Optimization of 2,3,5-trisubstituted pyridine derivatives as potent allosteric Akt1 and Akt2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.12.040

This letter shows inhibitor SAR on a pyridine series of allosteric Akt inhibitors to optimize enzymatic and cellular potency. We have optimized 2,3,5-trisubstituted pyridines to give potent Akt1 and Akt2 inhibitors in both enzyme and cell based assays. In addition, we will also highlight the pharmacokinetic profile of an optimized inhibitor that has low clearance and long half-life in dogs.

Full text of DOI:10.1016/j.bmcl.2007.12.040

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2194–2197
Optimization of 2,3,5-trisubstituted pyridine derivatives
as potent allosteric Akt1 and Akt2 inhibitors
John C. Hartnett,a,* Stanley F. Barnett,b Mark T. Bilodeau,a Deborah Defeo-Jones,b
George D. Hartman,a Hans E. Huber,b Raymond E. Jones,b Astrid M. Kral,b
Ronald G. Robinsonb and Zhicai Wua
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 29 October 2007; revised 14 December 2007; accepted 17 December 2007
Available online 23 December 2007
Abstract—This letter shows inhibitor SAR on a pyridine series of allosteric Akt inhibitors to optimize enzymatic and cellular
potency. We have optimized 2,3,5-trisubstituted pyridines to give potent Akt1 and Akt2 inhibitors in both enzyme and cell based
assays. In addition, we will also highlight the pharmacokinetic profile of an optimized inhibitor that has low clearance and long half-
life in dogs.
Ó 2007 Elsevier Ltd. All rights reserved.
Akt (protein kinase B) is a serine threonine kinase
N
O
Akt1 IC50 = 762 nM
Akt2 IC50 = 3139 nM
Akt3 IC50 > 50,000 nM
Cell Akt1 IC50 > 50,000 nM
Cell Akt2 IC50 > 50,000 nM
belonging to the AGC superfamily of kinases. There
are three isoforms of the enzyme with >80% homology
to one another. Akt is a critical downstream effector in
the PI3K signal transduction pathway. This pathway
is frequently activated in tumors by growth factor over-
expression and mutation in tumor suppressor PTEN
(phosphatase and tensin homologue). Activation of
Akt can lead to cell growth, survival, and tumor pro-
gression.1–3 Thus, the selective inhibition of Akt1 and
Akt2 by small molecule inhibitors could be critical in
the development in novel cancer therapy.
N
N
NH
NC
1
2
N
O
Akt1 IC50 = 273 nM
Akt2 IC50 = 150 nM
Akt3 IC50 > 50, 000 nM
Cell Akt1 IC50 > 50,000 nM
Cell Akt2 IC50 > 50,000 nM
N
N
NH
N
N
NH
N
N
A novel series of potent and selective Akt kinase inhib-
itors based on 2,3,5-trisubstituted pyridine inhibitors
was reported in a recent letter (Fig. 1).4b The optimiza-
tion of molecule 1 was accomplished through library
synthesis. It was discovered that the conversion of the
nitrile moiety in 1 to the tetrazole moiety in 2 led to a
more potent compound. Further optimization of the
compounds containing the tetrazole moiety led to com-
pound 3.
Akt1 IC50 = 210 nM
N
Akt2 IC50 = 105 nM
N
N
Akt3 IC50 > 50, 000 nM
Cell Akt1 IC50 > 50,000 nM
Cell Akt2 IC50 > 50,000 nM
N
N
N
NH2
NH
N
N
3
Figure 1. Structures of Akt1 and Akt2 inhibitors.
There were two important discoveries with the 2,3,5-tri-
substituted pyridines. The first was these molecules dis-
played selectivity over the Akt3 isozyme. The second
was this series displayed a different selectivity profile
than compounds previously reported.4a,c When this ser-
ies was substituted with a heterocycle in the 5 position, it
was more Akt2 potent than Akt1.
Keywords: Inhibitors; Akt1; Akt2; Enzymatic; Cellular.
*
Corresponding author. Tel.: +1 215 652 3035; fax: +1 215 652
7310; e-mail: john_hartnett@merck.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.040

J. C. Hartnett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2194–2197
2195
Two different reaction schemes were developed to vary
both the heterocycle on the western portion of the mol-
ecule in the final step (Scheme 1) as well as the basic
amine moiety on the eastern portion in the final step
(Scheme 2).
-
N+
PF6
d
a.b.c
N
+
Cl
N
O
N
Cl
6
In Scheme 1 the synthesis of the pyridine core is shown.
In this scheme the penultimate step was to vary the basic
amine moiety and then convert the nitrile to a heterocy-
cle in the final step. Condensation of the ketone 4 with
the iminium6 salt 5 afforded the pyridine core 6.7 Using
palladium coupling conditions, the chlorine of 6 was
converted to the nitrile in compound 7. Bromination
of the methyl group in 7 gave bromomethyl 8, which
could be displaced with a variety of amines to synthesize
molecules such as 9. Finally, the nitrile was then con-
verted to a heterocycle in the final step. In this sequence
thiosemicarbazide was used under acidic conditions to
make the aminothiadiazole, 10d.8
5
4
Br
N
e
NC
NC
8
7
N
N
f
N
g
NH
NC
N
9
In a similar sequence (Scheme 2) intermediate 6 was cou-
pled to a variety of heterocycles using a Suzuki reaction.
First, a boronic acid was formed from 6 under palla-
dium coupling conditions in the microwave. Then, in
the same pot, a heteroaryl bromide was added along
with an aqueous base and Pd(PPh3)4 to form molecule
11. With the western heterocycle in place, the methyl
group in 11 was converted to the bromomethyl 12.
The bromide was then displaced by basic amine moieties
to provide molecules 13a–c.
N
N
N
N
NH
S
H2N
N
N
10d
Scheme 1. Reagents and conditions: (a) t-BuOK, THF, 0 °C! rt, 1 h;
(b) 5, THF, 45 °C, 3 h; (c) NH4OAc, THF, 70 °C, 18 h, 36%; (d)
Zn(CN)2, Pd2(dba)3, dppf, Zn, DMA, 150 °C, 20 h, 89%; (e) NBS,
(BnO2)2, CDCl3, 20 h, 70 °C, 93%; (f) 2-(5-piperidin-4-yl-1H-pyrazol-
3-yl)pyridine, DIPEA, 1:1 MeOH THF, rt, 1 h, 57%; (g) thiosemicar-
bazide, TFA, 60 °C, 16 h, 79%.
Early efforts to find a heterocyclic replacement for the
tetrazole in the western portion led to the discovery of
the aminothiadiazole. Like the tetrazole, it could be eas-
ily synthesized in high yield from the nitrile in 9. It was
found to have similar potency to the tetrazole enzymat-
ically, but more importantly it helped to gain cell po-
tency for these 2,3,5-trisubstituted pyridines. In Table
1, the aminothiadiazole moiety was used as a template
on the western portion to vary the basic amine moiety
on the eastern portion.
Br
N
a
N
N
b
Cl
Het
Het
6
11
12
Table 1 shows how various different functionalities were
substituted and the molecules still retained potency both
enzymatically and in cells. In compounds 10a–b, 2-pyr-
idine and 4-pyridine substitution displayed similar activ-
ity to one another. In molecule 10c, the pyrazole was
substituted with a triazole and potency was maintained.
Also the C-linked pyrazoles were replaced with N-linked
pyrazoles 10d–e. Although some of the potency in cells
was lost, the compounds retained their enzymatic po-
tency. Changing the structure from biaryl to bicyclic re-
sulted in the discovery of 10f and 10g. 10g contains a
very similar eastern amine portion to 2. Although 10g
lacks the potency in cells that the others in the table ex-
hibit, it had far better cell potency than 2. On the other
hand, 10f (having a similar eastern portion to 3) showed
similar potency to the other molecules in the table both
enzymatically and in cells, but more importantly, it also
showed much better potency in cells than 3. Finally, we
replaced the tertiary amines with a secondary amine.
This molecule, 10h, retained the desired enzymatic and
cellular activity of all the molecules in this table.
N
N
c
N
N
Het = Heteroaryl
Het
N
NH2
N
13a-c
Scheme 2. Reagents and conditions: (a) bis(pinacolato)diboron,
KOAc, Pd2(dba)3, P(Cy)3, 160 °C, 1 h microwave, then Het-Br, 2 M
aq Na2CO3, Pd(PPh3)4, 150 °C, 0.5 h microwave, 59–69%; (b) NBS,
(BnO2)2, CDCl3, 68 °C, 20 h, 100%; (c) 1-piperidin-4-yl-1H-pyrazol-
o[3,4-d]pyrimidin-4-amine, DIPEA, 1:1 MeOH THF, rt, 1 h, 35–100%.
Although these leads exhibited excellent potency against
the Akt1 and Akt2 isozymes, they lacked activity in
cells.5 To improve the activity of this series, the overall
physical properties of these molecules needed to be im-
proved. This was achieved by replacing the acidic tetra-
zole moiety with non-acidic heterocycles and by
optimizing the eastern basic amine moiety.

2196
J. C. Hartnett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2194–2197
Table 1. Structures and Akt1/Akt2 enzymatic and cellular inhibition
of aminothiadiazolopyridines 10a–h
Table 2. Structures and Akt1/Akt2 enzymatic and cellular inhibition
of heterocycle replacements 13a–e
R2
N
N
N
R1
N
N
N
Het
S
N
H2N
NH2
N
N
N
Compound NR1R2
AKT1/2 enzymatic and cell inha
Compound
Het
Akt1/2 enyzmatic and cell inha
Akt1 IC50 = 1629 nM
Akt2 IC50 = 370 nM
N
N
13a
NH
Cell Akt1 IC50 = 10,000 nM (1)
Cell Akt2 IC50 = 8110 nM (1)
N
Akt1 IC50 = 123 nM
Akt2 IC50 = 38 nM
10a
Akt1 IC50 = 264 nM
Akt2 IC50 = 64 nM
Cell Akt1 IC50 = 407 nM
Cell Akt2 IC50 = 153 nM
N
S
13b
13c
13d
13e
Cell Akt1 IC50 = 1650 nM
Cell Akt2 IC50 = 3239 nM
N
N
N
N
NH
Akt1 IC50 = 95 nM
Akt2 IC50 = 28 nM
Akt1 IC50 = 306 nM
10b
N
N
Akt2 IC50 = 62 nM
Cell Akt1 IC50 = 1954 nM
Cell Akt2 IC50 = 3459 nM
Cell Akt1 IC50 = 734 nM
Cell Akt2 IC50 = 300 nM
N
N
N
Akt1 IC50 = 180 nM
NH
Akt2 IC50 = 121 nM
Cell Akt1 IC50 = 288 nM (1)
Cell Akt2 IC50 = 1061 nM (1)
N
Akt1 IC50 = 85 nM
Akt2 IC50 = 22 nM
N
N
O
10c
N
Cell Akt1 IC50 = 516 nM
Cell Akt2 IC50 = 330 nM
Akt1 IC50 = 182 nM
Akt2 IC50 = 101 nM
N
N
HN
Cell Akt1 IC50 = 890 nM (1)
Cell Akt2 IC50 = 666 nM (1)
N
N
N
Akt1 IC50 = 214 nM
Akt2 IC50 = 90 nM
a Average of at least two measurements. For determinations of n = 1
number is shown in parentheses. Enzyme and Cell protocol.5 All
compounds >50,000 nM versus SGK except for 13a where no data
are available.
10d
N
N
Cell Akt1 IC50 = 1352 nM (1)
Cell Akt2 IC50 = 1349 nM (1)
N
N
Akt1 IC50 = 172 nM
Akt2 IC50 = 51 nM
Table 3. Pharmacokinetic data for 10fa
10e
Cell Akt1 IC50 = 1378 nM
Cell Akt2 IC50 = 2028 nM
N
PK parameter
Dog
t1/2 (h)
Cl (ml/min/kg)
Vdss (L/kg)
3.62
1.37
0.41
N
N
N
Akt1 IC50 = 126 nM
Akt2 IC50 = 22 nM
a Compound dosed iv as a solution in DMSO at a concentration of
0.25 mpk iv.
10f
N
NH2
Cell Akt1 IC50 = 621 nM
Cell Akt2 IC50 = 416 nM
N
western heterocyclic portion of the molecule needed to
be done. Since 10f showed our desired enzymatic and
cell activity, its eastern portion was used as a template
to examine other heterocycles in the western portion.
These molecules are shown in Table 2. Using the Suzuki
chemistry developed in Scheme 2, compounds 13a–c
were synthesized. Although these leads maintained enzy-
matic activity, they lost the cell potency of the aminothi-
adiazole. An oxadiazole, 13d, and a triazole, 13e, were
also synthesized. Although these molecules showed bet-
ter cell activity than 13a–c, they were not superior to the
aminothiadiazole 10f.
N
O
N
NH
Akt1 IC50 = 434 nM
Akt2 IC50 = 73 nM
10g
10h
N
Cell Akt1 IC50 = 12,650 nM
Cell Akt2 IC50 = 3957 nM
N
H
NH2
Akt1 IC50 = 114 nM
Akt2 IC50 = 30 nM
O
Cell Akt1 IC50 = 618 nM
Cell Akt2 IC50 = 472 nM
a Average of at least two measurements. For determinations of n = 1
number is shown in parentheses. Enzyme and Cell protocol.5 All
compounds >50,000 nM versus SGK except for 10h where no data
are available.
Due to our desired potency enzymatically and in cells,
compound 10f was chosen for further characterization.
First, this compound showed selectivity against a variety
of other kinases. It was >10,000 nm versus PKA, PKC,
and PKCa. Next, this compound displayed good cas-
Although the aminothiadiazole showed the desired
activity from the start, a thorough SAR study of the
pase
3
induction activity (ꢀ6-fold induction at

J. C. Hartnett et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2194–2197
2197
2000 nM).9 In addition, 10f showed a desired pharmaco-
kinetic profile in dog (Table 3).
Friebe, W.; Thomas, U.; Huber, R.; Engh, R. A.; Masjost,
B. J. Med. Chem. 2004, 47, 1375.
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.
In summary, the search to find compounds that exhib-
ited better cell potency than compounds 1–3 led us to
many heterocyclic replacements with significantly im-
proved cell potency over the acidic tetrazole. The eastern
basic amine moiety was also optimized, and this led us
to our most optimal molecule 10f which showed a de-
sired pharmacokinetic profile in dogs.
4. (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) Wu, Z.; Bilodeau, M. T.; Hartman, G. D.;
Robinson, R. G.; Fu, S.; Barnett, S. F.; Defeo-Jones, D.;
Jones, R. E.; Kral, A. M.; Huber, H. E.; Kohl, N. E.
Bioorg. Med. Chem. Lett., in press.
5. For Akt enzyme assay details and the proposed model for
the allosteric mode of inhibition of these inhibitors, see: (a)
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; For details on the Akt IPKA cell assay and
the caspase-3 assay see: (b) Defeo-Jones, D.; Barnett, S. F.;
Fu, S.; Hancock, P. J.; Haskell, K. M.; Leander, K. R.; M
Avoy, E.; Robinson, R. G.; Duggan, M. E.; Lindsley, C.
W.; Zhao, Z.; Huber, H. E.; Jones, R. E. Mol. Cancer Ther.
2005, 4, 271.
6. Compound 8 obtained from the Merck internal sample
collection.
7. Marcoux, J.-F.; Marcotte, F.-A.; Wu, J.; Dormer, P. G.;
Davies, I. W.; Hughes, D.; Reider, P. J. J. Org. Chem. 2001,
66, 4194.
8. Ishikawa, T. et al. Bioorg. Med. Chem. Lett. 2003, 11, 2427.
9. 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 effector caspase 3 in compound-treated and
untreated cells values are reported as the fold induction.
For details, see Ref. 5.
Acknowledgments
The authors thank Dr. Art Coddington, Dr. Charles W.
Ross III, and Harri Ramjit for obtaining HRMS data
(accurate mass measurements).
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.
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.
Acad. Sci. USA 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. USA 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.;