Design driven HtL: The discovery and synthesis of new high efficacy β2-agonists

Article abstract of DOI:10.1016/j.bmcl.2011.04.135

The design and synthesis of a new series of high efficacy β₂-agonists devoid of the key benzylic alcohol present in previously described highly efficacious β₂-agonists is reported. A hypothesis for the unprecedented level of efficacy is proposed based on considerations of β₂-adrenoceptor crystal structure, other biophysical data and modeling studies.

Full text of DOI:10.1016/j.bmcl.2011.04.135

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4027–4031
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design driven HtL: The discovery and synthesis of new high efficacy b2-agonists
Michael J. Stocks a, , Lilian Alcaraz a, Andrew Bailey a, Roger Bonnert a, Elaine Cadogan b, Jadeen Christie a,
⇑
Stephen Connolly a, Anthony Cook a, Adrian Fisher a, Alice Flaherty a, Stephen Hill a, Alexander Humphries a,
Anthony Ingall a, Stephen Jordan b, Mandy Lawson b, Alex Mullen a, David Nicholls b, Stuart Paine c,
Garry Pairaudeau a, Stephen St-Gallay c, 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:
The design and synthesis of a new series of high efficacy b2-agonists devoid of the key benzylic alcohol
present in previously described highly efficacious b2-agonists is reported. A hypothesis for the unprece-
dented level of efficacy is proposed based on considerations of b2-adrenoceptor crystal structure, other
biophysical data and modeling studies.
Received 30 March 2011
Revised 27 April 2011
Accepted 28 April 2011
Available online 20 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
LABA
b2-Agonists
Adrenoreceptor agonist
Asthma
COPD
Inhaled
The b2-adrenoceptor (b2AR) is a member of the class A, G-pro-
tein coupled receptor (GPCR) family and is widely distributed in
the respiratory tract and particularly in airway smooth muscle.
Agonists of this receptor are effective bronchodilators1 and their
use has been pivotal in the treatment of asthma2 and chronic
obstructive pulmonary disease COPD.3 The ‘first generation’ b2-
agonists (e.g., salbutamol (pEC50 6.7, intrinsic activity (IA–relative
to formoterol IA = 1) 0.8), terbutaline (pEC50 5.5, IA 0.7) and iso-
prenaline (pEC50 7.4, IA 1.0) were classed as short duration ago-
nists requiring multiple daily dosing. The subsequent ‘second
generation’ compounds (e.g., salmeterol or formoterol) exhibited
longer duration of action amenable to twice-daily dosing. Formo-
terol is characterized as a high efficacy agonist (pEC50 9.3, IA 1.0)
with a rapid onset of action,4 whereas salmeterol is reported as a
low efficacy b2-agonist (pEC50 9.1, IA 0.6) with a slower onset of ac-
tion.5 More recently, ‘third generation’ long acting b2-agonists6 de-
signed to have once a day duration of action have been described
and a number (e.g., olodaterol (pEC50 9.9, IA 0.9))7 are in late stage
clinical development.8 Notably, indacaterol (pEC50 7.9, IA 0.8)9 has
recently been approved for treatment of COPD in Europe (Fig. 1).
A screening of betamimetic compounds from AstraZeneca’s pre-
vious sibenadet (Viozan™-a dual DA2/b2-agonist) program10 re-
vealed an interesting set of lead compounds amenable to further
study (Table 1).
Interestingly, 1 and 2 were shown to be low-efficacy b2-ago-
nists, whereas 3 was shown to be a high-efficacy b2-agonist, high-
lighting the need of the benzylic hydroxyl function in promoting
b2AR agonism. A medicinal chemistry program was established
to search for high efficacy agonists in the class of bothdes-hydroxyl
and hydroxy b2-agonists.11 Our initial SAR demonstrated that it is
advantageous to have the requirement for both the phenol and sec-
ondary phenethylamine to achieve high-efficacy b2-agonists (Table
2).
Compound 7 was shown to be a weak but high-efficacy (IA 1.4)
b2-agonist and it was postulated that substitution of the amide
nitrogen may well lead to enhancement in both the potency and
efficacy of the series. Indeed, exploration through the substitution
of the amide group rapidly highlighted a new series of highly effi-
cacious b2-agonists similar in potency and intrinsic activity to the
corresponding C-1 hydroxy series (Table 3).
Increasing the size of the alkyl group on the amide (1, 2, 8, 9, 10,
and 11) showed an increase in both potency and efficacy. Compar-
ing the activities of the hydroxyl and des-hydroxy series showed
comparable levels of potency and efficacy (11, 12).
⇑
Corresponding author. Tel.: +44 1509 645916; fax: +44 1509 645571.
E-mail address: mike.stocks@astrazeneca.com (M.J. Stocks).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.135

4028
M. J. Stocks et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4027–4031
OH
OH
OH
H
N
H
N
H
N
HO
HO
HO
HO
OH
OH
Salbutamol
Terbutaline
Isoprenaline
OH
H
OH
H
N
N
O
OMe
HO
HO
HO
NHCHO
OH
Salmeterol
Formoterol
OH
H
N
H
HO
N
OMe
O
O
HO
HN
HN
O
Indacaterol
HO
Olodaterol
H
N
O
S
O
O
S
N
H
Sibenadet
O
Figure 1. Examples of first, second and third generation b2-agonists.
Table 1
Potency and intrinsic activity at the human b2AR
R1
O
H
N
O
N
R2
HO
S
N
H
O
Entry
R1
R2
H
Me
Me
b2 potency# (pEC50
)
Intrinsic activity (IA)*
1
2
3
H
H
OH
8.8
9.3
9.7
0.3
0.7
1.0
#
b2 Receptor agonism was performed in H292 cells (bronchial epithelial cell line) expressing the human b2 adrenergic receptor. Functional activity was determined by
measuring accumulation of intracellular cAMP using AlphaScreenTM. The compounds were incubated for 1 h at 22 °C and the data expressed as intrinsic activity relative to
formoterol. pEC50 is the negative logarithm of the molar drug concentration that produces a cAMP response equal to 50% of its maximal response.
*Intrinsic activity measured relative to formoterol (IA = 1).
Table 2
Potency and intrinsic activity at the human b2AR
R2
N
O
R4
R3
N
O
R5
R1
O
S
N
H
O
Entry
R1
R2
R3
R4
R5
b2 potency (pEC50
)
Intrinsic activity (IA)
4
5
6
7
Me
H
H
H
Me
H
H
H
Me
H
H
H
Me
H
Me
Me
H
n/a#
n/a#
8.1
0.2
1.4
H
Me
nBu
6.9
#
n/a reflects a compounds with a b2 potency (pEC50) of <6.0.

M. J. Stocks et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4027–4031
4029
in receptor activation.14 Mutagenesis studies have shown an aspar-
tic acid residue 113, which is conserved in the cationic neurotrans-
mitter family of receptors, is involved in binding15 as well as two
serine residues,16 which are conserved in receptors binding cate-
chols, with the para phenolic hydroxyl forming an interaction with
serine 207 and the meta phenolic hydroxyl binding interacting
with serine 204. This characterization of the binding site allowed
modeling agonist ligands into the inactive conformation of the b2
adrenergic receptor structure.17 We propose a hypothesis that
hydrogen bonds between the protein and the catechol hydroxyls,
the b-hydroxl and the basic amine of agonists hold the ligand in
place with its aromatic group in an orientation such that it induces
a conformational shift in the tryptophan that leads to receptor acti-
vation. Conversely antagonists or inverse agonists (which generally
have a longer chain length between the basic amine and the aro-
matic group) stabilize the inactive conformation of the tryptophan.
Modeling studies suggested the movement of the tryptophan
opens up a pocket (christened the ‘agonist pocket’) that accommo-
dates the amide substituents R2 from Tables 1 and 3, and broadly
explains the structure activity relationship since this pocket is gen-
erally lipophilic (see Fig. 2). Thus, it can be postulated that the high
efficacy agonism in the des-hydroxyl compounds can be explained
by the group R2 occupying the agonist pocket requiring an induced
Table 3
Potency and intrinsic activity at the human b2AR
R1
O
H
N
N
O
C-1
S
R2
HO
N
H
O
Entry
R1
R2
Function (pEC50
)
Intrinsic activity (IA)
8
9
10
11
12
H
H
H
H
Et
Pr
iPr
nBu
nBu
9.0
8.1
8.1
8.8
9.1
0.7
0.9
0.6
0.9
1.0
OH
Receptor activation is believed to involve a rearrangement of
the helical bundle12 causing relatively large conformational shifts
remote to the ligand binding site. Closer to the binding site is a
highly conserved tryptophan residue 286, suggesting it has a func-
tional role,13 and its reorientation from the indole plane being par-
allel with the bilayer normal to perpendicular has been implicated
Figure 2. Shows on the left the X-ray structure of b2AR bound with a partial agonist carazolol (yellow) and on the right the model of the b2AR complexed with Compound 11
(magenta). The surface of the active sites are displayed in gray. The key aspartic acid and serine residues are displayed along with tryptophan 286 in orange and showing the
different orientations between the X-ray structure and the model. It is proposed that movement of the tryptophan opens up the ’agonist pocket’ which is occupied by the R2
substituents—a butyl side chain in the case of compound 11.
Figure 3. Dose response of 11 in guinea pig histamine-induced bronchoconstriction model. The potency was measured in vivo using intratracheal (i.t.) administration. Guinea
pigs were dosed with compounds and 2 h later anaesthetised and their respiratory resistance was measured after administering intravenous histamine. The dose of
compound that inhibited 80% of the bronchoconstriction induced by histamine (ED80) was calculated from the dose-response curve and was used in the resulting duration of
action studies.

4030
M. J. Stocks et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4027–4031
NH2
HO
S
N
H
O
13
O
O
Br
b)
a)
O
S
O
O
S
S
N
H
N
H
N
H
O
O
O
14
15
16
OH
OH
Br
NH2
c)
d)
O
S
HO
S
N
H
N
H
O
96% ee
17
O
18
Scheme 1. Reagents and conditions: (a) acetyl chloride, AlCl3, CH2Cl2 (83%); (b) (i) 48% aq HBr reflux (83%); (ii) Hunig’s base, BnBr, NMP, 65 °C (47%),
(iii) phenyltrimethylammonium tribromide, THF, 65 °C (52%); (c) (1R,2S)-(+)-cis-1-amino-2-indanol, BH3.THF (81%); (d) (i) NaN3, NaI, DMSO, 65 °C (85%) (ii) H2, Pd/C,
ethanol; (iii) Pd black, formic acid (65%) or H2, Pd/C, MeOH + 10% concd HCl (92%).
fit of the conserved tryptophan 286 resulting in receptor
activation.
of deprotection and re-protection followed by bromination affor-
ded the bromo ketone (16) that was selectively reduced21 to afford
the chiral bromohydrin (17) in greater than 96% enantiomeric ex-
cess. Displacement with sodium azide followed by a 2-step hydro-
genation procedure afforded (18) as a stable crystalline compound
(Scheme 1).
The amino compounds (13 and 18) were then used in the syn-
thesis of the final compounds where examples 11 and 12 are
shown as representative examples (Scheme 2).22
Compound 11 was progressed to the guinea pig histamine-in-
duced bronchoconstriction model—a well characterized species
for modeling human lung disease. Bronchodilators offering protec-
tion in this model have historically shown good efficacy in man
with a comparable duration of action.18 Compound 11 was tested
at various concentrations and demonstrated a dose-dependent
protection against histamine-induced brochoconstriction with an
ED80 of 2.6
of action and in this model formoterol (ED80 of 1.4
salmeterol (ED80 of 0.7 g/kg) gave protection up to 12 h, where
as 11 only gave protection up to 2 h and, as a consequence, was
not progressed further (Fig. 3).
The synthesis of compounds (1–12) required the synthesis of
both the known compound (13)19 and (18). Benzthiazolone
(14)20 was acylated with acetyl chloride to afford (15). A sequence
l
/kg. The ED80 dose was used to determine the duration
A new series of potent and highly-efficacious b2-agonists have
been described that are devoid of the key 1-hydroxy group pre-
viously considered important to achieve full agonism. Consider-
ation of the b2AR crystal structure, other biophysical data and
modeling studies has led to the proposal that the unprecedented
level of efficacy within the described series is achieved by an
induced fit where a lipophilic pocket is opened up after move-
ment of a key tryptophan (286) in the b2AR crystal structure.
Compound 11 was shown to be highly efficacious, however
when dosed in vivo in a guinea pig trachea model the compound
showed limited duration of action. Work is on-going within our
laboratories to build on this key observation to deliver new b2-
agonists to enable once a day dosing in man.
l
g/kg) and
l
O
HO
O
O
O
a)
N
O
19
20
nBu
Supplementary data
HO
H
N
nBu
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.135.
R
O
H
N
b)
N
O
References and notes
nBu
HO
S
1. For a recent review see: Glossop, P. A.; Price, D. A. Annu. Rep. Med. Chem. 2006,
41, 237.
2. National Asthma Education and Prevention Oprogram. Expert Panel Report II.
NHLBI, NIH Publication #97-4051, 1997.
11 R = H
N
H
12 R = OH
O
3. Global Initiative for Obstructive Lung Disease: Global strategy for the diagnosis,
management and prevention of chronic obstructive pulmonary disease.
National Heart, Lung and Blood Institute, Bethesda 2000.
Scheme 2. Reagents and conditions: (a) (i) (19) + (COCl)2, CH2Cl2, DMF (one drop),
rt 2 h, (ii) add (20), NEt3, CH2Cl2, (iii) (COCl)2; DMSO, Et3N, À70 °C to rt; (b) (13) or
(18) + acetic acid (1 equiv), NaCNBH3, MeOH 30–45%.

M. J. Stocks et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4027–4031
4031
4. Matthys, H. Respiration 2001, 68, 432.
5. Johnson, M. Med. Res. Rev. 1995, 15, 225.
10. Austin, R. P.; Barton, P.; Bonnert, R. V.; Brown, R. C.; Cage, P. A.; Cheshire, D. R.;
Davis, A. M.; Dougall, I. G.; Ince, F.; Pairaudeau, G.; Young, A. J. Med. Chem. 2003,
46, 3210.
11. Beattie, D.; Bradley, M.; Brearley, A.; Charlton, S. J.; Cuenoud, B. M.; Fairhurst, R.
A.; Gedeck, P.; Gosling, M.; Janus, D.; Jones, D.; Lewis, C.; McCarthy, C.;
Oakman, H.; Stringer, R.; Taylor, R. J.; Tuffnell, A. Bioorg. Med. Chem. Lett. 2010,
20, 5302.
12. Rosenbaum, D. M.; Rasmussen, S. G. F.; Kobilka, B. K. Nature 2009, 459, 356.
13. Oliveira, L.; Paiva, P. B.; Paiva, A. C. M.; Vriend, G. Proteins 2003, 52, 553.
14. Crocker, E.; Eilers, M.; Ahuja, S.; Hornak, V.; Hirshfeld, A.; Sheves, M.; Smith, S.
O. J. Mol. Biol. 2006, 357, 163.
15. Strader, C. D.; Sigal, I. S.; Candelore, M. R.; Rands, E.; Hill, W. S.; Dixon, R. A. F. J.
Biol. Chem. 1988, 263, 10267.
6. For some recent examples see: (a) Procopiou, P. A.; Barrett, V. J.; Bevan, N. J.;
Biggadike, K.; Butchers, P. R.; Coe, D. M.; Conroy, R.; Edney, D. D.; Field, R. N.;
Ford, A. J.; Guntrip, S. B.; Looker, B. E.; McLay, I. M.; Monteith, M. J.; Morrison, V.
S.; Mutch, P. J.; Richards, S. A.; Sasse, R.; Smith, C. E. J. Med. Chem. 2009, 52,
2280; (b) Brown, A. D.; Bunnage, M. E.; Glossop, P. A.; James, K.; Jones, R.; Lane,
C. A. L.; Lewthwaite, R. A.; Mantell, S.; Perros-Huguet, C.; Price, D. A.;
Trevethick, M.; Webster, R. Bioorg. Med. Chem. Lett. 2008, 18, 1280; (c)
Brown, A. D.; Bunnage, M. E.; Glossop, P. A.; Holbrook, M.; Jones, R. D.; Lane, C.
A. L.; Lewthwaite, R. A.; Mantell, S.; Perros-Huguet, C.; Price, D. A.; Webster, R.
Bioorg. Med. Chem. Lett. 2007, 17, 6188; (d) Brown, A. D.; Bunnage, M. E.;
Glossop, P. A.; James, K.; Jones, R.; Lane, C. A. L.; Lewthwaite, R. A.; Mantell, S.;
Perros-Huguet, C.; Price, D. A.; Trevethick, M.; Webster, R. Bioorg. Med. Chem.
Lett. 2007, 17, 4012; (e) Bouyssou, T.; Hoenke, C.; Rudolf, K.; Lustenberger, P.;
Pestel, S.; Sieger, P.; Lotz, R.; Heine, C.; Büttner, F. H.; Schnapp, A.; Konetzki, I.
Bioorg. Med. Chem. Lett. 2010, 20, 1410; (f) Hoenke, C.; Bouyssou, T.;
Tautermann, C. S.; Rudolf, K.; Schnapp, A.; Konetzki, I. Bioorg. Med. Chem. Lett.
2009, 19, 6640; (g) Bouyssou, T.; Rudolf, K.; Hoenke, C.; Lustenberger, P.;
Schnapp, A.; Konetzki, I. Bioorg. Med. Chem. Lett. 2009, 19, 5237.
16. Strader, C. D.; Candelore, M. R.; Hill, W. S.; Sigal, I. S.; Dixon, R. A. F. J. Biol. Chem.
1989, 264, 13572.
17. Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G. F.; Thian, F. S.;
Kobilka, T. S.; Choi, H.-J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C.
Science (NY) 2007, 318, 1258.
18. Walland, A.; Palluk, R.; Burkard, S.; Hammer, R. Eur. J. Pharmacol. 1997, 330,
213.
7. Bouyssou, T.; Hoenke, C.; Rudolf, K.; Lustenberger, P.; Pestel, S.; Sieger, P.; Lotz,
R.; Heine, C.; Buettner, F. H.; Schnapp, A.; Konetzki, I. Bioorg. Med. Chem. Lett.
2010, 20, 1410.
19. Giles, M. E.; Thomson, C.; Eyley, S. C.; Cole, A. J.; Goodwin, C. J.; Hurved, P. A.;
Morlin, A. J. G.; Tornos, J.; Atkinson, S.; Just, C.; Dean, J. C.; Singleton, J. T.;
Longton, A. J.; Woodland, I.; Teasdale, A.; Gregertsen, B.; Else, H.; Athwal, M. S.;
Tatterton, S.; Knott, J. M.; Thompson, N.; Smith, S. J. Org. Process Res. Dev. 2004,
8, 628.
20. Engel, W.; Trummlitz, G.; Eberlein, W.; Schmidt, G.; Engelhardt, G.;
Zimmermann, R. Ger. Offen. 1981, DE 3017977.
21. Fairhurst, R. A. 2005, WO2005074924.
8. Davies, S. L.; Castaner, J. Drugs Fut. 2005, 30, 1219.
9. (a) Roig, J.; Hernando, R.; Mora, R. Open Respir. Med. J. 2009, 3, 27; (b) Baur, F.;
Beattie, D.; Beer, D.; Bentley, D.; Bradley, M.; Bruce, I.; Charlton, S. J.; Cuenoud,
B.; Ernst, R.; Fairhurst, R. A.; Faller, B.; Farr, D.; Keller, T.; Fozard, J. R.; Fullerton,
J.; Garman, S.; Hatto, J.; Hayden, C.; He, H.; Howes, C.; Janus, D.; Jiang, Z.; Lewis,
C.; Loeuillet-Ritzler, F.; Moser, H.; Reilly, J.; Steward, A.; Sykes, D.; Tedaldi, L.;
Trifilieff, A.; Tweed, M.; Watson, S.; Wissler, E.; Wyss, D. J. Med. Chem. 2010, 53,
3675.
22. Bailey, A.; Bonnert, R.; Flaherty, A.; Pairaudeau, G.; Stocks, M. 2007,
WO2007027133; Bailey, A.; Bonnert, R.; Connolly, S.; Ingall, A.; Pairaudeau,
G.; Stocks, M. 2007, WO2007027134.