Rapid assembly of diverse and potent allosteric Akt inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.10.023

This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a pro

Full text of DOI:10.1016/j.bmcl.2007.10.023

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2211–2214
Rapid assembly of diverse and potent allosteric Akt inhibitors
Zhicai Wu,a,* Ronald G. Robinson,b Sheng Fu,b Stanley F. Barnett,b
Deborah Defeo-Jones,b Raymond E. Jones,b Astrid M. Kral,b Hans E. Huber,b
Nancy E. Kohl,b George D. Hartmana and Mark T. Bilodeaua
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 28 August 2007; revised 3 October 2007; accepted 5 October 2007
Available online 17 October 2007
Abstract—This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate.
Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed
a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.
Ó 2008 Published by Elsevier Ltd.
Akt is a serine/threonine kinase that is a key regulator of
apoptosis and directly phosphorylates several proteins
that are part of the cell survival machinery.1,2 Akt has
also been shown to be involved in the regulation of cell
cycle progression, cell proliferation, and cell growth.1,2
Thus Akt activation plays a critical role in tumorigenesis.
This is supported by the common observation of dysreg-
ulation of Akt in the development of human cancer.2a–f,3
Therefore, Akt inhibitors have potential as a new thera-
peutic treatment for cancer and may induce apoptosis
alone or in combination with standard cancer
chemotherapeutics.
This sensitization was not reversed by over-expression
of a functionally active Akt3. In addition, compound 1
is PH domain-dependent and also specific for Akt over
other closely related kinases. Our goal is to develop
highly specific Akt inhibitors for therapeutic use that
are devoid of off-target activities.
Me Me
NH2
N
O
1
N
N
N
N
N
N
2
NH
N
H
3
The three isoforms of human Akt (Akt1, Akt2, and
Akt3) have an amino terminal pleckstrin homology
(PH) domain and a kinase domain separated by a 39-
amino acid hinge region. They share more than 80%
of their amino acid identities.2a,b,4 In addition, Akt be-
longs to the AGC family of kinases and shares high
homology with PKA and PKC. Therefore, it is a chal-
lenge to develop isozyme selective and Akt specific (vs
the AGC family of kinases) inhibitors. Recently, leading
compound 1 was found to reversibly inhibit the activity
as well as the activation of Akt1 and Akt2, but not
Akt3.5–7 It was shown that inhibition of both Akt1
and Akt2, but not Akt1 or Akt2 alone, was sufficient
to maximally sensitize tumor cells to apoptotic stimuli.
1
2
Akt1 IC50 = 58 nM
Akt2 IC50 = 210 nM
AKt3 IC50 > 2,119 nM
Akt1 IC50 = 3,400 nM
Akt2 IC50 = 2,3000 nM
AKt3 IC50 > 50,000 nM
Optimization of potency for 1 by library synthesis led to
compound 2 which induced apoptosis,5–7 but displayed
a high molecular weight and limited solubility. To elim-
inate these problems as well as improve potency and
physical properties, we modified the quinoxaline tem-
plate. We communicate here a rapid assembly of potent
Akt1 and Akt2 dual inhibitors with diversified core
structures from a common intermediate.
Keywords: Akt inhibitor; Allosteric; Rapid assembly; Cancer; Kinase.
*
The earlier SAR studies of quinoxalines pointed out the
importance of 2,3-diphenyl substituents and the N-1
Corresponding author. Tel.: +1 2156526331; fax: +1 2156527310;
e-mail: zhicai_wu@merck.com
0960-894X/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.10.023

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Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2211–2214
Table 1. Akt activities of tetrasubstituted pyridines
MeO
Me
OMe
Me
CH3
CH3
O
N
O
NC
NH2
Me2N
N
O
O
DMF, 110 ˚C
85%
R
N
NaH, DMF
71%
3
4
N
NH
CH3
NC
Ph
CH3
9
H
N
NBS, CHCl3
Cl
N
POCl3
O
benzoylperoxide, reflux
110 ˚C
90%
NC
NC
6
5
Compound
R
Akt1 IC50 (nM)
Akt2 IC50 (nM)
N
O
DIPEA, THF/MeOH
Br
8
Cl
NH2
253 47
495 240
2249 915
8457 975
5548 1660
7356 5104
2062 1021
8141 2584
15390 692
37720 8779
Cl
NC
N
N
Cl
N
NH
9a
9b
9c
9d
HN
O
NHMe
NMe2
OMe
NC
N
NH
8
7
52% for two steps
Scheme 1. Synthesis of the common intermediate 8.
encouraging results (Akt1 IC50 = 253 nM, Akt2
IC50 = 7356 nM). However, its analogs, aminopyridines
(9a–c) or alkoxypyridine (9d), offered lower potency
against both Akt1 and Akt2.
heteroatom.5 Taken together with the known versatility
of 2-chloro-3-cyanopyridine functionality,8 we envi-
sioned pyridine 8 (Scheme 1) as a common intermediate
to generate analogs with different heterocyclic cores.
Compound 8 was easily obtained in five steps from
ketone 39 (Scheme 1). Condensation of 3 with N,N-
dimethylformamide dimethylacetal gave 4, which
cyclized with 2-cyanoacetamide to afford pyridone 5.
Treatment of 5 with phosphorus oxychloride produced
chloropyridine 6. Radical bromination followed by dis-
placement with suitably substituted amines generated
the proposed intermediate 8.
Table 2. Akt activities of pyrazolopyridines
N
O
R
N
N
N
NH
N
Ph
H2N
10
Compound 8 served as a powerful common intermediate
and afforded different heterocyclic systems in just one
further step including: (a) tetrasubstituted aminopyri-
dines or alkoxypyridines (9) via the displacement of
the chlorine atom; (b) pyrazolopyridines (10) through
the reaction with hydrazines; (c) furopyridines (11)
via the reaction with glycolates; and (d) pyridopyrimi-
dines (12) through the reaction with amidines (Scheme 2).
Compound
R
H
Akt1 IC50 (nM) Akt2 IC50 (nM)
10a
10b
10c
86
121 22
9
7356 5104
556 26
1228 26
Me
CH2CH2OH 131 12
N
HN
10d
21
6
839 53
The Akt activities10 of selected tetrasubstituted pyri-
dines are shown in Table 1. Chloropyridine 8 provided
R
Table 3. Akt activities of pyridopyrimidines
R'HN or R'O
NC
N
N
O
9
R
N
N
R'OH or R'NH2
R
N
NH
R
R
N
NH
NH2
tert-BuOK
R'
N
R'
N
N
N
Cl
N
N
R'NHNH2
R'
NH2
N
EtOH
130 ˚C
NC
NH2
H2N
12
DMF, 140 ˚C
8
10
12
O
HO
OMe
KOMe, DMF, 130 ˚C
Compound
R
Akt1 IC50
(nM)
Akt2 IC50 Caspase 3-fold
induction*
R
(nM)
N
MeO
O
O
12a
12b
12c
OH
NH2
H
244 146
107 76
81 48
577 300
274 148
259 150
1.4
2.2
2.7
H2N
11
* @ 4000 nM.
Scheme 2. Synthesis of different core structures from 8.

Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2211–2214
2213
We then turned our attention to bicyclic cores. Shown in
N
Table 2 are the Akt activities of selected pyrazolopyri-
dines. In this series, the Akt1 potency was greatly im-
proved with the best compound (10d) displaying an
Akt1 IC50 = 21 nM. However, its Akt2 IC50 was still
839 nM. These compounds were not active in cells as
they were not able to sensitize LNCaP cells toward
apoptosis in a caspase 3 induction assay (no induction
was observed at 4000 nM in the presence of TRAIL).11
Their weak Akt2 activities might be responsible for the
lack of cell potency.
N
N
MeO
O
O
HN
H2N
F
11
Fused [6,6] pyridopyrimidines also yielded satisfactory
results. Shown in Table 3 are pyridopyrimidines with
different 2-substituents. These compounds offer potent
activity against Akt1 and Akt2. They also showed good
cell activity in the caspase-induction assay.11 The 2-non-
substituted 12c and the 2-amino substituted 12b dis-
played better intrinsic potency and greater fold
induction in the caspase 3 assay.
We also investigated furopyridines as another6,5 fused
system represented by compound 11. This compound
gave good Akt activity: Akt1 IC50 = 40 18 nM, Akt2
IC50 = 237 123 nM. More importantly, it displayed
good caspase 3 induction activity (fold induction = 5.2
at 4000 nM).11 Further quantitative measurement of
its ability to inhibit Akt phosphorylation in cells showed
encouraging results: Akt1 IC50 = 256 nM and Akt2
IC50 = 1200 nM).12
With the initial encouraging results of 2-unsubstituted
pyridopyrimidines, we further optimized this series by
modifying the terminal benzimidazolone group (Table
4). Clearly, the terminal groups had a great impact on
the compounds’ Akt activity. While purine (13a) and
Table 4. Optimization of pyridopyrimidines
N
N
N
R
N
NH2
Compound
R
IC50 (nM)
Cell IC50 (nM)
Akt1
Akt2
Akt1
1834
Akt2
5084
O
N
N
NH
12c
81 48
129 58
69 31
259 150
N
13a
13b
696
5
nd
nd
N
NH2
N
Me
N
N
281 26
221 63
344 112
N
HN
13c
13d
13e
18
21
3
6
239 18
277
463
295
1811
1198
468
F
N
NH
85 30
N
N
N
N
14 0.1
99
8
NH2
N

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Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2211–2214
Table 5. Akt1 fold-selectivity of pyridopyrimidines
Compound Akt2 Akt3 Akt1-dPH SGK
Acad. Sci. U.S.A. 1996, 93, 3636; (d) Haas-Kogan, D.;
Shalev, N.; Wong, M.; Mills, G.; Yount, G.; Stokoe, D.
Curr. Biol. 1998, 8, 1195; (e) Staal, S. P. Proc. Natl. Acad.
Sci. U.S.A. 1987, 84, 5034; (f) Brognard, J.; Clark, A. S.;
Ni, Y.; Dennis, P. A. Cancer Res. 2001, 61, 3986; (g)
Kozikowski, A. P.; Sun, H.; Brognard, J.; Dennis, P. A. J.
Am. Chem. Soc. 2003, 125, 1144; (h) Breitenlechner, C. B.;
Wegge, T.; Berillon, L.; Graul, K.; Marzenell, K.; Friebe,
W.; Thomas, U.; Huber, R.; Engh, R. A.; Masjost, B. J.
Med. Chem. 2004, 47, 1375.
PKA
>2800 nd
PKC
nd
13c
13e
13
7
105
40
>2800
>3400
>3400 >2700 1862
methylbenzimidazole (13b) gave similar results to benz-
imidazolone (12c), fluorobenzimidazole (13c), pyridopy-
razole (13d), and isopurine (13e) displayed much more
potent enzyme activities against Akt1 (IC50 6 20 nM)
and Akt2 (IC50 ꢀ 100 nM). Further, they were also very
active at inhibiting Akt phosphorylation in cells.12 It
should be noted that pyridopyrimidine 13c and furo-
pyridine 11 share the same terminal fluorobenzimidazole
group and have similar Akt activity. Further, these com-
pounds generally have good solubility in common or-
ganic solvents and acidic aqueous solution (pH < 4).
3. (a) Hsu, J. H.; Shi, Y.; Hu, L. P.; Fisher, M.; Franke, T. F.;
Lichtenstein, A. Oncogene 2002, 21, 1391; (b) Page, C.; Lin, H.;
Jin, Y.; Castle, V. P.; Nunez, G.; Huang, M.; Lin, J. Anticancer
Res. 2000, 20, 407; (c) Cheng, J. Q.; Lindsley, C. W.; Cheng, G.
Z.; Yang, H.; Nicosia, S. V. Oncogene 2005, 24, 7482.
4. Masure, S.; Haefner, B.; Wesselink, J. J.; Hoefnagel, E.;
Mortier, E.; Verhasselt, P.; Tuytelaars, A.; Gordon, R.;
Richardson, A. Eur. J. Biochem. 1999, 265, 353.
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.
6. 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.
7. Defeo-Jones, D.; Barnett, S. F.; Fu, S.; Hancock, P. J.;
Haskell, K. M.; Leander, K. R.; McAvoy, E.; Robinson,
R. G.; Duggan, M. E.; Lindsley, C. W.; Zhao, Z.; Huber,
H. E.; Jones, R. E. Mol. Cancer Ther. 2005, 4, 271.
8. (a) Witherington, J.; Bordas, V.; Gaiba, A.; Garton, N. S.;
Naylor, A.; Rawlings, A. D.; Slingsby, B. P.; Smith, D. G.;
Takle, A. K.; Ward, R. W. Bioorg. Med. Chem. Lett. 2003,
3055, and references therein; (b) Pochat, F.; Lavelle, F.;
Fizames, C.; Zerial, A. Eur. J. Med. Chem. Chim. Ther.
1987, 135; (c) Schoenfeld, F.; Troschuetz, R. Heterocycles
2001, 1979, and references therein.
Initial pharmacokinetic studies in dogs showed promis-
ing profiles for the pyridopyrimidines. For example,
compound 13c had a long half-life (t1/2 = 2.8 h) with a
low clearance (Cl = 5.6 mL/min/kg).13
We also examined the selectivity of these compounds for
Akt1 versus Akt2, Akt3, and other closely related ki-
nases. Akt1 fold selectivities for 13c and 13e over related
kinases are shown in Table 5. In general, these com-
pounds behaved similarly to compound 2. They dis-
played a modest level of selectivity against Akt2 and a
high level of selectivity against Akt3. Their activity
against Akt1 required the presence of the PH domain
and they were not active against other closely related ki-
nases such as SGK, PKA, and PKC.
In summary, we have rapidly synthesized four classes of
compounds from a common pyridine intermediate.
Among them, furopyridines and pyridopyrimidines have
been identified as potent dual inhibitors of Akt1 and
Akt2. In addition, pyridopyrimidine compounds have
shown a promising pharmacokinetic profile and an
excellent selectivity profile. Future investigation will fo-
cus on further improving potency to develop clinical
candidates for the treatment of cancer. Efforts along this
line will be reported in due course.
9. Renault, O.; Dallemagne, P.; Rault, S. Org. Prep. Proced.
Int. 1999, 31, 324.
10. Akt IC50 represents biochemical inhibition of peptide
phosphorylation with a full length of Akt enzyme.
Detection was performed by homogeneous time resolved
fluorescence (HTRF) using an europium chelate (Perkin-
Elmer) [Eu(K)]-labeled phospho(S21)-GSK3a antibody
(Cell Signaling Technologies) and streptavidin-linked
XL665 fluorophore which binds to the biotin moiety on
the substrate peptide (biotin-GGRARTSSFAEPG). For
detail, see Ref. 6. Values are reported as single determi-
nations or as the average of at least two determina-
tions standard deviation.
Acknowledgments
The authors thank Dr. Art Coddington, Dr. Chales
Ross, and Dr. Harri Ramjit for mass spectral analyses.
11. Caspase assay measures the ability of Akt inhibitors to
sensitize LNCaP cells to tumor necrosis factor related
apoptosis inducing ligand (TRAIL) induced apoptosis. As
a surrogate for apoptosis induction we determine the
enzymatic activity of the effector caspase 3 in compound-
treated and untreated cells and values are reported as the
fold induction. For details, see Ref. 7.
12. Cell Akt IC50 represents the ability of inhibitors to block
the phosphorylation of Akt isozymes in C33 A cells
(human cervical carcinoma). For detail, see Ref. 7. Values
are reported as single determinations or as the average of
at least two determinations standard deviation.
References and notes
1. For excellent reviews on Akt, see: (a) Graff, J. R. Expert
Opin. Ther. Targets 2002, 6, 103; (b) Nicholson, K. M.;
Anderson, N. G. Cell. Signalling 2002, 14, 381; (c) Li, Q.;
Zhu, G.-D. Curr. Top. Med. Chem. 2002, 2, 939; (d)
Barnett, S. F.; Bilodeau, M. T.; Lindsley, C. W. Curr. Top.
Med. Chem. 2005, 5, 109.
2. (a) Hanks, S.; Hunter, T. FASEB 1995, 9, 576; (b) Zinda,
M. J.; Johnson, M. A.; Paul, J. D.; Horn, C.; Konicek, B.
W.; Lu, Z. H.; Sandusky, G.; Thomas, J. E.; Neubauer, B.
L.; Lai, M. T.; Graff, J. R. Clin. Cancer Res. 2001, 7, 2475;
(c) Cheng, J. Q.; Ruggeri, B.; Klein, W. M.; Sonoda, G.;
Altomare, D. A.; Watson, D. K.; Testa, J. R. Proc. Natl.
13. Compound dosed 0.25 mpk iv as a solution in DMSO and
the data are the average of two dogs.