Discovery and optimization of a series of 3-(3-phenyl-3H-imidazo[4,5-b] pyridin-2-yl)pyridin-2-amines: Orally bioavailable, selective, and potent ATP-independent Akt inhibitors

Article abstract of DOI:10.1021/jm300276x

This paper describes the implementation of a biochemical and biophysical screening strategy to identify and optimize small molecule Akt1 inhibitors that act through a mechanism distinct from that observed for kinase domain ATP-competitive inhibitors. With the aid of an unphosphorylated Akt1 cocrystal structure of 12j solved at 2.25 ?, it was possible to confirm that as a consequence of binding these novel inhibitors, the ATP binding cleft contained a number of hydrophobic residues that occlude ATP binding as expected. These Akt inhibitors potently inhibit intracellular Akt activation and its downstream target (PRAS40) in vitro. In vivo pharmacodynamic and pharmacokinetic studies with two examples, 12e and 12j, showed the series to be similarly effective at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice.

Full text of DOI:10.1021/jm300276x

Article
pubs.acs.org/jmc
Discovery and Optimization of a Series of 3-(3-Phenyl-3H-
imidazo[4,5-b]pyridin-2-yl)pyridin-2-amines: Orally Bioavailable,
Selective, and Potent ATP-Independent Akt Inhibitors
Mark A. Ashwell, Jean-Marc Lapierre, Christopher Brassard, Karen Bresciano, Cathy Bull,
Susan Cornell-Kennon, Sudharshan Eathiraj, Dennis S. France, Terrence Hall, Jason Hill, Eoin Kelleher,
Sampada Khanapurkar, Darin Kizer, Steffi Koerner, Jeff Link, Yanbin Liu, Sapna Makhija, Magdi Moussa,
Nivedita Namdev, Khanh Nguyen, Robert Nicewonger, Rocio Palma, Jeff Szwaya, Manish Tandon,
Uma Uppalapati, David Vensel, Laurie P. Volak, Erika Volckova, Neil Westlund, Hui Wu, Rui-Yang Yang,
and Thomas C. K. Chan*
ArQule Inc., 19 Presidential Way, Woburn, Massachusetts 01801, United States
ABSTRACT: This paper describes the implementation of a biochemical and biophysical screening
strategy to identify and optimize small molecule Akt1 inhibitors that act through a mechanism distinct
from that observed for kinase domain ATP-competitive inhibitors. With the aid of an unphosphorylated
Akt1 cocrystal structure of 12j solved at 2.25 Å, it was possible to confirm that as a consequence of
binding these novel inhibitors, the ATP binding cleft contained a number of hydrophobic residues that
occlude ATP binding as expected. These Akt inhibitors potently inhibit intracellular Akt activation and
its downstream target (PRAS40) in vitro. In vivo pharmacodynamic and pharmacokinetic studies with
two examples, 12e and 12j, showed the series to be similarly effective at inhibiting the activation of Akt and an additional
downstream effector (p70S6) following oral dosing in mice.
approach were customized to maximize the likelihood of
identifying molecules exhibiting this mode of inhibition in the
hit-generation phase of the program by utilizing a suite of
biophysical and biochemical assays in parallel.
INTRODUCTION
■
Akt (protein kinase B or PKB) is a serine/threonine kinase that
functions as a key signaling node in the PI3K/Akt/mTOR
pathway and plays a central role in cell proliferation, migration,
survival, and angiogenesis.1 Oncogenic activation of Akt signal
transduction has been described for a variety of human cancers,
and it is therefore a compelling target for therapeutic
intervention.2 Akt1, Akt2, and Akt3 are three members of the
Akt kinase family, each exhibiting specific patterns of expression
in both normal and tumor tissues.3
Small molecule inhibitors of Akt are emerging with a few
having advanced into clinical trials.4 With one notable
exception5 most of the Akt inhibitors described to date are
ATP-competitive and, because of the similarity of the ATP
binding sites of the AGC kinase family, are generally poorly
selective against closely related kinases. Furthermore they show
little selectivity among the Akt isoforms.
We recently described a novel inhibition mode for c-Met
kinase6 that led to a new approach for the design and discovery
of inhibitors that stabilize autoinhibited conformations of
kinases.7 After characterization of the role of hydrophobic
residues within the ATP-binding cleft in the autoinhibitory
state, ATP-independent FGFR kinase inhibitors were identified
and biologically characterized. Herein, we provide an expansion
of this concept through the enablement of an in silico design
and screening strategy to identify Akt1 inhibitors with an
analogous mechanism of action. The goal was to identify
inhibitors that utilized the intrinsic negative regulatory function
of hydrophobic clusters in the ATP-binding cleft to inhibit the
kinase activity of Akt1. The design and implementation of this
The Akt kinase family members, Akt1, Akt2, and Akt3, have
three conserved domains, a N-terminal pleckstrin homology
domain (PH domain), an ATP binding kinase domain, and a
hydrophobic motif containing a C-terminal regulatory domain.8
While there is high homology between the catalytic kinase
domains of the three Akt isoforms and other AGC family
members, the unphosphorylated Akt2 isoform has been shown
to adopt a very distinct inactive conformation compared to
other AGC-kinase family members. In the published crystal
structure of Akt2,9 there is a disordered αC-helix, and the ATP-
binding cleft of Akt2 collapses into hydrophobic clusters that
results in the steric occlusion of ATP from the binding site. It
was hypothesized that the PH domain in Akt1 may play a role
in stabilizing this inactive conformation. Indeed, unlike in Akt2,
when a Lys-C limited proteolytic cleavage of Akt1 was
attempted, it did not lead to a separable PH domain, indicating
that the PH-domain stabilization of the unphosphorylated,
inactive Akt1 was likely stronger than observed in Akt2. This
suggested that maximal stabilization of the autoinhibited forms
of Akt might necessitate the utilization of the full-length
unphosphorylated forms of the enzymes. To optimally evaluate
the ability of compounds to bind to Akt1 in the inactive state,
several biophysical methods that included indirect affinity mass
Received: February 27, 2012
© XXXX American Chemical Society
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a
Scheme 1
a
Reagents and conditions: (a) R3NH2, DIEA, DMSO or 1,4-dioxane, 80 °C, overnight; (b) R4CHO, Na2S2O4, DMSO, MeOH, 100 °C, overnight.
a
Scheme 2
a
Reagents and conditions: (a) anilines, DIEA, DMSO or 1,4-dioxane, 80 °C, overnight; (b) R4CHO, Na2S2O4, DMSO, MeOH, 100 °C, overnight;
(c) 4.0 M HCl in 1,4-dioxane, 1,4-dioxane, room temperature, overnight.
a
Scheme 3
a
Reagents and conditions: (a) tert-butyl 4-aminobenzylcarbamate, tert-butyl 3-aminobenzylcarbamate or ethyl 2-(4-aminophenyl)acetate, 1,4-
dioxane, diisopropylethylamine, 80 °C, overnight; (b) 2-aminonicotinaldehyde, Na2S2O4 (85%), DMSO, MeOH, 100 °C, overnight.
spectrometry (IA-MS),10 thermal shift assay (TSA),11,12 and
surface plasmon resonance (SPR)13 were employed.
in the activation loop by the membrane-localized phosphoinosi-
tide-dependent kinase 1 (PDK1). Full activation of Akt requires
phosphorylation of Ser473 on the HM domain by an upstream
kinase, the mTORC2 complex.14 Therefore, the unphosphory-
lated full length protein constructs (Akt1 (1−480), Akt2 (1−
481), and Akt3 (1−479)) were preincubated with potential
inhibitors followed by the addition of the activating kinases
PDK1 and MAPKAPK2 (a functional surrogate for mTORC2-
mediated phosphorylation), lipid vesicles, and ATP prior to the
addition of substrate. To remove the possibility that inhibitors
of PDK1 and MAPKAK2 would complicate data analysis,
In order to determine the functional consequence of
stabilizing the autoinhibitory conformation, unphosphorylated
full length Akt proteins were used in the Akt biochemical
assays. Biochemical inhibition was determined following in situ
activation after preincubation with test compounds. The
activation of Akt involves translocation to the plasma
membrane and binding to phosphoinositides. The interaction
of phosphoinositides with the PH domain of Akt induces a
conformational change that enables phosphorylation of Thr308
B
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a
Scheme 4
a
Reagents and conditions: (a) LiOH, THF−MeOH−H2O, room temperature; (b) aniline or substituted anilines, HBTU, TEA, DMF, room
temperature; (c) arylboronic acid, Pd(PPh3)4, saturated NaHCO3 solution, toluene−ethanol, 100 °C, overnight.
counterscreens against these two kinases were employed.15
Finally, in the screening phase of the project, the activated
catalytic domain (Akt1 (104−480)) was employed as a cross-
screen.
salts 5a, 5f, 5g, and the intermediate for preparing 8g. The
ammonium intermediate 5a was coupled with benzoic acid
under standard HBTU conditions to provide amide 8a. 8b and
8c were obtained using the appropriate acids and 1,1′-
carbonyldiimidazole (CDI). Reaction of 5a with acetyl chloride
gave 8d. 8e was prepared by coupling 5f with benzoic acid
using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI)
as the coupling reagent. Finally, 5g was converted into 8f using
benzoyl chloride in the presence of NaOH. Further
modification of the halogen substituents of 8e and 8f using
phenyl- or 3-pyridylboronic acids under standard Suzuki
conditions gave 12d and 12f, respectively. Reductive amination
of 5a with benzaldehyde in the presence of tetramethylammo-
nium triacetoxyborohydride provided amine 8h. 9 was prepared
from 8e using zinc coupling. Reaction of 8e with cyclo-
pentylzinc bromide in the presence of Pd[P(tBu)3]2 and
potassium tert-butoxide in THF gave 9 in a modest yield.18
Amides 12e and 12g−k were prepared by reversing the
sequence of Suzuki coupling and amide formation from that
used for compounds 12d and 12f. The boc protected
intermediates 4a, 6c, and 6d (Scheme 6) provided 10c−f
following a Suzuki coupling reaction. Removal of the boc group
gave the hydrochloride salts 11a−d which were converted into
the amides 12e and 12g−k using CDI as the coupling reagent
and the corresponding acids.
SYNTHESIS
■
Starting from commercially available 2-chloro-3-nitropyridine
(1a), 5-bromo-2-chloro-3-nitropyridine (1b), and 2,6-dichloro-
3-nitropyridine (1c), 2-chloro displacement with the appro-
priate aniline in DMSO or dioxane with base provided
compounds 2a−n in good yields (Schemes 1−3) . Compound
3a−h (Scheme 1) were prepared by condensation of 2a−e with
the corresponding pyridylaldehydes. The one-pot reductive
cyclization was carried out using sodium hydrosulfite in a
mixture of DMSO and methanol at 100 °C. In a similar manner
4a−c, 5c, and 5e (Scheme 2) were synthesized from
intermediates 2f−i. Removal of the boc protection from 4a,
4b, and 4c was effected by treatment with 4 M HCl in dioxane
at room temperature to provide 5a, 5b, and 5d, respectively.
Intermediates 6a−e (Scheme 3) were prepared using
standard displacement and cyclization chemistry as described
in Schemes 1 and 2.
As shown in Scheme 4, the two ester intermediates 6a and
6b were converted into phenylacetamides 7 and 12a−c. Acid
6a was first hydrolyzed to the lithium carboxylate salt 6f which
was subsequently coupled with aniline using HBTU to yield the
amide 7. Alternatively, the 5-chloro substituent of 6b was
reacted with either 3-acetamidophenylboronic acid or 3-
pyridylboronic acid using a Suzuki reaction.16,17 Hydrolysis to
the corresponding lithium salts 6g and 6h followed by coupling
to the appropriate anilines gave the desired amides 12a−c.
Boc protected aminomethyl intermediates 4a and 6c−e were
converted into a series of amides and amines as shown in
Schemes 5 and 6. Removal of the boc protecting group from 4a
and 6c−e with 4 M HCl in dioxane provided the hydrochloride
RESULTS AND DISCUSSION
■
To demonstrate that ATP did not bind to the full length
unphosphorylated Akt1 (1−480) and that a representative
ATP-competitive inhibitor also did not bind to this form of the
protein, binding affinity and biochemical inhibition were
measured.
The ATP mimetic, AMP-PNP, did not bind to and/or
stabilize the full length inactive Akt1 (Table 1), indicating that
the protein is in a conformation that precludes ATP binding.
C
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a
Scheme 5
a
Reagents and conditions: (a) 4.0 M HCl in 1,4-dioxane, 1,4-dioxane, room temperature, overnight; (b) acids, HBTU, TEA, DMF, room
temperature; (c) benzoic acid, 1,1′-carbonyldiimidazole, DMA, room temperature; (d) acetyl chloride, DIEA, DCM; (e) benzoic acid, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, diisopropylethylamine, DCM, room temperature; (f) benzoyl chloride, NaOH, water,
room temperature; (g) benzaldehyde, Me4N(OAc)3BH, TEA, AcOH, DCE, room temperature, 20 h; (h) cyclopentylzinc bromide, Pd(PtBu3)2,
KOtBu, THF, microwave, 100 °C, 15 min; (i) arylboronic acid, Pd(PPh3)4, aqueous NaHCO3, ethanol−toluene, reflux, overnight.
Similarly, the ATP-competitive inhibitor A67456319 did not
bind and/or stabilize the full length inactive Akt1, thereby
providing additional support that the assays employing this
form of Akt1 would be suitable for the identification of non-
ATP competitive inhibitors. Both AMP-PNP and the ATP-
competitive inhibitor showed stronger affinity for the
unphosphorylated truncated kinase domain (residues 144−
480) (Table 1).
These data are consistent with the underlying assumption
that the unphosphorylated full length Akt1 adopts an
autoinhibitory conformation and that ligands that bind to and
stabilize this conformation can be experimentally differentiated
from ATP-competitive ligands.
While A674563 and AMP-PNP do not bind to the full length
inactive Akt1 protein used in the biophysical assay (Table 1),
A674563 is a biochemical inhibitor in the substrate
phosphorylation assay employing the same full length inactive
Akt1 protein. This indicates that in order for A674563 to show
inhibition, the conformation of the Atk1 protein must change.
As A674563 is an ATP-competitive inhibitor and the ATP
mimic AMP-PNP does not bind to the full length inactive Akt1
protein, it is likely that the protein moves to an active
conformation during the course of the biochemical assay so that
ATP can bind (and phosphorylate the substrate) and thus
A674563 can compete for this conformation.
Initially we employed indirect affinity mass spectrometry (IA-
MS) to identify binders to the unphosphorylated Akt1 (1−480)
from our compound collection. The compounds selected for
screening were from three distinct sources. Library 1 consisted
of approximately 7000 members across multiple scaffolds
selected to represent active kinase inhibitors (those most likely
to be type 1 or type 2 inhibitors). The second library, library 2,
comprised approximately 5000 members of multiple scaffolds
designed to target inactive conformations of kinases other than
type 2 inhibitor designs. The final collection of molecules (1000
members across more than 11 scaffolds) was designed in silico
to be specifically tailored toward the stabilization of the inactive
conformations of Akt1.20
Compounds from libraries 1 and 2 were pooled and screened
in mixtures of 20 compounds per well by the IA-MS technique,
and the compounds binding to the protein were identified by
their unique mass fingerprint using a hyphenated SEC/LC/MS
protocol. This rapid screen resulted in the identification of
more than 1200 binders. In order to provide a ranking of these
binders, a confirmatory binding screen using a thermal shift
assay was employed. From monitoring of the shift in the
D
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a
Scheme 6
a
Reagents and conditions: (a) arylboronic acid, Pd(PPh3)4, aqueous NaHCO3, ethanol−toluene, reflux, overnight; (b) 4.0 M HCl in 1,4-dioxane,
1,4-dioxane, room temperature, overnight; (c) acids, 1,1′-carbonyldiimidazole, DMA, room temperature.
a
Table 1. Biochemical and Biophysical Results for Two Reference Compounds
thermal shift assay, ΔTm (°C)
Trp fluorescence quench assay, Kd (μM)
biochemical assay, IC50 (μM)
Akt1 (1−480)
ligand
Akt1 (1−480)
no shift
no shift
Akt1 (144−480)
1.9
1.3
Akt1 (1−480)
Akt1 (144−480)
b
AMP-PNP
A674563
NB
3.3
2.1
>100
0.27
b
NB
a
See Experimental Section for assay details. The Tm shifts were measured at the following concentrations: AMP-PNP at 1.2 mM and A674563 at 15
b
μM. NB = no binding.
protein melting temperature (Tm) in the presence and absence
of the ligand, the degree of stabilization was determined. A shift
greater than 0.6 °C in the protein Tm (3 × STD) was taken as
indicative of compound binding. Previous studies have shown
that the degree of stabilization correlates well with the binding
affinity of the inhibitor. The in silico designed Akt1 library
(library 3) was directly screened in the thermal shift assay.
From the combined thermal shift analysis, approximately 80
compounds were identified as stabilizers of the full length
unphosphorylated form of Akt1 (Table 2). While the protein
stabilization for these initial hits was generally modest (ΔTm ≈
1 °C), 11 distinct scaffolds were identified as stabilizers with
low biochemical inhibition. In addition, several additional
scaffolds contained a number of stabilizers of Akt for which no
biochemical inhibition could be detected.
biochemical inhibition of both inactive and preactivated
enzymes by these analogues, it was clear that the assays were
providing differentiation at the substructure level.
This differentiation within scaffold 24 was clearly demon-
strated by comparing the profiles of 3b (a 3-(3H-imidazo[4,5-
b]pyridin-2-yl)pyridin-2-amine subseries member) and 3e (a 2-
(pyridinyl)-3H-imidazo[4,5-b]pyridine subseries member). Rel-
atively small structural modifications on the central 3H-
imidazo[4,5-b]pyridine core provided quite distinct biological
profiles against Akt1 (Table 3). For example, while 3b binds to
(ΔTm = 1.9 °C) and biochemically inhibits the full length Akt1
(IC50 = 3.9 μM), the structurally related analogue 3e neither
binds to nor inhibits Akt1. When 3b and 3e were tested for
their inhibition of the preactivated kinase domain of Akt1
(104−480), 3e was an Akt1 inhibitor (IC50 = 5.9 μM) while 3b
did not show any appreciable inhibition (6.3% inhibition at 30
μM). The lack of inhibition of the preactivated kinase domain
indicated that the subseries represented by 3b was inhibiting
Akt1 in a manner distinct from that of ATP-competitive
ligands.
Scaffolds were prioritized for optimization, and four were
advanced to the hit-to-lead stage of the project based on their
binding and biochemical inhibition profile. Two out of the four
selected scaffolds originated from library 2 and two from library
3.
Herein we describe the SAR and optimization of one of those
scaffolds. Scaffold 24 contained several structurally related
analogues including 2-(pyridinyl)-3H-imidazo[4,5-b]pyridine
and 3-(3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine deriva-
tives. By comparison of the thermal stabilization with
In order to gain a better understanding of the structure−
activity relationships (SARs) of scaffold 24, a small set of
compounds were prepared in the hit-to-lead phase of the
program (Table 3). Replacement of the 4-pyridine ring of 3e
with the 2-aminopyridine ring of 3b gave 3d which did not bind
E
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a
3d, although the reason for the lack of biochemical inhibition
for 3g was not apparent. Replacement of the 4-pyridine ring in
3e with the 2-amino pyridine group provided 3c which was also
a nonbinder and more than a 10-fold weaker inhibitor than 3b.
In addition, comparison of 3a and 3f, which were both
biophysically and biochemically inactive, further confirmed the
importance of the 2-aminopyridine substituent on the 3H-
imidazo[4,5-b]pyridine core. The importance of the para-
substitution for activity was clearly demonstrated by 3h.
Movement of the tert-butyl group to the meta position resulted
in a loss of all activity against Akt1.
From these results, it was apparent that the screening
paradigm employed had successfully separated two closely
related series of Akt1 inhibitors by their mechanism of
inhibition. The next objective was to increase the potency of
the 2-(pyridinyl)-3H-imidazo[4,5-b]pyridine series, since this
class of compounds met our screening requirements of binding
to and preventing the activation of Akt1.
Having established a preference for para-substituents
(compare 3h, a nonbinder, to 3b) on the pendent phenyl
ring, replacement of the tert-butyl group was examined (Table
3). Compound 5d indicated that the tert-butyl group could be
replaced with a smaller more polar substituent, such as an
aniline group, to provide a concomitant increase in both
binding and biochemical inhibition (ΔTm = 4.1 °C which
corresponds to a Kd of approximately 0.5 μM, IC50 = 0.74 μM).
The acetamide 5c further demonstrated that this position could
be modified, although no apparent improvement over 5d was
observed. Replacement of the undesirable aniline to the more
polar benzylamine 5a provided an improvement over 3b with
decreased protein stabilization when compared to the aniline
5d. 5b confirmed that the newly introduced benzylamine group
also required the presence of the 2-amino group in the pyridine
ring for binding and submicromolar inhibition. Its removal
resulted in a loss of binding and a decrease in inhibition.
Compared to 3b, the acetamide 5c had a 3-fold lower IC50, and
so additional amides were prepared. The benzamide group of
5e (Table 4) increased the stabilization by more than 3 °C over
the simple aniline 5d, and similar improvements were observed
for the benzylamide 8a and the reverse amide 7.
Table 2. Hit Generation Results
library
size
no. of TSA
actives
no. of biochemical
actives
Akt1 IC50
scaffold
(μM)
c
1
1109
106
382
776
170
81
2
3
1
5
9
1
1
19
1
2
7
2
2
1
1
2
3
2
5
2
0
1
NA
2
4.3−8.9
c
5
0
NA
7
2
14−72
9
36
0
12−100
c
10
11
17
18
19
20
22
24
28
29
30
31
32
NA
c
14
0
NA
69
6
16−100
>100
19
1
8
2
16−100
9.4−100
20
5
c
6
0
NA
313
15
1
1.5−16
50−100
>100
3
9
1
32
3
12−71
c
196
9
0
NA
c
0
NA
b
33
151
40
2
10−100
22−100
b
34
2
b
a
See Experimental Section for assay details. Scaffolds from the in
silico directed homology methodology. NA = not active.
c
to or inhibit full length Akt1. As 3g demonstrated binding to
Akt1, albeit somewhat reduced (ΔTm = 0.9 °C), a negative
effect on binding of the bromine substituent was ruled out in
Table 3. Akt1 Biochemical and Biophysical Results for
a
Scaffold 24
The steric requirements for this benzamide improvement in
profile were further probed by analogues 8b, 8h, and 8c, and in
each case a decrease in activity compared to 5e was observed.
The loss in activity for the acetamide 8d and the meta-
positional isomer of 8a, 8g, showed clearly the sensitivity of this
position to modification with a strong preference for an
aromatic ring attached to the para-position with a short
nonbasic linker.
Having identified 7 and 8a as strong binders to Akt1 with
IC50 below 0.03 μM, a number of AGC-kinase family members
were added to a minipanel screen together with representatives
from CAMK, CMGC, STE, and TKs.21 At a screening
concentration of 5 μM, 7 and 8a showed no inhibition above
25% of any kinase in the panel, demonstrating their selectivity
for Akt and providing support for the further optimization of
the inhibitors.
To assess the isoform selectivity of the inhibitors, both
binding affinity and biochemical inhibition were assessed using
unphosphorylated forms of Akt2 (1−479) and Akt3 (1−481)
proteins. These additional assays were employed in the lead
optimization phase of the program to monitor the effect of
chemical modification on the isoform selectivity. In vitro
cellular activity was assessed in two cell lines, A2780 (ovarian)
a
See Experimental Section for assay details. The compounds were
tested at 30 μM in the thermal shift assay.
F
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the same cell lines was also assessed using a standard MTS
assay.
Table 4. Akt1 Structure−Activity Relationship for Para-
a
Substituents
The initial results from the cell-based experiments to assess
the ability of the compounds to inhibit phosphorylation of Akt
were encouraging, as compounds of modest potency were able
to induce cellular pharmacodynamic changes (Table 5). Both
8a and 7 were cell-penetrant as shown by their ability to reduce
the levels of phosphorylation of Ser473 and Thr308 in A2780
cells (IC50 < 0.5 μM). However, this did not result in an
expected reduction of the phosphorylation of the downstream
target PRAS40 (IC50 > 10 μM). In the AN3CA cells, both 8a
and 7 were approximately 10-fold less potent at inhibiting the
phosphorylation of Ser473, and no significant inhibition of
phosphorylation of Thr308 or PRAS40 (IC50 > 10 μM) was
apparent. Additionally, both compounds failed to induce
appreciable growth inhibition in either cell line. These data
clearly suggested that increased in vitro potency against Akt1
and possibly Akt2 and/or Akt3 would be required if more
pronounced cellular effects were to be obtained.23
In order to achieve the necessary improvements in Akt
inhibition, additional sites on the 3-(3H-imidazo[4,5-b]pyridin-
2-yl)pyridin-2-amine core were modified (Table 6). It was
found that a lipophilic substituent at the 6-position of the 3-(3-
phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine core
generally improved binding to Akt isoforms, although some
groups such as the cyclopentyl of 9 (Table 6) increased binding
affinity to Akt2 rather than Akt1 when compared to 7 or 8a.
However, with a 3-pyridyl substituent at the 6-position a
substantial increase in Akt1 stabilization (ΔTm = 9.2 °C) and
inhibition (IC50 = 8 nM) was observed for 12e when compared
to 8a. A similar increase in binding to Akt2 (ΔTm = 4.8 °C)
resulted in a 22-fold increase in inhibition (IC50 = 30 nM).
Introduction of a meta-F group into the phenyl ring of the
benzamide group of 12e provided 12g, which demonstrated a
further increase in binding to Akt2 (ΔTm = 5.4 °C) with no
significant change in the biochemical inhibition. As shown in
Table 5, the two most potent 6-substituted analogues, 12e and
its meta-F analogue 12g, were also potent cellular inhibitors of
the activation of Akt (antibodies recognize all forms of the
corresponding phospho-Akt) in both human cancer cell lines.
a
See Experimental Section for assay details. The compounds were
tested at 30 μM in the thermal shift assay.
and AN3CA (endometrial), expressing all three Akt isoforms.
The effects of the inhibitors on the phosphorylation of Ser473
and Thr308, and downstream Akt-mediated phosphorylation of
PRAS40,22 were measured 2 h after treatment by Western blot
analysis. Additionally, the inhibition of cellular proliferation in
a
Table 5. Cellular Potency of Inhibitors
p-Akt, IC50 (μM)
S473
T308
p-PRAS40 (T246), IC50 (μM)
MTS, IC50 (μM)
A2780 AN3CA
compd
A2780
AN3CA
A2780
AN3CA
A2780
AN3CA
7
0.21
2.5
1.3
>10
>10
>10
3.1
>10
>10
>10
>10
>10
1.5
57
70
8a
0.41
5.6
0.37
0.17
0.21
0.054
0.015
0.055
0.68
0.062
0.067
0.20
0.14
0.18
0.032
0.69
>10
>100
65
>100
60
8e
<0.012
0.27
2.5
>10
9
3.7
9.4
2.1
65
33
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
0.046
0.12
0.047
0.09
0.069
0.16
0.08
1.4
0.049
0.26
0.051
1.1
0.31
0.14
0.48
1.8
6.0
3.3
9.4
>100
>100
23
2.1
1.7
8.8
>100
>100
13
0.065
0.33
2.5
0.98
1.5
0.10
0.091
4.2
0.95
0.74
0.58
0.91
0.41
0.25
1.3
0.034
0.054
0.13
3.2
0.046
0.11
>10
0.044
0.095
0.049
0.17
>10
0.82
0.84
>10
0.22
2.1
19
13
1.6
9.5
2.2
10
1.5
5.5
2.0
10
0.068
0.027
0.19
0.045
0.34
a
See Experimental Section for assay details.
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xenografts, and the inhibition of the phosphorylation of Akt as
well as the downstream kinase p70S6 (Thr389) was evaluated
following treatment with the inhibitor. In addition, tumor and
plasma samples were collected and the concentration of 12e
was determined. As shown in Table 8, 2 h after oral solution
Table 6. Structure−Activity Relationship of 6-Substituted
a
Analogues
Table 8. In Vivo Pharmacokinetic and Pharmacodynamic
a
Results For 12e and 12j
% inhibition
plasma
tumor
concn
(μM)
po dose time p-Akt
p-Akt p-p70S6 concn
compd (mg/kg) (h) (S473) (T308) (T389)
(μM)
12e
12j
100
250
2
2
4
82
62
51
25
57
67
76
90
86
14.1
2.0
8.4
1.2
0.6
1.2
a
See Experimental Section for assay details.
dosing at 100 mg/kg, 12e reduced the phosphorylation of Akt
(Ser473) by 82% and the phosphorylation of p70S6 by 76% in
tumors. Plasma levels of 12e at the 2 h time point were 14.1
μM with the tumor concentrations slightly lower (8.4 μM).
These positive results demonstrated that this chemical series
could provide a selective Akt1 inhibitor with good druglike
properties capable of inhibiting pharmacodynamic markers in
vivo following oral delivery. However, the lack of cellular
potency in the AN3CA MTS assay (IC50 > 100 μM) needed to
be addressed in order to demonstrate in vivo tumor growth
inhibition.
Increasing the inhibition of cellular proliferation became the
goal of the next phase of optimization and was achieved by the
introduction of substituents at the 5-position of the 3-(3-
phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine core
(Table 9). This proved to be a very effective strategy for
achieving dramatic improvements in Akt1 binding as shown by
the range of Tm values (increase in ΔTm from 9.7 to 13.0 °C), a
significant increase compared to the 6-positional isomers.
Interestingly, in most cases this did not directly translate into a
similar increase in the Akt1 biochemical potency as had been
observed with the 5-substituents. For example, 12j had the
same Akt1 biochemical inhibition as 12g (IC50 = 5 nM), yet the
ΔTm upon binding of 12j was 13 °C compared to the 9.4 °C
for 12g. A similar trend was also apparent for Akt2, a possible
explanation for which is discussed below.
a
See Experimental Section for assay details. The compounds were
tested at 15 μM in the thermal shift assay.
Specifically, 12e demonstrated an approximately 70-fold
decrease in IC50 for the inhibition of phosphorylation of Ser473
(IC50 = 80 nM) and marked improvement in the potency in
inhibiting Thr308 phosphorylation (IC50 = 91 nM) in the
AN3CA cell line, compared to 8a (IC50 > 10 μM). A further
improvement in cellular potency for 12g compared to 12e was
observed in both cell lines. Additionally, it was also apparent
that an aromatic 6-substituent (phenyl or 3-pyridyl) provided
cellular inhibition of the downstream marker, phospho-
PRAS40, in the AN3CA cancer cell line, the first time such a
pharmacodynamic effect had been demonstrated in the
campaign. However, this pharmacodynamic effect was not
accompanied by a concomitant increase in antiproliferative
potency. Indeed, the most potent 6-substituted Akt1 inhibitor
in Table 6 (12g IC50 = 5 nM) showed only modest (IC50 > 10
μM, Table 5) activity in the cell proliferation assay in the two
human cancer cell lines.
The in vitro ADME profile of 12e (Table 7) was satisfactory
with good microsomal stability and a lack of CYP450
inhibition. Despite the weak effect on cellular proliferation,
12e was selected for proof-of-concept pharmacokinetic and
pharmacodynamic studies in vivo.
In a mouse pharmacokinetic study (oral dose at 100 mg/kg,
iv at 2.5 mg/kg), 12e was well absorbed with good oral
bioavailability (F = 46%). For the pharmacodynamic assess-
ment, NCr-M nude mice were implanted with AN3CA tumor
The crystal structure of nonphosphorylated Akt1 (1−446)
was solved in complex with 12j to assist the SAR
optimization.24 In the cocrystal structure the N-terminal PH
domain and the C-terminal kinase domains were tightly
associated in a “closed” conformation (Figure 1A). The overall
structure of Akt1 is similar to a reported crystal structure of
Akt1 (1−443) bound to an allosteric inhibitor which was
published25 during the course of this work. Crucially, however,
a
Table 7. In Vitro ADME Properties of Selected Compounds
CYP 450, IC50 (μM)
liver microsomal stability at 1.0 μM, T1/2 (min)
compd
1A2
2C19
2C8
2C9
2D6
3A4
HLM
MLM (F)
MLM (M)
7
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
47.6
60
>60
>60
>60
>30
>60
>60
>60
>30
8a
>10
>10
>10
12e
12j
>10
>10
>10
>30
>30
1−10
1−10
1−10
a
See Experimental Section for assay details.
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a
Table 9. Structure−Activity Relationship of 5-Substituted Analogues
a
See Experimental Section for assay details. The compounds were tested at 15 μM in the thermal shift assay.
the important structural element of this work, the 2-
aminopyridine group of the (3-phenyl-3H-imidazo[4,5-b]-
pyridin-2-yl)pyridin-2-amine core, makes direct hydrogen
bond interactions with the loop of β4 which are distinctly
different. Additionally the αC-helix of the kinase domain is
disordered.
nitrogen around the aromatic ring disrupts this and leads to a
loss of binding (Table 3). The plane of the pendent phenyl ring
is perpendicular to the core and forms a direct hydrophobic
interaction with the conserved Tyr272 residue of the AGC
family.
Visual inspection of the cocrystal structure shows that
substituents other than those at the para position will be
detrimental to binding. In addition, the amide linker does not
participate in any direct interaction with Akt1. Rather, it
functions to orientate the terminal phenyl ring for a
hydrophobic interaction with Ile84 of the PH domain. This
likely explains the need for an additional hydrophobic ring for
optimal binding and inhibition and a rationale as to why there is
little preference for the amide or reverse amide (Table 4). The
improved binding of the 5-substituents over the 6-substituents
and the preference for the phenylacetamide group in the meta-
position are due to a combination of complementary matching
of the phenyl ring with a nonpolar region, which is made up of
the aliphatic side chains of several amino acid residues, and a
direct hydrogen bond interaction of the carbonyl of the
phenylacetamide with the NH main chain atoms of the Trp80
residue.
A rationalization of the observed Akt isoform binding
specificity for these inhibitors is apparent also. The Val270
residue provides a hydrophobic interaction with the core
(Figure 1C). In Akt2, this residue is also valine, while in Akt3
the corresponding residue is the sterically larger isoleucine
(Figure 1D). The additional methyl group in Akt3 is expected
to require a reorientation in order to bind the inhibitors in a
similar manner.
As expected, the kinase domain adopts a DFG-out
conformation similar to the inactive form of the Akt2 catalytic
domain structure. The activation loop in the kinase domain is
displaced outward because of a direct interaction of the PH
domain which is in an inward orientation with the C-lobe such
that the phosphoinositide binding region is buried at the
interface. 12j is bound at an allosteric pocket formed between
the kinase domain and the PH domain. The ATP-binding
region is occupied with nonpolar residues which are associated
as tight clusters. The cleft is closed with a “hydrophobic lock”
which may function to sterically exclude the binding of both
ATP and ATP-competitive inhibitors (Figure 1B). The
interdomain interactions stabilize the autoinhibitory conforma-
tion of Akt1 which is likely induced by 12j binding, as the
majority of the inhibitor is buried in a groove between the PH
and kinase domains. The conserved tryptophan (Trp80) in the
PH domain makes a key hydrophobic interaction with the core
of 12j, and the ring-nitrogen is bridged to the aspartic acid
residue of the DFG-motif by a water molecule mediated
hydrogen bond network. The plane of the imidazopyridine ring
of the core is hydrophobically stacked between Trp80 and the
E-helix residues Val270 and Leu264 (Figure 1C).
Solving the structure with 12j assisted in the SAR
optimization of this and other series of Akt1 inhibitors. The
position of hydrogen donor and acceptor groups present within
the 2-aminopyridine moiety of the 3-(3H-imidazo[4,5-b]-
pyridin-2-yl)pyridin-2-amine is critical for the inhibitor
interaction. Removal of the amine and/or moving the pyridine
The cocrystal structure of 12j bound to Akt 1 shows that the
inhibitor occupies a deep cavity at the interface of kinase and
the PH domains. A tight network of inhibitor interactions with
Akt suggests that the binding requires extensive rearrangement
I
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Figure 1. Cocrystal structure of Akt1 (2−446) in complex with 12j (PDB accession code 4EJN). (A) 12j binds deep in the cavity between the kinase
and PH domains and bridges the two domains with extensive polar and nonpolar interactions. The tight association of the PH domain with the
kinase domain prevents the A-loop from adopting an active conformation. (B) A close view of the ATP binding region is shown to indicate the
hydrophobic residues organized into hydrophobic clusters with steric exclusion of ATP binding in the cleft. 12j binds beneath the hydrophobic
clusters, and the aminopyridyl group binds to the loop of the β4-strand and αC-helix through hydrogen bonding interactions. The plane of
imidazopyridine core is matched to that of the indole ring of Trp80 of the PH domain. (C) Sequence alignment of the Akt isoform in a region that
may play a role in isoform specificity. (D) The side chain of Val270 essentially complements the hydrophobic interaction with the aromatic core of
12j. Substitution of Val270 with Ile as in Akt3 is likely to have a negative impact on Akt3 binding, favoring Akt1 and Akt2 selectivity.
of residues at the interface that facilitates binding of the
inhibitor deeply between the interdomains. The impact of this
conformational change upon binding was measured by
following the kinetics of the interaction using SPR. A slow
on-rate (3.7 × 104 M−1 s−1) and off-rate (1.0 × 10−4 s−1)
indicate strong binding of 12j to Akt1 (koff/kon = 2.7 nM),
which is in agreement with the large thermal stabilization of 13
°C measured by the thermal shift assay. The measured on-rate
is several orders of magnitude lower than the rate of a diffusion
governed process, suggesting that the binding likely induces a
large conformational rearrangement in Akt1. This is consistent
with the structural data and may contribute to the contraction
of the dynamic range of the biochemical assay for the most
potent binders of the 5-substituted analogues. The mechanism
of action of 12j with Akt1 was studied with respect to ATP. 12j
showed mixed uncompetitive/noncompetitive type kinetics
with respect to ATP. In addition the inhibition of Akt1 by 12j
was compared to that of the ATP-competitive ligand A674563
by determining the fold decrease in IC50 with increasing
concentrations of ATP (up to 1 mM) (Figure 2). As expected,
the fold decrease is considerably less with 12j, as is to be
expected with its mechanistic profile.
On the basis of the in vitro biochemical profile, the stronger
binding affinity, its slow off-rate, and superior cellular
pharmacodynamic profile (especially in AN3CA cells), 12j
was advanced into broader kinase panel screening and further
in vivo studies.
In a broad kinase panel screen of 297 kinases, 12j showed
excellent Akt selectivity with no kinase being inhibited at
greater than 17%.26 In vitro ADME profiling results (Table 7)
showed 12j to have good microsomal stability and a reasonable
CYP450 inhibition profile. An oral solution formulation of 12j
allowed a dosing at 250 mg/kg, and two time points (2 and 4
h) were selected for plasma and tumor sampling. As shown in
Table 8, the inhibition of phosphorylation of the downstream
marker p70S6 was excellent at both time points (90% at 2 h
J
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were eluted from the HPLC column (Maccel 120-10-C18 SH, 10 μm,
20 mm i.d. × 50 mm) at 88 mL/min with an acetonitrile/water
gradient using 0.1% TFA as a modifier. Nuclear magnetic resonance
(1H NMR) spectra were recorded on a Varian Mercury plus NMR
1
spectrometer operating at 400.13 MHz frequencies for H, using a 5
1
mm ASW PFG probe capable of detecting H, 13C, 31P, 15N nuclei.
The proton chemical shifts (ppm) were referenced to the
tetramethylsilane standard (0 ppm). NMR data are reported with
these descriptions: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad peak.
N-(4-(tert-Butyl)phenyl)-3-nitropyridin-2-amine (2a). 2-
Chloro-3-nitropyridine 1a (1.0 g, 6.31 mmol), 4-(tert-butyl)aniline
(1.0 g, 6.72 mmol), and DIEA (1.74 mL, 10.0 mmol) in DMSO (20
mL) were stirred at 80 °C for 16 h. DMSO was removed under
reduced pressure to give a brown solid (2.2 g) which was purified by
column chromatography (1% MeOH/DCM, silica gel) to give 2a
Figure 2. Inhibition of Akt1 by 12j and the ATP competitive inhibitor
A674563 as a function of ATP concentration.
1
(1.55 g, 89%) as a red solid. Mp 156−158 °C. H NMR (400 MHz,
and 86% at 4 h) as was the degree of inhibition of
pAkt(Thr308) (57% at 2 h, 67% at 4 h). The inhibition of
pAkt(Ser473) was also robust, albeit somewhat less pro-
nounced than had been observed for 12e. Unfortunately, the
measured plasma and tumor concentrations of 12j at both time
points were low especially compared to the concentrations
observed for 12e which had been dosed at the lower level of
100 mg/kg. The final phase of the optimization was then
focused upon improving the druglike properties of these potent
and selective Akt inhibitors.
In conclusion, through the implementation of a screening
paradigm designed to thoroughly annotate Akt1 kinase
inhibitors by their mode of inhibition, a novel series of Akt1
inhibitors were discovered and advanced through lead
optimization. SAR-guided optimization of the selected series
using in vitro biophysical and biochemical assessment provided
highly selective Akt inhibitors. In vivo characterization
employed both pharmacokinetic and pharmacodynamic anal-
yses and characterized lead inhibitors as orally bioavailable, well
tolerated with robust inhibition of the activation of Akt as well
as a downstream substrate, p70S6. Further optimization of this
series is anticipated to yield additional biologically active Akt
inhibitors with antitumor activity in appropriate animal models
and will be detailed in the near future.
DMSO-d6) δ 9.92 (s, 1H), 8.51 (dd, J = 14.5, 5.6 Hz, 1H), 7.54 (d, J =
8.4 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 6.96 (dd, J = 8.1, 4.6 Hz, 1H),
1.29 (s, 9H). MS m/z 272 [M + H]+.
N-(4-((3-Nitropyridin-2-yl)amino)phenyl)acetamide (2g). 2g
was prepared from 1a as described for 2a using N-(4-aminophenyl)-
1
acetamide. Mp 177−180 °C. H NMR (400 MHz, DMSO-d6) δ 9.93
(s, 1H), 9.91 (s, 1H), 8.50 (dd, J = 8.3, 1.8 Hz, 1H), 8.47 (dd, J = 4.5,
1.8 Hz, 1H), 7.53 (d, J = 2.4 Hz, 4H), 6.93 (dd, J = 8.3, 4.5 Hz, 1H),
2.02 (s, 3H). MS m/z 273 [M + H]+.
tert-Butyl (4-((3-Nitropyridin-2-yl)amino)phenyl)carbamate
(2h). 2h was prepared from 1a as described for 2a using tert-butyl
1
(4-aminophenyl)carbamate. Mp 162−165 °C. H NMR (400 MHz,
DMSO-d6) δ 9.88 (s, 1H), 9.32 (s, 1H), 8.50 (dd, J = 8.2, 1.8 Hz, 1H),
8.45 (dd, J = 4.5, 1.7 Hz, 1H), 7.48 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 8.9
Hz, 2H), 6.92 (dd, J = 8.4, 4.5 Hz, 1H), 1.46 (s, 9H). MS m/z 331 [M
+ H]+.
tert-Butyl 4-((5-Bromo-3-nitropyridin-2-yl)amino)-
benzylcarbamate (2l). 2l was prepared from 2-chloro-3-nitro-5-
bromopyridine (1b) as described for 2a using tert-butyl 4-amino-
benzylcarbamate. Mp 178−180 °C. 1H NMR (400 MHz, DMSO-d6) δ
9.95 (s, 1H), 8.65 (m, 1H), 8.54 (s, 1H), 7.51 (d, J = 6.4 Hz, 2H), 7.23
(d, J = 6.4 Hz, 2H), 4.21−4.12 (m, 2H), 1.41 (s, 9H). MS m/z 423 [M
+ H]+.
tert-Butyl 4-((6-Chloro-3-nitropyridin-2-yl)amino)-
benzylcarbamate (2m). 2m was prepared as described for 2a
using 2-chloro-3-nitro-6-chloropyridine (1c) and tert-butyl 4-amino-
1
benzylcarbamate. Mp 103−106 °C. H NMR (400 MHz, CDCl3) δ
EXPERIMENTAL SECTION
10.24 (s, 1H), 8.45 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 6.2 Hz, 2H), 7.31
(d, J = 8.2 Hz, 2H), 6.79 (d, J = 8.6 Hz, 1H), 4.31 (d, J = 5.8 Hz, 2H),
1.46 (s, 9H). MS m/z 379 [M + H]+.
■
Chemistry. All reagents and solvents were purchased from
commercially available sources and used without further purification.
All reactions were carried out according to the indicated procedures
and conditions. Reactions were monitored by LC/MS analysis and/or
thin-layer chromatography (TLC) on silica-coated glass plates (EMD
silica gel 60 F254) with the indicated eluent. The compounds were
visualized by UV light (254 nm). LC/MS analysis was performed on a
Shimadzu HPLC/UV (214 nm and/or 254 nm wavelength) system
coupled to ELSD (Sedex 75, Sedere) and MS (ZQ, Micromass)
detectors. Compounds were dissolved in 100% DMSO and separated
on Zorbax SB-C8 rapid resolution cartridge (30 mm × 4.6 mm, 3.5
μm, 3 mL/min flow rate) using acetonitrile/water mobile phase with
0.1% TFA as a modifier. The gradient started at 5% acetonitrile and
linearly increased to 95% acetonitrile in 1.9 min, with 0.3 min hold at
95% and re-equilibration to the original conditions in a total of 2.5
min. The purity of the compounds selected for in vivo studies was
determined using an Agilent HP1100 system with a YMC C18 column
(4.6 mm × 100 mm, 5 μm, 1 mL/min flow rate) and acetonitrile/
water mobile phase with 0.1% TFA modifier. The gradient started at
2% acetonitrile, linearly increased to 30% acetonitrile in 7 min,
followed by a linear increase to 80% acetonitrile by 12 min and a hold
at 80% by 16 min. Purity was determined at 254 nm wavelength as
>95%. Library compounds were purified by reverse phase chromatog-
raphy on a preparative LC/UV/MS system (high throughput
purification) using a mass triggered fractionation method. Compounds
3-(4-(tert-Butyl)phenyl)-2-(pyridine-4-yl)-3H-imidazo[4,5-b]-
pyridine (3a). N-(4-(tert-Butyl)phenyl)-3-nitropyridin-2-amine 2a
(1.52 g, 5.6 mmol), isonicotinaldehyde (0.82 g, 7.61 mmol), sodium
dithionite (2.45 g, 14.1 mmol), methanol (15 mL), and DMSO (15
mL) were combined. The mixture was shaken at 100 °C for 18 h. The
methanol was removed under reduced pressure. The solution was
diluted with DMSO (40 mL) and H2O (40 mL) and filtered to yield a
brown solid (2.2 g). Purification by column chromatography (100%
DCM to 5% MeOH/DCM, silica gel) gave 3a (0.76 g, 41%) as a pale
yellow solid. Mp 184−186 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.62
(dd, J = 3.8, 2.0 Hz, 2H), 8.39 (dd, J = 3.3, 1.4 Hz, 1H), 8.29 (dd, J =
3.4, 2.0 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.49 (dd, J = 3.8, 2.1 Hz,
2H), 7.44 (dd, J = 5.0, 3.0 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 1.35 (s,
9H). MS m/z 329 [M + H]+. Anal. Calcd for C21H2ON4: C, 76.8; H,
6.14; N, 17.06. Found: C, 76.53; H, 6.15; N, 17.13.
3-(1-(4-(tert-Butyl)phenyl)-1H-benzo[d]imidazol-2-yl)-
pyridin-2-amine (3b). 3b was prepared as described for 3a using 2a
1
and 2-aminonicotinaldehyde. Mp 175−180 °C. H NMR (400 MHz,
acetone-d6) δ 8.44−8.42 (dd, J = 4.7, 1.2 Hz, 1H), 8.26−8.23 (dd, J =
8.2,1.6 Hz, 1H), 8.05−8.02 (dd, J = 6.3, 1.6 Hz, 1H), 7.75−7.73 (dd, J
= 7.8, 1.6 Hz, 1H), 7.70−7.68 (dd, J = 6.6, 2.3 Hz, 2H), 7.57−7.55
(dd, J = 8.6, 1.9 Hz, 2H), 7.48−7.44 (dd, J = 8.2, 4.7 Hz, 1H), 6.82−
6.78 (dd, J = 7.8, 6.3 Hz, 1H), 1.41 (s, 9H). MS m/z 344 [M + H]+.
K
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Anal. Calcd for C21H21N5·1.5TFA·0.1 acetone: C, 56.10; H, 4.48; N
13.46. Found: C, 56.32; H, 4.25; N, 13.70.
residue was purified by column chromatography (0−20% methanol/
DCM, silica gel) to give 4a as a yellow solid (2.0 g, 72%). Mp 141−
1
3-(3-(2-Methoxybenzyl)-3H-imidazo[4,5-b]pyridin-2-yl)-
pyridin-2-amine (3c). Step 1: N-(2-Methoxybenzyl)-3-nitropyridin-
2-amine (2b) was prepared as described for 2a using 1a and (2-
methoxyphenyl)methanamine. Step 2: 3c was prepared from 2b as
142 °C. H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 4.4 Hz, 1H),
8.19 (d, J = 8.4 Hz,1H), 7.99−7.98 (m, 1H), 7.51−7.40 (m, 1H),
7.39−7.37 (m, 5H), 7.21−7.19 (m, 1H), 7.00 (bs, 2H), 6.41−6.38 (m,
1H), 4.21 (d, J = 6.0 Hz, 2H), 1.41 (s, 9H). MS m/z 417 [M + H]+.
tert-Butyl 4-(2-(Pyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-yl)-
benzylcarbamate (4b). A solution of 2f (0.5 g, 1.45 mmol), sodium
dithionite (1.5 g, 8.6 mmol), and pyridine-3-carboxaldehyde (0.16 mL,
1.74 mmol) in DMSO (5 mL) and methanol (2.5 mL) was heated at
100 °C for 24 h. The mixture was cooled to room temperature and
methanol removed under reduced pressure. H2O (10 mL),
tetrahydrofuran (15 mL), and saturated sodium carbonate (30 mL)
were added followed by di-tert-butyl dicarbonate (1 g). After being
stirred at room temperature for 3 h, the mixture was poured into ethyl
acetate (150 mL), washed with H2O (300 mL), and dried over
anhydrous magnesium sulfate. The solids were removed by filtration,
and the supernatant was evaporated to dryness under reduced
pressure. The yellow oil was purified by column chromatography (70−
100% ethyl acetate in hexanes, silica gel) to yield 4b as an off-white
solid (0.27 g, 46%). Mp 182−183 °C. 1H NMR (400 MHz, CDCl3) δ
8.84 (d, J = 0.8 Hz, 1H), 8.63 (dd, J = 4.6, 1.6 Hz, 1H), 8.42 (dd, J =
4.6, 1.6 Hz, 1H), 8.18 (dd, J = 4.6, 1.6 Hz, 1H), 7.93 (m, 1H), 7.47−
7.45 (m, 2H), 7.37−7.29 (m, 4H), 5.3 (s, 1H), 4.41 (d, J = 6.0 Hz,
2H), 1.48 (s, 9H). MS m/z 402 [M + H]+.
1
described for 3a using 2-aminonicotinaldehyde. Mp 175−182 °C. H
NMR (400 MHz, acetone-d6) δ 9.2 (s,1H), 8.48−8.46 (dd, J = 4.7, 1.6
Hz, 1H), 8.20−8.16 (dd, J = 7.8, 1.2 Hz, 1H), 8.14−8.07 (m, 2H),
7.43−7.40 (dd, J = 7.8, 4.7 Hz, 1H), 7.28−7.24 (m, 1H), 7.03−6.97
(dd, J = 9.0, 0.78 Hz, 2H), 6.89−6.86 (dd, J = 1.6, 0.7 Hz, 1H), 6.83−
6.79 (m, 1H), 5.7 (s, 2H), 3.8 (s, 3H). MS m/z 332 [M + H]+. Anal.
Calcd for C19H17N5O·1.22TFA·0.5 acetone: C, 54.78; H, 3.94; N,
14.79. Found: C, 54.79; H, 3.84; N, 14.81.
3-(6-Bromo-3-(2-methoxybenzyl)-3H-imidazo[4,5-b]pyridin-
2-yl)pyridin-2-amine (3d). Step 1: 5-Bromo-N-(2-methoxybenzyl)-
3-nitropyridin-2-amine (2c) was prepared as described for 2a using 1b
and (2-methoxyphenyl)methanamine. Step 2: 3d was prepared from
2c as described for 3a using 2-aminonicotinaldehyde. Mp 179−181
°C. 1H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 2H), 8.08 (dd, J = 4.8,
1.8 Hz, 1H), 7.55 (dd, J = 7.6, 1.8 Hz, 1H), 7.25−7.16 (m, 1H), 6.97
(d, J = 8.0 Hz, 1H), 6.79−6.66 (m, 3H), 6.64−6.51 (m, 2H), 5.44 (s,
2H), 3.72 (s, 3H). MS m/z 410 [M + H]+.
6-Bromo-3-(2-methoxybenzyl)-2-(pyridin-4-yl)-3H-imidazo-
[4,5-b]pyridine (3e). 3e was prepared as described for 3a using 2c
and isonicotinaldehyde. Mp 167−172 °C. 1H NMR (400 MHz,
acetone-d6) δ: 8.86−8.83 (dd, J = 4.7, 1.9 Hz, 2H), 8.52−8.39 (dd, J =
46.2, 2.3 Hz, 2H), 7.95−7.93 (dd, J = 4.7, 1.9 Hz, 2H), 7.30−7.23 (m,
1H), 7.02−6.99 (d, J = 7.8 Hz, 1H), 6.82−6.79 (m, 2H), 5.74 (s, 2H),
3.77 (s, 9H). MS m/z 395 [M + H]+. Anal. Calcd for
C19H15BrN4O·1.02TFA·0.15 acetone: C, 49.61; H, 3.28; N, 10.77.
Found: C, 49.59; H, 2.95; N, 10.76.
3-(4-(tert-Butyl)phenyl)-2-(pyridin-3-yl)-3H-imidazo[4,5-b]-
pyridine (3f). 3f was prepared as described for 3a using 2a and
nicotinaldehyde. Mp 171−175 °C. 1H NMR (400 MHz, DMSO-d6) δ
8.69 (d, J = 2.2 Hz, 1H), 8.60 (dd, J = 4.8, 1.6 Hz, 1H), 8.34 (dd, J =
4.7, 1.4 Hz, 1H), 8.24 (dd, J = 8.0, 1.4 Hz, 1H), 7.93 (m, 1H), 7.56 (d,
J = 2.2 Hz, 2H), 7.44 (dd, J = 8.0, 4.8 Hz, 1H), 7.42−7.35 (m, 3H),
1.33 (s, 9H). MS m/z 329 [M + H]+. Anal. Calcd for
C21H2ON4·0.03H2O: C, 76.68; H, 6.15; N, 17.03. Found: C, 76.68;
H, 6.25; N, 16.87.
3-(6-Bromo-3-(4-(tert-butyl)phenyl)-3H-imidazo[4,5-b]-
pyridine-2-yl)pyridine-2-amine (3g). Step 1: 5-Bromo-N-(4-(tert-
butyl)phenyl)-3-nitropyridin-2-amine (2d) was prepared as described
for 2a using 1b. Step 2: 3g was prepared from 2d as described for 3a
using 2-aminonicotinaldehyde. Mp 129−131 °C. 1H NMR (400 MHz,
DMSO-d6) δ 8.50 (d, J = 2.1 Hz, 1H), 8.41 (d, J = 2.1 Hz, 1H), 8.00
(dd, J = 4.8, 1.9 Hz, 1H), 7.55 (d, J = 8.7 Hz, 3H), 7.36 (d, J = 8.6 Hz,
3H), 7.23 (dd, J = 7.7, 1.9 Hz, 1H), 6.94 (s, 3H), 6.41 (dd, J = 7.7, 4.8
Hz, 1H), 1.33 (s, 9H). MS m/z 422 [M + H]+.
tert-Butyl (4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]-
pyridin-3-yl)phenyl)carbamate (4c). 4c was prepared as described
for 4a using 2h. Mp 215−218 °C. 1H NMR (400 MHz, DMSO-d6) δ
9.60 (s, 1H), 8.30 (dd, J = 4.8, 1.5 Hz, 1H), 8.17 (dd, J = 8.0, 1.5 Hz,
1H), 7.97 (dd, J = 4.8, 1.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.36 (dd,
J = 8.0, 4.8 Hz, 1H), 7.28 (d, 2H), 7.20 (dd, J = 7.7, 1.8 Hz, 1H), 7.01
(s, 2H), 6.40 (dd, J = 7.7, 4.8 Hz, 1H), 1.47 (s, 9H). MS m/z 403 [M
+ H]+.
3-(3-(4-(Aminomethyl)phenyl)-3H-imidazo[4,5-b]pyridin-2-
yl)pyridin-2-amine Hydrochloride (5a). To a solution of 4a (0.7 g,
1.7 mmol) in methanol (5 mL) was added HCl (4 M in dioxane, 7.2
mL, 28.8 mmol), and the mixture was stirred at room temperature for
4 h. Concentration of the solution under reduced pressure gave 5a
(0.82 g, quantitative yield) as the hydrochloric acid salt. Mp 255−257
°C. 1H NMR (400 MHz, DMSO-d6) δ 8.44 (m, 3H), 8.30 (dd, J = 8.0,
1.4 Hz, 1H), 8.12 (dd, J = 6.2, 1.6 Hz, 1H), 7.87 (d, J = 6.6 Hz, 1H),
7.65 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.48 (dd, J = 8.0, 4.7
Hz, 1H), 6.85 (dd, J = 7.1, 6.6 Hz, 1H), 4.12 (d, J = 5.6 Hz, 2H), 3.81
(s, 2H). MS m/z 317 [M
+
H]+. Anal. Calcd for
C18H16BrN6·4.6HCl·4.2H2O·0.8Et2O: C, 41.12; H, 6.02; N, 13.58.
Found: C, 41.13; H, 6.02; N, 13.58.
(4-(2-(Pyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)-
methanamine (5b). 5b was prepared as described for 5a using 4b.
1
Mp 265−270 °C. H NMR (400 MHz, DMSO-d6) δ 8.82 (d, J = 1.6
Hz, 1H), 8.75 (dd, J = 4.6, 1.6 Hz, 1H), 8.61 (s, 3H), 8.4 (dd, J = 4.6,
1.6 Hz, 1H), 8.31 (dd, J = 4.6, 1.6 Hz, 1H), 8.13 (m, 1H), 7.71−7.65
(m, 3H), 7.57 (d, J = 8.0 Hz, 2H), 7.47 (dd, J = 8.0, 4.6 Hz, 1H), 4.13
(dd, J = 11.4, 5.5 Hz, 2H). MS m/z 302 [M + H]+. Anal. Calcd for
C18H16N6·2.07HCl·3.14H2O: C, 49.89; H, 5.43; N, 16.16. Found: C,
49.90; H, 5.43; N, 15.51.
N-(4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)phenyl)acetamide (5c). 5c was prepared as described for 3a using
2g and 2-aminonicotinaldehyde. Mp 258−261 °C. 1H NMR (400
MHz, DMSO-d6) δ 10.16 (s, 1H), 8.31 (dd, J = 4.8, 1.4 Hz, 1H), 8.18
(dd, J = 8.0, 1.4 Hz, 1H), 7.98 (dd, J = 4.8, 1.8 Hz, 1H), 7.69 (d, J =
8.8 Hz, 2H), 7.37 (dd, J = 8.0, 4.8 Hz, 1H), 7.33 (d, J = 8.8 Hz, 2H),
7.20 (dd, J = 7.7, 1.8 Hz, 1H), 7.02 (s, 2H), 6.41 (dd, J = 7.7, 4.8 Hz,
1H), 2.07 (s, 3H). MS m/z 345 [M + H]+.
3-(3-(3-(tert-Butyl)phenyl)-3H-imidazo[4,5-b]pyridin-2-yl)-
pyridin-2-amine (3h). Step 1: N-(3-(tert-Butyl)phenyl)-3-nitro-
pyridin-2-amine (2e) was prepared as described for 2a using 3-(tert-
butyl)aniline. Step 2: 3h was prepared from 2e as described for 3a
using 2-aminonicotinaldehyde. Mp 198−200 °C. 1H NMR (400 MHz,
DMSO-d6) δ 8.41 (t, J = 1.6 Hz, 1H), 8.11 (dd, J = 4.7, 1.6 Hz, 1H),
8.08 (dd, J = 12.1, 1.6 Hz, 1H), 7.53−7.50 (m, 2H), 7.40−7.30 (m,
2H), 7.18 (d, J = 1.6 Hz, 1H) 7.03 (d, J = 7.8 Hz, 1H), 6.62 (s, 2H),
6.36−6.33 (m, 1H), 1.23 (s, 9H). MS m/z 344 [M + H]+.
tert-Butyl 4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]-
pyridin-3-yl)benzylcarbamate (4a). Step 1: tert-Butyl 4-((3-
nitropyridin-2-yl)amino)benzylcarbamate (2f) was prepared as
described for 2a and using tert-butyl 4-aminobenzylcarbamate. Step
2: To a solution of 2f (3.3 g, 9.6 mmol) in DMSO (80 mL) and
methanol (14 mL) was added 2-aminonicotinaldehyde (1.3 g, 10.6
mmol) and Na2S2O4 (85%, 4.9 g, 24 mmol). The mixture was heated
at 100 °C for 15 h. After cooling to room temperature, the mixture was
diluted with DCM (200 mL) and washed with H2O and brine (200
mL). The organic phase was separated and dried over Na2SO4. After
filtration the solvent was removed under reduced pressure. The
3-(3-(4-Aminophenyl)-3H-imidazo[4,5-b]pyridin-2-yl)-
pyridin-2-amine Hydrochloride (5d). 5d was prepared as described
1
for 5a using 4c. Mp 210−215 °C. H NMR (400 MHz, DMSO-d6) δ
8.41 (dd, J = 4.7, 1.5 Hz, 1H), 8.28 (dd, J = 8.1, 1.5 Hz, 1H), 8.12 (dd,
J = 6.2, 1.6 Hz, 1H), 7.91 (dd, J = 7.5, 1.6 Hz, 1H), 7.45 (dd, J = 8.0,
4.7 Hz, 2H), 7.31−7.19 (m, 1H), 6.89 (dd, J = 7.5, 6.3 Hz, 1H). MS
L
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m/z 303 [M + H]+. Anal. Calcd for C17H14N6·3.13HCl·0.16 dioxane:
C, 49.21; H, 4.31; N, 19.52. Found: C, 49.21; H, 4.50; N, 19.53.
N-(4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)phenyl)benzamide (5e). Step 1: N-(4-((3-Nitropyridin-2-yl)-
amino)phenyl)benzamide (2i) was prepared as described for 2a using
N-(4-aminophenyl)benzamide. Step 2: To a solution of 2i (0.67 g, 0.2
mmol) in DMSO (0.7 mL) and methanol (0.7 mL) were added
Na2S2O4 (0.35 g, 0.4 mmoL) and 2-aminonicotinaldehyde (0.29 g,
0.24 mmoL). The mixture was heated at 100 °C for 24 h, cooled to
room temperature, diluted with EtOAc (20 mL), and washed with
H2O (10 mL) and brine (10 mL). The organic layer was separated and
concentrated. The residue was purified by column chromatography
(0−20% MeOH−DCM, silica gel) to give 5e as a light yellow solid
3-(3-(4-(Aminomethyl)phenyl)-5-chloro-3H-imidazo[4,5-b]-
pyridin-2-yl)pyridin-2-amine Hydrochloride (5g). 5g was pre-
pared from 6d as described for 5a. Mp 208−211 °C. H NMR (400
MHz, DMSO-d6) δ 8.48 (brs, 2H), 8.32 (d, J = 8.4 Hz, 2H), 8.15 (d, J
= 6.2 Hz, 1H), 7.93 (d, J = 7.4 Hz, 1H), 7.69−7.67 (m, 2H), 7.58−
7.53 (m, 3H), 6.90 (t, J = 7.0 Hz, 1H), 4.08 (d, J = 5.2 Hz, 2H). MS
m/z 351 [M + H]+.
1
2-(4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)phenyl)-N-phenylacetamide (7). Step 1: To a solution of 6a (7.2
g, 19.3 mmol) in THF (40 mL) and MeOH (10 mL) was added
LiOH·H2O (0.97 g, 1.2 mmol) in H2O (10 mL). The mixture was
stirred at room temperature for 6 h. The solvent was evaporated under
reduced pressure to yield lithium 2-(4-(2-(2-aminopyridin-3-yl)-3H-
imidazo[4,5-b]pyridin-3-yl)phenyl)acetate (6f) (7.3 g) which was used
in the next step without further purification. Step 2: To a solution of 6f
(3.5 g, 10.0 mmol) and aniline (1.36 mL, 14.9 mmol) in DMF (40
mL) was added HBTU (5.6 g, 14.9 mmol). The mixture was stirred at
room temperature for 6 h and concentrated to half volume. DCM
(300 mL) and saturated NaHCO3 solution (80 mL) were added. The
organic layer was separated and washed with brine (80 mL). After 30
min the precipitate was removed by filtration, washed with a mixture
of DCM: H2O (1:3) (20 mL) and dried to give 7 as a white solid (2.8
1
(0.09 g, 11%). Mp 222−224 °C. H NMR (400 MHz, DMSO-d6) δ
10.48 (s, 1H), 8.38 (dd, J = 4.7, 1.4 Hz, 1H), 8.25 (dd, J = 8.0, 1.4 Hz,
1H), 8.05 (dd, J = 5.7, 1.6 Hz, 1H), 7.98−7.90 (m, 5H), 7.62 (dd, J =
15.3, 7.8 Hz, 2H), 7.54 (t, J = 7.3 Hz, 2H), 7.47−7.40 (m, 3H), 6.75−
6.67 (m, 1H). MS m/z 407 [M + H]+.
Ethyl 2-(4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]-
pyridin-3-yl)phenyl)acetate. (6a). Step 1: Ethyl 2-(4-((3-nitro-
pyridin-2-yl)amino)phenyl)acetate (2j) was prepared as described for
2a using ethyl 2-(4-aminophenyl)acetate and used without further
purification. Step 2: To a solution of 2j (5.5 g, 18.3 mmol) and 2-
aminopyridine-3-carboxaldehyde (2.7 g, 21.9 mmol) in DMSO (60
mL) and MeOH (10 mL) was added Na2S2O4 (7.9 g, 45.7 mmol).
The mixture was heated at 100 °C for 24 h. EtOAc (300 mL) and
H2O (100 mL) were added, and the organic layer was separated,
washed with brine (80 mL), dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure. The residue was
purified by column chromatography (EtOAc/DCM (1:1), silica gel) to
give 6a as a pale yellow solid (2.7 g, 40%). Mp 138−140 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.34−8.32 (dd, J = 5.2 Hz, 1.6 Hz, 1H),
8.23−8.20 (dd, J = 8.0, 1.6 Hz, 1H), 8.01−7.99 (dd, J = 4.8, 2.0 Hz,
1H), 7.44−7.38 (m, 5H), 7.22−7.19 (dd, J = 8.4, 1.6 Hz, 1H), 6.98 (s,
2H), 6.42−6.38 (dd, J = 8.0, 5.2 Hz, 1H), 4.12 (q, J = 7.6 Hz, 2H),
3.78 (s, 2H), 1.21 (t, J = 6.8 Hz, 3H). MS m/z 374 [M + H]+.
Ethyl 2-(4-(2-(2-Aminopyridin-3-yl)-5-chloro-3H-imidazo-
[4,5-b]pyridin-3-yl)phenyl)acetate (6b). Step 1: Ethyl 2-(4-((6-
chloro-3-nitropyridin-2-yl)amino)phenyl)acetate (2k) was prepared as
described for 2a using 1c and ethyl 2-(4-aminophenyl)acetate and
used without further purification. Step 2: 6b was prepared from 2k as
described for 6a. Mp 138−140 °C. 1H NMR (400 MHz, DMSO-d6) δ
8.60 (d, J = 8.0 Hz, 1H), 8.00−7.99 (dd, J = 4.8, 2.0 Hz, 1H), 7.47−
7.39 (m, 5H), 7.22−7.19 (dd, J = 8.4, 1.6 Hz, 1H), 6.90 (s, 2H), 6.41−
6.38 (dd, J = 8.0, 5.2 Hz, 1H), 4.12 (q, J = 7.6 Hz, 2H), 3.78 (s, 2H),
1.21 (t, J = 6.8 Hz, 3H). MS m/z 408 [M + H]+.
1
g, 67%). Mp 251−253 °C. H NMR (400 MHz, DMSO-d6) δ 10.24
(s, 1H), 8.32−8.30 (dd, J = 4.4, 1.2 Hz, 1H), 8.22−8.19 (dd, J =
8.4,1.6 Hz, 1H), 8.01−7.99 (dd, J = 4.8, 1.6 Hz, 1H), 7.63−7.61 (d, J =
8.4 Hz, 2H), 7.49−7.47 (d, J = 8.4 Hz, 2H), 7.40−7.37 (m, 3H), 77.31
(t, J = 7.6 Hz, 2H), 7.25−7.22 (dd, J = 8.4, 1.6 Hz, 2H), 6.99 (s, 2H),
6.42−6.39 (dd, J = 7.6, 4.4 Hz, 1H), 4.12 (q, J = 7.6 Hz, 2H), 3.78 (s,
2H), 1.21 (t, J = 6.8 Hz, 3H). MS m/z 421 [M + H]+.
N-(4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)benzyl)benzamide (8a). A mixture of benzoic acid (0.11 g, 0.91
mmol), DIEA (1.15 mL, 6.60 mmol), and HBTU (0.34 g, 0.91 mmol)
in anhydrous DMF (2 mL) was stirred at room temperature for 5 min
before 5a (0.40 g, 0.824 mmol) was added. The mixture was stirred at
room temperature for 2 h, diluted with EtOAc (20 mL), and washed
with H2O (20 mL × 3). The organic layer was concentrated to half-
volume and the precipitate removed by filtration and rinsed with
EtOAc. Drying under vacuum yielded 8a (0.24 g, 62%). Mp 226−228
1
°C. H NMR (400 MHz, DMSO-d6) δ 9.14 (t, J = 6.0 Hz, 1H), 8.31
(dd, J = 4.8, 1.5 Hz, 1H), 8.20 (dd, J = 8.0, 1.4 Hz, 1H), 7.99 (dd, J =
4.8, 1.8 Hz, 1H), 7.92 (dd, J = 5.3, 3.3 Hz, 2H), 7.60−7.34 (m, 6H),
7.24 (dd, J = 7.7, 1.8 Hz, 1H), 6.98 (s, 2H), 6.41 (dd, J = 7.6, 4.8 Hz,
1H), 4.57 (d, J = 6.0 Hz, 2H). MS m/z 421 [M + H]+.
N-(4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)benzyl)cyclohexanecarboxamiden (8b). Cyclohexanecarbox-
ylic acid (0.11 g, 0.82 mmol) and CDI (0.13 g, 0.82 mmol) in
DMA (4 mL) were stirred for 1 h at room temperature. This mixture
was added to a solution of 5a (0.29 g, 0.81 mmol) in DMA (4 mL)
and triethylamine (0.47 mL, 3.2 mmol) over 10 min. The mixture was
stirred for 2 h at room temperature before the addition of H2O (8
mL). The precipitate formed was removed by filtration, washed with
H2O and ether to yield 8b (0.21 g, 61%). Mp 160−162 °C. 1H NMR
(400 MHz, DMSO-d6) δ 8.31 (d, J = 3.4 Hz, 2H), 8.20 (d, J = 8.0 Hz,
1H), 7.99 (d, J = 4.2 Hz, 1H), 7.47−7.27 (m, 4H), 7.21 (d, J = 7.6 Hz,
1H), 6.98 (s, 2H), 6.40 (dd, J = 7.6, 4.9 Hz, 1H), 4.33 (d, J = 5.9 Hz,
2H), 2.19 (t, J = 11.6 Hz, 1H), 1.71 (m, 5H), 1.61−1.28 (m, 3H),
1.25−0.99 (m, 3H). MS m/z 427 [M + H]+. Anal. Calcd for
C25H26N6O·1.17H2O·0.38Et2O: C, 66.95; H, 6.81; N, 17.66. Found:
C, 66.96; H, 6.56; N, 17.67.
tert-Butyl 4-(2-(2-Aminopyridin-3-yl)-6-bromo-3H-imidazo-
[4,5-b]pyridine-3-yl)benzylcarbamate (6c). 6c was prepared
1
from 2l as described for 6a. Mp 176−178 °C. H NMR (400 MHz,
DMSO-d6) δ 8.50 (d, J = 2.0 Hz, 1H), 8.40 (d, J = 2.4 Hz, 1H), 7.99
(dd, J = 4.7, 2.0 Hz, 1H), 7.48 (t, J = 5.6 Hz, 1H), 7.40−7.31 (m, 3H),
7.23 (dd, J = 7.8, 2.0 Hz, 1H), 6.97 (s, 2H), 6.40 (dd, J = 7.7, 4.4 Hz,
1H), 4.121 (d, J = 6.2 Hz, 2H), 1.41 (s, 9H). MS m/z 495 [M + H]+.
tert-Butyl 4-(2-(2-Aminopyridin-3-yl)-5-chloro-3H-imidazo-
[4,5-b]pyridin-3-yl)benzylcarbamate (6d). 6d was prepared from
2m as described for 6a. Mp 235−237 °C. 1H NMR (400 MHz,
DMSO-d6) δ 8.26 (d, J = 8.6 Hz, 1H), 7.98 (dd, J = 4.7, 1.9 Hz, 1H),
7.48−7.44 (m, 2H), 7.38 (s, 4H), 7.21 (dd, J = 7.4, 1.9 Hz, 1H), 6.92
(s, 2H), 6.39 (dd, J = 7.6, 4.8 Hz, 1H), 4.21 (d, J = 6.2 Hz, 2H), 1.41
(s, 9H). MS m/z 451 [M + H]+.
N-(4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
tert-Butyl 3-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]-
pyridine-3-yl)benzylcarabamate (6e). Step 1: tert-Butyl 3-((3-
nitropyridin-2-yl)amino)benzylcarbamate (2n) was prepared as
described for 2a using tert-butyl 3-aminobenzylcarbamate and used
without further purification. Step 2: 6e was prepared from 2n as
yl)benzyl)-2-phenylacetamide (8c). 8c was prepared as described
1
for 8b using 2-phenylacetic acid. Mp 158−160 °C. H NMR (400
MHz, DMSO-d6) δ 8.64 (t, J = 6.4 Hz, 2H), 8.31 (dd, J = 4.7, 1.3 Hz,
1H), 8.20 (dd, J = 8.0, 1.2 Hz, 1H), 8.00 (dd, J = 4.8, 1.8 Hz, 1H),
7.63−7.31 (m, 5H), 7.30−7.03 (m, 5H) 6.97 (s, 2H), 6.40 (dd, J = 7.5,
5.0 Hz, 1H), 4.36 (d, J = 6.0 Hz, 2H), 3.53 (s, 2H). MS m/z 435 [M +
H]+. Anal. Calcd for C26H22N6O·0.03EtOAc·0.32Et2O: C, 71.41; H,
5.56; N, 18.24. Found: C, 71.41; H, 5.29; N, 18.22.
1
described for 6a. Mp 90−93 °C H NMR (400 MHz, DMSO-d6) δ
8.30 (dd, J = 4.7, 1.4 Hz, 1H), 8.23−8.15 (m, 1H), 7.97 (dd, J = 4.8,
1.8 Hz, 1H), 7.50−7.29 (m, 5H), 7.19−7.01 (m, 4H), 6.35 (dd, J =
7.7, 4.8 Hz, 1H), 4.15 (d, J = 6.2 Hz, 2H), 1.35 (s, 9H). MS m/z 417
[M + H]+.
N-(4-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)benzyl)acetamide (8d). A solution of 5a (0.10 g, 0.21 mmol) in
M
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DCM (1 mL), DIPEA (0.29 mL, 1.6 mmol), and acetyl chloride 0.024
g, 0.31 mmol) was heated at 50 °C for 2 h. The mixture was diluted
with EtOAc (20 mL) and washed three times with H2O (10 mL). The
organic layer was evaporated under reduced pressure and the residue
purified by column chromatography (0−20% MeOH/DCM, silica gel)
chromatography (3−20% methanol/EtOAc, silica gel) to yield 9 as a
1
yellow solid (0.027 g, 28%). Mp 210−211 °C. H NMR (DMSO-d6)
400 MHz δ 8.28 (d, J = 2.0 Hz, 1H), 8.05 (dd, J = 6.0, 2.0 Hz, 1H),
7.96 (d, J = 2.0 Hz, 1H), 7.82−7.84 (m, 5H), 7.34−7.37 (m, 2H),
7.09−7.12 (m, 1H), 6.62 (bs, 2H), 6.37−6.40 (m, 1H), 4.74 (d, J = 5.1
Hz, 2H), 3.14−3.19 (m, 1H), 2.13−2.18 (m, 2H), 1.85−1.89 (m, 8H).
MS m/z 489 [M + H]+.
1
to yield 8d (0.014 g, 19%). Mp 257−259 °C. H NMR (400 MHz,
DMSO-d6) δ 8.45 (t, J = 6.0 Hz, 1H), 8.31 (dd, J = 4.6, 1.4 Hz, 1H),
8.20 (dd, J = 8.4, 1.6 Hz, 1H), 8.00 (dd, J = 4.8, 2.0 Hz, 1H), 7.4−7.37
(m, 6H), 7.22 (dd, J = 7.8, 1.8 Hz, 1H), 6.97 (s, 2H), 6.40 (dd, J = 8.0,
4.8 Hz, 1H), 4.33 (d, J = 6.0 Hz, 2H), 1.9 (s, 3H). MS m/z 359 [M +
H]+.
N-(4-(2-(2-Aminopyridin-3-yl)-6-phenyl-3H-imidazo[4,5-b]-
pyridin-3-yl)benzyl)benzamide (12d). 8e (0.15 g, 0.3 mmol) and
phenylboronic acid (0.073 g, 0.3 mmol) were suspended in a mixture
of EtOH and toluene (1:1, 10 mL). A solution of aqueous NaHCO3
(sat.) was added (1 mL). The mixture was degassed with nitrogen for
5 min. Tetrakis(triphenylphosphine)palladium(0) (0.02 g, 0.017
mmol) was added to the mixture and heated to 100 °C for 16 h.
After cooling to room temperature, the mixture was diluted with DCM
(50 mL) and H2O (20 mL). The organic layer was separated, washed
with brine (20 mL), and concentrated to 15 mL in vacuo. MeOH (2
mL) was added and the mixture purified by column chromatography
(3−20% MeOH in DCM, silica gel). The solid was triturated with hot
DCM (20 mL) and filtered to give 12d as a tan solid (0.039 g, 26%).
N-(4-(2-(2-Aminopyridin-3-yl)-6-bromo-3H-imidazo[4,5-b]-
pyridin-3-yl)benzyl)benzamide (8e). Step 1: 3-(3-(4-
(Aminomethyl)phenyl)-6-bromo-3H-imidazo[4,5-b]pyridin-2-yl)-
pyridin-2-amine hydrochloride (5f) was prepared as described for 5a
from 6c. Step 2. 5f (4.12 g, 8.76 mmol), benzoic acid (1.49 g, 12.2
mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (5.06 g, 29.2
mmol), and DIEA (25 mL) in DCM (50 mL) were stirred at room
temperature for 16 h. The mixture was diluted with DCM (10 mL)
and washed with H2O (3 × 40 mL). The organic layer was
concentrated under reduced pressure to an orange oily solid (6.2 g)
and purified by column chromatography (75% EtOAc/hexanes to
100% EtOAc, silica gel) to yield 8e as an off-white solid (2.84 g, 65%).
1
Mp 248−250 °C. H NMR (400 MHz, DMSO-d6) δ 9.15 (t, J = 6.1
Hz, 1H), 8.61 (d, J = 2.1 Hz, 1H), 8.46 (d, J = 2.0 Hz, 1H), 8.01 (dd, J
= 4.8, 1.8 Hz, 1H), 7.97−7.87 (m, 2H), 7.85−7.71 (m, 2H), 7.64−7.35
(m, 10H), 7.27 (dd, J = 7.7, 1.8 Hz, 1H), 7.03 (s, 2H), 6.43 (dd, J =
7.7, 4.8 Hz, 1H), 4.59 (d, J = 6.0 Hz, 2H). MS m/z 497 [M + H]+.
Ethyl 2-(4-(5-(3-Acetamidophenyl)-2-(2-aminopyridin-3-yl)-
3H-imidazo[4,5-b]pyridin-3-yl)phenyl)acetate (10a). 10a was
prepared from 6b as described for 12d using 3-acetamidophenylbor-
1
Mp 195−197 °C. H NMR (400 MHz, DMSO-d6) δ 9.11 (t, J = 5.8
Hz, 1H), 8.47 (d, J = 2.4 Hz, 1H), 8.36 (d, J = 2.4 Hz, 1H), 7.97 (dd, J
= 5.0, 1.9 Hz, 1H), 7.88 (d, J = 7.1 Hz, 2H), 7.54−7.36 (m, 8H), 7.22
(dd, J = 7.8, 1.9 Hz, 1H), 6.92 (s, 2H), 6.39 (dd, J = 7.8, 5.1 Hz, 1H),
4.54 (d, J = 6.3 Hz, 2H). MS m/z 499 [M + H]+. Anal. Calcd for
C25H19BrN6O·0.07EtOAc: C, 60.06; H, 3.90; N, 16.62. Found: C,
60.03; H, 3.30; N, 16.37.
1
onic acid. Mp 93−95 °C. HNMR (400 MHz, DMSO-d6) δ 10.08 (s,
1H), 8.29−8.27 (d, J = 8.4 Hz, 1H), 8.10 (s. 1H), 8.00 (dd, J = 4.4, 1.6
Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.11−7.65 (dd, J = 18.0, 6.8 Hz,
2H), 7.45 (s, 4H), 7.38 (t, J = 8.0 Hz, 1H), 7.19−7.17 (dd, J = 8.0, 2.0
Hz, 1H), 6.92 (s, 2H), 6.42−6.38 (dd, J = 8.0, 4.8 Hz, 1H), 4.13 (q, J =
7.2 Hz, 2H), 3.79 (s, 2H), 2.06 (s, 3H), 1.21 (t, J = 6.8 Hz, 3H). MS
m/z 507 [M + H]+.
N-(3-(2-(2-Aminopyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-
yl)benzyl)benzamide (8g). Step 1: 6e was deprotected as described
for 5a from 4a and the product used without further purification. Step
2: 8g was prepared from the product from step 1 as described for the
1
preparation of 8a from 5a. Mp 106−107 °C. H NMR (400 MHz,
DMSO-d6) δ 9.07 (br t, J = 6.0 Hz, 1H), 8.32 (dd, J = 4.7, 1.4 Hz, 1H),
8.21 (dd, J = 8.0, 1.4 Hz, 1H), 7.95 (dd, J = 4.9, 1.8 Hz, 1H), 7.88−
7.81 (m, 1H), 7.58−7.35 (m, 5H), 7.35−7.29 (m, 1H), 7.23 (dd, J =
7.7, 1.7 Hz, 1H), 7.18 (s, 1H), 6.37 (dd, J = 7.7, 4.9 Hz, 1H), 4.51 (d, J
= 6.0 Hz, 2H). MS m/z 421 [M + H]+.
2-(4-(5-(3-Acetamidophenyl)-2-(2-aminopyridin-3-yl)-3H-
imidazo[4,5-b]pyridin-3-yl)phenyl)-N-phenylacetamide (12a).
Step 1: To a solution of 10a (0.50 g, 0.99 mmol) in THF (6 mL)
and MeOH (2 mL) was added a solution of LiOH·H2O (0.046 g, 1.08
mmol) in H2O (2 mL). The mixture was stirred at room temperature
for 6 h. The solvent was removed under reduced pressure to give 6g
(0.55 g) which was used without further purification. Step 2: To a
solution of lithium 2-(4-(5-(3-acetamidophenyl)-2-(2-aminopyridin-3-
yl)-3H-imidazo[4,5-b]pyridin-3-yl)phenyl)acetate (6 g) (0.1 g, 0.207
mmol) and aniline (31 μL, 0.331 mmol) in DMF (1 mL) was added
HBTU (0.14 g, 0.372 mmol). The mixture was stirred at room
temperature for 6 h. The solvent was removed under reduced pressure
and the residue purified by preparative HPLC (H2O, 0.05 M TFA/
ACN, 0.05 M TFA, 0−100% ACN) to give 12a as a yellow solid (22
3-(3-(4-((Benzylamino)methyl)phenyl)-3H-imidazo[4,5-b]-
pyridin-2-yl)pyridin-2-amine (8h). To a suspension of 5a (0.361 g,
0.72 mmol) in DCE (10 mL) and triethylamine (0.703 mL, 5.04
mmol) was added acetic acid (0.5 mL, 8.73 mmol) followed by
benzaldehyde (0.07 mL, 0.66 mmol). The mixture was stirred at room
temperature for 30 min before tetramethylammonium triacetoxybor-
ohydride (0.28 g, 1.08 mmol) was added After 16 h additional
tetramethylammonium triacetoxyborohydride (0.47 g, 1.80 mmol) was
added. After 4 h the mixture was diluted with saturated aqueous
Na2CO3 (50 mL) and DCM (50 mL). The organic layer was
separated, dried over sodium sulfate, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy (100% DCM to 10% MeOH in DCM, silica gel) to give 8h as a
1
mg, 19%). Mp 166−168 °C. HNMR (400 MHz, DMSO-d6) δ 10.25
(s, 1H), 10.03 (s, 1H), 8.33 (d, J = 8.8 Hz, 1H), 8.12 (s, 1H), 8.06−
8.04 (dd, J = 5.6, 1.6 Hz, 1H), 7.71−7.61 (m, 5H), 7.51 (s, 4H), 7.39
(t, J = 8.0 Hz, 1H), 7.33 (t, J = 8.0 Hz, 2H), 7.06 (t, J = 7.2 Hz, 1H),
6.72 (t, J = 6.0 Hz, 1H), 3.77 (s, 2H), 2.04 (s, 3H). MS m/z 554 [M +
H]+. Anal. Calcd for C33H27N7O2·1.82TFA: C, 57.82; H, 3.82; N,
12.88. Found: C, 57.84; H, 3.93; N, 12.93.
1
light yellow solid (0.084 g, 30%). Mp 151−152 °C. H NMR (400
MHz, DMSO-d6) δ 8.38−8.25 (m, 1H), 8.23−8.13 (m, 1H), 8.01−
7.91 (m, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.43−7.26 (m, 7H), 7.26−7.14
(m, 2H), 6.99 (s, 2H), 6.37 (ddd, J = 7.6, 4.8, 0.7 Hz, 1H), 3.72 (d, J =
16.6 Hz, 4H). MS m/z 407 [M + H]+.
Ethyl 2-(4-(2-(2-Aminopyridin-3-yl)-5-(pyridin-3-yl)-3H-
imidazo[4,5-b]pyridin-3-yl)phenyl)acetate (10b). 10b was pre-
pared from 6b as described for 12d using 3-pyridylboronic acid. Mp
N-(4-(2-(2-Aminopyridin-3-yl)-6-bromo-3H-imidazo[4,5-b]-
pyridin-3-yl)benzyl)benzamide (9). Under a nitrogen atmosphere
cyclopentylzinc bromide (0.5 M, 0.6 mL, 0.3 mmol), Pd(tBu3P)2 (10.2
mg, 0.02 mmol), and potassium tert-butoxide (22.4 mg, 0.2 mmol)
were added to a solution of 8e (0.10 g, 0.2 mmol) in tetrahydrofuran
(3 mL). The reaction mixture was degassed with nitrogen for 30 min
and heated to 100 °C for 15 min in a microwave. After cooling, the
mixture was filtered through Celite and diluted with DCM (5 mL),
H2O (5 mL), and EDTA (100 mg, 0.34 mmol). The organic layer was
separated, washed with brine (3 mL), and dried over Na2SO4. The
solids were removed by filtration, and the solvent was evaporated
under reduced pressure. The residue was purified by column
1
155−157 °C. HNMR (400 MHz, DMSO-d6) δ 9.24 (m, 1H), 8.60−
8.58 (dd, J = 4.8, 1.2 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.32 (d, J = 8.0
Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 8.01 (dd, J = 5.2, 2.0 Hz, 1H),
7.52−7.44 (m, 5H), 7.22−7.20 (dd, J = 7.6, 2.0 Hz, 1H), 6.99 (s, 2H),
6.43−6.39 (dd, J = 6.8, 4.4 Hz, 1H), 4.13 (q, J = 7.2 Hz, 2H), 3.80 (s,
2H), 1.21 (t, J = 6.8 Hz, 3H). MS m/z 451 [M + H]+.
tert-Butyl 4-(2-(2-Aminopyridin-3-yl)-6-(pyridin-3-yl)-3H-
imidazo[4,5-b]pyridin-3-yl)benzylcarbamate (10c). 10c was
prepared from 6c as described for 12d using 3-pyridylboronic acid.
1
Mp 95−100 °C. H NMR (400 MHz, DMSO-d6) δ 8.98 (d, J = 1.9
Hz, 1H), 8.64 (d, J = 2.0 Hz, 1H), 8.59 (dd, J = 4.7, 1.6 Hz, 1H), 8.54
N
dx.doi.org/10.1021/jm300276x | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(d, J = 2.0 Hz, 1H), 8.20 (m, 1H), 7.98 (m, 1H), 7.52 (m, 2H), 7.48−
7.34 (m, 4H), 7.23 (m, 1H), 7.01 (s, br, 2H), 6.38 (m, 1H), 4.19 (m,
2H), 1.38 (s, 9H). MS m/z 494 [M + H]+.
N-(4-(2-(2-Aminopyridin-3-yl)-5-(pyridin-3-yl)-3H-imidazo-
[4,5-b]pyridin-3-yl)benzyl)benzamide (12f). Step 1: To a solution
of 5g (8.9 g, 25.5 mmol) in H2O (50 mL) was added NaOH (1.51 g,
38.25 mmol) and benzoyl chloride (5.35 g, 38.25 mmol). The mixture
was stirred at room temperature for 18 h. The precipitate was removed
by filtation and washed with H2O and ether to yield N-(4-(2-(2-
aminopyridin-3-yl)-5-chloro-3H-imidazo[4,5-b]pyridin-3-yl)benzyl)-
benzamide (8f) (2.9 g 84%) which was used without further
purification. Step 2: 12f was prepared from 8f as described for 12d
tert-Butyl 4-(2-(2-Aminopyridin-3-yl)-5-phenyl-3H-imidazo-
[4,5-b]pyridin-3-yl)benzylcarbamate (10e). 10e was prepared
from 6d as described for 12d. Mp 109−112 °C. 1H NMR (400
MHz, DMSO-d6) δ 8.24 (d, J = 8.4 Hz, 1H), 8.02−7.96 (m, 4H),
7.48−7.37 (m, 8H), 7.19 (d, J = 8.0 Hz, 1H), 6.99 (bs, 2H), 6.38 (m,
1H), 4.22 (d, J = 5.6 Hz, 2H), 1.39 (s, 9H). MS m/z 493 [M + H]+.
3-(3-(4-(Aminomethyl)phenyl)-6-(pyridin-3-yl)-3H-imidazo-
[4,5-b]pyridin-2-yl)pyridin-2-amine Hydrochloride (11a). 11a
was prepared from 10c as described for 5a. Mp 223−225 °C. 1H NMR
(400 MHz, MeOH-d4) δ 9.38 (d, J = 2.0 Hz, 1H), 9.11 (d, J = 2.0 Hz,
1H), 8.95−8.93 (m, 1H), 8.89 (d, J = 2.0 Hz, 1H), 8.78 (m, 1H),
8.83−8.25 (m, 1H), 8.06−8.04 (m, 1H), 7.88−7.86 (m, 1H), 7.77−
7.66 (m, 4H), 6.86−6.83 (m, 1H), 4.23 (m, 2H). MS m/z 394 [M +
H]+.
1
using 3-pyridylboronic acid. Mp 253−256 °C. H NMR (400 MHz,
DMSO-d6) δ 9.23 (s, 1H), 9.16 (t, J = 5.8 Hz, 1H), 8.58 (d, J = 7.1 Hz,
1H), 8.37 (d, J = 8.2 Hz, 1H), 8.31 (d, J = 8.2 Hz, 1H), 8.08 (d, J = 8.6
Hz, 1H), 7.95 (m, 1H), 7.93 (d, J = 8.6 Hz, 1H), 7.48−7.55 (m, 6H),
7.24 (d, J = 7.8 Hz, 1H), 6.97 (s, 2H), 6.42 (m, 1H), 4.62 (d, J = 6.2
Hz, 2H). MS m/z 498 [M
+
H]+. Anal. Calcd for
C30H23N7O·0.17EtOAc: C, 71.90; H, 4.79; N, 19.13. Found: C,
71.16; H, 4.67; N, 19.12.
N-(3-(3-(4-(Aminomethyl)phenyl)-2-(2-aminopyridin-3-yl)-
3H-imidazo[4,5-b]pyridin-5-yl)phenyl)acetamide Hydrochlor-
ide (11b). Step 1: tert-Butyl 4-(5-(3-acetamidophenyl)-2-(2-amino-
pyridin-3-yl)-3H-imidazo[4,5-b]pyridin-3-yl)benzylcarbamate (10d)
was prepared from 6d as described for 12d using 3-acetamidophe-
nylboronic acid. Step 2: 10d (0.23 g, 0.42 mmol) was suspended in
EtOAc (5 mL) and hydrochloric acid in dioxane (4M, 10 mL) added.
The mixture was stirred at 56 °C for 4 h. After the mixture was cooled
to room temperature, the precipitate was collected by filtration to give
11b hydrochloride salt as a yellow solid (0.24 g, 100%). Mp 245−248
N-(4-(2-(2-Aminopyridin-3-yl)-6-(pyridin-3-yl)-3H-imidazo-
[4,5-b]pyridin-3-yl)benzyl)-3-fluorobenzamide (12g). 12g was
prepared from 11a as described for 8b using 3-fluorobenzoic acid. Mp
1
258−260 °C. H NMR (400 MHz, DMSO-d6) δ 9.25 (t, J = 6.0 Hz,
1H), 8.01 (s, 1H), 9.01 (d, J = 1.6 Hz, 1H), 8.51 (d, J = 3.2 Hz, 1H),
8.58 (d, J = 2.0 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 3.2 Hz,
1H), 7.78 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 10.4 Hz, 1H), 7.56−7.41
(m, 6H), 7.27 (d, J = 8.0 Hz, 1H), 7.02 (s, 2H), 6.43 (t, J = 6.8 Hz,
2H), 4.59 (s, 2H). MS m/z 516 [M + H]+. Anal. Calcd for
C30H22N7O·0.1H2O·0.09DMA: C, 69.43; H, 4.42; N, 18.91. Found: C,
69.37; H, 4.37; N, 19.04.
1
°C. H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 8.47 (s, 3H),
8.35 (d, J = 8.4 Hz, 2H), 8.17 (s, 1H), 8.12 (d, J = 6.2 Hz, 1H), 7.89
(d, J = 8.2 Hz, 2H), 7.67−7.60 (m, 5H), 7.37 (t, J = 8.0 Hz, 1H),
6.88−6.85 (m, 1H), 4.10 (m, 2H), 2.04 (s, 3H). MS m/z 450 [M +
H]+.
3-(3-(4-(Aminomethyl)phenyl)-5-phenyl-3H-imidazo[4,5-b]-
pyridin-2-yl)pyridin-2-amine Hydrochloride (11c). 11c was
prepared from 10e as described for 11b, step 2. Mp 215−218 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.52 (bs, 5H), 8.35 (d, J = 8.4 Hz,
N-(4-(5-(3-Acetamidophenyl)-2-(2-aminopyridin-3-yl)-3H-
imidazo[4,5-b]pyridin-3-yl)benzyl)benzamide (12h). 12h was
prepared from 11b as described for 8b using benzoic acid. Mp
1
205−208 °C. H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.13
(t, J = 6.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.06 (s, 1H), 7.98−7.96
(m, 1H), 7.91 (d, J = 7.2 Hz, 2H), 7.82 (d, J = 8.0 Hz, 1H), 7.68 (d, J
= 8.8 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.55−7.43 (m, 6H), 7.35 (t, J
= 8.0 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 6.87 (s, 2H), 6.39 (dd, J = 7.6,
4.8 Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H), 2.02 (s, 3H). MS m/z 554 [M +
H]+.
1H), 8.13 (d, J = 6.0 Hz, 1H), 8.04 (m, 3H), 7.89 (m, 1H), 7.65 (dd, J
= 8.4, 1.6 Hz, 4H), 7.48−7.38 (m, 3H), 6.89 (m, 1H), 4.11 (d, J = 5.6
Hz, 2H). MS m/z 393 [M + H]+.
N-(4-(2-(2-Aminopyridin-3-yl)-5-phenyl-3H-imidazo[4,5-b]-
2-(4-(5-(3-Acetamidophenyl)-2-(2-aminopyridin-3-yl)-3H-
imidazo[4,5-b]pyridin-3-yl)phenyl)-N-(3-fluorophenyl)-
acetamide (12b). 12b was prepared as described for 12a using 3-
pyridin-3-yl)benzyl)benzamide (12i). 12i was prepared from 11c
1
as described for 8b using benzoic acid. Mp 237−240 °C. H NMR
1
(400 MHz, DMSO-d6) δ 9.13 (t, J = 8.4 Hz, 1H), 8.23 (d, J = 8.4 Hz,
1H), 8.00−7.90 (m, 6H), 7.52−7.36 (m, 10H), 7.18 (d, J = 7.6 Hz,
2H), 6.92 (s, 2H), 6.39 (dd, J = 4.8, 2.8 Hz, 1H), 4.58 (d, J = 6.0 Hz,
2H). MS m/z 497 [M + H]+.
fluoroaniline. Mp 150−152 °C. HNMR (400 MHz, DMSO-d6) δ
10.49 (s, 1H), 10.08 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H), 8.13 (s, 1H),
8.06 (dd, J = 4.8 Hz, 1.6 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.71−7.61
(m, 4H), 7.52 (s, 4H), 7.41−7.33 (m, 3H), 6.89 (t, J = 8.4 Hz, 1H),
6.74 (t, J = 7.2 Hz, 1H), 3.79 (s, 2H), 2.04 (s, 3H). MS m/z 572 [M +
H]+. Anal. Calcd for C33H26FN7O2·1.6TFA·0.47ACN: C, 57.68; H,
3.78; N, 12.88. Found: C, 57.70; H, 3.58; N, 13.53.
N-(4-(5-(3-Acetamidophenyl)-2-(2-aminopyridin-3-yl)-3H-
imidazo[4,5-b]pyridin-3-yl)benzyl)-3-fluorobenzamide (12j).
12j was prepared from 11b as described for 8b using 3-fluorobenzoic
1
acid. Mp 174−187 °C. H NMR (400 MHz, DMSO-d6) δ 10.03 (s,
2-(4-(2-(2-Aminopyridin-3-yl)-5-(pyridin-3-yl)-3H-imidazo-
[4,5-b]pyridin-3-yl)phenyl)-N-phenylacetamide (12c). Step 1:
Lithium 2-(4-(2-(2-aminopyridin-3-yl)-5-(pyridin-3-yl)-3H-imidazo-
[4,5-b]pyridin-3-yl)phenyl)acetate (6h) was prepared from 10b as
described for 6g and used without further purification. Step 2: 12c was
prepared from 6h as described for 12a and obtained as a yellow solid.
1H), 9.22 (t, J = 6.0 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.06 (s, 1H),
7.97 (dd, J = 4.8, 1.6 Hz, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.0
Hz, 1H), 7.72−7.66 (m, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.55−7.51 (m,
1H), 7.45 (s, 3H), 7.41−7.32 (m, 2H), 7.18 (dd, J = 7.6, 1.6 Hz, 1H),
6.86 (br, 2H), 6.38 (dd, J = 8.0, 4.8 Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H),
2.01 (s, 3H). MS m/z 572 [M + H]+. Anal. Calcd for
C33H26FN7O2·1.4HCl·0.1 dioxane·0.15Et2O: C, 63.55; H, 4.66; N,
15.26. Found: C, 63.91; H, 4.36; N, 15.38.
1
Mp 162−164 °C. HNMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H),
9.33 (s, 1H), 8.72 (d, J = 4.0 Hz, 1H), 8.63 (d, J = 8.0 Hz, 1H), 8.43
(d, J = 8.4 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.13−8.09 (dd, J = 6.4,
2.0 Hz, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.72 (dd, J = 8.4, 5.2 Hz, 1H),
7.62 (d, J = 8.0 Hz, 1H), 7.57−7.54 (m, 4H), 7.33 (t, J = 6.0 Hz, 2H),
7.06 (t, J = 7.6 Hz, 1H), 6.82 (t, J = 6.8 Hz, 1H), 3.78 (s, 2H). MS m/z
498 [M + H]+. Anal. Calcd for C30H23N7O·3.26TFA: C, 50.46; H,
3.04; N, 11.28. Found: C, 50.47; H, 2.97; N, 11.25.
tert-Butyl 4-(2-(2-Aminopyridin-3-yl)-5-(pyridin-3-yl)-3H-
imidazo[4,5-b]pyridin-3-yl)benzylcarbamate (10f). 10f was
prepared from 6d as described for 12d using 3-pyridylboronic acid.
1
Mp 223−225 °C. H NMR (400 MHz, DMSO-d6) δ 9.23 (d, J = 1.7
Hz, 1H), 8.59 (dd, J = 4.7, 1.5 Hz, 1H), 8.41−8.34 (m, 1H), 8.32 (d, J
= 8.3 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 8.01 (dd, J = 4.8, 1.8 Hz, 1H),
7.52−7.37 (m, 5H), 7.22 (dd, J = 7.7, 1.8 Hz, 1H), 7.00 (s, 2H), 6.42
(dd, J = 7.6, 4.9 Hz, 1H), 4.24 (d, J = 6.1 Hz, 2H), 1.41 (s, 9H). MS
m/z 494 [M + H]+.
N-(4-(2-(2-Aminopyridin-3-yl)-6-(pyridin-3-yl)-3H-imidazo-
[4,5-b]pyridin-3-yl)benzyl)benzamide (12e). 12e was prepared
from 11a as described for 8b using benzoic acid. Mp 257−260 °C. 1H
NMR (400 MHz, DMSO-d6) δ 9.15 (t, J = 5.6 Hz, 1H), 9.01−9.00
(m, 1H), 8.67−8.66 (m, 1H), 8.63−8.62 (m, 1H), 8.58−8.57 (m, 1H),
8.22−8.19 (m, 1H), 8.02−8.00 (m, 1H), 7.95−7.92 (m, 2H), 7.57−
7.45 (m, 8H), 7.29−7.27 (m, 1H), 7.03 (s, br, 2H), 6.45−6.42 (m,
1H), 4.59−4.58 (m, 2H). MS m/z 497 [M]+.
N-(4-(2-(2-Aminopyridin-3-yl)-5-(pyridin-3-yl)-3H-imidazo-
[4,5-b]pyridin-3-yl)benzyl)-3-fluorobenzamide (12k). Step 1: 3-
(3-(4-(Aminomethyl)phenyl)-5-(pyridin-3-yl)-3H-imidazo[4,5-b]-
pyridin-2-yl)pyridin-2-amine (11d) was prepared as described for 5a
O
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from 10f and was used without further purification. Step 2: 12k was
prepared from 11d as described for 8b using 3-fluorobenzoic acid. Mp
236−234 °C. H NMR (400 MHz, DMSO-d6) δ 9.26 (t, J = 6.0 Hz,
1H), 9.23 (d, J = 1.7 Hz, 1H), 8.59 (dd, J = 4.7, 1.6 Hz, 1H), 8.44−
8.34 (m, 1H), 8.32 (d, J = 8.3 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.01
(dd, J = 4.8, 1.9 Hz, 1H), 7.85−7.77 (m, 1H), 7.77−7.69 (m, 1H),
7.62−7.52 (m, 1H), 7.52−7.46 (m, 5H), 7.46−7.37 (m, 1H), 7.24 (dd,
J = 7.7, 1.8 Hz, 1H), 6.97 (s, 2H), 6.43 (dd, J = 7.7, 4.8 Hz, 1H), 4.62
(d, J = 6.0 Hz, 2H). MS m/z 516 [M + H]+.
with the following parameters: capillary voltage 3.5 V, cone voltage 40
V, source temperature 120 °C, desolvation temperature 200 °C.
Binders were identified by the detection of their molecular weight at
the appropriate retention time compared with the mixtures of
compound standards dissolved in DMSO and analyzed with the
same analytical method.
1
Thermal Shift Binding Assay (Akt1 (1−480), AKT2 (1−481),
and Akt3 (1−479)). The binding affinities of test compounds to the
full length inactive Akt isoforms were assessed by a thermal shift
binding assay. Aliquots of compounds in DMSO (l μL at 375 μM)
were placed in a V-bottom 96-well PCR plate and incubated for 30
min with 24 μL of the protein (1.5 μM) in 25 mM Tris buffer, pH 7.5,
100 mM NaCl, 0.1% 2-mercaptoethanol, premixed with environ-
mentally sensitive fluorescent dye Sypro Orange (12.5×). The plates
were centrifuged for 3 min at 2000 rpm and 25 °C before analysis on
an IQ5 real time PCR machine. The thermal stability of the protein−
ligand complex was monitored by changes in Sypro Orange
fluorescence while subjecting the binding mixture to a temperature
gradient (30 to 75 at 1 °C/min heating rate). Shifts in the protein
melting temperature (ΔTm) greater than 0.6 °C compared to the
protein alone were indicative of inhibitor binding. The reported
thermal shift data are a geometric mean of at least two measurements
with typical STD of <10%.
Tryptophan Fluorescence Quench Assay. Binding of AMP-
PNP and A674563 to Akt1 constructs (Akt1 (1−480) and Akt1 (144−
480)) was detected by measuring the intrinsic tryptophan
fluorescence. The Akt1 proteins (2 μM) were titrated with increasing
concentrations of AMP-PNP or A674563. The change in intrinsic
emission was measured on a Tecan microplate reader at an excitation
wavelength of 295 nm and emission wavelength of 345 nm. The
binding measurements were performed in a buffer solution containing
25 mM Tris, pH 7.5, 0.1 M NaCl, and 2% DMSO. The magnitude of
increase in tryptophan fluorescence (ΔF) was calculated as ΔF = F −
F0, where F and F0 are the emission intensities in the presence and
absence of ligand, respectively. Dissociation constants (Kd) were
Protein Expression and Purification. Full length Akt isoform
constructs (Akt1 (2−480), Akt2 (2−481), and Akt3 (2−479)) and
ΔPH-Akt1 (144−480) were expressed using a baculovirus vector in
Sf9 cells. The frozen cell pellets were thawed and resuspended in lysis
buffer containing 25 mM Tris, pH 8.0, 100 mM NaCl, 25 mM
imidazole, 10% glycerol, protease inhibitors (Sigma no. S8820). Cells
were lysed by sonication. The lysate was centrifuged, and the clarified
supernatant was loaded onto a Ni-NTA affinity column. Following the
washing with buffer containing 25 mM Tris, pH 8.0, 500 mM NaCl,
and 25 mM imidazole, the protein was eluted in buffer containing 25
mM Tris, 100 mM NaCl, and 400 mM imidazole, pH 8.0. The protein
was concentrated and purified on a Superdex 200 gel filtration column.
The concentrated protein (10 mg/mL) was stored in buffer containing
25 mM Tris, pH 8.0, 100 mM NaCl, and 10% glycerol at −80 °C.
Protein Characterization (Akt1, Akt2, and Akt3). The
phosphorylation state of Thr308 and Ser473 in Akt1 (1−480) was
analyzed as described below. Proteins were subjected to a solution-
phase digest, and the phosphorylation state of the peptides was
determined by LC/MS and LC/MS/MS analysis. Protein (50 μg) was
diluted in 50 mM ammonium bicarbonate buffer, pH 8, containing 5
mM DTT, 3 M urea, 5% acetonitrile, and endoprotease (trypsin, 1:20
w/w or GluC, 1:10 w/w ratio), and digested overnight. The digest was
separated on a BEH C18 column (1.0 mm × 50 mm, 1.7 μm)
equilibrated at 40 °C and mass-analyzed on a QTOF mass
spectrometer. The elution was performed using a water/acetonitrile
gradient with 0.1% formic acid modifier, starting from 2% and reaching
40% acetonitrile in 14 min, followed by 40−50% acetonitrile from 14
to 16 min, 50−80% acetonitrile from 16 to 18 min, held at 80% for 2
min, and re-equilibriated at 2% acetonitrile for another 3 min. Mass
spectra of peptides were deconvoluted by the MaxEnt3 software
package (Masslynx, Waters). For the inactive Akt proteins, no
phosphorylation was detected on the Thr308/Thr309/Thr305 site,
and less than 10% of the Ser473/Ser474/Ser472 was detected as
phosphorylated (in Akt1, Akt2, and Akt3, respectively). Following
activation with PDK1 and MAPKAP2 in the presence of lipids, MS
analysis identified 60% of Thr308 and 80% of Ser473 as
phosphorylated.
High Throughput Binding Screen by Indirect Affinity Mass
Apectrometry (IA-MS) (Mixed Pool). The compound library was
plated into 96-well plates as mixtures of 20 compounds per well in
DMSO. Test compounds in DMSO were mixed with buffer consisting
of 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.1% 2-β-
mercaptoethanol and incubated with inactive full length Akt1 (1−480)
for 30 min at room temperature. The final binding mixture (25 μL
volume) contained 10 μM Akt1 (1−480) protein, compound pool (10
μM each), and a 4% final DMSO concentration. Protein-bound
inhibitors were separated from the unbound inhibitors by fast
centrifugation at 4 °C through a size exclusion gel in a 96-well plate
format. Controls that contained the compound pool (10 μM each) in
buffer were used to verify that no inhibitor passed through the gel in
the absence of protein carrier. The eluent from the size exclusion
column (∼25 μL) was mixed with 30 μL of an internal standard in
DMSO/H2O (1:1) and analyzed for the presence of the small
molecular weight inhibitor by LC/MS. Test samples were injected
onto the Acquity UPLC instrument equipped with a BEH C18 column
(1.0 mm × 50 mm, 1.7 μm) and separated at 0.1 mL/min using a
water/acetonitrile gradient with 0.1% formic acid modifier, starting at
10% and reaching 90% acetonitrile in 5 min, held at 90% for 1 min,
and re-equilibriating at 10% acetonitrile for another 3 min. UPLC flow
was directed to a QTOF mass spectrometer (Waters) equipped with
an ESI probe, which was operated in the full scan monitoring mode
obtained from a fit to the hyperbolic binding equation, ΔF/F0
=
Fmax[L]/([L] + Kd), where Fmax corresponds to the fluorescence
intensity at saturation.
Surface Plasmon Resonance (SPR). SPR measurements were
performed on a Reichert SR7500DC SPR system (Reichert
Technologies, Depew, NY). Unphosphorylated Akt1 (1−480) was
exchanged into sodium acetate buffer (10 mM, pH 5.2) and
immobilized onto a Reichert CMD 500k chip using standard amine
coupling methodology. Specifically, a solution containing 40 mg of
EDC (Sigma) and 10 mg of NHS (Sigma) dissolved in 0.9 mL of
doubly distilled H2O was injected over the surface for 7 min. A 182
μg/mL solution of Akt1 (1−480) in 10 mM sodium acetate buffer
with 1 mM DTT was subsequently injected over the sensor surface for
about 10 min, achieving an immobilization response level of 8400
μRIU. Lastly, the surface was deactivated/blocked with an injection of
1 M ethanolamine (Sigma), pH 8.5, with 1 mM DTT for 10 min. The
running buffer for immobilization was 25 mM HEPES, pH 7.4, 150
mM NaCl, 0.05% Tween 20, and 1 mM DTT. Kinetic analysis was
performed at 25 °C in 25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM
MgCl2, 5 mM DTT, and 5% DMSO. Test compound was prepared in
the buffer used for kinetic analysis at 31.25, 62.5, 125, and 500 nM and
injected over the sample and reference flow cells at 50 μL/min. The
association and dissociation phases were each monitored for 3 min,
and the test compound concentrations were injected from low to high
without regeneration (kinetic titration approach). Data were fit to a
1:1 kinetic titration model.
Lipid Vesicles Preparation. Lipid vesicles were prepared from
PtdIns(3,4,5)P3, dipalmitoyl, and a synthetic phospholipid blend of
DOPS/DOPC. A 1.0 mg/mL solution of PtdIns(3,4,5)P3 and a 16.7
mg/mL suspension of DOPS/DOPC in 10 mM Hepes, pH 7.4, were
prepared immediately before use. The DOPS/DOPC suspension (800
μL) and the PtdIns(3,4,5)P3 solution (964 μL) were mixed and then
diluted in 18.2 mL of 10 mM Hepes, pH 7.4, and mixed well. This
mixture, (400 μM DOPS/400 μM DOPC/40 μM PtdIns(3,4,5)P3),
P
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was sonicated on ice for 30 s at 75% power until the mixture was
clarified. Aliquots were prepared and frozen at −80 °C until use.
Akt1 (1−480), Akt2 (1−481), and Akt3 (1−479) α Screen
Assays. Akt activity was assayed using a GSK3-derived biotinylated
peptide substrate, crosstide (biotin-GRPRTSSFAEG), and the
AlphaScreen (amplified luminescent proximity homogeneous assay)
technology. Test compounds and controls were prepared in 10%
DMSO at 10-fold the desired final concentration and added to each
well of a reaction plate (Corning 96-well half-area solid white
nonbinding surface plate) in a volume of 2.5 μL. Full-length
unphosphorylated Akt was diluted in assay buffer (50 mM Tris, pH
8.0, 0.02 mg/mL BSA, 10 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.2
mM Na3VO4, 1 mM DTT, 0.1 mM β-glycerophosphate, and 0.2 mM
NaF) and added to each well in a volume of 17.5 μL for a final
concentration in the 25 μL mixture of 8 nM (Akt1), 63 nM (Akt2), or
13 nM (Akt3). After a 20 min preincubation at room temperature, the
kinase reaction was initiated by the addition of 5 μL of the activation
mixture diluted in assay buffer containing biotinylated crosstide,
PDK1, MAPKAPK2, DOPS/DOPC, PtdIns(3,4,5)P3, and ATP for
final concentrations of 60 nM biotinylated crosstide, 0.1 nM (Akt1,
Akt3) or 0.3 nM (Akt2) PDK1, 0.7 nM (Akt1), 1.3 nM (Akt2), or 0.4
nM (Akt3) MAPKAPK2, 5.5 μM DOPS, 5.5 μM DOPC, 0.5 μM
PtdIns(3,4,5)P3, and 50 μM (Akt1, Akt2) or 18 μM (Akt3) ATP. The
plates were incubated for 30 min at room temperature. The reaction
was stopped in the dark by the addition of 10 μL of stop/detection
mixture prepared in the assay buffer containing EDTA, AlphaScreen
streptavidin donor and protein A acceptor beads, and phospho-AKT
substrate antibody for final concentrations of 10 mM EDTA, 500 ng/
well of both AlphaScreen streptavidin donor beads and protein A
acceptor beads, and phospho-AKT substrate antibody at a final
dilution of 1:350. Assay plates were incubated for 90 min at room
temperature in the dark, and the plates were read on a Perkin-Elmer
Envision multilabel plate reader (excitation wavelength of 640 nm,
emission wavelength of 570 nm). All IC50 values reported are the
geometric mean of at least n = 2 determinations.
μL. PDK1 was diluted in assay buffer (50 mM Tris, pH 8.0, 0.02 mg/
mL BSA, 10 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.1 mM
Na3VO4, 1 mM DTT) and added to each well in a volume of 17.5 μL
for a final concentration in the 25 μL mixture of 6.0 nM. After a 20
min preincubation at room temperature, the kinase reaction was
initiated by the addition of 5 μL of an activation mixture diluted in
assay buffer containing biotinylated PKA(Thr197), DOPS/DOPC,
PtdIns(3,4,5)P3 and for final concentrations of 0.5 μM biotinylated
PKA(Thr197), 5.5 μM DOPS, 5.5 μM DOPC, 0.5 μM PtdIns(3,4,5)-
P3, and 2 μM ATP. The plates were incubated for 60 min at room
temperature, and then the reaction was stopped in the dark by the
addition of 10 μL of stop/detection mixture prepared in assay buffer
containing EDTA, AlphaScreen streptavidin donor and protein A
acceptor beads, and phospho-AKT substrate antibody for final
concentrations of 10 mM EDTA, 500 ng/well of both AlphaScreen
streptavidin donor beads and protein A acceptor beads, and phospho-
PKA C (Thr197) antibody at a final dilution of 1:945. Assay plates
were incubated for 60 min at room temperature in the dark, and the
plates were read on a Perkin-Elmer Envision multilabel plate reader
(excitation wavelength of 640 nm, emission wavelength of 570 nm).
All IC50 values reported are the geometric mean of at least n = 2
determinations.
Cellular Antiproliferation Assay (MTS). Antiproliferative cellular
assays were conducted using the CellTiter nonradioactive cell
proliferation assay developed by Promega utilizing the production of
formazan from a tetrazolium compound by live cells. AN3CA and
A2780 cells were obtained from ATCC. AN3CA cells were cultured in
DMEM, and A2780 cells were cultured in RPMI supplemented with
10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2
mM glutamine at 37 °C under 5% CO2. Cells were plated in 96-well
plates at 2000−10000 cells/well, cultured for 24 h, and treated with
test compound to a final DMSO concentration no greater than 0.5%
v/v for 72 h. PMS stock reagent (0.92 mg/mL in DPBS) was diluted
20-fold in MTS stock reagent (2 mg/mL in DPBS), and this MTS/
PMS mixture was diluted 5-fold into each well of the 96-well plate.
Plates were incubated for 3−4 h, and the absorbance of formazan was
measured at 490 nm. Data were normalized to untreated controls.
Dose response curves were fit to a four-parameter logistic equation,
and IC50 values were determined. All IC50 values reported are the
geometric mean of at least two independent determinations.
Akt1 (104−480). Reactions with Akt1 (Carna 01-101) enzyme in
the activated form were performed as above in the AlphaScreen format
with the following modifications. PDK1, MAPKAPK2, and the lipid
vesicle mixture were omitted from the reaction. Active Akt1 was added
for a final reaction concentration of 0.1 nM.
MAPKAPK2 Assay. MAPKAPK2 activity was assayed using the
CisBio KinEASE HTRF assay technology. Test compounds and
control (staurosporine) were prepared in 10% DMSO at 10-fold the
desired final concentration and added to each well of a reaction plate
(Corning 96-well half-area solid black nonbinding surface plate) in a
volume of 2.5 μL. MAPKAPK2 was diluted into assay buffer (50 mM
Tris, pH 8.0, 0.02 mg/mL BSA, 10 mM MgCl2, 1 mM EGTA, 10%
glycerol, 0.1 mM Na3VO4, and 1 mM DTT) and added to each well in
a volume of 17.5 μL for a final concentration in the 25 μL mixture of
1.7 nM. After a 20 min preincubation at room temperature, the kinase
reaction was initiated by the addition of 5 μL of an activation mixture
diluted in assay buffer containing biotinylated STKS1 and ATP for
final concentrations of 50 nM biotinylated STKS1 and 5 μM ATP.
The plates were incubated for 30 min at room temperature and then
stopped by the addition of 25 μL of HTRF detection buffer containing
phospho-STK antibody-Eu3+-cryptate and streptavidin labeled XL665
antibody at dilutions of 1:200 and 1:2647, respectively. Final assay
dilutions of phospho-STK antibody-Eu3+-cryptate and streptavidin
labeled XL665 antibody were 1:400 and 1:5294, respectively. Assay
plates were incubated for 60 min at room temperature, and the plates
were read on a Perkin-Elmer Envision multilabel plate reader
(excitation of 320 nm, emission I of 665 nm, emission II of 615 nm).
PDK1 Assay. PDK1 activity was assayed using the PKA(Thr197)
derived biotinylated peptide substrate, biotin-RTWT*LCG, and the
AlphaScreen technology. Test compound and controls (N-[2-
(diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-
ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide (sutent) and
staurosporine were prepared in 10% DMSO at 10-fold the desired final
concentration and added to each well of a reaction plate (Corning 96-
well half-area solid white nonbinding surface plate) in a volume of 2.5
In Vitro Western Blot Pharmacodynamic Assay (p-Ser473, p-
Thr308, p-PRAS40). Cellular activity was determined by measuring
the phosphorylation status of Akt Ser473, Thr308, and its substrate
phospho-PRAS40 using A2780 and AN3CA cell lines. A2780 and
AN3CA cells were plated in six-well plates at 60−75% confluency and
incubated at 37 °C overnight in 5% CO2. At 75−90% confluency, cells
were treated with test compound, following a 3-fold dilution scheme,
for 1 h with a final DMSO concentration of less than 0.2% v/v. Cells
were stimulated with 100 ng/mL EGF and 100 nM insulin for 15 min.
The medium was removed, and 150 μL of 1× E-Page loading buffer
(catalog no. EPBUF-01, Invitrogen) was added. Cells were scraped
and transferred to a deep well 96-well plate and lysed by sonication at
medium power four to six times in 30 s bursts. Samples were run on
EPage gels and transferred to PVDF membranes using the iBlot
system (Invitrogen). Membranes were washed for 3 min in distilled
H2O, blocked for 30 min in Odyssey blocking buffer (catalog no. 927-
40000, LiCor), and then incubated overnight at 4 °C in Odyssey
blocking/TBST buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.05%
Tween 20) (1:3) with antibodies against phospho-Akt (catalog no.
9271 and catalog no. 9275, Cell Signaling Technology), phospho-
PRAS40 (catalog no. 2640, Cell Signaling Technology), and β-actin
(catalog no. A2228, Sigma). After three washes for 5 min each in
TBST buffer, membranes were incubated with the appropriate
secondary antibodies labeled with near-infrared or Alexa Fluor dyes
(catalog no. 611-132-122 goat anti-rabbit IgG H + L, catalog no. 610-
132-121 goat anti-mouse IgG H + L, Rockland catalog no. A21076
goat anti-rabbit IgG H + L, catalog no. A21057 goat anti-mouse IgG H
+ L, Molecular Probes). After 1 h of incubation, membranes were
washed three times for 5 min each and then scanned using an Odyssey
infrared scanner (LiCor). The intensity of the bands was quantified
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using the accompanying software. Data were normalized to untreated
controls. Dose response curves were fitted to a four-parameter logistic
equation, and IC50 values were determined.
Determination of 12j and 12e Concentrations in Mouse
Plasma and AN3CA Tumors. CD-1 mouse plasma (sodium heparin
as anticoagulant) from Bioreclamation (Hicksville, NY) was used for
standard curve preparation and plasma sample dilution when
necessary. Tumor tissues (density was treated as 1 mg/mL) were
weighed and diluted with 10 mM NH4OAc at volumes 9× the tumor
volume (i.e., 450 μL buffer was added to a 50 mg tumor). The
mixtures were homogenized using a hand-held homogenizer, and
vehicle treated mouse tissue homogenates were used to prepare
standard curves. Plasma and tissue standards were prepared by spiking
5 μL of 10× acetonitrile spiking solutions into 50 μL of plasma or
tumor tissue to produce standards from 1 to 2000 ng/mL
concentrations. Study plasma samples (5 μL) were diluted with 45
μL (1:10) of blank mouse plasma. A total of 50 μL plasma was added
to 400 μL of acetonitrile containing 50 ng/mL reserpine (internal
standard). Tumor homogenate (50 μL) was added without further
dilution to 400 μL of acetonitrile containing 50 ng/mL reserpine. The
mixtures were vortexed, sonicated for 2 min, and then centrifuged at
3000 rpm for at least 10 min. Supernatant (200 μL) was transferred to
a shallow 96-well plate and solvent removed under nitrogen gas, 30
°C. The dried samples were reconstituted in 150 μL of 10%
acetonitrile in HPLC water for LC/MS/MS analysis.
LC/MS/MS Analysis of Plasma and Tumor Samples. Analysis
was performed using a Waters Quattro Micro triple quadrupole mass
spectrometer (Milford, MA) equipped with an ESI source. The
samples were delivered to the mass spectrometer ion source by an
Agilent 1100 binary HPLC pump (Santa Clara, CA), and samples were
injected using a LEAP CTC PAL autoinjector (Carrboro, NC). A
Waters SymmetryShield RP18 (2.1 mm × 100 mm, 3.5 μm, Milford,
MA) reverse-phase HPLC column was used for separation. Mobile
phases contained water (A) and acetonitrile (B) both modified with
0.1% formic acid. The HPLC gradient was started and maintained at
10% mobile phase A for the first 0.5 min and was increased to 80%
mobile phase B over the next 6.5 min and held there for 0.8 min. At
7.81 min, the mobile phase was returned to 10% mobile phase A and
maintained there until the end of the run at 10 min. Flow rates were
set at 0.2 mL/min. Transitions ions monitored were as follows: for 12j
they were m/z 572.1 → 391.0, 432.9, and 530.0 (all three summed for
quantification); for 12e they were m/z 498.0 → 272.2 and 377.2 (both
summed for quantification); for reserpine it was m/z 609.2 → 195.2.
Peak areas for all analytes and the internal standard (reserpine) were
determined using Waters MassLynx, version 4.1, software (Waters,
Milford, MA). Peak area ratios were next calculated by dividing the
peak area of analyte by the reserpine peak area for all test samples.
Standard curves were fit with a linear equation and with weighting of
1/x2. All R2 values for linear fit for plasma and tissues were >0.98.
When appropriate, AUC values were estimated with the linear
trapezoidal method using WinNonlin (version 5.2) to calculate oral
bioavailability.
Cross-Species NADPH-Dependent Microsomal Stability
Assay. Human and CD-1 mouse, liver microsomes (0.25 mg/mL),
3.3 mM MgCl2, and NADPH-regenerating system (0.4 units/mL
G6PDH, 1.3 mM NADP+, and 3.3 mM G6P) were incubated with 1
μM compound for 0, 3, 6, 10, 15, or 30 min. The incubations were
stopped with acetonitrile containing warfarin (internal standard), and
the samples were analyzed by LC/MS/MS. Peak area ratios were used
to determine percent remaining at each time point compared to time
zero. Half-lives were estimated using the equation for monoexponen-
tial decay. Midazolam and propranolol (female mouse only) were used
as positive controls.
CYP450 Inhibition Assay. Human liver microsomes (0.25 mg/
mL), probe substrates [3 μM midazolam (3A4), 5 μM bufuralol
(2D6), 100 μM tolbutamide (2C9), 10 μM paclitaxel (2C8), 80 μM S-
mephenytoin (2C19), and 50 μM phenacetin (1A2)], 3.3 mM MgCl2,
1 mM NADPH, and compound (0.1−10 μM) dissolved in DMSO
(0.1% final) were incubated for 10 min, at which time acetonitrile
containing reserpine (internal standard) was used to stop the reaction.
Solvents were evaporated under a constant stream of nitrogen gas at
35 °C and then reconstituted in 100 μL of water for LC/MS/MS
analysis. A cocktail of inhibitors was used as a positive control and
consisted of 0.5 μM ketoconazole (3A4), 1 μM sulfaphenazole (2C9),
1 μM quinidine (2D6), 2 μM quercetin (2C8), 1 μM nootkatone
(2C19), and 0.5 μM α-naphthoflavone (1A2). Incubations containing
only DMSO (no compound) served as the 100% activity control.
Percent inhibition was calculated based on the signal for the 100%
activity control, and IC50 values were either estimated or determined
using fit model 205 (one site dose response) in XLfit4 (Guildford,
Surrey, U.K.).
Materials and Methods for in Vivo Studies. Six-week old NCr
female nude mice were purchased from Taconic Farms (Germantown,
NY) and allowed to acclimate for more than 2 weeks. Mice were
housed in sterile microisolator cages, five mice per cage, and received
food and water ad libitum.
In Vivo Pharmacokinetic Studies. Naive NCr female nude mice
were utilized for a definitive PK study with compound 12e. Oral
administration (po) was at 100 mg/kg in NMP/DMA/Tween 80/
PEG 400 (10:10:10:70). Time points for the oral arm were 0.5, 1, 2, 4,
8, 24, and 48 h after a single dose with three mice per time point. An iv
arm (tail vein injection with a 27G needle) was administered at 2.5
mg/kg in DMA/propyleneglycol/cremophore/20% HPBCD in 0.2 M
acetate buffer, pH 5.0 (10:30:10:50). Time points were 5, 15, 30 min
and 1, 2, 4, 8, and 24 h after administration. The dosing volume for all
groups was 10 mL/kg. At specific time points mice were euthanized
with CO2 inhalation. Whole blood was retrieved via cardiac puncture
and placed in heparin coated tubes. Plasma was collected and placed
on dry ice until analysis.
Pharmacodynamic Analysis of the Akt Pathway in Vivo (p-
Ser473, p-Thr308, p-P70s6). Frozen tumor tissues were ground in
mortars with liquid nitrogen. The tissue powder was lysed in buffer
consisting of 50 mM Tris-HCl (pH 7.5), 20 mM NaCl, 6 mM EDTA,
15 mM EGTA, 1% NP-40, and a protease/phosphatase inhibitor
cocktail. Samples were run on EPage gels and transferred to PVDF
membranes using the iBlot system (Invitrogen). Membranes were
washed for 3 min in distilled H2O, blocked for 30 min in Odyssey
blocking buffer (catalog no. 927-40000, LiCor), and then incubated
overnight at 4 °C in Odyssey blocking/TBST buffer (50 mM Tris, pH
7.4, 150 mM NaCl, 0.05% Tween 20) (1:3) with antibodies against
phospho-Akt (Ser473 and Thr308) (catalog no. 9271 and catalog no.
9275, Cell Signaling Technology), phospho-p70S6 (Thr389) (catalog
no. 9206, Cell Signaling Technology), and β-actin (catalog no. A2228,
Sigma). After three washes for 5 min each in TBST buffer, membranes
were incubated with the appropriate secondary antibodies labeled with
near-infrared or Alexa Fluor dyes (catalog no. 611-132-122), goat anti-
rabbit IgG H + L (catalog no. 610-132-121), goat anti-mouse IgG H +
L (Rockland or catalog no. A21076), goat anti-rabbit IgG H + L
(catalog no. A21057), goat anti-mouse IgG H + L (Molecular Probes).
After 1 h of incubation, membranes were washed three times for 5 min
Acute Pharmacokinetic (PK) and Pharmacodynamic (PD)
Assays in Vivo. AN3CA cells were purchased from ATCC. Cells
were cultured in sterile RPMI supplemented with 10% fetal bovine
serum. Mice were implanted subcutaneously in the upper right
shoulder flank with 5 × 106 cells suspended in sterile Hanks buffered
salt solution (HBSS) in 0.2 mL volume. Mice were monitored and
randomized when tumor size ranged between 300 and 500 mg. Tumor
measurements and body weights were collected once with electronic
calipers and balance. The tumor weight (mg) was calculated from the
following equation: [(length)(width)2]/2. This formula was also used
to calculate tumor volume, assuming unit density 1 mg = 1 mm3. Mice
received a single administration of selected compound dissolved in
NMP/DMA/Tween 80/PEG400 (10:10:10:70) or 0.1% Tween 80 in
1% HPMC. Dosing volume was 10 mL/kg. At designated time points
after administration, mice were euthanized with CO2 inhalation and
tumor tissue was trisected. Two-thirds went immediately into tubes on
dry ice, while one-third was place in 10% formalin for further analysis.
Whole blood was harvested via cardiac puncture and placed in heparin
coated tubes. Plasma was retrieved and placed in an Eppendorf tube
on dry ice until analysis.
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each and then scanned using the Odyssey infrared scanner (LiCor).
The intensities of the bands were quantitated using the accompanying
software.
Human Mesenchymal-Epithelial Transcription Factor (c-Met) Protein
Autophosphorylation by ARQ 197. J. Biol. Chem. 2011, 286, 20666−
20676.
Crystallization, Data Collection, and Refinement. A 6xHis-
Akt1 (2−446) with a TEV protease site construct was cloned in a
baculovirus vector and expressed in Sf9 cells. To aid the crystallization,
three mutations were introduced on the linker region (E114A, E115A,
and E116A) in the Akt1 (2−446) construct. The protein was purified
with a similar protocol as described for the Akt isoforms described
above and the 6xHis tag was removed using TEV protease. Cocrystals
of 12j were obtained with the following conditions: 16% butanol, 10
mM ammonium sulfate, 0.1% 2-mercaptoethanol, 15% ethylene glycol,
50 mM Tris, pH 7.5. Diffraction data were collected at NSLS on the
X29 beamline, and the data were processed using Mosflm and scaled
using Scala. The structure was solved by molecular replacement using
the Phaser program with search models consisting of Akt2 (1MRV)
and the Akt1 PH domain (2UVM). The initial model was built using
ARP/wARP program, and the model was subsequently improved
through iterative cycles of manual model building with Coot and
refinement with ARP/wARP or Refmac5.
(7) Eathiraj, S.; Palma, R.; Hirschi, M.; Volckova, E.; Enkeleda, N.;
Castro, J.; Chen, C.-R.; Chan, T. C. K.; France, D. S.; Ashwell, M. A. A
Novel Mode of Protein Kinase Inhibition Exploiting Hydrophobic
Motifs of Autoinhibited Kinases. J. Biol. Chem. 2011, 286, 20677−
20687.
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Hemmings, B. A.; Downward, J.; Parker, P. J.; Larijani, B.
Intramolecular and Intermolecular Interactions of Protein Kinase B
Define Its Activation in Vivo. PLoS Biol. 2007, 5, e95.
(9) Huang, X.; Begley, M.; Morgenstern, K. A.; Gu, Y.; Rose, P.;
Zhao, H.; Zhu, X. Crystal Structure of an Inactive Akt2 Kinase
Domain. Structure 2003, 11, 21−30.
(10) Muckenschnabel, I.; Falchetto, R.; Mayr, L. M.; Filipuzzi, I.
SpeedScreen: Label-Free Liquid Chromatography−Mass Spectrome-
try-Based High-Throughput Screening for the Discovery of Orphan
Protein Ligands. Anal. Biochem. 2004, 324, 241−249.
(11) Lo, M. C.; Aulabaugh, A.; Jin, G.; Cowling, R.; Bard, J.;
Malamas, M.; Ellestad, G. Evaluation of Fluoresence-Based Thermal
Shift Assays for Hit Identification in Drug Discovery. Anal. Biochem.
2004, 332, 153−159.
(12) Pantoliano, M.; W., Petrella, E. C.; Kwasnoski, J. D.; Lobanov,
V. S.; Myslik, J.; Graf, E.; Carver, T.; Asel, E.; Springer, B. A.; Lane, P.;
Salemme, F. R. High-Density Miniaturized Thermal Shift Assays as a
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(13) Nordin, H.; Jungnelius, M.; Karlsson, R.; Karlsson, O. P. Kinetic
Studies of Small Molecule Interactions with Protein Kinases Using
Biosensor Technology. Anal. Biochem. 2005, 340, 359−368.
(14) Bozulic, L.; Hemmings, B. A. PIKKing on PKB: Regulation of
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261.
AUTHOR INFORMATION
Corresponding Author
*Phone: (781) 994-0467. E-mail: tchan@arqule.com.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
PI3K/Akt/mTOR, phosphoinositide 3-kinase/protein kinase
B/mammalian target of rapamycin; PH, pleckstrin homology;
HM, hydrophobic motif; AMP-PNP, adenylyl imidodiphos-
phate, tetralithium salt; HCl, hydrochloric acid; HBTU, O-
benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophos-
phate; CDI, 1,1′-carbonyldiimidazole; EDCI, 1-(3-dimethyla-
minopropyl)-3-ethylcarbodiimide; NaOH, sodium hydroxide;
Tm, protein melting temperature; STD, standard deviation;
MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphen-
yl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PRAS40, pro-
line-rich Akt substrate, 40 kDa; p70S6, ribosomal protein S6
kinase; SPR, surface plasmon resonance; IA-MS, indirect
affinity mass spectrometry; SEC/LC/MS, size exclusion/liquid
chromatography/mass spectrometry; TSA, thermal shift assay,
MLM, mouse liver microsome; HLM, human liver microsome;
Kd, dissociation constant
(15) All 3-(3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine analogues
tested in the two counterscreens had IC50 values greater than 10 μM.
(16) Chang, L. C. W.; Kunzel, J. K. F. D.; Mulder-Krieger, T.;
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Clerin, V.; Chen, L.; Qiang-Yuan Liu, Q.-Y.; Feingold, I.; Huselton, C.;
Azam, F.; Goos-Nilsson, A.; Wilhelmsson, A.; Nambib, P.; Wrobela, J.
Further Modification on Phenyl Acetic Acid Based Quinolines as Liver
X Receptor Modulators. Bioorg. Med. Chem. 2007, 15, 3321−3333.
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Lin, J.; Marantz, Y.; Melendez, R.; Mohanty, P.; Shacham, S.; Zhang,
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T
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