398
A. M. Birch et al. / Bioorg. Med. Chem. Lett. 17 (2007) 394–399
Table 4. Pharmacokinetic data for compounds
Compound
Rat clearance
(mL/min/Kg)
Rat Vdss
(L/Kg)
Rat
t1/2
Bioavail.
(%)
Caco-2 A–B
cm/s · 10ꢀ6
Caco-2 B–A
cm/s · 10ꢀ6
h
11
16
28
33
1.2
5.1
2.5
2.4
1.4
1.4
2.2
1.9
16
35
12
12
4
7
14
10
29
5.4
13
11
41–94
>100
17
29
Compounds dosed at 2.1–4.0 mg/kg (iv) and 8.1–8.9 mg/kg (po) to male AP Wistar rats.
G. K.; Wasilko, D. J.; Jayvardhan, P. Mol. Cell 2000, 6,
139.
istration of glucagon increases hepatic glucose output
through cAMP mediated activation of GPa.
4. Oikonomakos, N. G.; Skamnaki, V. T.; Tsitsanou, K.
E.; Gavalas, N. G.; Johnson, L. N. Structure 2000, 8,
575.
5. Rath, V. L.; Ammirati, M.; Danley, D. E.; Ekstrom, J. L.;
Gibbs, E. M.; Hynes, T. R.; Mathiowetz, A. M.; McPh-
erson, R. K.; Olson, T. V.; Treadway, J. L.; Hoover, D. J.
Chem. Biol. 2000, 7, 677.
6. Whittamore, P. R. O.; Addie, M. S.; Bennett, S. N. L.;
Birch, A. M.; Butters, M.; Godfrey, L.; Kenny, P. W.;
Morley, A. D.; Murray, P. M.; Oikonomakos, N. G.;
Otterbein, L. R.; Pannifer, A. D.; Parker, J. S.; Readman,
K.; Siedlecki, P. S.; Schofield, P.; Stocker, A.; Taylor, M.
J.; Townsend, L. A.; Whalley, D. P.; Whitehouse, J.
Bioorg. Med. Chem. Lett. 2006, 16, 5567.
7. Birch, A. M.; Morley, A. D., WO 074532, 2003.
8. Rosauer, K. G.; Ogawa, A. K.; Willoughby, C. A.;
Ellsworth, K. P.; Geissler, W. M.; Myers, R. W.; Deng,
Q.; Chapman, K. T.; Harris, G.; Moller, D. E. Bioorg.
Med. Chem. Lett. 2003, 13, 4385.
Compounds were dosed po to between four and six ani-
mals in each group (basal blood glucose was around 5–
6 mM) and did not affect pre-glucagon challenge blood
glucose at doses which inhibited the glucagon response.
When dosed at 12.5 lM/kg (dose volume 5 mL/kg)
45 min prior to the glucagon, compound 33 caused a
46% lowering of the induced glucose increase, measured
90 min after the compound dose.
In summary, optimisation of a series of 1-substituted
3,4-dihydro-2-quinolone GPa inhibitors resulted in
compound 33, which exhibits good potency in vitro
and in vivo, a good DMPK profile and improved phys-
ical properties.
Acknowledgments
9. Protein crystals kindly provided by N.G.O were grown
using published procedures.15 On soaking the inhibitors
(2.5 mM and 5 mM for compounds 11 and 16, respective-
ly, for 30 min), these crystals underwent a space group
change from tetragonal to orthorhombic symmetry, such
that the dimer dyad is no longer crystallographic. Crystals
could be flash-cooled to 100 K directly from the stabiliza-
tion buffer which contained 10 mM BES, pH 6.7, 0.1 mM
EDTA and 35% DMSO. Diffraction data were collected at
SRS, Daresbury, at 100 K. The crystals of the complex
with 11 have space group P212121, unit cell 114.1, 125.2,
Thanks to Sue Freeman and Simon Poucher for super-
vision of bioscience screening and to Sue Loxham, Jo
deSchoolmeester, Gemma Convey, Julie Bartlett and
Jenny Thomas for technical assistance. DMPK interpre-
tation and technical assistance were contributed by
Mike Walker, Julie Evans and Louise Finch. Richard
Pauptit is thanked for establishing the crystallography
collaboration with the Oikonomakos group, who have
kindly provided protein and crystals, and Claire Mins-
hul is thanked for the soaking experiments. Thanks to
the crystallography team at AstraZeneca for synchro-
˚
128.4 A. 144,829 unique reflections from 492,418 obser-
˚
vations to 1.9 A give 81.3% completeness with
Rmerge = 9.3%. The final R-factor is 22.8% (Rfree using
5% of data is 26.9%). The mean temperature factor is
ˆ
tron data collection and analysis, and to Sian Rowsell
for initiating structure refinements.
2
38.2 A for protein atoms and 39.1 A for the ligand.
2
˚
˚
Crystals of the complex with 16 have space group P212121
˚
and unit cell 113.9, 124.4, 128.3 A. 143,632 unique
˚
reflections from 330,353 observations to 1.9 A give
Supplementary data
82.5% completeness with Rmerge = 10.2%. The final model
has an R-factor of 24.3% (Rfree using 5% of data is 28.5%).
Mean atomic temperature factor for the protein is 23.6
Supplementary data associated with this article can be
2
˚
and for the inhibitor is 37.2 A . Data analysis and
structure solution used programs from the CCP4 suite.16
The inhibitors were modelled into electron density using
Quanta2000 (Accelrys). The model was refined using CNX
(Accelrys) and Refmac516. Crystallographic data (exclud-
ing structure factors) for the structures in this paper have
been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication numbers CCDC
2IEG and 2IEI. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK [fax: +44 (0)1223 336033 or
e-mail: deposit@ccdc.cam.ac.uk].
References and notes
1. McCormack, J. G.; Westergaard, N.; Kristiansen, M.;
Brand, C. L.; Lau, J. Curr. Pharm. Des. 2001, 7,
1451.
2. Treadway, J. L.; Mendys, P.; Hoover, D. J. Exp. Opin.
Invest. Drugs 2001, 10, 439.
3. Rath, V. L.; Ammirati, M.; LeMotte, P. K.; Fennell, K.;
Mansour, M. N.; Danley, D. E.; Hynes, T. R.; Schulte,
10. Enantiomeric purity was determined throughout the
syntheses either by NMR estimation with (R)-(+)-1,10-bi-