Development of potent, allosteric dual Akt1 and Akt2 inhibitors with improved physical properties and cell activity

Article abstract of DOI:10.1016/j.bmcl.2007.11.015

This letter describes the development of potent, allosteric dual Akt1 and Akt2 inhibitors with improved aqueous solubility (～18 mg/mL) that translates into enhanced cell activity and caspase-3 induction.

Full text of DOI:10.1016/j.bmcl.2007.11.015

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 49–53
Development of potent, allosteric dual Akt1 and Akt2
inhibitors with improved physical properties and cell activity
Zhijian Zhao,a,* Ronald G. Robinson,b Stanley F. Barnett,b Deborah Defeo-Jones,b
Raymond E. Jones,b George D. Hartman,a Hans E. Huber,b
Mark E. Duggana and Craig W. Lindsleya,c
aDepartment of Medicinal Chemistry, Technology Enabled Synthesis Group, 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
cVICB Program in Drug Discovery, Department of Pharmacology, Vanderbilt Medical Center, Nashville, TN 37232, USA
Received 4 September 2007; revised 2 November 2007; accepted 7 November 2007
Available online 13 November 2007
Abstract—This letter describes the development of potent, allosteric dual Akt1 and Akt2 inhibitors with improved aqueous solubil-
ity (ꢀ18 mg/mL) that translates into enhanced cell activity and caspase-3 induction.
Ó 2007 Elsevier Ltd. All rights reserved.
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 PI3K/Akt is a major contributor to tumorigene-
sis and a promising target for cancer therapy.3 However,
the development of inhibitors of Akt as small molecule
therapeutics for the treatment of cancer has been hin-
dered 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
homology.1–3
ical properties which afforded only modest activity in
cell-based IPKA Akt assays (Akt1 IC50 = 305 nM,
Akt2 IC50 = 2100 nM, Akt3 IC50 = >10,000 nM).6 These
issues limited the utility of 1 for further in vivo experi-
ments. In this letter, we describe the optimization of 1
to provide potent allosteric Akt1 and Akt2 dual inhibi-
tors with improved solubility, physical properties, and
cellular activity.
Early work in this series indicated that substitution in
the C6 or C7 positions was tolerated and provided im-
proved potency; however, the regioisomeric quinoxa-
lines 2 displayed distinct and variable inhibition
against the individual Akt isozymes (Table 1).4a For in-
stance, 2a and 2b displayed balanced inhibition of Akt1
and Akt2, but 2a did not inhibit Akt3 whereas 2b did.
Despite significant increases in intrinsic potency with
2c and 2d, all of these analogs were not cell permeable
likely due to zwitterionic character.4 As a result, we fo-
Recent reports from our laboratories disclosed a series of
novel, allosteric Akt kinase inhibitors that displayed high
levels of Akt isozyme selectivity and PH-domain depen-
dent inhibition.4 Significantly, these allosteric inhibitors
were postulated to lock Akt into a closed conformation
which not only inhibited the activity of the kinase, but
also prevented the activation (phosphorylation) of
Akt.4,5 This was observed both in vitro and in vivo with
the dual Akt1 and Akt2 inhibitor 1 (Akt1 IC50 = 58 nM,
Akt2 IC50 = 210 nM, Akt3 IC50 = 2200 nM), thereby
representing a fundamentally new mechanism of kinase
inhibition (Fig. 1).4,5 Despite this notable advance, 1 pos-
sessed limited solubility in most vehicles and poor phys-
N
O
N
N
N
N
NH
N
H
1
Keywords: Kinases; Akt; Inhibitors; Cancer.
*
Figure 1. Allosteric dual Akt1 and Akt2 inhibitor 1.
Corresponding author. E-mail: zhijian_zhao@merck.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.015

50
Z. Zhao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 49–53
Table 1. Structures and activities for quinoxalines 2
tions and the same allosteric mechanism of Akt inhibi-
tion as our earlier series.4,6 In general, cyclic diamines,
such as 5a, were selective for Akt2 but provided only
modest inhibition. Aliphatic diamines afforded more bal-
anced inhibition of both Akt 1 and Akt2; however, the
nature of the diamine had dramatic effects on the degree
of individual Akt isozyme inhibition. For instance, 5c
and 5d show a 2- to 3-fold preferential inhibition of
Akt2 over Akt1, a pattern of inhibition previously ob-
served only in our pyridine series of allosteric Akt inhib-
itors.4b Interestingly, the addition of a single methyl
group to 5e (IC50s: Akt1 = 5028 nM, Akt2 = 4182 nM,
and Akt3 > 10,000 nM) affords 5f, a submicromolar
inhibitor (IC50s: Akt1 = 715 nM, Akt2 = 816 nM, and
Akt3 = 5200 nM). Despite similar in vitro potency and
uniformly improved aqueous solubility (>10 mg/mL), a
number of analogs of 5 showed a significant potency shift
in cells. With respect to cell potency, compound 5i stood
out with a minimal potency shift in our cell-based IPKA
assay (IC50s: Akt1 = 526 nM, Akt2 = 730 nM, and
Akt3 > 10,000 nM).
N
O
7
6
N
N
N
NH
R3
2
Compound
R3
Akt1
a
IC50
(nM)
Akt2
a
IC50
(nM)
Akt3
a
IC50
(nM)
2a
2b
2c
2d
6-COOH
7-COOH
240
166
63
281
388
65
>50,000
3200
1228
6-(2H-Tetrazole)
7-(2H-Tetrazole)
20
144
1613
a Average of at least three measurements; enzyme protocol5; all com-
pounds >50,000 nM versus PKA, PKC, SGK.
cused our attention on increasing the aqueous solubility
and cell penetration of 2 by other means, in particular,
through the incorporation of amine-containing substitu-
ents in the C6/C7 positions.
Prior to further evaluation, 5i was separated into pure
C6 and C7 regioisomers, 6 and 7, respectively (Fig. 2).
The C7 regioisomer 7 proved to be more potent in an
enzymatic as well as in the cell-based IPKA assay than
the C6 regioisomer 6 as displayed in Figure 2. Nota-
bly, for 7, this was the first case in which there was
such a negligible shift between the in vitro IC50s and
those determined in the cell-based IPKA (LNCAP
cells) assay in our program. In addition, both 6 and
7 were dependent on the PH-domain for Akt inhibi-
tion, non-competitive with ATP and selective versus
the AGC family of kinases (>50 lM vs PKA, PKC,
SGK).
As shown in Scheme 1, our initial strategy focused on a
microwave-assisted condensation of benzil 3 with 3,4-
diaminobenzoic acid 4 to deliver regioisomeric quinoxa-
lines 2a/2b.4,7 The isomers could be separated by HPLC;
however, initial libraries employed a 1:1 mixture of 2a/2b
to afford, after amide coupling protocols, 5. As shown in
Table 2, this 48-membered library provided compounds
that maintained potency, variable Akt isozyme inhibi-
NH2
Based on the improvements in solubility and cell activity
with the amine-tethered quinoxalines 5, we synthesized
additional libraries that incorporated other polar sub-
stituents in place of basic amines. Following the same
basic synthetic route outlined in Scheme 1, a number
of potent compounds (represented by a general structure
8) resulted from this endeavor (Table 3). While in vitro
potency, physical properties, and aqueous solubility re-
mained favorable in 8a–8f, these analogs suffered signif-
icant 10- to 20-fold potency shifts in the cell-based
IPKA assay affording micromolar levels of inhibition,
which prevented further in vivo experiments.
N
O
O
O
HO2C
NH2
N
NH
4
(a)
3
R1
N
H
N
O
N
N
R2
7
6
N
NH
HOOC
(b)
Because of its minimal potency shift between in vitro
and cell-based IPKA assay, 7 was selected for further
evaluation. By way of comparison, our earlier proof of
concept (POC) molecule 1 was 10-fold less potent on
Akt2 in the IPKA assay than 7, had poor solubility in
most vehicles, and had no measurable aqueous solubil-
ity.4,6 Despite a molecular weight of 654, 7 possessed
good physical properties. As the di-HCl salt, 7 was sol-
uble (ꢀ18 mg/mL) in 98% saline. Based on these data, 7
was evaluated in caspase-3 assays as a surrogate for
apoptosis induction. Similar to 1, 7 significantly in-
creased caspase-3 activity (6- to 10-fold) with a variety
of chemotherapeutic agents (herceptin, camptothecin,
doxorubicin, and TRAIL) and against a number of tu-
2a/2b
R1
N
N
O
R2
N
N
7
6
N
NH
O
5
Scheme 1. Synthesis of amine-tethered quinoxalines 5. Reagents and
conditions: (a) EtOH/HOAc (9:1), 160 °C, 10 min, microwave, 87%;
(b) i—PS-DCC, HOBt, DCM:DIEA (9:1); ii—MP–CO23À, 75–95%. All
compounds purified by mass-guided HPLC.7

Z. Zhao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 49–53
Table 2. Structures and activities for quinoxalines 5
51
R1
N
O
7
6
N
N
N
R2
N
NH
O
5
Compound
Akt1
a
IC50
(nM)
Akt2
a
IC50
(nM)
Akt3
a
IC50
(nM)
Solubility
in 98%
salineb
(mg/mL)
R1
N
R2
MeN
O
5a
5b
5c
5d
5e
5f
>10,000
461
805
910
92
>10,000
7200
10
N
8
N
N
H
2
N
173
5400
n.d.
10
N
N
H
2
H
720
225
4182
816
423
3097
390
2411
2040
6400
Me2N
N
H
N
5028
715
>10,000
5200
n.d.
n.d.
9
N
H
H
N
N
H
5g
5h
5i
521
6800
N
N
H
Me2N
N
6381
560
>10,000
7800
n.d.
18
Et2N
N
H
N
N
5j
4391
8494
n.d.
n.d.
n.d.
N
N
5k
n.d.
N
N
a Average of at least three measurements; enzyme protocol5; all compounds assayed as a 1:1 C6:C7 mixture; all compounds >50,000 nM versus PKA,
PKC, SGK.
b Solubilities were determined at pH 7.4; n.d., not determined.
mor cell lines (LnCaP, HT29, MCF7, and A2780). As
shown in Figure 3, 7 afforded a similar level of induction
of caspase-3 activity as 1, but at a 3-fold lower dose
(4 lM for 7 vs 12 lM for 1) reflecting the improved
aqueous solubility and cell activity despite roughly
equivalent in vitro enzyme inhibition profiles.
effects in rodents which precluded further in vivo exper-
iments from being conducted.
In summary, we have developed novel, allosteric Akt1
and Akt2 dual kinase inhibitors with improved aqueous
solubility (ꢀ18 mg/mL) and balanced in vitro and cell
IPKA activity that translate directly into significant
improvements in caspase-3 activation (apoptosis).
Importantly, the degree and selectivity of inhibition of
Akt1 and Akt2 is dependent on both the nature of the
C6/C7 substituent and the regiochemistry. Further eval-
uation of this class of allosteric inhibitors and structural
Encouraged by the profile of 7, a mouse PD study was
undertaken to see if 7 could potentially allow for a 24-
hour tolerability (Akt1 and Akt2 knockdown) study.4,6
As with 1, 7 inhibited Akt1 and Akt2 phosphorylation
in vivo. However, 7 also induced significant behavioral

52
Z. Zhao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 49–53
Table 3. Structures and activities for quinoxalines 8
6000
5000
4000
3000
2000
1000
0
(--) )TTrarialil
( +(+)Trail(0.5ug/ml)
6.6 fold increase
R3
N
O
7
R4
N
N
N
NH
O
N
6
3 fold increase
8
Compound
Akt1 Akt2 Akt3
a
R3
a
a
IC50
(nM) (nM) (nM)
IC50
IC50
N
R4
H
8a
8b
8c
8d
8e
8f
624
460
185
150
231
398
316
254
4500
7200
4600
7400
HO
N
Control
7 (2μM)
7 (4μM)
Figure 3. Caspase-3 assay: LnCaP cells treated with 7 alone or in
combination with TRAIL. Each experiment employed either 2 lM or
4 lM total dose of 7.
OH
H
N
Ph
HO
N
H
3
Acknowledgments
H2NO2S
The authors wish to thank Dr. Sandor Varga and Dr.
Steven Pitzenberger for extensive NMR work to confirm
the structural assignments of the regioisomeric quinoxa-
line analogs, and Dr. Charles W. Ross III for obtaining
HRMS data (accurate mass measurements).
NH
H
N
8053 1268 >10,000
HO
O
H
N
574
651
5200
References and notes
a Average of at least three rneasurements; enzyme protocol5; all com-
pounds assayed as a 1:1 C6:C7 mixture; all compounds >50,000 nM
versus PKA, PKC, SGK.
1. For recent reviews on Akt, see: (a) Barnett, S. F.; Bilodeau,
M. T.; Lindsley, C. W. Curr. Top. Med. Chem. 2005, 5, 109;
(b) Cheng, J. Q.; Lindsley, C. W.; Cheng, G. Z.; Yang, H.;
Nicosia, S. V. Oncogene 2005, 24, 7482; (c) Kim, D.; Cheng,
G. Z.; Lindsley, C. W.; Yang, H.; Cheng, J. Q. Curr. Opin.
Investig. Drugs 2005, 6, 1250.
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. 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.
N
O
N
N
N
NH
H
N
Et2N
O
6
Enzyme assay (IC50s)
Akt1 = 458 nM
Cell-based IPKA assay (IC50s)
Akt1 = 368 nM
Akt2 = 617 nM
Akt2 = 359 nM
Akt3 = 7,400 nM
Akt3 = 8,000 nM
O
N
O
Et2N
N
N
H
N
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.
NH
N
7
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.
Enzyme assay (IC50s)
Akt1 = 138 nM
Akt2 = 212 nM
Cell-based IPKA assay (IC50s)
Akt1 = 253 nM
Akt2 = 276 nM
Akt3 = 7,200 nM
Akt3 >10,000 nM
Figure 2. Allosteric dual Akt1 and Akt2 inhibitors 6 and 7.
5. For Akt enzyme assay details and the proposed model for
the allosteric mode of inhibition of these inhibitors, see:
Barnett, S. F.; Defeo-Jones, D.; Fu, S.; Hancock, P. J.;
refinements are in progress and will be reported in due
course.

Z. Zhao et al. / Bioorg. Med. Chem. Lett. 18 (2008) 49–53
53
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.
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.
6. For details on the Akt IPKA assay, the caspase-3 assays,
and in vivo experimental details, see: Defeo-Jones, D.;
7. Leister, W. H.; Strauss, K.; Wisnoski, D.; Zhao, Z.;
Lindsley, C. J. Comb. Chem. 2003, 5, 322.