Non-peptidic substrate-mimetic inhibitors of Akt as potential anti-cancer agents

Article abstract of DOI:10.1016/j.bmc.2008.09.058

Akt has emerged as a critical target for the development of anti-cancer therapies. It has been found to be amplified, overexpressed, or constitutively activated in numerous human malignancies with oncogenesis derived from the simultaneous promotion of cell survival and suppression of apoptosis. A valuable alternative to the more common ATP-mimetic based chemotherapies is a substrate-mimetic approach, which has the potential advantage of inherent specificity of the substrate-binding pocket. In this paper we present the development of high affinity non-peptidic, substrate-mimetic inhibitors based on the minimum GSK3β substrate sequence. Optimization of initial peptidic leads resulted in the development of several classes of small molecule inhibitors, which have comparable potency to the initial peptidomimetics, while eliminating the remaining amino acid residues. We have identified the first non-peptidic substrate-mimetic lead inhibitors of Akt 29a-b, which have affinities of 17 and 12 μM, respectively. This strategy has potential to provide a useful set of molecular probes to assist in the validation of Akt as a potential target for anti-cancer drug design.

Full text of DOI:10.1016/j.bmc.2008.09.058

Bioorganic & Medicinal Chemistry 17 (2009) 1764–1771
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Non-peptidic substrate-mimetic inhibitors of Akt as potential anti-cancer
agents
Katherine J. Kayser-Bricker a, , Matthew P. Glenn a, Sang Hoon Lee a, Said M. Sebti b,d,à
,
Jin Q. Cheng b,c,d,§, Andrew D. Hamilton a,
*
a Department of Chemistry, Yale University, 225 Prospect Street, PO Box 208107, New Haven, CT 06520-8107, USA
b Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
c Molecular Oncology Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
d Department of Oncologic Sciences, University of South Florida College of Medicine, Tampa, FL 33612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Akt has emerged as a critical target for the development of anti-cancer therapies. It has been found to be
amplified, overexpressed, or constitutively activated in numerous human malignancies with oncogenesis
derived from the simultaneous promotion of cell survival and suppression of apoptosis. A valuable alter-
native to the more common ATP-mimetic based chemotherapies is a substrate-mimetic approach, which
has the potential advantage of inherent specificity of the substrate-binding pocket. In this paper we pres-
ent the development of high affinity non-peptidic, substrate-mimetic inhibitors based on the minimum
GSK3b substrate sequence. Optimization of initial peptidic leads resulted in the development of several
classes of small molecule inhibitors, which have comparable potency to the initial peptidomimetics,
while eliminating the remaining amino acid residues. We have identified the first non-peptidic sub-
Received 5 August 2008
Revised 17 September 2008
Accepted 24 September 2008
Available online 27 September 2008
Keywords:
Akt
Protein kinase B
Substrate-mimetic
Cancer
strate-mimetic lead inhibitors of Akt 29a–b, which have affinities of 17 and 12 lM, respectively. This
strategy has potential to provide a useful set of molecular probes to assist in the validation of Akt as a
Inhibitor
potential target for anti-cancer drug design.
Ó 2009 Published by Elsevier Ltd.
1. Introduction
lar side of the plasma membrane.18–20 Akt then undergoes dual
phosphorylation by membrane associated protein kinases PDK1
Akt/protein kinase B (PKB) has been shown to be a widely ex-
pressed Ser/Thr protein kinase whose persistent activation leads
to human oncogenesis.1–13 Its role in cancer and chemoresistance
is accomplished by the concomitant promotion of cell growth,
migration, and angiogenesis as well as the suppression of the apop-
totic pathway.14–16 There has been significant interest in Akt for its
structural and functional properties as well as its implications in
the area of cancer therapy.
and other kinases on Thr308 in the activation loop and Ser473 in
the C-terminal hydrophobic motif, respectively.21 This dual phos-
phorylation induces a conformational change in the enzyme to
its activated form, which incorporates an ATP binding site as well
as a substrate-binding site.
Akt directly phosphorylates substrates that are involved in the
regulation of cellular functions such as cellular proliferation, tran-
scription, migration, apoptosis, cellular differentiation, and metab-
olism.22,23 One of the first cellular substrates discovered for Akt
was glycogen synthase kinase 3 (GSK3), which plays a key role in
metabolism and insulin signaling pathways. GSK3 is negatively
regulated by Akt via insulin stimulated phosphorylation of GSK3
that results in its inactivation and consequent activation of glyco-
gen synthase.5 The disregulation of Akt kinase activity has been de-
tected in a number of human malignancies including ovarian,
breast, thyroid, and colon cancers.24 Amplification or overexpres-
sion of Akt results in the up-regulation of cell growth and the
down-regulation of apoptosis. The cellular levels of PIP3 regulate
the activity of PDK-1, which is responsible for Akt activation. The
levels of these phosphoinositides are dependent on the activity of
PI3K and a set of phosphatases PTEN and SHIP.25 Tumor suppressor
PTEN negatively regulates the activity of Akt by converting PIP3
The Akt family consists of three members, Akt1 (PKB
a), Akt2
(PKBb), and Akt3 (PKB ). Numerous cellular stimuli result in Akt
c
activation including molecules that regulate tyrosine kinase activ-
ity and G-protein-linked receptors, and phosphatase inhibitors.5,17
Direct activation of Akt is mediated by phosphoinositide-3 kinase
(PI3K) which generates phosphatidylinositol-3,4,5-triphosphate
(PIP3), a lipid second messenger which binds to the pleckstrin
homology (PH) domain of Akt and translocates it to the intracellu-
* Corresponding author. Tel.: +1 203 432 5570; fax: + 1 203 432 3221.
E-mail address: Andew.Hamilton@yale.edu (A.D. Hamilton).
Tel.: +1 203 432 8966; fax: +1 203 432 6144.
à
Tel.: +1 813 745 6734; fax: +1 813 745 6748.
Tel.: +1 813 745 6915; fax: +1 813 745 3829.
§
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.09.058

K. J. Kayser-Bricker et al. / Bioorg. Med. Chem. 17 (2009) 1764–1771
1765
back to PIP2. PTEN deletions and mutations are prevalent in a vari-
ety of human cancers.17 Inhibition of Akt activity has been shown
to suppress cell growth and induce apoptosis in tumor cell lines
derived from various organs possessing constitutively activated
Akt.24
Herein we describe the first non-peptidic, substrate-mimetic
inhibitor of Akt developed through systematic rigidification and
replacement of the remaining amino acid residues. Further struc-
tural refinement included the incorporation of essential binding
groups into organic scaffolds to increase rigidity and to deliver im-
proved potency and selectivity.
The majority of small molecule kinase inhibitors to date target
the ATP binding pocket and there have been few reports targeting
the substrate-binding site. ATP mimetics have met with much suc-
cess, however selective binding within this pocket remains chal-
lenging as these inhibitors compete with the many ATP utilizing
enzymes possessing similar contact regions as well as with high
cellular concentrations of ATP.26–29 Substrate-mimetics offer a
promising strategy for the design of selective inhibitors of Akt as
they can potentially exploit sequence specificity.30–34 The sub-
strate-binding region has evolved to recognize specific substrate
sequences and therefore offers a larger number of potential inter-
actions for a properly designed inhibitor than the corresponding
ATP pocket. The inherent design challenges present in substrate-
mimetics are the large binding pocket and extended binding con-
formation of many natural substrates. We recently described the
development of substrate-mimetic inhibitors of Akt based on the
consensus sequence and the structure of an enzyme bound sub-
strate.35 These preliminary studies demonstrated that limited
structural modification of the initial peptidic substrate can over-
come these challenges and provide peptidomimetic inhibitors with
increasing lipophilicity, rigidity, and potency as well as decreasing
the size and peptidic nature of the inhibitors.
2. Results and discussion
Modifications of our earlier preliminary peptidomimetic struc-
tures focused on three main areas: the N-terminal hydrophilic do-
main, the central region, and C-terminal substituents. The
efficiencies of the inhibitors in disrupting Akt function was tested
utilizing a fluorescence polarization assay system.38 The lowest se-
quence homology in Akt substrates exists within the dipeptide se-
quence adjacent to the phosphorylated serine/threonine residue. In
1 this region is replaced by a 4-aminobenzoic acid (Abz) spacer.
The contacts within this region are mainly hydrophobic, therefore
a variety of hydrophobic substituents projected from the central
phenyl spacer was explored (Table 1). Incorporation of a phenyl
substituent at R2 provides 5 with a slight increase in activity when
compared to previously reported inhibitor 2.35 Docking studies
suggested that the phenyl substituent is able to access the Thr
pocket previously exploited in the design of inhibitor 1. Truncation
of the N-terminus of the inhibitors (7–9) resulted in a modest de-
crease in affinity, but a desirable decrease in molecular weight and
peptidic character of the inhibitors. The study of the central por-
tion of the inhibitor confirmed the importance of the projection
of substituents into the Thr binding pocket.
Our substrate-mimetic design was based on the truncated
GSK3b substrate sequence, GRPRTTSF, utilizing the recently pub-
lished X-ray crystal structure of an activated Akt ternary complex
with GSK3b and an ATP analogue.36 Our design approach focused
on reducing the entropy cost of the extended binding conforma-
tion, accessing a large unoccupied hydrophobic pocket adjacent
to the C-terminus, and eliminating non-essential amino acid resi-
dues. From this we identified inhibitor 1 with in vitro inhibition
Flexible ligand docking (GOLD)37 of lead peptidomimetics iden-
tified several potential conformationally restrained replacements
for the Val-Phe-Bn C-terminal sequence, which remove two of
the three remaining amino acids. A simple cyclic constraint such
as in quinazolines 15a–b projects appended hydrophobic groups
into adjacent hydrophobic pockets while maintaining the N-termi-
nal and central inhibitor/Akt interactions (Scheme 1). Inhibitor 15a
of IC50 = 14 l
M (Fig. 1).35
has similar affinity (IC50 = 112 lM) to the corresponding inhibitor 9
containing the Val-Phe dipeptide, but contains two fewer stereo-
centers. Careful consideration of the potential binding site contacts
made by the three key areas of the peptidomimetic inhibitors pro-
vided guidance in the design of non-peptidic substrate-mimetics.
Inhibitor 21ba was designed using GOLD37 to incorporate
important binding elements from the previous studies (Fig. 2).
The guanidine group is directly projected into the Arg pocket via
Table 1
Investigations of central hydrophobic contacts
Compound
IC50 (l
M)a
2b
3
4
5
6
Ac-
Ac-
Ac-
Ac-
Ac-
G
G
G
G
G
R
R
R
R
R
P
P
P
P
R
R
R
R
R
R
R
R
-H
-H
V
V
V
V
V
V
V
V
F
F
F
F
F
F
F
F
-Bn 28 8
-CH3 -H
-Ph
-H
-H
-Ph
-H
-Bn 50 10
-Bn >500
-Bn 16 5
-Bn 78 14
-Bn 288 100
-Bn 173 31
-Bn 119 36
-H
-Ph
-Nap
-H
-Ph
-H
P
7
Ac-
Ac-
Ac-
8
9b
-H
a
Reported values are an average of three independent binding curves utilizing an
IMAPÒ Akt Assay Kit (Molecular Devices). Reactions were conducted in wells with
20.0
DTT, 100 nM 5FAM-GRPRTSSFAEG-COOH, 5
stock solution). Reaction mixtures were incubated for 1 h at room temperature and
then quenched with 60 of the IMAP-binding solution. The reactions were
lL of 10 mM Tris–HCl (pH 7.2), 10 mM MgCl2, 0.1% BSA, 0.05% NaN3, 1 mM
lM ATP, Akt1, and inhibitor (DMSO
lL
equilibrated for 1 h at room temperature then data points were collected and
analyzed.
Figure 1. Flexible ligand docking GOLD37 of lead peptidomimetic inhibitor
1
(previously reported)35 (green) against the solvent-accessible surface area of Akt
(Gradient color scale- red hydrophobic, blue hdrophilic).
b
Previously reported inhibitors.35

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K. J. Kayser-Bricker et al. / Bioorg. Med. Chem. 17 (2009) 1764–1771
Table 2
an ethylenediamine linker that extends the correct distance be-
tween the aromatic spacer and the arginine binding pocket of
Akt. The Thr pocket can be accessed by direct projection of substit-
uents from Abz, shown here as a simple phenyl substituent. Finally,
the 4-aminoaniline provides a C-terminal rigid scaffold to project
various hydrophobic substituents into the pockets of Akt, with
21ba possessing two benzyl substituents. This small molecule sub-
Optimization of C-terminal and central substituents
Compound
R1
R2
IC50 (l
M)a
21aa
–H
96 20
84 21
strate-mimetic of Akt has an IC50 of 84 lM, which is comparable or
better than our previous peptidomimetic inhibitors, and is signifi-
cantly more rigid and impervious to proteases.
This non-peptidic scaffold design easily allowed an extensive
exploration of the different binding groups, beginning with the
C-terminal hydrophobic interactions in series 21aa–21bi (Table
2, Scheme 2). This series suggests that the two pockets are exten-
sive and able to accommodate large hydrophobic substituents
(21bd, 21bf, 21bh). Inhibitor 21bi with a 4-cyanobenzyl functional
group is the most potent inhibitor in this series having an IC50 of
21ba
21bb
21bc
21bd
21be
21bf
215 73
152 44
84
7
19 lM. Secondly, a range of substituents were added to explore
the role of contacts within the Thr pocket via the projection of
functionality directly off Abz to provide inhibitors 21aa and
22aa–fa (Table 2, Scheme 3). Inhibitor 21aa, which lacks the phe-
nyl substituent and the ability to make contacts within this region,
is slightly less potent than the biphenyl derivative. This suggests
that optimization at this position could lead to increased potency.
The addition of H-bond donors and acceptors here did not lead to
increased affinity (22aa and 22ba), however, larger hydrophobic
groups, such as 2-naphthyl, led to a two-fold increase in affinity
55 13
F
43
28
23
5
8
4
21bg
with inhibitor 22fa having an IC50 of 44 lM.
These series of non-peptidic substrate-mimetic inhibitors pro-
vided valuable information concerning the nature of the three
binding pockets within the active site of Akt. To further optimize
our inhibitors, the best substituents at the two positions were
combined in an effort to increase potency (21cg and 21ci). Inhibi-
tor 21ci, which incorporates the best C-terminal functionality, 4-
cyanobenzyl, and the best central element, 2-naphthyl, is the most
potent non-peptidic inhibitor of this scaffold series with an IC50 of
21bh
21bi
21cg
21ci
19 10
CN
CN
38
17
4
2
17 lM, a slight improvement from phenyl derivative 21bi. To in-
crease the stability and rigidity of 21cg and 21ci, the amide analogs
22aa
22ba
22ca
22da
22ea
253 31
158 19
83 10
NHCOCH3
CO2CH3
78
52
44
9
8
6
CN
22fa
a
Reported values are an average of three independent binding curves utilizing an
IMAPÒ Akt Assay Kit (Molecular Devices). Reactions were conducted in wells with
20.0
DTT, 100 nM 5FAM-GRPRTSSFAEG-COOH, 5
stock solution). Reaction mixtures were incubated for 1 h at room temperature and
then quenched with 60 of the IMAP-binding solution. The reactions were
equilibrated for 1 h at room temperature then data points were collected and
analyzed. (Inhibitors 21aa–ci synthesized via Scheme 2, Inhibitors 22aa–22fa
synthesized via Scheme 3).
l
L of 10 mM Tris–HCl (pH 7.2), 10 mM MgCl2, 0.1% BSA, 0.05% NaN3, 1 mM
lM ATP, Akt1, and inhibitor (DMSO
l
L
Figure 2. Flexible ligand docking GOLD37 of inhibitor 21ba (green) against the
solvent-accessible surface area of Akt (Gradient color scale—red hydrophobic, blue
hydrophilic).

K. J. Kayser-Bricker et al. / Bioorg. Med. Chem. 17 (2009) 1764–1771
1767
Scheme 1. Reagents and conditions: (a) i—NaNCO; ii—NaOH; iii—HCl, 53%; (b) CO2 1 atm, DBU, THF, 82%; (c) K2CO3, BnBr, DMF, 81%, 91%; (d) Pd/C, H2 35 psi, CH3OH, 88%,
81%; (e) i—LDA THF; ii—4-nitro benzoyl chloride ꢁ78–50 °C, 76%, 81%; (f) Pd/C, H2 35 psi, 1:1 CH3OH/CH2Cl2, 80%, 86%; (g) AcArg(Pmc)-OH, DIC, CH2Cl2, cat. DMAP; (h) TFA, 5%
thioanisole, 1% TIPS, 1% H2O, 4%, 5% (Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; DIC, diisopropyl carbodiimide).
Scheme 2. Reagents and conditions: (a) R1B(OH)2, PdCl2(dppf), K2CO3, DMF, 100 °C, 71–82% (16a = methyl 4-aminobenzoate); (b) i—N-Boc-2-aminoacetaldehyde, AcOH,
MeOH, 4 Å mol. sieves; ii—NaCNBH3, 68–100%; (c) i—LiOH, THF, H2O; ii—TFA, 70–100%; (d) BisBocPCH, Et3N, MeOH, 61–71%; (e) RBr, K2CO3, NaI, DMF, 61–100%; (f)
SnCl2ꢀ2H2O, EtOAc, reflux, 19–92%; (g) i—DIC, cat DMAP, DCM; ii—TFA, 26–76% (BisBocPCH, N,N-bis-tert-butoxy-carbonyl-1H-pyrazole-1-carboxamidine).
29a–b were synthesized, which also led to a further increase in po-
tency (IC50’s = 17 M and 12 M, respectively) (Scheme 4).
3. Synthesis
l
l
The initial non-peptidic substrate-mimetic design was success-
ful and optimization of the scaffold provided inhibitors 29a–b that
are comparable to our previous lead 1. Further optimizations fo-
cused on increasing rigidity by the addition of a ring constraint
through an indole-aryl scaffold 36a–b (Fig. 3). The indole deriva-
tive 36a is comparable to 21aa as both lack access to the Thr pocket
and possess C-terminal benzyl substituents. The inclusion of an in-
dole scaffold provided a slight decrease in affinity in 36a (Scheme
5). Consistent with the previous scaffold, the addition of the C-ter-
minal 4-cyanobenzyl substituent in 36b provided a four-fold in-
Peptidomimetics 2–9 were synthesized via solid phase peptide
synthesis. Suzuki couplings employing various boronic acids and
aryl bromides were performed to provide intermediates that dis-
played hydrophobic substituents from the aromatic spacer (Abz).
The simple quinazoline scaffolds were readily derived from com-
mercially available starting materials. The synthesis of the quina-
zolines cores 10a–b was accomplished by the cyclization of 4-
nitroanthranilic acid by the reaction with sodium isocyanate or
cyclization employing a carbon dioxide atmosphere with catalytic
DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) from 4- and 5-nitro pre-
cursors, respectively (Scheme 1).39–41 Alkylation was followed by
crease in affinity from 126 to 32 lM.

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K. J. Kayser-Bricker et al. / Bioorg. Med. Chem. 17 (2009) 1764–1771
Scheme 3. Reagents and conditions: (a) LiOH, THF, H2O, 100%; (b) DIC, cat DMAP, DCM; (c) R1B(OH)2, PdCl2(dppf), K2CO3, DMF, 100ꢀC, 62–78%; (d) i—N-Boc-2-
aminoacetaldehyde, AcOH, MeOH, 4 Å mol. sieves; ii—NaCNBH3; (e) TFA, 41–69%; (f) BisBocPCH, Et3N, MeOH, 51–88%; (g) TFA, 40–99%.
Scheme 4. Reagents and conditions: (a) Boc-Gly-OH, DIC, cat. DMAP, DCM, 45%; (b) i—LiOH, THF, H2O; ii—TFA, 90%; (c) BisBocPCH, Et3N, MeOH, 54%; (d) i—20g or 20i, DIC, cat
DMAP, DCM; ii—TFA, 57%.
employing PdCl2(dppf) as a catalyst (16a commercially available)
followed by reductive amination utilizing N-Boc-aminoacetalde-
hyde provided 17a–c. A series of deprotections followed by gua-
nidinylation of the resulting amine afforded the N-terminal
portions of the inhibitor 18a–c. The C-terminal hydrophobic por-
tion of the molecule was synthesized via alkylation of 4-nitroani-
line with the corresponding bromide and subsequent reduction
of the nitro group utilizing tin (II) chloride to afford 20a–i. Cou-
pling of 18a–c and 20a–i followed by Boc deprotection under
acidic conditions gave final inhibitors 21aa–ci. Inhibitors 29a–b
were derived from a similar synthesis, but in place of the reductive
amination step, 16c was reacted with Boc-Gly-OH to provide the
amide intermediate 27 which was manipulated in a similar man-
ner to provide 29a–b (Scheme 4).
The synthesis of inhibitors 22aa–fa used a late stage Suzuki
coupling to provide faster access to a number of derivatives at
the R1 position, while keeping R2 as a benzyl substituent (Scheme
3). Commercially available methyl-4-amino-3-bromobenzoate was
saponified under basic conditions followed by amide bond forma-
tion with 20a to provide 24a. This intermediate was then reacted
with different boronic acid derivatives with PdCl2(dppf) as a cata-
lyst to provide 25aa–fa. A series of functional group transforma-
tions similar to Scheme 2 provided inhibitors 22aa–fa.
Figure 3. Flexible ligand docking GOLD37 of inhibitor 36a (green) against the
The indole scaffold was readily derived from commercially
solvent-accessible surface area of Akt (Gradient color scale—red hydrophobic, blue
available 4-iodoaniline and Boc-Gly-OH, which were reacted to
form iodo-amide 30 (Scheme 5). Sonagashira cross-coupling of
30 and ethynyl–trimethyl-silane (TMS–acetylene) followed by
removal of the silyl protecting group afforded terminal alkyne
hydrophilic).
reduction of the nitro group followed by coupling with 4-nitro-
benzoyl chloride via anilide formation to provide 13a–b. Reduction
to the aniline, coupling with AcArg(Pmc)-OH, and deprotection of
the guanidine protecting groups afforded 15a–b.
A convergent synthesis starting from methyl-4-amino-3-bro-
mobenzoate or methyl-4-aminobenzoate and 4-nitroaniline pro-
vided non-peptidic inhibitors 21aa–ci (Scheme 2). Suzuki
coupling of the bromoaniline with the corresponding boronic acid
31.
A consecutive Sonagashira cross-coupling with 2-iodo-4-
nitroaniline followed by cycloisomerization employing catalytic
copper (II) acetate42 afforded indole scaffold 33. Reduction of
the nitro to the amine followed by alkylation with the corre-
sponding bromide provided 35a–b. A series of functional group
transformations similar to Schemes 1 and 2 provided inhibitors
36a–b.

K. J. Kayser-Bricker et al. / Bioorg. Med. Chem. 17 (2009) 1764–1771
1769
Scheme 5. Reagents and conditions: (a) Boc-Gly-OH, DIC, cat. DMAP, DCM, 100%; (b) i—TMS–acetylene, CuI, Pd(PPh3)2Cl2, Et3N; ii—K2CO3, MeOH, 74%; (c) CuI, Pd(PPh3)2Cl2,
2:1 Et3N/THF, 82%; (d) Cu(OAc)2, DMF, 120ꢀC, 3 days, 66%; (e) H2 (1 atm), Pd/C, MeOH, 92%; (f) RBr, K2CO3, DMF, 100%, 78%; (g) i—1:1 TFA/DCM; ii—BisBocPCH, Et3N, MeOH,
71%, 62%; (h) 1:1 TFA/DCM, 32%, 20%.
in d (ppm) relative to tetramethylsilane. All coupling constants
are described in Hz. Analysis and purification by revere phase HPLC
(rpHPLC) were performed using either a Waters 2487 dual k UV
detector with a Waters 1525EF binary pump using a Phenomenex
4. Conclusion
Akt is an attractive target for developing novel cancer therapies
and there exists a need for isoform-specific inhibitors to validate
them as potential targets for anti-cancer drug design. The sub-
strate-mimetic approach is a valuable alternative to ATP-mimetic
based chemotherapies due to the inherent specificity of the sub-
strate-binding pocket. Preliminary peptidomimetic substrate-mi-
metic studies provided the foundation for the development of
small molecule inhibitors by elucidating critical inhibitor/Akt
interactions. The published X-ray crystal structure36 along with
flexible ligand docking (GOLD)37 and extensive structure–activity
relationships allowed the design of the first non-peptidic sub-
strate-mimetic inhibitors of Akt. Through optimizations of the var-
ious substituents we improved the affinity of the non-peptidic
inhibitors to provide more potent inhibitors than the initial pepti-
domimetics. These substrate-mimetics are more rigid, lipophilic
inhibitors that lack amino acids and are significantly smaller in size
than the GSK3b peptide.
Luna 5
l
C18(2) 250 ꢂ 21 mm column run at 20 mL/min (prepara-
tive), or a Waters 2487 dual k UV detector with a Waters 1525 bin-
ary pump using a Microsorb-MV 300 Å C18 250 ꢂ 4.6 mm column
run at 1 mL/min (analytical), using gradient mixtures of water with
0.1% trifluoroacetic acid (TFA) (A) and 10:1 acetonitrile/water (B)
with 0.1% TFA. Compound purity was confirmed by analytical
rpHPLC using a linear gradient from 100% A to 100% B with chang-
ing solvent composition over either: (I) 20 minutes or (II) a linear
gradient from 90% A to 80% B over 25 min or (III) a linear gradient
from 100% A to 60% B over 20 min. All gradients started after an ini-
tial 2 min of 100% A. Preparative HPLC purifications were per-
formed using Method
I and product fractions were always
lyophilized to dryness. High resolution mass (HRMS) determina-
tions were performed at the University of Illinois at Urbana-Cham-
paign Mass Spectrometry Center on a Varian MAT-CH-5.
5.1.2. General coupling/deprotection procedure
5. Experimental
The required acid (1.0 equiv) was added in one portion to a
solution of DIC (1.0 equiv) in CH2Cl2 (5 mL/mmol), and the result-
ing solution stirred at room temperature for 5 min under a nitro-
gen atmosphere. The required amine (2.0 equiv) was then
dissolved in a solution of CH2Cl2 (5 mL/mmol) and added to the
activated acid drop-wise in one portion followed by the addition
of DMAP (catalytic). The resulting solution was stirred for 20 h,
then diluted with CH2Cl2 (100 mL/mmol) and washed successively
with sat. NaHCO3 (100 mL/mmol) and brine (100 mL/mmol). The
organic layer was dried over Na2SO4, and the solvent was removed
under reduced pressure to afford the desired amide. The amide was
then dissolved in TFA (1 mL/mmol) and stirred at room tempera-
ture for 1 h. The TFA was evaporated under reduced pressure and
the crude product was dissolved in acetonitrile/H2O and purified
by preparatory HPLC column chromatography to afford the desired
amide as the TFA salt.
5.1. Synthesis and spectroscopic data for representative
compounds
5.1.1. General
All air and/or moisture sensitive reactions were carried out un-
der a positive pressure of nitrogen in flame-dried glassware. Sol-
vents dimethylformamide (DMF), tetrahydrofuran (THF),
dichloromethane, and toluene were obtained from commercial
sources and dried on an Innovative Technology SPS-400 dry sol-
vent system. Flash column chromatography was performed on sil-
ica gel (40–63 lm) under a pressure of about 4 psi. Analytical thin
layer chromatography (TLC) was carried out on glass plates coated
with 0.25 mm of silica gel with 254 nm fluorescent indicator (Ba-
ker, Si250F). 1H and 13C spectra were recorded on a Bruker AM-
500 or Bruker AM-400 spectrometer. Chemical shifts are reported

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K. J. Kayser-Bricker et al. / Bioorg. Med. Chem. 17 (2009) 1764–1771
5.1.3. 6-(2-Guanidino-ethylamino)-biphenyl-3-carboxylic acid
{4-[bis-(4-cyano-benzyl)-amino]-phenyl}-amide (21bi)
Reaction of 18b (0.050 g, 0.100 mmol) and 20i (0.034 g,
0.100 mmol) according to procedure above, yield after preparatory
HPLC (47%, tan powder). 1H NMR (DMSO, 500 MHz) d 9.63 (1H, s,
NH), 7.82 (1H, dd, J = 8.7 and 2.1 Hz, Ar-H), 7.79 (4H, d, J =
8.1 Hz, Ar-H), 7.66 (1H, d, J = 2.1 Hz, Ar-H), 7.63 (1H, b, NH),
7.52–7.01 (14H, m, Ar-H and NH), 6.81 (1H, d, J = 8.7 Hz, Ar-H),
6.61 (2H, d, J = 9.1 Hz, Ar-H), 5.19 (1H, b, NH), 4.78 (4H, s, CH2),
3.31–3.27 (4H, m, CH2); 13C NMR (DMSO, 500 MHz) d 164.2,
156.9, 147.0, 145.3, 143.5, 138.4, 132.3, 129.7, 129.0, 128., 128.7,
127.6, 127.3, 125.9, 122.3, 122.0, 118.8, 112.5, 109.4, 109.2, 54.2,
41.4, 39.7. HRMS (ESI) m/z calculated for C38H34N8OH+ 619.2934.
Found 619.2933. rpHPLC tR: condition (I) 13.930 (II) 13.193 min,
purity 100%.
5.1.7. {[4-(5-Amino-1H-indol-2-yl)-phenylcarbamoyl]-methyl}-
carbamic acid tert-butyl ester (34)
To a solution of 33 (0.200 g, 0.49 mmol) in anhydrous CH3OH
(5 mL) was added 10% Pd/C (0.010 g). The reaction was placed un-
der a hydrogen balloon (1 atm) and purged three times under high
vacuum to provide a complete hydrogen atmosphere then stirred
at room temperature for 4 hours. The reaction mixture was then
filtered through a celite pad and the filtrate was evaporated under
reduced pressure. The crude product was purified by FCC eluting
with 80% EtOAc: 20% hexanes to afford 34 as a gray solid
(0.170 g, 92%). 1H NMR (DMF, 500 MHz) d 11.03 (1H, s, NH),
10.12 (1H, s, NH), 7.84–7.77 (4H, m, Ar-H), 7.14 (1H, d, J = 8.3
Hz, Ar-H), 6.99 (1H, br, NH), 6.82 (1H, s, Ar-H), 6.62–6.61 (2H, m,
Ar-H), 4.58 (2H, br, NH2), 3.94 (2H, s, CH2), 1.44 (9H, s, CH3); 13C
NMR (DMF, 500 MHz) d 169.5, 157.5, 143.0, 139.5, 138.8, 132.7,
131.4, 129.4, 126.2, 120.6, 113.4, 112.3, 104.4, 98.1, 79.4, 45.4,
29.0. HRMS (ESI) m/z calculated for C21H24N4O3H+ 381.1927. Found
381.1928.
5.1.4. N-{4-[Bis-(4-cyano-benzyl)-amino]-phenyl}-4-(2-
guanidino-ethylamino)-3-naphthalen-2-yl-benzamide (21ci)
Reaction of 18c (0.040 g, 0.073 mmol) and 20i according to pro-
cedure above, yield after preparatory HPLC (37%, white powder).
1H NMR (MeOD, 500 MHz) d 7.98 (1H, d, J = 8.4 Hz, Ar-H), 7.92–
7.86 (4H, m, Ar-H), 7.77 (1H, d, J = 1.9 Hz, Ar-H), 7.67–7.66 (4H,
m, Ar-H), 7.56–7.36 (9H, m, Ar-H), 6.89 (1H, d, J = 8.7 Hz, Ar-H),
6.67 (2H, d, J = 8.9 Hz, Ar-H), 4.77 (4H, s, CH2), 3.44 (2H, t, J =
6.0 Hz, CH2), 3.38 (2H, t, J = 6.0 Hz, CH2); 13C NMR (MeOD,
500 MHz) d 168.5, 158.9, 149.3, 146.5, 137.4, 135.3, 134.3, 133.6,
131.4, 130.6, 130.1, 129.8, 129.4, 129.1, 129.0, 128.8, 128.7,
128.4, 127.6, 127.4, 124.4, 124.0, 119.8, 114.6, 111.8, 110.6, 56.2,
43.0, 41.8. HRMS (ESI) m/z calculated for C42H36N8OH+ 669.3090.
Found 669.3109. rpHPLC tR: condition (I) 14.128 (II) 13.549 min,
purity 100%.
5.1.8. [(4-{5-[Bis-(4-cyano-benzyl)-amino]-1H-indol-2-yl}-
phenylcarbamoyl)-methyl]-carbamic acid tert-butyl ester (35b)
To a solution of 34 (0.107 g, 0.281 mmol) in dry DMF (1.5 mL)
was added anhydrous potassium carbonate (0.077 g, 0.562 mmol)
and 4-(bromomethyl)benzonitrile (0.110 g, 0.562 mmol). The
resulting reaction was placed under a nitrogen atmosphere and
stirred at room temperature for 3 h. The DMF was evaporated un-
der reduced pressure and the residue was dissolved in EtOAc
(30 mL) and washed with 10% citric acid (30 mL) and brine
(30 mL) and the organic layer was dried over Na2SO4 and filtered.
The solvent was removed under reduced pressure and the crude
product was purified by FCC on silica gel eluting with 60% EtOAc:
40% hexanes (78%, light blue solid). 1H NMR (DMSO, 500 MHz) d
11.11 (1H, s, NH), 9.99 (1H, s, NH), 7.78 (4H, d, J = 8.0 Hz, Ar-H),
7.70 (2H, d, J = 8.5 Hz, Ar-H), 7.62 (2H, d, J = 8.5 Hz, Ar-H), 7.49
(4H, d, J = 8.0 Hz, Ar-H), 7.17 (1H, d, J = 8.8 Hz, Ar-H), 7.05 (1H, t,
J = 5.9 Hz, NH), 6.72–6.66 (2H, m, Ar-H), 6.55 (1H, s, Ar-H), 4.71
(4H, s, CH2), 3.73 (2H, d, J = 5.9 Hz, CH2), 1.40 (9H, s, CH3); 13C
NMR (DMSO, 500 MHz) d 168.09, 155.82, 145.80, 141.57, 137.93,
137.76, 132.17, 131.33, 129.36, 127.92, 127.23, 125.11, 119.14,
118.81, 111.57, 109.30, 104.00, 97.27, 79.06, 77.94, 55.52, 43.69,
28.11. HRMS (ESI) m/z calculated for C37H34N6O3H+ 611.2771.
Found 611.2758.
5.1.5. N-{4-[Bis-(4-cyano-benzyl)-amino]-phenyl}-4-(2-
guanidino-acetylamino)-3-naphthalen-2-yl-benzamide (29b)
Reaction of 28 (0.041 g, 0.073 mmol) and 20i according to pro-
cedure above, yield after preparatory HPLC (58%, white powder).
1H NMR (MeOD, 500 MHz) d 8.01–7.91 (7H, m, Ar-H), 7.68–7.66
(4H, m, Ar-H), 7.57–7.41 (9H, m, Ar-H), 6.70 (2H, d, J = 9.1 Hz,
Ar-H), 4.79 (4H, s, CH2), 3.95 (2H, s, CH2); 13C NMR (MeOD,
500 MHz) d 168.3, 167.6, 159.4, 146.7, 146.4, 138.4, 136.8, 135.1,
134.4, 133.6, 131.3, 130.2, 129.6, 129.5, 129.3, 129.0, 128.8,
128.5, 127.9, 127.7, 126.0, 125.9, 125.9, 124.3, 119.8, 114.5,
111.8, 56.1, 45.2. HRMS (ESI) m/z calculated for C42H34N8O2H+
683.2883. Found 683.2872. rpHPLC tR: condition (I) 13.799 (II)
12.997 min, purity 100%.
5.1.9. N-(4-{5-[Bis-(4-cyano-benzyl)-amino]-1H-indol-2-yl}-
phenyl)-2-guanidino-acetamide (36b)
A solution of 35b (0.120 g, 0.200 mmol) in 1:1 CH2Cl2:TFA was
stirred at room temperature for 1 h to remove the Boc protecting
group. The solvent was removed under reduced pressure and the
crude amine was dried under high vacuum for 1 h. The amine
was then dissolved in anhydrous CH3OH (5 mL) and triethylamine
5.1.6. {[4-(5-Nitro-1H-indol-2-yl)-phenylcarbamoyl]-methyl}-
carbamic acid tert-butyl ester (33)
In a pressure vessel, 32 (0.940 g, 2.29 mmol) and copper (II) ace-
tate (0.083 g, 0.46 mmol) were dissolved in dry DMF (10 mL). The
reaction was placed under a nitrogen atmosphere and purged three
times under high vacuum to provide a complete nitrogen atmo-
sphere. The vessel was then sealed and stirred for 72 h at 120ꢀC.
The reaction was then cooled to room temperature and the DMF
was evaporated under reduced pressure and the residue was dis-
solved in EtOAc (100 mL) and washed with brine (1 ꢂ 100 mL).
The organic suspension was then filtered to afford the desired in-
(279 lL, 2.00 mmol) and N,N’-bis(tert-butoxycarbonyl)-1H-pyra-
zole-1-carboxyamidine (0.093 g, 0.300 mmol) were added and
the resulting solution was stirred for 24 h. The CH3OH was evapo-
rated under reduced pressure and the concentrates were dissolved
in EtOAc (20 mL) and washed consecutively with 10% citric acid
(20 mL) and brine (20 mL). The organic layer was dried over so-
dium sulfate (Na2SO4) and the solvent was removed under reduced
pressure. The amine was purified by FCC eluting with 40% EtOAc:
60% hexanes to yield N,N0-Bis-tert-butoxycarbonyl-N00-(4-{5-[Bis-
(4-cyano-benzyl)-amino]-1H-indol-2-yl}-phenyl)-2-guanidino-acet-
amide (62%, light blue oil). 1H NMR (CDCl3, 400 MHz) d 11.37 (1H,
s, NH), 9.80 (1H, s, NH), 8.98 (1H, t, J = 5.0 Hz, NH), 8.48 (1H, s, NH),
7.58–7.26 (12H, m, Ar-H), 7.15 (1H, d, J = 8.7 Hz, Ar-H), 6.86 (1H, s,
Ar-H), 6.68 (1H, d, J = 8.2 Hz, Ar-H), 6.55 (1H, s, Ar-H), 4.59 (4H, s,
CH2), 4.13 (2H, d, J = 5.0 Hz, CH2), 1.52 (9H, s, CH3), 1.50 (9H, s,
CH3); 13C NMR (CDCl3, 400 MHz) d 166.7, 162.4, 156.9, 152.7,
dole 33 as
a
yellow solid (0.498 g, 66%). 1H NMR (DMSO,
400 MHz) d 12.24 (1H, s, NH), 10.13 (1H, s, NH), 8.51 (1H, d,
J = 2.2 Hz, Ar-H), 7.99 (1H, dd, J = 9.0 and 2.2 Hz, Ar-H), 7.85 (2H,
d, J = 8.7 Hz, Ar-H), 7.72 (2H, d, J = 8.7 Hz, Ar-H), 7.53 (1H, d,
J = 9.0 Hz, Ar-H), 7.11–7.08 (2H, m, Ar-H and NH), 3.75 (2H, d,
J = 6.1 Hz, CH2), 1.40 (9H, s, CH3); 13C NMR (DMSO, 400 MHz) d
168.3, 155.9, 141.3, 140.8, 140.2, 139.1, 128.0, 126.0, 125.7,
119.2, 116.6, 116.6, 111.4, 99.9, 78.0, 43.7, 28.1. HRMS (ESI) m/z
calculated for C21H22N4O5H+ 411.1668. Found 411.1675.

K. J. Kayser-Bricker et al. / Bioorg. Med. Chem. 17 (2009) 1764–1771
1771
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crude product was purified by HPLC. Yield of 36b after preparatory
HPLC (20%, pink TFA salt). 1H NMR (DMSO, 500 MHz, 330 K) d
10.98 (1H, s, NH), 10.08 (1H, s, NH), 7.75–7.71 (6H, m, Ar-H),
7.63 (2H, d, J = 8.7 Hz, 1H), 7.51–7.50 (5H, m, Ar-H and NH),
7.20–7.18 (4H, Ar-H and NH), 6.82 (1H, d, J = 2 Hz, Ar-H), 6.74
(1H, dd, J = 8.8 and 2 Hz, Ar-H), 6.56 (1, s, Ar-H), 4.68 (4H, s,
CH2), 4.07 (2H, d, J = 6.1 Hz, CH2); 13C NMR (DMSO, 500 MHz,
330 K) d 165.6, 157.4, 145.4, 141.6, 137.5, 131.8, 131.5, 129.1,
127.8, 127.5, 124.9, 119.2, 118.3, 111.9, 111.8, 111.3, 109.2,
104.7, 97.3, 55.6, 43.9. HRMS (ESI) m/z calculated for C33H28N8OH+
553.2464. Found 553.2491. rpHPLC tR: condition (I) 11.432 (II)
8.610 min, purity 97%.
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Acknowledgments
K.J.K. thanks Sanofi-Aventis for their funding of the American
Chemical Society division of Medicinal Chemistry predoctoral fel-
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Supplementary data
32. Luo, Y.; Smith, R. A.; Guan, R.; Liu, X. S.; Klinghofer, V.; Shen, J. W.; Hutchins, C.;
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.09.058.
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