Discovery of 2,3,5-trisubstituted pyridine derivatives as potent Akt1 and Akt2 dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.12.062

This letter describes the discovery of a novel series of dual Akt1/Akt2 kinase inhibitors, based on a 2,3,5-trisubstituted pyridine scaffold. Compounds from this series, which contain a 5-tetrazolyl moiety, exhibit more potent inhibition of Akt2 than Akt1.

Full text of DOI:10.1016/j.bmcl.2004.12.062

Bioorganic & Medicinal Chemistry Letters 15 (2005) 905–909
Discovery of 2,3,5-trisubstituted pyridine derivatives as potent
Akt1 and Akt2 dual inhibitors
Zhijian Zhao,a,* William H. Leister,a Ronald G. Robinson,b Stanley F. Barnett,b
Deborah Defeo-Jones,b Raymond E. Jones,b George D. Hartman,a Joel R. Huff,a
Hans E. Huber,b Mark E. Duggana and Craig W. Lindsleya
aDepartment of Medicinal Chemistry, Technology Enabled Synthesis Group, 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 22 October 2004; revised 16 December 2004; accepted 21 December 2004
Available online 19 January 2005
Abstract—This letter describes the discovery of a novel series of dual Akt1/Akt2 kinase inhibitors, based on a 2,3,5-trisubstituted
pyridine scaffold. Compounds from this series, which contain a 5-tetrazolyl moiety, exhibit more potent inhibition of Akt2 than
Akt1.
Ó 2005 Elsevier Ltd. All rights reserved.
N
The serine/threonine kinase Akt (PKB) phosphorylates
an ever increasing list of downstream substrates that
promote cell survival, growth and block pro-apoptotic
signals.1,2 Numerous studies have shown that dysregula-
tion of Akt is a major contributor to tumorigenesis and
a potential target for cancer therapy.3 However, the
development of inhibitors of Akt as small molecule thera-
peutics for the treatment of cancer has been hindered by
a lack of Akt specific inhibitors (versus the AGC family
of kinases) and isozyme selective (Akt1, Akt2, and
Akt3) Akt inhibitors due to high sequence identity
homology.1–3
N
O
O
N
N
N
N
N
N
N
NH
NH
N
H
1
3
2
4
N
N
O
O
H
N
O
H3C
O
N
N
N
NH
NH
N
N
H
Figure 1. Structures of Akt1 and Akt2 inhibitors.
In a recent letter, we reported a novel series of potent
and selective allosteric Akt kinase inhibitors based on
a 2,3-diphenylquinoxaline core 1 (Fig. 1).4 An optimized
dual Akt1/Akt2 inhibitor 2 (Fig. 1) was developed
through an iterative analog library synthesis approach
that was shown to sensitize tumor cells to apoptotic
stimuli and inhibit the phosphorylation of both Akt1
and Akt2 in vitro (Table 1). Importantly, these Akt
inhibitors displayed selectivity versus the closely related
AGC family (PKA, PKC, SGK) of kinases as well as
selectivity with respect to the individual Akt isozymes.
Table 1. Akt activities of 1–4
Compd Akt1 IC50 (nM)a Akt2 IC50 (nM)a Akt3 IC50 (nM)a
1
2
3
4
295
58
2057
210
>50,000
2200
760
>20,000
>20,000
325
>20,000
>20,000
All compounds >50,000 nM versus PKA, PKC, SGK.
a Average of at least three measurements; enzyme protocol.5
The observed selectivity has been attributed to an allo-
steric mode of binding, noncompetitive with ATP, where-
in Akt inhibition is dependent on the presence of the
pleckstrin homology (PH) domain.5 In the same letter,
Keywords: Pyridine; Tetrazole; Akt; Inhibitor; Kinase.
*
Corresponding author. Tel.: +1 215 652 5892; fax: +1 215 652 6345;
e-mail: zhijian_zhao@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.062

906
Z.Zhao et al./ Bioorg.Med.Chem.Lett.15 (2005) 905–909
we also reported on the discovery of a series of 5,6-di-
phenyl-pyrazin-2(1H)-one derivatives that, selectively
inhibited either Akt1 or Akt2 (3 and 4, respectively,
Table 1). These isozyme selective inhibitors demon-
strated, in caspase-3 assays, that inhibition of both
Akt1 and Akt2 were required for a maximal apoptotic
response.4
A similar sequence was applied to advanced intermedi-
ate 137 to deliver quinoline 6 (Scheme 2). As summa-
rized in Table 2, the in vitro Akt1 inhibition activity
of quinoline 5 (IC50 Akt1 = 365 nM) is almost equal to
that of the quinoxaline 1 (Table 1, IC50 Akt1 = 295 nM).
The Akt2 inhibition by 5 (Table 2, IC50 Akt2 =
1205 nM) is even more potent than 1 (Table 1, IC50
Akt2 = 2057 nM). Interestingly, regioisomer 5 is 8-fold
more potent than the regioisomeric quinoline 6 (Table
2, IC50 Akt1 = 3136 nM) for Akt1 inhibition, and this
trend is even more pronounced with respect to Akt2
inhibition, wherein 5 is 30 times more potent than 6
(Table 2, IC50 Akt2 = 33,660 nM). The large difference
in the activities of regioisomers 5 and 6 clearly indicates
the importance of the nitrogen position in the quinoline
core. Importantly, neither quinoline 5 nor 6 displayed
inhibition against Akt3, PKA, PKC or SGK, or the del-
ta-PH Akt mutants, suggesting the allosteric mode of
inhibition observed with 1–4 was maintained.
Despite this notable advance, 1–4 possessed poor phys-
ical properties which translated into poor cellular po-
tency. In order to conduct additional in vivo studies,
dual Akt1/Akt2 inhibitors with improved physical prop-
erties and cellular potency were required. In this letter,
we disclose results of our efforts directed toward the
development of Akt inhibitors with improved physical
properties and the discovery of a series of dual Akt1/
Akt2 inhibitors based on a 2,3,5-trisubstituted pyridine
scaffold.
One way to improve the physical properties of the 2,3-
diphenylquinoxaline series 1 is to incorporate a more ba-
sic nitrogen into the core ring system, that is, convert the
quinoxaline core to a quinoline core. Based on this idea,
the two quinoline regioisomers 5 and 6 (Fig. 2) were pre-
pared wherein either the N4 or N1 nitrogen of 1, respec-
tively, was replaced with carbon. The synthesis of 5 is
shown in Scheme 1.
The dual Akt1/Akt2 inhibition observed with quinoline
5 prompted us to further increase the basicity of the core
heterocyclic template. In an effort to both reduce mole-
cular weight and provide a basic nitrogen, capable of
salt formation for solubility studies, the quinoline core
was truncated to provide a pyridine template. Based
on the SAR generated within 1–4, our initial design
placed a cyano group in the 5-position to provide a
handle for further manipulation.
Microwave-assisted Suzuki coupling6 of iodide 97 gener-
ated 10, which was converted into triflate 11. Then, a
second microwave-assisted Suzuki coupling, employing
4-formylphenyl boronic acid, followed by a polymer-
supported reductive amination sequence8 afforded quino-
line 5.
As shown in Scheme 3, applying the same chemistry em-
ployed for the synthesis of quinoline 5, a library of
cyanopyridine derivatives 7 (32 compounds total, 7a–g
shown in Table 3) was prepared from 16.7 It should
be mentioned that the use of polymer-supported
N
N
O
O
N
OH
OTf
N
N
a
b
NH
NH
N
N
N
13
5
6
14
Figure 2. Structures of quinolines 5 and 6.
CHO
N
O
c
N
NH
N
N
H
H
N
O
N
OTf
N
O
I
a
b
15
6
11
9
10
Scheme 2. Reagents and conditions: (a) (CF3SO2)2O, pyridine, 0 °C–
rt, overnight, 90%; (c) (4-formylphenyl)boronic acid, Pd(dppf)Cl2,
Cs2CO3, THF–H2O, microwave 150 °C, 10 min, 92%; (c) 1-piperidin-
4-yl-1,3-dihydro-2H-benzimidazol-2-one, MP-BH3(CN), 94%.
CHO
N
O
N
N
c
c
N
NH
12
5
Table 2. Activities of quinolines 5 and 6
Compd Akt1 IC50 (nM)a Akt2 IC50 (nM)a Akt3 IC50 (nM)a
Scheme 1. Reagents and conditions: (a) PhB(OH)2, Pd(dppf)Cl2,
Cs2CO3, THF–H2O, microwave 150 °C, 10 min, 91%; (b)
(CF3SO2)2O, pyridine, 0 °C–rt, overnight, 93%; (c) (4-formylphen-
yl)boronic acid, Pd(dppf)Cl2, Cs2CO3, THF–H2 O, microwave 150 °C,
10 min, 87%; (d) 1-piperidin-4-yl-1,3-dihydro-2H-benzimidazol-2-one,
MP-BH3(CN), 95%.
5
6
365
3136
1205
33,660
>50,000
>50,000
Both compounds >50,000 nM versus PKA, PKC, SGK.
a Average of at least three measurements; enzyme protocol.5

Z.Zhao et al./ Bioorg.Med.Chem.Lett.15 (2005) 905–909
907
CHO
To our delight, the truncated cyanopyridine core deriva-
tives maintain the Akt1/Akt2 inhibitory activities.
Comparison of the cyanopyridine 7a (Table 3, IC50
Akt1 = 762 nM, Akt2 = 3139 nM) with the analogous
N
OH
N
OTf
N
a
b
NC
NC
NC
2,3-diphenylquinoxaline
1 (Table 1, IC50 Akt1 =
16
18
17
295 nM, Akt2 = 2057 nM) and 2,3-diphenylquinoline 5
(Table 2, IC50 Akt1 = 365 nM, Akt2 = 1205 nM), shows
a maintenance of potency and selectivity. Moreover, the
tetrazolylpyridine 8a (Table 4, IC50 Akt1 = 273 nM,
Akt2 = 157 nM) is a more potent inhibitor of both
Akt1 and Akt2 as compared to 1 and 5. Remarkably,
the Akt2 inhibition by tetrazolylpyridine 8a is 14 and
8 times more potent than that of 1 and 5, respectively.
When comparing the profiles of Akt1/Akt2 inhibition
in Tables 3 and 4, it is interesting that the conversion
of the cyano group to a tetrazole ring enhances Akt2
activity 6–24 times and Akt1 activity 1–7 times. It
should be noted that with the exception of 8b and 8c,
all other tetrazolylpyridine derivatives in Table 4 are
more potent inhibitors of Akt2 than Akt1. This is in
contrast to our previous observations in the 2,3-diphen-
ylquinoxalines, quinolines, and cyanopyridines wherein
inhibition of Akt1 was more pronounced than Akt2.
As with our other series of Akt inhibitors 1–6, 7a–g
and 8a–g displayed no inhibition of Akt3, PKA, PKC
or SGK, or the delta-PH Akt mutants, suggesting the
R1
R1
N
R2
N
R2
N
N
c
d
N
NC
N
NH
N
7a-h
8a-h
Scheme 3. Reagents and conditions: (a) (CF3SO2)2O, pyridine, 0 °C–
rt, overnight, 91%; (b) (4-formylphenyl)boronic acid, Pd(dppf)Cl2,
Cs2CO3, THF–H2O, microwave 150 °C, 10 min, 87%; (c) amine, MP-
BH3(CN), 82–95%; (d) NaN3, ZnBr2, H2O, microwave 185 °C, 20 min,
75–91%.
cyanoborohydride reductive amination sequence greatly
enhanced the parallel synthesis of the cyanopyridine li-
brary 7. Utilizing a microwave enhanced [3 + 2] cycload-
dition of nitrile with NaN3 under Sharpless conditions,9
the cyanopyridine library 7 was rapidly converted to an-
other tetrazolylpyridine library 8 (32 compounds total,
8a–g shown in Table 4).
Table 3. Structures and Akt1/Akt2 inhibition of cyanopyridines 7
R2
N
R1
N
NC
7
Compd
NR1R2
Akt1 IC50 (nM)a
Akt2 IC50 (nM)a
3139
Akt3 IC50 (nM)a
>50,000
N
O
7a
762
N
NH
HN
N
N
7b
7c
1288
865
4811
6650
>50,000
>50,000
N
S
H
N
N
N
F
N
H
N
7d
2874
11,410
>50,000
Me
N
N
H
N
7e
7f
1471
4397
8740
>50,000
>50,000
N
N
N
N
13,000
N
N
7g
7407
18,360
>50,000
All compounds >50,000 nM versus PKA, PKC, SGK.
a Average of at least three measurements; enzyme protocol.5

908
Z.Zhao et al./ Bioorg.Med.Chem.Lett.15 (2005) 905–909
Table 4. Structures and Akt1/Akt2 inhibition of tetrazolylypyridines 8
R2
N
N
R1
N
N
N
NH
8
Compd
NR1R2
Akt1 IC50 (nM)a
Akt2 IC50 (nM)a
Akt3 IC50 (nM)a
>50,000
N
O
8a
273
157
N
NH
HN
N
N
8b
8c
177
239
248
>50,000
>50,000
N
S
H
N
1720
N
N
F
N
H
N
8d
8e
8f
763
422
>50,000
>50,000
>50,000
Me
N
N
H
N
3323
4237
1501
1956
N
N
N
N
N
N
8g
6915
2746
>50,000
All compounds >50,000 nM versus PKA, PKC, SGK.
a Average of at least three measurements; enzyme protocol.5
allosteric mode of inhibition observed with 1–4 was once
again maintained.
In conclusion, we have discovered a novel series of po-
tent, allosteric Akt (PKB) kinase inhibitors based on a
2,3,5-trisubstituted pyridine core with improved aque-
ous solubility, cell permeability and reduced molecular
weight relative to our previously reported inhibitors.
Further refinements to the pyridine core and in vivo
experiments with pyridine analogs are in progress and
will be reported in due course.
Importantly, both the cyanopyridine analogs 7a–g and
the tetrazolylpyridine analogs 8a–g, displayed excellent
aqueous solubility at pH 4.5; however, while 7a–g were
cell permeable, 8a–g were not cell permeable possibly
due to zwitterionic character and afforded no inhibition
of the Akt isozymes in our cell-based IP assay.10 To fur-
ther test this theory, 8a was alkylated with methyl iodide
to provide 19, thereby removing any zwitterionic char-
acter (Scheme 4). This modification did improve cell per-
meability, and 19 was active in the cell-based IP assay;
however, inhibition of Akt1 was diminished 8-fold
(Akt1 IC50 = 1772 nM) while inhibition of Akt2 was re-
duced by only 3-fold (Akt2 IC50 = 613 nM).
Acknowledgments
The authors wish to thank Dr. Sandor Varga for exten-
sive NMR work to confirm the structural assignments of
the quinoline and pyridine derivatives, and Dr. Charles
W. Ross III for obtaining HRMS data (accurate mass
measurements).
N
O
N
N
NH
a
8a
N
N
References and notes
N
N
CH3
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Opin.Ther.Targets 2002, 6, 103; (b) Nicholson, K. M.;
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Zhu, G.-D. Curr.Top.Med.Chem. 2002, 2, 939.
19
Scheme 4. Reagents and conditions: (a) MeI, MP-CO32À, CH2Cl2,
0 °C, 48%.

Z.Zhao et al./ Bioorg.Med.Chem.Lett.15 (2005) 905–909
909
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the allosteric mode of inhibition of these inhibitors, see:
Barnett, S. F.; Defeo-Jones, D.; Fu, S.; Hancock, P. J.;
Haskell, K. M.; Jones, R. E.; Kahana, J. A.; Kral, A.;
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in press.
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10. Similar behavior was observed with a carboxylate in the 5-
position, which was alleviated when the corresponding
methyl ester analog was tested, providing further evidence
of zwitterionic character affording poor cell permeability
in this series.