Synthesis and structure-activity relationships of long-acting β2 adrenergic receptor agonists incorporating arylsulfonamide groups

Article abstract of DOI:10.1021/jm801016j

A series of saligenin alkoxyalkylphenylsulfonamide β₂ adrenoceptor agonists were prepared by reacting a protected saligenin oxazolidinone with alkynyloxyalkyl bromides, followed by Sonogashira reaction, hydrogenation, and deprotection. The meta-substituted primary sulfonamide was more potent than the paraand the ortho-analogues. Primary sulfonamides were more potent than the secondary and tertiary analogues. The onset and duration of action in vitro of selected compounds was assessed on isolated superfused guinea pig trachea. Sulfonamide 29b had the best profile of potency, selectivity, onset, and duration of action on both guinea pig trachea and human bronchus. Furthermore, 29b was found to have low oral bioavailability in rat and dog and also to have long duration of action in an in vivo model of bronchodilation. Crystalline salts of 29b were identified that had suitable properties for inhaled administration. A proposed binding mode for 29b to the β₂-receptor is presented.

Full text of DOI:10.1021/jm801016j

2280
J. Med. Chem. 2009, 52, 2280–2288
Synthesis and Structure-Activity Relationships of Long-acting ꢀ2 Adrenergic Receptor Agonists
Incorporating Arylsulfonamide Groups
Panayiotis A. Procopiou,*,† Victoria J. Barrett,‡ Nicola J. Bevan,‡ Keith Biggadike,† Peter R. Butchers,‡ Diane M. Coe,†
Richard Conroy,† Dean D. Edney,§ Rita N. Field,† Alison J. Ford,‡ Stephen B. Guntrip,† Brian E. Looker,† Iain M. McLay,|
Michael J. Monteith,§ Valerie S. Morrison,‡ Peter J. Mutch, Stephen A. Richards,# Rosemary Sasse,‡ and Claire E. Smith
Departments of Medicinal Chemistry, Asthma Allergy Biology, Synthetic Chemistry, Computational Structural Chemistry, Drug Metabolism and
Pharmacokinetics, Analytical Chemistry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, SteVenage,
Hertfordshire SG1 2NY, United Kingdom
ReceiVed August 12, 2008
A series of saligenin alkoxyalkylphenylsulfonamide ꢀ2 adrenoceptor agonists were prepared by reacting a
protected saligenin oxazolidinone with alkynyloxyalkyl bromides, followed by Sonogashira reaction,
hydrogenation, and deprotection. The meta-substituted primary sulfonamide was more potent than the para-
and the ortho-analogues. Primary sulfonamides were more potent than the secondary and tertiary analogues.
The onset and duration of action in vitro of selected compounds was assessed on isolated superfused guinea
pig trachea. Sulfonamide 29b had the best profile of potency, selectivity, onset, and duration of action on
both guinea pig trachea and human bronchus. Furthermore, 29b was found to have low oral bioavailability
in rat and dog and also to have long duration of action in an in vivo model of bronchodilation. Crystalline
salts of 29b were identified that had suitable properties for inhaled administration. A proposed binding
mode for 29b to the ꢀ2-receptor is presented.
Introduction
siveness and constriction lead to the symptoms of the disease,
which are cough, wheezing, breathlessness, and chest tightness.
There are two major categories of medicines used in asthma
treatment: bronchodilators and anti-inflammatories. Bronchodi-
lators may be ꢀ2 adrenoceptor agonists or in some cases
theophylline. Anti-inflammatories may be corticosteroids or
nonsteroidal anti-inflammatories. Inhaled ꢀ2-agonists are the
most effective bronchodilators, and inhaled corticosteroids are
the mainstay anti-inflammatory treatment for asthma, offering
proven benefits in reducing the burden of this disease.7,8 There
are two classes of ꢀ2-agonists: the short-acting (first-generation)
and the long-acting (second-generation) agonists. The short-
acting agonists, typified by salbutamol (1), have a rapid onset
of action and relieve symptoms for 3-6 h. The two currently
prescribed inhaled long-acting ꢀ2-agonists are salmeterol (2) and
formoterol (3). A racemic mixture of salmeterol xinafoate (1-
hydroxy-2-naphthoic acid) salt marketed by GlaxoSmithKline
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.9,10 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.10,11 We
are interested in identifying inhaled ꢀ2-agonists with extended
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. Bronchoscopy studies of patients
with asthma have shown that airway inflammation may lead to
long-term structural changes in airway wall components.2 The
smooth-muscle dysfunction is evidenced by exaggerated bron-
choconstriction, bronchial hyperresponsiveness, excessive pro-
liferation (hyperplasia), and excessive growth (hypertrophy) of
the airway smooth-muscle cells3 and their release of pro-
inflammatory mediators.4 Increased vascularity in the subepi-
thelial layers contributes to the thickening of the airway wall,
leading to airway narrowing as the smooth muscle contracts.
Airway inflammation is the result of the recruitment and
activation of a range of inflammatory cells, including mast cells,
eosinophils, and T-lymphocytes and the release of mediators
that perpetuate the inflammatory cycle causing edema formation
and epithelial damage.5 Airway remodelling processes are
thought to be mediated by angiogenic (blood vessel develop-
ment) mediators such as vascular endothelial growth factor and
basic fibroblast growth factor.2,6 The bronchial hyper-respon-
* To whom correspondence should be addressed. Phone: (+44)1438
762883. Fax: (+44)1438 768302. E-mail: pan.a.procopiou@gsk.com.
†
Department of Medicinal Chemistry, GlaxoSmithKline Medicines
Research Centre.
‡
Department of Asthma Allergy Biology, GlaxoSmithKline Medicines
Research Centre.
§
Department of Synthetic Chemistry, GlaxoSmithKline Medicines
Research Centre.
|
Department of Computational Structural Chemistry, GlaxoSmithKline
Medicines Research Centre.
Department of Drug Metabolism and Pharmacokinetics, GlaxoSmith-
Kline Medicines Research Centre.
Department of Analytical Chemistry GlaxoSmithKline Medicines
Research Centre.
duration of action that are suitable for once-daily dosing as the
third-generation products from this therapeutic class. Although
salmeterol and formoterol show quite different profiles of
#
10.1021/jm801016j CCC: $40.75
2009 American Chemical Society
Published on Web 03/24/2009

Long-Acting ꢀ2 Adrenergic Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2281
Scheme 1a
Figure 1
relaxation of both guinea pig trachea and human bronchus, both
are used successfully as twice-daily bronchodilators in the clinic.
In the search for a once-daily ꢀ2 agonist, candidates have been
sought, showing reduced systemic exposure/improved thera-
peutic index and/or an inherently longer duration of action at
the ꢀ2 adrenoceptor. Receptor desensitization is an autoregu-
latory process associated with ꢀ-adrenoceptor activation, which
operates as a safety device to prevent overstimulation of
receptors on excessive ꢀ-agonist exposure. As desensitization
results from agonist occupancy and can be inhibited by
antagonists, it follows that a lower intrinsic efficacy agonist
would be less prone to induce receptor desensitization than a
high intrinsic efficacy agonist.12 Indeed, this has been demon-
strated clinically with ꢀ2-agonists, where a degree of bronchodi-
lator tolerance was observed with the high-efficacy agonist
formoterol on chronic exposure13 but not with the lower intrinsic
efficacy agonist salmeterol.14 Recently, the mechanism of the
difference in desensitization between salmeterol and formoterol
has been reported.15 Following phosphorylation, the formoterol-
stimulated receptor binds ꢀ-arrestin and internalizes, whereas
this does not occur with salmeterol. In addition to desensitization
processes, ꢀ-agonists, acting through the cyclic AMP pathway,
also dramatically modulate ꢀ2-receptor gene expression. Cells
treated with salmeterol, however, had no such down-regulating
effect on ꢀ2-receptor gene expression.16 It was therefore decided
to use our own salmeterol arylethanolamine (saligenin) phar-
macophore as the starting point for this project. It was reported
that a significant contribution to the cardiovascular effects of
inhaled salmeterol seen at doses higher than those used clinically
results from the major fraction of the dose (80-90%) that is
swallowed and is available for absorption.17 Therefore, reducing
the oral bioavailability of our target compounds was considered
critical to achieving a once-daily ꢀ2 agonist with an improved
safety profile. Substitution on the right-hand side phenyl ring
of salmeterol was known to retain ꢀ2 agonist affinity.18 It was
hypothesized that a polar substituent, such as urea, sulfonamide,
or heterocycle, which contravened the Lipinski rules,19 might
be expected to show reduced oral bioavailability. Herein we
describe our efforts in identifying a novel class of sulfonamide
containing ꢀ2-agonists (Figure 1). The profile of the target
molecule was a potent, homochiral, and selective ꢀ2-agonist with
intrinsic activity similar to or better than that of salmeterol,
improved therapeutic index over salmeterol, which might offer
safe once daily bronchodilation in the clinic. The (R)-enantiomer
of salmeterol was reported to be the more potent isomer,9 so
from the outset it was decided to prepare homochiral compounds
with the (R)-configuration.
a Reagents and conditions: 50% NaOH, Bu4NHSO4, (a) 1,7-dibromo-
heptane;(b)1,6-dibromohexane;(c)1,5-dibromopentane;(d)1,4-dibromobutane.
Scheme 2a
a Reagents and conditions: (a) sodium sulfite, acetone, water, 71 °C, 3 h;
(b) POCl3, sulfolane, MeCN, 80 °C, 2 h; (c) aq NH3, THF.
Recently, we have published a versatile and enantioselective
route to (R)-salmeterol, which utilizes the oxazolidinone moiety
as a protecting group for both the amino and hydroxyl groups
present in ꢀ2-agonists in order to block further reaction
(dialkylation on nitrogen) and concurrently moderate the
reactivity of the amine nitrogen toward electrophiles.20 The
general synthetic route to the target sulfonamides (Figure 1) is
shown in Scheme 3. Alkylation of the homochiral (R)-
oxazolidinone 1120 with the alkynyl bromides 5-8 provided
the corresponding alkynes 12-15. Sonogashira coupling21 of
alkynes 12-15 with iodo- (9) or bromo-arenesulfonamides (10)
gave alkynyl arenesulfonamides 16-19, which were saturated
by catalytic hydrogenation to alkyl arenesulfonamides 20-23.
The oxazolidinone ring of 20-23 was cleaved using our mild,
anhydrous conditions of potassium trimethylsilanolate in THF20
to provide the amino alcohols 24-27, which were then
hydrolyzed with aqueous acetic acid to give the saligenin
sulfonamides 28-31 as their acetate salts. The compounds were
initially assayed as the acetate salts, and subsequently the most
promising compounds were converted into crystalline salts
suitable for inhalation. The (S)-enantiomer of the meta-primary
sulfonamide 29b (32) was obtained from resolution by chiral
HPLC of a mixture of (R)- and (S)- isomers of 25b, followed
by hydrolysis of the acetonide protecting group by heating in
aqueous acetic acid. Alternatively, (S)-29b could be prepared
via the (S)-enantiomer of oxazolidinone 11, which was prepared
from the parent (S)-amino alcohol22 and 1,1-carbonyldiimidazole
in THF. The synthesis of sulfonamide 33, the formoterol
analogue of 29b, is shown in Scheme 4. Treatment of the (R)-
epoxide 3423 with the benzylamine 35 (prepared by reaction of
Chemistry. The intermediate alkynyl bromides18 5-8 were
prepared by phase transfer catalyzed alkylation of alkynols 4
(n ) 1-4) with excess of the appropriate dibromoalkanes (3
equiv) in order to minimize the bis-alkylation products (Scheme
1).
The intermediate iodo- or bromo-arenesulfonamides (9 and
10, respectively) were prepared by reaction of the respective
sulfonyl chlorides with the appropriate amine or ammonia. The
sulfonamide 9j was prepared from 3-iodobenzyl bromide by
displacement of bromide with sodium sulfite, followed by
conversion to the sulfonyl chloride and then reaction with
aqueous ammonia (Scheme 2).

2282 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Procopiou et al.
Scheme 3a
pressure gave 33, isolated as its fumarate salt. Finally, (R,R)-
formoterol was prepared as a standard according to Hett’s
published procedure.23
Results and Discussion
All compounds in Table 1 were tested for their ability to cause
cyclic AMP accumulation in Chinese hamster ovary (CHOa)
cells transfected with human ꢀ1, ꢀ2, or ꢀ3 adrenoceptors. Agonist
activity was assessed by measuring changes in intracellular
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 isoprenaline. 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. Test compounds were considered as selective if
the difference between ꢀ2 and ꢀ1 pEC50 value or ꢀ2 and ꢀ3 pEC50
value was better than that of (R,R)-formoterol, that is, values
greater than 2.
The primary sulfonamide analogues 29a-c were very potent
agonists, with the meta-analogue (29b) being significantly more
potent than the para-(29c) and the ortho-(29a) analogues. The
monomethyl sulfonamide 29d appears slightly more potent than
the dimethyl sulfonamide 29e, but both analogues were less
potent than 29b. Similarly, the more lipophilic iso-propyl
secondary sulfonamide 29f was equipotent to the monomethyl
analogue 29d but still more potent than the dimethyl analogue
29e. The cyclohexylsulfonamide 29g, however, was found to
be nearly as potent as 29b. They both possessed similar IA,
however 29b was more selective against both ꢀ1 and ꢀ3
adrenoceptors. The cyclic tertiary sulfonamides derived from
piperidine (29h) and morpholine (29i) possessed similar potency
to that of the dimethyl analogue 29e. The (S)-enantiomer of
29b (32), in common with other known ꢀ2 agonists, was found
to be significantly less potent than the corresponding (R)-
enantiomer.9,24-27 The homologous primary sulfonamide 29j
was considerably less potent than its parent 29b. All saligenin
sulfonamides were found to have IA < 0.77. The para-
sulfonamide 29c possessed the highest IA, whereas the tertiary
sulfonamides 29e, 29i, and 32 possessed the lowest IA. Finally,
the sulfonamide 33 with the formoterol arylethanolamine
pharmacophore was as potent as 29b and as expected had a
higher IA, however, 29b had better selectivity against ꢀ1 and
ꢀ3 receptors.
a Reagents and conditions: (a) NaH (1.4 equiv), 5-8 (1.5 equiv), DMF,
2 h; (b) 9 or 10 (1.1 equiv), CuI (0.1 equiv), (PPh3)2PdCl2 (0.06 equiv),
MeCN-Et3N (1:1), 17 h; (c) H2, PtO2, THF, 2 h; (d) KOSiMe3 (4 equiv),
THF, 70 °C, 2.5 h; (e) aqueous AcOH (1:2), 70 °C, 0.5 h.
Scheme 4a
The optimal position of the ether oxygen in the chain was
found to be six carbons from the basic nitrogen, as is the case
for salmeterol. The analogue with seven carbon atoms in the
chain (28b) was less potent, and the analogue with five (30b)
even less potent. The analogue with four carbon atoms in the
chain (31b) was almost devoid of any activity in CHO cells.
The hexyl side chain does allow for enormous conformational
freedom, which makes difficult any accurate prediction of the
active spatial orientation in this region of the molecule. The
six atom distance between the basic nitrogen and ether oxygen
is present, however, in a diverse number of other ꢀ2 agonists
including salmeterol, formoterol, picumeterol, salmefamol,
carmoterol, and sibenadet (Figure 2). This observation has been
noted and commented upon by other groups.28,29 A notable
exception is indacaterol, which lacks the ether oxygen at the
a Reagents and conditions: (a) 120 °C; (b) 9b, CuI (0.16 equiv),
(PPh3)2PdCl2 (0.12 equiv), MeCN-Et3N (1:1), 2 h; (c) H2, 10% Pd/C,
Pd(OH)2, EtOH, 100 psi.
benzylamine with the alkyne bromide 6) gave 36, which was
then coupled with 9b to provide the alkyne 37. Hydrogenation
over a mixture of 10% Pd/C and Pearlman’s catalyst and under
a Abbreviations: CHO, Chinese hamster ovary; cAMP, adenosine 3′,5′-
monophosphate; COPD, chronic obstuctive pulmonary disease; DMA,
dimethyl acetamide; MDCK, Madin-Darby canine kidney cells.

Long-Acting ꢀ2 Adrenergic Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2283
Table 1. Stimulation of cAMP Accumulation in CHO Cells Expressing Human ꢀ2, ꢀ1, and ꢀ3 Adrenoceptors
a
a
a
reg. no.
ꢀ2 pEC50
IA (L95%conf-U95% conf)b
n
ꢀ1 pEC50
n
ꢀ2-ꢀ1
ꢀ3 pEC50
n
ꢀ2-ꢀ3
1
2
3
4
5
6
7
8
29a.AcOH
29b.cinnamate
29c.AcOH
29d.AcOH
29e.AcOH
29f.AcOH
29 g.AcOH
29 h.AcOH
29i.AcOH
9.1 ( 0.1
9.8 ( 0.2
9.3 ( 0.2
9.2 ( 0.1
9.0 ( 0.1
9.3 ( 0.1
9.7 ( 0.1
9.0 ( 0.1
8.9 ( 0.1
8.0 ( 0.1
8.8 ( 0.1
8.6 ( 0.1
8.1 ( 0.1
<4.6
0.35 (0.34-0.39)
0.54 (0.50-0.59)
0.77 (0.66-0.90)
0.35 (0.29-0.42)
0.32 (0.26-0.39)
0.36 (0.32-0.41)
0.57 (0.53-0.61)
0.45 (0.42-0.49)
0.32 (0.27-0.39)
0.28 (0.24-0.32)
0.32 (0.29-0.36)
0.63 (0.58-0.69)
0.25 (0.22-0.28)
ND
14
4
6.6 ( 0.1
6.5 ( 0.1
6.3 ( 0.2
6.3 ( 0.1
6.2 ( 0.1
6.0 ( 0.1
7.0 ( 0.1
6.9 ( 0.1
6.2 ( 0.1
5.3 ( 0.1
6.0 ( 0.1
5.9 ( 0.0
5.9 ( 0.0
<4.1
16
4
2.5
3.3
3.0
2.9
2.8
3.3
2.7
2.1
2.7
2.7
2.8
2.7
2.2
ND
2.2
-0.7
3.5
1.9
6.3 ( 0.1
5.8 ( 0.2
5.9 ( 0.1
6.1 ( 0.1
6.2 ( 0.1
6.5 ( 0.1
7.3 ( 0.1
6.8 ( 0.1
6.2 ( 0.1
4.7 ( 0.1
5.6 ( 0.1
6.5 ( 0.0
5.6 ( 0.1
5.7 ( 0.1
6.7 ( 0.1
7.4 ( 0.0
5.9 ( 0.0
7.6 ( 0.0
14
4
9
14
14
16
14
16
14
6
10
4
4
10
6
659
849
653
2.8
4.0
3.4
3.1
2.8
2.8
2.4
2.2
2.7
3.3
3.2
2.1
2.5
ND
3.1
0.0
3.7
1.7
10
11
10
18
16
18
14
7
9
4
4
12
6
11
11
12
11
13
15
14
7
9
4
4
12
6
9
10
11
12
13
14
15
16
17
18
32.AcOH
29j.free base
28b.sulfamate
30b.AcOH
31b.AcOH
33.fumarate
isoprenaline
2
9.8 ( 0.2
7.4 ( 0.0
9.6 ( 0.0
9.3 ( 0.0
0.8 (0.75-0.85)
1.0
0.37 (0.36-0.38)
0.97 (0.97-0.98)
7.6 ( 0.0
8.1 ( 0.0
6.1 ( 0.0
7.4 ( 0.0
767
929
791
641
656
670
(R,R)-3.fumarate
a Human ꢀ1, ꢀ2 and ꢀ3 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 L95%conf ) lower 95% confidence limit. U95%conf ) upper 95% confidence limit.
Table 2. Onset and Duration of Action of Selected Compounds on
Isolated Superfused Guinea Pig Trachea (Minimum n ) 2)
shift
(3 h)
entry
compd
pEC50 onset time (min) shift (1 h)
1
2
3
4
5
6
7
8
29a.AcOH
29b.AcOH
29c.AcOH
28b.AcOH
29f.AcOH
33.fumarate
salmeterol
(R,R)-3.fumarate
8.6
9.1
8.7
8.6
8.0
10.4
8.3
9.5
6.6
10.8
7.0
8.9
6.0
18.9
27.6
10
48
7.0
75
8.3
8.4
10
3.7
4.2
1.1
>1940
7.1
9.8
3.0
3.1
1.0
20
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 (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 and similar
to formoterol. The isopropyl analogue 29f was the only
compound with a lower potency than salmeterol. The ortho-
sulfonamide had a formoterol-like profile; the remaining sul-
fonamides, however, had a unique profile, intermediate between
that of salmeterol and that of formoterol. These compounds
showed a biphasic washout profile on guinea pig isolated
superfused trachea. On removal of the agonist from the
superfusing fluid, the tissue showed a degree of recovery,
followed by no further recovery over 3 h. In contrast, tissues
showed little or no recovery to salmeterol upon washout. The
effects of formoterol were progressively reversed upon washing,
whereas the effects of salbutamol and isoprenaline were rapidly
and fully reversed.
On the basis of the data in Tables 1 and 2, sulfonamide 29b,
which had high potency, fast onset of action, long duration of
action, and selectivity for ꢀ2 over ꢀ1 and ꢀ3 receptors, was
progressed to pharmacokinetic studies. In Table 3, selected
physicochemical and structural parameters for 29b are shown
and contrasted to those for salmeterol. log D (at pH 6.4) for
sulfonamide 29b was found to be 0.52 compared with 1.06 for
salmeterol, its polar surface area (PSA) was calculated to be
142 Å, the total number of hydrogen bond donors and acceptors
was 13, its molecular weight was 494.7, the number of rotatable
bonds was 15, and the non-hydrogen atom count was 34. The
passive membrane permeability (Pexact) of 29b across MDCKII-
Figure 2
end of the six-atom chain and displays lower binding affinity
for the human ꢀ2 receptor.25
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.30 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 ꢀ2
adrenoceptor. 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, 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 potency, the
onset time, and the duration after 1 and 3 h is presented. 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. Duration of action was determined
by measuring the recovery of electrically induced contraction
following washout of agonist. This was expressed as the
rightward shift in the agonist concentration-effect curve
following 1 and 3 h of washout (EC50 for test compound after

2284 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Procopiou et al.
Table 3. Physicochemical, Structural Parameters, and MDCKII Permeability for 29b and Salmeterol
compd
29b
salmeterol
MW
log D6.4
PSA (Å2)
H-bond acceptors
H-bond donors
rotatable bonds
heavy atoms
Pexact (nm s-1)
494.7
415.6
-0.52
1.06
142
82
8
5
5
4
15
16
34
30
4.5
119
Table 4. Rat and Dog Pharmacokinetic Data for 29b Acetate
dose Clp Vdss T1/2 Cmax
(mg/kg) (mL/min/kg) (L/kg) (h) (ng/mL) F%
Computational docking of compounds to the homology model
were carried out using the FLO+ docking algorithm39 and
incorporating flexibility in the side chains interacting with the
ligand. Wherever possible, the “interacting” residues identified
by receptor mutagenesis and molecular modeling studies were
utilized to guide the docking. These are the catechol mimic with
the serine residues 204 and 207 on the fifth transmembrane
spanning domain (TM5),40 the benzylic alcohol with the chirally
discriminating Asn-293 on TM6,41 and the protonated amine
with Asp-113 on TM3.42 Tyr-308 on TM7 was identified as
the major amino acid interacting with the methylene groups near
the protonated amine of salmeterol conferring the ꢀ2 selectiv-
ity.43 In addition, Tyr-308 has been identified as the residue
defining the interactions of the amine substituents of formoterol
and carmoterol.44 The ether oxygen in the side chain of
salmeterol interacts with Tyr-316.43 The 4-phenylbutyl ether
moiety of salmeterol consequently orients itself into an interac-
tion with the amino acids around positions 149-158 on TM4,
which define the key residues of the exosite binding pocket.33
Sulfonamide 29b was docked in the above model, and for most
residues, the interactions were clearly observed in the docking.
Ser-207 was found to H-bond to the phenolic hydroxyl, while
Ser-204 and Ser-203 were positioned 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 a strong charge interaction. The ether oxygen in the
chain was seen to be positioned within H-bonding range of Asn-
318 (TM6), while the meta-sulfonamido group was seen to be
ideally situated to form H-bonds to both Ser-120 (TM3) and
Asn-322 (TM6) (Figure 3). These latter interactions are
important as they might provide a rational explanation for the
biphasic behavior of 29b on guinea pig isolated airway
preparations. In this model, direct interactions were neither seen
for Tyr308 or Tyr318. The sulfonamide tail was found to project
down toward the “exosite binding pocket” near the base of TM4,
but again no direct interaction was possible. The model was
superimposed and compared with the recent crystal structure
species
rat
route
intravenous
oral
2
5
79
16
2.3
<1
<1
dog intravenous 0.05
oral 0.25
20
1.9 2.1
6
12
MDR1 cells in the presence of a potent P-glycoprotein inhibitor
was measured as 4.5 nm s-1, whereas salmeterol was 119 nm
s-1. On the basis of all these data, 29b was expected to have
lower absorption than salmeterol, hence the swallowed fraction
of the inhaled dose was not expected to have a major
contribution to the overall systemic exposure of 29b. In vitro
metabolic stability was measured as a ratio vs verapamil in
human microsomes, and 29b (ratio 0.9) was found to have
moderate turnover, similar to that of salmeterol (ratio 1.2).
Pharmacokinetic studies in vivo with 29b were initiated and
compared with salmeterol. The acetate salt of 29b was dosed
as a solution orally and intravenously to rats and dogs, and the
data are presented in Table 4. High plasma clearance and a
moderately long half-life was observed in both species, and oral
bioavailability was low in the dog (mean 12%) and very low in
the rat (<1%). This oral bioavailability was much lower than
that previously reported for salmeterol (66-78% in dog, and
10-12% in rat)31 and strengthened the confidence that the
swallowed fraction of the inhaled dose in humans would be
unlikely to contribute to the systemic exposure of 29b.
As mentioned above, the washout profile of sulfonamide ꢀ2
agonists on guinea pig isolated airway preparations was biphasic
in nature. When the ꢀ2-adrenoceptor antagonist, sotalol, was
perfused over the trachea during the washout of 29b, there was
a rapid and full reversal of ꢀ2 agonist relaxation. However, upon
withdrawl of sotalol, reassertion of the relaxant effects was seen,
suggesting retention of 29b in the vicinity of the ꢀ2 adrenoceptor.
Bradshaw et al. suggested that the mechanism by which
salmeterol exhibits long activity was due to the anchoring of
the agonist to an “exosite” present in the cell membrane adjacent
to the ꢀ2-adrenoceptor32 but subsequently shown to be in TM4
of the receptor.33 This hypothesis was supported by the Nials
et al. studies.30 In these studies, the residual effects of salmeterol
(after washout) were reversed by sotalol. On withdrawal of
sotalol, full relaxant effect was reasserted. This suggested that
salmeterol was bound in an area where it could associate with
the receptor causing relaxation but not readily be washed off,
i.e., “exosite” bound. In the current study, the effect of sotalol
on 29b showed a similar profile. This suggests that the second
phase of 29b activity is by a mechanism similar to that of
salmeterol in that it is bound somewhere in the cell membane
where it can readily associate with the receptor but is not easily
washed out.
Modeling Studies. A homology model was prepared to assist
in the understanding of the SAR for the sulfonamides. The
model was built using the bovine rhodopsin structure as the
template34 and applying the methods of Blaney.35 This model
was build several years ago, when this research work was
underway, and well before the recent publication of the structure
of the human ꢀ2 receptor, which was crystallized in a lipid
environment when bound to the inverse agonist carazolol.36-38
Figure 3. Postulated interactions of the meta-sulfonamide 29b with
the ꢀ2 receptor. Portions of TM6 are omitted for clarity.

Long-Acting ꢀ2 Adrenergic Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2285
for the ꢀ2 receptor with carazolol bound (PDB ID 2RH1).36-38
All the proposed interacting residues were similarly placed in
the model and the crystal structure.
High resolution positive ion mass spectra were acquired on a
Micromass Q-Tof 2 hybrid quadrupole time-of-flight mass spec-
trometer. The elemental composition was calculated using Mass-
Lynx v4.0 for the [M + H]+.
A variety of salts of 29b were prepared and examined for
crystallinity, and two such salts were identified, the cinnamate
(mp 127-128 °C) and triphenylacetate (mp 99-102 °C).45
Cinnamic and triphenylacetic acids are not precedented in
marketed inhaled products and therefore warrant further toxi-
cological investigation. The cinnamate salt (solubility 0.382 mg/
mL at pH 7.4) was screened for stability in a range of humidity
and thermal conditions, for lactose compatibility, and for particle
size after micronization and was found to be suitable for inhaled
administration. The cinnamate salt of 29b was also investigated
in vivo in histamine-induced bronchospasm in the guinea pig
in a plethysmograph chamber (Buxco) and found to be about
7-fold more potent than salmeterol (EC90 8 and 55 µM,
respectively). At an equi-effective (EC90) dose, the duration of
action of 29b-cinnamate (time to 50% recovery) was longer
than that of salmeterol (18 h vs 12 h) when administered as a
nebulized solution in a DMA/saline vehicle using micronized
material.46 In conscious guinea pigs, 29b-cinnamate demon-
strated a wider (>600) therapeutic index compared with salme-
terol (>10) when comparing nebulized concentrations effective
at preventing histamine-induced bronchoconstriction with those
inducing significant decreases in mean arterial pressure. Fur-
thermore 29b-cinnamate reversed spasmogen-induced contrac-
tion of human isolated bronchial preparations with an onset time
of 3 min and potency similar to that seen on guinea pig trachea
(EC50 ) 2.4 nM), whereas salmeterol had an onset time of 36
min and potency of 5 nM. The sulfonamide 29b-cinnamate was
therefore selected for further development.
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 containing 0.1% (v/v) formic acid. The conditions 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 and 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.
General Procedure for the Alkylation of Oxazolidinone 11.
(5R)-3-[6-(But-3-ynyloxy)hexyl]-5-(2,2-dimethyl-4H-1,3-benzo-
dioxin-6-yl)-1,3-oxazolidin-2-one (13). (5R)-5-(2,2-dimethyl-4H-
1,3-benzodioxin-6-yl)-1,3-oxazolidin-2-one20 (11) (10 g, 40 mmol)
in DMF (100 mL) was added dropwise to a stirred suspension of
sodium hydride (60% oil dispersion, 2.33 g, 58.2 mmol) in DMF
(50 mL) with stirring under nitrogen and maintaining the internal
temperature at 0 °C. Stirring was continued at 0-5 °C for 1 h. The
mixture was recooled to 0 °C, and a solution of 6-bromohexyl but-
3-ynyl ether (6) (14.7 g, 63.0 mmol) in DMF (50 mL) was added
over 1 min. The mixture was then stirred at 20-30 °C for 2 h.
Then 2 M HCl (9 mL) was added and the mixture was partitioned
between water and Et2O. The aqueous layer was extracted with
more Et2O, and the combined organic layers were washed twice
with brine. After drying, the solution was concentrated and the
residue was purified by column chromatography on silica gel (600
g) eluting with Et2O-petroleum ether (bp 40-60 °C) (1:2), (1:1)
and then Et2O to give 13 (13.88 g, 86%) as a waxy solid: ES +ve
1
m/z 402 (M + H)+. H NMR δ (CDCl3) 7.12 (1H, dd, J 8 and 1
Hz), 7.00 (1H, d, J 1 Hz), 6.83 (1H, d, J 8 Hz), 5.39 (1H, t, J 8
Hz), 4.83 (2H, s), 3.85 (1H, t, J 9 Hz), 3.54 (2H, t, J 7 Hz), 3.45
(2H, t, J 7 Hz), 3.40 (1H, t, J 9 Hz), 3.38-3.20 (2H, m), 2.45 (2H,
dt, 3 and 7 Hz), 1.98 (1H, t, J 3 Hz), 1.62-1.52 (4H, m), 1.54
(6H, s), 1.42-1.30 (4H, m). Anal. C, H, N.
General Procedure for the Sonogashira Coupling of Alkynes
12-15 with Aryl Iodides 9 or Aryl Bromides 10. Reactions with
iodides were carried out at room temperature; however, reactions
with bromides were performed at 80 °C.
Conclusion
Incorporation of the sulfonamide functionality on the right-
hand side phenyl ring of (R)-salmeterol has given a series of
very potent human ꢀ2 adrenoceptor agonists. The primary
sulfonamide at the meta-position (29b) was identified as the
analogue that fulfilled all the criteria of potency, selectivity,
rapid onset, and long duration of action in vivo, low oral
bioavailability, and higher than salmeterol therapeutic index.
The cinnamate salt of 29b was found to have suitable properties
for inhaled administration and was therefore selected as a
candidate for further development. The interest in the pharma-
ceutical industry for the development of the third-generation
ꢀ2 agonists for the treatment of asthma and COPD is
continuing25,47-51 and will intensify as data from clinical trials
become available in the next few years.
3-[4-({6-[(5R)-5-(2,2-Dimethyl-4H-1,3-benzodioxin-6-yl)-2-
oxo-1,3-oxazolidin-3-yl]hexyl}oxy)but-1-ynyl]benzenesulfona-
mide (17b). Compound 13 (1.79 g, 4.46 mmol) was stirred with
9b (1.4 g, 5.1 mmol) in MeCN-Et3N (1:1, 42 mL) under nitrogen
for 10 min. Cuprous iodide (83 mg, 0.43 mmol) and dichlorobis-
(triphenylphosphine)palladium(II) (192 mg, 0.27 mmol) were added,
and the mixture was stirred for 17 h under nitrogen at 21 °C. The
mixture was evaporated to dryness, and the residue was purified
by chromatography on silica gel (250 g) eluting with EtOAc-
petroleum ether (bp 40-60 °C) (3:7), then (1:1), then (3:1), and
finally EtOAc to give 17b (2.35 g, 95%) as a colorless glass: ES
+ve m/z 557 (M + H)+. 1H NMR δ (CDCl3) 7.95 (1H, t, J 1 Hz),
7.80 (1H, dt, J 8 and 1 Hz), 7.55 (1H, dt, J 8, 1 Hz), 7.40 (1H, t,
J 8 Hz), 7.12 (1H, dd, J 8, 2 Hz), 7.01 (1H, d, J 2 Hz), 6.83 (1H,
d, J 8 Hz), 5.39 (1H, t, J 8 Hz), 5.29 (2H, br s), 4.84 (2H,s), 3.84
(1H, t, J 8 Hz), 3.62 (2H, t, J 6 Hz), 3.50 (2H, t, J 6 Hz), 3.40 (1H,
dd, J 8, 7.5 Hz), 3.35-3.18 (2H, m), 2.68 (2H, t, 6 Hz), 1.63-1.50
(4H, m), 1.53 (6H, s), 1.50-1.30 (4H, m). Anal. C, H, N, S.
General Procedure for Hydrogenation of Alkynes 16-19.
3-[4-({6-[(5R)-5-(2,2-Dimethyl-4H-1,3-benzodioxin-6-yl)-2-oxo-
1,3-oxazolidin-3-yl]hexyl}oxy)butyl]benzenesulfonamide (21b).
Compound 17b (2.35 g, 4.22 mmol) was stirred with platinum oxide
(0.3 g) in THF (30 mL) under hydrogen for 2 h. The catalyst was
removed by filtration using a filter aid, and the filter cake was
leached with EtOAc. The combined filtrates were passed through
silica gel (200 g) in EtOAc, and the eluate was evaporated to give
21b (2.32 g, 98%) as a colorless gum: ES +ve m/z 561 (M + H)+.
1H NMR δ (CDCl3) 7.76 (1H, s), 7.71 (1H, dt, J 6, 2 Hz),
Experimental Section
Organic solutions were dried over anhydrous MgSO4. 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
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 ES -ve). Column chromatography was
performed on Merck Kieselgel 60 (article 9385) or Biotage
prepacked silica gel cartridges containing KP-Sil run on a flash
1
12i chromatography module. H NMR spectra were recorded at
400 MHz unless otherwise stated and 13C NMR at 100 MHz. The
chemical shifts are expressed in ppm relative to tetramethylsilane.

2286 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Procopiou et al.
7.41-7.35 (2H, m), 7.11 (1H, dd, J 8, 2 Hz), 7.00 (1H, d, J 2 Hz),
6.81 (1H, d, J 8 Hz), 5.39 (1H, t, J 8 Hz), 5.27 (2H, s), 4.83 (2H,s),
3.85 (1H, t, J 9 Hz), 3.45-3.35 (5H, m), 3.35-3.20 (2H, m), 2.68
(2H, t, 8 Hz), 1.74-1.63 (2H, m), 1.62-1.48 (8H, m), 1.53 (6H,
s), 1.42-1.29 (4H, m).
t, J 6 Hz), 3.41 (2H, t, J 6 Hz), 3.08-2.89 (4H, m), 2.72 (2H, t, J
7 Hz), 1.76-1.52 (8H, m), 1.43-1.33 (4H, m). Anal. C, H, N, S.
29b-Cinnamate Salt. Cinnamic acid (0.3 g, 2 mmol) was added
to a solution of 28b free base (1.0 g, 2 mmol) in MeOH (5 mL) at
room temperature. The solution was stirred for 5 min before being
concentrated under reduced pressure. Water (10 mL) was added to
the residue, and the resulting suspension was stirred at room
temperature for 24 h. The solid was collected by filtration and
recrystallized from EtOH (5 mL) to give 29b-cinnamate (0.54 g,
General Procedure for the Cleavage of Oxazolidinone Rings
with KOSiMe3. 3-{4-[(6-{[(2R)-2-(2,2-Dimethyl-4H-1,3-benzodi-
oxin-6-yl)-2-hydroxyethyl]amino}hexyl)oxy]butyl}benzenesulfon-
amide (25b). A solution of 21b (0.43 g, 0.76 mmol) in THF (10
mL) was treated with potassium trimethylsilanolate (90% pure,
0.43 g, 3 mmol), and the mixture was stirred at 70 °C under nitrogen
for 2.5 h. The mixture was partitioned between CH2Cl2 and pH
6.4 phosphate buffer, and the aqueous layer was extracted with
more CH2Cl2. The combined organic layers were washed with
water, dried, and concentrated. The residue was purified by column
chromatography on silica gel (60 g), eluting successively with
EtOAc-petroleum ether (bp 40-60 °C) (1:1), EtOAc, MeOH-EtOAc
(1:9 to 1:4) to give 25b (0.286 g, 70%): ES +ve m/z 535 (M +
H)+. 1H NMR δ (CDCl3) 7.77-7.72 (2H, m), 7.43-7.35 (2H, m),
7.11 (1H, br d, J 8 Hz), 7.00 (1H, br s), 6.78 (1H, d, J 8 Hz), 4.83
(2H, s), 4.70 (1H, dd, J 9, 3 Hz), 3.42 (2H, t, J 6 Hz), 3.40 (2H,
t, J 6 Hz), 2.87 (1H, dd, J 12, 4 Hz), 2.75-2.60 (5H, m), 1.76-1.66
(2H, m), 1.65-1.46 (6H, m), 1.53 (6H, s), 1.40-1.30 (4H, m).
1
42%) as a white solid: mp 127-128 °C. H NMR δ (500 MHz;
CD3OD) 7.74 (1H, s), 7.71 (1H, d, J 7.5 Hz), 7.50 (2H, d, J 7 Hz),
7.41 (3H, m), 7.36-7.28 (4H, m), 7.17 (1H, dd, J 2, 8 Hz), 6.79
(1H, d, J 8 Hz), 6.51 (1H, d, J 16 Hz), 4.89 (1H, dd, J 4, 9.5 Hz),
4.66 (2H, s), 3.43 (2H, t, J 6 Hz), 3.39 (2H, t, J 6 Hz), 3.10 (2H,
m), 3.00 (2H, m), 2.70 (2H, t, J 7 Hz), 1.68 (4H, m), 1.55 (4H, m),
1.40 (4H, m). 13C NMR δ (125 MHz; CD3OD) 175.3, 156.3, 145.1,
144.9, 141.2, 137.2, 133.3, 133.0, 130.1, 130.0, 129.8, 128.9, 128.5,
127.1, 127.0, 126.8, 126.2, 124.6, 116.0, 71.6, 71.5, 70.2, 60.9,
55.4, 49.0, 36.3, 30.5, 30.1, 29.0, 27.5, 27.2, 26.8. Anal. C, H, N,
S.
2-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)hexyl]oxy}butyl)benzenesulfonamide (29a)-
1
Acetate Salt. ES +ve m/z 495 (M + H)+. H NMR δ (CD3OD)
includes 7.97 (1H, dd, J 8, 1 Hz), 7.52 (1H, dt, J 8, 1 Hz), 7.42
(1H, d, J 8 Hz), 7.38 (1H, d, J 2 Hz), 7.33 (1H, dt, J 8, 1 Hz), 7.18
(1H, dd, J 8, 2 Hz), 6.82 (1H, d, J 8 Hz), 4.68 (2H, s), 3.52 (2H,
t, J 6 Hz), 3.48 (2H, t, J 6 Hz), 3.17-3.12 (6H, m), 1.97 (6H, s),
1.82-1.68 (6H, m), 1.68-1.58 (2H, m), 1.48-1.42 (4H, m). HRMS
found: 495.2531 C25H38N2O6S requires 495.2529.
4-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)hexyl]oxy}butyl)benzenesulfonamide (29c)-
Acetate Salt. ES +ve m/z 495 (M + H)+. 1H NMR δ (DMSO-d6)
includes 7.72 (2H, d, J 8 Hz), 7.38 (2H, d, J 8 Hz), 7.27 (1H, d, J
2 Hz), 6.99 (1H, dd, J 8, 2 Hz), 6.69(1H, d, J 8 Hz), 4.57 (1H, dd,
J 9, 4.5 Hz), 3.38-3.28 (4H, m), 2.69-2.55 (6H, m), 1.90 (3H, s),
1.65-1.55 (2H, m), 1.53-1.35 (6H, m), 1.35-1.23 (4H, m). HRMS
found: 495.2521 C25H38N2O6S requires 495.2529.
3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)hexyl]oxy}butyl)-N-methylbenzenesulfona-
mide (29d)-Acetate Salt. ES +ve m/z 509 (M + H)+. 1H NMR δ
(CD3OD) 7.68-7.60 (2H, s), 7.53-7.43 (2H, m), 7.34 (1H, br d,
J 2 Hz), 7.13 (1H, dd, J 8, 2 Hz), 6.78 (1H, d, J 8 Hz), 4.64 (2H,
s), 3.45 (2H, t, J 6 Hz), 3.41 (2H, t, J 6 Hz), 3.13-2.93 (4H, m),
2.73 (2H, t, J 8 Hz), 2.49 (3H, s), 1.92 (3H, s), 1.77-1.64 (4H,
m), 1.63-1.53 (4H, m), 1.46-1.35 (4H, m). HRMS found:
509.2680 C26H40N2O6S requires 509.2685.
3-{4-[(6-{[(2S)-2-(2,2-Dimethyl-4H-1,3-benzodioxin-6-yl)-2-hy-
droxyethyl]amino}hexyl)oxy]butyl}benzenesulfonamide (ent 25b).
Resolution of 3-{4-[(6-{[2-(2,2-dimethyl-4H-1,3-benzodioxin-6-yl)-
2-hydroxyethyl]amino}hexyl)oxy]butyl}benzenesulfonamide (403
mg) on an HPLC Chiralcel OJ column using 40% ethanol-heptane
afforded ent 25b (96 mg). Analytical HPLC Chiralcel OJ column
(25 cm × 4.6 mm) RT for ent 25b ) 22.9 min, (40% EtOH-
heptane, flow rate 1 mL/min, detected at 215 nm). RT for 25b )
15.3 min.
General Procedure for Acetal Hydrolysis. 3-(4-{[6-({(2R)-2-
Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)-
hexyl]oxy}butyl)benzenesulfonamide (29b) Acetate Salt. A so-
lution of 25b (283 mg, 0.53 mmol) in acetic acid (8 mL) and water
(4 mL) was heated at 70 °C for 35 min before evaporating to
dryness. The residue was re-evaporated twice with toluene to give
29b-acetate salt (318 mg) as a gum: ES +ve m/z 495 (M + H)+.
1H NMR δ (DMSO-d6) 7.65-7.63 (2H, m), 7.47 (1H, t, J 8 Hz),
7.43 (1H, t, J 8 Hz), 7.26 (1H, d, J 2 Hz), 7.18 (1H, d, J 8 Hz),
7.00 (1H, dd, J 8, 2 Hz), 6.70 (1H, d, J 8 Hz), 4.59 (1H, dd, J 9,
4 Hz), 4.46 (2H, s), 3.36 (2H, t, J 6 Hz), 3.33 (2H, t, J 6 Hz),
2.72-2.58 (6H, m), 1.90 (3H, s), 1.65-1.55 (2H, m), 1.55-1.35
(6H, m), 1.35-1.30 (4H, m). Analytical chiral HPLC RT ) 21.3
min, 99.5% (Chirobiotic column 25 cm × 4.6 mm, flow rate 0.8
mL/min, MeOH-AcOH-triethylamine (1000:0.05:0.2) at 10 °C,
detected at 210 nm).
3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)hexyl]oxy}butyl)-N,N-dimethylbenzene-
1
sulfonamide (29e)-Acetate Salt. ES +ve m/z 523 (M + H)+. H
3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)hexyl]oxy}butyl)benzenesulfonamide (29b
Free Base). The acetate salt was converted to the free base by
passing through an SCX-2 ion exchange cartridge, eluting with
MeOH and then with 0.67 M NH3 in MeOH. The ammoniacal
fractions were concentrated under reduced pressure to give 29b-
NMR δ (CD3OD) 7.64-7.56 (2H, m), 7.54-7.50 (2H, m), 7.34
(1H, br d, J 2 Hz), 7.16 (1H, dd, J 8, 2 Hz), 6.78 (1H, d, J 8 Hz),
4.64 (2H, s), 3.45 (2H, t, J 6 Hz), 3.41 (2H, t, J 6 Hz), 3.13-2.98
(2H, m), 2.75 (2H, t, J 8 Hz), 2.65 (6H, s), 1.95 (3H, s), 1.76-1.52
(8H, m), 1.45-1.36 (4H, m). HRMS found: 523.2840 C27H42N2O6S
requires 523.2842.
1
free base as a colorless gum. H NMR δ (CD3OD) 7.73 (1H, s),
3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)hexyl]oxy}butyl)-N-isopropylbenzenesulfona-
7.70 (1H, dt, J 6, 2 Hz), 7.45-7.40 (2H, m), 7.28 (1H, d, J 2 Hz),
7.10 (1H, dd, J 8, 2 Hz), 6.75 (1H, d, J 8 Hz), 4.69 (1H, dd, J 9,
4 Hz), 4.64 (2H, s), 3.44 (2H, t, J 6 Hz), 3.40 (2H, t, J 6 Hz), 2.78
91H, dd, J 12, 9 Hz), 2.74-2.68 (3H, m), 2.67-2.56 (2H, m),
1.75-1.65 (2H, m), 1.63-1.45 (6H, m), 1.40-1.29 (4H, m).
29b-Triphenylacetate Salt. Triphenylacetic acid (115 mg, 0.4
mmol) was added to a solution of 29b-free base (198 mg, 0.4 mmol)
in EtOH (3 mL), and the suspension was warmed to effect
dissolution. The solvent was removed under reduced pressure to
give a gum, which was dissolved in EtOAc (3 mL) and allowed to
stand at 20 °C for 3 d. The crystals were collected by filtration and
recrystallized from EtOAc to give 29b-triphenylacetate salt (91 mg)
as a white solid: mp 99-102 °C. 1H NMR δ (CD3OD) 7.75-7.68
(2H, m), 7.44-7.40 (2H, m), 7.33 (1H, br s), 7.29-7.26 (6H, m),
7.22-7.09 (10H, m), 6.77 (1H, d, J 8 Hz), 4.65 (2H, s), 3.45 (2H,
1
mide (29f)-Acetate Salt. ES +ve m/z 537 (M + H)+. H NMR δ
(CD3OD) 7.72-7.63 (2H, m), 7.48-7.40 (2H, m), 7.34 (1H, d, J
2 Hz), 7.16 (1H, dd, J 8, 2 Hz), 6.78 (1H, d, J 8 Hz), 4.64 (2H, s),
3.44 (2H, t, J 6 Hz), 3.40 (2H, t, J 6 Hz), 3.13-2.96 (4H, m), 2.73
(2H, t, J 8 Hz), 1.95(3H, s), 1.74-1.64 (4H, m), 1.60-1.50 (4H,
m), 1.46-1.35 (4H, m), 0.99 (6H, d, J 6 Hz). HRMS found:
537.2999 C28H44N2O6S requires 537.2998.
N-Cyclohexyl-3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hy-
droxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)benzenesulfon-
1
amide (29g)-Acetate Salt. ES +ve m/z 577 (M + H)+. H NMR
δ (CD3OD) 7.72-7.63 (2H, m), 7.47-7.40 (2H, m), 7.34 (1H, d,
J 2 Hz), 7.16 (1H, dd, J 8, 2 Hz), 6.78 (1H, d, J 8 Hz), 4.65 (2H,
s), 3.44 (2H, t, J 6 Hz), 3.42 (2H, t, J 6 Hz), 3.13-2.95 (5H, m),

Long-Acting ꢀ2 Adrenergic Receptor Agonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2287
2.73 (2H, t, J 8 Hz), 1.95 (3H, m), 1.76-1.61 (17H, m), 1.45-1.07
(5H, m). HRMS found: 577.3314 C31H48N2O6S requires 577.3311.
1.55-1.38 (6H, m), 1.37-1.23 (4H, m). HRMS found: 495.2529
C25H38N2O6S requires 495.2529.
3-{6-[4-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)butoxy]hexyl}benzenesulfonamide (31b)-
Acetate Salt. ES +ve 495 m/z (M + H)+. 1H NMR δ (DMSO-d6)
7.66-7.61 (2H, m), 7.49-7.40 (2H, m), 7.26 (1H, d, J 2 Hz), 6.98
(1H, dd, J 8, 2 Hz), 6.69 (1H, d, J 8 Hz), 4.53 (1H, t, J 6 Hz), 4.46
(2H, s), 3.33 (2H, t, J 6 Hz), 2.68-2.53 (8H, m), 1.89 (3H, s),
1.63-1.53 (2H, m), 1.53-1.38 (6H, m), 1.35-1.25 (4H, m). HRMS
found: 495.2529 C25H38N2O6S requires 495.2529.
2-(Hydroxymethyl)-4-{(1R)-1-hydroxy-2-[(6-{4-[3-(piperidin-
1-ylsulfonyl)phenyl]butoxy}hexyl)amino]ethyl}phenol (29h)-
1
Acetate Salt. ES +ve m/z 563 (M + H)+. H NMR δ (CD3OD)
7.58-7.46 (4H, m), 7.33 (1H, br d, J 2 Hz), 7.15 (1H, dd, J 8, 2
Hz), 6.77 (1H, d, J 8 Hz), 4.64 (2H, s), 3.44 (2H, t, J 6 Hz), 3.40
(2H, t, J 6 Hz), 3.13-2.98 (4H, m), 2.94 (4H, t, J 6 Hz), 2.74 (2H,
t, J 7 Hz), 1.95 (3H, s), 1.78-1.52 (12H, m), 1.45-1.35 (6H, m).
HRMS found: 563.3151 C30H46N2O6S requires 563.3155.
3-(4-{[6-({(2R)-2-[3-(Formylamino)-4-hydroxyphenyl]-2-hy-
droxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide com-
pound with (2E)-But-2-enedioic Acid (2:1) (33). A solution of
37 (320 mg, 0.47 mmol) in EtOH (15 mL) was hydrogenated in
the presence of 10% palladium on carbon (50 mg) and palladium
hydroxide on carbon (100 mg) at 100 psi for 18 h. The catalyst
was removed by filtration over celite, and the filtrate was evaporated
to dryness. The residual oil was purified by chromatography on a
Biotage (8 g) cartridge eluting with CH2Cl2-EtOH-0.88 aqueous
ammonia (25:8:1) to give 33-free base (118 mg, 49%), which was
dissolved in MeOH (20 mL) and treated with fumaric acid (13.3
mg, 0.11 mmol). The mixture crystallized to give 33-fumarate salt
2-(Hydroxymethyl)-4-{(1R)-1-hydroxy-2-[(6-{4-[3-(morpholin-
4-ylsulfonyl)phenyl]butoxy}hexyl)amino]ethyl}phenol (29i)-
1
Acetate Salt. ES +ve m/z 565 (M + H)+. H NMR δ (CD3OD)
7.60-7.52 (4H, m), 7.34 (1H, br d, J 2 Hz), 7.15 (1H, dd, J 8, 2
Hz), 6.78 (1H, d, J 8 Hz), 4.65 (2H, s), 3.66 (4H, t, J 5 Hz), 3.45
(2H, t, J 6 Hz), 3.41 (2H, t, J 6 Hz), 3.15-2.96 (4H, m), 2.92 (4H,
t, J 5 Hz), 2.76 (2H, t, J 8 Hz), 1.92 (3H, s), 1.75-1.64 (4H, m),
1.63-1.51 (4H, m), 1.46-1.34 (4H, m). HRMS found: 565.2943
C29H44N2O7S requires 565.2947.
[3-(4-{[6-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]-ethyl}amino)hexyl]oxy}butyl)phenyl]methanesulfon-
1
amide (29j). ES +ve m/z 509 (M + H)+. H NMR δ (CD3OD)
1
(80 mg, 56%): ES +ve m/z 508 (M + H)+. H NMR δ (DMSO-
7.30-7.15 (5H, m), 7.10 (1H, dd, J 8, 2 Hz), 6.75 (1H, d, J 8 Hz),
4.69 (1H, dd, J 9, 4 Hz), 4.64 (2H, s), 4.29 (2H, s), 3.43 (2H, t, J
7 Hz), 3.42 (2H, t, J 7 Hz), 2.80 (1H, dd, J 12, 9 Hz), 2.72 (1H,
dd, J 12, 4 Hz), 2.69-2.60 (4H, m), 1.72-1.62 (2H, m), 1.62-1.49
(6H, m), 1.42-1.30 (4H, m). HRMS found: 509.2690 C26H40N2O6S
requires 509.2685.
d6) 9.54 (1H, s), 8.25 (1H, s), 8.03 (1H, d, J 2 Hz), 7.63 (1H, s),
7.62 (1H, d, J 8 Hz), 7.44 (1H, t, J 8 Hz), 7.40 (1H, d, J 8 Hz),
6.88 (1H, dd, J 8, 2 Hz), 6.81 (1H, d, J 8 Hz), 6.40 (2H, s), 4.60
(1H, dd, J 9, 4 Hz), 3.34 (2H, t, J 6.5 Hz), 3.31 (2H, t, J 6.5 Hz),
2.78-2.61 (6H, m), 1.66-1.54 (2H, m), 1.54-1.40 (6H, m),
1.29-1.18 (4H, m). HRMS found: 508.2478 C25H38N3O6S requires
508.2481.
3-(4-{[6-({(2S)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)hexyl]oxy}butyl)benzenesulfonamide (32)-
Acetate Salt. ES +ve m/z 495 (MH)+. 1H NMR δ (CD3OD)
7.76-7.68 (2H, m), 7.46-7.40 (2H, m), 7.34 (1H, d, J 2 Hz), 7.16
(1H, dd, J 8, 2 Hz), 6.78 (1H, d, J 8 Hz), 4.64 (2H, s), 3.45 (2H,
t, J 6 Hz), 3.42 (2H, t, J 6 Hz), 3.23-2.97 (4H, m), 2.72 (2H, t, J
8 Hz), 1.94 (3H, s), 1.78-1.65 (4H, m), 1.64-1.52 (4H, m),
1.45-1.36 (4H, m). HRMS found: 495.2522 C25H38N2O6S requires
495.2529. Analytical chiral HPLC RT ) 23.0 min, 99.9% (Chi-
robiotic column 25 cm × 4.6 mm, flow rate 0.8 mL/min,
MeOH-AcOH-triethylamine (1000:0.05:0.2) at 10 °C, detected
at 210 nm).
Acknowledgment. We thank Bill J. Leavens for collecting
the HRMS data, Steve Jackson for the resolution of 25b, Eric
G. Hortense for the analytical chiral HPLC, Steve M. Evans,
Isobel Hackney, and Kevin J. Norton for technical assistance.
Supporting Information Available: NMR spectral data for 5,
7, 8, 9d, 9i, 9j, 10e-10h, 12, 13, 14, 15, 16b, 17a-17j, 18b, 19b,
20b, 21a-21j, 22b, 23b, 24b, 25a-25j, 26b, 27b, 29b,
29b-cinnamate, 35, 36, 37, biological screens, pharmacokinetic
studies, cell permeability, HPLC retention times and purity of target
compounds, microanalytical data for 13, 17b, 29b-triphenylacetate
and 29b-cinnamate, LCMS traces for 28b, 29c, 29g, 29j, and 33.
This material is available free of charge via the Internet at http://
pubs.acs.org.
3-(3-{[7-({(2R)-2-Hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-
phenyl]ethyl}amino)heptyl]oxy}propyl)benzenesulfonamide (28b)-
1
Acetate Salt. ES +ve 495 (M + H)+. H NMR δ (DMSO-d6)
7.67-7.63 (2H, m), 7.47 (1H, t, J 8 Hz), 7.43 (1H, br d, J 8 Hz),
7.27 (1H, d, J 2 Hz), 7.00 (1H, dd, J 8, 2 Hz), 6.70 (1H, d, J 8
Hz), 4.61 (1H, dd, J 9, 4 Hz), 4.46 (2H, s), 3.35 (2H, t, J 7 Hz),
3.34 (2H, t, J 7 Hz), 2.73-2.60 (5H, m), 1.89 (3H, s), 1.85-1.77
(2H, m), 1.54-1.40 (4H, m), 1.35-1.24 (6H, m).
References
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28b-Sulfamic Acid Salt. A solution of 28b-free base (15 g, 30
mmol) in MeOH (250 mL) was treated with sulfamic acid (2.94 g,
30 mmol) at room temperature and the mixture was stirred for 1 h.
The solvent was removed under reduced pressure, and the residue
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