J. F. Blake et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5607–5612
5611
xenografts implanted in female nude mice showed that p-PRAS40
Cl
Cl
Cl
NH2
NH2
NH2
levels decreased to 15% of control at 1 h (normalized to total Erk)
following ip injection of 20 mg/kg 54 (Fig. 4). At 4 h, the p-PRAS40
levels were still only 40% of control, but were starting to recover.
O
N
O
N
O
N
N
N
N
N
N
N
Plasma levels of 54 at 1 h were ca. 3.8
0.2 M at 4 h, which was consistent with the observed PD effect.
Following the initial proof-of-concept results, we undertook tol-
lM, and were less than
l
erability studies in preparation for more advanced tumor growth
inhibition experiments. Unfortunately, subcutaneous (sc) injection
of 54 in male CD-1 mice ranging from 20 to 60 mg/kg uniformly
produced death by ca. 4 h. Screening of 54 against a broad panel
of kinases found the compound displayed potent inhibition versus
N
H
N
H
N
H
N
46
N
N
60
61
13
1.2
4.3
PKA/Akt1
CaMKIV, PKA, PDK1, PKC(
(>90% inhibition at 1
M).11 To see if the poor tolerability was
due to the overall pharmacophore rather than an on target effect,
we evaluated 59, a weak inhibitor (cell IC50 = 11.8 M) at doses
c, bI, g, h), p70S6K, ROCK1, and AMPK
Figure 5. Prototype compounds with variable PKA/Akt1 selectivity. Ratio is
l
calculated from PKA/Akt1 enzyme IC50s.
l
ranging from 20 to 100 mg/kg, via sc injections. This compound
was well tolerated, suggesting that the overall scaffold was not
inherently problematic. The kinase inhibition profile of 59 showed
potent inhibition of PKA, MSK1, and p70S6K (>90% inhibition at
Ala230 of Akt1; this change may allow adjustment of the gate-
keeper and surrounding residues to accommodate a larger ligand
in Akt1. Also, the substitution of Leu (PKA) for Met281 at the base
of the hinge region tends to afford less space in PKA. Given the
above profiles and tolerability issues, we decided to focus our ef-
forts on alternative hinge binding cores that offered a better kinase
selectivity profile; forthcoming papers will address these selectiv-
ity issues.
We have described the discovery and optimization of a series a
pyrrolopyrimidine based pan inhibitors of Akt. The compounds dis-
play potent enzyme and cell based inhibition of our primary tar-
gets Akt1/2/3. Additionally, the compounds display excellent
knockdown of p-PRAS40 in tumor xenografts, combined with high
solubility and good ADME properties. While this profile is encour-
aging, the lack of selectivity against related kinases, in particular
PKA and ROCK1/2 render them poorly tolerated and unsuitable
for development as therapeutic agents.
1 lM). Interestingly, 55 (cell IC50 = 4.2 lM) was not tolerated using
the above paradigm (MTD <20 mg/kg, ip). Kinase panel screening
of 55 revealed significant inhibition of PKA IC50 = 1 nM, with
MSK1, PRK2, PRKG1, PrKX, ROCK1/2, Rsk1-4, CHK2, p70S6K, and
MRCKb all showing greater than 90% inhibition at 1 lM. This sug-
gested that some off-target activity, likely kinase-related, is the
cause of the poor tolerability of these inhibitors, and that develop-
ment of a selective Akt inhibitor would be better tolerated. Sup-
porting this notion, MK-2206, which is
a highly selective
allosteric Akt inhibitor, is reported to be well tolerated in preclin-
ical animal models.12
As documented above, the pyrrolopyrimidines tended to posses
potent inhibition of undesirable kinases (i.e., ROCK1/2, PKA, etc.),13
though a few examples did show a more selective profile. In partic-
ular, 61 showed PKA IC50 = 278 nM, with PKA/Akt1 = 13 (Fig. 5).
Interestingly, selectivity (PKA/Akt1) in this series increases from
1.2 for the 5-methyl (46), to 4.3 for the 6-methyl (60). For the qui-
nazoline series of compounds, the PKA/Akt1 ratio tended to be ca. 7
for most analogs, though 25 showed a slightly higher ratio of 12.
All of these results suggested that increased steric bulk near the
gatekeeper residue tended to improve the selectivity profile, rela-
tive to PKA. We note that PKA has a valine residue equivalent to
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
1. Cheng, J. Q.; Lindsley, C. W.; Cheng, G. Z.; Yang, H.; Nicosia, S. V. Oncogene 2005,
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K. P.; Klumperman, J.; Friedman, L. S.; Lin, K. J. Cell Biol. 2008, 183, 101.
6. Inhibition of Akt1, Akt2, Akt3, and PKA was determined using the IMAP format.
A detailed description of the assay format is provided in the Supplementary
data.
7. PDB Code 1O6K: Yang, J.; Cron, P.; Good, V. M.; Thompson, V.; Hemmings, B. A.;
Barford, D. Nat. Struct. Biol. 2002, 9, 940.
8. Docking studies were performed using the Glide program (Glide V4.5;
Schrödinger, L.L.C.: New York, NY, 2005) Friesner, R. A.; Banks, J. L.; Murphy,
R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley,
M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. J. Med. Chem. 2004, 47,
1739.
9. The cell-based assay monitored the phosphorylation of Thr246 on PRAS40 in
LNCaP cells. A detailed description of the assay format is provided in the
Supplementary data.
10. Wen, S.; Stolarov, J.; Myers, M. P.; Su, J. D.; Wigler, M. H.; Tonks, N. K.; Durden,
D. L. Proc. Natl. Acad. Sci. 2001, 98, 4622.
Figure 4. Evaluation of 54 dosed 20 mg/kg ip in the U87 human glioblastoma
11. The inhibition against
a panel of kinases was determined at Upstate,
xenograft model, results of four experiments. p-PRAS40 is normalized to total Erk.
Charlottesville, VA (256 total kinases screened).