The discovery of long-acting saligenin β2 adrenergic receptor agonists incorporating hydantoin or uracil rings

Article abstract of DOI:10.1016/j.bmc.2011.05.064

A series of novel, potent and selective human β₂ adrenoceptor agonists incorporating a hydantoin or a uracil ring on the right-hand side phenyl ring of (R)-salmeterol is presented. Hydantoin 12a had long duration of action in vitro on guinea pi

Full text of DOI:10.1016/j.bmc.2011.05.064

Bioorganic & Medicinal Chemistry 19 (2011) 4192–4201
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The discovery of long-acting saligenin b2 adrenergic receptor agonists
incorporating hydantoin or uracil rings
Panayiotis A. Procopiou a, , Victoria J. Barrett b, Nicola J. Bevan b, Peter R. Butchers b, Richard
⇑
Conroy a, Amanda Emmons b, Alison J. Ford b, Séverine Jeulin a, Brian E. Looker a, Gillian E. Lunniss a,
Valerie S. Morrison b, Peter J. Mutch c, Rossana Perciaccante a, Mark Ruston 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:
A series of novel, potent and selective human b2 adrenoceptor agonists incorporating a hydantoin or a
uracil ring on the right-hand side phenyl ring of (R)-salmeterol is presented. Hydantoin 12a had long
duration of action in vitro on guinea pig trachea, and 12 h in guinea pigs in vivo at its EC90 25 lM. It
had lower oral absorption than salmeterol in rats, and lower bioavailability than salmeterol in vivo in
both rats and dogs (2% and 5%, respectively). An improved method for measuring the absorbed fraction
of analogues dosed to rats, which considers the glucuronidated fraction is presented. Compound 12a was
metabolised in human liver microsomes and hepatocytes to the active hydantoic acid 12m.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 28 April 2011
Revised 26 May 2011
Accepted 30 May 2011
Available online 21 June 2011
Keywords:
Asthma
COPD
b2-Agonist
Saligenin
Hydantoin
1. Introduction
are used in maintenance therapy. The short-acting agonists have
a rapid onset of action and relieve symptoms for 3–6 h. The two
Asthma is a chronic disease affecting 300 million people world-
wide, characterised by an increase in inflammatory cell popula-
tions in the epithelium and submucosa of the airways.1 There are
two main components of asthma pathophysiology, airway inflam-
mation and smooth muscle dysfunction, which are treated by two
major categories of medicines: anti-inflammatory drugs and bron-
chodilators. Inhaled corticosteroids are used to treat the inflamma-
tory component of asthma. Inhaled b2-agonists are the most
effective bronchodilators, offering proven benefits in reducing the
burden of this disease.2,3 Chronic obstructive pulmonary disease
(COPD) is the fourth leading cause of death in the US, affecting
5% of its population. Bronchodilators such as inhaled b2-agonists
and muscarinic antagonists are currently the mainstay of treat-
ment for COPD and combinations with inhaled corticosteroids
are known to reduce the incidence of exacerbations. There are
two classes of b2-agonists: the short-acting (first generation) ago-
nists, such as salbutamol (1) (Chart 1), which are used in rescue
therapy, and the long-acting (second generation) agonists which
currently prescribed inhaled long-acting b2-agonists are salmeterol
(2) and formoterol (3). Salmeterol has lower intrinsic activity than
salbutamol, a delayed onset of action, and 12 h duration, which is
independent of dose.4,5 In contrast, formoterol has high intrinsic
efficacy, onset time similar to salbutamol, and a dose-dependent
duration of action.5,6 In the last 10 years there has been great inter-
est within the pharmaceutical industry in the discovery of a third
generation, once daily b2 adrenoceptor agonist to be used in new
combination therapies for the treatment of asthma and COPD. Nov-
artis have recently published data on their clinical candidate, ind-
acaterol (4),7 which has now been approved for the treatment of
COPD in Europe. A review by Glossop and Price summarises the
progress up to 2006 made by pharmaceutical companies in identi-
fying inhaled b2-agonists with extended duration of action.8 The
Pfizer group have published three papers9–11 describing their
earlier candidates, and more recently on their clinical candidate
PF-610355 (5).12 Our group has published two papers one on sul-
fonamides, including our first candidate 6,13 and the second on
antedrugs, including the clinical candidate vilanterol (7).14 Finally
the Boehringer-Ingelheim group have published three papers
including their clinical candidate olodaterol (8).15–17
⇑
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.2011.05.064

P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
4193
OH
OH
H
H
N
N
HO
HO
O
HO
HO
2
1
OH
H
N
OH
H
N
HO
N
OMe
HO
3
NHCHO
OH
4
O
H
N
H
N
O
5
6
OH
HO
NHSO2Me
OH
H
N
SO2NH2
O
O
HO
HO
Cl
OH
H
N
7
O
HO
Cl
HO
O
HN
OH
O
H
N
8
OMe
OH
Chart 1. Clinically used b2 agonists, and recent development candidates.
A major fraction of the dose (80–90%) of an inhaled drug is
swallowed and liable to be absorbed from the gastro-intestinal
tract.18 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.13 A second approach (the antedrug ap-
proach) could be to introduce metabolic instability to the molecule
to facilitate its conversion to inactive metabolites following sys-
temic absorption from either the GI tract or the lung.14 Better still,
a combination of the two approaches may potentially deal with
both the inhaled and swallowed fractions of each dose. In this re-
port we present our studies in identifying alternative, non-sulfon-
amide, b2 adrenoceptor agonists with reduced oral absorption.
Some of the physicochemical parameters that influence oral
absorption are molecular weight, lipophilicity, membrane perme-
ability, the number of hydrogen-bond donors and acceptors, con-
formational flexibility and solubility.19 Substitution on the right-
hand side phenyl ring of salmeterol with the polar sulfonamide
group was found to enhance b2 agonist activity.13 It was hypothe-
sised that an alternative polar substituent, such as a heterocycle,
with increased number of hydrogen-bond donors and acceptors
which contravened the Lipinski rules,20 might be expected to show
reduced oral bioavailability. Hydantoin and uracil rings, which pos-
sess two hydrogen-bond acceptor groups and two hydrogen-bond
donors, were considered as good starting points for investigation.
We have demonstrated that introduction of the polar sulfonamide
group brings about longer duration of action.13 It was therefore
hypothesised that introduction of the hydantoin or uracil ring
might also bind in a similar way to the sulfonamide group of 6
and hence have similarly long duration of action.
2. Chemistry
The aminoalcohol 9 with the R configuration was prepared by
reduction of the ketone 1021 with borane-dimethylsulfide and
(R)-2-methyl-CBS-oxazaborolidine as catalyst, followed by cata-
lytic hydrogenation of the resulting azido alcohol 11 (Scheme 1).
The enantiomeric ratio of 11 was determined by 1H NMR spectros-
copy using Pirkle’s alcohol22 at 750 MHz and found to be 9:1.
The b2-adrenoceptor agonists 12 were prepared using the syn-
thetic route outlined in Scheme 2, which involved Sonogashira
coupling of acetylene 1313 with aryl iodides 14, reaction of the
resulting bromide 15 with 2 equiv of amino alcohol 9 in order to
minimise bis-alkylation,21 catalytic hydrogenation of the mono-
alkylated product 16, and finally deprotection of 17 with aqueous
acetic acid.
The substituted aryl iodides 14 were obtained by the methods
shown in Scheme 3.23,24 Hydantoins 14a–c were prepared from
the appropriate regioisomeric iodoaniline and ethyl isocyanatoac-
etate, base-hydrolysis of the resulting ester 18, followed by cyclisa-
tion under acidic conditions. The analogue 14d was prepared from
BOC-a-methyl-alanine and 3-iodoaniline, followed by cyclisation
with sodium hydride in DMF. The regioisomeric hydantoin 14e

4194
P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
OH
O
OH
N3
N3
NH2
O
O
O
a
b
O
O
O
10
11
9
Scheme 1. Synthesis of (R)-enantiomer of aminoalcohol 9. Reagents: (a) BH3ÁSMe2, (R)-2-methyl-CBS-oxazaborolidine, 68%; (b) H2, 10% Pd/C, EtOH, 78%.
R
R
b
a
Br
I
O
Br
O
14
13
15
R
OH
OH
H
N
H
N
c
O
O
O
O
R
16
17
O
O
OH
H
N
R
d
O
HO
HO
12
O
O
O
O
O
O
N
NH
Me
N
NH
N
NH
N
NH
N
NH
O
N
NH
O
O
O
O
O
O
Me
d
e
f
g
h
a meta-isomer
b ortho-isomer
c para-isomer
O
O
O
O
O
N
N
N
NH
N
NH
N
NH
NH2
CO2H
N
N
H
H
O
O
Cl
O
O
O
m
i
j
k
l
Scheme 2. Synthetic route to analogues 12 and list of substituents R present in compounds 12 and 14–17. Reagents: (a) Pd(PPh3)2Cl2, CuI, i-Pr2EtN, DMF; (b) 9 (2 equiv),
DMF; (c) H2, PtO2, EtOAc; (d) AcOH–H2O (3:1), 75 °C.
was prepared by reaction of 3-iodophenylurea 19 and ethyl
chloroacetate in the presence of sodium hydride. The homologues
of 14a and 14e (14f and 14g, respectively) were prepared by sim-
ilar methods starting from 3-iodobenzylamine, whereas the ana-
logue 14h was prepared from 3-iodobenzyl bromide and
parabanic acid (20). The hydantoin 14i was prepared from 14e
by alkylation with 2-bromoacetamide in the presence of sodium
hydride. The pyrimidinedione 14j was prepared from urea 19
and diethyl malonate, followed by reaction of the resulting 21 with
phosphorus oxychloride, whereas the regioisomeric pyrimidinedi-
one 14l was obtained by reacting 19 with methyl 3,3-dimethoxy-
propionate (22) in the presence of sodium hydride. The de-
chlorination of 14j was not attempted as hydrogenolysis would
have concurrently removed the iodo group. The chloro group was
therefore removed at a later stage, at the hydrogenation of 16j to
give 17k. Finally hydantoic acid 12m was made by hydrolysis of
17a, followed by aqueous acetic acid deprotection.
and the potency is reported as pEC50 values (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 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. The relative instability of the compounds following oral
absorption and first-pass metabolism in the liver was assessed by
incubating test compounds with human liver microsomes (P450
content = 125 pmol/mL) at a compound concentration of 5 lM for
30 min at 37 °C. Compound turnover was expressed as a ratio rel-
ative to the assay standard, verapamil. Salmeterol (2) which had a
slightly higher turnover ratio (1.2) was also included as a standard.
All the compounds tested showed high b2 receptor agonist activity
(substantially more potent than the standard, isoprenaline). The
meta-hydantoin 12a was more potent agonist than the ortho-
(12b) and para- (12c) analogues, as was previously discovered in
the sulfonamide series.13 Furthermore 12a had higher selectivity
for b2 over b1 and b3 receptors than the regioisomers 12b and
12c, and also had a higher turnover ratio, and hence all other ana-
logues reported herein are in the meta- series. Substitution in the
3. 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 b1, b2 or b3 adrenoceptors. Agonist activity
was assessed by measuring changes in intracellular cyclic AMP,

P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
4195
H
N
O
a
b,c
e
O
N
O
O
NH2
N
H
N
O
H
I
I
O
O
I
14a-c
18a-c
d
O
O
N
H
N
I
NH2
I
N
H
I
NH
14d
O
f
O
O
I
N
NH
14e
I
N
NH2
H
19
O
H
N
O
a
b,c
O
H
N
H
14f
N
O
NH2
N
I
I
I
O
O
H
N
O
g
h,i
O
H
N
O
14g
N
I
O
I
O
H
N
O
N
e
HN
O
NH
O
Br
O
I
I
20
14h
O
NH2
O
Br
e
14e
+
I
N
O
N
NH2
14i
O
N
O
O
O
j
k
19
19
I
NH
Cl
I
N
NH
O
O
O
14j
21
O
e
I
N
NH
MeO
O
O
14l
OMe O
22
Scheme 3. Synthesis of substituted aryl iodides 14. Reagents: (a) EtO2CCH2NCO, DCM; (b) NaOH, H2O, EtOH; (c) 2 M HCl; (d) tetramethylfluoroformamidinium
hexafluorophosphate, i-Pr2EtN, DCM; (e) NaH, DMF; (f) ClCH2CO2Et, NaH, DMF; (g) BrCH2CO2Et, i-Pr2EtN, DMSO; (h) NaNCO, 1 M HCl; (i) concd HCl; (j) EtO2CCH2CO2Et, NaH,
MeOH; (k) POCl3.
C5-position of the hydantoin ring (12d) was tolerated, although the
IA was reduced slightly, and in addition its turnover ratio was low-
er than that of 12a. The regioisomeric hydantoin 12e was less po-
tent and more stable in microsomes than 12a, whereas the
homologous hydantoins 12f and 12g, although they were more
rapidly turned over in microsomes, had lower intrinsic activities
than their respective parents 12a and 12e. Introduction of an addi-
tional carbonyl group as in the parabanic acid derivative 12h was
well tolerated; however, the intrinsic activity and the selectivity
for b2 were slightly reduced. Introduction of the acetamide moiety
(12i) improved the b2 potency, selectivity and turnover ratio; how-
ever, it lowered its intrinsic activity. The uracil derivative 12k pos-
sessed excellent potency, intrinsic activity and selectivity, whereas
the isomeric analogue 12l had lower intrinsic activity and selectiv-
ity. Finally, the hydantoic acid 12m had good potency, intrinsic
activity and selectivity, but it was metabolically more stable than
12a with a turnover ratio of 0.55.
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 dura-
tion of action.25 Test compounds were investigated for their ability
to inhibit the contraction of guinea pig trachea strips expressed as
a measure of the functional response at the b2 adrenoceptor. Tis-
sues were contracted electrically, agonist was perfused over the

4196
P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
Table 1
Stimulation of cAMP Accumulation in CHO Cells Expressing Human b2, b1 and b3 adrenoceptors, and human liver microsomal turnover ratio against verapamil
a
a
b
a
c
Compound
b2 pEC50 (n)
IA
b1 pEC50 (n)
b2–b1
b3 pEC50 (n)
b2–b3
Turnover ratiod
1
2
3
4
5
6
7
8
12aÁAcOH
12b
9.2 0.1 (6)
8.6 0.1 (3)
8.7 0.1 (2)
9.1 0.2 (4)
8.6 0.1 (7)
8.9 0.2 (2)
9.2 0.0 (2)
9.1 0.1 (4)
9.6 0.2 (4)
9.1 0.1 (4)
8.9 0.1 (2)
9.0 0.2 (2)
7.4 0.0 (767)
9.6 0.0 (929)
9.3 0.0 (791)
0.40
0.49
0.55
0.32
0.50
0.33
0.42
0.32
0.36
0.70
0.49
0.52
1.0
7.1 0.2 (4)
6.7 0.1 (6)
7.1 0.1 (2)
6.7 0.1 (4)
6.7 0.1 (6)
6.9 0.2 (6)
7.1 0.0 (3)
7.3 0.1 (4)
6.8 0.1 (2)
6.6 0.1 (8)
7.3 0.1 (8)
6.9 0.6 (2)
8.1 0.0 (641)
6.1 0.0 (656)
7.4 0.0 (670)
2.1
1.9
1.6
2.4
1.9
2.0
2.1
1.8
2.8
2.5
1.6
2.1
À0.7
3.5
1.9
7.0 0.1 (4)
6.7 0.1 (8)
7.5 0.1 (2)
7.2 0.1 (4)
6.5 0.1 (6)
7.4 0.1 (4)
7.2 0.0 (4)
7.6 0.0 (4)
7.1 0.1 (2)
6.4 0.0 (8)
7.3 0.0 (8)
7.0 0.4 (2)
7.4 0.0 (659)
5.9 0.0 (849)
7.6 0.0 (653)
2.1
1.9
1.2
1.9
2.1
1.5
2.0
1.5
2.5
2.7
1.6
2.0
0.0
3.7
1.7
0.86
0.46
0.30
0.48
0.40
1.62
0.75
NDe
0.91
NDe
NDe
0.55
NDe
1.2
12cÁAcOH
12dÁAcOH
12eÁAcOH
12fÁAcOH
12gÁAcOH
12hÁHCO2H
12iÁAcOH
12k
9
10
11
12
13
14
15
12lÁAcOH
12m
Isoprenaline
2
(R,R)-3Áfumarate
0.37
0.97
NDe
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 relative to verapamil, where verapamil has a turnover ratio of 1.
ND, not determined.
c
d
e
tissue until maximum relaxation was achieved, and onset of action
determined. Perfusion of the agonist was then ceased, tissue con-
tinued to be perfused with buffer, and duration of action
determined by the time taken for the contractile response to re-
establish. The more potent analogues with the highest IA, selectiv-
ity, and highest turnover ratio (12a, e, k, m) were screened on iso-
lated electrically-stimulated superfused guinea pig trachea and
their potency, onset-time and in vitro duration after 1 and 3 h is
presented and contrasted to salmeterol and (R,R)-formoterol (Table
2). Potency was expressed in absolute terms (concentration re-
quired to induce 50% inhibition, EC50). Onset of action was calcu-
lated as the time taken for EC50 concentration to achieve 50%
maximum relaxant effect. Duration of action was determined by
measuring the recovery of electrically induced contraction follow-
ing washout of agonist. This was expressed as the rightward shift
in the agonist concentration-effect curve following 1 h and 3 h of
washout (EC50 for test compound after 60 or 180 min of wash-
out/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 (formoterol-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 salmeterol. The hydantoins 12a and 12e had low shift
values indicating a salmeterol-like duration of action, whereas ura-
cil 12k and hydantoic acid 12m had a different profile, intermedi-
ate between that of salmeterol and that of formoterol. Hydantoin
12a was more potent on both CHO cells and guinea pig trachea,
had a significantly shorter onset time (26 min vs 44 min), and
was metabolised more rapidly (0.86 vs 0.40) than 12e. For these
reasons 12a was progressed to pharmacokinetic studies in rats
and dogs and the data is shown in Table 3. The gut appears to con-
tribute significantly to phase II first-pass metabolism of salmeterol
in the rat, potentially limiting the discrimination window for com-
pounds in this model. Salmeterol is subject to conjugation as it is
absorbed (90% glucuronidated), thus the measurement of free
salmeterol in the hepatic portal vein is a large underestimate of
compound actually absorbed. Up to 30 times higher levels of
salmeterol were detected in hepatic portal and systemic blood
after deconjugation with b-glucuronidase. Deconjugation of hepa-
tic portal blood samples with b-glucuronidase and measurement of
total parent compound was therefore used to increase the resolu-
tion between well and poorly absorbed compounds. Compounds
were classified as less absorbed than salmeterol if the levels of
HPV—CP AUC was <7000 ng min/mL following a 2 mg/kg oral dose,
salmeterol-like if the levels were 7000–23,000 ng min/mL, and bet-
ter absorbed than salmeterol if AUC was >23,000 ng min/mL.
Deconjugated hepatic portal vein samples from rats dosed with
salmeterol and 12a indicated that 12a had lower absorption than
salmeterol (AUC <2340 ng min/mL). Furthermore 12a had a half-
life of 0.3 h and high plasma clearance of 120 mL/min/kg (140%
liver blood flow) in rats. Its half-life in dogs was 2 h, and its clear-
ance was moderate (42% liver blood flow). Low oral bioavailability
of 12a was observed in vivo in both rats (2%) and dogs (5%) (Table
3). This oral bioavailability was much lower than that previously
reported for salmeterol (66–78% in dog, and 10–12% in rat)26 and
strengthened the confidence that the swallowed fraction of the in-
haled dose in humans would be unlikely to contribute to the sys-
temic exposure of 12a.
The acetate salt of 12a was also investigated in vivo in the gui-
nea pig in a whole animal plethysmograph chamber (Buxco) using
histamine-induced bronchospasm and found to have similar po-
Table 2
The potency, onset time and duration of action of selected compounds on isolated
tency to salmeterol (EC90 25 lM and 50 lM, respectively, nebulizer
superfused guinea pig trachea (minimum n = 2)
concentration). At an equi-effective (EC90) dose, the duration of ac-
tion of 12a acetate (time to 50% recovery) was similar or slightly
longer than that of salmeterol (12 h vs 10 h) when administered
as a nebulised solution in a dimethyl acetamide-saline vehicle. Fur-
thermore the duration of 12a was extended to 18 h when the dose
was increased to 10-fold the EC90. Hydantoin 12a was then pro-
gressed towards a salt selection screen to identify a stable, non-
hygroscopic and crystalline salt, suitable for inhaled delivery.
Entry
Compound
pEC50
Onset
time (min)
Shift (1 h)
Shift (3 h)
1
2
3
4
5
6
12aÁAcOH
9.1
8.6
9.7
9.0
8.3
9.5
26.4
44
9.0
14
27.6
10
3.7
2.9
15
19
1.0
20
3.9
3.3
77
35
1.1
12eÁAcOH
12kÁAcOH
12mÁAcOH
Salmeterol
(R,R)-3Áfumarate
>1940

P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
4197
Table 3
Rat and dog pharmacokinetic data for 12a acetate
Species
Rat
Route
Dose (mg/kg)
Clp (mL/min/kg)
120
Vdss (L/kg)
T
(h)
Cmax (ng/mL)
F%
½
Intravenous
Oral
Intravenous
Oral
0.25
2.0
0.052
0.25
2
0.3
2.0
2
5
Dog
13
0.8
12
Unfortunately 12a free base was found to be unstable in the solid
state to storage at 4 °C over 18 days [13% dimerisation with loss of
water; LCMS ES+ve m/z 1009 (2MÀ18)+]. Furthermore the hydan-
toin ring was unstable to nucleophilic solvents, such as methanol,
forming slowly the methyl ester of the hydantoic acid 12m. The
acetic acid salt of 12a, which was amorphous, appeared to be sta-
ble; however, no other salts were found to be crystalline. Finally,
during drug metabolite identification studies it was discovered
that the potent hydantoic acid 12m was a minor metabolite of
12a in human liver microsomes and hepatocytes. Further progres-
sion of 12a was halted as we were interested only in analogues
that rapidly metabolised to inactive metabolites.
to 30 °C towards the end of the addition and was cooled in ice-
water bath to maintain the temperature at 20 °C. The mixture
was stirred for 2 h at 20 °C and turned orange colour while a white
precipitate formed. The reaction mixture was divided into two
equal portions and each portion was diluted with EtOAc (1.2 L)
and water (3 L), the phases were separated and the aqueous phase
extracted with more EtOAc (1 L). The combined organic solutions
were washed with saturated sodium bicarbonate (2 L), brine (2 L)
and dried (MgSO4). The solvent was removed under reduced pres-
sure, the resulting orange solid was dissolved in dichloromethane,
and filtered through silica (1 kg) eluting with dichloromethane.
Appropriate fractions were combined and evaporated under re-
duced pressure to give 10 (310 g, 97%) as a pale yellow solid: MS
TSP+ve m/z 248 (M+H)+; 1H NMR d (CDCl3) 7.72 (1H, dd, J 8.6,
2 Hz), 7.63 (1H, d, J 2 Hz), 6.88 (1H, d, J 8.6 Hz), 4.89 (2H, s), 4.48
(2H, s), 1.56 (6H, s).
4. Conclusion
Incorporation of a hydantoin or a uracil ring on the right-hand
side phenyl ring of (R)-salmeterol has provided a series of novel,
very potent and selective human b2 adrenoceptor agonists. Hydan-
toin 12a had long duration of action in vitro on guinea pig trachea,
5.2. (1R)-2-Azido-1-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)etha
nol (11)
and 12 h in guinea pigs in vivo at its EC90 25 lM. It had lower oral
(R)-Tetrahydro-1-methyl-3,3-diphenyl-1H,3H-pyrrolo[1,2-c]
[1,3,2]oxazaborole solution in toluene (1 M, 7.5 mL) was added
to THF (75 mL) and the solution was cooled to 0 °C. Borane–
THF complex (1 M solution in THF, 125 mL) was added and the
mixture was stirred under nitrogen for 15 min. A solution of
10 (24.7 g, 0.1 mol) in THF (250 mL) was added dropwise over
1.5 h at 5 °C. The mixture was stirred for a further 1 h and then
cautiously treated with 2 M HCl (100 mL). The reaction mixture
was extracted with ether and the organic layer was washed with
2 M HCl, saturated aq NaHCO3, brine and dried (MgSO4). The sol-
vent was removed under reduced pressure and the residue was
chromatographed on a Biotage column eluting with diethyl
ether–petroleum ether (40–60 °C) (1:9; 1:1) to give 11
(16.99 g, 68%) as a white solid: MS ES+ve m/z 250 (M+H)+; 1H
NMR d (CDCl3) 7.10 (1H, dd, J 8, 2 Hz), 6.95 (1H, d, J 2 Hz),
6.78 (1H, d, J 8 Hz), 4.78 (2H, s), 4.75 (1H, m), 3.42 (1H, dd, J
13, 8 Hz), 3.33 (1H, dd, J 13, 4 Hz), 2.90 (1H, d, J 4 Hz), 1.52
(6H, s). C12H15N3O3 requires C, 57.8; H, 6.1; N, 16.9. Found: C,
57.9; H, 6.0; N, 16.5.
absorption than salmeterol in rats, and lower bioavailability than
salmeterol in vivo in both rats and dogs (2% and 5%, respectively).
However 12a was metabolised in human liver microsomes and
hepatocytes to the active hydantoic acid 12m, and no crystalline
salts suitable for inhaled delivery were identified.
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 spectrom-
eter using electrospray positive and negative mode (ES+ve and
ESÀve). Column chromatography was performed on Merck Kiesel-
gel 60 (art. 9385), or Biotage pre-packed silica gel cartridges con-
taining 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 tetramethyl-
silane. 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.
5.3. (1R)-2-Amino-1-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)eth
anol (9)
A solution of 11 (16.99 g, 68.2 mmol) was hydrogenated over
10% Pd–C (1 g) in EtOH (300 mL). The catalyst was collected by fil-
tration, and washed with EtOH. The combined filtrate and wash-
ings were evaporated under reduced pressure and the residue
was triturated in ether to give 9 (5.86 g, 38%) as a white solid.
The mother liquors were chromatographed on a Biotage column
eluting with toluene–EtOH–0.88 aqueous ammonia (85:14:1) to
20
5.1. 2-Azido-1-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)ethan
one (10)
give a further batch of 9 (5.99 g, 39%) as a white solid: [a]
D
21.4 (c 1.26 in MeOH); MS ES+ve m/z 206 (MHÀH2O)+; 1H NMR d
(CDCl3) 7.11 (1H, dd, J 8.2 Hz), 6.99 (1H, br s), 6.79 (1H, d, J
8 Hz), 4.82 (2H, s), 4.54 (1H, dd, J 8.4 Hz), 2.96 (1H, dd, J 13.4 Hz),
2.77 (1H, dd, J 13.8), 1.54 (6H, s), 1.90–1.20 (3H, br). The enantio-
meric ratio was determined by 1H NMR (750 MHz) using Pirkle’s
2-Bromo-1-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)ethanone27
(370 g, 1.29 mol) in DMF (2.3 L) was treated portionwise with so-
dium azide (87.06 g, 1.33 mol) over 30 min. The temperature rose

4198
P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
alcohol [2,2,2-trifluoro-1-(9-anthracenyl)ethanol] (20 mg) on the
crude product before chromatography, and was found to be
(10:90) [4.30 ppm (minor isomer); 4.26 ppm (major isomer)].
(3H, m), 7.12 (1H, dd, J 8, 2 Hz), 7.00 (1H, br s), 6.78 (1H, d, J
8 Hz), 4.83 (2H, s), 4.61 (1H, dd, J 9, 3 Hz), 4.09 (2H, s), 3.61 (2H,
t, J 7 Hz), 3.49 (2H, t, J 6.5 Hz), 2.82 (1H, dd, J 12, 4 Hz), 2.70–2.55
(6H, m), 1.64–1.30 (10H, m), 1.53 (6H, s).
C12H17NO3 requires C, 64.6; H, 7.7; N, 6.3. Found: C, 64.8; H, 7.7;
N, 6.2.
5.8. 3-(3-{4-[(6-{[(2R)-2-(2,2-Dimethyl-4H-1,3-benzodioxin-6-
yl)-2-hydroxyethyl]amino}hexyl)oxy]butyl}imidazolidine-2,4-
dione (17a)
5.4. Ethyl N-{[(3-iodophenyl)amino]carbonyl}glycinate (18a)
A solution of 3-iodoaniline (9.39 g, 42.9 mmol) in dichloro-
methane (75 mL) was treated with ethyl isocyanatoacetate
(7.21 mL, 64.3 mmol) at 0 °C and the mixture was stirred for 2 h
and allowed to warm to 20 °C. EtOH (10 mL) was added and the
mixture was stirred for 15 h. The solvents were evaporated under
reduced pressure and the residue was triturated in Et2O to give
18a (12.9 g, 86%) as an off-white solid: MS ES+ve m/z 349
(M+H)+; 1H NMR d (CDCl3) 7.74 (1H, t, J 2 Hz), 7.35 (1H, br d, J
8 Hz), 7.29–7.24 (2H, m), 6.97 (1H, t, J 8 Hz), 5.72 (1H, br), 4.21
(2H, q, J 7 Hz), 4.04 (2H, br s), 1.30 (3H, t, J 7 Hz).
A solution of 16a (368 mg, 0.67 mmol) was hydrogenated over
platinum oxide (100 mg) in EtOAc (100 mL) over 3 h. The catalyst
was collected by filtration, washed with EtOAc and EtOH. The com-
bined filtrate and washings were evaporated under reduced pres-
sure to give 17a as a colourless gum: MS ES+ve m/z 554 (M+H)+;
1H NMR d (CDCl3) 7.37 (1H, t, J 7 Hz), 7.23–7.16 (3H, m), 7.11
(1H, d, J 8 Hz), 7.00 (1H, br s), 6.78 (1H, br d, J 8 Hz), 4.83 (2H, s),
4.63 (1H, dd, J 9, 3.5 Hz), 4.07 (2H, s), 3.41 and 3.38 (2t, 2H each,
J 6.5 Hz), 2.83 (1H, dd, J 12, 4 Hz), 2.71–2.56 (6H, m), 1.75–1.45
(10H, m), 1.53 (6H, s), 1.40–1.28 (4H, m).
5.5. 3-(3-Iodophenyl)imidazolidine-2,4-dione (14a)
5.9. 3-[3-(4-{[6-([(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxyme
thyl)phenyl]ethyl}amino) hexyl]oxy}butyl)phenyl]imidazoli
dine-2,4-dione acetate (12a)
A solution of ethyl N-{[(3-iodophenyl)amino]carbonyl}glycinate
(9.42 g, 27 mmol) in DMF (60 mL) was treated with sodium hy-
dride (60% oil dispersion, 1.2 g, 30 mmol) at 20 °C. After 2 h the
reaction mixture was treated with aqueous 2 M HCl (200 mL)
and stirred overnight. The solid was collected by filtration, washed
with water and dried to give 14a (6.9 g, 84%) as a white solid: MS
ES+ve m/z 320 (M+NH4)+; 1H NMR d (DMSO-d6) 8.36 (1H, s), 7.76
(2H, m), 7.40 (1H, br d, J 8 Hz), 7.28 (1H, t, J 8 Hz), 4.05 (2H, s).
A solution of 17a (417 mg, 0.75 mmol) in acetic acid (20 mL)
and water (5 mL) was heated to 75 °C for 30 min before evaporat-
ing to dryness under reduced pressure. The residue was purified by
chromatography on a Biotage cartridge (40 g) eluting with CH2Cl2–
MeOH–2 M NH3 in MeOH (85:10:5). Appropriate fractions were
combined and evaporated to dryness. Acetic acid (4 mL) was added
and the mixture was evaporated under reduced pressure. The res-
idue was dissolved in EtOAc and re-evaporated under reduced
pressure (twice). This process of dissolving and re-evaporating
was repeated with diethyl ether, followed by acetone to give 12a
(290 mg, 67%) as a white foam: LCMS RT = 2.44 min; ES+ve m/z
514 (M+H)+; 1H NMR (DMSO-d6+D2O) 7.36 (1H, t, J 8 Hz), 7.28
(1H, br s), 7.20 (1H, d, J 8 Hz), 7.10 (1H, s), 7.09 (1H, d, J 8 Hz),
7.04(1H, dd, J 8, 2 Hz), 6.73 (1H, d, J 8 Hz), 4.73 (1H, dd, J 5,
8 Hz), 4.45 (2H, s), 4.08 (2H, s), 3.33 and 3.30 (2t, 2H each, J
7 Hz), 2.98–2.90 (2H, m), 2.86 (2H, t, J 7 Hz), 2.59 (2H, t, J 7 Hz),
1.85 (3H, s), 1.62–1.40 (8H, m), 1.30–1.20 (4H, m); HRMS ES+ve
m/z 514.2917. C28H40N3O6 requires 514.2917.
5.6. 3-(3-{4-[(6-Bromohexyl)oxy]but-1-ynyl}phenyl)imidazoli
dine-2,4-dione (15a)
A mixture of 3-(3-iodophenyl)imidazolidine-2,4-dione (0.75 g,
2.5 mmol), 6-bromohexyl but-3-ynyl ether13 (1.3 g, 3.0 mmol),
bis(triphenylphosphine)palladium dichloride (87 mg, 0.12 mmol)
in DMF (10 mL) was treated with diisopropylethylamine (3 mL)
and copper(I) iodide (23 mg, 0.12 mmol), and the mixture was stir-
red under nitrogen for 24 h. The solvents were removed under re-
duced pressure, the residue was diluted with EtOAc and washed
with aqueous 2 M HCl, aqueous dilute ammonia, brine and dried
(MgSO4). The solvent was removed under reduced pressure, the res-
idue was purified on two Bond Elut 10 g silica cartridges eluting with
dichloromethane, Et2O, and EtOAc to give a mixture of 15a and the
5.10. 3-(2-Iodophenyl)-2,4-imidazolidinedione (14b)
corresponding
iodide,
3-(3-{4-[(6-iodohexyl)oxy]but-1-ynyl}
phenyl)imidazolidine-2,4-dione, (760 mg, ratio 22:3) as a brown
oil: 1H NMR d (CDCl3) 7.46–7.31 (4H, m), 6.53 (1H, br s), 4.11 (2H,
s), 3.62 (2H, t, J 7 Hz), 3.49 (2H, t, J 7 Hz), 3.40 (2H, t, J 7 Hz), 2.68
(2H, t, J 7 Hz), 1.90–1.80 (2H, m), 1.65–1.55 (2H, m), 1.51–1.35
(4H, m), and 3.18 (2H, t, J 7 Hz) for the iodide minor component.
MS ES+ve m/z 303 (M+H)+; 1H NMR d (DMSO-d6) 8.43 (1H, br s),
8.04 (1H, br d, J 8 Hz), 7.58 (1H, br t, J 8 Hz), 7.43 (1H, br d, J 8 Hz),
7.30 (1H, br t, J 8 Hz), 4.19 (2H, s).
5.11. 3-(4-Iodophenyl)-2,4-imidazolidinedione (14c)
5.7. 3-(3-{4-[(6-{[(2R)-2-(2,2-Dimethyl-4H-1,3-benzodioxin-6-
yl)-2-hydroxyethyl]amino}hexyl)oxy]but-1-ynyl}phenyl)imidaz
olidine-2,4-dione (16a)
MS ES-ve m/z 301 (MÀH)À; 1H NMR d (DMSO-d6) 8.35 (1H, br s),
7.83 (2H, d, J 8.5 Hz), 7.18 (2H, d, J 8.5 Hz), 4.05 (2H, s).
5.12. 3-(3-Iodophenyl)-5,5-dimethyl-2,4-imidazolidinedione
(14d)
A mixture of 15a and 3-(3-{4-[(6-iodohexyl)oxy]but-1-ynyl}
phenyl)imidazolidine-2,4-dione (760 mg, 22:3, 1.8 mmol total
alkylating agent), 9 (800 mg, 3.6 mmol, 2 equiv) in DMF (5 mL)
was stirred at 20 °C for 22 h. The solvent was removed under re-
duced pressure and the residue was diluted with EtOAc and
washed with water, brine and dried (MgSO4). The solution was
concentrated under reduced pressure and the residue was purified
by chromatography on a Biotage cartridge (40 g) eluting with
dichloromethane–MeOH–1 M ammonia in methanol (98:1:
1–95:4:1) to give 16a as a yellow gum (368 mg, 37%): MS ES+ve
m/z 550 (M+H)+; 1H NMR d (CDCl3) 7.45 (1H, br s), 7.40–7.30
MS ES+ve m/z 331 (M+H)+; 1H NMR d (DMSO-d6) 8.60 (1H, br s),
7.80 (1H, t, J 2 Hz), 7.77 (1H, dt, J 8, 2 Hz), 7.42 (1H, br d, J 8 Hz),
7.28 (1H, t, J 8 Hz), 1.40 (6H, s).
5.13. 1-(3-Iodophenyl)-2,4-imidazolidinedione (14e)
MS ES+ve m/z 303 (M+H)+; 1H NMR d (DMSO-d6) 11.26 (1H, br
s), 8.06 (1H, br s), 7.55 (1H, br d, J 8 Hz), 7.46 (1H, br d, J 8 Hz), 7.16
(1H, t, J 8 Hz), 4.42 (2H, s).

P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
4199
5.14. 3-[(3-Iodophenyl)methyl]-2,4-imidazolidinedione (14f)
5.23. 1-[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxy
methyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]-2,4-imida
zolidinedione formate salt (12e)
1H NMR d (DMSO-d6) 8.17 (1H, s), 7.65 (2H, m), 7.28 (1H, br d, J
8 Hz), 7.14 (1H, t, J 8 Hz), 4.49 (2H, s), 3.99 (2H, s).
LCMS RT = 2.47 min; ES+ve m/z 514 (M+H)+; 1H NMR d (CD3OD)
8.27 (1H, s), 7.47 (1H, br s), 7.37–7.32 (2H, m), 7.26 (1H, t, J 8 Hz),
7.14 (1H, dd, J 8.2 Hz), 6.97 (1H, d. J 8 Hz), 6.77 (1H, d, J 8 Hz), 4.83
(1H, overlapping with CD3OH), 4.64 (2H, s), 4.42 (2H, s), 3.44–3.42
(4H, m), 3.12–3.06 (2H, m), 3.02 (2H, t, J 8 Hz), 2.64 (4H, m), 1.77–
1.63 (4H, m), 1.63–1.54 (4H, m), 1.47–1.37 (4H, m).
5.15. 1-[(3-Iodophenyl)methyl]-2,4-imidazolidinedione (14g)
1H NMR d(DMSO-d6) 10.89 (1H, br s), 7.65 (2H, m), 7.30 (1H, br
d, J 8 Hz), 7.16 (1H, t, J 8 Hz), 5.76 (2H, s), 4.40 (2H, s).
5.16. 1-[(3-Iodophenyl)methyl]-2,4,5-imidazolidinetrione (14h)
5.24. 3-{[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxy
methyl)phenyl]ethyl}amino) hexyl]oxy}butyl)phenyl]methyl}-
2,4-imidazolidinedione acetate salt (12f)
MS ES+ve m/z 348 (M+NH4)+; 1H NMR d (CD3OD) 7.97 (1H, br s),
7.76 (1H, br s), 7.65 (1H, d, J 8 Hz), 7.38 (1H, d, J 8 Hz), 7.11 (1H, t, J
8 Hz), 4.68 (2H, s).
LCMS RT = 2.44 min; ES+ve m/z 528 (M+H)+; 1H NMR d (CD3OD)
7.36 (1H, br s), 7.25–7.09 (5H, m), 6.80 (1H, d, J 8 Hz), 4.87 (1H,
overlapping with CD3OH), 4.67 (2H, s), 4.61 (2H, s), 4.00 (2H, s),
3.46 (2H, t, J 7 Hz), 3.44 (2H, t, J 7 Hz), 3.17–3.08 (2H, m), 3.04
(2H, t, J 7 Hz), 2.63 (2H, t, J 7 Hz), 1.99 (3H, s), 1.78–1.54 (8H, m),
1.50–1.40 (4H, m).
5.17. 2-[3-(3-Iodophenyl)-2,5-dioxo-1-imidazolidinyl]acetam
ide (14i)
MS ES+ve m/z 360 (M+H)+; 1H NMR d (CD3OD) 8.24 (1H, br s),
7.85 (1H, br s), 7.73 (1H, br d, J 8 Hz), 7.65 (1H, br d, J 8 Hz), 7.44
(1H, br s), 7.35 (1H, t, J 8 Hz), 4.69 (2H, s), 4.16 (2H, s).
5.25. 1-{[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxym
ethyl)phenyl]ethyl}amino) hexyl]oxy}butyl)phenyl]methyl}-
2,4-imidazolidinedione acetate salt (12g)
5.18. 6-Chloro-3-phenyl-2,4(1H,3H)-pyrimidinedione (14j)
MS ES+ve m/z 349/351 (M+H)+; 1H NMR d (DMSO-d6) 12.5 (1H,
br s), 7.77 (1H, br d, J 8 Hz), 7.69 (1H, br s), 7.31 (1H, br d, J 8 Hz),
7.26 (1H, t, J 8 Hz), 6.00 (1H, br s).
LCMS RT = 2.42 min; ES+ve m/z 528 (M+H)+; 1H NMR d (CD3OD)
7.32 (1H, br s), 7.26 (1H, t, J 7.5 Hz), 7.17–7.07 (4H, m), 6.77 (1H, d,
J 8 Hz), 4.83 (1H, dd, J 9, 4 Hz), 4.64 (2H, s), 4.47 (2H, s), 3.80 (2H, s),
3.43 (2H, t, J 7 Hz), 3.41 (2H, t, J 7 Hz), 3.13–3.05 (2H, m), 2.99 (2H,
t, J 7 Hz), 2.63 (2H, t, J 7 Hz), 1.91 (3H, s), 1.74–1.52 (8H, m), 1.44–
1.38 (4H, m).
5.19. 1-(3-Iodophenyl)-2,4(1H,3H)-pyrimidinedione (14l)
MS ES+ve m/z 315 (M+H)+; 1H NMR d (DMSO-d6) 11.5 (1H, br s),
7.85 (1H, br s), 7.80 (1H, br d, J 8 Hz), 7.72 (1H, d, J 8 Hz), 7.46 (1H,
br d, J 8 Hz), 7.29 (1H, t, J 8 Hz), 5.66 (1H, d, J 8 Hz).
5.26. 1-{[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxym
ethyl)phenyl]ethyl}amino)hexyl] oxy}butyl)phenyl]methyl}-
2,4,5-imidazolidinetrione formate salt (12h)
5.20. 3-[2-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxyme
thyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]-2,4-imidazo
lidinedione acetate salt (12b)
LCMS RT = 2.56 min; ES+ve m/z 542 (M+H)+; 1H NMR d (CD3OD)
8.45 (1H, s), 7.34 (1H, br s), 7.25–7.08 (5H, m), 6.77 (1H, d, J 8 Hz),
6.67 (2H, s), 4.64 (2H, s), 3.45–3.37 (4H, m), 3.16–3.07 (2H, m), 3.02
(2H, t, J 8 Hz), 2.61 (2H, t, J 7.5 Hz), 1.76–1.52 (8H, m), 1.45–1.37
(4H, m).
LCMS RT = 2.08 min; ES+ve m/z 514 (M+H)+; 1H NMR d (CD3OD)
7.40–7.32 (3H, m), 7.32–7.25 (1H, m), 7.15 (2H, d, J 8 Hz), 6.79 (1H,
d, J 8 Hz), 4.84 (1H, dd, J 9, 4 Hz), 4.65 (2H, s), 4.17 (2H, s), 3.41 (4H,
t, J 7 Hz), 3.19 (2H, m), 2.99 (2H, t, J 8 Hz), 2.53 (2H, t, J 7.5 Hz), 1.94
(3H, s), 1.76–1.51 (8H, m), 1.47–1.35 (4H, m).
5.27. 2-{3-[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-
(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]-
2,5-dioxo-1-imidazolidinyl}acetamide acetate salt (12i)
5.21. 3-[4-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxym
ethyl)phen yl]ethyl}amino)hexyl]oxy}butyl)phenyl]-2,4-imi
dazolidinedione acetate salt (12c)
MS ES+ve m/z 571 (M+H)+; 1H NMR d (CD3OD) 7.54 (1H, br s),
7.40 (1H, br d, J 8 Hz), 7.34 (1H, br s), 7.29 (1H, t, J 8 Hz), 7.15
(1H, dd, J 8, 2 Hz), 7.01 (1H, d, J 8 Hz), 6.78 (1H, d, J 8 Hz), 4.85
(1H, overlapping with CD3OH), 4.65 (2H, s), 4.48 (2H, s), 4.24
(2H, s), 3.49–3.40 (4H, m), 3.15–3.05 (2H, m), 3.00 (2H, t, J 8 Hz),
2.67 (2H, t, J 7.5 Hz), 1.94 (3H, s), 1.76–1.53 (8H, m), 1.47–1.36
(4H, m).
LCMS RT = 2.34 min; ES+ve m/z 514 (M+H)+; 1H NMR d (CD3OD)
7.37 (1H, br s), 7.34–7.26 (4H, m), 7.18 (1H, dd, J 8, 2 Hz), 6.81 (1H,
d, J 8 Hz), 4.87 (1H, dd, J 9.4 Hz), 4.67 (2H, s), 4.12 (2H, s), 3.47 (2H,
t, J 7 Hz), 3.45 (2H, t, J 7 Hz), 3.16–3.08 (2H, m), 3.02 (2H, t, J 8 Hz),
2.71 (2H, t, J 8 Hz), 1.99 (3H, s), 1.77–1.56 (8H, m), 1.47–1.39 (4H,
m).
5.28. 3-[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymet
hyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]-2,4(1H,3H)-
pyrimidinedione acetate salt (12k)
5.22. 3-[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxyme
thyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]-5,5-dime
thyl-2,4-imidazolidinedione acetate salt (12d)
LCMS RT = 2.21 min; ES+ve m/z 526 (M+H)+; 1H NMR d (CD3OD)
7.48 (1H, br d, J 8 Hz), 7.37 (1H, t, J 8 Hz), 7.33 (1H, br s), 7.25 (1H,
br d, J 7 Hz), 7.14 (1H, d, J 7 Hz), 7.05 (1H, br s), 7.01 (1H, br d, J
7 Hz), 6.77 (1H, d, J 8 Hz), 5.78 (1H, d, J 7 Hz), 4.64 (2H, s), 3.47–
3.38 (4H, m), 3.08–3.01 (2H, m), 2.95 (2H, t, J 8 Hz), 2.68 (2H, t, J
7 Hz), 1.92 (3H, s), 1.75–1.52 (8H, m), 1.45–1.33 (4H, m).
MS ES+ve m/z 542 (M+H)+; 1H NMR d (CD3OD) 7.39–7.34 (2H,
m), 7.25–7.13 (4H, m), 6.78 (1H, d, J 8 Hz), 4.85 (1H, overlapping
with CD3OH), 4.65 (2H, s), 3.48–3.40 (4H, m), 3.12–2.97 (4H, m),
2.69 (2H, t, J 7 Hz), 1.93 (3H, s), 1.76–1.52 (8H, m), 1.49 (6H, s),
1.46–1.37 (4H, m).

4200
P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
5.29. 1-[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxyme
thyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]-2,4(1H,3H)-
pyrimidinedione acetate salt (12l)
were used to calculate pharmacokinetic parameters from plasma
concentration versus time profiles.
5.34. Metabolite identification study
LCMS RT = 2.36 min; ES+ve m/z 526 (M+H)+; 1H NMR d (CD3OD)
7.63 (1H, d, J 8 Hz), 7.42 (1H, t, J 8 Hz), 7.37 (1H, d, J 2 Hz), 7.31 (1H,
br d, J 8 Hz), 7.28 (1H, br s), 7.21 (1H, br d, J 8 Hz), 7.18 (1H, dd, J 8,
2 Hz), 6.80 (1H, d, J 8 Hz), 5.80 (1H, d, J 8 Hz), 4.88 (1H, dd, J 4,
9 Hz), 4.68 (2H, s), 3.47 (2H, t, J 7 Hz), 3.45 (2H, t, J 7 Hz), 3.15–
3.07 (2H, m), 3.05–2.98 (2H, m), 2.72 (2H, t, J 7 Hz), 2.00 (3H, s),
1.78–1.55 (8H, m), 1.50–1.35 (4H, m).
Compound 12a at 25 lM was incubated with human liver
microsomes and hepatocytes and analysed by LCMS. Samples were
mixed with an equal volume of acetonitrile, centrifuged, and the
supernatant transferred to auto-sampler vials for analysis. Aliquots
(15
lL) were injected into a reverse phase gradient LCMS system
(10 cm  2.1 mm i.d. Luna C18
3
l
m column) and eluted compo-
nents detected by electrospray mass spectrometry using a Quattro
LC mass spectrometer. Two metabolites were detected: 12m
(ES+ve m/z 532 (M+H)+ (parent + 18), and a hydroxylation metab-
olite (ES+ve m/z 530 (M+H)+ (parent + 16). There was no evidence
for the presence of any phase II metabolites.
5.30. N-({[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxy
methyl)phenyl]ethyl}amino) hexyl]oxy}butyl)phenyl]amino}
carbonyl)glycine acetate salt (12m)
LCMS RT = 2.46 min; ES+ve m/z 532 (M+H)+; 1H NMR d (CD3OD)
7.33 (1H, br s), 7.25 (1H, br s), 7.16–7.08 (3H, m), 6.80–6.75 (2H,
m), 4.87 (1H, dd, J 9.5, 3.8 Hz), 4.63 (2H, s), 3.81 (2H, s), 3.45–
3.35 (4H, m), 3.12–3.02 (2H, m), 2.98 (2H, t, J 8 Hz), 2.55 (2H, t, J
7.5 Hz), 1.96 (3H, s), 1.74–1.49 (8H, m), 1.42–1.32 (4H, m).
Acknowledgments
We thank Mr. Bill J. Leavens for collecting the HRMS data, Mr.
Inder Bhamra for scaling up the preparation of 10, Miss Isobel
Hackney and Miss Sara C. Hughes for the in vivo work on 12a.
5.31. Pharmacokinetic studies in rats
A. Supplementary data
Male Han Wistar rats were fasted for 18 h prior to dose admin-
istration. 12a acetate salt was formulated as a solution in DMSO-
PEG 200-distilled water (5:30:65, v/v/v) for oral dosing, and
DMSO-PEG 200-saline (10:45:45, v/v/v) for intravenous dosing.
Rats were dosed orally by gavage tube or intravenously via a tail
vein at nominal dose levels of 2.0 mg 12a base/kg or 0.25 mg 12a
base/kg, respectively. Blood samples were taken at 0.03 (intrave-
nous only), 0.08, 0.25, 0.5, 0.75, 1, 2, 4 and 8 h post dose (n = 2 ani-
mals/time-point). Plasma was prepared from blood by
centrifugation, and analysed for 12a content by LC–MS/MS. Non-
compartmental methods were used to calculate pharmacokinetic
parameters from plasma concentration versus time profiles.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.05.064. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
1. Barnes, P. J. Nat. Rev. Immunol. 2008, 8, 183.
2. Barnes, P. J. J. Allergy Clin. Immunol. 1998, 102, 531.
3. Boushey, H. A. J. Allergy Clin. Immunol. 1998, 102, S5.
4. Johnson, M. Med. Res. Rev. 1995, 15, 225.
5. Matthys, H. Respiration 2001, 68, 432.
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. Glossop, P. A.; Price, D. A. Ann. Rep. Med. Chem. 2006, 41, 237.
9. Brown, A. D.; Bunnage, M. E.; Glossop, P. A.; James, K.; Jones, R.; Lane, C. A. L.;
Lewthwaite, R. A.; Mantell, S.; Perros-Huguet, C.; Price, D. A.; Trevethick, M.;
Webster, R. Bioorg. Med. Chem. Lett. 2007, 17, 4012.
10. Brown, A. D.; Bunnage, M. E.; Glossop, P. A.; Holbrook, M.; Jones, R. D.; Lane, C.
A. L.; Lewthwaite, R. A.; Mantell, S.; Perros-Huguet, C.; Price, D. A.; Webster, R.
Bioorg. Med. Chem. Lett. 2007, 17, 6188.
11. Brown, A. D.; Bunnage, M. E.; Glossop, P. A.; James, K.; Jones, R.; Lane, C. A. L.;
Lewthwaite, R. A.; Mantell, S.; Perros-Huguet, C.; Price, D. A.; Trevethick, M.;
Webster, R. Bioorg. Med. Chem. Lett. 2008, 18, 1280.
12. 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.
13. 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.
5.32. Oral absorption study in rats
Male Han Wistar rats were dosed discretely via the oral route at
a nominal dose of 2 mg base/kg. Animals were provided with food
and water ad libitum. Terminal blood samples were taken from
two animals per time point at intervals up to 2 h. Samples were ta-
ken from the hepatic portal vein (HPV) and by cardiac puncture
(CP) from each animal and centrifuged immediately to yield plas-
ma for analysis. The plasma samples were store at À20 °C until
prepared for analysis. Prior to analysis, samples were defrosted
and vortex mixed. Sample (200
acetate buffer (50 mM, 200 L) and b-glucuronidase (5 mg/mL,
100 L). Samples were incubated overnight in a shaking water bath
lL) was mixed with pH 5 sodium
l
l
heated to 37 °C. Following incubation, samples were extracted by
protein precipitation and analysed by LC–MSMS.
5.33. Pharmacokinetic studies in dogs
14. 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.
15. Bouyssou, T.; Rudolf, K.; Hoenke, C.; Lustenberger, P.; Schnapp, A.; Konetzki, I.
Bioorg. Med. Chem. Lett. 2009, 19, 5237.
16. Hoenke, C.; Bouyssou, T.; Tautermann, C. S.; Rudolf, K.; Schnapp, A.; Konetzki, I.
Bioorg. Med. Chem. Lett. 2009, 19, 6640.
17. 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.
Female beagle dogs (n = 2), were fasted for 18 h prior to dose
administration. Compound 12a acetate salt was formulated as a
solution in DMSO-PEG 200-distilled water (5:30:65, v/v/v) for oral
dosing, and DMSO-PEG 200-saline (10:45:45, v/v/v) for intrave-
nous dosing. Dogs were dosed orally by gavage tube or intrave-
nously via the cephalic vein at nominal dose levels of 0.25 mg
12a base/kg or 0.052 mg 12a base/kg, respectively in a crossover
design. Blood samples were taken from the jugular vein at 0.03
(intravenous only), 0.08, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6 and 8 h post
dose. Plasma was prepared from blood by centrifugation, and ana-
lysed for 12a content by LC–MS/MS. Non-compartmental methods
18. Bennett, J. A.; Harrison, T. W.; Tattersfield, A. E. Eur. Respir. J. 1999, 13, 445.
19. Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.
J. Med. Chem. 2002, 45, 2615.

P. A. Procopiou et al. / Bioorg. Med. Chem. 19 (2011) 4192–4201
4201
20. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
25. Nials, A. T.; Sumner, M. J.; Johnson, M.; Coleman, R. A. Br. J. Pharmacol. 1993,
108, 507.
21. Procopiou, P. A.; Morton, G. E.; Todd, M.; Webb, G. Tetrahedron: Asymmetry
2001, 12, 2005.
26. 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,
22. Pirkle, W. H.; Sikkenga, D. L.; Pavlin, M. S. J. Org. Chem. 1977, 42, 384.
23. Coe, D. M.; Monteith, M. J.; Procopiou, P. A. WO2002070490A1.
24. Guntrip, S. B.; Procopiou, P. A. WO2004022547A1.
1022.
27. Skidmore, I. F.; Lunts, L. H. C.; Finch, H.; Naylor, A.; Campbell, I. B. DE3513885,
1985.