A multivalent approach to the discovery of long-acting β2- adrenoceptor agonists for the treatment of asthma and COPD

Article abstract of DOI:10.1016/j.bmcl.2011.11.072

A multivalent approach was applied to the design of long-acting inhaled β₂-adrenoceptor agonists. A series of dimeric arylethanolamines based on the short acting β₂-adrenoceptor agonist albuterol were prepared, varying the nature and length of the linker between the basic nitrogens. None of the C₂-symmetric dimers demonstrated increased potency, however dimer 5j, derived from 4-phenethylamine, was found to have increased binding potency in vitro relative to the parent monomer. Optimization of this structure led to the identification of 22 (milveterol) which demonstrates high potency in vitro and long duration of action in a guinea pig model of bronchoprotection.

Full text of DOI:10.1016/j.bmcl.2011.11.072

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1213–1218
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A multivalent approach to the discovery of long-acting b2-adrenoceptor
agonists for the treatment of asthma and COPD
⇑
John R. Jacobsen , Seok Ki Choi, Jesse Combs, Eric J.L. Fournier, Uwe Klein, Juergen W. Pfeiffer,
G. Roger Thomas, Cecile Yu, Edmund J. Moran
Theravance, Inc., 901 Gateway Blvd., South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A multivalent approach was applied to the design of long-acting inhaled b2-adrenoceptor agonists. A
series of dimeric arylethanolamines based on the short acting b2-adrenoceptor agonist albuterol
were prepared, varying the nature and length of the linker between the basic nitrogens. None of the
C2-symmetric dimers demonstrated increased potency, however dimer 5j, derived from 4-phenethylamine,
was found to have increased binding potency in vitro relative to the parent monomer. Optimization of
this structure led to the identification of 22 (milveterol) which demonstrates high potency in vitro and
long duration of action in a guinea pig model of bronchoprotection.
Received 1 November 2011
Revised 18 November 2011
Accepted 18 November 2011
Available online 25 November 2011
Keywords:
b2-Adrenoceptor agonist
Inhaled
Ó 2011 Elsevier Ltd. All rights reserved.
LABA
Bronchodilator
Milveterol
Multivalent approach
Inhaled b2-adrenoceptor agonists are highly effective bronchod-
ilators with significant utility in the treatment of both asthma and
chronic obstructive pulmonary disease (COPD).1 The introduction
40 years ago of short-acting agents such as albuterol has led to the
common use of this therapy for as-needed relief of acute
bronchoconstriction. The more recently discovered long-acting b2-
adrenoceptor agonists (LABAs) salmeterol (1) and formoterol (2)
(Fig. 1) have found broad utility when administered twice-daily
for the management of more severe disease, often in combination
with inhaled corticosteroids (commonly in a single inhaler device
for both asthma and COPD)1,2 or long-acting muscarinic antagonists
(for COPD).2,3 Longer lasting agents suitable for once-daily dosing
have been sought to improve disease control and patient compli-
ance4 and a large number of once-daily LABAs have been recently
reported.5–18
The physiological basis for the observed duration of action of
LABAs is somewhat controversial and remains an active area of
research.19,20 In vitro studies have demonstrated that salmeterol
and formoterol behave differently despite their similar duration
of action in man. The long duration of formoterol is explained by
the ‘diffusion microkinetic model’ which asserts that the cellular
membrane acts as a reservoir for the lipophilic drug molecule,
maintaining high local concentrations of drug long after concentra-
tions decline in the surrounding medium. This hypothesis does not
fully explain the in vitro properties of salmeterol, and interaction
with an ‘exosite’21,22 has been proposed for this drug. The key
structural elements of the lipophilic ‘tail’ of salmeterol responsible
for potency and duration of action have been explored,23 and
Alikhani et al. have extended this exploration to derivatives of for-
moterol and identified potent and long-acting compounds contain-
ing phenylalkyl ‘tails’.24
The dimeric b2-agonist hexoprenaline (Fig. 2) has demonstrated
efficacy as a bronchodilator in humans, but has limited duration of
action.25 Like hexoprenaline, salmeterol displays two aryl groups
joined by a linear linker, although one of these is an unsubstituted
phenyl group. We became interested in the possibility that a
second terminal b2-agonist head group might make an energeti-
cally favorable interaction with the exosite or other recognition
site within the b2-receptor and afford compounds with long intrin-
sic duration of action. Based on this hypothesis, we took a multiva-
lent approach26,27 to the identification of novel b2-agonists. We
anticipated that by optimization of the primary binding group
(b2-agonist head group) as well as ‘linker’ length and composition
we could generate highly potent compounds. This approach has
been used successfully in the identification of agonists for the
5-HT4 receptor28 (a member of the same G-protein coupled
receptor family as the b2-adrenoceptor).
The catechol ‘head group’ found in isoprenaline and hexopren-
aline is known to be rapidly metabolized and has not been success-
fully employed in the design of long-acting b2-agonists. We
therefore focused initially on the saligenin head group (found in
salmeterol) as a primary binding group. Based on the known
⇑
Corresponding author.
E-mail address: jjacobsen@theravance.com (J.R. Jacobsen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.072

1214
J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1213–1218
Table 1
OH
H
H
N
OH
b2-Adrenoceptor binding potency of dimeric ligands29
H
N
O
OH
OH
H
H
HO
O
HO
1
N
N
HN
L
2
HO
O
HO
HO
OH
OH
Figure 1. Structures of the b.i.d. LABAs salmeterol (1), and formoterol (2).
Formoterol is a racemic mixture of the (R,R) (shown above) and (S,S) isomers.
Compound
L
b2 pIC50 SD (n)
1 (salmeterol)
Albuterol
—
—
8.3 0.2 (39)
5.7 0.2 (12)
OH
(CH2)4
(CH2)6
5a
5b
*
*
*
*
5.0 (1)
5.1 (1)
OH
OH
H
N
H
N
HO
HO
HO
HO
N
H
OH
OH
5c
3.6 (1)
*
*
*
*
hexoprenaline
isoprenaline
5d
4.7 (1)
*
Figure 2. Structures of isoprenaline hexoprenaline.
5e
5.3 (1)
*
5f
5.0 (1)
4.3 (1)
4.7 (1)
structure–activity relationships of b2-agonists, we selected the ba-
sic nitrogen as the ‘point of attachment’ for dimer formation (as in
hexaprenaline), maintaining the secondary amine (a key element
of the b2-agonist pharmacophore). We explored dimeric structures
displaying two b2-agonist ‘head groups’ tethered with a variety of
diamine linkers (Scheme 1).
*
*
*
*
*
5g
5h
*
*
5i
4.0 (1)
Compounds5a–k were derived from the corresponding diamines
4a–k by condensation with arylglyoxal 3 followed by reduction of
*
*
the intermediate
(Scheme 1).
Linker length was varied from 3 to 9 atoms between basic nitro-
gen atoms, corresponding to a range of 9–15 atoms between the
terminal aryl groups (compare to 14 atoms found between the aryl
groups of salmeterol). Many linkers contained branching (alkyl or
a-iminoketones with borane-dimethylsulfide
5j
6.6 0.3 (12)
5.7 (1)
*
*
*
5k
Structure
OH
H
N
aryl) b- or c- to the basic nitrogens. This substitution is known to
confer selectivity for the b2 receptor over b1. As shown in Table
1, all of the C2-symmetrical dimers tested in a radioligand displace-
ment assay afforded lower binding potency for the b2-adrenocep-
tor than albuterol. Intriguingly, the asymmetrically substituted
5k matched the binding potency of albuterol and 5j showed
approximately 10-fold higher potency at the receptor. To investi-
gate this scaffold further, we prepared derivatives 7 and 8 in which
the hydroxy- and hydroxymethyl- substituents were removed
from one or both of the ‘head-groups’ (Scheme 2).
Compound 8 afforded a further enhancement in potency. De-
spite approximately 10-fold lower binding potency than salmeter-
ol for the receptor, compound 8 was found to be equipotent with
salmeterol in a functional assay for b2-adrenoceptor agonist activ-
ity (Table 2). For further optimization of this scaffold we adopted
this functional agonist assay in cells heterologously expressing
the human recombinant b2-adrenoceptor as the primary screen.
We explored a number of modifications to the linker portion of
compound 8 (Schemes 3–6). Substitution of the aniline nitrogen for
oxygen (10) did not impact potency nor did the addition of gem
-dimethyl substitution adjacent to the secondary amine (11).
Methylation of the aniline (12) resulted in decreased potency and
tying the phenethyl linkage into an indane ring (13) (comparable
7
8
6.6 0.6 (2)
N
H
OH
OH
H
N
7.4 0.4 (34)
HO
HO
N
H
OH
to the structure of indacaterol) decreased potency by more than
10-fold relative to 8. A survey of aryl group substitutions (15a–j,
Table 3, prepared as described Scheme 7) failed to identify any
compounds with improved potency over the unsubstituted parent
compound 8, and most substitutions resulted in substantially de-
creased potency.
To further assess compound 8 as a lead, we focused on the syn-
thesis of single diastereomers. Based on the well-established stereo-
chemical preference of the b2-adrenoceptor, we focused exclusively
on compounds possessing the (R)-configuration at the a-position of
the b2-agonist head group. Compounds 17-(R) and 17-(S) were
prepared by alkylation of bis-benzyl protected phenethylamine
with (R)- and (S)-styrene oxide, respectively, under strongly basic
conditions (Scheme 8). Reaction of 17-(R) and 17-(S) with epoxide
H2N
NH2
L
O
O
OH
OH
H
N
H
N
4a-k
OH
OH
OMe
a
L
HO
HO
HO
HO
OH
OH
b,c
5a-5k
O
OMe
O
3
Scheme 1. Reagents and conditions: (a) 48% HBr/DMSO, 16 h, 59%; (b) diamines 4a–4k, THF or CH2Cl2, rt, 6 h then concentrate under vacuum; (c) BH3ÁMe2S, THF, CH2Cl2,
0–50 °C followed by MeOH.

J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1213–1218
1215
H
H
a
b
Boc N
H2N
Boc N
N
H
NH2
b, e
NH2
6
OH
c, d, e
OH
H
N
OH
H
N
HO
HO
N
H
N
7
8
OH
H
OH
Scheme 2. Reagents and conditions: (a) Boc2O, MeOH; (b) phenylglyoxal, MeOH followed by NaCNBH3, MeOH; (c) TFA, CH2Cl2 followed by NaOH; (d) glyoxal 3 THF, rt, 12 h
then concentrate under vacuum; (e) BH3ÁMe2S, THF, 0–50 °C followed by MeOH.
Table 2
18 followed by reduction of the methyl ester and removal of the
benzyl protecting groups afforded the diastereomers 21-(R,R) and
In vitro agonist potency of b2-agonists30
21-(R,S). Both diastereomers were potent b2-agonists with the
(R,R)-isomer showing approximately threefold greater potency than
the (R,S)-diastereomer (Table 4).
Having established the preferred stereochemical configuration
for the ‘tail’ we investigated alternatives to the saligenin head
group. Compounds 22 and 23 (Fig. 3) were prepared by methods
similar to that shown in Scheme 8 using the appropriate (R)-epox-
ides (omitting the reduction in Scheme 8, step e). Compound 26
Compound
b2 pEC50 SD (n)
1 (Salmeterol)
Albuterol
8
10
11
12
13
9.5 0.2 (332)
7.6 0.2 (458)
9.5 0.5 (12)
9.4 0.1 (4)
9.5 0.2 (4)
8.7 0.1 (4)
8.3 0.1 (6)
O
O
Br
a, b
Bn O
HO
OH
Bn
N
H
N
OH
O
OMe
d,e
O
OMe
f, g, h
9
HO
HO
O
Bn O
HO
OH
OH
10
Bn
HN
c
H2N
OH
OH
Scheme 3. Reagents and conditions: (a) K2CO3, CH3CN, BnBr; (b) Br2, CHCl3; (c) PhCHO, toluene, DMF, concentrate to dryness followed by NaBH4, MeOH, THF; (d) NaHCO3,
DMF, 12 h, rt, followed by NaBH4 0 °C to rt; (e) LiAlH4, THF; (f) Cs2CO3, CH3CN, a-bromoacetophenone, 80 °C followed by 12 h at 45 °C; (g) NaBH4, MeOH 0 °C to rt; (h) H2, 5%
Pd/C, MeOH.
OH
H
N
H
f, g, h
a, b
Boc N
H2N
c, d, e
H2N
HO
HO
N
H
11
N
H
NH2
OH
OH
Scheme 4. Reagents and conditions: (a) H2SO4, HNO3, À10 °C; (b) NaOH followed by H2, 10% Pd/C, EtOH; (c) Boc2O, THF; (d) K2CO3,
a-bromoacetophenone, DMF; (e) NaBH4,
MeOH; (f) TFA, CH2Cl2 followed by NaOH; (g) glyoxal 3 THF, toluene, rt to 60 °C, 1 h then concentrate under vacuum; (h) BH3ÁMe2S, THF, rt to 60 °C followed by MeOH.
OH
H
N
e, f
c, d
H2N
a, b
NC
NC
HO
HO
N
N
H
NH2
N
12
OH
OH
OH
Scheme 5. Reagents and conditions: (a) K2CO3,
a-bromoacetophenone, DMF; (b) NaBH4, MeOH; (c) MeI, NaHCO3, DMF; (d) LiAlH4, THF; (e) glyoxal 3 toluene, rt, 1 h then
concentrate under vacuum; (f) BH3ÁMe2S, THF, 0–60 °C followed by MeOH.
OH
H
N
Boc
HN
e, f, g, h
Boc
HN
c, d
NO2
a, b
N
H
H2N
OH
HO
HO
N
H
13
OH
Scheme 6. Reagents and conditions: (a) HNO3, À30 °C; (b) Boc2O, CH2Cl2, DIEA; (c) H2, 10% Pd/C, THF; (d) K2CO3,
TFA, CH2Cl2 followed by NaOH; (f) bromide 9, DIEA, 0 °C to rt; (g) LiAlH4, THF; (h) H2, 10% Pd/C, EtOH.
a-bromoacetophenone, DMF followed by NaBH4, MeOH; (e)

1216
J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1213–1218
Table 3
Compounds 22 and 23 both had b2-agonist potencies comparable
to 21-(R,R) with compound 26 demonstrating slightly lower po-
tency (Table 4). At this stage we conducted more extensive in vitro
profiling. In addition to confirming b2/b1 selectivity (>300-fold for
both 21-(R,R) and 22), we measured agonist potency in a BEAS-2B
cell line which endogenously expresses the b2-adrenoceptor, also
allowing us to measure intrinsic activity relative to isoproterenol.
Potencies of compounds 21-(R,R), 21-(R,S), 22 and 23 were all
approximately 10-fold lower in this cell line relative to the
recombinant cell line used for primary screening. In contrast to the
partial agonist salmeterol (1), compounds 21-(R,R), 21-(R,S), 22
and 23 were all full agonists displaying intrinsic activities of 72%
or greater.
In vitro b2-agonist potency of aryl-substituted analogs of 830
OH
H
N
Ar
OH
HO
HO
N
H
Compound
Ar
b2 pEC50 SD (n)
15a
15b
15c
15d
15e
15f
15g
15h
15i
4-Chlorophenyl
4-Fluorophenyl
2,4-Dimethoxylphenyl
2,5-Dimethoxyphenyl
4-Hydroxyphenyl
4-Methylphenyl
4-t-Butylphenyl
4-Isopentyl
8.8 0.1 (4)
8.7 0.1 (4)
7.4 0.1 (2)
8.2 0.1 (2)
8.1 0.1 (2)
8.6 0.1 (4)
7.3 0.0 (2)
7.2 0.1 (2)
7.4 0.1 (2)
7.3 0.0 (4)
Compounds 21-(R,R) and 22 were investigated in a guinea pig
in vivo bronchoprotection model.32 Following nebulized dosing,
both compounds showed excellent bronchoprotective potency
(measured 1.5 h after dosing, data not shown). Because a high dose
4-Biphenyl
2-Naphthyl
15j
of salmeterol (300 lg/mL) provides bronchoprotection lasting
>24 h in this model, a 48-h time point was selected to provide con-
fidence that these compounds would be suitable for once-daily
dosing in man. At a concentration of 30 lg/mL, compound 22
maintained significant bronchoprotection at 48 h, whereas salme-
terol and 21-(R,R) were indistinguishable from the water control
(Fig. 4). 21-(R,R) and 22 have similar physicochemical properties,34
was prepared using an unprotected diamine tail (24) which was
prepared by direct alkylation of 4-phenethylamine with (R)-styrene
oxide under strongly basic conditions using DMPU to solubilize the
anilide anion (Scheme 9). Reaction of 24 with (R)-bromohydrin 25
followed by removal of the silyl protecting group afforded 26.
Table 4
In vitro agonist potency, intrinsic activity and selectivity of b2-agonists
Compound
Recombinant cell lines30
Endogenous cell line (BEAS-2B)31
b2 pEC50 SD (n)
b1 pEC50 SD (n)
b2 pEC50 SD (n)
IA (%) SD (n)
1 (Salmeterol)
9.5 0.2 (332)
10.0 0.2 (43)
9.5 0.2 (6)
10.2 0.2 (24)
9.9 0.1 (3)
6.6 0.2 (161)
6.6 0.3 (10)
6.6 0.3 (6)
7.6 0.2 (34)
nd
9.3 0.2 (344)
8.9 0.2 (11)
8.4 0.3 (4)
9.3 0.3 (20)
8.9 0.3 (4)
nd
34 10 (339)
72 10 (11)
87 20 (3)
87 9 (26)
92 19 (4)
nd
21-(R,R)
21-(R,S)
22
23
26
9.4 0.1 (2)
nd
nd: not determined.
IA: intrinsic activity.
OH
H
N
H
b, c, d
H
a
Boc N
Boc N
Ar
OH
HO
HO
N
H
Ar
N
NH2
H
OH
15a-j
14a-j
6
Scheme 7. Reagents and conditions: (a) K2CO3, CH3CN, ArC(@O)CH2Br, 30 °C, 16 h followed by NaBH4, THF, MeOH; (b) TFA, CH2Cl2 followed by NaOH; (c) glyoxal 3 THF, rt,
12 h then concentrate under vacuum; (d) BH3ÁMe2S, THF, 0–50 °C followed by MeOH.
O
HO
Bn
HN
Bn
HN
O
18
O
H2N
c
a, b
d
*
OH
NH2
N
Bn
NH
Bn
17-(
17-(
R)
16
S
)
Bn
N
OH
OH
Bn
N
H
N
OH
f
e
*
OH
*
OH
Bn
Bn
HO
HO
N
H
O
N
Bn
*
OH
O
N
Bn
HO
19-(R,R
)
20-(R,R
)
21-(R,R
)
O
O
19-(R,S
)
20-(R,S
)
21-(R,S
)
Scheme 8. Reagents and conditions: (a) benzaldehyde, toluene, concentrate under vacuum at 45 °C; (b) NaBH4, THF, MeOH; (c) n-BuLi, THF, (R)- or (S)-styrene oxide, À78 °C;
(d) epoxide 18, toluene, reflux; (e) LiAlH4, THF, 0 °C; (f) H2, 10% Pd/C, EtOH.

J. R. Jacobsen et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1213–1218
1217
OH
H
H
N
OH
H
N
HO
N
H
HO
N
H
HN
OH
HN
OH
O
23
O
22
Figure 3. Compounds 22 and 23.
OTBDMS
Br
Cl
H2N
TBDMSO
Cl
OH
H
N
H
N
25
c
a
Cl
H2N
H2N
Cl
H2N
b
N
H
N
H
H2N
N
H
NH2
Cl
OH
OH
Cl
OH
26
24
Scheme 9. Reagents and conditions: (a) DMPU, NaN(TMS)2 followed by (R)-styrene oxide, followed by water; (b) bromohydrin 25, DIEA, DMF, 105 °C; (c) TBAF, THF.
9. Bouyssou, T.; Hoenke, C.; Rudolf, K.; Lustenberger, P.; Pestel, S.; Sieger, P.; Lotz,
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Figure 4. Bronchoprotection in a guinea pig model measured 48 h after dosing of
water (control), 1, 21-(R,R) or 22.33
15. Stocks, M. J.; Alcaraz, L.; Bailey, A.; Bonnert, R.; Cadogan, E.; Christie, J.;
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sis for their different durations in this model warrants further
study. Based on its long duration in this model, compound 22 (mil-
veterol) was selected for further studies including safety assess-
ment and subsequent progression into clinical development.
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Acknowledgments
The authors would like to thank reviewers at Theravance for
their comments and suggestions on this article.
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[3H]dihydroalprenolol in the presence of increasing concentrations of
unlabeled test compound (90 min at room temperature). Binding reactions
were stopped by rapid filtration over filter plates; bound radioactivity was
determined by liquid scintillation counting.
30. In order to determine the agonist potencies of test compounds, the ability of
compounds to stimulate cAMP accumulation in whole cells overexpressing
cloned human b2- or b1-adrenoceptor was determined. cAMP concentrations
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were measured by
PerkinElmer). Agonist selectivities were calculated from the ratio of the
agonist potencies EC50
a
homogeneous radioimmunoassay (Flashplate,
mixture of gases (5% CO2, 21% O2, 74% N2). Post-treatment, pulmonary
resistance was measured in anesthetized, spontaneously-breathing animals
using a head-out plethysmograph. Bronchoconstriction was induced by
.
31. In order to determine the agonist potency and intrinsic activity of test
increasing doses of intravenous acetylcholine (ACh) and the dose of ACh
needed to double the baseline resistance (PD2) was calculated. At this
concentration, compounds 1, 21-(R,R), and 22 provide initial PD2 values
(measured 1.5 h after dosing), which are statistically significant relative to the
water control, but not statistically different from each other.
compounds on the human b2-adrenoceptor, the ability of compounds to
stimulate cAMP accumulation in
a human bronchial epithelial cell line
endogenously expressing the b2-adrenoceptor (BEAS-2B cells) was
determined. cAMP concentrations were measured by
radioimmunoassay (Flashplate, PerkinElmer).
a homogeneous
34. Calculated logD values (pH 7.4) for compounds 21-(R,R) and 22 are: cLogD
(21-(R,R)) = 1.5 and cLogD (22) = 0.9. Other studies such as those described in
32. Thomas, G. R.; Pfeiffer, J.; Yu, C.; Radke, E.; Shelton, D.; Yang, A.; Sutherland, K.;
Thibodeaux, H.; Renner, T.; McCullough, J.; Pulido-Rios, T.; Choi, S.-K.; Moran,
E. American Thoracic Society Meeting, 2004.
Refs. 5,6 have shown
a correlation between duration of action and
hydrophobicity, but the lower cLogD of the longer acting 22 is inconsistent
with this trend and not viewed as a likely basis for the long duration of
action.
33. Male guinea pigs were exposed for 10 min in a whole body exposure chamber
to nebulized aqueous solutions of test compound (30 lg/mL) delivered in a