1216
K. G. Pike et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1212–1216
the lipid PIP2 as substrate. More detailed description of the assay conditions
In summary, we have shown how iterative SAR investigations
24
are described in Ref.
have been used to optimize the cellular potency, aqueous solubility
and hERG potency of lead compound Ku-0063794 to deliver the
clinical candidate AZD8055. Further optimization focused on
reducing turnover in human hepatocytes and resulted in the iden-
tification of the clinical candidate AZD2014, which was believed to
offer a reduced human PK risk compared to AZD8055. AZD8055
and AZD2014 are potent and selective dual mTORC1 and mTORC2
inhibitors which show a differentiation from rapamycin in vitro
and have demonstrated dose-dependent tumor growth inhibition
in mouse xenograft models. AZD8055 was evaluated in a phase I
clinical study in patients with advanced tumors but is no longer
in clinical development. AZD2014 is currently in phase I.
15. MDA-MB-468 cells were exposed for 2 h to increasing concentrations of
compound. At the end of the incubation period cells were fixed, washed and
probed with antibodies against S473 pAKT or against S235/236 phosphorylated S6
(pS6). Levels of phosphorylation were assessed using an Acumen laser
scanning cytometer (TTP Labtech). Figures quoted are the mean of at least 3
determinations and the standard error in the mean (pIC50) was <0.3.
16. Pike, K. G.; Menear, K.; Gomez, S.; Malagu, K.; Hummersone, M.; Pass, M.;
Duggan, H.; Hickson, I.; Chresta, C.; Davies, B.; Martin, N.; Smith, G. Presented
at the 241st National Meeting of the American Chemical Society: Anaheim, CA,
March 2011; Abstract MEDI#188.
17. Assessments of aqueous solubility were made after an incubation of 24 h in pH
7.4 phosphate buffer. After centrifugation, analysis of the supernatant liquid
was performed by LC–UV to quantify the amount of compound in solution.
17
Further details are contained in Ref.
.
18. Buttar, D.; Colclough, N.; Gerhardt, S.; MacFaul, P. A.; Phillips, S. D.; Plowright,
A.; Whittamore, P.; Tam, K.; Maskos, K.; Steinbacher, S.; Steuber, H. Bioorg. Med.
Chem. 2010, 18, 7486.
19. Redfern, W. S.; Carlsson, L.; Davis, A. S.; Lynch, W. G.; MacKenzie, I.; Palethorpe,
S.; Siegl, P. K. S.; Strang, I.; Sullivan, A. T.; Wallis, R.; Camm, A. J.; Hammond, T.
G. Cardiovasc. Res. 2003, 58, 32; Waring, M. J.; Johnstone, C.; McKerrecher, D.;
Pike, K. G.; Robb, G. Med. Chem. Commun. 2011, 2, 775.
20. Monolayers of MDCKII (Madin Darby Canine Kidney Cells) cells transfected
with the human MDR1 (Multi-Drug Resistance 1) transporter protein were
cultured in transwells and used to study the permeability and efflux potential
Acknowledgments
The authors would like to thank Christine Chresta and Tom Har-
ding for their help in preparing the biological data presented and
Dr. Kin Tam for his help preparing the physicochemical data
presented.
of compounds at a concentration of 10 lM.
21. Keogh, J. P.; Kunta, J. R. Eur. J. Pharm. Sci. 2006, 27, 543.
References and notes
22. Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard, D. J.; Curran, K.; Brooijmans, N.;
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23. ClogP values were calculated using the commercially available package ClogP
v4.3 from BioByte Corp., 201 W. 4th Str., #204 Claremont, CA 91711-4707.
24. Chresta, C. M.; Davies, B. R.; Hickson, I.; Harding, T.; Cosulich, S.; Critchlow, S.
E.; Vincent, J. P.; Ellston, R.; Jones, D.; Sini, P.; James, D.; Howard, Z.; Dudley, P.;
Hughes, G.; Smith, L.; Maguire, S.; Hummersone, M.; Malagu, K.; Menear, K.;
Jenkins, R.; Jacobsen, M.; Smith, G. C. M.; Guichard, S.; Pass, M. Cancer Res.
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Meeting: Chicago, 2012; Abstract 917. Further details of the in vitro and in vivo
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26. The plasma protein binding of compounds was measured using equilibrium
dialysis technique and is quoted as the Fraction unbound (fu). Values for
AZD8055 and AZD2014 were generated in undiluted plasma at a range of drug
concentrations using radiolabelled material ([3H]-AZD8055 or [14C]-AZD2014);
no concentration dependency was observed. A more detailed description of the
equilibrium dialysis assay conditions are described in Chiou, W. L.; Robbiel, G.;
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27. Mouse clearance values were obtained following an IV dose of 10
CD-1 out bred mice and rat clearance values were obtained following an IV
dose of either 1 mol/kg (Ku-0063794) or 4 mol/kg (AZD8055 and AZD2014)
to CR-Han Wistar rats. The compounds were dosed as a solution. Bioavailability
values were calculated following an oral dose of 20 mol/kg in mouse and
either 6 mol/kg (Ku-0063794) or 20 mol/kg (AZD8055 and AZD2014) in rat.
lmol/kg to
l
l
l
l
l
14. Inhibition of mTOR kinase was evaluated in a high-throughput assay using
screen capture complex technology (Perkin-Elmer) with recombinant
truncated FLAG-tagged mTOR (amino acids 1362–2549, expressed in HEK293
cells). The activity of the lipid kinases, class I PI3Ks , b, , d was measured with
a
28. Hummersone, M. G.; Gomez, S.; Menear, K. A.; Smith, G. C. M.; Malagu, K.;
Duggan, H. M. E.; Cockcroft, X.-L. F.; Hermann, G. J. WO2007060404, 2007.
29. Duggan, H. M. E.; Leroux, F. G. M.; Malagu, K.; Martin, N. M. B.; Menear, K. A.;
Smith, G. C. M. WO2008023161, 2008.
a
a
c