The discovery of long-acting saligenin β2 adrenergic receptor agonists incorporating a urea group

Article abstract of DOI:10.1016/j.bmc.2011.08.043

A series of novel, potent and selective human β₂ adrenoceptor agonists incorporating a urea moiety on the terminal right-hand side phenyl ring of (R)-salmeterol is presented. Urea 9j had long duration of action in vitro on guinea pig trachea, and also in vivo similar to that of salmeterol. It had lower oral absorption and bioavailability than salmeterol in both rat and dog. It had a turnover ratio similar to salmeterol, with no evidence for formation of any aniline metabolites in human liver microsomes and hepatocytes. However no crystalline salts suitable for inhaled delivery were identified.

Full text of DOI:10.1016/j.bmc.2011.08.043

Bioorganic & Medicinal Chemistry 19 (2011) 6026–6032
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The discovery of long-acting saligenin b2 adrenergic receptor agonists
incorporating a urea group
Panayiotis A. Procopiou a, , Victoria J. Barrett b, Alison J. Ford b, Brian E. Looker a, Gillian E. Lunniss a,
⇑
Deborah Needham a, Claire E. Smith c, Graham Somers c
a Department of Medicinal Chemistry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
b Department of Respiratory Biology, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
c Department of Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2011
Revised 19 August 2011
Accepted 19 August 2011
Available online 26 August 2011
A series of novel, potent and selective human b2 adrenoceptor agonists incorporating a urea moiety on
the terminal right-hand side phenyl ring of (R)-salmeterol is presented. Urea 9j had long duration of
action in vitro on guinea pig trachea, and also in vivo similar to that of salmeterol. It had lower oral
absorption and bioavailability than salmeterol in both rat and dog. It had a turnover ratio similar to
salmeterol, with no evidence for formation of any aniline metabolites in human liver microsomes and
hepatocytes. However no crystalline salts suitable for inhaled delivery were identified.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Asthma
COPD
b2-Agonist
Saligenin
Urea
1. Introduction
the last 10 years there has been great interest within the pharma-
ceutical industry in the discovery of a once-daily b2 adrenoceptor
Asthma is a chronic disease affecting 300 million people world-
wide, characterised by an increase in inflammatory cell population
in the epithelium and submucosa of the airways.1 There are two
main components of asthma pathophysiology, airway inflamma-
tion and smooth muscle dysfunction leading to two major catego-
ries of medicines used in asthma treatment: anti-inflammatory
drugs and bronchodilators. Inhaled corticosteroids are used to treat
the inflammatory component of asthma, whereas inhaled b2-
agonists are the most effective bronchodilators, offering proven
benefits in reducing the burden of this disease.2,3 Chronic obstruc-
tive pulmonary disease (COPD) is the fourth leading cause of death
and is projected to rise to third place by 2020. Bronchodilators such
as inhaled b2-agonists and muscarinic antagonists are currently the
mainstay of treatment for COPD and combinations with inhaled
corticosteroids are known to reduce the incidence of exacerba-
tions. The two twice-daily prescribed inhaled b2-agonists for the
treatment of asthma are salmeterol (1) and formoterol (2) (Chart 1).
Salmeterol has intrinsic activity of 0.37 versus isoprenaline, a de-
layed onset of action, and a dose-independent duration of action,4,5
whereas formoterol is a high intrinsic activity agonist (0.97) with a
short onset of action, and a dose-dependent duration of action.5,6 In
agonist to be used in new combination therapies for the treatment
of asthma and COPD. There are currently at least five candidates in
clinical development, the two more advanced candidates are Nov-
artis’ indacaterol (3),7 which has already been approved for use in
COPD in some markets, and GlaxoSmithKline’s vilanterol (4),8
which is in late phase 3 clinical trials. The remaining three devel-
opment candidates are Boehringer-Ingelheim’s olodaterol (5),9
Pfizer’s candidate PF-610355 (6),10 and Chiesi’s carmoterol (7).11
Our group published three papers, one on sulfonamides including
GlaxoSmithKline’s first candidate 8,12 on our clinical candidate
vilanterol (4),8 and on hydantoins.13
A major fraction of the dose (up to 90%) of an inhaled drug is
swallowed and liable to be absorbed from the gastro-intestinal
tract.14 Thus, one approach to improve the therapeutic index could
be to alter the physicochemical properties of the drug and make it
less prone to absorption. Our group has published two papers
using this approach, one on sulfonamides including our first candi-
date 8,12 and on hydantoins.13 A second approach (the antedrug
approach) could be to introduce metabolic instability to the mole-
cule to facilitate its conversion into inactive metabolites following
systemic absorption from either the GI tract or the lung. Better still,
a combination of the two approaches may potentially deal with
both the inhaled and swallowed fractions of each dose.8 In this
report we present our studies in identifying urea b2 adrenoceptor
⇑
Corresponding author. Tel.: +44 1438 762883; fax: +44 1438 768302.
E-mail address: pan.a.procopiou@gsk.com (P.A. Procopiou).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.043

P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 6026–6032
6027
OH
H
OH
H
N
N
O
HO
HO
OMe
HO
1
2
NHCHO
OH
Cl
H
N
OH
H
O
N
O
O
HO
HO
HO
Cl
4
HN
3
O
O
OH
H
N
H
N
O
OH
H
N
HN
OH
HO
NHSO2Me
OMe
6
5
OH
OH
OH
H
H
N
N
SO2NH2
O
HO
HO
OMe
HO
8
HN
7
O
Chart 1. Clinically used b2 agonists, and recent development candidates.
agonists with reduced oral absorption. Some of the physicochemi-
cal parameters that influence oral absorption are molecular weight,
lipophilicity, membrane permeability, the number of hydrogen-
bond donors and acceptors, conformational flexibility and solubil-
ity.15 Substitution on the right-hand side phenyl ring of salmeterol
with the polar sulfonamide group was found to enhance b2 agonist
affinity.12 It was hypothesised that a polar substituent, such as a
urea, with increased number of hydrogen-bond donors and accep-
tors which contravened the Lipinski rules,16 might be expected to
show reduced oral bioavailability. We have demonstrated that
introduction of either a sulfonamide group,12 or a heterocyclic ring
such as hydantoin13 brings about longer duration of action. It was
therefore hypothesised that introduction of the urea group might
also bind in a similar way to the sulfonamide group of 8 and hence
have similarly long duration of action.
provide ethanolamines 19. A final aqueous acetic acid hydrolysis
of the acetonide protecting group gave the required target com-
pounds 9e,f.
The substituted aryl iodides/bromides 11 were obtained by
reaction of the appropriate iodo/bromo-aniline/benzylamines with
the corresponding isocyanate/sodium cyanate.19 The synthesis of
the intermediate aryl bromide 22 used in the synthesis of 9j is
shown in Scheme 3. Nitroaniline 20 was brominated in acetic acid
with bromine to provide 2-bromo-4-methyl-6-nitroaniline (21),20
which was diazotised and reduced to give 2221 in 62% yield over
the two steps.
Completion of the synthesis of 9j is shown in Scheme 4. Reac-
tion of acetylene 16 with bromide 22 provided 23, which was con-
verted to 24 by simultaneous hydrogenation of the acetylene and
reduction of the nitro group, formation of the primary urea 18j
with sodium cyanate, cleavage of the oxazolidinone ring with
potassium trimethylsilanolate to 19j, and finally hydrolysis of the
acetonide group as before, gave 9j.
2. Chemistry
Lastly the ureido-amides 9g and 9h were obtained by reaction
of the hydantoin 2513 with ammonia or cyclopentylamine, respec-
tively (Scheme 5).
The target compounds 9 were synthesised by two general routes,
the first one was a linear synthetic route outlined in Scheme 1. This
involved Sonogashira coupling of the acetylene 1012 with aryl
iodides/bromides 11, reaction of the resulting bromide 12 with
2 equiv of the amino alcohol 1313 in order to minimise bis-alkyl-
ation,17 catalytic hydrogenation of the mono-alkylated product 14,
and finally hydrolysis of the acetonide protecting group with aque-
ous acetic acid provided 9a–d,i.
The second route, outlined in Scheme 2, used (R)-2-oxazolidi-
none protected saligenin 1518 and bypassed the requirement of
using greater than stoichiometric quantities of the valuable chiral
amino-alcohol moiety, unlike the previous route where excess 13
was needed to minimise bis-alkylation on nitrogen. Thus, 15 was
alkylated with the bromoacetylene 1012 in the presence of sodium
hydride in DMF to give the intermediate 16, which was coupled
with the appropriate aryl iodide/bromide 11 using Sonogashira
conditions to provide acetylene intermediates 17. The latter were
reduced by catalytic hydrogenation over platinum oxide, the
oxazolidinone ring of the resulting saturated intermediates 18
were hydrolysed by our previously reported KOTMS method18 to
3. Results and discussion
Compounds in Table 1 were tested for their ability to cause cyc-
lic AMP accumulation in Chinese hamster ovary (CHO) cells trans-
fected with human b1, b2 or b3 adrenoceptors. Agonist activity was
assessed by measuring changes in intracellular cyclic AMP, and the
potency is reported as pEC50 values (negative log10 molar concen-
tration 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 iso-
prenaline. By definition, isoprenaline’s intrinsic activity is 1. The
IA for formoterol was 0.97, whereas that of salmeterol was 0.37.
Preferred compounds had IA > 0.37. Required selectivity for b2 over
b1 and b3 adrenoceptors was set as better than that of (R,R)-formo-
terol. Microsomal instability of the compounds towards high

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P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 6026–6032
I(Br)
O
a
O
Br
R
Br
10
12
11
R
OH
NH2
O
R
c,d
OH
13
H
O
N
O
O
14
b
O
OH
H
N
R
O
HO
HO
9
a
b
c
3-NHCONH2
f
3-CH2NHCONHEt
3-NHCONHEt
3-NHCONH-cC6H11
4-NHCONHEt
g
h
i
3-NHCONHCH2CONH2
3-NHCONHCH2CONH-cC5H9
3-NHCONH2-5-CF3
d
e
3-CH2NHCONH2
j
3-NHCONH2-5-CH3
Scheme 1. Synthetic route to analogues 9 via 13 and list of substituents R present in compounds 9, 11, 12, 14, 17, 18 and 19. Reagents: (a) Pd(PPh3)2Cl2, CuI, Et3N, DMF; (b) 13
(2 equiv), DMF; (c) H2, PtO2, EtOAc; (d) AcOH–H2O (3:1), 75 °C.
O
O
N
O
a
O
O
b
H
H
NH
O
O
11
10
15
16
O
O
O
O
N
O
O
d
O
c
H
H
N
O
R
O
O
18
O
O
17
R
OH
e
H
N
9
O
O
R
O
19
Scheme 2. Synthetic route to analogues 9 via 15. Reagents: (a) NaH, DMF; (b) Pd(PPh3)2Cl2, CuI, Et3N, MeCN; (c) H2, PtO2, EtOH-EtOAc; (d) KOTMS, THF, 65 °C; (e) AcOH–H2O
(3:1), 75 °C.
tested were potent b2 receptor agonists (substantially more potent
Me
Me
Me
than the standard, isoprenaline), and some were more potent than
salmeterol and formoterol. The primary urea and two secondary
ureas 9a–c had similar b2 receptor agonist activity. However, 9c
had lower intrinsic activity, inferior b1 and b3 selectivity, and was
rejected despite its high metabolic turnover ratio. The para-ethylu-
rea 9d had lower IA than the meta-ethylurea 9b and hence all other
analogues reported herein are in the meta-series. This observation
confirms our previous findings within other salmeterol series.12,13
The homologous urea 9e was equipotent to its parent 9a and had a
high turnover ratio, however, it had a lower intrinsic activity and
was therefore not investigated further. The homologous urea 9f
was equipotent to its parent 9b and was very selective for b2 over
b1 and b3. Its intrinsic activity was lower than that of salmeterol
and therefore not investigated any further. Introduction of an
additional amide group as in the derivatives 9g and 9h caused a
a
b
NO2
Br
NO2
Br
NO2
NH2
NH2
20
21
22
Scheme 3. Synthetic route to compound 22. Reagents: (a) Br2, AcOH, 94%; (b)
NaNO2, H2SO4, EtOH, 66%.
first-pass metabolism in the liver was assessed by incubating sam-
ples with human liver microsomes (P450 content = 125 pmol/mL)
at a compound concentration of 5 lM for 30 min at 37 °C. Com-
pound turnover was expressed as a ratio relative to the assay stan-
dard, verapamil. Salmeterol which had a slightly higher turnover
ratio (1.2) was also included as a standard. All the compounds

P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 6026–6032
6029
O
O
O
O
O
O
H
N
a
b
d
H
N
16
O
O
O
22
O
O
23
Me
24 R=H
c
18j R=H2NCO
Me
Me
RNH
O2N
OH
e
H
N
9j
O
N
H
NH2
O
19j
O
Scheme 4. Synthetic route to analogues 9j. Reagents: (a) Pd(PPh3)2Cl2, CuI, Et3N, DMF, 43%; (b) H2, PtO2, EtOAc; EtOH, 99%; (c) NaNCO, AcOH, H2O, 75%; (d) KOTMS, THF,
65 °C, 85%; (e) AcOH–H2O (3:1), 75 °C, 77%.
OH
OH
O
a
H
N
O
N
HO
HO
NH
25
O
O
H
N
NH2 9g
N
H
N
H
O
HO
HO
O
O
OH
H
N
H
N
b
9h
O
N
N
H
HO
HO
H
25
O
Scheme 5. Synthetic route to analogues 9g and 9h. Reagents: (a) 2 M NH3 in MeOH, 10%; (b) c-C5H9NH2, EtOH, 48%.
Table 1
Stimulation of cAMP Accumulation in CHO Cells Expressing Human b2, b1 and b3 adrenoceptors, and human liver microsomal turnover ratio against verapamil
Turnover ratiod
a
a
b
a
c
Entry
Compound
b2 pEC50 (n)
IA
b1 pEC50 (n)
b2–b1
b3 pEC50 (n)
b2–b3
1
2
3
4
5
6
7
8
9a.AcOH
9b.AcOH
9c.AcOH
9d.AcOH
9e.AcOH
9f.AcOH
9g.HCO2H
9h.AcOH
9i.AcOH
9j.AcOH
Isoprenaline
1
9.1 0.1 (4)
9.0 0.0(3)
0.41
0.46
0.35
0.37
0.36
0.32
0.39
0.30
0.34
0.72
1.0
7.2 1.0 (2)
7.5 0.3 (2)
8.1 0.0 (2)
7.5 0.1 (11)
8.1 0.2 (5)
7.5 0.1 (4)
7.5 0.1 (4)
8.0 0.1 (4)
7.3 0.1 (3)
1.9
1.5
0.9
1.7
1.2
1.7
2.1
1.5
1.7
2.4
À0.7
3.5
1.9
3.0
8.1 0.0 (2)
6.8 0.1 (2)
8.6 0.1 (2)
7.9 0.1 (12)
7.8 0.1 (8)
7.5 0.1 (4)
7.0 0.1 (4)
7.9 0.0 (4)
7.6 0.1 (4)
7.4 0.1 (24)
7.4 0.0 (659)
5.9 0.0 (849)
7.6 0.0 (653)
6.1 0.2 (8)
1.0
2.2
0.4
1.4
1.5
1.7
2.6
1.6
1.4
2.5
0.0
3.7
1.7
3.3
0.97
0.81
2.18
1.37
1.64
0.72
NDe
NDe
1.34
1.17
NDe
1.2
9.0 0.2 (2)
9.3 0.3 (6)
9.3 0.2 (4)
9.2 0.2 (4)
9.6 0.1 (5)
9.5 0.1 (2)
9.0 0.3 (6)
9.8 0.1(17)
7.4 0.0 (767)
9.6 0.0 (929)
9.3 0.0 (791)
9.4 0.0 (8)
9
10
11
12
13
14
7.4
0.1 (24)
8.1 0.0 (641)
6.1 0.0 (656)
7.4 0.0 (670)
6.4 0.1 (8)
0.37
0.97
0.69
(R,R)-2.fumarate
4.AcOH
NDe
1.65
a
Human b1, b2 and b3 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.
b
Selectivity for b2 over b1 expressed as pEC50 at b2 receptor—pEC50 at b1.
Selectivity for b2 over b3 expressed as pEC50 at b2 receptor—pEC50 at b3.
Compound turnover in human liver microsomes expressed as a ratio ralative to verapamil, where verapamil has a turnover ratio of 1.
ND not determined.
c
d
e
significant increase in the b2 receptor agonist activity by compari-
son to 9a. Furthermore the selectivity of both compounds was sig-
nificantly increased. In particular 9g possessed one of the highest
selectivity ratios of all the compounds in Table 1. The intrinsic
activity of 9h was lower than salmeterol, therefore this compound
was rejected too. The intrinsic activity of 9g was marginally higher
than salmeterol and the compound was retained for further inves-
tigation. Introduction of a trifluoromethyl substituent to the phe-
nyl ring bearing the urea moiety (9i) increased the metabolic
turnover ratio, but reduced the IA, and did not offer any advantage.
The methyl substituted primary urea 9j possessed the highest b2
potency of all the compounds in Table 1, highest b2 selectivity over
b1 and b3, similar turnover ratio to salmeterol, and a higher intrin-
sic activity than salmeterol. Furthermore it compared favourably
with vilanterol (Table 1). Urea 9j improved on all criteria of po-
tency, IA, b2 selectivity and turnover ratio, and therefore presented
a very attractive profile.
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

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P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 6026–6032
Table 2
The potency, onset time and duration of action of selected compounds on isolated superfused guinea pig trachea (minimum n = 2), and lipophilicity measurements
a
Entry
Compound
pEC50
Onset time (min)
Shift (1 h)
Shift (3 h)
clogP
logD6.4
1
2
3
4
5
6
9a.AcOH
9b.AcOH
9g.HCO2H
9j.AcOH
Salmeterol
(R,R)-2.fumarate
8.6
8.9
9.6
8.8
8.3
9.5
13.5
15
9
10
27.6
10
5
7
19
149
2
1.1
>1940
1.8
2.6
0.8
2.3
3.0
1.3
À0.28
0.5
11
78
1
1.0
20
À0.78
0.2
1.06
NDb
a
logD6.4 measured logD at pH 6.4.
ND not determined.
b
Table 3
Han Wistar rat and Beagle dog pharmacokinetic data for 9j acetate
Species
Rat
Route
Dose (mg/kg)
Clp (mL/min/kg)
Vdss (L/kg)
T
(h)
F%
½
Intravenous
Oral
Intravenous
Oral
0.25
2.0
0.05
0.25
83
2.0
0.9
<5
6
Dog
6–19
0.55
1–3
duration of action.22 Test compounds were investigated for their
ability to inhibit the contraction of guinea pig trachea strips ex-
pressed as a measure of the functional response at the b2 adreno-
ceptor. Tissues were contracted electrically, agonist was perfused
over the tissue until maximum relaxation was achieved, and onset
of action determined. Perfusion of the agonist was then ceased, tis-
sue 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 potency, the onset time and the duration
after 1 and 3 h of the more promising compounds (9a,b,g,j) is pre-
sented and contrasted to salmeterol. Potency was expressed in
absolute terms (concentration required to induce 50% inhibition,
EC50). Onset of action was calculated as the time taken for an
EC50 concentration to achieve 50% maximum relaxant effect. Dura-
tion of action was determined by measuring the recovery of elec-
trically induced contraction following washout of agonist. This
was expressed as the rightward shift in the agonist concentra-
tion–effect curve following 1 and 3 h 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 (after 1 h) and 1 (after 3 h)
indicate no washout (a salmeterol-like profile). Shift values of
about 20 and >300 indicate slow continuous washout (formoter-
ol-like profile). Shift values of infinity indicate rapid and complete
washout (isoprenaline-like profile). All the compounds in Table 2
had potency at the guinea pig trachea higher than that of salmeter-
ol. Onset times were faster than salmeterol and closer to that of
formoterol. Clearly the urea 9g despite its very high potency on
both CHO cells and guinea pig trachea, had a significantly shorter
duration of action. The shorter duration of action was related to
the lower lipophilicity of 9g (logD6.4–0.78, clogP 0.82, Table 2) as
we have previously discovered,8,12 and others have recently dis-
closed.23,24 From the remaining three compounds 9j had shift val-
ues of 1 and 2, a duration in vitro very similar to salmeterol.
Furthermore 9j had a better overall profile of potency on both
CHO cells and guinea pig trachea, b2 selectivity, faster onset time,
and higher metabolic turnover ratio. For these reasons 9j was pro-
gressed to pharmacokinetic studies in rats and dogs and the data is
shown in Table 3. The compound had lower oral absorption (AUC
4811 ng min/mL) than salmeterol in rats (deconjugated HPV mod-
el).13 In the male Han Wistar rat 9j had a moderate volume of dis-
tribution (2 L/kg) and a high plasma clearance (83 mL/min/kg,
100% liver blood flow) resulting in a short half-life of less than
1 h. In the female beagle dog 9j had a low volume of distribution
(0.55 L/kg) and a low to moderate plasma clearance (<67% liver
blood flow). Low oral bioavailability was observed in both the rat
(<5%) and the dog (6%) (Table 3). This oral bioavailability was much
lower than that previously reported for salmeterol (66–78% in dog,
and 10–12% in rat)25 and strengthened the confidence that low sys-
temic exposure would be expected from the swallowed fraction of
the inhaled dose of 9j in humans. In addition to the low oral
absorption of 9j in the rat, first pass metabolism in the gastrointes-
tinal tract may also contribute to the resultant low oral bioavail-
ability. Deconjugated rat plasma samples, following intravenous
or oral administration were analysed in an attempt to identify pos-
sible metabolites of 9j, such as the aniline derived from cleavage of
the primary urea moiety. However no aniline metabolite of 9j was
detected.
The acetate salt of 9j was also investigated in vivo in histamine-
induced bronchospasm in the guinea pig in a plethysmograph cham-
ber (Buxco) and found to have similar potency to salmeterol (EC90 10
and 50 lM respectively, nebuliser concentration). At an equi-effec-
tive (EC90) dose, the duration of action of 9j acetate (time to 50%
recovery) was similar to that of salmeterol (7 vs 8 h) when adminis-
tered as a nebulised solution in a dimethyl acetamide-saline vehicle.
Furthermore the duration of 9j was extended to 18 h when the dose
was increased to 10-fold the EC90. Urea 9j was progressed towards a
salt selection screen to identify a stable, non-hygroscopic and crys-
talline salt, suitable for inhaled delivery. Unfortunately 9j acetic acid
salt was a gum, and no developable crystalline salts were identified,
whereas the free base was an amorphous solid.
4. Conclusion
Incorporation of a urea group on the right-hand side phenyl ring
of (R)-salmeterol has provided a series of potent and selective hu-
man b2 adrenoceptor agonists. Urea 9j had duration of action on
guinea pig trachea, and also in vivo similar to that of salmeterol
and vilanterol. It had lower oral absorption and bioavailability than
salmeterol in both rat and dog. It had a turnover ratio similar to
salmeterol, with no evidence for formation of any aniline metabo-
lites. No crystalline salts suitable for inhaled delivery were identi-
fied; for this reason coupled with the steady progress of vilanterol
in clinical trials, compound 9j was not progressed any further.

P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 6026–6032
6031
(2 L), to give 23 (2.86 g, 43%): MS ES+ve m/z 537 (M+H)+; 1H NMR d
(CDCl3) 8.04 (1H, br s), 7.93 (1H, br s), 7.51 (1H, br s), 7.12 (1H, br
d, J 8 Hz), 7.00 (1H, br s), 6.84 (1H, d, J 8 Hz), 5.39 (1H, t, J 8 Hz),
4.84 (2H, s), 3.85 (1H, t, J 8 Hz), 3.63 (2H, t, J 7 Hz), 3.49 (2H, t, J
6 Hz), 3.43–3.20 (3H, m), 2.69 (2H, t, J 7 Hz), 2.42 (3H, s), 1.65–
1.51 (4H, m), 1.54 (6H, s), 1.47–1.31 (4H, m).
5. Experimental
Organic solutions were dried over anhydrous MgSO4. TLC was
performed on Merck 0.25 mm Kieselgel 60 F254 plates. Products
were visualised under UV light and/or by staining with aqueous
KMnO4 solution. LCMS analysis was conducted on a Supelcosil
LCABZ+PLUS column (3.3 cm  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+ve and ESÀve).
Column chromatography was performed on Merck Kieselgel 60
(art. 9385), or Biotage pre-packed silica gel cartridges containing
KP-Sil run on a flash 12i chromatography module. 1H NMR spectra
were recorded at 400 MHz unless otherwise stated. The chemical
shifts are expressed in ppm relative to tetramethylsilane. All animal
studies were ethically reviewed and carried out in accordance with
Animals (Scientific Procedures) Act 1986 and the GSK Policy on the
Care, Welfare and Treatment of Laboratory Animals.
5.4. (5R)-3-(6-{[4-(3-Amino-5-methylphenyl)butyl]oxy}hexyl)-
5-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)-1,3-oxazolidin-2-one
(24)
(5R)-5-(2,2-Dimethyl-4H-1,3-benzodioxin-6-yl)-3-(6-{[4-(3-
methyl-5-nitrophenyl)but-3-ynyl]oxy}hexyl)-1,3-oxazolidin-2-
one (3.18 g, 5.92 mmol) was stirred with platinum oxide (515 mg)
in ethanol (50 mL) and EtOAc (10 mL) under hydrogen for 2.5 h.
The catalyst was removed by filtration through a pad of celite.
The filtrate was evaporated to dryness to give 24 (2.99 g, 99%):
MS ES+ve m/z 511 (M+H)+; 1H NMR d (CDCl3) 7.12 (1H, br d, J
8.5 Hz), 7.00 (1H, br s), 6.84 (1H, d, J 8.5 Hz), 6.41 (1H, br s), 6.34
(2H, br s), 5.39 (1H, t, J 8 Hz), 4.84 (2H, s), 3.85 (1H, t, J 9 Hz),
3.70–3.45 (2H, br), 3.44–3.35 (6H, m), 3.35–3.20 (1H, m), 2.49
(2H, t, J 7 Hz), 2.22 (3H, s), 1.71–1.46 (14 H, m), 1.43–1.30 (4H, m).
5.1. 2-Bromo-4-methyl-6-nitroaniline (21)20
5.5. N-{3-[4-({6-[(5R)-5-(2,2-Dimethyl-4H-1,3-benzodioxin-6-
yl)-2-oxo-1,3-oxazolidin-3-yl]hexyl}oxy)butyl]-5-
methylphenyl}urea (18j)
4-Methyl-2-nitroaniline (131.2 g, 862 mmol) was suspended in
glacial acetic acid (1.25 L) and bromine (54 mL, 1.05 mol) added
over 1 h at ambient temperature. The reaction mixture was stirred
for 1 h, poured into water (7.5 L) and the suspension stirred for
30 min. The solid was filtered, washed with water (5 Â 1 L) and
dried to give 21 (187.2 g, 94%) as an orange solid: 1H NMR d (CDCl3)
7.94 (1H, s), 7.56 (1H, s), 6.47 (2H, br s), 2.27 (3H, s).
A suspension of potassium cyanate (952 mg, 11.7 mmol) in
water (23 mL) was slowly added to a solution of (5R)-3-{6-[4-(3-
amino-5-methylphenyl)butoxy]hexyl}-5-(2,2-dimethyl-4H-1,3-
benzodioxin-6-yl)-1,3-oxazolidin-2-one (2.99 g, 5.86 mmol) in
glacial acetic acid (23 mL) containing water (11 mL) at ꢀ0 °C under
nitrogen. The mixture was warmed to room temperature over
ꢀ2 h and then stirred at room temperature for 20 min. The reaction
mixture was evaporated to dryness and the residue purified by
chromatography on a Biotage (90 g) cartridge eluting with 20%
ethyl acetate–cyclohexane (1 L), 25% ethyl acetate–cyclohexane
(0.5 L), 50% ethyl acetate–cyclohexane (1 L), 60% ethyl acetate–
cyclohexane (1 L), 70% ethyl acetate–cyclohexane (1 L), and finally
ethyl acetate (1 L). Appropriate fractions were combined and puri-
fied further by chromatography on a Biotage cartridge (90 g) elut-
ing with a gradient of 50–80% ethyl acetate–cyclohexane to give
18j (2.45 g, 75%): MS ES+ve m/z 554 (M+H)+; 1H NMR d (CDCl3)
7.15-7.05 (2H, m), 7.00 (1H, br s), 6.84 (1H, d, J 8 Hz), 6.76 (1H,
br s), 6.71 (1H, br s), 5.41 (1H, t, J 8 Hz), 4.84 (2H, s), 4.80 (2H,
br), 3.87 (1H, t, J 9 Hz), 3.48–3.23 (7H, m), 2.55 (2H, t, J 8 Hz),
2.29 (3H, s), 1.73–1.49 (15H, m), 1.48–1.32 (4H, m); 13C NMR d
(CDCl3) 158.7, 151.5, 143.9, 139.2, 138.9, 132.7, 126.1, 124.9,
122.8, 120.0, 118.8, 117.6, 100.0, 70.4, 69.6, 61.2, 55.6, 49.0, 36.0,
29.6, 28.7, 26.7, 26.3, 25.2, 25.0, 21.8.
5.2. 3-Bromo-5-nitrotoluene (22)21
2-Bromo-4-methyl-6-nitroaniline (20.5 g, 88.7 mmol) was sus-
pended in ethanol (105 mL) and sulfuric acid (s.g. 1.84, 14 mL)
added portionwise. The solution was heated to 73 °C and sodium
nitrite (13.7 g, 198.5 mmol) added over 25 min, maintaining the
temperature at 73–78 °C for 30 min. The reaction mixture was
cooled and then poured into water (700 mL). The solid was col-
lected by filtration, washed with water and the product purified
by steam distillation to give 22 (12.6 g, 66%) as a yellow solid: 1H
NMR d (CDCl3) 8.19 (1H, br s) 7.99 (1H, br s), 7.66 (1H, br s), 2.47
(3H, s); 13C NMR d (CDCl3) 148.7, 141.6, 138.1, 123.9, 122.7,
122.5, 21.1; IR mmax 1533, 1342, 740 cmÀ1
.
5.3. (5R)-5-(2,2-Dimethyl-4H-1,3-benzodioxin-6-yl)-3-(6-{[4-(3-
methyl-5-nitrophenyl)-3-butyn-1-yl]oxy}hexyl)-1,3-oxazolidin-
2-one (23)
A degassed solution of anhydrous tetrahydrofuran (80 mL) and
triethylamine (10 mL) was treated with 1-bromo-3-methyl-5-nitro-
benzene (2.69 g, 12.4 mmol), dichloro bis(triphenylphosphine) pal-
ladium(II) (613 mg, 0.87 mmol) and cuprous iodide (308 mg,
1.6 mmol). The resultant mixture was then purged with nitrogen
and heated to 70 °C. After 10 min, a solution of (5R)-3-[6-(but-3-
ynyloxy)hexyl]-5-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)-1,3-
oxazolidin-2-one (5 g, 12.4 mmol) in anhydrous degassed THF
(20 mL) was added and the reaction mixture stirred at 70 °C for
3 h. The cooled reaction mixture was evaporated to dryness and
the residue was suspended in diethyl ether and filtered through cel-
ite. The filtrate was concentrated under reduced pressure and the
residue was purified by chromatography on a Biotage cartridge
(90 g), eluting with 20% ethyl acetate in cyclohexane (1 L), 25% ethyl
acetate in cyclohexane (0.5 L), 30% ethyl acetate in cyclohexane
5.6. N-(3-{4-[(6-{[(2R)-2-(2,2-Dimethyl-4H-1,3-benzodioxin-6-
yl)-2-hydroxyethyl]amino}hexyl)oxy]butyl}-5-methylphenyl)-
urea (19j)
To a solution of N-{3-[4-({6-[(5R)-5-(2,2-dimethyl-4H-1,3-ben-
zodioxin-6-yl)-2-oxo-1,3-oxazolidin-3-yl]hexyl}oxy)butyl]-5-
methylphenyl}urea (2.45 g, 4.42 mmol) in anhydrous THF
(100 mL) was added potassium trimethylsilanolate (2.27 g,
17.7 mmol). The reaction was stirred under nitrogen at 65 °C for
45 min. The cooled reaction mixture was then diluted with water
and extracted into ethyl acetate, the organic layer was dried
(MgSO4) and filtered. The filtrate was evaporated to dryness and
the residue purified by chromatography on a Biotage (90 g) car-
tridge, eluting with CH2Cl2, 0–100% ethyl acetate in cyclohexane

6032
P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 6026–6032
3. Boushey, H. A. J. Allergy Clin. Immunol. 1998, 102, S5.
4. Johnson, M. Med. Res. Rev. 1995, 15, 225–257.
5. Matthys, H. Respiration 2001, 68, 432.
gradient, followed by 0–8% MeOH (containing trace of aqueous
ammonia) in dichloromethane gradient to give 19j (1.97 g, 85%):
MS ES+ve m/z 528 (M+H)+; 1H NMR d (CDCl3) 7.16 (1H, br), 7.11
(1H, br d, J 8 Hz), 7.00 (1H, br s), 6.96 (1H, br s), 6.88 (1H, br s),
6.78 (1H, d, J 8 Hz), 6.74 (1H, br s), 4.99 (2H, s), 4.82 (2H, s), 4.64
(1H, dd, J 9, 3 Hz), 3.44–3.35 (4H, m), 2.82 (1H, dd, J 12, 3 Hz),
2.73–2.57 (4H, m), 2.55 (2H, t, J 8 Hz), 2.27 (3H, s), 1.71–1.44 (15
H, m), 1.40–1.30 (4H, m).
6. Johnson, M. Am. J. Respir. Crit. Care Med. 1998, 158, S146.
7. Baur, F.; Beattie, D.; Beer, D.; Bentley, D.; Bradley, M.; Bruce, I.; Charlton, S. J.;
Cuenoud, B.; Ernst, R.; Fairhurst, R. A.; Faller, B.; Farr, D.; Keller, T.; Fozard, J. R.;
Fullerton, J.; Garman, S.; Hatto, J.; Hayden, C.; He, H.; Howes, C.; Janus, D.; Jiang,
Z.; Lewis, C.; Loeuillet-Ritzler, F.; Moser, H.; Reilly, J.; Steward, A.; Sykes, D.;
Tedaldi, L.; Trifilieff, A.; Tweed, M.; Watson, S.; Wissler, E.; Wyss, D. J. Med.
Chem. 2010, 53, 3675.
8. Procopiou, P. A.; Barrett, V. J.; Bevan, N. J.; Biggadike, K.; Box, P. C.; Butchers, P.
R.; Coe, D. M.; Conroy, R.; Emmons, A.; Ford, A. J.; Holmes, D. S.; Horsley, H.;
Kerr, F.; Li-Kwai-Cheung, A.-M.; Looker, B. E.; Mann, I. S.; McLay, I. M.;
Morrison, V. S.; Mutch, P. J.; Smith, C. E.; Tomlin, P. J. Med. Chem. 2010, 53, 4522.
9. Bouyssou, T.; Hoenke, C.; Rudolf, K.; Lustenberger, P.; Pestel, S.; Sieger, P.; Lotz,
R.; Heine, C.; Büttner, F. H.; Schnapp, A.; Konetzki, I. Bioorg. Med. Chem. Lett.
2010, 20, 1410.
5.7. N-[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-
(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-5-
methylphenyl]urea (9j) acetate salt
10. Glossop, P. A.; Lane, C. A. L.; Price, D. A.; Bunnage, M. E.; Lewthwaite, R. A.;
James, K.; Brown, A. D.; Yeadon, M.; Perros-Huguet, C.; Trevethick, M. A.;
Clarke, N. P.; Webster, R.; Jones, R. M.; Burrows, J. L.; Feeder, N.; Taylor, S. C. J.;
Spence, F. J. J. Med. Chem. 2010, 53, 6640.
11. Kikkawa, H.; Naito, K.; Ikezawa, K. Jpn. J. Pharmacol. 1991, 57, 175.
12. Procopiou, P. A.; Barrett, V. J.; Bevan, N. J.; Biggadike, K.; Butchers, P. R.; Coe, D.
M.; Conroy, R.; Edney, D. D.; Field, R. N.; Ford, A. J.; Guntrip, S. B.; Looker, B. E.;
McLay, I. M.; Monteith, M. J.; Morrison, V. S.; Mutch, P. J.; Stephen, A.; Richards,
S. A.; Sasse, R.; Smith, C. E. J. Med. Chem. 2009, 52, 2280.
13. Procopiou, P. A.; Barrett, V. J.; Bevan, N. J.; Butchers, P. R.; Conroy, R.; Emmons,
A.; Ford, A. J.; Jeulin, S.; Looker, B. E.; Lunniss, G. E.; Morrison, V. S.; Mutch, P. J.;
Perciaccante, R.; Ruston, M.; Smith, C. E.; Somers, G. Bioorg. Med. Chem. 2011,
19, 4192.
14. Bennett, J. A.; Harrison, T. W.; Tattersfield, A. E. Eur. Respir. J. 1999, 13, 445.
15. Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.
J. Med. Chem. 2002, 45, 2615.
16. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
17. Procopiou, P. A.; Morton, G. E.; Todd, M.; Webb, G. Tetrahedron: Asymmetry
2001, 12, 2005.
18. Coe, D. M.; Perciaccante, R.; Procopiou, P. A. Org. Biomol. Chem. 2003, 1,
1106.
19. Blake, K.; Coe, D. M.; Procopiou, P. A. WO2003072539A1.
20. Carlin, R. B.; Foltz, G. E. J. Am. Chem. Soc. 1956, 78, 1992.
21. O’Neill, P. M.; Willock, D. J.; Hawley, S. R.; Bray, P. G.; Storr, R. C.; Ward, S. A.;
Park, B. K. J. Med. Chem. 1997, 40, 437.
N-(3-{4-[(6-{[(2R)-2-(2,2-Dimethyl-4H-1,3-benzodioxin-6-yl)-
2-hydroxyethyl]amino}hexyl)oxy]butyl}-5-methylphenyl)urea
(1.972 g, 3.73 mmol) was stirred with glacial acetic acid (50 mL)
and water (25 mL) at 80 °C for 45 min. The resultant reaction mix-
ture was cooled, evaporated to dryness and the residue was dis-
solved in MeOH and
a minimum amount of 2 M aqueous
ammonia in methanol. The solution was applied to a Biotage car-
tridge (90 g) and eluted with a gradient of 0–15% [(10% concen-
trated aqueous ammonia in methanol)–dichloromethane].
Appropriate fractions were combined and evaporated and the res-
idue was dissolved in acetic acid and then re-evaporated under re-
duced pressure to give 9j.AcOH (1.4 g, 77%): LC–MS RT = 2.36 min,
ES+ve m/z 488 (M+H)+; 1H NMR d (CD3OD) 7.35 (1H, br s), 7.16 (1H,
br d, J 8 Hz), 7.06 (1H, br s), 6.97 (1H, br s), 6.80 (1H, d, J 8 Hz), 6.68
(1H, br s), 4.83 (1H, t, J 6.5 Hz), 4.67 (2H, s), 3.46 (2H, t, J 6.5 Hz),
3.44 (2H, t, J 6.5 Hz), 3.02 (2H, d, J 6.5 Hz), 2.92 (2H, t, J 7.5 Hz),
2.56 (2H, t, J 7.5 Hz), 2.27 (3H, s), 1.92 (3H, s), 1.73–1.54 (8H, m),
1.47–1.37 (4H, m).
Acknowledgements
22. Nials, A. T.; Sumner, M. J.; Johnson, M.; Coleman, R. A. Br. J. Pharmacol. 1993,
108, 507.
23. Beattie, D.; Bradley, M.; Brearley, A.; Charlton, S. J.; Cuenoud, B. M.; Fairhurst, R.
A.; Gedeck, P.; Gosling, M.; Janus, D.; Jones, D.; Lewis, C.; McCarthy, C.;
Oakman, H.; Stringer, R.; Taylor, R. J.; Tuffnell, A. Bioorg. Med. Chem. Lett. 2010,
20, 5302.
We thank Ms. Lynn Crawford for scaling up the preparation of 5,
Mrs. Valerie S. Morrison for the guinea pig trachea data, Miss Isobel
Hackney and Miss Sara C. Hughes for the in vivo work on 9j.
24. Pérez, D.; Crespo, M.; Solé, L.; Prat, M.; Carcasona, C.; Calama, E.; Otal, R.;
Gavaldá, A.; Gómez-Angelats, M.; Miralpeix, M.; Puig, C. Bioorg. Med. Chem. Lett.
2011, 21, 1545.
References and notes
25. Manchee, G. R.; Barrow, A.; Kulkarni, S.; Palmer, E.; Oxford, J.; Colthup, P. V.;
Maconochie, J. G.; Tarbit, M. H. Drug Metab. Dispos. 1993, 21, 1022.
1. Barnes, P. J. Nat. Rev. Immunol. 2008, 8, 183.
2. Barnes, P. J. J. Allergy Clin. Immunol. 1998, 102, 531.