From libraries to candidate: The discovery of new ultra long-acting dibasic β2-adrenoceptor agonists

Article abstract of DOI:10.1016/j.bmcl.2011.10.049

Libraries of dibasic compounds designed around the molecular scaffold of the DA₂/β₂ dual agonist sibenadet (Viozan) have yielded a number of promising starting points that have been further optimised into novel potent and selective target molecules with required pharmacokinetic properties. From a shortlist, 31 was discovered as a novel, high potency, and highly efficacious β₂-agonist with high selectivity and a duration of action commensurable with once daily dosing.

Full text of DOI:10.1016/j.bmcl.2011.10.049

Bioorganic & Medicinal Chemistry Letters 22 (2012) 689–695
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
From libraries to candidate: The discovery of new ultra long-acting dibasic
b2-adrenoceptor agonists
Lilian Alcaraz a, , Andrew Bailey a, Elaine Cadogan b, Stephen Connolly a, Robert Jewell a, Stephen Jordan b,
⇑
Nicholas Kindon a, Andrew Lister a, Mandy Lawson b, Alexander Mullen a, Ian Dainty b, David Nicholls b,
Stuart Paine c, Garry Pairaudeau a, Michael J. Stocks a, Phillip Thorne a, Alan Young b
a Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, UK
b Department of Bioscience, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, UK
c Department of Physical and Metabolic Sciences, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, Leicestershire LE11 5RH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Libraries of dibasic compounds designed around the molecular scaffold of the DA2/b2 dual agonist sibe-
nadet (Viozan™) have yielded a number of promising starting points that have been further optimised
into novel potent and selective target molecules with required pharmacokinetic properties. From a short-
list, 31 was discovered as a novel, high potency, and highly efficacious b2-agonist with high selectivity
and a duration of action commensurable with once daily dosing.
Received 27 July 2011
Revised 13 October 2011
Accepted 14 October 2011
Available online 20 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
b2-Agonist
Adrenoceptor agonist
LABA
Inhaled
Duration
COPD
Asthma
Inhaled bronchodilators are the most commonly used drugs for
the treatment of bronchial asthma and chronic obstructive pul-
monary disease (COPD).1 The ability of bronchodilators to relax
smooth muscle tissue benefits patients with improvements in
symptom control, dyspnea, exacerbations,2 physical activity and
quality of life.3 Short-acting bronchodilators are the recommended
first-line therapy for mild asthma and COPD, with long-acting in-
haled bronchodilators being used in more severe, uncontrolled
respiratory diseases.4,5 Different long acting agents are available
in the form of b2-agonist6 and anticholinergic compounds.7 To pro-
vide effective disease control the long-acting b2 adrenoceptor ago-
nists (LABAs) formoterol and salmeterol require twice daily dosing
(Fig. 1). The muscarinic receptor antagonist, tiotropium, however is
used once daily to provide 24 h disease control. Recent develop-
ment activity has been directed towards obtaining ultra-long
acting beta agonists (uLABAs) with 24 h duration of action that
are expected to improve patient compliance and enhance clinical
outcomes.4,8 In terms of safety, tolerability and lung function
improvement, favourable clinical results have been demonstrated
for indacaterol, now accepted in Europe,9 with other uLABA
compounds in development such as vilanterol and olodaterol
awaiting investigation.8 To achieve greater efficacy it is likely that
future approaches will involve combinations of agents with en-
hanced and matched duration of action to prolong disease control
and improve patient outcomes. The number of recent publications
describing the discovery of novel series of compounds as potential
once-a-day b2-agonists clearly highlights the level of activity and
interest in this field of research.10 Here, we describe a novel series
of compounds which combine extended duration of action with
potential for reduced systemic exposure and rapid onset of effect.11
In parallel with other activities in this area within our laborato-
ries,12 and in an attempt to build on our experience gained during
the research program that led to the discovery of the dual DA2/b2-
agonist sibenadet (Viozan™),13 a focused library of compounds
that contained the benzothiazolone head group, a lipophilic por-
tion and a second basic group was designed. In addition to poten-
tial increase in affinity and efficacy, it was postulated that the
introduction of a second basic group would give rise to a set of
physicochemical properties that could be advantageous in terms
of lung residency, due to the expected increase in the terminal vol-
ume of distribution (Vz).12b,14 Based around this concept, a com-
pound library of generic structure 1, which encompassed a set of
nine scaffolds, was prepared and tested in a cAMP b2 functional as-
say (Table 1).
⇑
Corresponding author. Tel.: +44 1279645645; fax: +44 1279645646.
E-mail address: dr.lilian.alcaraz@gmail.com (L. Alcaraz).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.049

690
L. Alcaraz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 689–695
OH
OH
H
H
N
N
O
O
O
HO
H
O
HN
H
HO
Salmeterol
O
O
Formoterol
Cl
O
OH
OH
H
N
H
N
Cl
HO
H
O
H
N
O
H
Vilanterol
Indacaterol
OH
H
N
H
N
O
S
O
O
O
HO
O
H
O
S
H
N
N
H
O
O
Olodaterol
Sibenadet
Figure 1. Examples of b2-agonists.
Table 1
Generic structures and spot test results for libraries 1–9
R1
N
H
N
R2
HO
S
0,1
N
H
1
O
Library
1
Scaffold
No. of compds
112
No. of actives @1
l
Ma
Library
6
Scaffold
No. of compds
35
No. of actives @1 l
Ma
13
1
2
3
25
59
4
1
7
8
73
56
0
1
4
5
76
62
2
0
9
24
0
a
Stimulation of cAMP accumulation in H292 cells expressing human b2. Cut off for hits: >20% activation at 1
activation.
lM compared to formoterol standard assigned 100%

L. Alcaraz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 689–695
691
Table 2
b2 binding and functional data for representative examples from libraries 1, 2 and 4 and representative pairs of des-hydro/hydroxy examples
R
H
R1
N
N
Scaffold
R2
HO
S
N
H
R=H or OH
O
Compound
2
3
4
5
6
7
R1
Cl
N
N
N
R2
Cl
Cl
F
N
O
NH
NH
NH
NH
N
Scaffold entry
R = H
R = H
1
1
1
1
2
4
a
pEC50 (IA)
pIC50
7.0 (0.4)
6.3
7.1 (0.3)
5.9
6.8 (0.3)
7.0
6.8 (0.3)
6.9
8.4 (0.3)
7.9
6.5 (0.2)
6.1
b
Compound
9
10
11
12
13
14
R1
Ph
N
R2
MeO
Cl
Cl
NH
NH
N
N
NH
NH
Scaffold entry
R = H
R = OH
1
6.7
8.7
9.5 (1.0)
2
9.2
9.7
9.3 (0.8)
2
7.5
8.4
9.0 (0.6)
4
7.8
7.2
8.0 (0.8)
5
9.1
8.7
8.4 (0.6)
7
7.5
8.0
9.2 (0.9)
b
pIC50
pIC50
b
a
pEC50 (IA)
a
Stimulation of cAMP accumulation in H292 cells expressing human b2, pEC50 is the negative logarithm of the molar drug concentration that produces a cAMP response
equal to 50% of its maximal response and IA is the intrinsic activity measured relative to formoterol (IA = 1).
b
Compound affinities were measured by competition with a radioligand for binding to membranes expressing the b2 recombinant receptor using radiolabelled iodocy-
anopindolol. pIC50 is the negative logarithm of the molar drug concentration that causes 50% reduction in specific binding of the radioligand to the receptor.
The hit-rate in the spot test cAMP b2-agonist assay was rela-
tively low with the majority of the hits obtained from two scaffolds
(entries 1 and 2; 11% and 16% hit-rate, respectively) and only a few
compounds were worth progressing to a full dose response curve.
These results are however consistent with the fact that designing
compounds with high level of agonism (intrinsic activity (IA) >
0.6, compared to formoterol (IA = 1.0)) within the des-hydroxy
benzothiazolone head group series had historically been challeng-
ing.13 Typical results have been summarised in Table 2 (com-
pounds 2–7). Generally, apart from compound 6, compounds
displayed moderate potency and were weak partial agonists with
a maximum intrinsic activity (IA) of 0.4.
observed.15 Some representative examples can be found in Table 2
(compounds 9–14). Generally, it was found that both the level of
affinity or efficacy of the corresponding hydroxy compounds could
not be predicted from the binding activity of the corresponding
des-hydroxy analogues and that potency in the series was not dri-
ven by lipophilicity. In addition, probably due to their more struc-
turally rigid nature, the limited numbers of biphenyl-linked
analogues made, with the exception of 14, were found to be less
potent and/or less efficacious (compounds 12 and 13). The same
was true for bulky tertiary amines on the meta-phenyl scaffold
11. As a consequence, it was decided to focus lead optimisation
around the mono-phenyl lead compounds 9 and 10. A range of
analogues was prepared and key in vitro results are summarised
in Table 3.
Whilst this work was underway, in a related series,12b it was
found that the introduction of the benzylic hydroxyl group to a
compound of low efficacy such as 7 could, in some cases, turn an
antagonist into a potent agonist 8 (cAMP pEC50 8.8, IA 0.9).
The first part of our screening cascade was designed to identify
potent and efficacious compounds displaying good level of selec-
tivity against the closely related
a1-adrenergic, b1-adrenergic,
and D2 dopamine receptors with a minimum target of 30- to 50-
fold selectivity for the b2-receptor. Balancing the overall profile
of the compounds, while maintaining a good selectivity profile
has been one of the main challenges of this program, although
selectivity against the D2 receptor was generally good in this series.
Early examples, as exemplified by compounds 15 and 16, were rel-
atively lipophilic and, although displaying both high potency and
efficacy as well as promising selectivity, were found to inhibit cyto-
chrome P450 enzymes 2D6 and 3A4 as well as the hERG channel at
sub-micromolar levels. A range of replacements for the 2,6-dichlo-
rophenyl portion was explored leading to selective sub-nanomolar
analogues both in the para- and meta-positions (compounds 17–
26), whereas the ortho-isomer appear to reduce b2 potency whilst
R
H
N
S
N
H
HO
S
N
H
7: R=H: pIC50 6.8; IA<0.1 @ 10
8: R=OH: pIC50 8.7; pEC50 8.8 IA 0.9
µM
O
In the light of these results, the libraries were screened in a b2
binding assay to identify high affinity antagonists. From the
screening campaign, a range of potent binders with varied struc-
tures were identified and, from these, the corresponding hydroxyl
analogues were prepared. In most cases, a substantial gain in both
potency and efficacy over the parent des-hydroxy compounds was

692
L. Alcaraz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 689–695
Table 3
Summary of in vitro biological data for compounds 15–50
R9 R4 R5
R2
N
n
R4 R5 R6 R7 R8 R9
OH
H
R6
R7
N
N
X
( )n
R3
1
1
H
H
Cl
Cl
H
H
H
H
Cl
H
H
H
D
E
F
G
H
I
Ar
Y
R1
R8
1
1
1
H
H
H
F
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
D-U
1
2
Me
H
H
H
H
H
H
H
H
H
H
H
H
N
J
O
P
Q
R
S
T
0
0
0
0
0
0
0
H
H
Cl
F
H
H
H
H
H
H
H
H
H
H
H
N
HO
HO
K
S
HO
HO
H
HN
N
H
H OMe
H OMe
H
O
O
H
Me
H
H
H
H
H
H
H
N
N
A
B
C
( )m
H
H
H
OEt
H
L: m=0
M: m=1
N: m=2
H
H
H
OMe
H
H
U
OMe H
H
R2
N
a
b
b
b
b
Compd
Ar
R1
X
Subst.
Y
b2 pEC50 (IA)
b2 pIC50
a1D pIC50
b1 pIC50
D2 pIC50
LogD16
R3
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
C
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
(CH2)2
CH2
CH2O
CH2
CH2
CH2
CH2
Meta
Para
Meta
Meta
Para
Meta
Para
Para
Meta
Para
Meta
Para
Ortho
Para
Meta
Meta
Meta
Para
Meta
Meta
Meta
Meta
Meta
Meta
Meta
Meta
Para
CH2
D
D
E
F
F
G
G
H
K
K
M
M
F
9.3 (0.8)
9.5 (0.9)
9.6 (1.0)
9.8 (0.9)
9.4 (0.9)
9.5 (1.0)
9.5 (1.0)
9.6 (0.8)
9.5 (0.7)
9.6 (0.8)
9.3 (0.9)
9.7 (1.0)
8.8 (0.8)
9.4 (0.9)
9.3 (0.6)
9.0 (0.4)
9.2 (0.8)
8.5 (1.0)
9.5 (0.9)
9.2 (0.9)
9.2 (0.9)
9.0 (1.0)
9.1 (0.8)
8.5 (0.8)
8.5 (0.6)
8.7 (0.8)
7.8 (1.0)
9.2 (0.7)
7.4 (0.8)
8.9 (1.0)
9.1 (0.8)
8.6 (0.8)
9.4 (1.0)
7.5 (0.5)
8.6 (0.8)
8.1 (0.9)
9.7
8.4
9.2
9.3
8.7
9.1
8.7
8.7
9.2
8.7
8.6
8.5
7.9
9.7
9.1
9.3
8.2
6.9
8.7
9.0
7.8
8.2
8.3
7.6
7.6
7.6
6.5
8.4
ND
8.2
ND
8.1
8.8
6.2
7.2
6.6
7.2
7.1
7.3
6.9
7.3
7.1
8.1
7.9
6.4
7.0
6.7
7.6
6.7
7.5
6.8
6.6
6.7
7.0
6.8
7.1
6.5
7.0
6.8
7.5
6.6
6.3
6.2
6.0
ND
7.4
7.4
7.6
7.6
6.3
6.4
7.1
7.9
6.4
6.8
7.2
6.4
6.6
6.5
6.4
7.1
6.5
6.8
<6.1
8.7
6.3
7.7
7.2
7.3
5.9
8.0
8.2
7.1
7.2
8.0
7.0
6.7
6.8
6.1
6.5
ND
8.1
7.7
6.7
7.2
<5.5
5.5
5.9
6.6
6.0
6.2
6.4
5.7
<6.1
6.2
5.7
6.4
<6.1
5.8
5.6
6.1
5.6
6.5
<5.6
6.0
5.6
6.6
6.2
5.3
6.1
6.1
5.9
6.0
6.1
<5
3.2
2.3
1.6
1.1
1.3
0.8
0.8
1.1
1.1
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
0.4c
À0.1
À0.3
1.6
I
J
1.2
1.1
N
Q
Q
O
P
L
T
U
S
R
À1.2c
1.1
0.2
1.9
0.7
0.3c
0.9
0.9
1.1
1.5
0.0
NH–(CH2)2–OMe
NH–(CH2)2–OMe
Pyrolidine
Pyrolidine
D
D
F
Q
H
M
Q
À0.5
Meta
Para
5.5
ND
7.1
6.9
6.4
6.0
<5.1
<5.0
5.7
0.8
À0.4c
1.7c
2.4
Meta
Meta
Meta
Meta
Para
Me
H
H
H
H
1.9
0.9
(CH2)2
CH2
CH2
À1.1c
À0.1c
0.7c
Meta
Para
(S)–A
H
CH2
ND = Not Determined.
a
Stimulation of cAMP accumulation in H292 cells expressing human b2, pEC50 is the negative logarithm of the molar drug concentration that produces a cAMP response
equal to 50% of its maximal response and IA is the intrinsic activity measured relative to formoterol (IA = 1).
b
Compound affinities were measured by competition with radioligand for binding to membranes expressing various recombinant receptors: Adrenergic b1-receptor with
radiolabelled iodocyanopindolol, adrenergic a1D with radiolabelled Prazosin and dopamine D2 (S-isomer) using radiolabelled spiperone. pIC50 is the negative logarithm of the
molar drug concentration that causes 50% reduction in specific binding of the radioligand to the receptor.
c
ACD LogD.

L. Alcaraz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 689–695
693
Table 4
dramatically increasing b1 affinity (compound 27 compared to 18
iv PK profiles of selected examples
and 19). Removal of one of the chlorines led to a compound of sim-
ilar b1 potency but of lower b1 affinity (compound 17 compared to
15). Replacement of the chlorine atom for fluorine (compounds 18
and 19), methoxy (compounds 20 and 21), proton (compound 22),
or replacement of the terminal phenyl ring with heterocyclic sys-
tems such as indole (compounds 23 and 24) or pyridine (com-
pounds 25 and 26) all provided high potency and more polar
analogues. Their selectivity was generally good, except for two
para-isomers (compounds 21 and 22) that have increased affinity
Compound GPT pEC50 (IA) Cl (ml/min/kg) Vz (l/kg) T1/2 (h) LogD15
13
14
18
24
25
31
40
9.6 (0.9)
9.3 (0.9)
9.6 (0.9)
9.7 (0.8)
9.5 (0.9)
9.3 (0.9)
8.5 (0.8)
19
23
6
18
23
20
18
22
29
8.2
29
41
22
28
15
14
16
18
20
14
18
À0.3
0.8
1.1
0.4a
À0.1
1.1
0.0
a
ACD predicted.
for the
a1-receptor. Addition of a methyl group on the phenethyl
linker (compound 28 compared to 22) did not improve the
compound’s overall profile, whereas its extension by one carbon
(compounds 29, and 30 compared 25) led to a decrease in efficacy.
Less basic N-benzylic analogues were explored (compounds 31–
39). In this sub-series, it was rapidly found that the para-substi-
tuted analogues were generally less potent at the b2-receptor
and, as a consequence, analogues were prepared with the meta-
substitution pattern (compound 31 compared to 32). These find-
ings are analogous to that observed by researchers at Almirall in
their urea series.10a meta-Substituted N-benzyl-linked compounds
were generally of similar potencies and efficacies to that of the cor-
responding N-phenethyl analogues (compounds 31 compared to
20, 33–15, 34–18 and 35–25). The 2- and 3-methoxy analogues
had similar in vitro profiles (compound 31 compared to 36)
whereas the 4-methoxy compound had reduced b1 selectivity
(compound 37 compared to 31). Increased steric bulk on the
ortho-ether substituent (compound 38 compared to 31) or alkyl-
ation of the benzylic nitrogen (compound 39 compared to 31) led
to loss of potency with an additional decrease in efficacy in the
case of the tertiary amine. Some alkyl analogues were also pre-
pared (compounds 40 to 43). In these cases, a marked difference
in b2 potency in favour of the meta-substituted analogues was also
observed (compounds 40 compared to 41 and 42 to 43) and the
pyrrolidine analogue had an excellent selectivity profile (com-
pound 42). Additional SAR around the central phenyl ring was gen-
erated (compounds 44–47). One atom chain extension on the left
hand side through addition of a methylene group led to slight de-
crease in primary potency and increase of D2 affinity, whilst not
affecting either a1 or b1 affinities (compound 44 compared to
15), whereas primary b2 potency was reduced when using an oxy-
gen linker (compound 46 compared to 18). Introduction of an al-
pha-methyl group next to the amine nitrogen did not affect the
profile of the compound (compound 45 compared to 15) and the
Duration of Action Studies
100
80
60
40
20
0
**
**
Formoterol
Salmeterol
Indacaterol
Compound 31
*
*
**
**
**
*
*
*
*
-20
0
4
8
12
16
20
24
Time post i.t. (hr)
Figure 2. Duration of action of compound 31 versus standards dosed i.t. at ED80 in
guinea pigs (formoterol 1 g/kg, salmeterol 1 g/kg, indacaterol 14 g/kg and 31
g/kg).
l
l
l
7
l
in vivo duration in a bronchoprotection guinea pig model following
intra-tracheal (i.t.) dosing (cf. plasma T1/2 > 10 h for 24 h dura-
tion).12b As it can be seen from Table 4, compounds present similar
long terminal half-lives that are achieved through a combination of
moderate clearances (Cl) and high terminal volumes of distribution
(Vz). The exception being 18, for which the long half-life is a result
of a relatively low clearance and moderate volume of distribution.
The fact that all compounds have a similar pharmacokinetic
(PK) profile regardless of their lipophilicity implies that the dibasic
nature dictates the disposition of the compounds. The PK for these
compounds can be described by a three-compartmental PK model
formed of blood, tissues and a ‘deep compartment’. We define this
‘deep compartment’ as specific tissues that have higher affinity for
these dibasic compounds compared to other tissues and speculate
that the ‘deep compartment’ is significantly comprised of lyso-
somes. It is therefore hypothesised that the terminal half-lives
are largely controlled by the rate of diffusion of the compounds
from this compartment back to the blood.
use of phenethyl linker on the right hand side increased a1 affinity
(compound 47 compared to 31). Finally, changes in the b2 head-
group amine for the saligenin or 8-hydroxycarbostril moiety, or
reversal of chirality of the
a-hydroxyl group generally decreased
potency and/or efficacy of the compounds (compounds 48 com-
pared to 22, 49 to 26 and 50 to 31).
Based on its overall profile, 31 was selected for further progres-
sion. First, the lung half-life (T1/2 10 h) obtained from i.t. dosing
was predicted from the rat iv dosing. The extended i.t. half-life of
31 was measured in dog (T1/2 48 h) giving confidence of potential
for u.i.d. dosing. The low oral availability of 31 in both rat and
dog (F < 2%) will limit any systemic exposure due to swallowed
fraction. The potency of 31 hydrochloride salt was measured
Based on encouraging in vitro profiles as well as good level of
potency and intrinsic activity in guinea pig tissues (GPT, tracheal
rings contracted with methacholine in an organ bath), selected
compounds were progressed to in vivo rat intra-venous (iv) phar-
macokinetic (PK) profiling in order to determine their terminal
half-lives (Table 4). Indeed, in the course of this project it was
found that in vivo rat plasma iv T1/2 can be used as a predictor of
O
H
HO
O
HO
a-c
d
O
N
O
O
O
51
52
53
Scheme 1. Reagents and conditions: (a) NBS, benzoyl peroxide, CHCl3, reflux, (66%); (b) BH3, THF, 0 °C, (66%); (c) NaOEt, 2-nitropropane, EtOH, rt, (50%); (d) (1) 2-
methoxybenzylamine, cat. TsOH, toluene, Dean–Stark, reflux; (2) NaBH4, EtOH, rt; (3) BOC2O, CH2Cl2 (75% over the three steps).

694
L. Alcaraz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 689–695
O
OH
O
H
N
O
N
H
a
b, c
OH
N
53
HO
NH2
S
O
O
N
H
.2 CH2(COOH)2
54
O
.AcOH
31
HO
S
N
H
55
O
Scheme 2. Reagents and conditions: (a) Dess–Martin, DCM, rt; (b) 1 equiv HCl, NaCNBH3, MeOH, rt (50%); (c) (i) TFA, rt (ii) 2 equiv malonic acid, MeCN/EtOH, stirred at rt for
5 days (94%).
in vivo using intratracheal (i.t.) administration in a well established
histamine-induced bronchonstriction guinea pig model.17,18 The
duration of action of 31 and reference compounds (formoterol,
salmeterol and indacaterol) were measured in the guinea pig by
related receptors and to have a duration of action, in a bronchocon-
striction guinea pig model, that is superior to that of both formo-
terol and salmeterol, and similar to that of indacaterol. Based on
the promising profile, compound 31 was progressed into develop-
ment for further studies as the bis-malonate salt.
the i.t. route ( Fig. 2).19 Compound 31, when dosed at 7
lg/kg
(ED80, free base equivalent), retained 64% bronchoprotection at
24 h post dose. This bronchoprotection was significantly different
to that of control animals, clearly longer than the duration of action
of both formoterol and salmeterol, and was similar to that of ind-
acaterol. The good selectivity observed in the binding assays (Table
3) was confirmed in relevant functional assays (b1 pEC50 7.2 IA 0.9
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.049.
(CHO cells, cAMP),
pEC50 6.6 IA 0.4 (HEK cells, GTP
a
1D pEC50 6.9 IA 0.3 (HEK cells, Calcium flux), D2
References and notes
cS)), further profiling through a pa-
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100 nM, and activities at hERG (patch clamp) and cytochrome
P450s (5 isoforms, binding) were all inferior to 10 lM.
Since the hydrochloride salt of 31 did not possess optimal prop-
erties, extensive work was carried out in order to identify alterna-
tive crystalline salts. From these studies, the di-malonate salt
emerged as having good solid state properties and was selected
for further progression.20
Compounds 2–50 have been synthesised using similar proce-
dures to that employed for the preparation of compound 31. The
side chain was prepared in five steps from the commercially avail-
able 3-methylphenyl acetic acid 5 as outlined in Scheme 1. Bro-
mination of 51 with N-bromo succinimide was followed by
reduction of the acid to the corresponding alcohol. The resulting
benzyl bromide was then converted into the benzaldehyde 52
using the Hass–Bender procedure.21 Finally, reduction of the imine
formed from aldehyde 52 and 2-methoxybenzylamine followed by
BOC protection yielded 53.
Intermediate 53 was converted through to 31 as shown in
Scheme 2. Dess–Martin oxidation in dichloromethane was found
to give a smooth conversion to aldehyde 54 that was used imme-
diately after work up in the next step. It is worth noting that oxi-
dation with other reagents such as PCC also gave a good yields of
aldehyde 54 however, an inseparable side product resulting from
oxidative cleavage of the enol form of the resulting phenacetalde-
hyde giving benzaldehyde22 proved problematic. The coupling
through reductive amination of aldehyde 54 and amine 5512 was
carried out using sodium cyanoborohydride in methanol followed
by removal of the BOC protecting group using a 50% solution of tri-
fluoroacetic acid in dichloromethane for 20 min at room tempera-
ture to avoid racemisation of the hydroxyl group. The ‘free base’ of
31 was precipitated from an aqueous solution by treatment with
aqueous ammonia and compound 31 crystallised as its bis-malon-
ate salt which was found to be the most suitable for pharmaceuti-
cal development and was selected for progression.
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efficacious at the b2-receptor, to have good selectivity over closely

L. Alcaraz et al. / Bioorg. Med. Chem. Lett. 22 (2012) 689–695
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lg/kg of histamine, ED80 dose, was calculated from a dose–
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lg/kg. Propanolol (1 mg/kg iv), a b2-
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activity was mediated via b2-receptors.
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