Discovery of potent and cell-active allosteric dual Akt 1 and 2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.05.085

This paper describes the improvement of cell potency in a class of allosteric Akt 1 and 2 inhibitors. Key discoveries include identifying the solvent exposed region of the molecule and appending basic amines to enhance the physiochemical properties of the

Full text of DOI:10.1016/j.bmcl.2008.05.085

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4186–4190
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent and cell-active allosteric dual Akt 1 and 2 inhibitors
a
a
b
b
b
Tony Siu a, , Jun Liang , Jeannie Arruda , Yiwei Li , Raymond E. Jones , Deborah Defeo-Jones ,
*
Stanley F. Barnett b, Ronald G. Robinson b
a Department of Medicinal Chemistry, Merck Research Laboratories, Merck & Co., 3535 General Atomics Court, San Diego, CA 92129, USA
b Department of Cancer Research, 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:
This paper describes the improvement of cell potency in a class of allosteric Akt 1 and 2 inhibitors. Key
discoveries include identifying the solvent exposed region of the molecule and appending basic amines to
enhance the physiochemical properties of the molecules. Findings from the structure–activity relation-
ships are discussed.
Received 3 March 2008
Revised 18 May 2008
Accepted 19 May 2008
Available online 24 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Akt
Kinase
Allosteric
Cell active
The PI3K/Akt pathway has attracted much attention in the can-
cer research community due to its involvement in multiple cell
survival, growth, energy metabolism, and proliferation pathways.1
Akt, also known as Protein Kinase B (PKB), is a serine/threonine ki-
nase that plays a crucial role in the regulation of the PI3K/Akt path-
way. Akt exists as three isoforms: 1, 2, and 3, all of which contain a
pleckstrin homology (PH) domain in addition to the kinase cata-
lytic domain. Interaction of phosphatidyinositol-3,4,5-triphos-
phate with the PH domain recruits Akt to the membrane from
the cytoplasm. Upon translocation to the membrane, Akt under-
goes a conformational change and is phosphorylated by phospho-
inositide-dependent kinase 1 (PDK1) at threonine 308 and by
phosphoinositide-dependent kinase 2 (PDK2) at serine 473 for full
activation.
Support for the use of Akt kinase as a molecular target for can-
cer is provided by human cancers with mutations that upregulate
the PI3K/Akt pathway strongly. Most notable is the loss of function
of PTEN, a phosphatase and negative regulator of Akt, that is de-
leted or mutated in >50% of human cancers.2 Several therapeutic
strategies can be envisioned due to the many roles Akt plays in
neoplastic transformation. Because Akt is a crucial component of
the survival pathway, inhibition of the enzyme through small mol-
ecule perturbation in synergy with chemotherapy could poten-
tially sensitize cancer cells to undergo apoptosis.3 In addition,
inhibition of Akt kinase could have the benefit of reversing chemo-
therapeutic and radiation therapy resistance. This mechanism of
action would have a substantial impact on a broad spectrum of
cancers. Furthermore, small molecule inhibition of Akt kinase as
a monotherapy could inhibit tumorigenesis through deregulation
of the cell growth and proliferation pathways that Akt controls.1b
In previous letters, Merck Research Laboratories have reported a
strategy for targeting Akt kinase 1 and 2 using selective allosteric
inhibitors.4 This strategy involves targeting the PH domain as op-
posed to the catalytic ATP-binding domain of Akt. By designing
molecules that bind allosterically, these inhibitors have the advan-
tage of achieving kinase specificity. As a result of this selectivity,
the likelihood of encountering toxic adverse effects due to inhibit-
ing multiple kinases should be minimized.
Merck Research Laboratories have recently published a series of
bisphenyl analogs represented by 1 and 2 (Fig. 1) as allosteric
inhibitors of Akt.4e Most notable is pyridopyrimidine 1, which pos-
sesses potent intrinsic and cellular activity against Akt 1. However,
1 suffers from modest cellular Akt 2 activity. Our focus was to ex-
pand on these initial findings to obtain potent cell-based active
dual Akt 1 and 2 inhibitors.4e Continuing our strategy to obtain
selective kinase activity by targeting the allosteric site of Akt, we
report in this letter a detailed structure activity relationship based
on a novel scaffold of Akt inhibitors with potent enzymatic and
cell-based activities against Akt 1 and 2.
Table 1 shows data for a wide range of pyridopyrimidine
amines, which were tested against Akt 1 and 2. Included are the
Akt IC50 values which are derived from biochemical inhibition of
synthetic peptide phosphorylation, as well as Akt cell IC50 values
which represent inhibition of phosphorylation of Akt isozymes in
C33a cancer cells.5 Our strategy centered on preserving the pyrido-
* Corresponding author at present address: Department of Drug Optimization,
Merck Research Laboratories, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA
02115, USA.
E-mail address: tony_siu@merck.com (T. Siu).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.085

T. Siu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4186–4190
4187
N
N
H
H
H
N
N
N
MeS
N
N
N
O
N
N
N
N
N
H2N
2
1
Akt 1/2 IC50 = 6.0/94 nM
Akt 1/2 IC50 = 15/90 nM
Cell Akt 1/2 IC50 = 20/899 nM
Cell Akt 1/2 IC50 = 78/388 nM
Figure 1. Merck Akt allosteric inhibitors.
Table 1 (continued)
Table 1
SAR on pyridopyrimidine analogs
Enzyme IC50 (nM)
Cell IC50 (nM)
N
H
N
Akt1
Akt2
Akt1
Akt2
R
N
N
N
N
N
N
N
N
79
154
208
223
H
14
Enzyme IC50 (nM)
Cell IC50 (nM)
HO
Akt1
Akt2
Akt1
Akt2
HN
25
10
5
67
22
73
16
23
40
6
61
23
35
15
20
13
3
421
70
N
F
15
F
20
252
75
462
N
N
3
N
16
N
18
5
93
43
36
19
229
243
4
N
N
684
145
196
177
19
O
17
N
5
O
HO
N
5
N
N
7
28
38
45
102
297
17
42
168
184
18
6
O
O
N
S
O
O
7
N
N
7
19
O
S
O
NA
N
N
N
18
6
N
N
8
20
HN
HN
N
O
45
8
77
25
46
36
101
44
N
N
N
N
9
21
N
N
N
19
9
138
31
4
125
17
10
6
735
41
N
10
11
22
HO
HN
N
17
9
47
27
75
36
125
52
N
N
23
N
N
N
4
39
N
12
N
N
24
HN
N
43
29
194
199
N
12
9
27
13
25

4188
T. Siu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4186–4190
O
O
NH2
NHBoc
N
a,b
c
d,e
N
N
N
Cl
Cl
27
O
OMe
26
29
N
N
H
H
N
N
N
N
f,g
N
N
N
N
N
N
N
N
N
N
OMe
31
32
N
HN
H
N
N
N
N
N
N
H
Steps
N
N
N
N
30
N
NH2
O
N
N
Cl
O
33
34
O
28
NH2
NHBoc
O
N
N
NO2
Steps
N
h,i
j,k
N
N
Cl
N
N
35
36
37
N
H
N
N
N
N
N
N
N
N
38
Scheme 1. Synthesis of regioisomers. Reagents and conditions: (a) (BOC)2O, DMAP, Et3N, CH2Cl2; (b) i— t-BuLi, THF, À78 °C; ii—DMF; (c) 28, 25% sodium methoxide in MeOH,
100 °C; (d) 1 N HCl; (e) 30, NaBH(OAc)3, DMF; (f) i—4 N HCl/THF; ii—POCl3, MeCN, reflux; (g) methyl-piperazine 100 °C, dioxane; (h) methyl-piperazine, 100 °C, dioxane; (i)
H2, 5% Pd/C, EtOAc; (j) (BOC)2O, DMAP, Et3N, CH2Cl2; (k) i— t-BuLi, THF, À78 °C; ii—DMF.
pyrimidine biphenyl core and introducing a variety of amines in
the northwest region of the molecule.
investigation of a set of piperazines (9-13) led to improved cell-
based potency versus Akt 2 with modestly improved intrinsic po-
tency. Notably, we were able to obtain compounds with cellular
activity less than 100 nM for Akt 1 and 2 as exemplified by com-
pounds 10 (cell Akt 1/2 IC50 = 36/44 nM) and 12 (cell Akt 1/2
IC50 = 36/52 nM). The 2,5-dimethyl-substituted piperazine 13, de-
signed to rotate the piperazine out plane, displayed a 2- to 4-fold
loss in cellular potency compared to the methyl piperazine 10. This
result suggests that the conformation of the piperazine ring does
not contribute significantly to potency. The flexible acyclic di-
methyl amino analog 14 suffered a loss in both intrinsic and cellu-
lar potency compared to the piperazines 9–13, signaling the
importance of the conformational constraint of the basic nitrogen.
To investigate whether the basic nitrogen of the piperazine sub-
stituent is important to the activity of our inhibitors, we examined
To address the binding environment, we first probed the region
with lipophilic substituents such as difluoro pyrrolidine 3 and
piperidine 4. These analogs provided modest intrinsic and cell
based potencies. In contrast, the incorporation of a polar hetero-
atom in the piperidine in the form of morpholine 5 led to slight
gains in intrinsic potency (Akt 1/2 IC50 = 5/43 nM) with a 2-fold
improvement in cellular activity in Akt 2 (cell Akt 1/2 IC50 = 19/
243 nM) compared to 1. Several other oxygen-based analogs such
as 6 and 7 were explored; however, the cell-based potencies for
Akt 2 for both of them were similar to 5.
Although the morpholine analogs were not at the desired po-
tency level, these results provided an impetus to explore this re-
gion of the molecule with more hydrophilic substituents. Further

T. Siu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4186–4190
4189
Table 2
related to 12 were prepared (Scheme 1). Compound 32 was syn-
thesized starting with 2-chloro-4 amino pyridine. Boc protection
followed by lithiation and quenching with DMF afforded 27. Fried-
lander cyclization with sodium methoxide in methanol and 28 pro-
duced 29 with methoxy replacement at the 2-position of the
pyridine. Hydrolysis of the acetal with acidic conditions liberated
the aldehyde and reductive amination with 30 installed the piper-
idine triazole subunit to afford 31. Hydrolysis of the 2-methoxy
pyridine under strong acid followed by chlorination with POCl3
and displacement with methyl piperazine provided 32. Regioisomer
34 was synthesized using the same sequence of steps, but instead
starting with 2-chloro-4-amino pyridine 33. Regioisomer 38 was
synthesized starting with 2-chloro-5-nitro-pyridine 35 by dis-
placement of the chloride with methyl piperazine and reduction
of the nitro group with palladium on carbon under a balloon atmo-
sphere of hydrogen to afford amine 36.7 The amine was protected
with Boc anhydride followed by lithiation and trapping with DMF
to afford aldehyde 37. This intermediate was carried on to 38 using
the same synthetic sequence as for compound 32.
SAR of methyl piperazine regioisomers
N
1
2
H
X
4
N
N
X
N
N
N
X
N
N
3
Compound
Enzyme Akt 1/2 (nM)
9/27
Cell Akt 1/2 (nM)
36/52
N
N
N
12
N
N
32
285/539
NA
N
N
Orientation of the methyl piperazine in any position other than
position 2 (12) dramatically reduced the overall intrinsic potency
of these inhibitors (Table 2). For example, positioning the methyl
piperazine at positions 1 (34) or 4 (32) led to a 30- to 80-fold loss
in intrinsic potency against Akt 1 and a 20- to 130-fold lost in po-
tency against Akt 2. Interestingly, although orienting the methyl
piperazine at position 3 (38) diminished Akt 1 activity; a 13-fold
selectivity for Akt 2 activity over Akt 1 was observed. It is worth
noting that the southwest region of the molecule could potentially
be exploited for Akt 2-selective inhibitors. Maximal contact with
the solvent exposed region is likely obtained by orienting the
hydrophilic piperazine at the 2 position of the A ring.
In summary, potent dual inhibitors of Akt 1 and 2 such as 12, 23,
and 24 were achieved by introducing modifications to the pyrido-
pyrimidine core. Basic amines attached to the core were key to
achieving cellular activity while maintaining the intrinsic potency.
Optimal activity was restricted to a regiospecific northwest region
of the molecule which presumably projects toward solvent after
binding to the enzyme.
N
N
34
38
720/3570
NA
N
N
740/59
924/133
N
N
several analogs modulating its basicity. Increasing the basicity by
moving the nitrogen out of the ring (16) maintained the overall
intrinsic and cellular potency. Alternatively, reduction of the basic
nitrogen of the piperazine by capping the basic nitrogen as an
amide (18), sulfonamide (19), or urea (20) had minimal effects
on the intrinsic potency. However, the cell-based potency of the
compounds was diminished by 3- to 4-fold compared to 12, indi-
cating that the basicity of the second nitrogen is crucial to cellular
penetration properties. A notable exception is compound 21.
Although the basicity of the nitrogen piperazine is reduced due
to the amide moiety, the extended dimethylamino group seems
to help maintain the cellular activity.
Encouraged by the increased activity of methyl piperazine (12),
we proposed appending more aqueous solubilizing groups that ex-
tend further out such as compounds 23–25. In general, introduc-
tion of polar functionalities on the piperazine moiety was well
tolerated and conferred better overall intrinsic potency. Most nota-
ble was 24 with single digit intrinsic potency against Akt 1 and 2
(Akt 1/2 IC50 = 4/4 nM) and good overall cellular potency (cell Akt
1/2 IC50 = 10/39 nM). These results suggest that the northwest re-
gion of our inhibitors is exposed to an aqueous environment as op-
posed to a hydrophobic pocket. The enhanced intrinsic potency
from appending polar functional groups arises possibly from the
favorable charge interactions at the solvent interface. In addition,
the overall better solubility of the inhibitors might contribute to
their enhanced intrinsic and cell-based potencies. This rationale
is consistent with properties of other kinase inhibitors with
water-solubilizing groups.6
Acknowledgments
The authors thank Drs. Christopher Cox, Christopher Dinsmore,
and Philip Sanderson, for careful reading of this manuscript.
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5. Detection was performed by homogenous time-resolved fluorescence (HTRF)
using a europium chelate. IC50 values are reported as the averages of at least two
independent determinations; standard deviations are within 25–50% of IC50
values. For Akt enzyme 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.; Nhas, D. D.; Robinson, R.; Huber, H. E.
Biochem. J. 2005, 385, 399. For details on the Akt IPKA assay also see Ref. 3.
Values are reported as single determinations standard deviations..
6. (a) Chen, P.; Doweyko, A. M.; Norris, D.; Gu, H. H.; Spergel, S. H.; Das, J.; Moquin,
R. V.; Lin, J.; Wityak, J.; Iwanowicz, E. J.; McIntyre, K. W.; Shuster, D. J.; Behnia,
K.; Chong, S.; de Fex, H.; Pang, S.; Pitt, S.; Shen, D. R.; Thrall, S.; Stanley, P.; Kocy,
O. R.; Witmer, M. R.; Kanner, S. B.; Schieven, G. L.; Barrish, J. C. J. Med. Chem.
7. The introduction of the piperazine by displacement with the activating nitro-
group was necessary because all efforts to displace the chloride of compound 39
N
H
N
N
N
N
N
N
failed.
Cl
39