Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinyl carbamates as potent and long acting muscarinic antagonists

Article abstract of DOI:10.1016/j.bmcl.2011.03.096

Novel quaternary ammonium derivatives of N,N-disubstituted (3R)-quinuclidinyl carbamates have been identified as potent M₃ muscarinic antagonists with long duration of action in an in vivo model of bronchoconstriction. These compounds have also presented a high level of metabolic transformation (human liver microsomes). The synthesis, structure-activity relationships and biological evaluation of these compounds are reported.

Full text of DOI:10.1016/j.bmcl.2011.03.096

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3457–3461
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinyl
carbamates as potent and long acting muscarinic antagonists
Maria Prat a, , María Antonia Buil a, Maria Dolors Fernández b, Jordi Castro a, Juan Manuel Monleón a,
⇑
Laia Tort a, Gaspar Casals a, Manuel Ferrer a, Josep Maria Huerta a, Sònia Espinosa a, Manuel López a,
Victor Segarra a, Amadeu Gavaldà a, Montserrat Miralpeix a, Israel Ramos a, Dolors Vilella a,
Marisa González a, Mònica Córdoba a, Alvaro Cárdenas c, Francisca Antón d, Jorge Beleta a, Hamish Ryder e
a Almirall, R&D Centre, Laureà Miró 408-410, Sant Feliu de Llobregat, 08980 Barcelona, Spain
b Thomson Reuters, Provenza 398, 08025 Barcelona, Spain
c UCB Pharma S.A. , Avenue de l’Industrie, 1420 Braine-l’Alleud, Belgium
d Laboratorios Lesvi, S.L., Avenida Barcelona 69, Sant Joan Despí, 08970 Barcelona, Spain
e Cancer Research Technology Ltd., CRT Discovery Laboratories, 12 Rosemary Lane, Cambridge CB1 3LQ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel quaternary ammonium derivatives of N,N-disubstituted (3R)-quinuclidinyl carbamates have been
identified as potent M3 muscarinic antagonists with long duration of action in an in vivo model of bron-
choconstriction. These compounds have also presented a high level of metabolic transformation (human
liver microsomes). The synthesis, structure–activity relationships and biological evaluation of these com-
pounds are reported.
Received 31 January 2011
Revised 23 March 2011
Accepted 23 March 2011
Available online 3 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
(3R)-Quinuclidinyl carbamates
M3 Muscarinic antagonists
Long acting bronchodilators
Low systemic exposure
COPD
Chronic obstructive pulmonary disease (COPD) is a preventable
and treatable lung disease characterised by airflow limitation that
is not fully reversible.1,2 The major reversible component of airway
obstruction in COPD is believed to be cholinergic.3,4 Inhaled anti-
cholinergics, which increase bronchodilation by blocking musca-
rinic receptors on airway smooth muscle, are established
treatment options for COPD.5 Currently, only two inhaled musca-
rinic antagonists (Fig. 1) are available for the management of
symptomatic COPD patients: ipratropium, a short-acting agent
requiring up to four doses per day and tiotropium, a long-acting,
once-daily treatment.5–7
During the course of the Almirall research program to discover a
novel, long-acting muscarinic M3 antagonist for the inhaled treat-
ment of COPD, with a potentially improved risk-benefit profile
compared with currently marketed antimuscarinic agents,8 new
N,N-disubstituted (3R)-quinuclidinyl carbamates and their quater-
nary ammonium derivatives were identified as potent antimuscar-
inic compounds.
Several N,N-disubstituted carbamates of 3-quinuclidinol had
been described in the literature as compounds with potent
anticholinergic activity.9 During the 1990’s, researchers from
Yamanouchi Pharmaceuticals identified different series of quinuc-
lidinyl carbamates, represented by the compounds YM-905 and
YM-46303 (Fig. 2), as potent muscarinic antagonists with thera-
peutic potential for the treatment of smooth muscle contractility
and tone, for example in urinary and respiratory disorders.10–15
These Yamanouchi carbamates were described to have selectivity
for the M3 receptor in front of the M2 one.12–14 Compound YM-
905, solifenacin, is currently marketed for the treatment of urinary
incontinence.15
In this publication we will report the synthesis and biological
evaluation of a variety of novel N,N-disubstituted (3R)-quinucli-
dinyl carbamates. These compounds were prepared as tertiary
amines and evaluated for their muscarinic binding activities. Qua-
ternary ammonium derivatives of the more interesting examples
were prepared to obtain analogues with predicted low oral bio-
availability, adequate for inhaled administration.8,16 Although
quaternization limits the absorption across membranes,8,16 after
inhaled administration there could be observed anticholinergic
side effects associated with the binding of systemic available
⇑
Corresponding author. Tel.: +34 932 913816; fax: +34 933 128635.
E-mail address: maria.prat@almirall.com (M. Prat).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.096

3458
M. Prat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3457–3461
physiological effects could be tachycardia, dry mouth, urinary
Br
Br
retention and constipation.17 Whereas dry mouth is the most com-
mon one18,19 as a result of the systemic blockade of M1 and M3
receptors,20 tachycardia which results from blockade of cardiac
M2 receptors17,21 could be considered the most relevant one. For
this reason compounds with selectivity for the receptor M3 in front
of the M2 one would be safer. The influence of the quaternization in
the muscarinic binding activity profile and in the duration of action
in an in vivo model of bronchoconstriction will be described. The
plasma stability and the metabolism in human liver microsomes
of some selected analogues with long duration of action will also
be reported.
N
N
O
HO
H
O
H
O
S
O
O
HO
S
Tiotropium bromide
Ipratropium bromide
Figure 1. Inhaled muscarinic antagonists available for the treatment of COPD.
O
N
The synthetic route to the quinuclidine carbamate derivatives is
outlined in Scheme 1.
N
O
N
H
O
HCl
O
N
Disubstituted amines of general formula 2 that are not commer-
cially available were synthesized from amines of formula 1 accord-
ing to standard methods, for example by reductive alkylation using
the corresponding aldehyde, or by alkylation with the correspond-
ing bromide. Transformation of amines of formula 2 into their car-
bamoyl chloride derivatives 322 followed by reaction with the
sodium salt of (3R)-quinuclidinol in refluxing toluene led to carba-
mates of formula 4. Quaternary ammonium derivatives of formula
5 were obtained by reaction of selected (3R)-quinuclidinyl carba-
mates with a variety of bromoalkyl or bromoalkylaryl derivatives.
YM-905
Solifenacin succinate
YM-46303
Figure 2. Examples of quinuclidinyl carbamates.
compound to muscarinic receptors outside the respiratory tract.
Given the distribution of muscarinic receptors these unwanted
O
O
O
e
c
d
R1
a or b
R1
R1
R1
R1
N
O
NH
R2
N
O
NH2
N
Cl
R2
R2
R2
N
N
R3
Br
1
5
4
2
3
Scheme 1. Reagents and conditions: (a) (i) R2CHO, EtOH, molecular sieves 0.3 nm, 78 °C, (ii) NaBH4, 65–83%; (b) R2Br, K2CO3, toluene, 110 °C, 18–32%; (c) Triphosgene,
CH2Cl2, 10 °C to rt, 74–96%; (d) (3R)-quinuclidinol, Na, toluene, 110 °C, 18–33%; (e) R3Br, THF, 66 °C, 48–82%.
Table 1
Effects of R1 and R2 substitution on M3, M2, M1 muscarinic receptor binding activities for N,N-disubstituted-(3R)-quinuclidinyl carbamate derivatives
O
R1
N
O
R2
N
4
Compd
R1
R2
Activity: IC50 a(nM)
M2
Selectivityb
M3
M1
M2/M3
M1/M3
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph-CH2–
Ph-(CH2)2–
(2-Th)-CH2–
(3-Th)-CH2–
n-Propyl–
n-Butyl–
n-Pentyl–
Isopropyl–
Ph-CH2–
(2-Th)-CH2–
n-Butyl–
Ph-CH2–
Ph-CH2–
(3-F)-Ph-CH2–
(2,4,5-tri-F)-Ph-CH2–
(3,4,5-tri-F)-Ph-CH2–
796.8 (43.9)
1.2 (0.1)
3.6 (0.1)
0.6 (0.1)
1.0 (0.3)
71.1 (5.2)
4.3 (0.7)
1.7 (0.0)
659.3 (138.2)
22.5 (4.0)
5.9 (0.5)
17.7 (1.6)
74.7 (32.7)
15 (2.7)
2779.0c (1034.4)
3.7 (1.2)
42.1 (3.4)
1.8 (0.7)
4.7 (1.8)
349.3 (18.8)
22.9 (10.9)
13.6 (5.7)
1364.9 (17.2)
88.9 (11.6)
22.7 (3.2)
319.4 (6.0)
0.4 (0.1)
1 (0.4)
3.5
3.1
11.7
3.0
4.7
4.9
5.3
8.0
2.1
3.95
3.8
4.3
2.6
7.7
11.2
32.7
64.9
4.4
2.4
0.6
0.4
0.3
0.3
0.7
0.4
0.4
0.2
0.5
0.6
0.4
0.4
0.04
0.5
0.5
0.6
0.7
0.6
0.5
0.5
0.63
1.28
0.4 (0.3)
0.4 (0.1)
26.7 (4.8)
1.1 (0.1)
0.8 (0.4)
428.0 (5.8)
8.8 (0.2)
2.4 (0.4)
0.8 (0.5)
39.7 (5.4)
8.3 (1.7)
1.5 (0.3)
1 (0.1)
Ph–
(2-Th)-CH2-
(2-Th)-CH2–
(2-Th)-CH2–
(4-Me)-Ph–
(4-F)-Ph–
(3-F)-Ph–
(3-Me)-Ph–
(3-F)-Ph–
76.2 (21.9)
195.6 (33.2)
115.7 (61.0)
26.8 (15.9)
45.8 (21.0)
97.3 (15.0)
573.7 (45.3)
14.5 (2.6)
2.4 (0.6)
1.4 (0.1)
1.5 (0.1)
4q
0.9 (0.3)
63.5 (8.8)
3.0 (0.9)
0.22d(0.03)
2.65d(0.13)
Solifenacin
YM-46303
Tiotropium bromide
Ipratropium bromide
130.8 (27.1)
6.0 (1.2)
0.35d (0.01)
2.07d(0.03)
0.21d(0.04)
2.24d(0.16)
1.08
a
b
c
Values shown are the mean, n = 2 (SD), standard deviation is given in parentheses.
The selectivity is calculated as the ratio between the IC50 values.
The value shown is the mean of n = 1 (3510 nM) and n = 2 (2047 nM).
Values shown are the mean, n = 3 (SD), standard deviation is given in parentheses.
d

M. Prat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3457–3461
3459
Table 2
Effects of R3 substitution on the M3, M2, M1 muscarinic receptor binding activities and in vivo duration of action for novel quaternary N,N-disubstituted-(3R)-quinuclidinyl
carbamates
O
R1
N
O
R2
N
R3
Br
5
a
Compd
R1
R2
R3
_
Activity: IC50 (nM)
M2
Selectivityb
M2/M3 M1/M3
Duration of action
M3
M1
T (h)
4b
5a
5b
5c
5d
4d
5e
5f
5g
4e
5h
5i
4g
5j
5k
5l
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph–
Ph-CH2–
Ph-CH2–
Ph-CH2–
Ph-CH2–
1.2 (0.1)
17.7 (0.8)
0.3 (0.0)
0.6 (0.3)
1.0 (0.0)
0.6 (0.1)
18.6 (3.9)
0.5 (0.1)
0.3 (0.0)
1.0 (0.3)
0.6 (0.3)
0.2 (0.0)
4.3 (0.7)
19.0 (0.3)
0.8 (0.2)
0.6 (0.0)
0.8 (0.1)
1.4 (0.1)
0.9 (0.1)
1.2 (0.4)
1.5 (0.1)
0.6 (1.2)
0.35f (0.01)
2.07f (0.03)
3.7 (1.2)
0.4 (0.1)
15.1 (3.9)
1.5 (0.3)
1.4 (0.8)
1.7 (0.1)
0.4 (0.3)
9.2 (2.8)
1.6 (0.7)
0.4 (0.1)
0.4 (0.1)
2.0 (0.6)
0.4 (0.1)
1.1 (0.1)
33.3 (5.2)
2.5 (0.9)
2.0 (0.1)
1.4 (0.0)
1 (0.1)
3.1
2.9
13.3
12.7
3.9
3.0
1.8
6.4
3.7
0.3
0.85
5.0
2.3
1.7
0.7
0.5
3.2
1.3
0.4
3.3
2
1.5c
<2d
>6d
>6d
>6d
<4d
NEe
>6d
>6d
<4d
>6d
>6d
<1d
NEe
NEe
>6d
>6d
NEe
<6d
<6d
NEe
>6d
>6d
3.4g
Ph-(CH2)2–
Ph-(CH2)3–
2-Th-(CH2)3–
Ph-O-(CH2)2–
_
50.9 (0.6)
4.0 (1.2)
7.6 (0.6)
3.9 (0.3)
1.8 (0.7)
Ph-CH2–
(2-Th)-CH2–
(2-Th)-CH2–
(2-Th)-CH2–
(2-Th)-CH2–
(3-Th)-CH2–
(3-Th)-CH2–
(3-Th)-CH2–
n-Butyl–
n-Butyl–
n-Butyl–
n-Butyl–
n-Butyl–
(2,4,5-tri-F)-Ph-CH2–
(2,4,5-tri-F)-Ph-CH2–
(2,4,5-tri-F)-Ph-CH2–
(3,4,5-tri-F)-Ph-CH2–
(3,4,5-tri-F)-Ph-CH2–
CH3–
33.1 (6.5)
3.2 (0.5)
2-Th-(CH2)3–
Ph-O-(CH2)2–
_
2-Th-(CH2)3–
Ph-O-(CH2)2–
_
Ph-(CH2)2–
Ph-(CH2)3–
2-Th-(CH2)3–
Ph-O-(CH2)2–
_
Ph-(CH2)3–
Ph-O-(CH2)2–
_
1.1 (0.3)
4.7 (1.8)
8.4 (2.6)
1.6 (0.2)
4.7
14
8.0
22.9 (10.9)
120.8 (28.8)
17.7 (3.8)
10.9 (1.8)
8.9 (0.9)
45.8 (21.0)
29.8 (5.7)
28.0 (10.1)
97.3 (25.0)
14.4 (2.6)
0.21f (0.04)
2.24f (0.16)
5.3
6.3
0.2
1.7
3.1
3.3
1.7
0.7
3.3
2.0
0.6
1.8
0.63
1.28
22.1
18.2
11.1
32.7
33.1
23.3
64.9
24.0
0.6
5m
4p
5n
5o
4q
5p
Ph–
(3-Me)-Ph–
(3-Me)-Ph–
(3-Me)-Ph–
(3-F)-Ph–
(3-F)-Ph–
3.0 (0.3)
2.4 (0.5)
0.9 (0.3)
1.1 (0.9)
0.22f (0.03)
2.65f (0.13)
Ph-O-(CH2)2–
Tiotropium bromide
Ipratropium bromide
1.08
a
b
c
d
e
f
Values shown are the mean, n = 2 (SD), standard deviation is given in parentheses.
The selectivity is calculated as the ratio between the IC50 values.
Administered concentration of 3 mg/mL, n = 4–6 animals.
Administered concentration of 1 mg/mL, n = 4–6 animals.
NE = not evaluated.
Values shown are the mean, n = 3 (SD), standard deviation is given in parentheses.
Administered concentration of 0.3 mg/mL, n = 4–6 animals.
g
The results presented in Table 1 summarize the influence of R1
model described before, whereas the n-propyl (compound 4f)
and more clearly the isopropyl (compound 4i) derivatives didn’t
fit with one of the two hydrophobic-aromatic centers. For the
group of compounds where R1 is a 2-thienylmethyl group, com-
pound 4k, wherein R2 is also the 2-thienylmethyl substituent, pre-
sented the best potency at M3 receptor (IC50 M3: 5.9 nM). Among
the tested substituted analogues of compound 4b, the results ob-
tained for examples 4m and 4n suggested that the para-substitu-
tion in R1 was not favourable to activity. Compounds 4o, 4p, and
4q maintained the high M3 binding activity and their selectivity
for the M3 receptor over the M2 significantly increased. In general,
like the reference carbamates, examples of Table 1 showed less
activity at the M2 receptor in comparison with the activity at the
M3 one12–14 and the binding potencies for the M3 and M1 receptors
were in the same range.
and R2 substituents on the muscarinic binding activities (IC50) for a
series of compounds of formula 4. The IC50 values for solifenacin,
YM-46303, tiotropium bromide and ipratropium bromide were
also determined for reference.23
For the group of compounds where R1 is a phenyl group a signif-
icant influence of the R2 substituent was observed. Compound 4a,
wherein R2 is a phenyl group, presented low binding potencies at
the muscarinic M3, M2, M1 receptors. Replacement of the phenyl
substituent in R2 by a benzyl group (compound 4b), a 2-thienylm-
ethyl group (compound 4d) and a 3-thienylmethyl group (com-
pound 4e) resulted in a great improvement of the activity. In
order to understand the behaviour of these compounds, a qualita-
tive 4-point pharmacophore model (two hydrophobic-aromatic
rings, one hydrogen bond acceptor and one positive charged, pro-
tonated or quaternized, amine) was generated using a training
set of reference compounds.24 Benzyl and thienyl-methyl deriva-
tives presented a good overlap with the pharmacophore. By con-
trast phenyl derivative lacked one of the pharmacophoric centers.
Compound 4d wherein R2 is the 2-thienylmethyl substituent
showed the highest activity at the M3 receptor with an IC50 M3 of
0.6 nM, 10-fold lower than YM-46303 and in the range of tiotropi-
um bromide. For the subset of analogues wherein R2 is an alkyl
group, the best values were obtained for compounds 4g and 4h
(R2: n-butyl and n-pentyl, respectively). Both n-butyl and n-pentyl
derivatives presented a good overlap with the pharmacophore
To explore the SAR around R3, we made a selection of the non
quaternized derivatives previously described in Table 1. We chose
the most active analogues (compounds 4b, 4d, and 4e) and also
compound 4g as a representative example where R2 is an alkyl
group. Muscarinic receptor binding potencies (IC50) for their quat-
ernized derivatives are shown in Table 2.23
The evaluated derivatives of compound 4b and 4g presented
high M3 binding activities (IC50 61 nM) when R3 was a phenyl-
propyl, 2-thienylpropyl and phenoxyethyl chain. When R3 was
a phenethyl chain a loss of activity was observed suggesting that
a distance of three atoms between the nitrogen of the quinucli-

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M. Prat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3457–3461
dine and the phenyl or thienyl ring in R3 could be beneficial to
activity. In the case of compound 4d, its binding activity profile
was retained for the quaternized derivatives 5f and 5g (R3: 2-
thienylpropyl and phenoxyethyl, respectively). Derivatives of
compound 4e with these two chains also presented high M3
binding potencies. Among the tested examples, compounds 5b,
5g, and 5i presented IC50 M3 values comparable with that of tiot-
ropium bromide. As in the case of the non quaternized precur-
sors, the quaternary derivatives described in Table 2 showed
less activity at the M2 receptor in comparison with the activity
at the M3 subtype.
The in vivo duration of action was evaluated by measuring the
inhibition of bronchospasm induced by acetylcholine in anesthe-
tized guinea pigs at different time points.25 The results are shown
in Table 2 along with data for tiotropium bromide and ipratropium
bromide. Duration of action (T, h) was defined as the time taken to
recover 50% of the maximum inhibitory effect achieved by the test
compound.26
The non quaternized compounds 4b, 4d, 4e, and 4g had short
duration of action (1.5 h, <4 h, <4 h, <1 h, respectively). Quaterniza-
tion of compound 4b to obtain the derivatives 5b, 5c, and 5d (R3:
phenylpropyl, 2-thienylpropyl and phenoxyethyl respectively)
resulted in a longer duration of action (>6 h) similar to that of tiot-
ropium bromide. Quaternization of compounds 4d, 4e, and 4g with
the 2-thienylpropyl group, (compounds 5f, 5h, and 5l, respectively)
and the phenoxyethyl group (compounds 5g, 5i, and 5m, respec-
tively) also resulted in a longer duration of action (>6 h). In
summary, quaternization with the R3 chains phenylpropyl, 2-thie-
nylpropyl and phenoxyethyl, appeared to improve the duration of
action of these compounds relative to the corresponding unquat-
ernized precursors.
On the basis of the results of M3 binding potency and duration
of action, these preferred R3 chains were applied to the substituted
derivatives of compound 4b (carbamates 4p and 4q described in
Table 1). The synthesized quaternary compounds were evaluated
for their in vitro and in vivo activities. The results are presented
in Table 2. In comparison with their unquaternized precursors,
the evaluated quaternized derivatives retained their high activity
at the M3 receptor and their M2/M3 binding profile, but only com-
pound 5p showed a duration of action >6 h.
characterized. To evaluate its potential for low systemic class-
related effects in humans, preliminary stability studies in human
plasma and metabolism in human liver microsomes were per-
formed. The results are showed in Table 3. Tiotropium bromide
and ipratropium bromide were also evaluated for reference. All
the quaternized carbamates evaluated showed low level of degra-
dation in human plasma, but the level of transformation in the
metabolism assay was higher than for tiotropium bromide and
ipratropium bromide in all the examples.
The values of M3 muscarinic binding potency and duration of
action, for highlighted compounds of the Almirall ester series pre-
viously reported in the literature8 and for carbamate derivatives
described in Table 3 were comparable.23,26 A difference was ob-
served in their corresponding M3, M2, M1 binding activity profile.
In the case of ester derivatives the IC50 values for the three recep-
tors were similar,8 as in the case of tiotropium and ipratropium,
while in the carbamate derivatives certain degree of selectivity
for the M3 receptor in front of the M2 one was found (a range of
M2/M3 values from 3.7, compound 5g, to 18.2, compound 5l, is
shown in Table 3). Regarding to human plasma stability, a wide
range of values of percentage of degradation (depending on the
structure of the compound) was observed in the ester series8 while
the carbamate derivatives described in Table 3 presented low lev-
els of degradation, however their level of transformation in human
liver microsomes was high, from 79% (compound 5h) to 100%
(compound 5b).
In conclusion, novel quaternary ammonium derivatives of N,N-
disubstituted (3R)-quinuclidinyl carbamates have been identified
as potent antimuscarinic agents. They show high muscarinic M3
receptor binding activity, higher M2/M3 selectivity ratio than tiot-
ropium and ipratropium, long duration of action (comparable to
that of tiotropium bromide in an in vivo model of bronchoconstric-
tion), and higher level of metabolic transformation in human liver
microsomes than the reference compounds tiotropium and iprat-
ropium. Their predicted low oral absorption (quaternization of
the tertiary amino function minimize absorption across mem-
branes)8,16 and their observed high level of metabolism would
indicate that after inhaled administration the systemic exposition
of these compounds could be very limited, and the class-related
systemic effects very low.
A selection of compounds of Table 2 with higher M3 receptor
binding potencies and long duration of action were further
The potential of this series of compounds for the inhaled treat-
ment of COPD is currently under study and evaluation.
Table 3
In vitro studies of human plasma stability and metabolism in human liver microsomes for selected quaternary carbamates with higher M3 binding activities and longer in vivo
duration of action
a
Compound
Activity: IC50 (nM)
Selectivityb
In vivo duration
of action
T (h)
Stability
Metabolism
(% degradation 1 h)
(% transformation 30 min)
M3
M2
M1
M2/M3 M1/M3
Human plasmaa,c
Human microsomesa,d
5b
5c
5d
5f
5g
5h
5i
5l
0.3 (0.0)
0.6 (0.3)
1.0 (0.0)
0.5 (0.1)
0.3 (0.0)
0.6 (0.3)
0.2 (0.0)
0.6 (0.0)
0.35f (0.01)
2.07f (0.03)
4.0 (1.2)
7.6 (0.6)
3.9 (0.3)
3.2 (0.5)
1.1 (0.3)
8.4 (2.6)
1.6 (0.2)
10.9 (1.8)
0.21f (0.04)
2.24f (0.16)
1.5 (0.3)
1.4 (0.8)
1.7 (0.1)
1.6 (0.7)
0.4 (0.1)
2.0 (0.6)
0.4 (0.1)
2.0 (0.1)
0.22f (0.03)
2.65f (0.13)
13.3
12.7
12.7
6.4
3.7
14
8.0
18.2
0.6
5.0
2.3
2.3
3.2
1.3
3.3
2
3.3
0.63
1.28
>6e
>6e
>6e
>6e
>6e
>6e
>6e
>6e
>6e
3.4g
<5
<5
<5
6.2 (6.0)
2.7 (4.5)
<5
<5
<5
100 (0.0)
93 (0.18)
78 (0.41)
97 (1.36)
97 (0.37)
79 (0.07)
92 (0.85)
81 (0.06)
0.55 (0.36)
2.3 (2.4)
Tiotropium bromide
Ipratropium bromide
11.0 (7.5)
<5
1.08
a
b
c
d
e
f
Values shown are the mean, n = 2 (SD), standard deviation is given in parentheses.
The selectivity is calculated as the ratio between the IC50 values.
Experimental conditions: T = 37 °C, compound concentration = 5
Experimental conditions: T = 37 °C, compound concentration = 5
Administered concentration of 1 mg/mL, n = 4–6 animals.
l
l
M.
M, microsomes = 1 mg protein/mL.
Values shown are the mean, n = 3 (SD), standard deviation is given in parentheses.
Administered concentration of 0.3 mg/mL, n = 4–6 animals.
g

M. Prat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3457–3461
3461
13. Naito, R.; Takeuchi, M.; Morihira, K.; Hayakawa, M.; Ikeda, K.; Takemoto, T.
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Acknowledgments
We acknowledge the significant technical support provided by
Begoña Hernández, Manuel De Luca, Beatriz Lerga, Dolores Marín,
María Rosa Ortiz, Isabel Pagán, Julian Prieto, Enric Villanova (Almir-
all R&D Centre).
17. Lieberman, J. A. Prim. Care Companion J. Clin. Psychiatry. 2004, 6, 20.
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molecules were generated and ionized at pH = 7.4) were performed using
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26. Values of duration of action were determined as described in Ref. 8.