Allosteric inhibitors of Akt1 and Akt2: Discovery of [1,2,4]triazolo[3,4-f][1,6]naphthyridines with potent and balanced activity

Article abstract of DOI:10.1016/j.bmcl.2008.12.017

A series of [1,2,4]triazolo[3,4-f][1,6]naphthyridine allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been shown to have potent dual Akt1 and 2 cell potency. The representative compound 13 provided potent inhibitory activity against Akt1 and 2 in vivo in a mouse model.

Full text of DOI:10.1016/j.bmcl.2008.12.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 834–836
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Allosteric inhibitors of Akt1 and Akt2: Discovery of
[1,2,4]triazolo[3,4-f][1,6]naphthyridines with potent and balanced activity
b
b
b
a
c
c
Yiwei Li a, , Jun Liang , Tony Siu , Essa Hu , Michael A. Rossi , Stanley F. Barnett , Deborah Defeo-Jones ,
Raymond E. Jones c, Ronald G. Robinson c, Karen Leander c, Hans E. Huber c, Sachin Mittal d,
Nicholas Cosford b, Peppi Prasit b
*
a Department of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., 770 Sumneytown Pike, WP14-2, West Point, PA 19486, USA
b Department of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., 3535 General Atomics Court, San Diego, CA 92121, USA
c Department of Cancer Research, Merck Research Laboratories, Merck & Co., PO Box 4, West Point, PA 19486, USA
d Pharmaceutical Research and Development, Merck Research Laboratories, Merck & Co., PO Box 4, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of [1,2,4]triazolo[3,4-f][1,6]naphthyridine allosteric dual inhibitors of Akt1 and 2 have been
developed. These compounds have been shown to have potent dual Akt1 and 2 cell potency. The repre-
sentative compound 13 provided potent inhibitory activity against Akt1 and 2 in vivo in a mouse model.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 3 September 2008
Revised 3 December 2008
Accepted 3 December 2008
Available online 7 December 2008
Keywords:
Akt
Inhibitors
Allosteric
Cancer
Kinase
In vivo activity
The serine/threonine kinase Akt (PKB), a member of the AGC
super family of kinases, plays a key role in the regulation of cell
survival, proliferation and growth.1 Activation of Akt has been con-
sidered an important step in the development and maintenance of
cancers, and consequently the inhibition of Akt through small mol-
ecule perturbation could lead to novel oncology therapies.2,3
The Akt family kinases contain an N-terminal pleckstrin homol-
ogy (PH) domain in addition to the kinase catalytic domain. The ki-
nase domains of the Akt isozymes, denoted Akt1, Akt2, and
Akt3, are highly conserved but the PH domains are notably diver-
gent. Akt1 and Akt2 are ubiquitously expressed while Akt3 is the
major isozyme in tissues of neuroendocrine origin. Since each of
these Akt isozymes has distinct functions and expression profiles,
compounds that are isozyme selective may provide optimum im-
pact on cancer cell survival and minimum toxicity.4 Previous dis-
compound series with potent and balanced Akt1 and Akt2 activi-
ties in vivo and with an improved hERG activity profile.
A recent communication described the development of com-
pound 1 (Fig. 1).6b This potent Akt inhibitor exhibits in vivo inhibi-
tory activity against Akt1 and Akt2 in a mouse model and is
N
Akt1 IC50 = 3.5 nM
Akt2 IC50 = 42 nM
Cell Akt1 IC50 = 16 nM
Cell Akt2 IC50 = 266 nM
N
N
N
N
NH
HN
N
O
N
1
N
Akt1 IC50 = 5.6 nM
Akt2 IC50 = 70 nM
Cell Akt1 IC50 = 30 nM
Cell Akt2 IC50 = 1476 nM
N
N
NH
N
closures from these research laboratories have reported
a
2
strategy for targeting Akt isozyme 1 and 2 via allosteric inhibitors.
These molecules are PH-domain dependent and display excellent
selectivity against kinases outside of the Akt family.5,6 Continuing
our studies in this important research area we report in this
communication our successful efforts to develop a novel tricyclic
N
Akt1 IC50 = 95 nM
Akt2 IC50 = 28 nM
Cell Akt1 IC50 = 734 nM
Cell Akt2 IC50 = 300 nM
N
N
NH
S
H2N
N
N
N
3
* Corresponding author. Tel.: +1 215 652 9694; fax: +1 215 652 7310.
E-mail address: yiwei_li@merck.com (Y. Li).
Figure 1. Previously published balanced dual Akt1 and Akt2 inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.017

Y. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 834–836
835
efficacious in a tumor xenograft model. However, compound 1 pos-
sesses off-target binding affinity to the Ikr potassium channel hERG
(human Ether-a-go-go-Related Gene) (1: hERG binding IC50
= 5610 nM).6b Our objective was to develop compounds with atten-
uated hERG affinity and more balanced activity against the Akt1 and
Akt2 isozymes as a means to optimize in vivo efficacy. To achieve
these goals, we decided to revisit the naphthyridine compounds
that showed increased potency against the Akt1 isozyme and
through SAR exploration enhance the activity against the Akt2
isozyme.
Earlier work in these laboratories indicated that the regio-
chemistry of the two N atoms in the naphthyridine core had
significant impact on Akt1 and Akt2 activity. In the cases of
1,6-naphthyridin-5-one 1 and 1,5-naphthyridine 2, heteroatoms
occupying the southwest corner of the naphthyridine core were
shown to provide enhanced potency.6b The relationship between
the substitution pattern and potency can be further corrobo-
rated by earlier work exploring pyridine and other heterocycles.
In particular, studies on 2,3,5-substituted pyridine core showed
that Akt2 activity was more sensitive toward substitution at
the southwest corner.6a For example, compound 3 exhibits more
potent Akt2 activity relative to that of Akt1 with an aminothi-
adiazole moiety occupying the southwest corner. Based on our
SAR analysis, we envisioned that by merging the core structures
of compound 1 and 3 together to give a tricyclic structure we
could further enhance the inhibitory activity against the desired
Akt isozymes.
ble decrease of the cell activities (cell Akt1/2 IC50 = 265.5/
1213 nM).
To investigate whether polar functionality is required at the 7-
position of the tricyclic core, hydrogen (13), methyl (14) and chloro-
methyl (15) substituents were introduced. Interestingly, no signifi-
cant loss of activity was observed when a polar heteroatom was
replaced by a non-polar group such as methyl. Deterioration of cel-
lular activities, however, was noticed when the size of the substitu-
ents was increased.10 To further examine whether the southwest
portion of the molecule was exposed to an aqueous environment,
water solubilizing groups were introduced in 16 and 17. In both
cases a loss in potency was observed, suggesting that likely the
southwest corner of the inhibitors occupy an area that is at least
partially occluded.
Encouraged by the improved intrinsic and cell activity of the tri-
cylic compounds, we proceeded to explore variations of the pyridyl
triazole end groups. Replacement of the 2-pyridyl triazole end
group in 9 with a 2-pyridyl imidazole moiety led to compound
18 (Fig. 2). Although compound 18 exhibits a slight enhancement
in Akt2 enzyme activity (Akt 1/2 IC50 = 3.3/2.8 nM) relative to 9,
its cell potency did not show proportional improvement.
Table 2 shows hERG binding affinity data for selected tricyclic
compounds. As has been demonstrated in other Akt inhibitor series,
increase in polarity tends to attenuate hERG binding affinity.5h In
the tricyclic series, the more hydrophobic methyltriazole 14 and
chloromethyl 15 exhibit low micromolar hERG activity. The more
polar aminotriazole 9 and hydroxytriazole 10 display hERG binding
The chemistry that was utilized in this work is outlined in
Scheme 1. 1,6-naphthyridin-5-one 4 was converted to the 5-
affinity of IC50 = 5
significantly attenuated hERG binding affinity with an IC50 greater
than 10 M.
lM or greater. In particular, compound 13 shows
chloro-1,6-naphthyridine
5
by the treatment with POCl3.
l
Displacement of chlorine with hydrazine afforded the hydrazide
6. Treatment of 6 with diimidazole derivatives 87 (compound 9–
12) or orthoesters 7 (compound 13–17) provided the desired
[1,2,4]triazolo[3,4-f][1,6] naphthyridine analogs.
Due to improved cell potency and hERG binding affinity of com-
pound 13, tolerability and Akt inhibition in mice was assessed in a
PK/PD experiment.12 Under a mini-pump format, the levels of
Akt1 and Akt2 phosphorylation activities in mouse lung were mea-
sured while the drug concentration in the blood of the subject was
maintained at a steady level (Table 3). At a blood concentration of
To our gratification, improvement was observed with the tricy-
clic compounds. Compared to the lead naphthyridinone compound
1, introduction of a 7-amino-[1,2,4]triazolo[3,4-f][1,6]naphthyri-
dine core in 9 provided ꢀ4-fold increased enzymatic Akt2 activity
while maintaining similar Akt1 activity (Table 1). The improved
intrinsic potency of 9 translated to significantly enhanced cell
activities (cell Akt1/2 IC50 = 18.1/47.7 nM). The balanced activities
against Akt 1 and 2 and the improvement in the cellular activities
led to the continued exploration of the southwest corner of the tri-
cyclic core series.
Table 1 shows the analogs synthesized on the triazole core. Var-
iation of the heteroatom at the 7-position of the tricyclic core pro-
vided limited impact on Akt1 and Akt2 activity. Increasing polarity
in the form of urea such as 12 led to a loss in Akt1 and Akt2 intrin-
sic potency (Akt 1/2 IC50 = 8.5/24.1 nM) which translated to a nota-
1.8
bition of Akt2 in mouse lung. As a comparison, compound 1 was also
tested using the same format. At a blood concentration of 3.0 M,
lM, compound 13 provided 92% inhibition of Akt1 and 41% inhi-
l
compound 1 provided 85% inhibition of Akt1 while little Akt2 inhi-
bition was observed. These experiments showed a clear correlation
between the in vivo activities and the in vitro enzyme and cell
potencies of the Akt inhibitors, and the higher Akt2 inhibition levels
exhibited by compound 13 was likely due to its improved intrinsic
activity. Our attempts to achieve greater Akt2 inhibition level, how-
ever, were not successful as the blood concentration of compound
13 could not be increased with a larger dose.13 Overall, this study
showed that compound 13 was well tolerated and inhibition of both
Akt1 and Akt2 were seen in vivo at the blood levels achieved.
R
R
R
N
N
N
a
b
N
HN
N
Cl
O
NH
5
H2N
4
6
EtO
OEt
OEt
d
X
c
R =
N
R'
N
N
7
N
N
N
N
NH
N
8
R
R
N
N
N
N
R'
HX
X = O or NH
N
N
N
N
13-17
9-12
Scheme 1. Reagents and conditions: (a) POCl3, MeCN, reflux, 85% yield; (b) NH2NH2, dioxane, 75 °C, 65% yield; (c) 8, DMF, 85 °C, 84% yield; (d) 7, DMF, 85 °C, 70% yield.

836
Y. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 834–836
Table 1
This paper outlined the discovery of [1,2,4]triazolo[3,4-
[1,2,4]Triazolo[3,4-f][1,6]naphthyridine series SARa
f][1,6]naphthyridine inhibitors of Akt. Introduction of the tricyclic
structure significantly improved Akt2 intrinsic and cellular activi-
ties which led to the improved inhibition of Akt1 and Akt2 in a
mouse model.
N
N
N
N
NH
N
N
R
Ph
N
N
Acknowledgment
We thank Drs. Mark T. Bilodeau and Philip E. Sanderson, Mrs.
John C. Hartnett and Ahren I. Green for careful reading of this
manuscript.
R
Akt1 IC50
(nM)
Akt2 IC50
(nM)
Cell Akt1 EC50 Cell Akt2 EC50
(nM)
(nM)
H2N
HO
9
2.8
2.6
9.4
18.1
47.7
References and notes
10
11
12
13
14
15
6.7
10.4
24.1
10.0
14.6
10.5
27.0
17.0
167.7
107.6
1. (a) Cantley, L. C. Science 2002, 296, 1655; (b) Vivanco, I.; Sawyers, C. L. Nat. Rev.
Cancer 2002, 2, 489; (c) Hennessy, B. T.; Smith, D. L.; Ram, R. T.; Yiling, L.; Milla,
G. B. Nat. Rev. Drug Disc. 2005, 4, 989; (d) Cully, M.; You, H.; Levine, A. J.; Mak, T.
W. Nat. Rev. Cancer 2006, 6, 184.
2. (a) Li, J.; Simpson, L.; Takahashi, M.; Miliaresis, C.; Myers, M. P.; Tonks, N.;
Parsons, R. Cancer Res. 1998, 58, 5667; (b) Karakas, B.; Bachman, K. E.; Park, B.
H. Br. J. Cancer 2006, 94, 455; (c) Hynes, N. E.; Lane, H. A. Nat. Rev. Cancer 2005,
5, 341; (d) Engelman, J. A.; Zejnullahu, K.; Mitsudomi, T.; Song, Y.; Hyland, C.;
Park, J. O.; Lindeman, N.; Gale, C. M.; Zhao, X.; Christensen, J.; Kosaka, T.;
Holmes, A. J.; Rogers, A. M.; Cappuzzo, F.; Mok, T.; Lee, C.; Johnson, B. E.;
Cantley, L. C.; Janne, P. A. Science 2007, 316, 1039.
HS
10.0
8.5
H
H2N
N
265.5
5.0
1213
O
H
4.0
41.0
176.5
483.3
H3C
ClH2C
4.6
24.6
113.5
3. (a) Hennessy, B. T.; Smith, D. L.; Ram, P. T.; Lu, Y.; Mills, G. B. Nat. Rev. Drug
Discov. 2005, 988; (b) Lindsley, C. W.; Barnett, S. F.; Yaroschak, M.; Bilodeau, M.
T.; Layton, M. E. Curr. Top. Med. Chem. 2007, 1349.
26.7
N
4. (a) Chan, T. O.; Rittenhouse, S. E.; TSichilis, P. N. An Rev. Biochem. 1999, 68, 965;
(b) Barnett, S. F.; Bilodeau, M. T.; Lindsley, C. W. Curr. Top. Med. Chem. 2005, 5,
109; (c) Page, C.; Lin, J. J.; Jin, Y.; Castle, V. P.; Nunez, G.; Huang, M.; Lin, J. Y.
Anticaner Res. 2000, 20, 407; (d) Laine, J.; Kunstle, G.; Obata, T.; Noguchi, M. J.
Biol. Chem. 2002, 277, 3743.
16
17
9.9
55.1
62.5
376.1
N
H3C
OH
N
17.0
77.9
41.8
251.4
5. (a) Lindsley, C. W.; Zhao, Z.; Leister, W. H.; Robinson, R. G.; Barnett, S. F.; Defeo-
Jones, D.; Jones, R. E.; Hartman, G. D.; Huff, J. R.; Huber, H. E.; Duggan, M. E.
Bioorg. Med. Chem. Lett. 2005, 15, 761; (b) Zhao, Z.; Leister, W. H.; Robinson, R.
G.; Barnett, S. F.; Defeo-Jones, D.; Jones, R. E.; Hartman, G. D.; Huff, J. R.; Huber,
H. E.; Duggan, M. E.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2005, 15, 905; (c)
Zhao, Z.; Robinson, R. G.; Barnett, S. F.; Defeo-Jones, D.; Jones, R. E.; Hartman, G.
D.; Huber, H. E.; Duggan, M. E.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2008, 18,
49; (d) Wu, Z.; Hartnett, J. C.; Neilson, L. A.; Robinson, R. G.; Fu, S.; Barnett, S. F.;
Defeo-Jones, D.; Jones, R. E.; Kral, A. M.; Huber, H. E.; Hartman, G. D.; Bilodeau,
M. T. Bioorg. Med. Chem. Lett. 2008, 18, 1274; (e) Wu, Z.; Robinson, R. G.; Fu, S.;
Barnett, S. F.; Defeo-Jones, D.; Jones, R. E.; Kral, A. M.; Huber, H. E.; Kohl, N. E.;
Hartman, G. D.; Bilodeau, M. T. Bioorg. Med. Chem. Lett. 2008, 18, 2211; (f) Siu,
T.; Liang, J.; Arruda, J.; Li, Y.; Jones, R. E.; Defeo-Jones, D.; Barnett, S. F.;
Robinson, R. E. Bioorg. Med. Chem. Lett. 2008, 18, 4186; (g) Siu, T.; Liang, J.;
Arruda, J.; Li, Y.; Jones, R. E.; Defeo-Jones, D.; Barnett, S. F.; Robinson, R. G.
Bioorg. Med. Chem. Lett. 2008, 18, 4186; (h) Siu, T.; Li, Y.; Nagasawa, J.; Liang, J.;
Tehrani, L.; Chua, P.; Jones, R. E.; Defeo-Jones, D.; Barnett, S. F.; Robinson, R. G.
Bioorg. Med. Chem. Lett. 2008, 18, 4191.
H
Average of at least two measurements; enzyme protocol.8,9 All compounds
>50,000 nM versus PKA, PKC, SGK.
a
N
Akt1 IC50 = 2.8 nM
Akt2 IC50 = 9.4 nM
Cell Akt1 IC50 = 18.1 nM
Cell Akt2 IC50 = 47.7 nM
N
N
N
NH
N
N
N
H2N
N
N
9
N
Akt1 IC50 = 3.3 nM
Akt2 IC50 = 2.8 nM
Cell Akt1 IC50 = 18.3 nM
Cell Akt2 IC50 = 74.8 nM
N
N
N
H2N
6. (a) Hartnett, J. C.; Barnett, S. F.; Bilodeau, M. T.; Defeo-Jones, D.; Hartman, G. D.;
Huber, H. E.; Jones, R. E.; Kral, A. M.; Robinson, R. G.; Wu, Z. Bioorg. Med. Chem.
Lett. 2008, 18, 2194; (b) Bilodeau, M. T.; Balitza, A. E.; Hoffman, J. M.; Manley, P.
J.; Barnett, S. F.; Haskell, K.; Defeo-Jones, D.; Leander, k.; Jones, R. E.; Robinson,
R. G.; Smith, A. M.; Huber, H. E.; Hartman, G. D. Bioorg. Med. Chem. Lett. 2008,
18, 3178.
N
N
N
18
Figure 2. 2-Pyridyl end group modification.
7. Wu, Y.-Q.; Hamilton, S. K.; Wilkinson, D. E.; Hamilton, G. S. J. Org. Chem. 2002,
67, 7553.
8. 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, K.; Lee, L. L.;
Malinowski, J.; McAvoy, E. M.; Nahas, D. D.; Robinson, R.; Huber, H. E. Biochem.
J. 2005, 385, 399.
Table 2
hERG binding affinity for selected compoundsa
Compound
9
10
13
14
15
9. For all of these assays (enzyme Akt1 and Akt2 and cell Akt1 and Akt2 assays)
the standard deviation for a positive control is less than 50% of the IC50 value.
10. See also reference6b: The reason for the apparent disconnect between the
enzyme and cell activity for Akt2 isozyme is not well understood.
11. The hERG IC50 values were acquired by radioligand binding competition
experiments using membrane preparations from human embryonic kidney
cells that express hERG. For assay detail see Bell, I. M.; Gallicchio, S. N.; Abrams,
M.; Beshore, D. C.; Buser, C. A.; Culberson, J. C.; Davide, J.; Ellis-Hutchings, M.;
Fernandes, C.; Gibbs, J. B.; Graham, S. L.; Hartman, G. D.; Heimbrook, D. C.;
Homnick, C. F.; Huff, J. R.; Kassahun, K.; Koblan, K. S.; Kohl, N. E.; Lobell, R. B.;
Lynch, J. J., Jr.; Miller, P. A.; Omer, C. A.; Rodrigues, A. D.; Walsh, E. S.; Williams,
T. M. J. Med. Chem. 2001, 44, 2933.
hERG binding IC50 (nM)
5319
5312
>10,000
2778
2018
a
IC50 values are reported as the averages of at least two independent determi-
nations;11 standard deviations are within 25–50% of IC50 values.
Table 3
Inhibition of Akt1 and Akt2 in mouse lung with compound 13 and 1a
Compound Dose (mg/ Blood conc
% Akt1 inhibition
in lung
% Akt2 inhibition
in lung
mL)
(lM)
13
18
70
10
1.8 0.6
1.7 0.5
3.0 0.9
92
89 19
85
3
41 20
35 15
12. Experimental design: Subjects: Tumor (C33a cervical carcinoma cells) bearing
nude mice. Vehicle (25% hydroxypropyl-b-cyclodextrin); 13 hydrochloride
dosed via mini-pump (at 70 mg/mL and 18 mg/mL concentrations) at a rate of
1
6
4
7
a
8 lL/hr over 20 hrs. Blood draw for PK at 3, 6, 8, 11 and 20 h. Animals were
Compounds were delivered via mini-pump to nude mice (3 per concentration);
euthanized at 20 h time point.
13. Compound solubility issue was likely the cause for the non-linear
proportionality of drug concentrations versus dosage.
Blood concentrations are the average over the course of the experiment; Akt inhi-
bition data are from 20-h time points and the Akt phosphorylation activity was
determined by IP kinase assay in lung.