An investigation into the structure-activity relationships associated with the systematic modification of the β2-adrenoceptor agonist indacaterol

Article abstract of DOI:10.1016/j.bmcl.2012.07.096

The synthesis of a series of indacaterol analogues in which each of the three structural regions of indacaterol are modified in a systematic manner is described. Evaluation of the affinity of these analogues for the β₂-adrenoceptor identified t

Full text of DOI:10.1016/j.bmcl.2012.07.096

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6280–6285
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
An investigation into the structure–activity relationships associated
with the systematic modification of the b2-adrenoceptor agonist indacaterol
David Beattie, David Beer, Michelle E. Bradley, Ian Bruce, Steven J. Charlton, Bernard M. Cuenoud, Robin
⇑
A. Fairhurst , David Farr, John R. Fozard, Diana Janus, Elizabeth M. Rosethorne, David A. Sandham, David
A. Sykes, Alexandre Trifilieff, Katharine L. Turner, Elke Wissler
Novartis Institutes for BioMedical Research, Respiratory Diseases Area, Horsham, United Kingdom
Novartis Institutes for BioMedical Research, Respiratory Diseases Area, Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of indacaterol analogues in which each of the three structural regions of indaca-
terol are modified in a systematic manner is described. Evaluation of the affinity of these analogues for
the b2-adrenoceptor identified the 3,4-dihydroquinolinone and 5-n-butylindanyl analogues to demon-
Received 10 June 2012
Revised 27 July 2012
Accepted 30 July 2012
Available online 4 August 2012
strate the most similar profiles to indacaterol. An
a-methyl aminoindane analogue was discovered to
be 25-fold more potent than indacaterol, and functional studies revealed an atypical b2-adrenoceptor
activation profile for this compound consistent with that of a slowly dissociating ‘super agonist’.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
b2-Adrenoceptor agonists
COPD
Indacaterol
Binding kinetics
Bronchodilator
Inhaled b2-adrenoceptor agonists have become established as
one of the primary treatments for the respiratory conditions asth-
ma and chronic obstructive pulmonary disease (COPD).1 In the evo-
lution of these treatments the introduction of combination
products, in which a corticosteroid and a long-acting b2-adreno-
ceptor agonist are co-formulated in a single inhalation device, have
been shown to be more effective treatments in comparison to the
equivalent monotherapies.2 This increase in effectiveness is pri-
marily attributed to improved patient compliance. Presently the
inhaled long-acting b2-adrenoceptor agonists, and associated
fixed-dose combination products, in clinical use are suitable for
twice-daily administration in the majority of patient populations.
As a strategy to further improve patient compliance, the next gen-
eration of inhaled treatments for asthma and COPD are currently
being developed to be suitable for once-daily dosing.3 The most ad-
vanced of these next generation inhaled treatments is indacaterol
1, which has recently been approved as a once-daily inhaled treat-
ment for COPD. As well as having been shown to be an effective
once-daily bronchodilator in man, 1 also exhibits a fast onset of ac-
tion, and has been shown to be well tolerated.4
a broad range of disciplines. The b2-adrenoceptor having recently
been the first human GPCR to have a single crystal X-ray structure
solved, two decades after being the first GPCR to be cloned.5 In this
letter, we describe the synthesis and biological activity for a series
of close analogues of 1 to assist in understanding the structure–
activity relationships (SAR) surrounding the agonist activity, and
to further probe the structural features regulating the interaction
with the b2-adrenoceptor.
Me
HN
HN
HO
HO
2
1
In addition to the medical utility associated with ligands tar-
geted at the b2-adrenoceptor, this receptor has also long been at
the forefront of G-protein coupled receptor (GPCR) research across
N
H
O
OH
OH
OH
Figure 1. Structural relationship between indacaterol 1 and adrenaline 2, high-
lighting the three structural regions: catechol mimetic, ethanolamine linker and
amino substituent (shown in green, red and blue respectively).
⇑
Corresponding author.
E-mail address: robin.fairhurst@novartis.com (R. A. Fairhurst).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.096

D. Beattie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6280–6285
6281
R1
R2
i) or ii)
+
X
NH2
O
MeO
MeO
R1
R1
R2
O
O
iii)
N
H
N
H
R2
OH
R1 = Me, R2 = H 9
R1 = H, R2 = Me 10
R1 = Me, R2 = H 7
R1 = H, R2 = Me 8
O
Br
R1
R2
O
R1
R2
O
iv), v) or vi), vii)
viii), ix)
N
N
H
H
R3O
R3O
R1 = Me, R2 = H, R3 = MOM 11
R1 = H, R2 = Me, R3 = Bn 12
R1 = Me, R2 = H, R3 = MOM 5
R1 = H, R2 = Me, R3 = Bn 6
HN
HO
x), xi) or xii)
R1
R2
N
H
O
13
H2N
OH
R1 = Me, R2 = H 3
R1 = H, R2 = Me 4
Scheme 1. Synthesis of the 3- and 6-methylated analogues of indacaterol 3 and 4: (i) 6-methyl, cinnamyl chloride (X = Cl), K2CO3, acetone, 25 °C, 2 h (92%); (ii) 3-methyl, E-a-
methylcinnamic acid (X = OH), DCC, CH2Cl2, reflux, 18 h (51%); (iii) AlCl3, PhCl, 120 °C, 2 h (9–25%); (iv) 6-methyl, HBr, DMSO, 25 °C, 18 h; (v) MOM-Cl, K2CO3, DMF, 25 °C, 18 h
(80% 2 steps); (vi) 3-methyl, BnBr, K2CO3, acetone, reflux, 18 h (75%): (vii) Br2, NaOAc, AcOH, 25 °C, 2 h (99%); (viii) tributylvinyltin, Pd(PPh3)2Cl2, DMF, 90 °C, 24 h (69–80%);
(ix) titration with a solution of DMDO in acetone, acetone, 25 °C (60–90%); (x) 13, DMSO, 80 °C, 4–24 h (20–35%); (xi) 6-methyl, 3N HCl, iPrOH, THF, 40 °C, 18 h, then reversed
phase preparative chromatography11 (43%); (xii) 3-methyl, 1 atm H2, Pd/C, EtOH, 25 °C, 18 h, then reversed phase preparative chromatography11 (41%).
In common with the vast majority of b2-adrenoceptor agonists 1
can be aligned structurally to the endogenous ligand for the recep-
tor adrenaline 2, and the structure broken down into three sepa-
rate regions, as shown in Figure 1.6 These structural regions are:
the 8-hydroxyquinolinone catechol mimetic, as a replacement for
the catechol moiety of adrenaline; the ethanolamine linking group,
which remains unchanged; and the amino residue, in which the N-
methyl group of adrenaline is elaborated to the 5,6-diethyl indanyl
moiety in 1. To explore the SAR surrounding 1, close analogues
were targeted in which modifications were made individually to
all three of these structural regions. In this letter the preparation
and biological activity of these analogues are described.
Modifications to the catechol mimetic were made as described
below. Previously reported 8-hydroxyquinolinone catechol mi-
metic modifications in the context of b2-adrenoceptor agonists in-
clude: the introduction of a bromo or amino substituent into the
quinolinone 7-position, which resulted in a decrease in b2-adreno-
ceptor activity, and a series of 3,4-dihydro derivatives which
exhibited similar levels of activity to their parent compounds.7
Based upon these previous observations, we elected to explore
the effect of substitution of indacaterol at the quinolinone 3- and
6-positions, by the introduction of a methyl group, and to establish
the activity of the 3,4-dihydro derivative of indacaterol.
To prepare the methyl substituted analogues 3 and 4, a route
analogous to that reported for the preparation of 1 was selected
which required the preparation of the corresponding quinolinone
5-epoxide derivatives 5 and 6, as shown in Scheme 1.8 Both routes
started with the cyclisation of the appropriately methylated ani-
sole-cinnamide derivatives 7 and 8. This interesting cyclisation
reaction, in which benzene functions as the leaving group, pro-
duced the corresponding 8-hydroxyquinolinones 9 and 10 in mod-
est yields.9 For the 6-methylated analogue, direct bromination of 9
with hydrobromic acid in DMSO enabled the functionalisation of
the 5-position with a high degree of regeoselectivity which was
followed by protection of the phenol with a methoxymethyl
(MOM) residue to give 11.10 Alternatively, for the 3-methyl ana-
logue the phenol group in 10 was first protected as the benzyl
ether prior to bromination with bromine in acetic acid buffered
with sodium acetate to give 12. Further manipulation of the 5-po-
sition in both series was then carried out via a Stille coupling to

6282
D. Beattie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6280–6285
R4
R4
R6
R6
O
HN
HN
R5
R6
R6
R5
HO
i), ii)
iii)
ii), iii)
i)
1
R6
R6
R6
R5 = nBu, R6 = H 18
N
H
O
N
H
O
R5 = Me, R6 = Me 19
R6
14
OH
OH
15
R4 = 5,6-diethylindan-2-yl
O
R6
R6
R5
iv) or
OH
iv), v), vi), vii)
H
N
Scheme 2. Synthesis of the 3,4-dihydroquinolinone and deoxy analogues of
indacaterol 14 and 15: (i) 1 atm H2, Pd/C, EtOH, 25 °C, 15 days, then reversed
phase preparative chromatography11 (77%); (ii) SOCl2, CH3CN, reflux, 5 min; (iii)
NaBH4, EtOH, 25 °C, 18 h, then 6N HCl, reflux, 2 h, then reversed phase preparative
chromatography11 (11% for 2 steps).
N
R7
R5
R6
R6
R5 = nBu, R6 = H 20
R5 = nBu, R6 = H, R7 = H 22
R5 = Me, R6 = Me 21
R5 = Me, R6 = Me, R7 = Bn 23
R6
R5
R6
R6
O
O
O
S
HN
HN
S
HO
O
viii) or ix), x)
N
H
O
24
OH
N
H
O
Figure 2. Structure of sibenadet: an example of a b2-adrenoceptor agonist lacking
the benzylic alcohol from the enthanolamine-linker moiety. The three structural
regions are highlighted as described in Figure 1.
N
H
O
OH
R5 = nBu, R6 = H 16
BnO
R5 = Me, R6 = Me 17
Scheme 3. Synthesis of the indacaterol analogues 16 and 17 with alternative
indanyl aryl-substitution patterns: (i) 3-chloropropionyl chloride, AlCl3, CH3NO2,
25 °C, 1 h (90–95%); (ii) H2SO4, 90 °C, 1.5–4 h (60–64%); (iii) i-butyl nitrite, HCl,
CH3OH, 1–1.5 h (50–86%); (iv) 3 atm H2, Pd/C, H2SO4, AcOH, 25 °C, 24 h (54%); (v)
benzoyl chloride, Et3N, CH2Cl2, 25 °C, 1.5 h (20% 2 steps); (vi) NaBH4, CHCl3, CH3OH,
25 °C, 2 h (93%); (vii) 1 atm H2, Pd/C, H2SO4, AcOH, 25 °C, 18 h (86%); (viii) (R)-8-
benzyloxy-5-oxiranylquinolinone 24, dimethylacetamide, 180 °C (150 W) CEM
microwave reactor, 8 min (31%); (ix) 24, n-BuOH, 110 °C, 22 h (67%); (x) 1 atm
H2, Pd/C, CH3OH, rt, 4–18 h, then reversed phase preparative chromatography11
(12-41%).
introduce a 5-vinyl substituent which was followed by a dim-
ethydioxirane (DMDO) oxidation to give the racemic epoxides 5
and 6. Addition of 2-amino-5,6-diethylindane 138 to the epoxides
5 and 6 was followed by phenol deprotection, either through acid
catalysis or by hydrogenolysis as appropriate, to give the targeted
indacaterol analogues 3 and 4 as racemic mixtures.
Preparation of the 3,4-dihydro indacaterol analogue 14 was
readily achieved by hydrogenation of 1 over a palladium on carbon
catalyst for an extended period, as shown in Scheme 2.
Modifications to the ethanolamine linker were made as de-
scribed below. The report of sibenadet as a dual-acting b2-adreno-
ceptor and dopamine D2 receptor agonist attracted our attention
to the possibility that removal of the benzylic alcohol moiety from
the ethanolamine linker could lead to b2-adrenoceptor agonists
exhibiting good levels of activity, Figure 2.12 More recently the re-
port of a structurally related series of high intrinsic efficacy b2-
adrenoceptor agonists has further supported this concept.13 Addi-
tionally, modelling and mutation studies have proposed the inter-
action of the benzylic alcohol with the asparagine-293 residue of
the b2-adrenoceptor to contribute only a secondary interaction to
ligand binding.14 Taken together, these data support the possibility
for retaining a satisfactory level of affinity for the receptor upon
the deletion of this functionality. However, fluorescence lifetime
spectroscopy studies, comparing the structurally simple ligands
dopamine and adrenaline, have shown the benzylic alcohol to be
the key structural element driving a slow conformational change
in the b2-adrenoceptor.15 This slow change has been proposed to
be a key conformational switch for efficient G-protein activation.
To expand upon these observations, analogue 15 could provide a
useful tool compound for studying the role of the benzylic alcohol
in a more complex structural system.
reduction step with sodium borohydride to give the targeted deox-
ygenated analogue 15 in modest overall yield, as shown in
Scheme 2.
Modifications to the amino substituent were made as described
below. The SAR surrounding a homologous series of symmetri-
cally-substituted dialkylated indane derivatives leading to the
identification of 1 as the optimal example for use as a once-daily
inhaled-agent have previously been described.8 To further explore
the SAR surrounding the idanyl residue three separate approaches
to the modification of the 5,6- diethylaminoindane moiety were
explored: isomeric alkyl substitution patterns to the 5,6-diethyl
indanyl moiety, methylation at the a-position to the amino group,
and partial reduction of the benzene residue.
In the aryl dialkylated indane series the 4,7-diethyl indane iso-
mer of 1 has previously been shown to be less active compared to
the parent compound.8 To further explore the effect of the substi-
tution pattern in the aryl portion of the indane residue the isomeric
mono-substituted, 5-n-butyl analogue 16, and tetra-substituted,
4,5,6,7-tetramethyl indanyl analogue 17, were prepared as de-
scribed below. Thus, following the same basic route used to pre-
pare 1, n-butylbenzene and 1,2,3,4-teramethylbenzene were
converted to their corresponding indanone derivatives, 18 and
19, by sequential Friedel–Crafts acylation/intramolecular alkyl-
ation, as shown in Scheme 3.8 A subsequent condensation reaction
with isobutyl nitrite converts 18 and 19 to the corresponding keto
oximes 20 and 21. Direct reduction to the monosubstituted
To investigate the effect of removing the benzylic alcohol, 1 was
deoxygenated in a two step procedure by first forming the benzylic
chloride upon treatment with thionyl chloride. The intermediate
chloride was then used without purification in a subsequent

D. Beattie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6280–6285
6283
O
O
benzamide to the N-benzylated secondary amine 23. Addition of
the primary and secondary amines 22 and 23 to the chiral 8-ben-
zyloxy quinolinone epoxide 248 proceeded smoothly under micro-
wave and oil-bath heating respectively. Finally, debenzylation of
the coupled products gave the targeted derivatives 16 and 17.
Retention of the epoxide stereochemistry in these addition reac-
tions generates benzylic alcohols of the (R)-configuration, which
in the case of 22 results in a mixture of diastereoisomers epimeric
at the amino indane centre.
NH2
i)
ii), iii), iv), v)
Ph
Ph
H
N
H
N
vi), vii)
O
O
In related b2-adrenoceptor agonist series the introduction of a
26
methyl group in the a-position to the amino substituent has been
Ph
H
N
shown to increase the affinity for the receptor.16 To explore this
possibility the 2-methylindanyl indacaterol-analogue 25 was pre-
pared as shown in Scheme 4. Starting from 2-methylindan-1-one
a direct amination reaction with ammonia and potassium ferricy-
anide, under an inert atmosphere, generated the key quaternary
centre in good yield.17 Protection of the amine as the trifluoroace-
tamide was followed by hydrogenolysis of the ketone and N-pro-
tecting group interconversion to provide the benzamide 26. A
two step sequence of Friedel–Crafts acylation, with acetyl chloride,
and ketone hydrogenation was then repeated to introduce the 5,6-
diethyl residues with good overall regeoselectivity to give 27 in a
similar manner to that reported for the synthesis of 13.18 Deben-
zoylation of 27 was achieved following a two step sequence of
reduction and hydrogenolysis to give the key intermediate 5,6-
diethyl-2-methylaminoindane 28. Addition of 28 to the chiral 8-
benzyloxy quinolinone epoxide proceeded in modest yield under
microwave heating and was followed by debenzylation to give
the targeted analogue 25.
x), xi)
viii), ix)
O
27
HN
NH2
HO
xii), xiii)
28
N
H
O
OH
25
Scheme 4. Synthesis of the a-aminomethyl indane analogue of indacaterol 25: (i)
K3[Fe(CN)6], NH3(aq), 80 °C, 10 h (79%); (ii) (CF3CO)2O, Et3N, THF, 25 °C, 18 h, (66%);
(iii) 1 atm H2, Pd/C, H2SO4, AcOH, 25 °C, 18 h (82%); (iv) NaOH(aq), 25 °C, 1 h; (v)
benzoyl chloride, Et3N, CH2Cl2, 25 °C, 1 h (84% 2 steps); (vi) AcCl, AlCl3, CH3NO2,
25 °C, 4 h (98%); (vii) 1 atm H2, Pd/C, EtOH, 25 °C, 48 h (97%); (viii) AcCl, AlCl3,
CH3NO2, 25 °C, 3 h (35%); (ix) 1 atm H2, Pd/C, EtOH, 25 °C, 24 h (98%); (x) LiAlH4,
THF, reflux, 3 days (51%); (xi) 1 atm H2, Pd/C, EtOH, 25 °C, 18 h (94%); (xii) (R)-8-
benzyloxy-5-oxiranylquinolinone 24, n-BuOH, 100 °C PROLABO SynthewaveÒ 402
microwave reactor, 75 min (30%); (xiii) 1 atm H2, Pd/C, EtOH, 25 °C, 2 h (98%).
Partial saturation of the diethyl indane moiety in the form of the
thermodynamically more stable 4,7-dihydro derivative 29 was also
investigated. To prepare the analogue 29, Birch reduction of 2-ami-
no-5,6-diethyl aminoindane 13 gave the key 4,7-dihydro interme-
diate 30 in good yield, as shown in Scheme 5. Addition to the chiral
8-benzyloxy quinolinone epoxide 24 was achieved by first in situ
mono-N-silylating the amine 30, followed by heating with the
epoxide 24 in DMF.16 Subsequent debenzylation of the coupled
product through hydrogenolysis gave the targeted indacaterol ana-
logue 29 in modest yield.
The above indacaterol analogues were first screened in a radio-
ligand binding assay to assess their affinities for the human b2-
adrenoceptor. Additionally, affinity for the closely related human
b1-adrenoceptor was also measured, and these data are shown in
Table 1.19 The selectivity ratio determined between these two as-
says was found to be a useful ranking measure within this series.
However, functional activity has been shown to be a more appro-
priate measure of the absolute b1-adrenoceptor selectivity, and
measured in this way a much greater selectivity for the b2-adreno-
ceptor has been observed.8,20 Also included in Table 1 are calcu-
lated log D7.4 values for comparison with 1, as lipophilicity has
been shown to be one of the key parameters contributing to the
onset and duration of action profiles for this class of compound.8,21
In terms of the impact of the structural changes on the binding
affinity and relative b-adrenoceptor selectivity of the prepared ind-
acaterol analogues: introduction of the 3- and 6-methyl groups
into the quinolinone catechol mimetic resulted in a greater than
10-fold loss in b2-adrenoceptor activity when taking into account
the racemic nature of the derivatives 3 and 4 compared to indaca-
terol. Interestingly, introduction of the 3-methyl group in 4 re-
sulted in no change in affinity for the b1-adrenoceptor,
suggesting this modification may be a lead for gaining selectivity
in favour of the b1-adrenoceptor receptor. The 3,4-dihydroquinoli-
none derivative 14 exhibited a small three-fold reduction in affin-
ity for the b2-adrenoceptor, with no change in b1-adrenoceptor
activity, thus slightly reducing selectivity, but still making his ana-
logue of potential interest for further follow up studies.
HN
HO
i), ii), iii)
13
N
H
O
OH
29
Scheme 5.. Synthesis of the 4,7-dihydroindane derivative of indacaterol 29: (i) Li,
NH3, À78 °C, 3 h (88%); (ii) bis-(trimethylsilyl)-acetamide, DMF, 25 °C, 30 min, then
24, 80 °C, 36 h (21%); (iii) 1 atm H2, Pd/C, EtOH, 25 °C, 1 h, then reversed phase
preparative chromatography11 (37%).
aminoindane 22 proceeded efficiently, as previously reported, fol-
lowing direct hydrogenation of the keto oxime 20. For this exam-
ple, the absence of a plane of symmetry following reduction of
the keto oxime results in the 5-n-butyl aminoindane 22 being ob-
tained as the racemate. In contrast the keto oxime 21 was only re-
duced to the amino ketone under these conditions. To achieve an
efficient reduction to the amino indane 23 a three-step sequence
was required from this intermediate which involved N-benzoyla-
tion of the amino ketone, followed by sodium borohydride reduc-
tion of the carbonyl group. A subsequent hydrogenolysis of the
resulting benzylic alcohol was accompanied by reduction of the

6284
D. Beattie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6280–6285
Table 1
respectively. Both compounds were full agonists compared to for-
b-adrenoceptor binding affinities and clog D7.4 values
moterol at the highest concentrations tested. The similar potency
and rate of onset of action determined for the 3,4-dihydroquinoli-
none analogue 14 made this analogue of interest for further studies.
In line with the binding data, the deoxy derivative 15 was deter-
mined to be of reduced potency which has previously been linked
to a delayed onset of action in the guinea-pig tracheal-strip assay.8
To follow up on the promising in vitro profile of analogue 14, an
in vivo study was carried using the previously described metha-
choline-induced bronchoconstriction model in the rhesus mon-
key.19 From a dose response study an anti-bronchoconstrictor
Compound
b2 Ki (nM)
b1 Ki (nM)
b1/b2
clog D7.4
1
15.9
4.0
515
382
45
261
19.4
175
0.6
180
11.3
981
>19
0.59
4.1
1.8
13.4
2.4
3.0
2.4
3.4
3.5
3.1
3.8
3.0
2.8
3.5
3.0
sibenadet
3
4
14
15
16
17
25
29
3922
>10,000
227
183
478
259
421
1.9
133
3.2
1.7
76.6
ED80 of 53.2 lg/kg was determined for 14, making this analogue
four-fold less potent relative to 1. When compared at this equi-
effective ED80 anti-bronchoconstrictor dose level, the associated
systemic b2-adrenoceptor effects, as measured by increases in
heart rate, were greater and more persistent for 14. The observa-
tion of the potential for a greater systemic side-effect level with
14 made this analogue less attractive for further evaluation.
Removal of the benzylic hydroxyl from the ethanolamine linker
gave the analogue 15, and this modification lead to a greater than
10-fold loss of affinity for the b2-adrenoceptor in addition to a reduc-
tion in selectivity over the b1-adrenoceptor. As a further comparison
with sibenadet, the agonist activity at the dopamineD2 receptor was
also measured for 15.22 Although removal of the benzylic alcohol
leads to a more dopamine-like pharmacophore, an EC50 value
The high affinity a-methyl aminoindanyl analogue 25 was con-
sidered to be less well suited to advance as a development candidate
due to anticipating a high level of technical difficulty in producing an
inhaled formulation to deliver the anticipated low human-dose.
However, recent studies with a highly potent 5-hydroxy-benzoxaz-
inone series of b2-adrenoceptor agonists have revealed an atypical
‘super-agonist’ receptor activation profile (high potency and intrin-
sic efficacy).24 Additionally, for an example from this series such a
profile was shown to result in an extremely slow dissociation from
the b2-adrenoceptor. This kinetic profile was considered to be an
essential component to enabling the first agonist-bound human-
GPCR co-crystal structure to be determined with this compound.25
Thus from a mechanistic perspective, gaining a further understand-
ing of the receptor interaction with 25 was of interest. When com-
pared with 1 in a human bronchial smooth-muscle cell cAMP
assay: 25 was found to be 7.3-fold more potent with an EC50 of
0.28 nM and a relative intrinsic efficacy of 116% compared to
2.05 nM and 85% for 1. Having identified a high level of potency
and intrinsic efficacy for 25: kinetic binding studies at 37 °C, using
the protocol previously described to evaluate 1, showed an off-rate
driven kinetic Kd of 0.26 nM for 25.26 In comparison with 1 a similar
of > 10
lM was determined for 15. Similarly 1 was also inactive in
this dopamine D2 assay with an EC50 > 10
l
M, which was in contrast
to sibenadet which exhibited an EC50 value of 4.5 nM with an intrin-
sic efficacy of 106%. Therefore, the benzylic alcohol of 1 does make a
favourable contributon to the b2-adrenoceptor and to the b2/b1
selectivity. In contrast, the additional interactions of the extended
amino-substituent of sibenadet likely compensate for the absence
of the benzylic alcohol interaction with the b2-adrenoceptor, as well
as enhancing the interaction with the dopamine D2 receptor.
Modification of the amino substituent gave the isomeric tetra-
methyl derivative 17 and the 4,7-dihydroindanyl indacaterol deriv-
ative 29 which had comparable decreases in affinity and selectivity
when compared to 1. The 5-n-butylindanyl analogue 16, as a diaste-
roisomeric mixture epimeric at the 2-indanyl position, exhibited a
similar binding profile to 1, suggesting the potential for a similar
overall profile. However, the additional stereochemical complexity
in 16 reduced the attractiveness of this analogue for further follow
up studies. Introduction of the a-methyl group into the amino sub-
on was determined of 8.99 Â 107 MÀ1minÀ1, but the greater affinity
stituent gave the analogue 25 which led to the anticipated increase
in b2-adrenoceptor affinity, of greater than 10-fold relative to 1.
However, this increase in affinity was also accompanied by an even
greater relative increase in b1-adrenoceptor affinity and an overall
reduction in binding selectivity.
k
of 25 is derived from a 145-fold slower koff of 0.024 minÀ1. These
data highlight the marked impact on the interaction with the b2-
adrenoceptor for analogue 25 by the relatively small change of intro-
ducing an additional methyl group onto the a-carbon of the amino
Comparison of the calculated log D7.4 values for the analogues
in Table 1 indicates them all to be within one log unit higher than
that of 1, with the exception of the tetramethyl analogue 14 which
was predicted to be slightly lower. Based upon our previous obser-
vations, with this and related b2-adrenoceptor series, the lipophil-
icity predicted for the analogues in Table 1 would be anticipated to
be capable of delivering a similar duration and onset of action pro-
file to 1.8,21 However, in addition to lipophilicity a satisfactory level
of potency and intrinsic efficacy has also been shown to be a key
requirement for attaining the desired profile.8,23
To follow up on the most interesting analogues in Table 1, addi-
tional studies were carried out with the analogues 14, 15 and 25.
An assessment of the functional b2-adrenoceptor activity was made
using an electrically-stimulated guinea-pig tracheal-strip assay
with analogues 14 and 15, following the protocols previously de-
scribed for 1.19 Compared to 1, IC50 values of 3.9 and 240 nM were
determined for 14 and 15, making analogue 14 1.5-fold more potent,
and analogue 15 41-fold less potent. Analogue 14 also exhibited a
comparable onset, but shorter duration of action profile compared
to 1: 32.5 min and 2.3 h versus 30.0 min and 8.8 h respectively. In
contrast the less potent 15 exhibited a much slower onset of action,
but exhibited a comparable duration of action: 131 min and 9.3 h
substituent, when compared to 1.
In conclusion, from a number of close analogues of 1 the 3,4-
dihydroquinolinone and 5-n-butylindanyl analogues 14 and 16
were determined to exhibit the closest profiles to the parent com-
pound. However, the profiles of both analogues were deemed to be
less attractive as potential inhaled once-daily bronchodilator
candidates due to an inferior in vivo profile and increased stereo-
chemical complexity respectively. Interestingly, the highly potent
a-methyl analogue 25 showed an atypical activation profile, and
represents a further example of a slowly dissociating b2-adreno-
ceptor ‘super agonist’, albeit with a reduced b1-adrenoceptor bind-
ing selectivity when compared to 1.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012. 07.096.
References and notes
1. (a) Global Initiative for Asthma. Global Strategy for Asthma Management and
Prevention. Updated 2008. Available at: http://www.ginasthma.com/

D. Beattie et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6280–6285
6285
Guidelineitem.asp??l1=2&l2=1&intId=60. (b) Global Initiative for Chronic
Obstructive Lung Disease (GOLD). Global Strategy for the Diagnosis,
Management, and Prevention of Chronic Obstructive Pulmonary Disease.
13. Stocks, M. J.; Alcaraz, L.; Bailey, A.; Bonnert, R.; Cadogan, E.; Christie, J.;
Connolly, S.; Cook, A.; Fisher, A.; Flaherty, A.; Hill, S.; Humphries, A.; Ingall, A.;
Jordan, S.; Lawson, M.; Mullen, A.; Nicholls, D.; Paine, S.; Pairaudeau, G.; St-
Gallay, S.; Young, A. Bioorg. Med. Chem. Lett. 2011, 21, 4027.
14. Wieland, K.; Zuurmond, H. M.; Krasel, C.; IJzerman, A. P.; Lohse, M. J. Proc. Natl.
Acad. Sci. USA 1996, 93, 9276.
Updated
2007.
Available
at:
http://www.goldcopd.com/
Guidelineitem.asp?l1=2&l2=1&intId=1955.
2. Busse, W. W.; Shah, S. R.; Somerville, L.; Parasuraman, B.; Martin, P.; Goldman,
M. J. Allergy Clin. Immunol. 2008, 121, 1407.
3. Jacobsen, J. R. Future Med. Chem. 2011, 3, 1607.
15. Swaminath, G.; Xiang, Y.; Weon Lee, T.; Steenhuis, J.; Parnot, C.; Kobilka, B. K. J.
Bio. Chem 2004, 279, 686.
4. Tashkin, D. P. Expert Opin. Pharmacother. 2010, 11, 2077.
16. 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.
17. Farnum, D. G.; Carlson, G. R. Synthesis 1972, 191.
18. Prashad, M.; Hu, B.; Har, D.; Repic, O.; Blacklock, T. J.; Lohse, O. Org. Process Res.
Dev. 2006, 10, 135.
19. Battram, C.; Charlton, S. J.; Cuenoud, B.; Dowling, M. R.; Fairhurst, R. A.; Farr, D.;
Fozard, J. R.; Leighton-Davies, J. R.; Lewis, C. A.; McEvoy, L.; Turner, R. J.;
Trifilieff, A. J. Pharmacol. Exp. Ther. 2006, 371, 762.
5. (a) Dixon, R. A. F.; Kobilka, B. K.; Strader, D. J.; Benovic, J. L.; Dohlman, H. G.;
Frielle, T.; Bolanowski, M. A.; Bennett, C. D.; Rands, E.; Diehl, R. E.; Mumford, R.
A.; Slater, E. A.; Sigal, I. S.; Caron, M. G.; Lefkowitz, R. J.; Strader, C. D. Nature
1986, 321, 75; (b) Rasmussen, S. G.; Choi, H.-E.; Rosenbaum, D. M.; Kobilka, T.
S.; Thian, F. S.; Edwards, P. C.; Burghammer, M.; Ratnala, V. R. P.; Sanishvili, R.;
Fischetti, R. F.; Schertler, G. F. X.; Weis, W. I.; Kobilka, B. K. Nature 2007, 450,
383.
6. Waldeck, B. Eur. J. Phamacol. 2002, 445, 1.
7. (a) Yoshizak, S.; Yo, E.; Otsubo, J. Chem. Parm. Bull. 1978, 26, 1611; (b) Yoshizak,
S.; Tamada, S.; Yo, E.; Umezato, M. Chem. Parm. Bull. 1993, 41, 1305.
8. 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.;
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.; Alan Steward, A.; Sykes,
D.; Tedaldi, L.; Trifilieff, A.; Tweed, M.; Watson, S.; Wissler, E.; Wyss, D. J. Med.
Chem. 2010, 53, 3675.
20. Baker, J. G. Br. J. Pharmacol. 2010, 1048, 160.
21. Alikhani, V.; Beer, D.; Bentley, D.; Bruce, I.; Cuenoud, B. M.; Fairhurst, R. A.;
Gedeck, P.; Haberthuer, S.; Hayden, C.; Janus, D.; Jordan, L.; Lewis, C.; Smithies,
K.; Wissler, E. Bioorg. Med. Chem. Lett. 2004, 14, 4705.
22. Agonism was determined by the ability of test compounds to increase GTP
binding to membranes derived from Chinese Hamster Ovary cells expressing
the human dopamine D2 receptor. scintillation proximity assay with
GTP
35S was used as the readout. Intrinsic efficacy was based upon the
cS
A
9. Wang, T-C.; Chen, Y-L.; Lee, K-H.; Tzeng, C-C. Synthesis 1997, 87.
10. Srivastava, S. K.; Chauhan, P. M. S.; Bhaduri, A. P. Chem. Commun. 1996, 2679.
c
relative maximal responce compard to dopamine.
11. Preparative reversed phase chromatography was carried out using:
Phenomenex Luna C18 10
a
23. Rosethorne, E. M.; Turner, R. J.; Fairhurst, R. A.; Charlton, S. J. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 2010, 382, 255.
24. Hoenke, C.; Bouyssou, T.; Tautermann, C. S.; Rudolf, K.; Schnapp, A.; Konetzki, I.
Bioorg. Med. Chem. Lett. 2009, 19, 6640.
25. Rasmussen, S. G. F.; Choi, H-J; Fung, J. J.; Pardon, E.; Casarosa, P.; Chae, P. S.;
DeVree, B. T.; Rosenbaum, D. M.; Thian, T. S.; Kobilka, T. S.; Schnapp, A.;
Konetzki, A.; Sunahara, R. K.; Gellman, S. H.; Pautsch, A.; Steyaert, J.; Weis, W.
I.; Kobilka, B. K. Nature 2011, 469, 175.
l
M column, 150 Â 50 mm; eluing with a gradient
from 10% to 95% acetonitrile in water containing 0.1% trifluoroacetic acid; with
UV detection at 254 nM. Final products were obtained either: as
trifluoroacetate salts by direct evaporation; or as free bases by partial
evaporation, to remove organics, netralisation with NaHCO3, and extraction
into ethyl acetate.
12. Bonnert, R. V.; Brown, R. C.; Chapman, D.; Cheshire, D. R.; Dixon, J.; Ince, F.;
Kinchin, E. C.; Lyons, A. J.; Davis, A. M.; Hallam, C.; Harper, S. T.; Unitt, J. F.;
Dougall, I. G.; Jackson, D. M.; McKechnie, K.; Young, A.; Simpson, W. T. J. Med.
Chem. 1998, 41, 4915.
26. Sykes, D. A.; Charlton, S. J. Br. J. Pharmacol. 2012, 165, 2672.