Allosteric inhibitors of Akt1 and Akt2: A naphthyridinone with efficacy in an A2780 tumor xenograft model

Article abstract of DOI:10.1016/j.bmcl.2008.04.074

A series of naphthyridine and naphthyridinone allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been optimized to have potent dual activity against the activated kinase as well as the activation of Akt in cells. One molecu

Full text of DOI:10.1016/j.bmcl.2008.04.074

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 3178–3182
Allosteric inhibitors of Akt1 and Akt2: A naphthyridinone with
efficacy in an A2780 tumor xenograft model
Mark T. Bilodeau,a,* Adrienne E. Balitza,a Jacob M. Hoffman,a Peter J. Manley,a
Stanley F. Barnett,b Deborah Defeo-Jones,b Kathleen Haskell,b
Raymond E. Jones,b Karen Leander,b Ronald G. Robinson,b
Anthony M. Smith,a Hans E. Huberb and George D. Hartmana
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., PO Box 4, West Point, PA 19486, USA
bDepartment of Cancer Research, Merck Research Laboratories, Merck & Co., PO Box 4, West Point, PA 19486, USA
Received 31 January 2008; revised 22 April 2008; accepted 28 April 2008
Available online 1 May 2008
Abstract—A series of naphthyridine and naphthyridinone allosteric dual inhibitors of Akt1 and 2 have been developed. These com-
pounds have been optimized to have potent dual activity against the activated kinase as well as the activation of Akt in cells. One
molecule in particular, compound 17, has potent inhibitory activity against Akt1 and 2 in vivo in a mouse lung and efficacy in a
tumor xenograft model.
ꢀ 2008 Elsevier Ltd. All rights reserved.
N
The serine/threonine kinase Akt (PKB) plays a central
role in cancer cell growth and survival. It is a key down-
stream effector of phosphoinositide 30-kinase (PI3K)
and directly modulates a wide range of pro-apoptotic
and metabolism regulating proteins.1 Inhibition of Akt
is a significant therapeutic goal due to the prevalence
of activating mutations in the PI3K/Akt pathway.2,3
Previous papers have described potent dual Akt1 and
Akt2 inhibitors. These molecules are PH-domain depen-
dent inhibitors, prevent the activation of Akt, and dis-
play excellent selectivity versus kinases outside of the
Akt family as well as the highly homologous AGC fam-
ily of kinases.4
O
Akt1 IC50 = 58 nM
N
N
N
N
NH
Akt2 IC50 = 210 nM
N
H
Cell Akt1 IC50 = 200 nM
Cell Akt2 IC50 = 900 nM
1
O
N
O
Akt1 IC50 = 138 nM
N
HN
N
NH
Akt2 IC50 = 212 nM
N
Cell Akt1 IC50 = 527 nM
Cell Akt2 IC50 = 730 nM
NEt2
2
Figure 1. Previously published balanced dual Akt1 and Akt2
inhibitors.
obtained.4c,5 This Letter describes the subsequent suc-
cessful effort to invent a compound with sufficient in
vivo activity, pharmacokinetics and tolerability to be
used in proof-of-concept tumor xenograft studies.
The utility of previous allosteric Akt inhibitors for in
vivo studies was limited due to the inability to achieve
sustained high levels of inhibition of Akt1 and Akt2.
Though inhibition of Akt1 and 2 could be demonstrated
with compound 14a (Fig. 1), the high plasma levels nec-
essary for Akt1 and 2 inhibition could not be sustained
due to its low solubility and moderate potency. Com-
pound 2, though more soluble than 1, was poorly toler-
ated in mice and only limited plasma exposures could be
While the dibasic functionality in 2 gave the dihydro-
chloride salt good aqueous solubility at low pH, this
compound has a high MW (653 Da) and the dibasic
functionality may contribute to its poor tolerability.
The goal was to improve the potency of the lead series
and at the same time lower molecular weight by focusing
on the key structural elements of these molecules. These
efforts fell into two categories, variation of the quinoxa-
line core and broad exploration of alternate piperidines.
Keywords: Akt; Oncology.
*
Corresponding author. Tel.: +1 215 652 5304; fax: +1 215 652
7310; e-mail: mark_bilodeau@merck.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.074

M. T. Bilodeau et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3178–3182
3179
NHBoc
CHO
N
NHBoc
a, b
N
O
N
N
N
N
N
N
N
NH
N
NH
Cl
Ph
Cl
8
9
Ph
4
3
O
N
O
Akt1 IC50 = 295 nM
Akt2 IC50 = 2057 nM
Akt3 IC50 = 27,000 nM
Akt1 IC50 = 75 nM
Akt2 IC50 = 1075 nM
Akt3 IC50 = 11,280 nM
O
O
c
O
NC
10
11
Ph
Figure 2. Quinoxaline lead compounds.
R1
N
R2
d, e, f, g
N
9 + 11
Screening of alternate amines began around the quinox-
aline 3 (Fig. 2).4a The first break in this wide-ranging ap-
proach to find alternate piperidines was the
identification of the pyrazolylpiperidine motif in 4.6 This
compound displayed improved activity over 3 albeit
with still relatively weak Akt2 activity.7,8 It also had a
respectable MW (522 Da) and good physical properties
(logD = 2.71).
HN
Ph
12
O
Scheme 2. Reagents and conditions: (a) t-BuLi, ꢀ70 ꢁC, THF; (b)
DMF, 53% yield; (c) BnMgCl, 0 ꢁC to rt, 84% yield; (d) NaOMe,
MeOH, reflux, 55% yield; (e) 1 N HCl, THF, water, rt, 85% yield; (f)
HNR1R2, NaBH(OAc)3 (g) pyr-HCl, 150 ꢁC, HCl.
Alternate core heterocycles were also explored to at-
tempt to identify more potent and balanced dual activ-
ity. Quinoline and pyridine variations have been
described,4b and this Letter describes the exploration
of naphthyridine derivatives. Naphthyridines were ex-
pected to have improved physical properties, since the
pKa of 1,8-naphthyridine is 3.39, versus 1,4-quinoxaline
with a pKa of 0.60. The added basicity was hoped to
contribute to increased polarity and better solubility.
Previously it was shown that the position of one of the
core nitrogens was critical to potency.4b In the case of
1,8-naphthyridines, this would correspond to the posi-
tion of Nꢀ1, so 2-aryl-3-phenyl-[1,8]-naphthyridines
were targeted. Later we explored other 2-aryl-3-phe-
nyl-[1,x]-naphthyridines (vide infra).
methoxide led to the methoxynaphthyridine. Hydrolysis
of the acetal, reductive amination and demethylation
provided the 1,6-naphthyridinon-5-one 12.
Table 1 shows selected additional end groups that were
incorporated into this structure. The guiding principles
were to find balanced in vitro activity, good cell potency,
and to optimize physical properties by keeping molecu-
lar weight in check and introducing polar functionality
where possible. Pyridine regiochemistry had a moderate
impact on Akt1 and Akt2 activities, though the 2-substi-
tution in 7c provided 3-fold improved Akt1 activity rel-
ative to 7a. Increasing the polarity of the molecules with
the 4-pyridone (7d) functionality gave improved Akt2
activity. All these heterocyclic substituents impart im-
proved potency relative to phenyl (7e). Despite the sig-
nificant improvements in Akt1 and Akt2 activities
observed in this optimization of the amine end group,
the cell potency was still moderate. To attain the goal
of inventing a compound that inhibits Akt in vivo at
reasonable plasma levels further improvements in cell
activity were sought.
Schemes 1 and 2 show the synthesis of naphthyridines
described in this Letter. 3-Phenylnaphthyridin-2-ones
(5) were prepared by utilizing chemistry previously pub-
lished by this group.9 Scheme 1 illustrates the chemistry
for the 1,8-naphthyridine series but the same chemistry
was also utilized for the synthesis of 1,5- and 1,7-naph-
thyridines. The naphthyridinone 5 was converted to the
2-chloro-1,8-naphthyridine by treatment with POCl3.
Suzuki coupling with the appropriate boronic acid then
provided the aldehyde 6. Reductive amination afforded
the range of tertiary amines (7) described below.
A significant advance in the series was found when
exploration was done around the 2-pyridyl pyrazole.
When the pyrazole was replaced with a triazole (Table
2, compound 13) the potency was similar but the triazole
was more active in a caspase induction assay (vide
infra). Using this end group an investigation of naph-
thyridine isomers was performed (Table 2). While the
1,7-naphthyridine 14 gave comparable activity to the
1,8-isomer, increased Akt1 activity was found in
the 1,6-naphthyridine (15). The most notable isomer
proved to be the 1,5-naphthyridine 16, which possessed
significantly enhanced activity against Akt1 and Akt2
relative to 13. These improvements in Akt1 activity
against the activated kinase translated to significantly
improved inhibition of Akt1 phosphorylation in cells.
However, the apparent improvements in Akt2 activity
did not translate directly to improved Akt2 cell activity.
The reason for this disconnection is not well understood.
Scheme 2 illustrates the synthesis of 1,6-naphthyridines
and naphthyridinones. Lithiation of 810 and Quenching
with N,N-dimethylformamide provided the aldehyde 9.
The nitrile 1011 was treated with benzyl magnesium
chloride to provide the ketone 11. Condensation of the
aldehyde 9 with the ketone 11 in the presence of sodium
R1
O
H
N
N
N
O
N
N
c
N
N
R2
a,b
Ph
Ph
Ph
7 a-e
5
6
Scheme 1. Reagents and conditions: (a) POCl3 (1.5 equiv), MeCN,
reflux, 72% yield; (b) p-CHO-PhB(OH)2, PdP(t-Bu)3, Cs2CO3, diox-
ane, reflux, 72% yield; (c) HNR1R2, NaBH(OAc)3.
The final improvement in this progression occurred
when an additional polar element was added to the core.

3180
M. T. Bilodeau et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3178–3182
Table 1. 1,8-Naphthyridine series SAR
N
N
N
N
NH
Ph
R
R
Akt1 IC50 (nM)
Akt2 IC50 (nM)
260
Cell Akt1 IC50 (nM)
369
Cell Akt2 IC50 (nM)
1454
7a
7b
7c
7d
7e
120
250
44
N
900
280
83
nd
nd
N
149
132
nd
995
613
nd
N
34
N
H
O
470
1600
All compounds >50,000 nM versus PKA, PKC, SGK.
aAverage of at least three measurements; enzyme protocol.8,12
Previously in the quinoxaline series several examples of
potency-enhancing polar hydrogen bond donating sub-
stituents were identified including the fused imidazole
in 1.4a Here, polarity and hydrogen bond donating func-
tionalities were added directly to the core without signif-
icantly increasing molecular weight by creating a 1,6-
naphthyridin-5-one (17). This change resulted in
enhancement in both Akt1 and Akt2 activities. In par-
ticular, compound 17 shows significantly improved po-
tency while having a lower molecular weight (539 Da)
than 2.
spondingly lower levels of Akt inhibition were observed.
Overall, the extent of Akt inhibition responded well to
the blood levels of 17. Importantly, compound 17 was
well tolerated and high levels of inhibition of Akt1
and Akt2 were seen in vivo at readily achievable blood
levels.
The properties of compound 17 were sufficient to war-
rant a multiple-dose efficacy study in tumor xenograft
mice. A tumor xenograft efficacy study using the
A2780 ovarian carcinoma cell line in nude mice was con-
ducted. It included an experiment to determine pharma-
Consistent with the allosteric mode of inhibition, 17 is
dependent on the PH-domain for Akt inhibition, is
selective for Akt1/2 over Akt3 (IC50 = 1900 nM), and
is highly selective over other members of the AGC fam-
ily of kinases (>50 lM vs PKA, PKC, SGK). Com-
pound 17 has moderate activity in an hERG binding
assay (IC50 = 5610 nM) and is a substrate for human
P-glycoprotein (MDR1 Directional Transport Ratio (B
to A/A to B) = 17.6). It also showed good physical prop-
erties with a human plasma protein binding of 97.3%
and a logP = 3.51. It showed good pharmacokinetics
in rat with a low clearance of 4.6 mL/min/kg and a
half-life of 3.8 h.
cokinetics and in vivo Akt1 and 2 inhibition in lung and
13
tumor samples during the course of dosing
.
In the study, A2780 cells were implanted on day 1.
Animals were weighed on day 7, and dosing was com-
menced on day 12. Compound 17 was dosed subcutane-
ously at 50 mg/kg three times per day (at t = 0, 3, and
7 h), twice a week for five cycles. Figure 3 shows the tu-
mor weight data for vehicle and compound 17 treatment
(21 days from when animals were initially weighed). A
statistically significant decrease in mean tumor weight
as compared to the vehicle control was noted for mice
treated with 17 (p = 0.007).14 Compound 17 was well
tolerated and there was no significant effect on animal
weight relative to vehicle treated animals.
Due to the improved cell potency, physical properties,
and rodent pharmacokinetics of compound 17, tolera-
bility and Akt inhibition were assessed in mice. Using
an acute dosing schedule (IP dosing of 50 mg/kg at times
0, 3, and 8 h), administration of 17 was well tolerated in
mice and showed high levels of Akt inhibition in mouse
lung. The levels of Akt1 and Akt2 activities in mouse
lung were determined by isozyme-specific immunopre-
cipitation from lysates, followed by in vitro kinase as-
says (Table 3). At the 10 h timepoint, a blood
concentration of 13 lM of 17 resulted in 79% inhibition
of Akt1 and 59% inhibition of Akt2 in mouse lung. At
later timepoints lower blood concentrations, and corre-
On the day the study was terminated, three tumor
bearing vehicle mice were treated twice with
a
50 mg/kg dose of 17 (at 0 and 3 h). Two hours after
the last dose, blood, lung, and tumor tissues were ta-
ken from these animals as well as two control animals
for pharmacokinetic and pharmacodynamic analysis.
The extent of Akt1 and 2 inhibition was determined
by an IP kinase assay. Inhibition of Akt1 and 2 in
lung tissue was 80% and 75%, respectively. Inhibition
of Akt1 and 2 in tumor tissue was 95% and 54%,
respectively. The average blood concentration from

M. T. Bilodeau et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3178–3182
3181
Table 2. Naphthyridine core variation
N
N
NH
Het
N
Ph
N
R
Akt1 IC50 (nM)
45
Akt2 IC50 (nM)
220
Cell Akt1 IC50 (nM)
83
Cell Akt2 IC50 (nM)
N
N
13
14
15
16
17
1077
981
Ph
Ph
Ph
Ph
N
N
N
21
160
210
70
31
21
30
16
N
7.4
5.6
3.5
2679
1476
266
N
N
N
HN
42
Ph
O
All compounds >50,000 nM versus PKA, PKC, SGK.
aAverage of at least three measurements; enzyme protocol.8,12
Table 3. Inhibition of Akt1 and Akt2 in mouse lung with 17a
Time (h) Blood conc (lM) Akt1 % activity Akt2 % activity
activity in cells relative to Akt1. But the levels of Akt1
and 2 inhibition in vivo in this study have been con-
sistently observed in several related in vivo studies.
10
16
24
13.0
2.6
21
62 34
8
41 13
86 35
91 30
This Letter has outlined the invention of naphthyridine
and naphthyridinone inhibitors of Akt. In particular,
naphthyridinone 17 was identified as a potent dual
inhibitor of Akt1 and Akt2. This compound is well tol-
erated in at exposures that provide high levels of Akt1
and 2 inhibition in vivo. It has also been shown to inhi-
bit the growth of A2780 tumors in vivo when used as
monotherapy. Additional studies with 17 will be the
subject of subsequent publications.
0.7
100 25
Akt activity was determined by IP kinase assay at the given timepoints.
a Compound 17 was dosed 3 · 50 mg/kg IP at times 0, 3, and 8 h to
nude mice (3 per timepoint).
4
3
2
1
0
32
30
28
26
24
22
20
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.04.074.
Day
21
21
0
21
0
21
Treatment veh cmpd 17
vehicle
cmpd 17
References and notes
Figure 3. A2780 tumor xenograft study with 17 (solid line: median;
dotted line: mean; gray box: 25–75% confidence limits, bars: 5–95%
confidence limits).
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13. Experimental design: A2780 tumor cells (9 · 106 cells per
animal) were implanted subcutaneously into the left flank
of nude mice. The mice were weighed after 5 days (study
day 0). Animals were randomized into groups based on
tumor volume and treatment was initiated on study day 5
(12 days from implantation) when tumors were a size of
ꢁ100 cm3. Vehicle (25% hydroxypropyl-b-cyclodextrin);
17 hydrochloride dosed subcutaneously at 50 mg/kg 3· per
day (at t = 0, 3, and 7 h), twice a week for 5 cycles.
Animals were euthanized on study day 21 (16 days from
beginning of dosing). Animal weights exclude tumor
weight.
5. Upon dosing nude mice with 2 (di-HCl salt, 100 mg/kg ip)
significant agitation was observed. No alternate routes of
administration were found that showed desired tolerability
and exposure.
6. This end-group and optimizations of it were later success-
fully incorporated into other inhibitor motifs (see Ref. 4d–
4f).
7. For Akt enzyme assay details see: Barnett, S. F.;
Defeo-Jones, D.; Fu, S.; Hancock, P. J.; Haskell, K.
M.; Jones, R. E.; Kahana, J. A.; Kral, A.; Leander,
14. The mean tumor weight and standard error within each
dose group were estimated from an analysis of variance
(ANOVA) model. The effect of 17 on mean tumor weight
was compared to the vehicle group mean and compared
via a 1-sided a = 0.05 t-test.