Development of Pyridopyrimidines as Potent Akt1/2 Inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.01.054

This communication reports a new synthetic route of pyridopyrimidines to facilitate their structural optimization in a library fashion and describes the development of pyridopyrimidines that have excellent enzymatic and cell potency against Akt1 and Akt2.

Full text of DOI:10.1016/j.bmcl.2008.01.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1274–1279
Development of Pyridopyrimidines as Potent Akt1/2 Inhibitors
Zhicai Wu,a,* John C. Hartnett,a Lou Anne Neilson,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 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 7 November 2007; revised 3 January 2008; accepted 10 January 2008
Available online 19 January 2008
Abstract—This communication reports a new synthetic route of pyridopyrimidines to facilitate their structural optimization in a
library fashion and describes the development of pyridopyrimidines that have excellent enzymatic and cell potency against Akt1
and Akt2. This series also shows a high level of selectivity over other closely related kinases and significantly improved caspase-
3 activity with the more optimized compounds.
Ó 2008 Published by Elsevier Ltd.
Akt is a serine/threonine kinase that is a key regulator of
apoptosis, cell cycle progression, cell proliferation and
growth.1,2 Recently, inhibition of Akt kinase has been
recognized as a potential new therapeutic treatment
for cancer.1,3,4 It has been shown that inhibition of both
Akt1 and Akt2, but not Akt1 or Akt2 alone, is needed
to maximally sensitize tumor cells to certain apoptotic
stimuli.5 Much effort has been dedicated to develop
small molecule Akt1 and Akt2 dual inhibitors.5–9
pyrimidines obtained did not display optimal cell
potency.8 We communicate here our effort to improve
their cell potency as well as physical properties.
Since these compounds required the presence of PH do-
main to be active and they did not compete for ATP
binding site, it is unclear where and how they exactly
bind to Akt. We decided to use library synthesis to rap-
idly move the project. While it was found the lower right
6-phenyl group tolerated very limited modification, pre-
vious data showed position 2 could be substitued and
the terminal group attached to the piperidine ring had
a great impact on the Akt activity.8 However, the termi-
nal groups were introduced at the early stage of the syn-
thetic sequence, thus it was not possible to investigate
these groups in an efficient library fashion. To facilitate
the optimization process, a new synthetic route was
devised (Scheme 1). Hydroxylmethylpyrimidine 3 was
oxidized to give the aldehyde 4. This aldehyde then
underwent an aldol reaction with methyl phenylacetate
followed by a cyclization to produce a pyrimidylpyri-
done, which was transformed to the chloropyridopyrim-
idine 5 with the treatment of phosphorus oxychloride.
Suzuki coupling reaction of 5 and 4-formylbenzenebo-
ronic acid afforded aldehyde 6. Reductive amination of
6 with various amines provided the final product. Since
the terminal groups were incorporated at the last step of
this new synthetic scheme, they can be investigated in a
rapid library fashion.
N
N
N
N
N
N
N
N
2
N
N
HN
N
6
N
NH2
NH2
NH2
F
N
2
1
Akt1 IC50 = 18 nM, cell IC50 = 277 nM
Akt2 IC50 = 239 nM, cell IC50 = 1811 nM
Akt1 IC50 = 14 nM, cell IC50 = 295 nM
Akt2 IC50 = 99 nM, cell IC50 = 468 nM
Previously, we have reported the discovery of pyridyl-
pyrimidines as dual inhibitors of Akt1 and Akt2.8 These
compounds are pH domain dependent and also specific
for Akt over other closely related kinases. Therefore, it
should be possible to develop highly specific Akt inhib-
itors for therapeutic use that are devoid of off-target
activities. As represented by 1 and 2, the initial pyridine-
Keywords: Akt; Kinase; Cancer; Pyridopyrimidine.
*
Corresponding author. Tel.: +1 215 652 6331; fax: +1 215 652
7310; e-mail: zhicai_wu@merck.com
0960-894X/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.01.054

Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1274–1279
1275
OMe
, LHMDS
MeS
N
NH2
H
MeS
N
NH2
OH
MnO2
1)
O
N
N
CHCl3
92%
O
2) CH3CN, POCl3
80%
4
3
CHO
(HO)2B
CHO
MeS
N
N
Cl
MeS
N
N
N
N
Pd(P-t-Bu3)2
Cs2CO3, dioxane
6
5
76%
NR1R2
R1R2NH, NaBH(OAc)3
2% AcOH in ClCH2CH2Cl
MeS
N
N
N
7
Scheme 1. Synthesis of pyridopyrimidines.
In addition, 2-methylthio group can be easily trans-
formed to other functionalities, which allows SAR of
this position to be readily explored (Scheme 2). Methyl-
thio 6 was oxidized to sulfoxide 8 which was then dis-
placed with various nucleophiles including amines,
alkoxide or cyano groups to give 9. Compound 9 was
converted via reductive amination reactions to the final
product 10 with varied 2-substituents.
With the new template and optimal synthetic route, we
modified systematically the terminal groups by varying
the functional groups attached to the piperidine ring
and changing the piperidine to an acyclic amine. The
guiding principle was to improve in vitro activity and
cell potency and the amines are selected to optimize
physical properties by keeping molecular weight in
check and introducing polar functionality where possi-
ble. First, the Akt activities of selected pyridopyrimi-
dines with acyclic amines are shown in Table 1. The
very simple primary amide 12a gave a promising starting
point (Akt1 IC50 = 226 nM, Akt2 IC50 ꢀ 1300 nM).10
Replacing the amide with a piperazine ring (12b) de-
creased activities against both Akt1 and Akt2 3- to
5-fold. On the other hand, replacement with aromatic
heterocycles such as, imidazole (12c), triazole (12d), thi-
azole (12e) and pyridine (12f) resulted in slightly im-
proved Akt1 activity and similar Akt2 activity.
Apparently the H-bond donor or acceptor properties
of the heterocycles were not critical for these compounds
It should be noted that the substitution pattern is differ-
ent between this new generation of pyridopyrimidines 7
and 10 and the original compounds 1 and 2. Com-
pounds 7 and 10 have a 2-substituent while 1 and 2 con-
tain a 4-amino group. A direct comparison is made
between 2 and 11 in Figure 1. With the same terminal
groups, the Akt1 activity of 2-substituted 11 is 5-fold
more potent than that of 4-substituted 2 and their
Akt2 activity is similar.10 As a result, the newly gener-
ated 2-substituted pyridopyrimidine provided a suitable
template to develop Akt inhibitors.
CHO
CHO
O
S
N
N
MeS
N
N
m-CPBA
Me
N
N
CH2Cl2
71%
8
6
CHO
R1R2NH, NaBH(OAc)3
R
N
N
displacement
N
2% AcOH in ClCH2CH2Cl
9
NR1R2
R
N
N
N
10
Scheme 2. Synthesis of pyridopyrimidines with different 2-substituents.

1276
Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1274–1279
N
N
N
N
N
MeS
N
N
N
N
N
N
N
N
N
NH2
NH2
NH2
N
N
2
11
Akt1 IC50 = 14 nM, cell IC50 = 295 nM
Akt2 IC50 = 99 nM, cell IC50 = 468 nM
Akt1 IC50 = 4 nM, cell IC50 = 60 nM
Akt2 IC50 =44 nM, cell IC50 = 828 nM
Figure 1. Comparison of the new and original pyridopyrimidines.
Table 1. Akt activities of pyridopyrimidines with acyclic amines
R2
N
MeS
N
N
R1
N
O
12
Compound
R1
H
R2
Akt1 IC50 (nM)
226
Akt2 IC50 (nM)
1299
12a
NH2
N
12b
12c
12d
12e
H
H
H
H
765
8923
N
Me
N
N
N
NH
99 13
139
1242 215
1709
NH
N
S
198
1381
12f
H
90 20
1160 313
N
N
N
12g
12h
12i
H
137
1514
17470
1792
NH
N
Me
H
1521
162
Me
NH
N
N
12j
12k
12l
H
H
H
63 26
494 75
N
N
NH
N
N
46
15
2
3
529
1
NH2
S
O
NH2
90 19

Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1274–1279
1277
to inhibit Akt. Changing the chain length between the
amine nitrogen and the heterocycle from two to three also
made little difference (12g). Surprisingly, unlike the cyclic
tertiary piperidyl compound 11, tertiary amine was not
compatible with the acyclic version, leading to a great loss
of potency against both Akt1 and Akt2 (12h). Next we
investigated the effect of the substituent on the heterocy-
cle. While a methyl group had no effect on Akt inhibitory
ability (12i vs. 12d), a phenyl (12j) or an amino group
(12k) enhanced both Akt1 and Akt2 activities 2- to 3-fold.
Finally, we were pleased to find that an anilineketone ter-
minal group (12l) offered satisfactory results (Akt1
IC50 = 15 nM, Akt2 IC50 = 90 nM), with a 10-fold in-
crease in potency over the initial 12a. The compound
12l was also very potent in the cell based assay (Akt1 cell
IC50 = 78 nM, Akt2 cell IC50 = 388 nM).11
provided encouraging results (Akt1 IC50 < 100 nM,
Akt2 IC50 ꢀ 1000 nM). Further effort to explore more
acyclic functional groups (13c and 13d) offered no
improvement. However, when an aromatic heterocycle
was put on the piperidine ring directly (13e) or attached
to the amide nitrogen (13f), significant enhancement of
Akt1 activity was observed (IC50 less than 10 nM).
The Akt2 activity was also increased to about an IC50
of 300 nM for these compounds. Compounds 13e and
13f had good cell potency too. The results from 13e,
13f, and previous leading compounds represented by 1
and 2 indicated the potential to include two aromatic
heterocycles (13g, 13h and 13i). While the pyridylox-
adiazole produced a less potent compound (13g), the
more polar pyridylpyrazole (13h) and pyridyltriazole
(13i) with a hydrogen donor provided compounds with
excellent activity against Akt1 and Akt2 (Akt1
IC50 ꢀ 5 nM, Akt2 IC50 ꢀ 50 nM). These two com-
pounds were also shown to penetrate cells, and it is
worth noting the remarkable Akt1 cell potency of 13i
We also examined the terminal groups in the 4-position
of the piperidine and the results of selected examples are
shown in Table 2. The simple amide (13a) or urea (13b)
Table 2. Akt activities of pyridopyrimidines with 4-substituted piperidines
N
MeS
N
N
R
N
13
Compound
R
Intrinsic IC50 (nM)
Cell IC50 (nM)
Akt2
Akt1
29
Akt2
Akt1
NH2
O
13a
13b
5
4
928 118
560 107
9259
5296
O
81
1909 357
2106 60
5127 459
267 19
795 330
N
H
NHEt
H
N
NH2
Me
13c
13d
13e
13f
13g
13h
13i
91 20
nd
nd
nd
O
O
O
O
S
H
N
286
5
nd
N
H
NH2
8.5 1.5
9.7 0.5
79
1
1572 306
N
N
H
N
301
2
133
nd
1131
O
O
N
N
N
N
64
5
1304 374
nd
N
N
4.5 2.9
57
26
7
5
85 52
635 208
NH
H
N
N
3.8
1
9.3 2.6
589
8
N
N

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Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1274–1279
Table 3. Akt activities of pyridopyrimidines with various 2-substituents
N
R
N
N
N
N
N
HN
N
14
Compound
R
Akt1 IC50 (nM)
Akt2 IC50 (nM)
Cell
Akt1 IC50 (nM)
Akt2 IC50 (nM)
14a
14b
14c
14d
–OMe
–CN
23.6
107.2
42.6
2295
24 1.4
6.2
6.0 0.3
59 22
35
94.4 2.6
100 35
96.4 55
20.3 10.1
586 420
3706 2458
899 202
–CONH2
–NHMe
(Akt1 IPKA IC50 < 10 nM, Akt2 IC50 ꢀ 600 nM). How-
ever, this improvement in Akt1 cell activity did not
translate directly to Akt2 cell activity. The reason for
this disconnection is not well understood.
ary amine as the terminal group displayed promising
potency. Finally, modification of the piperidine and pyr-
idopyrimidine substituents resulted in compounds (13i
and 14d) with excellent potency and greatly improved
caspase-3 activity.
With successful modification of the western terminal
group, we turned our attention to the modification of
2-methylthio group. Several 2-substituents were exam-
ined with the best pyridyltriazole terminal group (Table
3). 2-methoxy (14a), 2-cyano (14b), 2-aminocarbonyl
(14c), and 2-methylamine (14d) all gave satisfactory
results. These substituents make the compounds more
polar with better physical properties, but did not de-
crease the Akt activities significantly. For example, the
methylamine 14d is comparable to 13i regarding to both
the intrinsic and cell potency.
Acknowledgments
The authors thank Dr. Art Coddington, Dr. Charles
Ross, and Dr. Harri Ramjit for mass spectral analyses.
References and notes
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We investigated the selectivity of these compounds for
Akt1 and Akt2 versus Akt3 and other closely related ki-
nases. In general, these compounds maintained the
excellent selectivity profiles of leading compounds 1
and 2. For example, 14d has an IC50 = 2474 nM for
Akt3 and not active against other closely related kinases
such as SGK, PKA, and PKC (IC50 > 50,000 nM).
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Compounds 13i and 14d significantly increased caspase-
3 activity in LnCaP cells treated in combination with
TRAIL (Table 4).12 Compared to compound 2 which
showed a 2-fold increase in caspase-3 at 2 lM, 13i and
14d gave a 3-fold induction at 0.1 lM.
In summary, we have described the development of pyr-
idopyrimidines that are potent and selective Akt1/2 dual
inhibitors. Compound 12l with a simple acyclic second-
Table 4. Fold increase of Caspase-3 activity in LnCaP cells with
TRAILa
Compound
0.1 lM
0.5 lM
1 lM
13i
14d
3.2-fold
3-fold
6.8-fold
6-fold
8-fold
7.8-fold
a Caspase-3 assay: LnCaP cells treated with compound at the given
compound concentration in combination with +/- TRAIL (0.5 lg/
mL) expressed as a fold difference versus TRAIL alone.

Z. Wu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1274–1279
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10. Akt IC50 represents biochemical inhibition of peptide
phosphorylation. Detection was performed by homoge-
neous time resolved fluorescence (HTRF) using an
europium chelate (Perkin-Elmer) [Eu(K)]-labeled phos-
pho(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. 5. Values are
reported as single determinations or as the average of at
least two determinations standard deviation.
11. Cell-based potency of Akt inhibitors was determined
in immunoprecipitation kinase assays (IPKA). IC50
values represent the ability of inhibitors to block the
phosphorylation of Akt isozymes in C33
A cells
(human cervical carcinoma). For detail see Ref. 6.
Values are reported as single determinations or as the
average of at least two determinations standard
deviation.
12. Choi-Sledeski, Y. M.; Kearney, R.; Poli, G.; Pauls, H.;
Gardner, C.; Gong, Y.; Becker, M.; Davis, R.; Spada, A.;
Liang, G.; Chu, V.; Brown, K.; Collussi, D.; Leadley, R.;
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ski, C.; Maignan, S.; Guilloreau, J. P.; Mikol, V. J. Med.
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9. Zhao, Z.; Robinson, R. G.; Barnett, S. F.; Defeo-Jones,
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