651813;
e-mail, charlotte.lane@pfizer.com.
a Abbreviations: ACh, acetylcholine; cAMP, cyclic adenosine mono-
phosphate; CHO, Chinese hamster ovary; COPD, chronic obstructive
pulmonary disease; DDI, drug-drug interaction; DoA, duration of
action; DPI, dry powder inhaler; DSC, differential scanning calori-
metry; DVS, dynamic vapor sorption; EFS, electrical field stimulation;
GPT, guinea pig trachea; LABA, long-acting β2-adrenoreceptor ago-
nist; PXRD, powder X-ray diffraction; sGAW, specific airway conduc-
tance; TGA, thermogravimetric analysis.
r
pubs.acs.org/jmc
Published on Web 08/30/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6641
Chart 2
Chart 3. Structures of Previously Disclosed Pfizer β2-Adrenoreceptor Agonists
Chart 4
The key sulfonamide headgroup 4 was prepared according to
literature methods.11 Selective N-mesylation to give 5 was
followed by O-silylation toafford the protected intermediate 6
(Scheme 2).
The monomethyl substituted linker 7 was prepared from
3-bromophenylacetic acid (Scheme 3). Esterification followed
by palladium-mediated coupling with isoprenyl acetate gave
the ketoesterintermediate8. Reductive aminationwithR-(R)-
methylbenzylamine gave the protected amino ester as a mix-
ture of diastereomers in a 4:1 ratio (R,R-9a/R,S-9b). Selective
crystallization of the desired R,R diastereomer 9a as the
hydrochloride salt was followed by transfer hydrogenation
to give the aminoester 7 in good yield and high enantiomeric
excess (Scheme 3).
The 1,1-dimethyl substituted linker 10 was prepared from
2,20-(1,3-phenylene)diacetic acid (Scheme 4). Esterification of
the diacid with ethanol gave the diester 11, which was equili-
brated with further diacid under acidic conditions to give a
statistical mixture of the desired monoester 12, together with
diacid and diester. Treatment of the monoester 12 with excess
methyl Grignard reagent in tetrahydrofuran gave the tertiary
alcohol 13. Standard Ritter conditions with acetonitrile were
used to convert the alcohol to the acetamide; however, sub-
sequent cleavage of the resulting acetamide proved problema-
tic.12 Treatment of the acetamide in refluxing sodium hydro-
xide in ethylene glycol resulted in decomposition, and acid
hydrolysis was very slow. Further examination of the literature
identified a modified Ritter procedure, using chloroaceto-
nitrile rather than acetonitrile as the nitrogen source.13 Yields
for the rearrangement reaction increased, and the resulting
R-chloroacetamide 14 was readily cleaved by treatment with
thiourea in refluxing acetic acid, followed by esterification to
give the aminoester 10 in good yield (Scheme 4).
met our criteria for progression, including high levels of
potency, long duration of action (DoA) in vitro and in vivo,
and suitable pharmacokinetics for inhaled delivery (low oral
absorption, rapid systemic clearance). However, while 3 pos-
sessed good material properties as its crystalline L-tartrate salt,
compatible with delivery from a dry powder inhaler, it was
generally found that securing suitable solid form characteris-
tics in this conformationally flexible and lipophilic series
presented a significant challenge. Therefore, as part of our stra-
tegy to identify additional development candidates, we sought
to identify a differentiated series of β2-adrenoreceptor agonists
with higher intrinsic crystallinity to simplify solid form identi-
fication and candidate selection.
In this paper we describe the discovery of a novel series
of β2-adrenoreceptor agonists that combine the pharmaco-
logy profile required for once-daily dosing potential with the
robust solid form attributes necessary to support conve-
nient delivery in a preferred dry-powder inhaler device. This
“inhalation by design” philosophy culminated in the disco-
very of compound 38 as a clinical candidate that is currently in
advanced phase II studies for the treatment of asthma and
COPD.
Chemistry
The target compounds in this paper have architecture
similar to that of 2 (Chart 3) while utilizing the sulfonamide
headgroup found in zinterol (Chart 4). The synthetic strategy
for preparation of these targets was based upon two primary
disconnections that lead to the key bromo alcohol “head”,
amino ester “linker”, and amine “tail” fragments (Scheme 1).
Coupling of 6 and the linkers 7 and 10 was effected by direct
displacement of the bromide in the absence of solvent to give
the key ester intermediates 15 and 16 (Scheme 5). The use of
solvent and triethylamine as base was investigated; however,
no advantage was found over the neat reaction. Ester hydro-
lysis of template 15 with aqueous lithium hydroxide gave the

6642 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Glossop et al.
Scheme 1. General Retrosynthetic Strategy for Preparation of Target Compounds
Scheme 2a
a Reagents: (a) MeSO2Cl, pyridine, DCM, 16.5 h, 88%; (b) TBDMSCl, imidazole, DMAP, DMF, 16 h, 86%.
Scheme 3a
a Reagents: (a) (i) MeOH, H2SO4; (ii) Bu3SnOMe, isoprenyl acetate, P(o-tolyl)3, Pd(OAc)2, toluene, 100 ꢀC, 5 h, 94%; (b) R-(R)-methylbenzylamine,
NaBH(OAc)3, AcOH, DCM, 48 h; (c) (i) HCl, MeOH; (ii) recrystallization, DIPE, MeOH, 40% (two steps); (d) 20% Pd(OH)2/C, NH4HCO2, MeOH,
75 ꢀC, 1.5 h, 100%.
Scheme 4a
a Reagents: (a) EtOH, AcCl, reflux, 18 h, 92%; (b) 2,20-(1,3-phenylene)diacetic acid, EtOH, 1,4-dioxane, HCl, reflux, 18 h, 27%; (c) MeMgCl, THF,
18 h, 100%; (d) chloroacetonitrile, H2SO4, AcOH, 4 h, 96%; (e) (i) thiourea, AcOH, EtOH, reflux, 16 h; (ii) HCl, MeOH, reflux, 16 h, 67%.
carboxylic acid intermediate 17, whereas transfer hydrogena-
tion of 16 afforded phenol 18. Subsequent debenzylation of 17
and ester hydrolysis of 18 afforded key acid intermediates 19
and 20, respectively. Target compounds were generally pre-
pared by active ester couplings of acids 17, 19, and 20 with
appropriate amines to provide 21a-38a, followed by full
deprotection to afford test compounds 21-38. In the majority
of cases, deprotection was achieved via ammonium fluoride
mediated desilylation (22, 24, 26-38), although some exam-
ples required additional debenzylation prior to final desilyla-
tion (21, 23, 25) (Scheme 6).
benzonitriles with borane, (2) reductive amination of benzal-
dehydes with allylamine followed by Pd-mediated deprotec-
tion with dimethylbarbituric acid, (3) amide coupling of benzoic
acids with ammonia followed by reduction with borane, and
(4) Pd-mediated Suzuki coupling of phenolic boronic acids with
Boc-protected halobenzylamines to afford bespoke biaryl
systems.14
Results and Discussion
Our strategy to identify a differentiated series of β2-adreno-
receptor agonists with higher intrinsic crystallinity focused
on retention of the key β2-pharmacophore combined with
additional chemical motifs known to predispose compounds
toward greater crystallinity. Sulfonamides were of particular
For target compounds 21-29 the amine coupling partners
were commercially available; however, for phenolic com-
pounds 30-38 some of the amines required de novo synthesis
according to one of four general methods: (1) reduction of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6643
Scheme 5a
a Reagents: (a) neat, 90 ꢀC, 16-72 h, 58-80%; (b) LiOH, THF, 48-72 h, 90-100%; (c) 20% Pd(OH)2/C, NH4HCO2, EtOH, 85 ꢀC, 3 h, 65-84%.
Scheme 6a
i
a Reagents: (a) R6R5NH, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, Pr2NEt, DMF, 18 h, 31-85%; (b) 20%
Pd(OH)2/C, NH4HCO2, EtOH, 85 ꢀC, 3 h; (c) NH4F, EtOH, H2O, 50 ꢀC, 18 h, 38-93%.
interest given their capacity for formation of hydrogen-bonding
interactions and salt bridges and their presence in the headgroup
of the β2-adrenoreceptor agonist zinterol (Chart 4).
were important considerations as design criteria to obtain
sufficient DoA to meet our objectives.7-9
Excellent β2-adrenoreceptor potency, agonism (equivalent
to isoprenaline), and selectivity were retained through incor-
poration of the sulfonamide motif, as illustrated by initial
target 21, confirming the need to fully explore this expression
ofournovel template(Table1). However, despite theexcellent
potency of 21, it was clear that incorporation of the sulfon-
amide group had led to significant reduction in lipophilicity
(log D < 2) that was inconsistent with our design strategy.
Consequently, a range of sulfonamide analogues were synthe-
sized to establish SAR, while incrementally increasing logD.
These data clearly demonstrated that a wide variety of amide
substitution was tolerated, including CH2 homologation (23,
24), aromatic substitution (25-28), and cycloalkyl substitu-
tion (29). Importantly, these analogues also demonstrated
that potency and selectivity were achievable with a range of
lipophilicity that extended beyond our design criteria (28;
log D = 2.4) and as such had potential to deliver long DoA in
vitro. Replacement of the (R)-methyl group adjacent to the
basic nitrogen with the gem-dimethyl moiety (21 vs 22)
retained the desired levels of both β2-adrenoreceptor potency
and selectivity over the β1-adrenoreceptor.8 Given the pre-
ference of the β2-adrenoreceptor for molecules with an (R,R)-
configuration (1, formoterol),7,17 these data further illustrate
that the (R)-methyl substituent makes the key interaction
regarding β2 potency and selectivity (driven by this β2 potency)
while the (S)-methyl substituent makes neither significantly
positive nor negative receptor-ligand interactions. Incorpora-
tion of gem-dimethyl substitution was also advantageous be-
cause it removed one of the two stereogenic centers from the
molecules, thereby reducing synthetic complexity. Overall, it
In addition to its potential to confer crystallinity, with the
sulfonamide moiety being one of the most effective H-bond
donors, it also had the potential to further reduce oral absorp-
tion across the gut wall in a manner consistent with our
“inhalation by design” objectives, namely, to minimize systemic
exposure and maximize therapeutic index (TI) through reduced
oral bioavailability (of the swallowed fraction following in-
halation) and increased hepatic clearance.8 There have been
numerous analyses investigating molecular properties that influ-
ence good oral absorption/bioavailability, and a feature that
most of them agree upon is the preference to minimize hydrogen
bonding character.15 One example of this type of analysis is
Lipinski’s so-called “rule-of-five” (Ro5), in which H-bond do-
nors (<5) and H-bond acceptors (<10) represent two of the four
rules suggestive of good oral absorption.16 Retaining the pro-
pensity for hydrogen bonding and molecular properties consis-
tent with non-“Ro5” compliance was therefore an imperative
part of our design strategy that sulfonamides also appeared to
complement. Consequently, we decided to focus our immediate
efforts on incorporating this headgroup into our novel series of
phenethylamide containing β2-adrenoreceptor agonists.
Initial amide analogues were prepared using the (R)-methyl-
phenethylamine linker found in development candidate 3, as a
design strategy to retain desirable properties such as high levels
of potency, long DoA, and suitable pharmacokinetics while
introducing the sulfonamide headgroup to assess its impact on
these properties and its potential for crystallinity. Project ex-
perience also taught us that relatively lipophilic compounds
(logD g 2) and molecular architecture similar to salmeterol

6644 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Glossop et al.
Table 1. β2 Potency, β1 Selectivity, clogP, log D, and In Vitro Guinea Pig Trachea Data for Compounds 21-29
a Potency and efficacy at human recombinant β2-adrenoceptors expressed in CHO cells assessed as elevations in cAMP. In this assay, all compounds
appeared to be full agonists in comparison to isoprenaline control. b Ratio of EC50 values generated at human recombinant β1- and β2-adrenoceptors
expressed in CHO cells assessed as elevations in cAMP. Ratio is quoted to 2 significant figures. c EC50 potency in the in vitro guinea pig trachea assay,
quoted relative to salmeterol, such that a compound twice as potent will be quoted as 0.5. d Duration of action (DoA) was calculated as the time taken in
hours for the EFS response to recover by 50% of the inhibition induced by an Emax concentration of the test compound. e n.d.: EC50 and potency relative
to salmeterol not determined to facilitate rapid assessment of duration of action at an estimated Emax concentration of test compound. f Incomplete
(<100%) recovery after 14 h. Fully reversible by selective β2 antagonist 2-butanol, (2R,3R)-rel-1-[2,3-(Dihydro-7-methyl-1H-inden-4-yl)oxy]-3-
[(1-methylethyl)amino]-2-butanol.
was demonstrated that levels of β2-adrenoreceptor potency and
selectivity comparable to those of previously developed series
(2, 3) were achievable in this sulfonamide template.8,9
and ethoxybenzylamide 26 (6.2 h, logD 1.6) vs methoxy-
benzylamide 25 (4.22 h, logD = 1.2). Moreover, dimethylbenzyl-
amide 27 (6.74 h, log D = 2.0) possessed similar DoA and
measured log D to both salmeterol and development candi-
date 3, while the yet more lipophilic 3,4-dichloro analogue 28
(8.34 h, logD = 2.4) demonstrated superior DoA to salme-
terol. In addition, 28 was essentially equipotent with salme-
terol, zinterol, and 3 in terms of their tracheal and cell based
EC50 data. Cyclohexylamide 29 also satisfied the general
lipophilicity-DoA relationship. These in vitro data provided
further evidence that this sulfonamide template could deliver
the overall β2-adrenoreceptor agonist pharmacology that we
required.
To assess the potential of these sulfonamide compounds to
deliver long DoA, the effect of 21, 24-29, zinterol, and
salmeterol on airway smooth muscle was investigated in vitro
using guinea pig tracheal strips (Table 1). The pharmacology
of β2 agonists in this model is well documented and correlates
well with clinical data, giving a measurement of potency,
efficacy, and DoA.18 As anticipated, benzylamide 21 was
found to have short in vitro DoA (3.24 h, log D = 1.0) when
compared to salmeterol (6.9 h, logD = 2.1) and similar in
vitro DoA when compared to zinterol (3.4 h, log D = 0.6),
probably due to its significantly reduced lipophilicity. Con-
sistent with our design hypothesis, tracheal data for com-
pounds 24-29 revealed that increasing lipophilicity in this
series resulted in the anticipated increase in DoA, for example,
phenpropylamide 24 (5.85 h, logD = 1.7) vs benzylamide 21,
Following this demonstration of pharmacology, we turned
to our attention to the key, overriding project objective:
obtaining high levels of intrinsic crystallinity to simplify solid
form identification and selection for inhaled dry-powder deli-
very. Consequently, a number of crystallization experiments

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6645
endogenous compounds and xenobiotics; consequently, it is a
major source of drug inactivation and therefore appeared
ideally suited to our objectives. Carboxylic acids and phenols
are among the most commonly glucuronidated functional
groups (although a variety of other moieties have precedent),
but we were concerned that the addition of carboxylic acids
to our template would negatively affect DoA as a result of
introducing significant polarity and ionization. In addition,
carboxylic acids give rise to potentially reactive acylglucuronide
metabolites that we felt were an unnecessary drug safety risk to
introduce, despite the anticipated low dose of these inhaled
agents (e1 mg). Consequently, we chose to incorporate phe-
nols as the metabolic handle to assess glucuronidation while
seeking to retain the desired crystallinity and β2-adrenoreceptor
pharmacology of sulfonamide analogues 21-29.
Figure 1. Small molecule X-ray crystal structure of compound 26.
The β2-adrenoreceptor agonist pharmacophore is predicted
to require an aromatic ring that makes hydrophobic inter-
actions with Phe-290, associated meta- and para-H bond
donors that form a hydrogen bonding network with Ser203,
-204, and -207, and a key amino alcohol motif that makes
hydrogen bonding and salt bridge interactions to Asn293 and
Asp113, respectively.20 As a result of these requirements, the
vast majority of β2-adrenoreceptor agonist headgroups con-
tain one or two phenols, as illustrated by the endogenous
ligand norepinephrine (adrenaline) and examples such as
salmeterol, formoterol, and zinterol. However, while there is
some evidence that phenols such as that present in salmeterol
are glucuronidated in rat, the extent of glucuronidation in
humans is much less significant.21 Consequently, it appeared
that incorporation of phenols for glucuronidation required
more bespoke medicinal chemistry design than use of simple
phenolic head groups. Given the critical β2-adrenoreceptor
agonism, crystallinity, and synthetic complexity imparted by
the sulfonamide headgroup, we decided to retain this frag-
ment in its entirety, choosing to introduce phenolic substitu-
tion on the more synthetically accessible benzylamide tail
portion. Phenolic analogues30-38 were subsequently synthe-
sized to assess the effect of different phenols on β2-adreno-
ceptor potency, selectivity, DoA, crystallinity, and glucuroni-
dation in this sulfonamide template (Table 2).
Phenolic substitution was generally well tolerated across a
variety of amide expressions and compared well with previous
sulfonamide SAR (Table 1). For example, excellent levels of
β2-adrenoceptor potency, agonism (equivalent to isoprenaline),
and selectivity were observed for chlorophenol analogues 30,
32, and 34 and dimethylphenol 35. These data illustrated that
both ortho- and para-phenols (32 vs 30) were equally potent
and selective. Dichlorophenol 33 and N-ethyl analogue 31
showed slightly reduced β2 potency (∼8- to 18-fold) and
consequently reduced selectivity over β1 activity, when com-
pared to analogues 32 and 30, respectively. Similarly, sterically
demanding phenols such as naphthol 36 and biaryl analogues
37 and 38 also displayed slightly reduced but good levels of β2
potency (β1 selectivity was also reduced for these analogues).22
Importantly, the β2 potency and selectivity of phenolic sulfon-
amide analogues 30-38 were demonstrated over a range of
lipophilicity that suggested the potential to deliver long DoA
in vitro.
were performed with a selection of compounds from Table 1,
using a variety of standard techniques and conditions. It soon
became apparent that isolation of crystalline material in this
series was readily achievable. In fact, crystallinity was such
that it enabled generation of single crystal X-ray structures,
as exemplified by compound 26 (Figure 1). The structural
information gleaned from this X-ray not only confirmed the
stereochemistry and solid state conformation but also
revealed a potential driving force for crystallization: intra-
molecular protonation of the basic amine with the acidic
sulfonamide proton. The formation of this internal salt, or
zwitterion, may provide the crucial intermolecular salt
bridge or hydrogen bonding interactions that order the
crystal lattice and induce crystallinity.
The zwitterionic potential of 26 and these sulfonamide
analogues in general is supported by 28, whose pKa values
were measured as 8.93 and 7.97 for the amine and sulfon-
amide, respectively. Compound 28 was also found to be crystal-
line by powder X-ray diffraction (PXRD) with additional
analysis by solid state Fourier transform infrared (FTIR)
spectroscopy providing evidence for strong absorption bands
at wavelengths characteristic of secondary ammonium
(R2NH2þ) N-H stretching/bending vibrations. This crystal-
line form of 28 possessed a high melting point (195 ꢀC) and
was nonhygroscopic (<1.3% water uptake at 90% relative
humidity), demonstrating that the key material properties for
compatibility with a dry-powder inhaler device could be
secured within this series.
As reported, compounds from previous saligenin series
(1, 2, and 3) displayed suitable pharmacokinetics for inhaled
delivery as a result of low oral absorption and rapid clearance
from the systemic compartment, primarily through phase I
cytochrome P450 (CYP) mediated metabolism.7-9 While we
were confident that this would be the case for sulfonamide
analogues 21-29, we were keen to explore the possibility of
introducing additional routes of metabolism to further maxi-
mize clearance, increase TI, and mitigate potential drug-
drug interactions (DDI). Following a review of non-P450
mediated metabolic pathways, we identified glucuronidation
as a clearance route of interest to us. Glucuronidation is a well
documented high capacity phase II metabolic pathway that
covalently conjugates glucuronic acid from UDPGA (uridine-
50-diphosphoglucuronic acid) to lipophilic substrates via UGT
enzymes (uridine-50-diphosphoglucuronosyl transferases).19
The glucuronide metabolites produced are polar, are water-
soluble, and consequently undergo renal or biliary elimination.
Glucuronidation is acknowledged as the major glycosylation
reaction and is responsible for the detoxification of both
The effect of 30-38 on airway smooth muscle was investi-
gated using the previously described in vitro guinea pig trachea
assay. Pleasingly, the majority of these phenolic analogues
displayed high levels of potency and long DoA (Table 2). The
exceptions to this were 34 (3.66h) and35 (4.59h), bothofwhich
had significantly shorter DoA. It is interesting to note that these

6646 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Glossop et al.
Table 2. β2 Potency, β1 Selectivity, clogP, log D, and In Vitro Guinea Pig Trachea Data for Compounds 30-38
a Potency and efficacy at human recombinant β2-adrenoceptors expressed in CHO cells assessed as elevations in cAMP. In this assay, all compounds
appeared to be full agonists in comparison to isoprenaline control. b Ratio of EC50 values generated at human recombinant β1- and β2-adrenoceptors
expressed in CHO cells assessed as elevations in cAMP. Ratio is quoted to 2 significant figures. c EC50 potency in the in vitro guinea pig trachea assay,
quoted relative to salmeterol, such that a compound twice as potent will be quoted as 0.5. d Duration of action (DoA) was calculated as the time taken in
hours for the EFS response to recover by 50% of the inhibition induced by an Emax concentration of the test compound. e n.d.: EC50 and potency relative
to salmeterol not determined to facilitate rapid assessment of duration of action at an estimated Emax concentration of test compound.
analogues were the most polar phenolic sulfonamides tested,
again demonstrating the important relationship between lipo-
philicity and DoA in this template. In the case of 35 (logD =
1.1), it appears that the two methyl groups are insufficient
lipophilic compensation for the addition of the polar phenol.
However, dichlorophenol 34 is essentially isolipophilic with its
regioisomer 33 but has a considerably lower log D (0.8 vs 2.0).
The rational explanation for this effect relates to differences in
ionization. Specifically, the two flanking chlorine atoms in 34
render the adjacent phenol much more acidic and prone
to ionization, as reflected by a lower measured log D. This
hypothetical level of acidity for 34 is supported by a measured
pKa value of 6.69, compared to chlorophenol 32 (9.22)
and dimethylphenol 35 (9.93). This trend is consistent with
widely understood electronic effects and clearly illustrates that
this 2,6-dichlorophenol motif is approximately 3 orders of
magnitude more acidic than a standard phenol and only 2
orders of magnitude less acidic than acetic acid. Nevertheless,
the majority of in vitro GPT data for 30-38 confirmed that this
phenolic sulfonamide template could deliver the β2-adreno-
receptor agonist pharmacology that we required, with obvious
potential for crystallinity.22
Compound 38 is essentially isolipophilic with salmeterol, so
it is possible that lipophilicity drives its DoA, as proposed for
both formoterol and salmeterol by the diffusion microkinetic
hypothesis23,24 and as recently illustrated for indacaterol.3
However, the receptor exosite hypothesis has also been pro-
posed to rationalize the long duration of action of salmeterol
and recentanalogues.1,18,25,26 High affinity bindingof the long
side chain deep into a hydrophobic core domain of the recep-
tor effectively anchors the molecule and allows the saligenin
headgroup to continuously activate the β2-adrenoceptor.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6647
Table 3. Pulmonary and Cardiovascular TI Data for 38, 1, Formoterol,
and Salmeterol following Intratracheal Solution Administration to
Anesthetized Dogs
lung
lung DoA
lung DoA at
TI c lung vs
CV
compd
38
ED50a (μg) at ED50 b (h) 10ꢀ ED50 b (h)
0 . 1
1
>8
>8
4
>8
>8
<6
<7
g10
10
1
1
formoterol
salmeterol
0.1
1
4
1
a ED50 is the intratracheal dose giving 50% inhibition of ACh induced
bronchoconstriction. b DoA was assessed by comparison of the airway
resistance at 1 h intervals for a single dose of the test compound relative
to the control ACh response. c TI (therapeutic index) is the inverse ratio
of lung ED50 dose compared to dose that caused the first increase in
CV parameters.
receptor agonists from this sulfonamide template were suitable
for inhaled dry-powder delivery, thus securing one aspect of our
key project objectives.
With encouraging in vitro pharmacology and solid form
data in hand, 38 was profiled in an anesthetized dog model of
bronchoconstriction to assess in vivo efficacy, DoA, and cardio-
vascular (CV) side effects (e.g., heart rate).27,28 Anesthetized
and artificially ventilated dogs were challenged with iv
bolus doses of acetylcholine (ACh) to establish a dose that
gave a transient 60-100% increase in lung resistance (RL).
A dose of either β2 agonist or vehicle (micelle) was then delivered
intratracheally (it.) before animals were rechallenged with
ACh to compare RL and CV parameters to the control ACh
response. In this model, 38 was found to be equipotent to for-
moterol (ED50 = 0.1 μg) and more potent than both salmeterol
and development candidate 1 (ED50 = 1 μg) (Table 3). At the
ED50 dose, 38 displayed similar intrinsic DoA to 1, superior to
both salmeterol and formoterol. Furthermore, a 10-fold higher
dose of 38 (1 μg) afforded even greater efficacy and DoA, as
illustrated against 1 μg of salmeterol (Figure 3). In addition,
hemodynamic data from these studies confirmed that while
salmeterol (1 μg) showed a trend toward increased heart rate,
38 did not induce tachycardia at the doses tested (Figure 3).
These comparative data to b.i.d. clinical agents in a well validated
in vivo model suggested encouraging potential for 38 to deliver
24 h duration of action in humans with a therapeutic index of at
least 10-fold.
Compound 38 was subsequently profiled through a variety
of in vitro pharmacokinetic (PK) assays (Table 4). Rapid
CYP-mediated metabolism in a human microsomal assay
(Clint = 54 (μL/min)/mg) and low to moderate cell perme-
ability with high transporter mediated efflux (Papp(A-B) <
1 ꢀ 10-6 cm/s, Papp(B-A) = 13 ꢀ 10-6 cm/s) as measured by
apical to basal flux rate through a monolayer of Caco-2 cells
were observed. These in vitro data suggested that 38 would
have low oral bioavailability due to poor absorption through
the gut lumen and high first pass metabolism and warranted
further progression of 38 to rat in vivo pharmacokinetic
studies (Table 4).29,30 Following iv administration the volume
of distribution was high (31 L/kg); however, with total
clearance exceeding liver blood flow (103 (mL/min)/kg) the
measured half-life was 3.5 h. Plasma protein binding in rat of
95% also gave very high unbound clearance (>2000 (mL/
min)/kg). Following po administration in rat, the oral bioa-
vailability was found to be <5%, consistent with predictions
from in vitro data. Furthermore, analysis of rat portal vein
samples confirmed that this was driven by fundamentally
poor intestinal absorption with portal bioavailability of 38
also of <5%.
Figure 2. Structural overlays of 38 with salmeterol (purple).
Although there is significantly less conformational flexibility
in the sulfonamide template compared to salmeterol, struc-
tural overlays suggest that salmeterol and 38 may be able to
adopt similar conformations (Figure 2), as previously pro-
posed for indole analogue 1.7 It can be seen that when the key
β2-adrenoceptor agonist headgroups are overlaid, the termi-
nal aromatic tail residues occupy similar regions of space.
Furthermore, the position of the amidemoietyin 38 may serve
to make receptor interactions similar to those made by the
ether oxygen atom in the salmeterol tail. On the basis of these
overlays, it can also be hypothesized that 38 retains the
potential to access the proposed receptor exosite and that this
drives its prolonged DoA.
In a manner consistent with initial sulfonamides such as 26
and 28, the solid formof phenolicanalogues30-38was highly
encouraging, with many compounds isolated as solids directly
from the final desilylation reaction by simple filtration. This
ease of isolation enabled rapid PXRD analysis and early solid
form assessment, identifying analogues such as 32, 33, 36, 37,
and 38 with good levels of crystallinity. PXRD data were of
such importance to us that these data were available for biaryl
38 prior to the emergence of biological data. Further solid
form analysis of these compounds using well-known techni-
ques such as DSC, TGA, and DVS identified 38 in particular
as a compound with the most suitable combination of pharma-
cology and material properties. 38 possessed a highly crys-
talline, nonhygroscopic (1.7% water uptake at 90% relative
humidity) solid form with high melting point (198 ꢀC), material
properties that were consistent with our solid form requirements.
With favorable material properties secured, the solid state
compatibility of 38 was assessed with lactose monohydrate,
the most common carrier excipient used in commercial dry
power inhalers (DPI). Material blended at a 1:100 ratio with
lactose monohydrate was shown to be stable after 8 weeks of
storage at 40 ꢀC and 75% relative humidity in open vials.
These data for 38 demonstrated that crystalline β2-adreno-

6648 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Glossop et al.
Figure 3. Respiratory and cardiovascular data for 38 and salmeterol following intratracheal administration to anesthetized dogs as solutions
in micelle vehicle. Data are expressed as the percent change from pretreatment values, taking the pretreatment value as 100. Data are mean (
SEM of n = 4/group.
The metabolites of 38 were determined from numerous in
vitro studies (human, rat, and dog hepatocytes and liver
microsomes) and an in vivo isolated perfused rat liver (IPRL)
study. The major metabolites observed were the glucuronide
conjugate consistent with phase II conjugation on the hydroxy
biphenyl moiety, the primary amide resulting from N-dealky-
lation of the biphenyl moiety, and the carboxylic acid resulting
from amidolysis. The sulfate conjugate of 38 was also observed
indicating additional phase II metabolism. Approximately 6%
of the dose of 38 was excreted unchanged in the bile. By use
of synthetic standards for analysis, none of these metabolites
were found to circulate in any in vivo samples (IPRL perfusate
or plasma). The extent of glucuronidation of 38 was further
assessed in vitro using glucuronidation specific human liver
microsome methodology and clinical standards similar to those
described in the literature.31 Under these conditions 38 had a
short half-life (Clint = 33 (μL/min)/mg) that suggested an
unbound intrinsic clearance of >10000 (mL/min)/kg. Glucuro-
nidation was also observed in vitro for analogues 32 and 36.
Factoring in liver blood flow and human plasma protein
binding, 38 was predicted to exhibit very high intrinsic clear-
ance in humans due to glucuronidation. This successful intro-
duction of phenolic glucuronidation as an additional route of
metabolism to maximize clearance, TI and mitigate potential
drug-drug interactions (DDI) was entirely consistent with our
design strategy.
systemic compartment was found to be significantly slower
than salmeterol, resulting in longer Tmax (0.5 h vs 0.1 h) and
lower systemic unbound Cmax (1.3 nM vs 23 nM). These diffe-
rences in absorption rate and exposure are illustrated by the it.
PK profiles of both 38 and salmeterol (Figure 4). It is worth
noting that the it. PK profile of salmeterol is identical to its iv
PK profile, further illustrating its rapid absorption from the
lung. In contrast, the it./iv PK profiles of 38 are markedly
different, resulting in the observed 18-fold lower systemic
unbound Cmax relative to salmeterol.
Overall, 38 exhibited an ideal pharmacokinetic profile for
an inhaled agent. In contrast to salmeterol, data following it.
administration indicated potential for delayed but complete
absorption from the lung in humans. Furthermore, the swal-
lowed fraction of the dose was unlikely to contribute to
systemic exposure or associated adverse events in humans
due to incomplete oral absorption and extensive hepatic first
pass extraction. In addition, high intrinsic clearance predicted
in humans via multiple routes of metabolism suggested that
unbound exposure of 38 arising from the lung absorbed dose
should be minimal. Glucuronidation was predicted to be a
major route of clearance of 38 in humans, leading to a low
potential for drug-drug interactions. This pharmacokinetic
profile suggested that, relative to salmeterol, compound 38
should demonstrate reduced systemically mediated adverse
eventsarising from boththe inhaledand swallowed fraction of
the dose following inhalation in humans.32
From a drug safety perspective, 38 showed no significant
affinity (>100 nM) when evaluated for broader pharmacolo-
gical activity against a wide ranging external panel of other
receptors, enzymes, andion channels. Furthermore, 38 had no
meaningful functional effect on in vitro cardiac ion channel
assaysatmicromolarconcentrations (e.g., hERGpatch clamp
IC50 > 10 μM). In vitro genetic toxicity testing and in vivo rat
toleration studies were also completed without observing any
meaningful adverse effects. The tussivity of 38 was evaluated
inaclinicallyvalidatedrabbit lungcoughmodel thatmeasures
action potentials from Aδ-fibres/rapidly adapting receptors
Additional studies in human liver microsomes with the
addition of the cytochrome P450 3A4 inhibitor ketoconazole
significantly reduced the intrinsic clearance of 38 (Clint < 10
(μL/min)/mg), suggesting that approximately 80% of phase I
metabolism was mediated by CYP3A4. This observation
was confirmed by experiments using expressed human CYP
enzymes. In the absence of CYP3A4 metabolism the most
significant CYP enzyme involved in the metabolism of 38
was found to be the polymorphically expressed enzyme
CYP2D6.
Following intratracheal (it.) solution administration of 38
(100 μg) to rats, the rate of absorption from the lung into the
Table 4. In Vitro and In Vivo Pharmacokinetic Data for Compound 38
in vitro PK
rat in vivo PK
Caco-2 Papp ꢀ 10-6 (cm/s)
iv
po
F (%)
compd
HLM Clint ((μL/min)/mg)
A-B
B-A
Vd (L/kg)
ClT ((mL/min)/kg)
103
t1/2 (h)
rat ppb (%)
95
38
54
<1
13
31
3.5
<5

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6649
Figure 4. Pharmacokinetic profiles for salmeterol and 38 following intratracheal and intravenous administration of 100 μg solution
formulations to rats.
Chart 5
(RAR).27 This enables early assessment of the potential to
cause cough, which is a key attrition risk for inhaled com-
pound development. Indacaterol, an inhaled long acting
β2-adrenoreceptor agonist that has been reported to cause
postinhalational cough in humans, was also found to be
tussive in this rabbit cough model.33 In contrast, both 38
and salmeterol (nontussive in humans) were inactive in this
model, thus providing confidence that 38 was unlikely to cause
Figure 5. Phase Ib data from healthy human volunteers for 38 and
cough in humans. Overall, 38 had a preclinical safety profile
salmeterol following inhalation of dry-powder formulations. Data
that suggested it should be well tolerated in humans at all
are expressed as the mean percent change in sGAW from baseline by
clinically relevant inhaled doses.
treatment.
Conclusion
Experimental Section
In conclusion, we have described our “inhalation by design”
Biology. In Vitro Potency and Selectivity at Recombinant
strategy to deliver a novel β2-adrenoceptor agonist (38) that has
Human β1- and β2-Adrenoreceptors Expressed in CHO Cells.
longer duration of action in preclinical models than salmeterol,
Chinese hamster ovary (CHO) cells separately expressing recombi-
with clear potential for once-daily inhaled administration in
humans via a dry-powder inhaler. Material properties were
secured early through incorporation of the sulfonamide moiety
in the headgroup as a key design feature to provide higher
intrinsic crystallinity and to simplify solid form selection.
Compound 38 exhibits an ideal preclinical pharmacokinetic
profile for an inhaled agent by virtue of delayed lung absorp-
tion, negligible oral bioavailability, and high intrinsic clearance
via multiple routes of metabolism, including extensive phenolic
glucuronidation. Furthermore, the preclinical safety profile of
38 suggests that it will be well tolerated in humans at all
clinically relevant doses. Given this exciting profile, 38 (PF-
610355) was nominated as a development candidate (Chart 5),
a novel once-daily inhaled β2-adrenoreceptor agonist for the
treatment of asthma and COPD. Recent data (sGAW) from
healthy volunteers in phase Ib clinical trials illustrate that 38
does indeed provide 24 h of bronchodilation from a single
inhaled dose, thus confirming 38 as a once-daily inhaled
β2-adrenoreceptor agonist (ultra-LABA) (Figure 5).34 Com-
pound 38 has achieved positive proof-of-concept (PoC) for the
treatment of asthma and COPD and is currently in advanced
phase II clinical studies.35 Further information relating to 38
will be reported in future disclosures.
nant human β1- and β2-adrenoceptors were generated using con-
ventional cloning and PCR techniques. Cells were suspended in
Flashplate stimulation buffer containing 0.5 mM IBMX (isobutyl-
methylxanthine), and 50 μL/well was added to the Flashplate
containing the test compound. Each plate also contained control
wells of isoprenaline (100 nM to measure maximum cAMP produc-
tion) and 1% DMSO (to measure basal cAMP production). Plates
were incubated at room temperature for 1 h. The cells were then
lysed by addition of 100 μL/well Flashplate detection buffer
containing 125I-cAMP at 0.18 μCi/mL and the plates incubated at
room temperature for a further 2 h prior to measurement of cAMP
produced in the assay. Data were expressed as a percentage of the
cAMP generated with isoprenaline and plotted against the con-
centration of agonist on a log scale. A four-parameter sigmoid fit
was used to generate EC50 and Emax (%).
In Vitro Potency and Duration of Action in Guinea Pig Isolated
Trachea. Tracheas were removed from male Dunkin-Hartley
guinea pigs (475-525 g), cut into strips, and mounted in 5 mL
organ baths in warmed (37 ꢀC) and aerated (95% O2/5% CO2)
Krebs solution (flow rate 1 mL/min) containing indomethacin
(3 mM) and guanethidine (10 mM) under 1 g weight initial
tension. Tissues were retensioned to 1 g every 15 min for 1 h and
then subjected to electrical field stimulation (EFS: 0.1 ms, 10 Hz
for 10s every 2 min) at submaximal voltage (10-30 V). A single
bolus concentration of test compound was added to each tissue

6650 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Glossop et al.
and left for 4 h. A concentration-response curve to test
compound was constructed across tissues, and data were ex-
pressed as percent inhibition of the control contraction size.
Following the 4 h incubation, tissues were washed initially at
90 mL/min for 1 min and then for 1 mL/min for the remainder of
the experiment (up to 16 h). Duration of action (DoA) was
calculated as the time taken in hours for the EFS response to
recover by 50% of the inhibition induced by the test compound.
In Vivo Potency and Duration of Action in the Anesthetized
Dog. Female beagle dogs (10-15 kg) were anesthetized with
chloralose/urethane (100 (mg/mL)/kg, iv infusion) and main-
tained throughout the experiment with continuous iv infusion
(1 (mL/kg)/h). The trachea was cannulated and connected to a
large animal constant volume respirator (Ugo Basile 5025).
Respiratory parameters (transpulmonary pressure (PTP) and
flow) were monitored using differential pressure transducers.
Blood pressure and left ventricular pressure were also moni-
tored. Signals were analyzed using Ponemah software package
PCR to determine lung resistance. Challenges of iv acetylcholine
(ACh) were administered in the right femoral vein. Cardio-
vascular (CV) and respiratory parameters were measured before
and after exposure to iv ACh (10 min intervals) to establish a
dose that gave a change in lung resistance of between 60% and
100% over basal.
(i) For the dose response study, up to five doses of test
compound or vehicle were given via intratracheal administration
at cumulative 1 h intervals. The established iv dose of ACh was
repeated 1 h after test compound administration to compare
airway resistance and CV parameters to the control ACh response.
(ii) For the duration of action study, one dose of test com-
pound or vehicle was administered intratracheally and ACh
challenge (iv) repeated at 1 h intervals for up to 8 h to compare
airway resistance and CV parameters to the control ACh
response.
solvent removed in vacuo to give an orange oil. The residue was
crystallized from hot toluene (100 mL) to give 5 as a pale pink solid,
33.7 g, 88% yield. 1H NMR (DMSO-d6, 400 MHz) δ: 2.93 (s, 3H),
3.52-3.66 (m, 2H), 4.74 (m, 1H), 5.19 (s, 2H), 7.11 (d, 1H),
7.19-7.22 (m, 1H), 7.33-7.36 (m, 2H), 7.40-7.43 (m, 2H), 7.56
(d, 2H), 8.95 (s, 1H) ppm. LRMS: m/z 398-400 [M - H]-.
N-[2-(Benzyloxy)-5-((1R)-2-bromo-1-{[tert-butyl(dimethyl)-
silyl]oxy}ethyl)phenyl]methanesulfonamide (6). Bromide 5 (21.5 g,
53.7 mmol) in N,N-dimethylformamide (125 mL) was treated with
imidazole (4.16 g, 75.2 mmol) and tert-butyl(dimethyl) silylchlo-
ride (9.73 g, 64.5 mmol), and the resulting solution was stirred at
room temperature for 16 h. The reaction mixture was diluted with
ethyl acetate (200 mL) and washed with water (2 ꢀ 100 mL). The
aqueous phases were combined and extracted with ethyl acetate
(100 mL). The combined organic extracts were washed with 2 M
hydrochloric acid (100 mL), dried (magnesium sulfate), and con-
centrated in vacuo. The residue was triturated twice from pentane/
ethyl acetate (200 mL, 1:1 by volume) and dried in vacuo to give 6
as a colorless solid, 23.7 g, 86% yield. 1H NMR (CDCl3, 400 MHz)
δ: -0.07 (s, 3H), 0.11 (s, 3H), 0.89 (s, 9H), 2.91 (s, 3H), 3.40-3.50
(m, 2H), 4.80-4.83 (m, 1H), 5.14 (s, 2H), 6.80 (bs, 1H), 6.98 (d,
1H), 7.12 (d, 1H), 7.36-7.44 (m, 5H), 7.52-7.54 (m, 1H) ppm.
Diethyl 2,20-(1,3-Phenylene)diacetate (11). 2,20-(1,3-Phenylene)-
diacetic acid (10.0 g, 51.0 mmol) was dissolved in ethanol (100 mL)
and the solution treated dropwise with catalytic acetyl chloride
(2.50 mL). The reaction mixture was stirred at reflux for 18 h and
then cooled to room temperature and concentrated in vacuo. The
residue was taken up in ethyl acetate (100 mL) and extracted with
sodium bicarbonate solution (3 ꢀ 50 mL) and brine (3 ꢀ 50 mL).
The organic phase was then dried (magnesium sulfate) and con-
centrated in vacuo. The residue was triturated with pentane to give
11, 11.8 g, 92% yield. 1H NMR (CDCl3, 400 MHz) δ: 1.31 (t, 6H),
3.65 (s, 4H), 4.20 (q, 4H), 7.24-7.36 (m, 4H) ppm. LRMS: m/z 251
[M þ H]þ.
Chemistry. Unless otherwise indicated, all reactions were
carried out under a nitrogen atmosphere, using commercially
available anhydrous solvents. “0.880 Ammonia” refers to com-
mercially available aqueous ammonia solution of 0.880 specific
gravity. Thin-layer chromatography was performed on glass-
backed precoated Merck silica gel (60 F254) plates, and com-
pounds were visualized using UV light, 5% aqueous potassium
permanganate, or chloroplatinic acid/potassium iodide solu-
tion. Silica gel column chromatography was carried out using
40-63 μm silica gel (Merck silica gel 60). Ion exchange chro-
matography was performed using Isolute strong cation ex-
change resin (SCX-2) cartridges which had been prewashed
with methanol. Proton NMR spectra were measured on a
Varian Inova 400 or Varian Mercury 400 spectrometer in the
solvents specified. In these NMR spectra, exchangeable protons
that appeared distinct from solvent peaks are also reported. Low
resolution mass spectra were recorded on either a Fisons Trio
1000, using thermospray positive ionization, or a Finnigan
Navigator, using electrospray positive or negative ionization.
High resolution mass spectra were recorded on a Bruker Apex II
FT-MS using electrospray positive ionization. Test compound
purity of g95% was determined using combustion analysis
conducted by Exeter Analytical U.K. Ltd., Uxbridge, Middlesex,
U.K. Optical rotations were determined at 25 ꢀC using a Perkin-
Elmer 341 polarimeter using the solvents and concentrations
specified.
[3-(2-Ethoxy-2-oxoethyl)phenyl]acetic Acid (12). A solution of
the diester 11 (44.3 g, 177 mmol) and 2,20-(1,3-phenylene)di-
acetic acid (59.2 g, 308 mmol) in ethanol (24 mL) and dioxane
(290 mL) was treated dropwise with 12 M hydrochloric acid
(4.90 mL, 58.8 mmol). The reaction mixture was stirred at reflux
for 18 h, then cooled to room temperature and concentrated to
low volume. The reaction mixture was diluted with toluene
(125 mL) and the resulting slurry filtered. The filtrate was
concentrated in vacuo and the residue taken up in water and
basified with sodium bicarbonate until neutral pH was ob-
tained. The mixture was diluted with ethyl acetate (200 mL),
and the organic layer was separated and washed with sodium
bicarbonate solution (5 ꢀ 30 mL) and brine (50 mL). The com-
bined aqueous extracts were acidified to pH 3 with 6 M hydro-
chloric acid and extracted with ether (3ꢀ30 mL). The organic
layers were combined, dried (magnesium sulfate), and concen-
trated in vacuo. The residue was triturated with pentane to give
1
12 as a colorless solid, 10.8 g, 27% yield. H NMR (CD3OD,
400 MHz) δ: 1.25 (t, 3H), 3.60 (m, 2H), 3.63 (m, 2H), 4.15 (q,
2H), 7.18-7.32 (m, 4H) ppm. LRMS: m/z 245 [M þ Na]þ.
[3-(2-Hydroxy-2-methylpropyl)phenyl]acetic Acid (13). Methyl-
magnesium chloride (51.0 mL of a 3 M solution in tetrahydrofuran,
153 mmol) was added dropwise to a stirred solution of 12 (11.6 g,
51.0 mmol) in tetrahydrofuran (300 mL) at 0 ꢀC under nitrogen.
The mixture was allowed to warm to room temperature overnight
with the formation of a thick white precipitate. Water (50 mL) and
2 M hydrochloric acid (80 mL) were cautiously added. The aqueous
layer was extracted with ethyl acetate (2 ꢀ 300 mL), the combined
organic layers were washed with brine (50 mL) and dried (sodium
sulfate), and the solvent was removed in vacuo to give 13 as a golden
oil, 11.2 g, 100% yield. 1HNMR(CDCl3, 400MHz) δ: 1.22(6H, s),
2.75 (2H, s), 3.63 (2H, s), 7.12-7.30 (4H, m) ppm. LRMS: m/z 209
[M þ H]þ.
N-{2-(Benzyloxy)-5-[(1R)-2-bromo-1-hydroxyethyl]phenyl}-
methanesulfonamide (5). A solution of (1R)-1-[3-amino-4-(benzyl-
oxy)phenyl]-2-bromoethanol 411 (30.8 g, 95.6 mmol) in dichloro-
methane (300 mL) was treated with pyridine (9.30 mL, 115 mmol).
The resulting solution was cooled to 5 ꢀC and a solution of methan-
esulfonyl chloride (7.80 mL, 101 mmol) in dichloromethane (10 mL)
added dropwise. The mixture was stirred at 5 ꢀC for a further 30 min
and then allowed to warm to room temperature over 16 h. The
reaction mixture was washed with 2 M hydrochloric acid (110 mL),
the organic phase separated, dried (magnesium sulfate), and the
(3-{2-[(Chloroacetyl)amino]-2-methylpropyl}phenyl)acetic Acid
(14). 2-Chloroacetonitrile (8.80 mL, 140 mmol) was added to a
solution of alcohol 13 (16.0 g, 70.0 mmol) in acetic acid (33 mL).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6651
The resulting solution was cooled to 0 ꢀC, treated with concen-
trated sulfuric acid (33 mL) and the reaction mixture allowed to
warm to room temperature. After 4 h the reaction mixture was
poured onto ice and basified with solid sodium carbonate. The
solution was extracted with ethyl acetate (2ꢀ500 mL) and the
combined organic extracts were dried (magnesium sulfate) and
concentrated in vacuo to give 14 as a colorless solid, 19.0 g, 96%
yield. 1H NMR (CDCl3, 400 MHz) δ: 1.36 (s, 6H), 3.02 (s, 2H),
3.62 (s, 2H), 3.95 (s, 2H), 6.19 (m, 1H), 7.06-7.31 (m, 4H) ppm.
LRMS: m/z 282 [M - H]-.
[3-(2-Amino-2-methylpropyl)phenyl]acetic Acid Methyl Ester
(10). A solution of amide 14 (5.10 g, 18.0 mmol), thiourea (1.60
g, 21.0 mmol), and acetic acid (18 mL) in ethanol (80 mL) was
heated to reflux under a nitrogen atmosphere for 16 h. The
reaction mixture was allowed to cool to room temperature and
filtered. The filtrate was concentrated in vacuo, the residue dis-
solved in methanol (150 mL), saturated with hydrogen chloride
gas, and the resulting solution heated to reflux for 16 h. The
mixture was concentrated in vacuo and the residue partitioned
between ethyl acetate (200 mL) and 5% aqueous sodium carbo-
nate solution (200 mL). The organic phase was washed with
brine (100 mL), dried (magnesium sulfate), and concentrated in
vacuo. The residue was purified by strong cation exchange resin,
eluting with methanol followed by a 2 M solution of ammonia in
methanol, to give 10 as a yellow oil, 2.68 g, 67% yield. 1H NMR
(CDCl3, 400 MHz) δ: 1.14 (s, 6H), 2.68 (s, 2H), 3.62 (s, 2H), 3.69
(s, 3H), 7.08-7.16 (m, 3H), 7.23-7.27 (m, 1H) ppm. LRMS: m/z
222 [M þ H]þ.
hydrofuran (50 mL) and toluene (50 mL) and the solvent removed
in vacuo to give 20 as a pale brown foam, 20.2 g, 100% yield. 1H
NMR (400 MHz, CD3OD) δ: -0.14 (s, 3H), 0.07 (s, 3H), 0.83 (s,
9H), 1.32 (m, 6H), 2.93 (m, 5H), 3.23 (m, 2H), 3.54 (m, 2H), 4.94
(m, 1H), 6.91 (d, 1H), 7.03-7.16 (m, 3H), 7.26 (m, 2H), 7.60 (m,
1H) ppm.
2-[3-(2-{[(2R)-2-{[tert-Butyl(dimethyl)silyl]oxy}-2-{4-hydroxy-
3-[(methylsulfonyl)amino]phenyl}ethyl]amino}-2-methylpropyl)-
phenyl]-N-[(40-hydroxybiphenyl-3-yl)methyl]acetamide (38a). A
solution of carboxylic acid 20 (2.80 g, 5.08 mmol) in N,N-
dimethylformamide (60 mL) was treated with 1-hydroxybenzo-
triazole hydrate (0.755 g, 5.59 mmol), 30-(aminomethyl)biphenyl-
4-ol hydrochloride14 (1.20 g, 5.08 mmol), 1-(3-dimethylaminopro-
pyl)-3-ethylcarbodiimide hydrochloride (1.07 g, 5.59 mmol), and
triethylamine (1.49 mL, 10.7 mmol). The resulting suspension was
stirred at room temperature for 18 h. The solvent was removed in
vacuo and the residue partitioned between dichloromethane
(100 mL) and saturated aqueous sodium bicarbonate solution
(50 mL). The organic phase was separated and the aqueous phase
extracted with dichloromethane/methanol (95:5 by volume, 2 ꢀ
20 mL). The combined organic extracts were washed with satu-
rated aqueous sodium chloride (100 mL), dried (sodium sulfate),
and the solvent was removed in vacuo. The residue was purified by
column chromatography on silica gel, eluting with dichloro-
methane/methanol/0.88 specific gravity ammonia (95:5:0.5, by
volume) to give 38a as a white foam, 2.10 g, 56% yield. 1H
NMR (400 MHz, CD3OD) δ: -0.19 (s, 3H), -0.02 (s, 3H), 0.79
(s, 9H), 0.99 (s, 3H), 1.02 (s, 3H), 2.56-2.66 (m, 3H), 2.80-2.83
(m, 1H), 2.85 (s, 3H), 3.53 (s, 2H), 4.40 (s, 2H), 4.65-4.68 (dd, 1H),
6.80-6.82 (d, 2H), 6.83-6.85 (d, 1H), 7.00-7.02 (d, 2H), 7.09 (s,
1H), 7.12-7.14 (d, 1H), 7.18-7.20 (m, 2H), 7.27-7.31 (dd, 1H),
7.33-7.40 (m, 5H) ppm. LRMS: m/z730 [M - H]-, 732[Mþ H]þ,
754 [M þ Na]þ.
(3-{2-[(2R)-2-(4-Benzyloxy-3-methanesulfonylaminophenyl)-
2-(tert-butyldimethylsilanyloxy)ethylamino]-2-methylpropyl}-
phenyl)acetic Acid Methyl Ester (16). Amine 10 (36.0 g, 153 mmol)
and bromide 6 (36.0 g, 70.8 mmol) were heated together at 85 ꢀC
for 72 h. The reaction mixture was cooled to room temperature and
purified by column chromatography on silica gel, eluting with
pentane/ethyl acetate (50:50, by volume) to give 16 as a pale yellow
oil, 37.2 g, 80% yield. 1H NMR (CDCl3, 400 MHz) δ: -0.15 (s,
3H), 0.00 (s, 3H), 0.83 (s, 9H), 1.01 (s, 3H), 1.04 (s, 3H), 2.57-2.97
(m, 7H), 3.59 (s, 2H), 3.68 (s, 3H), 4.68-4.72 (m, 1H), 5.09 (s, 2H),
6.79 (bs, 1H), 6.95 (d, 1H), 7.04-7.21 (m, 7H), 7.37-7.44 (m, 5H),
7.56 (d, 1H) ppm. LRMS: m/z 655 [M þ H]þ.
N-[(40-Hydroxybiphenyl-3-yl)methyl]-2-[3-(2-{[(2R)-2-hydroxy-
2-{4-hydroxy-3-[(methylsulfonyl)amino]phenyl}ethyl]amino}-2-
methylpropyl)phenyl]acetamide (38, PF-610355). Compound
38a (2.10 g, 2.87 mmol) was dissolved in ethanol (79.0 mL)
and treated with a solution of ammonium fluoride (1.06 g, 28.7
mmol) in water (53.0 mL). The reaction mixture was stirred at
40 ꢀC for 18 h, then cooled to room temperature. The reaction
mixture was filtered and washed with methanol/water (1:1 by
volume, 3 ꢀ 10 mL) and dried in vacuo to give 38 as a crystalline
white solid, 1.55 g, 87% yield. 1H NMR (400 MHz, DMSO-d6)
δ: 0.88 (s, 3H), 0.90 (s, 3H), 2.54-2.66 (m, 4H), 2.88 (s, 3H),
3.43 (s, 2H), 4.29-4.30 (d, 2H), 4.39-4.43 (dd, 1H), 6.79-
6.82 (m, 3H), 6.96-7.01 (dd, 2H), 7.07-7.17 (m, 5H),
7.27-7.31 (dd, 1H), 7.36-7.41 (m, 4H), 8.50-8.53 (dd, 1H)
ppm. HRMS: m/z 618.2607 [M þ H]þ, calcd 618.263 782. [R]D
Methyl (3-{2-[((2R)-2-{[tert-Butyl(dimethyl)silyl]oxy}-2-{4-
hydroxy-3[(methylsulfonyl)amino] phenyl}ethyl)amino]-2-methyl-
propyl}phenyl)acetate (18). A solution of 16 (36.8 g, 56.0 mmol)
in ethanol (550 mL) was treated with ammonium formate (16.0 g,
254 mmol) and 20% palladium hydroxide on carbon (1.50 g). The
resulting suspension was heated to 85 ꢀC for 2 h. Further 20%
palladium hydroxide on carbon (1.00 g) was added, and heating
continued for 1 h. The reaction mixture was cooled to room
temperature, filtered and the solvent removed in vacuo. The
residue was partitioned between ethyl acetate (500 mL) and 2 M
aqueous ammonia (100 mL). The organic phase was separated,
dried (magnesium sulfate) and the solvent removed in vacuo. The
residue was purified by column chromatography on silica gel,
eluting with dichloromethane/methanol/0.88 specific gravity am-
monia (95:5:0.5, by volume) to give 18 as a pale yellow oil, 20.6 g,
65% yield. 1H NMR (400 MHz, CDCl3) δ:-0.17 (s, 3H), -0.05 (s,
3H), 0.80 (s, 9H), 1.07 (s, 3H), 1.09 (s, 3H), 2.66-2.91 (m, 7H), 3.62
(d, 2H), 3.69 (s, 3H), 4.71-4.74 (m, 1H), 6.58 (d, 1H), 6.88 (dd,
1H), 7.05-7.14 (m, 3H), 7.21-7.25 (m, 1H), 7.30 (s, 1H) ppm.
LRMS: m/z 565 [M þ H]þ.
-14.8ꢀ (c 0.392, methanol). Anal. (C34H39N3O6S 0.3H2O) C, H,
3
N. HPLC analysis: g99.3% purity.
Acknowledgment. We thank Michele Coghlan, Christel
Jones, Sheena Patel, and Susan Summerhill for their contri-
butions to in vitro biology studies and John Adcock, Petra
Chaffe, Tim Davies, Louise Sladen, and Karen Wright for in
vivo biology studies. We also thank Ken Butcher, Sara
Chunn, Tim Hobson, Louise Marples, Michael Paradowski,
Dannielle Roberts, Keith Reeves, and Michelle Wilkes for
assistance with the chemistry programme. Yogesh Sabnis is
thanked for performing computational superpositions, Cheryl
Doherty is thanked for her contributions to the X-ray structural
information, and Fiona MacIntyre is also thanked for the
provision of clinical data.
(3-{2-[(2R)-2-(tert-Butyldimethylsilyloxy)-2-(4-hydroxy-3-
methanesulfonylaminophenyl)ethylamino]-2-methylpropyl}phenyl)-
acetic Acid (20). Ester 18 (20.6 g, 36.0 mmol) was dissolved in
tetrahydrofuran (150 mL) and the solution treated dropwise with
1 M aqueous lithium hydroxide (72.0 mL, 72.0 mmol). The
reaction mixture was stirred at room temperature for 72 h. The
reaction mixture was neutralized by addition of 1 M hydrochloric
acid (72.0 mL, 72.0 mmol) and concentrated in vacuo to low
volume. The aqueous phase was decanted and the residue washed
with water (2 ꢀ 50 mL). The residue was redissolved in tetra-
Supporting Information Available: Biology experimental
details for the in vivo rabbit lung Aδ-fiber cough model;
chemistry experimental details and analytical data for inter-
mediates 8, 9a, 7, 15, 17, 19, 21a-37a and test compounds
21-37; X-ray data collection, structure solution, and ORTEP

6652 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Glossop et al.
plot for compound 26; and general procedure for computational
superposition of 38 and salmeterol. This material is available
free of charge via the Internet at http://pubs.acs.org.
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