The design of a novel series of muscarinic receptor antagonists leading to AZD8683, a potential inhaled treatment for COPD

Article abstract of DOI:10.1016/j.bmcl.2013.09.092

A novel series of muscarinic receptor antagonists was developed, with the aim of identifying a compound with high M₃ receptor potency and a reduced risk of dose-limiting side effects with potential for the treatment of COPD. Initial compound mo

Full text of DOI:10.1016/j.bmcl.2013.09.092

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6248–6253
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The design of a novel series of muscarinic receptor antagonists
leading to AZD8683, a potential inhaled treatment for COPD
Antonio Mete a, , Keith Bowers b, , Richard J. Bull e, Helen Coope b, , David K. Donald a,
,
Katherine J. Escott c, , Rhonan Ford a, , Ken Grime d, Andrew Mather a, , Nicholas C. Ray e, Vince Russell f
⇑
a Department of 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 Emerging Innovations Unit, AstraZeneca R&D, Alderley Park, Cheshire SK10 4TF, UK
d Respiratory, Inflammation & Autoimmunity iMed, AstraZeneca R&D, Molndal, Sweden
e Argenta Discovery, Flex Meadow, Harlow CM19 5TR, UK
f Argenta Discovery, Stoke Poges, Slough SL2 4SY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of muscarinic receptor antagonists was developed, with the aim of identifying a compound
with high M3 receptor potency and a reduced risk of dose-limiting side effects with potential for the
treatment of COPD.
Initial compound modifications led to a novel cycloheptyl series, which was improved by focusing on a
quinuclidine sub-series. A wide range of N-substituents was evaluated to determine the optimal substi-
tuent providing a high M3 receptor potency, high intrinsic clearance and high human plasma protein
binding. Compounds achieving in vitro study criteria were selected for in vivo evaluation. Pharmacoki-
netic half-lives, inhibition of bronchoconstriction and duration of action, as well as systemic side effects,
induced by the compounds were assessed in guinea-pig models.
Received 22 August 2013
Revised 26 September 2013
Accepted 27 September 2013
Available online 4 October 2013
Keywords:
COPD
Muscarinic M3 antagonist
Bronchoconstriction
Salivation
Compounds with a long duration of action and good therapeutic index were identified and AZD8683
was selected for progression to the clinic.
Ó 2013 Elsevier Ltd. All rights reserved.
Muscarinic acetylcholine (ACh) receptors perform important
roles in the regulation of many physiological functions of the cen-
tral and peripheral nervous systems and are widely distributed
throughout the body.1–5 They belong to the G-protein-coupled
receptor family and the five different muscarinic receptor subtypes
currently known are denoted M1, M2, M3, M4 and M5.6,7 In the lung,
the M3 receptor sub-type is present on airway smooth muscles and
sub-mucosal glands and when activated by ACh released from
parasympathetic nerves, produces bronchoconstriction, mucus
secretion and subsequently an increase in airways resistance, as
observed in chronic obstructive pulmonary disease (COPD). Antag-
onism of the M3 receptor sub-type leads to improved lung function
and is an established part of the therapeutic management of
COPD.8,9 The muscarinic receptor antagonists currently used to
treat COPD (ipratropium, tiotropium (1.1) and aclidinium bro-
mide10(1.2)) and newer antagonists, such as glycopyrrolate11,12
(1.3) in late stage clinical development, are all non-selective across
the M1 to M5 receptor subtypes (Fig. 1). All these compounds are
quaternary ammonium salts and carry a fixed positive charge
Figure 1. Structure of muscarinic antagonists in clinical use or in development.
which imparts very low permeability and makes them poorly ab-
sorbed. These compounds are administered directly to the lung
by inhaled delivery and this, in combination with their poor per-
meability, limits their systemic exposure. All these compounds
contain an ester function, which can be metabolized upon systemic
exposure, further reducing their potential to cause side effects such
as tachycardia, via antagonism of cardiac M2 receptors. Tiotropium
⇑
Corresponding author. Tel.: +44 1625 514519.
E-mail address: jane.escott@astrazeneca.com (K.J. Escott).
A.M., K.B., H.C., D.K.D., R.F. and A.M. were former employees of AstraZeneca.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.092

A. Mete et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6248–6253
6249
to potent compounds in both the phenyl (2.3) and thiophene (2.4)
derivatives. In order to rapidly improve the properties of this novel
cycloheptyl series, effort was primarily focused on the quinuclidine
sub-series as N-substitution would lead to single isomers, whereas
N-alkylation of the piperidine series would give an isomeric mix-
ture at the quaternized center, which would introduce lengthy
and difficult separation steps.
The synthetic routes used to prepare the types of compounds
explored in this work are summarized in Scheme 1.16 The synthesis
started from cycloheptanone (i) which was reacted with phenyl
Grignard and the resulting tertiary alcohol was methylated
in situ to give (ii). The methoxy group of (ii) could be replaced by
a carboxylate under the influence of Na/K (sodium/potassium al-
loy) followed by quenching the resultant anion with solid CO2 to
give (iiia) and subsequent methylation gives the methyl ester (iiib).
This was then reacted with 3-(R)-quinuclidinol to afford the com-
pound 2.3. A similar approach could be used for derivatives con-
taining other aromatic rings. An alternative approach started
Figure 2. Novel series of muscarinic receptor antagonists.
is currently considered the bronchodilator of choice for the treat-
ment of COPD and is generally well tolerated with dry mouth being
the main reported side-effect.8,9 Tiotropium is a highly potent13
and long acting muscarinic receptor antagonist and any new mus-
carinic receptor antagonist developed for COPD should match tiot-
ropium for efficacy and duration of action whilst reducing the dry
mouth and other dose-limiting side effects.
with the ketone (iv) which was chlorinated to give (v). This a-chlo-
roketone rearranged under the influence of silver nitrate followed
by heating under basic conditions to afford the acid (iiia) which
was then methylated to give ester (iiib). The quinuclidine deriva-
tives, 2.3 and 2.4, (Fig. 2) could be reacted on the tertiary nitrogen
with a range of alkylating groups leading to the quaternized deriv-
atives of structural sub-types 2, 3 and 4 shown in Figure 3.
The choice of groups used as N-substituents determines the
physical and biological properties of the analogs listed in Table 2.
A wide range of groups were used and evaluated to find the opti-
mal substituent to afford a compound which met our desired crite-
ria of high potency at the M3 receptor together with high intrinsic
clearance and high hPPB. We set in vitro criteria of M3 pIC50 >9.5,13
We recently reported the discovery of a novel series of musca-
rinic antagonists which demonstrated a reduced side-effects pro-
file relative to tiotropium in pre-clinical studies.14 Our approach
was to achieve low systemic exposure with the M3 receptor antag-
onists by selecting compounds which possessed high intrinsic met-
abolic clearance coupled with high human plasma protein binding
(hPPB) in order to reduce the free concentration of any compound
reaching the plasma. In this paper, another series is described with
the aim of identifying a chemically differentiated compound which
also possesses high M3 receptor potency and low systemic expo-
sure and hence minimizes the risk of systemic side effects in vivo.
We have previously reported that compounds such as 2.1
(Fig. 2), which bear a methyl group in the position where tiotropi-
um, glycopyrrolate and aclidinium bromide have a hydroxy group,
exhibit antagonism of the M3 receptor13 coupled with high meta-
bolic clearance and high human plasma protein binding.15 How-
ever, in compounds such as 2.1, constructing the chiral center at
the quaternary carbon was synthetically very challenging and a
hindrance to rapid analog synthesis. Therefore, in order to main-
tain the promising biological profiles of these molecules but in a
chemically simpler series the cyclopentyl and the methyl groups
of 2.1 were combined into a cycloheptyl ring, thus eliminating
the chiral center and leading to the derivative 2.2. This type of
compound possessed good activity as M3 receptor antagonists (Ta-
ble 1). Replacement of the piperidine ring by quinuclidine also led
a
b
O
O
ii
R
O
i
c
f
O
iiia R=H
iiib R=Me
d
e
Cl
O
O
O
iv
v
O
N
2.3
Scheme 1. Synthetic routes to the novel series of muscarinic receptor antagonists.
Reagents and conditions. (a) PhMgBr, THF, RT, 0.5 h, 66%; then NaH, MeI, THF,
reflux, 78 °C h, 55%; (b) Na/K, À10 to 0 °C diethyl ether; solid CO2 À78 °C to rt, 75%;
(c) MeOH, HCl, reflux, 24 h, 80%(d) (3R)-quinuclidinol, (2 equiv), NaH (60% disp, 2
equiv), toluene, reflux, 70%; (e) SO2Cl2, reflux, 18 h, 95%; (f) AgNO3, water, 75 °C,
4.5 h then K2CO3, 90 °C, 1 h, 20%.
Table 1
In vitro biological data of known muscarinic antagonists compared to the new series
Compound
M3
pIC50
HM Cl_intb
min/mg)
(l
l/
hPPB (%) bound
a
1.1 Tiotropium
>10.3
<3
48
bromide
1.2 Glycopyrrolate
9.9
<3
<30
1.3 Aclidinium
>10.3
>145
No data (not stable in
bromide
plasma)
58
73
2.1
2.2
2.3
2.4
10.5
9.6
35
20
10.2
10.1
—
—
95
95
a
Binding estimates measured using recombinant human M3 receptor (n P2).
Intrinsic clearance in human liver microsomes.
b
Figure 3. Chemical sub-types made and profiled with data shown in Table 2.

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A. Mete et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6248–6253
Table 2
In vitro biological data for the novel muscarinic antagonists
a
b
Compound
R group
X
M3 pIC50
GPT pA2
HM Cl_intc
(
ll/min/mg)
hPPB (%) bound
LogPd
2.3
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
2.13
2.14
2.15
2.16
2.17
2.18
2.19
2.20
2.21
2.22
3.1
—
Me–
Ph-CH2–
3,4-di-F-Ph-CH2–
3-MeO-Ph-CH2–
Ph-(CH2)2–
5-Me-Thien-2-yl-(CH2)2–
3-Cl-Ph-(CH2)2–
4-CN-Ph-(CH2)2–
Ph-O-(CH2)2–
—
10.2
8.6
7.4
7.4
8.5
10.1
10.2
>10.4
8.5
9.8
9.9
10.2
>10.4
10.3
>10.5
9.8
9.0
9.3
10.3
9.7
9.6
9.9
7.0
>10.2
9.8
10.0
8.1
9.4
7.9
8.0
9.8
7.5
9.7
8.6
9.2
9.8
9.8
>9.9
9.6
9.3
—
—
—
—
—
9.5
9.5
9.6
—
9.4
8.4
9.1
9.9
10.0
9.8
—
—
34
68
55
—
95
73
—
—
—
99.3
99
99.5
—
4.6
2.1
3.4
3.4
3.3
3.3
3.5
3.9
2.8
3.4
3.7
3.4
2.0
1.6
2.4
2.4
2.9
1.4
3.3
1.0
3.3
3.8
1.3
1.2
1.6
1.4
1.8
1.8
1.8
2.8
2.7
2.7
2.5
2.8
2.0
2.2
0.3
2.3
2.5
3.1
3.0
3.1
3.8
3.9
2.9
1.0
2.7
0.8
1.74
1.6
2.8
2.8
3.4
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
S
105
130
72
49
106
120
75
57
55
135
77
50
63
82
19
27
17
15
150
26
37
39
25
66
—
55
—
40
39
32
—
70
43
49
51
—
96
97
91.5
88
94
97
95
96
94
96
82
99.4
99.7
—
82
97
95
94
—
85
—
96
—
Ph-CH2-O-(CH2)2–
Ph-O-(CH2)3–
4-Pyridyl-(CH2)3–
4-Pyridyl-(CH2)3–
2-Me-4-pyridyl-(CH2)3–
6-Me-3-Pyridyl-(CH2)3–
2,6-di-Me-3-pyridyl-(CH2)3–
1-Me-3-Pyrazolyl-(CH2)3–
2-Benzoxazolyl-(CH2)3–
H–
Ph–
3-F-Ph
-4-Morpholino
3-Isoxazolyl
5-Me-3-Isoxazolyl
3-Pyrazolyl
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
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
—
—
9.2
10.1
—
8.5
—
10.7
9.6
—
—
—
—
—
9.4
—
—
—
—
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
1,3,4-Thiazol-2-yl
1,2,4-Thiadiazol-5-yl
2-Oxazolyl
3.10
3.11
3.12
3.14
3.15
3.16
3.17
3.18
3.19
3.20
3.21
3.22
3.23
3.24
3.25
3.26
3.27
3.28
3.29
3.30
3.31
3.32
3.33
3.34
3.35
5-tButyl-3-isoxazolyl
2-Pyridyl (R enantiomer)
2-Pyridyl (S enantiomer)
3-Pyridyl
96
95
—
4-Pyridyl
5-Pyrimidyl
4-Pyrimidyl
3-Pyridazinyl
2-Pyrazinyl
3-F-2-Pyridyl
5-Me-2-Pyridyl
5-F-2-Pyridyl
4-Me-2-Pyridyl
6-CF3-2-Pyridyl
6-CF3-3-Pyridyl
6-Me-3-Pyridyl
2-Me-4-Pyrimidyl
6-Me-4-Pyrimidyl
6-Me-3-Pyridazinyl
6-CF3-3-Pyridazinyl
6-Cl-3-Pyridazinyl
5-Me-2-Pyrazinyl
6-Me-2-Pyrazinyl
6-Cl-2-Pyrazinyl
—
89
90
92.5
94
98.1
97.6
98.2
99.6
—
10.1
9.9
—
—
—
—
—
—
—
—
—
—
—
9.7
—
—
9.2
9.1
8.6
9.6
9.1
9.4
9.0
9.1
8.6
9.7
9.1
9.2
30
—
5
—
—
26
16
—
—
—
80
90
—
96
96
—
—
27
27
51
10.0
9.3
98.5
N
H
N
N
3.36
3.37
CH2
CH2
8.5
8.2
—
—
74
—
0.8
0.6
O
N
H
N
101
97.4
O
N
4.1
4.2
CH2
CH2
10.0
9.8
9.8
9.4
78
97.7
99
4.1
4.5
O
O
N
N
102
N
O
Ph
Ph
4.3
CH2
9.6
9.5
63
99
2.8

A. Mete et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6248–6253
6251
Table 2 (continued)
a
b
Compound
R group
X
M3 pIC50
GPT pA2
HM Cl_intc
103
(
ll/min/mg)
hPPB (%) bound
—
LogPd
4.4
4.5
CH2
9.4
—
3.2
4.4
N
S
N
Ph
Ph
CH2
CH2
7.6
—
133
79
—
N
N
O
N
4.6
10.1
8.5
99.5
3.6
2.8
Ph
N
O
4.7
CH2
9.5
9.7
97
96.5
Ph
a
Binding estimates measured using recombinant human M3 receptors (n P2).
Antagonist potency at the M3 receptor measured in guinea pig trachea (n = 2).
Intrinsic clearance in human liver microsomes.
b
c
d
LogP was calculated using ACD LogP software.
Table 3
In vivo pharmacokinetics, efficacy and mechanistic side effect of the novel series of muscarinic antagonists
Compound
IT-PK T½ h
BC ED80
(lg/kg) 2 or 4 h
BC % inhibition 24 h
BC % inhibition 48 h
Salivation EC50
(
lg/kg)
TI
Tiotropium
2.15
2.16
2.18
2.19
2.22
3.1
nd
10.2
19
43.7
19.2
9.3
11.4
19
26.6
25.6
42.4
23
56.7
13
0.3a
3a
1
1
—
>80c
31b
0
69c
—
—
—
—
—
—
—
—
84c
—
73c
—
—
0
<0.3
—
—
—
—
63
—
—
—
—
—
—
—
P10
P10
—
P10
P10
—
0
—
—
—
—
—
—
—
3.5
3.6
1a
3
0
>10
>30
>30
>10
>10
>30
>10
—
56b
—
3.12
3.19
3.20
3.32
4.1
1a
—
1
3
—
3
88c
89c
—
4.2
17
—
—
4.3
4.6
4.7
16.5
12.1
22
3
—
3
—
—
—
0
—
0
>30
—
>30
—
—
—
a
b
c
Measured ED80 dose.
P <0.05.
P <0.01 compared to control group.
guinea pig trachea pA2 >9,17 HM Cl_int >50, hPBB >95% bound, and
primarily compounds which matched these were considered for
progression into a range of in vivo models.
assays (3.5–3.35). However, the challenge in the latter structural
type was to achieve high hPPB (P95%). It became apparent that
in this type of compound a LogP value of >2.5 was typically re-
quired to attain high hPPB. A number of these compounds such
as 3.1, 3.2, 3.6, 3.12, 3.19, 3.20 and 3.32 did possess the desired
in vitro profile and were selected for evaluation in the in vivo mod-
els. Finally, a set of compounds were made in which the terminal
aryl was linked to the quinuclidine by an intervening heterocycle,
4.1–4.7, which acted as an amide isostere. Several of these analogs,
4.1, 4.2, 4.3, 4.6 and 4.7 combined the required in vitro properties
for progression into the in vivo studies.
The in vivo profiles of compounds which passed our pre-set
in vitro criteria are summarized in Table 3. These in vivo
guinea pig studies included determining pharmacokinetic half-
lives (PK T½ (lung)), duration of action (inhibition of bronchocon-
striction) and systemic side effects (inhibition of hypersecretion
(salivation)). Initially, the PK half-lives (IT-PK T½) of the com-
pounds were determined following intratracheal dosing.18 Com-
pounds with long half-lives were then evaluated in a guinea pig
model of bronchoconstriction (BC) to determine their ED80 dose
and then duration of action at 24 and/or 48 h post-dose.19 To test
duration of action, compounds were administered to the lung at
an ED80 dose (i.e., dose which produced ꢀ80% inhibition of metha-
Table 2 summarizes the range of compounds made and some
key in vitro data. The parent compound 2.3 was initially N-methyl-
ated to afford 2.5 which proved significantly less potent. A range of
N-benzyl substituted derivatives, 2.6–2.8, were also in general,
found to possess lower potency than the parent 2.3. However,
derivatives 2.9–2.22 in which an aromatic ring is linked to the
quinuclidine by chain lengths of two or more atoms exhibited a
range of potencies, with some having pIC50 values >10.3 at the
M3 receptor. Some of these compounds also exhibited high human
PPB and high HM Cl_int leading to 2.15, 2.16 and 2.18 being taken
forward for detailed in vivo evaluation (discussed later).
Another structural sub-type explored is exemplified by com-
pounds 3.1–3.37. In this compound set, the chain linking the aryl
group to the quinuclidine contains an amide function. A range of
aromatic and heterocyclic rings were evaluated to improve the
properties of the final molecules. However, when the aryl was a
phenyl ring (e.g., 3.3) the high potency exhibited at the M3 receptor
was not matched by the potency seen in the tissue-based guinea
pig trachea assay.17 When the aromatic ring was a range of more
polar heterocycles, similar potency was achieved in both of these

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A. Mete et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6248–6253
choline induced BC at 2 or 4 h).19 The ED80 dose was either mea-
sured or predicted from the guinea pig trachea potency data. Com-
pounds which had significant bronchoconstriction beyond 24 h
were then evaluated in a guinea pig pilocarpine-induced salivation
model to assess the peripheral side effects of the compounds at a
4 h timepoint.20 Data from the BC and salivation models were used
to establish a therapeutic index (TI) for bronchoprotection over sal-
ivation effects in the guinea pig. Our objective was to identify com-
pounds with a TI P10 as this would be a significant improvement
over tiotropium which has a narrow therapeutic window (TI <3) in
these models.
during this bronchodilator alliance between Pulmagen, Argenta
and AstraZeneca. The authors would also like to thank Katherine
Wiley, Sandy Nicol, Marilyn Mather, Sarah Lewis, Elizabeth Kin-
chin, Ian Millichip, Barry Teobald, Sasvinder Sidhu, Hemant Mistry,
Jaspal Singh, James Bird and Elizabeth Skidmore for technical con-
tributions to the work.
References and notes
1. Moulton, B. C.; Fryer, A. D. Br. J. Pharmacol. 2011, 163, 44.
2. Peretto, I.; Petrillo, P.; Imbimbo, B. P. Med. Res. Rev. 2009, 29, 867.
3. Laine, D. I. Expert Rev. Clin. Pharmacol. 2010, 3, 43.
4. Cazzola, M.; Matera, M. G. Br. J. Pharmacol. 2008, 155, 291.
5. Joos, G. F. Expert Opin. Investig. Drugs 2010, 19, 257.
The compounds bearing an alkyl-aryl substituted quinuclidine,
2.13–2.22 exhibited IT-PK half-lives ranging from 9 to 44 h.18 Sev-
eral of these compounds were taken into the guinea pig broncho-
constriction (BC) model but demonstrated limited or no efficacy
at 24 h post-dose. The compounds from the amide-linked sub-ser-
ies, 3.1–3.32, also had a range of IT-PK half-lives and a number
were taken into the BC model. In general, higher levels of efficacy
were observed at 24 h and long duration of action (>48 h) was seen
in this class of compound, when the IT-PK T½ was P23 h. From the
third series, 4.1 to 4.7 none of the compounds that were progressed
attained IT-PK T½ >24 h and indeed did not show significant effi-
cacy in the BC model at 48 h. The long duration of action in the
BC model for this compound series is thought to be mediated by
improved retention of compound in the lung as demonstrated by
the IT-PK half-lives >23h. However, the contribution of slow-off
rate at the M3 receptor to the duration action cannot be ruled
out and additional receptor kinetic studies would be required to
further understand the pharmacology profile of these compounds.
In addition, the systemic side effect profile of some of the more
potent compounds with long duration of action were tested using
the salivation model.20 Thus three compounds 3.6, 3.12 and 3.20
demonstrated a therapeutic index (TI) of at least 10-fold when
comparing their efficacy in the BC model seen at 4 h and the EC50
dose in the salivation model. The improved TI may be attributed
to high PPB and metabolic clearance limiting systemic exposure
in the salivation model. As the guinea pig bronchoconstriction
and salivation models are primarily mediated by M3 receptors then
the inhibitory effects of these compounds are thought to be due to
antagonism of the M3 receptor. In general, compounds from this
series are not selective for the M3 receptor (data not shown) over
other muscarinic receptors and so the contribution of other musca-
rinic receptors to the in vivo findings are unlikely but cannot be ex-
cluded at this time.
Compounds 3.12 and 3.20 demonstrated long duration of action
and a good TI and so their pharmaceutical properties were further
evaluated for development in inhalation delivery devices. The com-
pound chosen to progress to the clinic based on the balance of effi-
cacy, side effect profile and solid state properties (not discussed in
this paper) was 3.12.
In summary, we have reported on the design and improvement
of a novel series of M3 receptor antagonists which match a set of
criteria including in vitro potency and ADME properties (high
PPB and clearance). Key compounds were identified with optimal
in vivo properties demonstrating duration of action in the lung
and an improved therapeutic index over tiotropium in guinea pigs.
Our criteria were selected to improve on the biological profile of
existing M3 receptor antagonists, leading to the selection of com-
pound 3.12, AZD8683, which combined the required biological
profile with good pharmaceutical properties and was progressed
into the clinic for evaluation as a potential treatment for COPD.
6. Caulfield, M. P.; Birdasall, N. J. M. Pharmacol. Rev. 1998, 50, 279.
7. Eglen, R. M. Prog. Med. Chem. 2005, 43, 105.
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Pharmacol. Exp. Ther. 2009, 330, 660.
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Pharmacol. 1827, 2004, 5.
10. Gavalda, A.; Miralpeix, M.; Ramos, I.; Otal, R.; Carreno, C.; Vinals, M.; Domenech,
T.; Carcasona, C.; Reyes, B.; Vilella, D.; Gras, J.; Cortijo, J.; Morcillo, E.; Llenas, J.;
Ryder, H.; Beleta, J. J. Pharmacol. Exp. Ther. 2009, 331, 740.
11. Hansel, T. T.; Neighbour, H.; Erin, E. M.; Tan, A. J.; Tennant, R. C.; Maus, J. G.;
Barnes, P. J. Chest 1974, 2005, 128.
12. Villetti, G.; Bergamaschi, M.; Bassani, F.; Bolzoni, P. T.; Harrison, S.; Gigli, P. M.;
Geppetti, A. J. P.; Civelli, M.; Patacchini, R. Br. J. Pharmacol. 2006, 148, 291.
13. In vitro M3 radioligand binding assay: The affinity of a compound at the human
M
3 receptors was estimated from its ability to compete with specific [3H]NMS
binding to membranes from CHO-K1 cells expressing recombinant human M3
receptors as measured using a SPA assay format. Concentration-effect curves for
each compound (0.003À100 nM final concentrations) were constructed using
serial dilutions in ½-log unit intervals. For each assay plate, eight replicates were
obtained for [3H]NMS binding in the absence of test compound. Non-specific
binding of [3H]NMS was determined by replacing test compound with atropine
1 l
M. [3H]NMS was used at a concentration of 0.1 nM, which was below the
determined dissociation constant (Kd) for [3H]NMS, and about 10% of the
radioactivity was specifically bound to the membranes. The assay mixture was
incubated at room temperature for at least 16 h before counting to allow
[3H]NMS binding with muscarinic receptors to reach equilibrium; binding of
[3H]NMS was also reversible. Test compound inhibition was expressed as
percent inhibition relative to the specific radioligand binding for the plate.
14. Mete, A.; Bowers, K.; Chevalier, E.; Donald, D.; Edwards, E.; Escott, K.; Ford, R.;
Grime, K.; Millichip, I.; Teobald, B.; Russell, V. Bioorg. Med. Chem. Lett. 2011, 21,
7440.
15. Bailey, A.; Mete, A.; Pairaudeau, G.; Stocks, M.; Wenlock, M. WO2007123465,
2007, Chem. Abstr. 2007, 147, 502240. .
16. Ford, R.; Mather, A.; Mete, A.; Bull, R.; Skidmore, E. WO2009138707, 2009,
Chem. Abstr. 2009, 152, 12539. Synthetic conditions and yields for all new
compounds described herein are reported in these publications. New
compounds were identified and characterized by their 1H NMR and mass
spectra, chiral HPLC and elemental analyses.
17. In vitro guinea-pig trachea assay: The antagonist potency of compounds was
measured by the ability of compounds to inhibit methacholine-induced airway
smooth muscle contraction using a functional, guinea-pig trachea organ bath
assay. Dunkin Hartley guinea-pigs were sacrificed by cervical dislocation and
the trachea removed. Tracheal rings were prepared and then suspended in
10 mL organ baths containing
a modified Krebs solution (containing
Indomethacin at 2.8 M final concentration), gassed with 5% CO2, 95% O2 at
37 °C and tensioned to 1 g. The tracheal rings were left to equilibrate for 1 h,
washed and re-tensioned to 1 g. The rings were ‘primed’ with methacholine
(1 M), washed and left for a further 1 h. Then a cumulative methacholine
concentration response curve was constructed (3 Â 10À9 M to 3 Â 10À5 M).
Vehicle or test compound was then added to the baths and allowed to
equilibrate for 1 or 4 h. Then, a second extended cumulative concentration
response curve was constructed (3 Â 10À9
M
to 1 Â 10À3
M
final
concentration). Changes in isometric force were recorded and results were
expressed as % of maximum response in curve 1 for each individual tissue. A50
values were generated using calculated
% increase in tension at each
compound concentration. The A50 values for the vehicle control
concentration effect curve and test concentration effect curve were
determined and then used to calculate
compound concentration used.
a potency pA2 value at each
18. For pharmacokinetic studies, compounds were dosed to halothane
anaesthetised guinea pigs by intratracheal instillation at g/kg. For
1
l
terminal lung collection at 0.25, 0.5, 0.75, 1, 2, 4, 7, 12, 18, 24 h post-dosing,
the animals were euthanised by an intravenous overdose of barbiturate
(Euthatal 20 mL/kg) and the lungs removed and homogenized with phosphate
buffer and added to ice cold methanol. Aliquots of the supernatant were
analyzed by mass spectrometry/HPLC methodologies.
Acknowledgments
19. In vivo guinea-pig bronchoconstriction: The efficacy of compounds was measured
in vivo by the inhibition of methacholine induced bronchoconstriction in
Dunkin–Hartley guinea pigs. Dose solutions of test compounds were prepared in
saline (0.9% (w/v) sodium chloride) and delivered to the lung by either
Thanks to Dr. Harry Finch and Dr. Mary Fitzgerald of Pulmagen
Therapeutics (Synergy) Ltd for helpful discussions and guidance

A. Mete et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6248–6253
6253
intratracheal or intranasal instillation using a Penn–Century microsprayer
(Penn–Century Inc., Wyndmoor, PA, USA) under halothane anaesthesia. At
various time points after dosing (2, 4, 24 or 48 h), guinea pigs were terminally
anaesthetised with pentobarbitone, surgically prepared, ventilated (at
60 breaths/min at a tidal volume of 2 mL) and lung function measured by
changes in respiratory resistance in response to the bronchoconstrictor agent
20. In vivo salivation: The ability of compounds to inhibit salivation was measured
in vivo by the reduction of pilocarpine induced salivation in guinea-pigs.
Compounds or vehicle were administered intranasally in halothane-
anaesthetised guinea-pigs. At 4 h after dosing, guinea-pigs were terminally
anaesthetised (urethane). Once anaesthesia was established a gauze pad was
inserted into the mouth for 5 min to dry the mouth of residual saliva. A pre-
weighed pad was then inserted into the mouth for 5 min and weighed for a
measurement of baseline saliva production. Pilocarpine (0.6 mg/kg at a dose
methacholine (10 lg/kg, intravenous bolus), using the Flexivent lung function
system (EMMS, UK). After each administration of methacholine, the peak
resistance value was obtained and respiratory mechanics returned to baseline in
between administrations of methacholine. At the end of the experiments
animals were euthanized. Percentage inhibition of bronchoconstriction was
calculated for each compound treated group in relation to the appropriate
vehicle, time-matched control group.
volume of 2 mL/kg) was then administered subcutaneously.
A new pre-
weighed pad was inserted into the mouth immediately and replaced at 5 min
intervals for 15 min. The difference in weight of the pads pre and post the
5 min sampling period was used to calculate the amount of saliva collected
over a 15 min period.