A. Mete et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7440–7446
7445
Table 5
In vivo activity of the novel series of muscarinic antagonists
Compound
M3 pIC50
M2 pIC50
gp-IT PK T
h
BC ED80
(l
g/Kg) at 2 h
BC % inhibition at 24 h
Salivation EC50
(
lg/Kg)
TI
½
Tiotropium
5.8
10.3
>10
9.8
>10.4
10.1
9.5
10.2
9.9
10
10.2
—
9.0
—
—
—
—
—
—
—
8
8.8
10.5
8.1
26
26.3
21
20.2
16.9
18.0
0.3a
1
>80
<0.3
>10
>100
>30
nd
>30
nd
nd
<3
17
>10
>100
>10
—
>10
—
—
<10
—
5.14
5.16
5.28
5.32
5.46
6.2
1a
3a
1
50** (at 18 h)
74**
0
3
3
30*
28*
41**
59**
10
1
6.6
6.12
3a
1
<30
nd
9.8
a
Measured ED80 dose.
*
P <0.05.
**
P <0.01 compared to control group.
3. (a) Casarosa, P.; Bouyssou, T.; Germeyer, S.; Schnapp, A.; Gantner, F.; Pieper, M.
J. Pharmacol. Exp. Ther. 2009, 330, 660; (b) Gross, N. J. Chest 2004, 126, 1946;
ZuWallack, A. R.; ZuWallack, R. L. Expert Opin. Pharmacol. 2004, 5, 1827.
4. (a) Hansel, T. T.; Neighbour, H.; Erin, E. M.; Tan, A. J.; Tennant, R. C.; Maus, J. G.;
Barnes, P. J. Chest 2005, 128, 1974; (b) 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.
nea pig pilocarpine-induced salivation model was used to assess
the peripheral side effects of compounds at a 4 h timepoint.13 Data
from the BC and salivation models were used to establish a thera-
peutic index (TI) for bronchoprotection over salivation effects in
the guinea pig. Our objective was to identify compounds with a
TI >10 as this would be a significant improvement over tiotropium
which has a narrow therapeutic window (TI <3) in these models.
The complete set of in vivo data for leading compounds is summa-
rized in Table 5 together with data generated for tiotropium in the
same assays for comparison.
5. 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.
6. In vitro M3 and M2 radioligand binding assay: the antagonist affinity of a
compound at the human M3 or M2 receptors was estimated from its ability to
compete with specific [3H]NMS binding to membranes from CHO-K1 cells
expressing either recombinant human M3 or M2 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
The table shows that in this novel series we have identified a
group of compounds with good in vivo efficacy in the guinea pig
BC model that exhibit ED80 of between 1–3 lg/Kg. Duration of action
beyond 12 h was statisticallysignificant for a numberof compounds,
5.14, 5.16, 5.32, 5.46, 6.2 and 6.6. In accord with expectations from
our design criteria, most compounds in this series have an improved
salivation TI compared to tiotropium, with 5.14 standing out with a
TI >100. In addition, compound 5.14 exhibited ca. 10-fold selectivity
for M3 over M2. Taken together with its high hPPB and high HM
Cl_int, the data highlight compound 5.14 as having a promising
and differentiated profile relative to tiotropium and was selected
for progression into studies in Man for evaluation of its potential
as a treatment for COPD as AZD9164. The results of these studies will
be reported in due course.
In summary, we describe a strategy of starting from known M3
antagonists and modifying their chemical structures to arrive at a
novel, potent series. The design and screening strategies used were
tailored to identify optimized compounds which met our pre-set
target product profile criteria for in vivo PK, hPPB, human liver
microsomal intrinsic clearance and M2 selectivity. These character-
istics were selected to differentiate the novel compounds from cur-
rently used antagonists (e.g., tiotropium). This led to compounds
which combined an improved systemic side effect profile, as mea-
sured by a pilocarpine induced salivation model, with good dura-
tion of action.
[3H]NMS was determined by replacing test compound with atropine 1
[3H]NMS was used at
concentration of 0.1 nM, which was below the
lM.
a
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.
7. Bailey, A.; Donald, D. WO2006112778, 2006, Chem. Abstr. 2006, 145, 454929.
8. Bailey, A.; Mete, A.; Pairaudeau, G.; Stocks, M.; Wenlock, M. WO2007123465,
2007, Chem. Abstr. 2007, 147, 502240.
9. (a) Ford, R.; Mete, A.; Millichip, I.; Teobald, B. WO2008075005, 2008, Chem.
Abstr. 2008, 149, 104901; (b) Ford, R.; Mete, A.; Millichip, I.; Teobald, B.;
Kinchin, E.C. WO2009153536, 2009, Chem. Abstr. 2009, 152, 75267. 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 and chiral HPLC and elemental analyses.
10. 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
l
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 lM), washed and left for a further 1 h. Then a cumulative methacholine
concentration response curve was constructed (3 Â 10À9–3 Â 10À5 M). Vehicle
or test compound was then added to the baths and allowed to equilibrate for
1 h. Then, a second extended cumulative concentration response curve was
constructed (3 Â 10À9–1 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 a potency pA2 value at each
compound concentration used.
Acknowledgments
The authors would like to thank Elizabeth Kinchin, Andrew
Mather, Sasvinder Sidhu, Phil West, Hemant Mistry, Jaspal Singh
and James Bird for technical contributions to the work.
11. For pharmacokinetic studies, compounds were dosed to halothane
References and notes
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(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.
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12. In vivo guinea-pig bronchoconstriction: The efficacy of compounds was
measured in vivo by the inhibition of methacholine induced