Synthesis and structure-activity relationships of long-acting β2 adrenergic receptor agonists incorporating metabolic inactivation: An antedrug approach

Article abstract of DOI:10.1021/jm100326d

A series of saligenin β₂ adrenoceptor agonist antedrugs having high clearance were prepared by reacting a protected saligenin oxazolidinone with protected hydroxyethoxyalkoxyalkyl bromides, followed by removal of the hydroxy-protecting group, alkylation, and final deprotection. The compounds were screened for β₂, β₁, and β₃ agonist activity in CHO cells. The onset and duration of action in vitro of selected compounds were assessed on isolated superfused guinea pig trachea. Compound 13f had high potency, selectivity, fast onset, and long duration of action in vitro and was found to have long duration in vivo, low oral bioavailability in the rat, and to be rapidly metabolized. Crystalline salts of 13f (vilanterol) were identified that had suitable properties for inhaled administration. A proposed binding mode for 13f to the β₂-receptor is presented.

Full text of DOI:10.1021/jm100326d

4522 J. Med. Chem. 2010, 53, 4522–4530
DOI: 10.1021/jm100326d
Synthesis and Structure-Activity Relationships of Long-acting β2 Adrenergic Receptor Agonists
Incorporating Metabolic Inactivation: An Antedrug Approach
Panayiotis A. Procopiou,*,† Victoria J. Barrett,‡ Nicola J. Bevan,‡ Keith Biggadike,† Philip C. Box,† Peter R. Butchers,‡
Diane M. Coe,† Richard Conroy,† Amanda Emmons,‡ Alison J. Ford,‡ Duncan S. Holmes,† Helen Horsley,† Fern Kerr,†
Anne-Marie Li-Kwai-Cheung,† Brian E. Looker,† Inderjit S. Mann,^ Iain M. McLay,§ Valerie S. Morrison,‡
Peter J. Mutch, Claire E. Smith, and Paula Tomlin†
†Departments of Medicinal Chemistry, ‡Respiratory Biology, §Computational Structural Chemistry, and Drug Metabolism and
Pharmacokinetics, Respiratory CEDD, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY,
United Kingdom, and ^Synthetic Chemistry, GlaxoSmithKline, Old Powder Mills, Near Leigh, Tonbridge, Kent TN11 9AN, United Kingdom
Received March 11, 2010
A series of saligenin β2 adrenoceptor agonist antedrugs having high clearance were prepared by reacting
a protected saligenin oxazolidinone with protected hydroxyethoxyalkoxyalkyl bromides, followed by
removal of the hydroxy-protecting group, alkylation, and final deprotection. The compounds were
screened for β2, β1, and β3 agonist activity in CHO cells. The onset and duration of action in vitro of
selected compounds were assessed on isolated superfused guinea pig trachea. Compound 13f had high
potency, selectivity, fast onset, and long duration of action in vitro and was found to have long duration
in vivo, low oral bioavailability in the rat, and to be rapidly metabolized. Crystalline salts of 13f
(vilanterol) were identified that had suitable properties for inhaled administration. A proposed binding
mode for 13f to the β2-receptor is presented.
Introduction
a third generation once daily β2 adrenoceptor agonist to be used
as a part of a combination therapy for the treatment of asthma
and chronic obstructive pulmonary disease (COPDa). Novartis
has recently published data on their clinical candidate, indaca-
terol (4).7 A review by Glossop and Price summarizes the
progress up to 2006 made by other pharmaceutical companies
in identifying inhaled β2-agonists with extended duration of
action.8 More recently, the Pfizer group has published three
communications describing their studies in identifying long-
acting β2-adrenoceptor agonists such as 5-7.9-11 We have just
published our efforts in identifying the sulfonamide 8as our own
inhaled β2-agonist development candidate, which is suitable for
once-daily dosing.12 Finally the Boehringer-Ingelheim group has
published three communications on their benzo[1,4]oxazinone
alternative phenethanolamine.13-15 In this report, we outline
our studies in identifying our second development candidate.
In the search for a once-daily β2 agonist, candidates have
been sought showing reduced systemic exposure/improved
therapeutic index and/or an inherently longer duration of action
at the β2 adrenoceptor. Increasing the dose of a β2-agonist
would increase its duration of action but would also increase
unwanted cardiac side effects arising from excessive systemic
exposure. A major fraction of the dose (80-90%) of an inhaled
drug is swallowed and liable to be absorbed from the gastro-
intestinal tract.16 One approach to avoiding these consequences
is to investigate structural features which may have the potential
to minimize absorption, and this strategy was used in identify-
ing our first candidate.12 An alternative method to reducing
systemic exposure would be the “antedrug” approach. The
terms antedrug17 or “soft drug”18 were introduced to describe
drugs which act topically at the site of application but that are
transformed into inactive metabolites upon entry into the
systemic circulation. Our group has reported on glucocorticoid
Asthma is a common and chronic disease characterized by
an increase in inflammatory cell population in the epithelium
and submucosa of the airways.1 There are two major compo-
nents of asthma pathophysiology, airway inflammation and
smooth muscle dysfunction, leading to two major categories
of medicines used in asthma treatment: anti-inflammatories
and bronchodilators. Inhaled corticosteroids are the mainstay
anti-inflammatory treatment for asthma and β2-agonists are
the most effective bronchodilators, offering proven benefits in
reducing the burden of this disease.2,3 There are two classes of
β2-agonists: the short-acting (first generation) and the long-
acting (second generation) agonists. The short-acting ago-
nists, typified by salbutamol (1) (Chart 1), have a rapid onset
of action and relieve symptoms for 3-6 h. The two currently
prescribed inhaled long-acting β2-agonists are salmeterol (2)
and formoterol (3). Salmeterol xinafoate (1-hydroxy-2-
naphthoic acid) salt, a racemic mixture, marketed by Glaxo-
SmithKline, has lower intrinsic activity than salbutamol and
a delayed onset of action, but a duration of action of 12 h,
which is independent of dose.4,5 In contrast, formoterol
fumarate salt, marketed by Novartis is a single racemic
diastereoisomer (RR,SS), high intrinsic efficacy agonist with
onset of action similar to salbutamol, and a dose dependent
duration of action.5,6 In the last 10 years, there has been great
interest within the pharmaceutical industry in the discovery of
*To whom correspondence should be addressed. Phone: (þ44)1438
762883. Fax: (þ44)1438 768302. E-mail: pan.a.procopiou@gsk.com.
a Abbreviations: CHO, Chinese hamster ovary; cAMP, adenosine
30,50-monophosphate; COPD, chronic obstructive pulmonary disease;
DMA, dimethyl acetamide; MDCK MDR1, Madin-Darby canine
kidney cells (MDCK)-multidrug resistance 1 (MDR1).
r
pubs.acs.org/jmc
Published on Web 05/12/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4523
Chart 1. Clinically Used β2 Agonists and Recent Development Candidates
Chart 2. Hypothesis of Metabolic Inactivation of Salmeterol Analogues
antedrugs possessing a butyrolactone ring, which are ra-
pidly hydrolyzed by paraoxonase on entry into the systemic
circulation.19-22 Salmeterol is metabolized in humans by
benzylic hydroxylation, giving 9 (Chart 2).23,24 We there-
fore decided to investigate alternative β2 agonist structures
which might produce a less active or inactive metabolite in
addition to possessing the other requisite features of a long-
acting β2 agonist. The profile of the target molecule was a
potent, homochiral, and selective β2-agonist with intrinsic
activity similar to that of salmeterol, long duration of
action, with rapid metabolic inactivation, which might
offer safe once-daily bronchodilation in the clinic. Our
initial efforts involved replacement of the right-hand side
phenyl ring of β2 agonists with a γ-lactone moiety,25 which
would be expected to be rapidly hydrolyzed by paraoxonase
on entry into the systemic circulation. Such analogues were
found to have low efficacy. In a further approach, we inves-
tigated replacing a methylene group of the salmeterol chain
with an oxygen atom, as for example in compound 10.
Assuming that benzylic hydroxylation is still the main route
of metabolism, we postulated that the corresponding meta-
bolite would be an unstable hemiacetal (11), which may cleave
to the alcohol 12 and benzaldehyde (in the first instance).
Analogues such as12might belesspotent than10, whereasthe
smaller fragment would be expected to be either oxidized to
benzoic acid, or reduced to benzyl alcohol, both of which are
nontoxic and would be rapidly excreted.
The (R)-enantiomer of salmeterol is known to be the more
potent isomer, so from the outset it was decided to prepare
homochiral compounds with the (R)-configuration.4 The
optimal position of the ether oxygen in the chain of salmeterol
for both potency and duration is six carbon atoms from the

4524 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Procopiou et al.
Scheme 1a
a Reagents and conditions: (a) 1,6-dibromohexane (3 equiv), 50% aq NaOH (4.7 equiv), tetrabutylammonium bromide (0.02 equiv), 3 days, 63%;
(b) NaH, DMF, 1.5 h, 73-82%; (c) KOSiMe3 (4 equiv), THF, 80 ꢀC, 3 h, 76-98%; (d) AcOH-water (2:1), 70 ꢀC, 2 h, 36-95%; (e) H2, PtO2, EtOH,
EtOAc, 2 h, 99%; (f) NaOCN, AcOH, water, 20 ꢀC, 0.5 h, 38%; (g) MsCl, pyridine, 20 ꢀC, 2 h, 77%.
basic nitrogen,26 and confirmed by us with the related series of
sulfonamide salmeterol analogues.12 This part of the chain
was therefore retained for our investigations.
convergent route, where a late-stage common intermediate
was alkylated with a variety of substituted benzylic moieties,
followed by deprotection as outlined in Scheme 2. Thus tert-
butyl-dimethylsilyl monoprotected ethylene glycol was re-
acted with excess 1,6-dibromohexane under phase transfer
conditions, and the resulting bromide 19 was further reacted
with the anion of oxazolidinone 16 to provide 20. The silyl
protecting group was then removed using tetrabutylammo-
nium fluoride, giving the common intermediate alcohol 21. A
sample of 21 was deprotected first with KOTMS in THF and
then with aqueous AcOH to give the putative metabolite 22
[(R)-enantiomer of 12]. Generally, alcohol 21 was either alkyl-
ated with benzyl bromides 23f-h where available or it was
converted into the mesylate 24 and then reacted with sub-
stituted benzyl alcohols 25i-n (or protected derivatives there-
of), giving ethers 17. The latter were fully deprotected by the
two-step procedure as before, giving the target compounds
13a,d-n. The naphthyl derivative 29 was made in a similar
way from 26 (Scheme 2).
Chemistry
The first target compound to test the metabolic inactivation
hypothesis, but also for the retention of β2 agonism, was 13a
(theR-enantiomerof10). Thiswas prepared by first alkylating
2-benzyloxyethanol (14a) with excess 1,6-dibromohexane
under phase transfer conditions, followed by reaction of the
resulting bromide 15 with the homochiral (R)-oxazolidinone-
protected saligenin 1627 in the presence of sodium hydride to
give 17. Deprotection of oxazolidinone 17 using the mild
potassium trimethylsilanolate in THF conditions gave 18,27
which was further deprotected by aqueous acetic acid cleavage
of the acetonide moiety to provide 13a (Scheme 1).
The meta-nitro intermediate 17b was also prepared by this
approach. Thenitro group of17b was reduced using hydrogen
over PtO2, the resulting aniline 17c was then converted to the
primary urea 17d by treatment with acidic sodium cyanate, or
to the methanesulfonamide 17e by treatment with MsCl in
pyridine. Finally 17d,e were deprotected using KOTMS,
followed by aqueous AcOH to give 13d,e, respectively. In
principle, most analogues could have been prepared by this
linear approach, however, it was more efficient to use a
Intermediate (aminosulfonyl)benzyl alcohols 25k,l were
prepared from (aminosulfonyl)benzoic acid 30k,l respectively
by borane-THF reduction (Scheme 3). Alcohol 25k,lrequired
further protection as the bis-SEM protected sulfonamide in
order to block alkylation on the sulfonamide moiety. The
target sulfonamide derivatives 13k,l were obtained after alky-
lation with mesylate 24, cleavage of oxazolidinone ring with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4525
Scheme 2a
a Reagents and conditions: (a) NaH, DMF, 0.75 h; (b) TBDMS-Cl 0.75 h, 67%; (c) 1,6-dibromohexane (3 equiv), 50% aq NaOH (9.8 equiv),
tetrabutylammonium bromide (0.02 equiv), 7 days, 90%; (d) 16, NaH, DMF, 1.5 h, 61%; (e) TBAF-SiO2, (2 equiv), THF, 2 h, 87%; (f) KOSiMe3 (4
equiv), THF, 80 ꢀC, 3 h, 80-97%; (g) AcOH-water (2:1), 70 ꢀC, 0.5 h, 40-95%; (h) MsCl, iPr2NEt, DCM, 91%.
KOTMS, and final aqueous acetic acid deprotection, which
removed the acetonide and SEM groups, although a longer
heating time was required (Scheme 2). The commercially
available hydroxybenzyl alcohols 25m,n were selectively pro-
tected on the phenolic hydroxy group (Scheme 3) before they
were reacted with mesylate 24 and deprotected to give 13m,n.
Preferred compounds had IA > 0.37. The β1 adrenoceptors
are found in heart tissue, and their activation causes tachy-
cardia, whereas the β3 adrenoceptors are found in adipose
tissue and are thought to be involved in the regulation of lipid
metabolism. Required selectivity for β2 over β1 and β3 adre-
noceptors was set as better than that of (R,R)-formoterol. All
the compounds tested showed high affinity for the β2 receptor
(more potent than the standard, isoprenaline). It was impor-
tant to find from the outset of the project whether the
introduction of the second oxygen atom in the chain had a
deleterious effect on the potency against the β2 adrenoceptor.
The salmeterol analogue 13a was found to be less potent than
salmeterol (pEC50 at β2 8.6 vs 9.6), and this was also the case
with the sulfonamide pair 13l and 8 (pEC50 at β2 8.8 vs 9.8).
Despite the fact that there was a reduction by 10-fold in the
potency, it was explored whether further substitution in the
terminal phenyl ring might increase the potency and compen-
sate for the loss. Indeed, compounds 29 and 13e-h had the
highest β2 pEC50 values. The most potent and selective
analogues were 13f and 29, however, the latter was not as
Results and Discussion
Compounds in Table 1 were tested for their ability to cause
cyclic AMP accumulation in Chinese hamster ovary (CHO)
cells transfected with human β1, β2, or β3 adrenoceptors.
Agonist activity was assessed by measuring changes in intra-
cellular cyclic AMP, and the potency is reported in terms of
pEC50 (negative log10 molar concentration for half-maximal
response) ( SEM. The efficacy of the test compounds was
expressed as intrinsic activity (IA), which is defined as the
maximal response of the test compound, relative to the
maximum effect of the high intrinsic efficacy agonist isoprena-
line. By definition, isoprenaline’s intrinsic activity is 1. The IA
for formoterol was 0.97, whereas that of salmeterol was 0.37.

4526 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Procopiou et al.
selective for the β2 receptor as 13f. All test compounds in
Table 1, except 13h, had intrinsic activity higher than salme-
terol (IA > 0.37). Microsomal instability of the compounds
toward high first-pass metabolism in the liver was assessed by
incubating samples with human liver microsomes (P450 con-
tent = 125 pmol/mL) at a compound concentration of 5 μM
for 30 min at 37 ꢀC. Compound turnover was expressed as a
ratio relative to the assay standard, verapamil, where verapa-
mil has a turnover ratio of 1. Salmeterol, which had a slightly
higher turnover ratio (1.2), was also included as a standard.
The salmeterol analogue 13a had a turnover ratio of 1.3,
however, the analogue of 8, 13l, had a very low ratio and this is
probably a reflection of its reduced lipophilicity. All the
analogues prepared with lower clogP values showed lower
turnover ratios, for example, the sulfonamide 13k, reverse-
sulfonamide 13e, urea 13d, and the two phenols 13m and 13n.
Conversely, analogues with more lipophilic substituents were
metabolized to a greater extent, for example, the 2,6-dichloro-
(13f) and the naphthyl- (29) analogues. The putative metabo-
lite alcohol 22 devoid of an aryl terminus was approximately
1000-fold less potent than 13a at β2. The human liver micro-
somal incubation of the dichloro-analogue 13f was repeated
on a larger scale, and the β2 potency of the resulting mixture of
metabolites was found to be very low. The main metabolite
wasisolatedandconfirmedtobethe alcohol22. The metabolic
leaving group, 2,6-dichlorobenzoic acid or 2,6-dichlorobenzyl
Scheme 3a
a Reagents and conditions: (a) BH3-THF, 0-20 ꢀC, 73-74%;
(b) NaH (2.2 equiv), DMF, 20 ꢀC, 0.25 h, then SEM chloride (2 equiv),
20 ꢀC, 1 h, 38-61%; (c) NaH (1.1 equiv), DMF, 20 ꢀC, 0.15 h, then SEM
chloride (1 equiv), 20 ꢀC, 2 h, 36-70%.
Table 1. Stimulation of cAMP Accumulation in CHO Cells Expressing Human β2, β1 and β3 Adrenoceptors, Human Liver Microsomal Turnover Ratio
against Verapamil, and clogP
β2a pEC50 (n) IA (L95%conf-U95% conf)b β1a pEC50 (n) β2-β1 β3a pEC50 (n) β2-β3
Turnover ratioe clogP
c
d
compd
1
2
3
4
5
6
7
8
9
13a.AcOH
8.6 ( 0.1
(2)
0.44
0.41-0.47
0.52
7.5 ( 0.1
(4)
1.1
1.1
1.7
3.0
1.5
1.3
7.4 ( 0.1
(4)
1.2
2.3
2.5
3.3
1.3
1.1
1.3
0.3
0.2
1.65
2.3
1.0
0.5
3.2
3.5
3.4
2.8
2.9
0.0
0.0
1.6
1.8
3.5
-0.5
1.2
3.0
1.3
13d.AcOH
13e.AcOH
13f.AcOH
13g.AcOH
13h.AcOH
13i.AcOH
13j.AcOH
13k
8.8 ( 0.1
(4)
7.7 ( 0.1
(4)
6.5 ( 0.1
(4)
0.47-0.58
0.52
9.6 ( 0.5
(2)
7.9 ( 0.1
(4)
7.1 ( 0.0
(4)
0.52-0.52
0.69
9.4 ( 0.0
(8)
6.4 ( 0.1
(8)
6.1 ( 0.2
(8)
0.64-0.74
0.56
9.4 ( 0.1
(4)
7.9 ( 0.1
(4)
8.1 ( 0.0
(4)
ND
0.48-0.64
0.37
9.4 ( 0.1
(4)
8.1 ( 0.0
(4)
ND
8.3 ( 0.1
(4)
ND
1.1
1.0
1.0
0.0
0.2
0.4
0.3
1.9
0.30-0.45
0.42
8.6 ( 0.1
(2)
0.38-0.46
0.43
8.6 ( 0.0
(2)
ND
ND
0.40-0.46
0.60
8.7 ( 0.1
(2)
7.0 ( 0.1
(3)
1.7
1.5
6.4 ( 0.1
(3)
2.3
1.9
0.9
1.6
1.6
0.7
4.0
3.7
1.7
0.0
0.55-0.65
0.58
10 13l.AcOH
11 13m
8.8 ( 0.0
(2)
7.3 ( 0.0
(4)
6.9 ( 0.0
(4)
0.56-0.59
0.41
8.1 ( 0.1
(4)
7.1 ( 0.1
(4)
1.0
7.2 ( 0.1
(4)
0.33-0.49
0.66
12 13n.AcOH
12 29.AcOH
13 22.AcOH
14 8.cinnamate
9.1 ( 0.1
(2)
7.7 ( 0.1
(4)
1.4
7.5 ( 0.0
(4)
0.61-0.71
0.56
9.9 ( 0.0
(6)
7.9 ( 0.1
(8)
2.0
8.3 ( 0.1
(8)
0.48-0.63
0.62
5.6 ( 0.3
(7)
<4.6
(10)
>1.0
3.3
4.9 ( 0.3
(4)
0.52-0.73
0.54
9.8 ( 0.2
(4)
6.5 ( 0.1
(4)
5.8 ( 0.2
(4)
0.9
1.2
0.50-0.59
0.37
15
2
9.6 ( 0.0
6.1 ( 0.0
(656)
3.5
5.9 ( 0.0
(849)
(929)
9.3 ( 0.0
(791)
7.4 ( 0.0
(767)
0.36-0.38
0.97
16 (R,R)-3.fumarate
7.4 ( 0.0
(670)
1.9
7.6 ( 0.0
(653)
ND
0.97-0.98
1.00
17 isoprenaline
8.1 ( 0.0
(641)
-0.7
7.4 ( 0.0
(659)
0.15
a Human β1, β2, and β3 receptors expressed in CHO cells. pEC50 is the negative logarithm of the molar drug concentration that produces a cAMP
response equal to 50% of its maximal response. If n < 3, the range is quoted. b L95%conf = lower 95% confidence limit. U95%conf = upper 95%
confidencelimit. c Selectivity for β2 over β1 expressed as pEC50 at β2 receptor - pEC50 at β1. d Selectivityfor β2 over β3 expressedas pEC50 at β2 receptor -
pEC50 at β3. e Compound turnover in human liver microsomes expressed as a ratio ralative to verapamil, where verapamil has a turnover ratio of 1.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4527
Table 2. Onset and Duration of Action of Selected Compounds on
Electrically Stimulated Isolated Superfused Guinea Pig Trachea
(Minimum n = 2)
Table 3. Rat Pharmacokinetic Data for 13f Acetate in the Han Wistar
Rata
dose
Clp
Vdss
(mg/kg) (mL/min/kg) (L/kg) T1/2 (h) (ng/mL) F%
Cmax
entry
compd
onset time (min) shift (1 h) shift (3 h)
species
route
rat intravenous 0.25 123 4.4 0.8
oral
1
2
3
4
5
6
13a.AcOH
13f.AcOH
13 h.AcOH
29.AcOH
2
4.8
6.6
7.6
1.0
2.2
1.5
1.0
21
1.0
2
<2
<5
a Clp = plasma clearance; Vdss = volume of distribution at steady
state; F% = bioavailability.
6.9
2.4
4.2
1.1
10.2
27.6
10
(R,R)-3.fumarate
20
>1940
dataare presented inTable3. Highplasma clearance (123 mL/
min/kg), a moderate volume of distribution (4.4 L/kg) and a
short half-life (0.8 h) were obtained. Compound 13f was not
quantifiable in plasma at any time-point after oral dosing. A
maximum oral AUC was assumed to be the lower limit of
quantification (2 ng/mL), and the maximum oral bioavail-
ability calculated was 5%, which is therefore likely to be an
overestimate. This oral bioavailability was lower than that
previously reported for salmeterol (10-12% in rat)23 and
strengthened the confidence that the swallowed fraction of the
inhaled dose in humans would be unlikely to contribute to the
systemic exposure of 13f.
A variety of salts of 13f were prepared and examined
for crystallinity, and two such salts were identified, the
R-phenylcinnamate (mp 116-118 ꢀC) and triphenylacetate
(mp 132-134 ꢀC).30 The R-phenylcinnamic and triphenyl-
acetic acids are not precedented in marketed inhaled products
and therefore require further toxicological investigation. The
R-phenylcinnamate salt (solubility 0.190 mg/mL at pH 7.4)
and triphenylacetate salt (solubility 0.033 mg/mL at pH 7.4)
were screened for stability in a range of humidity and thermal
conditions, for lactose compatibility, for particle size after
micronization, and were found to be suitable for inhaled
development.
The acetate salt of 13f was also investigated in vivo in
histamine-induced bronchospasm in the guinea pig in a
plethysmograph chamber (Buxco) and found to have similar
potency to salmeterol (EC90 30 μM and 50 μM, respectively,
nebulizer concentration). At an equi-effective (EC90) dose, the
duration of action of 13f acetate (time to 50% recovery) was
similar to that of salmeterol (10 h) when administered as a
nebulized solution in a DMA/saline vehicle. Furthermore the
duration of 13f was extended to 17 h when the dose was
increased to 10-fold the EC90.31 This data suggests that 13f is a
long-acting β2 agonist in the guinea pig; prolonged broncho-
dilation over 24 h has now been demonstrated in asthmatic
patients, suggesting the potential for once daily administration.32
Finally, the binding of 13f to the receptor was examined by
docking in our model and compared with 8. This model was
built several years ago, when this research work was underway,
and well before the recent publication of the structure of the
human β2 receptor which was crystallized in a lipid environ-
ment when bound to the inverse agonist carazolol.33-35 The
model was superimposed and compared with the recent crystal
structure for the β2 receptor with carazolol bound (PDB ID
2rh1), and all the proposed interacting residues were similarly
placed in the model and the crystal structure. The following
interactions were clearly observed in the docking and were
identical with those reported for 8: Ser-207 was found to
H-bond to the phenolic hydroxyl, while Ser-204 and Ser-203
were found to interact with the primary benzylic hydroxyl. A
potential H-bonding interaction was identified between Asn-
293 and the asymmetric benzylic hydroxyl, while Asp-113 and
the amine were in proximity, allowing for a strong charge
interaction. The middle ether oxygen in the chain was seen to
alcohol, are not reported to have any toxicological issues
associated with them. The work describing the metabolite
identification will be published elsewhere.28
The pharmacology of salmeterol assessed on isolated super-
fused guinea pig trachea strips correlates well with clinical
data and gives a measurement of potency, efficacy, onset time,
and duration of action.29 Selected test compounds were there-
fore investigated for their ability to inhibit the contraction of
electrically stimulated guinea pig trachea strips expressed as a
measure of the functional response at the β2 adrenoceptor.
The onset time and duration of action in vitro of selected
compounds were assessed on guinea pig isolated superfused
trachea strips. Tissues were contracted electrically, different
concentrations of agonist were perfused over the tissue until
maximum relaxation was achieved, and onset of action was
determined. Perfusion of the agonist was then ceased, tissue
continued to be perfused with buffer and duration of action
determined by the time taken for the contractile response to
re-establish.
In Table 2, the onset time and duration after 1 and 3 h is
presented. Onset of action was calculated as the time taken for
an EC50 concentration to achieve 50% maximum relaxant
effect. Duration of action was determined by measuring the
recovery of electrically induced contraction following wash-
out of agonist. This was expressed as the rightward shift in the
agonist concentration-effect curve following 60 and 180 min
of washout (EC50 for test compound after 60 or 180 min of
washout/EC50 at equilibrium, time 0 min). With this analysis,
the greater the shift values the greater the recovery. Shift
values of 1 (1 h) and 1 (3 h) indicate no washout (a salmeterol-
like profile). Shift values of about 20 (1 h) and >300 (3 h)
indicate slow continuous washout (formoterol-like profile).
Shift values of infinity indicate rapid and complete washout
(isoprenaline-like profile). All the test compounds in Table 2
exhibited onset times faster than salmeterol. Two lipophilic
analogues, the 2,6-dichloro- (13f), and the 3,5-dimethyl-
analogue (13h) indicated very little washout and thus would
be expected to be long-acting bronchodilators.
On the basis of the data in Tables 1 and 2, the 2,6-dichloro
analogue 13f, which had high potency, fast onset, and long
duration of action, and selectivity for β2 over β1 and β3
receptors, was progressed to pharmacokinetic studies. The
passive membrane permeability (Pexact) of 13f across
MDCKII-MDR1 cells in the presence of a potent P-glyco-
protein inhibitor was measured as 34 nm s-1, whereas salme-
terolxinafoatewas 119 nm s-1. Theinvitro metabolicstability
of 13f was measured as a ratio vs verapamil in human
microsomes and was found to have higher turnover than
salmeterol, which would suggest that any absorbed fraction
of the dose would be rapidly metabolized. Pharmacokinetic
studies in vivo with 13f were initiated and compared with
salmeterol xinafoate. The acetate salt of 13f was dosed as a
solution orally and intravenously to Han Wistar rats, and the

4528 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Procopiou et al.
expressed in ppm relative to tetramethylsilane. High resolution
positive ion mass spectra were acquired on a Micromass Q-Tof 2
hybrid quadrupole time-of-flight mass spectrometer. The ele-
mental composition was calculated using MassLynx v4.0 for
the [M þ H]þ. All separations for HRMS were achieved using
a Phenomenex Luna C18(2) reversed phase column (150 mm ꢀ
2.1 mm, 3 μm particle size). Gradient elution was carried out with
the mobile phases as (A) water containing 0.1% (v/v) formic acid
and (B) MeCN containing0.1% (v/v) formicacid. Theconditions
for the gradient elution were initially 0% B for 2 min, increasing
linearly to 100% B over 25 min, remaining at 100% B for 2 min
then decreasing linearly to 0% B over 0.5 min, followed by an
equilibration period of 2.5 min prior to the next injection. The
flow rate was 0.4 mL/min, temperature controlled at 25 ꢀC with
an injection volume of 5 μL. All compounds screened in the
biological assays were either acetate salts or free-bases, which
were gums (or glasses). Their purity was examined by LCMS
analysis and was found to be g95%, unless otherwise specified.
The purity of crystalline salts was additionally assessed by
elemental microanalysis.
General Procedure for the Preparation of 17 from 24 and
Benzyl Alcohols 25. Benzyl alcohol 25 (0.38 mmol) was stirred
with sodium hydride (60% oil dispersion; 17 mg, 0.43 mmol)
in DMF (1 mL) under nitrogen for 10 min at 20 ꢀC and then 24
(120 mg, 0.25 mmol) in DMF (0.5 mL) was added. The mixture
was stirred at 20 ꢀC for 3 days and then phosphate buffer (pH 6.5,
15 mL) was added, followed by EtOAc (20 mL). The organic
phase was washed with water (2 ꢀ 20 mL), dried, and concen-
tratedunderreducedpressure. Theresidue waspurifiedbycolumn
chromatography on silica eluting with EtOAc-cyclohexane (1:1)
to give 17:
Figure 1. Postulated interactions of 13f with the β2 receptor, figure
generated by PyMOL (The PyMOL Molecular Graphics System,
Version 1.2b2, Schrodinger, LLC).
be positioned within H-bonding range of Asn-318 (TM7)
while the benzylic ether oxygen was seen to be ideally situated
to form H-bonds to both Ser-120 (TM3) and Asn-322 (TM7)
(Figure 1). The latter two interactions are similar to those seen
for the meta-sulfonamide group of 8.
Conclusion
A series of novel β2 agonist antedrugs were synthesized.
Incorporation of an oxygen atom at the homobenzylic posi-
tion of the right-hand side phenyl ring of (R)-salmeterol has
given a series of potent human β2 adrenoceptor agonists that
were rapidly metabolized by human liver microsomes. The
2,6-dichlorobenzyl analogue 13f fulfilled all the criteria of
potency, selectivity, rapid onset of action on guinea pig
trachea, long duration of action, rapid turnover, and in vivo
efficacy with a long duration that increased with increasing
dose. TheR-phenylcinnamate and triphenylacetatesalts of 13f
were found to have suitable properties for inhaled adminis-
tration and were therefore selected as candidates for further
development. Further preclinical pharmacokinetic and meta-
bolite identification28 and additional pharmacological31 data
for 13f will be reported elsewhere. Compound 13f.triphenyl-
acetate (vilanterol trifenatate) is currently undergoing clinical
trials for the treatment of COPD and asthma, as a combina-
tion with fluticasone furoate, and the clinical data from these
studies will be reported in due course.
(5R)-3-{6-[(2-{[(2,6-Dichlorophenyl)methyl]oxy}ethyl)oxy]-
hexyl}-5-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)-1,3-oxazoli-
din-2-one (17f). MS ES þve m/z 552/554 (M þ H)þ. 1H NMR δ
(CDCl3) 7.31 (2H, d, J 8 Hz), 7.20-7.14 (1H, m), 7.12 (1H, dd,
J 8, 2 Hz), 7.00 (1H, d, J 2 Hz), 6.83 (1H, d, J 8 Hz), 5.39 (1H, t,
J 8 Hz), 4.84 (2H, s), 4.82 (2H, s), 3.85 (1H, t, J 8 Hz),
3.72-3.66 (2H, m), 3.63-3.57 (2H, m), 3.46 (2H, t, J 6 Hz),
3.42-3.20 (3H, m), 1.62-1.48 (10H, m), 1.43-1.27 (4H, m).
General Two-Step Procedure for the Deprotection of 17. Step 1.
Cleavage of Oxazolidinone Ring with KOTMS. A solution of 17
(0.68 mmol) in THF (20 mL) was treated with potassium
trimethylsilanolate (2.7 mmol), and the mixture was stirred at
80 ꢀC under nitrogen for 3 h. The mixture was partitioned
between EtOAc and pH 6.4 phosphate buffer, and the aqueous
layer was extracted with more EtOAc. The combined organic
layers were dried and evaporated under reduced pressure to give
the expected product.
(1R)-2-({6-[(2-{[(2,6-Dichlorophenyl)methyl]oxy}ethyl)oxy]-
hexyl}amino)-1-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)ethanol (18f).
1
MS ES þve m/z 526/528 (M þ H)þ. H NMR δ (CDCl3) 7.30
(2H, d, J 8 Hz), 7.20-7.14 (1H, m), 7.12 (1H, dd, J 8, 2 Hz), 7.01
(1H, d, J 2 Hz), 6.78 (1H, d, J 8 Hz), 4.83 (2H, s), 4.82 (2H, s),
4.67-4.61 (1H, m), 3.72-3.67 (2H, m), 3.63-3.58 (2H, m), 3.46
(2H, t, J 6 Hz), 2.87 (1H, dd, J 12, 3 Hz), 2.74-2.58 (3H, m), 1.78
(2H, br), 1.60-1.46 (10H, m), 1.37-1.28 (4H, m).
Experimental Section
Organic solutions were dried over anhydrous Na2SO4. TLC
was performed on Merck 0.25 mm Kieselgel 60 F254 plates.
Products were visualized under UV light and/or by staining with
aqueous KMnO4 solution. LCMS analysis was conducted on a
Supelcosil LCABZþPLUS column (33 mm ꢀ4.6 mm) eluting
with 0.1% formic acid and 0.01 M ammonium acetate in water
(solvent A) and 0.05% formic acid and 5% water in acetonitrile
(solvent B), using the following elution gradient 0-0.7 min 0% B,
0.7-4.2 min 100% B, 4.2-5.3 min 0% B, 5.3-5.5 min 0% B at a
flow rate of 3 mL/min. The mass spectra were recorded on a
Fisons VG Platform spectrometer using electrospray positive and
negative mode (ES þve and ES -ve). Column chromatography
was performed on Merck Kieselgel 60 (art. 9385), or Biotage
prepacked silica gel cartridges containing KP-Sil run on a flash
Step 2. Acidic Hydrolysis of Acetonide (and Concurrent Re-
moval of Any Additional Acid-Labile Group). The crude product
from step 1 above (0.66 mmol) in acetic acid (12 mL) and water
(6 mL) was heated at 70 ꢀC for 2 h. The mixture was cooled to
20 ꢀC, and the solvent was removed under reduced pressure. The
residue was re-evaporated with MeOH (2 ꢀ 20 mL) to give 13.
2-(Hydroxymethyl)-4-((1R)-1-hydroxy-2-{[6-({2-[(phenylmethyl)-
oxy]ethyl}oxy)hexyl]amino}ethyl)phenol (13a) acetate salt. MS
1
ES þve m/z 418 (M þ H)þ. H NMR δ (CD3OD) 7.44-7.28
(6H, m), 7.23 (1H, dd, J 8, 2 Hz), 6.85 (1H, d, J 8 Hz), 4.73 (2H, s),
4.61 (2H, s), 3.72-3.64 (4H, m), 3.56 (2H, t, J 6 Hz), 3.21-3.03
(4H, m), 1.99 (3H, s), 1.84-1.73 (2H, m), 1.72-1.63 (2H, m),
1.56-1.44 (4H, m). HRMS found: 418.2586 C24H36NO5 requires
418.2588.
1
12i chromatography module. H NMR spectra were recorded
at 400 MHz unless otherwise stated. The chemical shifts are

Article
N-(3-{[(2-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)hexyl]oxy}ethyl)oxy]methyl}phenyl)urea (13d)
Acetate salt. MS ES þve m/z 476 (M þ H)þ. 1H NMR δ (CD3OD)
7.44 (1H, br s), 7.34 (1H, br s), 7.26-7.20 (2H, m), 7.16 (1H, dd,
J 8, 2 Hz), 7.00 (1H, br s), 6.78 (1H, d, J 8 Hz), 4.65 (2H, s), 4.51
(2H, s), 3.66-3.57 (4H, m), 3.49 (2H, t, J 6 Hz), 3.11-3.04 (2H,
m), 3.04-2.95(2H, m), 1.93(3H, s), 1.74-1.65 (2H, m), 1.64-1.56
(2H, m), 1.48-1.36 (4H, m).
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4529
J 9, 4 Hz), 4.65 (2H, s), 4.62 (2H, s), 3.75-3.69 (2H, m), 3.68-3.63
(2H, m), 3.52 (2H, t, J 6 Hz), 3.13-2.96 (4H, m), 1.92 (3H, s),
1.77-1.65 (2H, m), 1.65-1.58 (2H, m), 1.51-1.37 (4H, m).
4-[(1R)-2-({6-[(2-{[(3,5-Dimethylphenyl)methyl]oxy}ethyl)oxy]-
hexyl}-amino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol Acetate
Salt (13h). MS ES þve m/z 446 (M þ H)þ. 1H NMR δ
(CD3OD) 7.34 (1H, d, J 2 Hz) 7.15 (1H, dd, J 8, 2 Hz), 6.94
(2H, s), 6.91 (1H, s), 6.79 (1H, d, J 8 Hz), 4.85 (1H, dd, J 9, 4 Hz),
4.65 (2H, s), 4.45 (2H, s), 3.59 (4H, s), 3.48 (2H, t, J 6 Hz),
3.12-2.94 (4H, m), 2.28 (6H, s), 1.91 (3H, s), 1.76-1.65 (2H, m),
1.65-1.56 (2H, m), 1.50-1.36 (4H, m). HRMS, found: 446.2901
C26H40Cl2NO5 requires 446.2901.
2-(Hydroxymethyl)-4-[(1R)-1-hydroxy-2-({6-[(2-{[(4-methyl-
phenyl)methyl]oxy}ethyl)oxy]hexyl}amino)ethyl]phenol Acetate
Salt (13i). MS ES þve m/z 432 (M þ H)þ. 1H NMR δ (CD3OD)
7.34 (1H, d, J 2 Hz), 7.22 (2H, d, J 8 Hz), 7.18-7.11 (3H, m), 6.78
(1H, d, J 8 Hz), 4.86 (1H, dd, J 9, 4 Hz), 4.65 (2H, s) 4.49 (2H, s),
3.59 (4H, s), 3.48 (2H, t, J 6 Hz), 3.13-2.96 (4H, m), 2.31 (3H, s),
1.91 (3H, s), 1.76-1.65 (2H, m), 1.65-1.55 (2H, m), 1.47-1.36
(4H, m). HRMS found: 432.2743 C25H38Cl2NO5 requires
432.2744.
N-(3-{[(2-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]-ethyl}amino)hexyl]oxy}ethyl)oxy]methyl}phenyl)methane-
sulfonamide Acetate Salt (13e). MS ES þve m/z 511 (M þ H)þ. 1H
NMR δ(CD3OD) 7.49-7.41 (3H, m),7.32-7.23 (3H, m), 6.92 (1H,
d, J 8 Hz), 4.79 (2H, s), 4.67 (2H, s), 3.80-3.72 (4H, m), 3.64 (2H, t,
J 6 Hz), 3.29-3.20 (2H, m), 3.20-3.11 (2H, m), 3.08 (3H, s), 2.08
(3H, s), 1.89-1.79 (2H, m), 1.79-1.70 (2H, m), 1.61-1.52 (4H, m).
HRMS found: 511.24708 C25H39N2O7S requires 511.24725.
4-[(1R)-2-({6-[(2-{[(2,6-Dichlorophenyl)methyl]oxy}ethyl)oxy]-
hexyl}-amino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol (13f)
1
Acetate Salt. MS ES þve m/z 486/488 (M þ H)þ. H NMR δ
(CD3OD) 7.42-7.25 (4H, m), 7.16 (1H, dd, J 8, 2 Hz), 6.78 (1H,
d, J 8 Hz), 4.82(2H, s) 4.65 (2H, s), 3.72-3.64 (2H, m), 3.63-3.54
(2H, m), 3.48 (2H, t, J 6 Hz), 3.14-2.95 (4H, m), 1.93 (3H, s),
1.77-1.64 (2H, m), 1.64-1.52 (2H, m), 1.47-1.34 (4H, m).
HRMS found: 486.1806 C24H34Cl2NO5 requires 486.1809.
4-[(1R)-2-({6-[(2-{[(2,6-Dichlorophenyl)methyl]oxy}ethyl)oxy]-
hexyl}-amino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol (13f)
Triphenylacetate Salt. Triphenylacetic acid (1.81 g, 6.28 mmol)
was added to a solution of 4-((R)-2-{6-[2-(2,6-dichlorobenzyl-
oxy)-ethoxy]-hexylamino}-1-hydroxyethyl)-2-hydroxymethyl-
phenol (95% pure; 3.28 g, 6.41 mmol) in EtOH (20 mL), and the
mixture heated to 80 ꢀC to obtain a solution. The mixture was
allowedtocooltoambienttemperature, andthe resulting product
filtered, washed with a little ethanol, then dried in vacuo at 50 ꢀC
to afford 13f-triphenylacetate salt (4.3 g, 88%) as a white crystal-
line solid: mp (DSC) 131.9-134.2 ꢀC, [R]D 20 -14.6 (c 1.034 in
4-[(1R)-2-({6-[(2-{[(4-Chlorophenyl)methyl]oxy}ethyl)oxy]-
hexyl}amino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol Acetate
Salt (13j). MS ES þve m/z 452/454 (M þ H)þ. 1H NMR δ
(CD3OD) 7.36-7.28 (5H, m), 7.16 (1H, dd, J 8, 2 Hz), 6.78 (1H,
d, J 8 Hz), 4.82 (obscured by CD3OH), 4.65 (2H, s), 4.52 (2H, s),
3.65-3.56 (4H, m), 3.49 (2H, t, J 6 Hz), 3.14-2.95 (4H, m), 1.92
(3H, s), 1.76-1.65 (2H, m), 1.64-1.55 (2H, m), 1.46-1.35 (4H,
m). HRMS found: 452.2198 C24H35ClNO5 requires 452.2198.
4-{[(2-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]-ethyl}amino)hexyl]oxy}ethyl)oxy]methyl}benzenesulfon-
amide (13k). MS ES þve m/z497 (M þ H)þ. 1HNMRδ(CD3OD)
7.90 (2H, d, J 8 Hz), 7.56 (2H, d, J 8 Hz), 7.33 (1H, d, J 2 Hz), 7.15
(1H, dd, J 8, 2 Hz), 6.79 (1H, d, J 8 Hz), 4.78-4.73 (2H, m), 4.68
(2H, s), 4.67 (2H, s), 3.72-3.64 (4H, m), 3.52 (2H, t, J 6 Hz), 2.91-
2.78 (2H, m), 2.76-2.65 (2H, m), 1.67-1.53 (4H, m), 1.48-1.34
(4H, m). HRMS found: 495.2154 C24H35N2O7S requires 495.2165.
3-{[(2-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]-ethyl}amino)hexyl]oxy}ethyl)oxy]methyl}benzenesulfon-
amide acetate salt (13l). MS ES þve m/z 497 (M þ H)þ. 1H NMR
δ (CD3OD) 7.93 (1H, s), 7.81 (1H, br d, J 8 Hz), 7.58-7.48 (2H,
m), 7.36 (1H, d, J 2 Hz), 7.16 (1H, dd, J 8, 2 Hz), 6.79 (1H, d,
J 8 Hz), 4.85 (1H, dd, J 9, 4 Hz), 4.65 (2H, s), 4.63 (2H, s),
3.70-3.61 (4H, m), 3.51 (2H, t, J 6 Hz), 3.15-2.98 (4H, m), 1.94
(3H, s), 1.76-1.66 (2H, m), 1.65-1.57 (2H, m), 1.48-1.37 (4H, m).
4-[(1R)-1-Hydroxy-2-({6-[(2-{[(4-hydroxyphenyl)methyl]oxy}-
ethyl)oxy]hexyl}amino)ethyl]-2-(hydroxymethyl)phenol (13m).
1
MeOH). MS ES þve m/z 289, 486/488 (M þ H)þ. H NMR δ
(500 MHz, CD3OD) 7.47 (2H, m), 7.38 (8H, m), 7.28 (6H, tt,
J 7.1, 1.8 Hz), 7.22 (4H, m), 6.86 (1H, d, J 7.9 Hz), 4.94 (1H, dd,
J 9.7, 4.6 Hz), 4.91 (2H, s), 4.74 (2H, s), 3.79 (2H, m), 3.69 (2H, m),
3.56 (2H, t, J6.1 Hz), 3.10 (2H, m), 2.99 (2H, m), 1.72 (2H, m), 1.65
(2H, m), 1.45 (4H, m). 13C NMR δ (125 MHz, CD3OD) 180.1,
156.2, 147.7, 140.3, 137.9, 134.5, 133.0, 131.9, 131.6, 129.6, 128.9,
128.1, 127.1, 127.0, 126.7, 116.0, 72.1, 71.4, 71.3, 71.1, 70.1, 68.4,
60.9, 55.4, 48.9, 30.5, 27.4, 27.1, 26.8. Anal. found: C, H, N, Cl.
4-[(1R)-2-({6-[(2-{[(2,6-Dichlorophenyl)methyl]oxy}ethyl)oxy]-
hexyl}-amino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol (13f)
R-Phenylcinnamate Salt. R-Phenylcinnamic acid (249 mg, 1.1
mmol) was added to a solution of 4-((R)-2-{6-[2-(2,6-dichloro-
benzyloxy)-ethoxy]-hexylamino}-1-hydroxyethyl)-2-hydroxy-
methyl-phenol (540 mg, 1.1 mmol) in isopropyl alcohol (5 mL).
The solution was seeded with product and allowed to stir at
ambient temperature for 20 h. The product was filtered, washed
with a little isopropyl alcohol, then dried in vacuo at 50 ꢀC to
afford 13f-R-phenylcinnamate salt (0.56 g, 72%) as a crystalline
white solid: mp (DSC) 116.1-117.9 ꢀC, [R]D 20 -15.8 (c 1.172 in
1
MS ES þve m/z 434 (M þ H)þ. H NMR δ (CD3OD) 7.30
(1H, d, J 2 Hz), 7.18 (2H, d, J 8 Hz), 7.12 (1H, dd, J 8, 2 Hz),
6.79-6.73 (3H, m), 4.71 (1H, dd, J 9, 4 Hz), 4.66 (2H, s), 4.45
(2H, s), 3.59 (4H, s), 3.47 (2H, t, J 6 Hz), 2.85-2.59 (4H, m),
1.63-1.50 (4H, m), 1.43-1.32 (4H, m).
4-[(1R)-1-Hydroxy-2-({6-[(2-{[(3-hydroxyphenyl)methyl]oxy}-
ethyl)oxy]-hexyl}amino)ethyl]-2-(hydroxymethyl)phenol Acetate
Salt (13n). MS ES þve m/z 434 (M þ H)þ. 1H NMR δ (CD3OD)
7.34 (1H, d, J 2 Hz), 7.18-7.10 (2H, m), 6.81-6.76 (3H, m), 6.69
(1H,d,J8 Hz), 4.85 (1H, dd, J9,4Hz),4.65(2H,s),4.47(2H,s),3.60
(4H, s), 3.49 (2H, t, J 6 Hz), 3.14-2.97 (4H, m), 1.94 (3H, s),
1.76-1.65 (2H, m), 1.64-1.56 (2H, m), 1.49-1.37 (4H, m). HRMS
found: 434.2536 C24H36NO6 requires 434.2537.
2-(Hydroxymethyl)-4-((1R)-1-hydroxy-2-{[6-({2-[(1-naphthalenyl-
methyl)oxy]ethyl}oxy)hexyl]amino}ethyl)phenolAcetateSalt(29).
MSES þve m/z 468 (M þ H)þ. 1H NMRδ(CD3OD) 8.14 (1H, br
d, J 8 Hz), 7.86 (1H, dd, J 8, 2 Hz), 7.81 (1H, d, J 8 Hz), 7.54-7.45
(3H, m), 7.44-7.39 (1H, m), 7.33 (1H, d, J 2 Hz), 7.14 (1H, dd,
J 8, 2 Hz), 6.78 (1H, d, J 8 Hz), 4.99 (2H, s), 4.83 (1H, dd, J 8,
5 Hz), 4.65 (2H, s), 3.73-3.69 (2H, m), 3.63-3.60 (2H, m),
3.47 (2H, t, J 6 Hz), 3.14-2.89 (4H, m), 1.92 (3H, s), 1.69-1.62
(2H, m), 1.62-1.53 (2H, m), 1.43-1.35 (4H, m). HRMS found:
468.2744 C28H38NO5 requires 468.2745.
1
MeOH). MS ES þve m/z 225, 486/488 (M þ H)þ. H NMR δ
(300 MHz, CD3OD) 7.62 (1H, s), 7.48 (2H, m), 7.40 (5H, m), 7.26
(3H, m), 7.18 (3H, m), 7.08 (2H, m), 6.87 (1H, d, J 8.3 Hz), 4.94
(1H, dd, J 8.5, 5.7 Hz), 4.91 (2H, s), 4.74 (2H, s), 3.79 (2H, m),
3.69 (2H, m), 3.57 (2H, t, J 6.4 Hz), 3.14 (2H, m), 3.05 (2H, m),
1.77 (2H, m), 1.67 (2H, m), 1.48 (4H, m). 13C NMR δ (125 MHz,
CD3OD) 176.1, 156.2, 141.4, 140.3, 137.9, 137.6, 135.8, 134.5,
133.0, 131.6, 131.0, 130.9, 129.6, 129.3, 128.9, 128.6, 127.9, 127.0,
127.0, 72.0, 71.3, 71.1, 70.2, 68.4, 60.9, 55.4, 48.9, 30.4, 27.4, 27.1,
26.8. Anal. C, H, N, Cl.
4-[(1R)-2-({6-[(2-{[(2,5-Dichlorophenyl)methyl]oxy}ethyl)oxy]-
hexyl}-amino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol Acetate
1
Salt (13g). MS ES þve m/z 486/488 (M þ H)þ. H NMR δ
(CD3OD) 7.57 (1H, d, J 2 Hz), 7.38-7.32 (2H, m), 7.27 (1H, dd,
J8,2Hz),7.15(1H,dd,J8, 2 Hz), 6.78 (1H, d, J8Hz),4.85(1H,dd,

4530 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Procopiou et al.
4-[(1R)-1-Hydroxy-2-({6-[(2-hydroxyethyl)oxy]hexyl}amino)-
ethyl]-2-(hydroxymethyl)phenol (22) Acetate Salt. MS ES þve
m/z 328 (M þ H)þ. 1H NMR δ (CD3OD) 7.35 (1H, d, J 2 Hz),
7.25-7.07 (5H, m), 6.79 (1H, d, J 8 Hz), 4.65 (2H, s), 3.66 (2H,
t, J 5 Hz), 3.53-3.46 (4H, m), 3.15-2.98 (4H, m), 1.93 (3H, s),
1.78-1.66 (2H, m), 1.66-1.57 (2H, m), 1.49-1.36 (4H, m).
(14) Hoenke, C.; Bouyssou, T.; Tautermann, C. S.; Rudolf, K.;
Schnapp, A.; Konetzki, I. Use of 5-hydroxy-4H-benzo[1,4]oxazin-
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Acknowledgment. The authors would like to thank Dr.
Brian E. Looker, who has recently retired from GlaxoSmith-
Kline after 39 years service, for his great contribution to the
company and to this project in particular. We thank Bill J.
Leavens of Analytical Chemistry for collecting the HRMS data,
Dr. Steve M. Evans, Sara Hughes, Isobel Hackney, and Kevin J.
Norton of Respiratory Biology for technical assistance, and Dr.
Pamela J. Thomas of Computational Chemistry for checking
the modelling section and providing the picture of Figure 1.
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Supporting Information Available: Preparative details and
spectroscopic data for 14b, 15a, 15b, 17a-e, 19, 17h, 20, 21, 24,
17, 17g, 25k, 25l, 25k-bisSEM, 25l-bisSEM, 25m-SEM, 25n-
SEM, 17i, 17j, 17k-bisSEM, 17l-bisSEM, 17m-SEM, 17n-SEM,
27, 18a, 18d, 18e, 18g, 18h, 18i, 18j, 18k-bisSEM, 18l-bisSEM,
18m-SEM, 18n-SEM, 28, biological screens, pharmacokinetic
studies, cell permeability, table of LCMS purity, and retention
times and microanalytical data on 13f.triphenylacetate and 13f.
r-phenylcinnamate. This material is available free of charge via
the Internet at http://pubs.acs.org.
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Hancock, A. P.; Harker, A. J.; Irving, W. R.; Ioannou, C.;
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