Design, synthesis, and evaluation of 3,5-disubstituted 7-azaindoles as Trk inhibitors with anticancer and antiangiogenic activities

Article abstract of DOI:10.1021/jm3002982

Tropomyosin-related kinase A (TrkA) is considered a promising target in the development of a therapeutic treatment of cancer and pain. In this study, we designed and synthesized a series of novel 7-azaindole-based Trk kinase inhibitors through the structure-based design strategy. By varying the functional groups at the 3 and 5 positions of a 7-azaindole scaffold, we studied the structure-activity relationships (SAR) profiles and identified a series of potent Trk inhibitors. Representative derivatives showed desirable activity in cellular proliferation and apoptosis assays. Moreover, these inhibitors exhibited noteworthy antiangiogenic activity.

Full text of DOI:10.1021/jm3002982

Article
pubs.acs.org/jmc
Design, Synthesis, and Evaluation of 3,5-Disubstituted 7-Azaindoles
as Trk Inhibitors with Anticancer and Antiangiogenic Activities
Seunghee Hong,†,§ Jinhee Kim,†,§ Ju Hyeon Seo,‡,§ Kyung Hee Jung,‡ Soon-Sun Hong,*,‡
and Sungwoo Hong*,†
†Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea
‡Department of Biomedical Sciences, College of Medicine, Inha University, Incheon, 400-712, Korea
S
* Supporting Information
ABSTRACT: Tropomyosin-related kinase A (TrkA) is
considered a promising target in the development of a
therapeutic treatment of cancer and pain. In this study, we
designed and synthesized a series of novel 7-azaindole-based
Trk kinase inhibitors through the structure-based design
strategy. By varying the functional groups at the 3 and 5
positions of a 7-azaindole scaffold, we studied the structure−
activity relationships (SAR) profiles and identified a series of
potent Trk inhibitors. Representative derivatives showed
desirable activity in cellular proliferation and apoptosis assays.
Moreover, these inhibitors exhibited noteworthy antiangiogenic activity.
Trks could produce antiangiogenic activity useful against
human cancers. Despite their potential as cancer therapeutic
targets, only a few Trk inhibitors have been studied to explore
their effect on major cancer diseases.11−16 Herein, we report the
identification of new 7-azaindole-based Trk inhibitors that
demonstrate anticancer and antiangiogenic effects in human
breast cancer cells. The design strategy for improving their
potency using docking simulations in ATP-binding sites to
analyze the binding mode in detail was also discussed.
INTRODUCTION
■
Receptor tyrosine kinases (RTKs) are transmembrane
receptors that phosphorylate tyrosine residues with specificity
in various protein substrates. RTKs play an important role in
cell signaling and are involved in a variety of normal and cancer
related processes including cell migration, proliferation,
survival, angiogenesis, and metastasis. The RTK family includes
tropomyosin-related kinases (Trk's) that serve as receptors for
neurotrophin and play a critical role in the development and
maintenance of the central and peripheral nervous systems.1
This kinase subfamily includes three homologous isoforms:
TrkA, TrkB, and TrkC.2 Defective TrkA signaling observed in
patients of various ethnic groups has been shown to have the
effect of preventing them from being able to adequately
perceive painful stimuli. Besides their role in the formation and
maintenance of neuronal cells, a growing body of experimental
evidence has indicated that Trks are involved in malignant
transformation, metastasis, survival, migration, and invasion
signaling in a variety of human cancers including breast, colon,
pancreatic, papillary thyroid prostate, and lung cancers and
neuroblastoma.3−8
In addition, recent studies have indicated that Trk
compounds play important roles in angiogenesis and in the
expression of HIF-1α and VEGF.9,10 Angiogenesis, the
formation of new blood vessels from preexisting one, is a
crucial step for tumor growth, invasion, and metastasis. The
transcription factor hypoxia-inducible factor 1 (HIF-1) is a
major regulator of tumor cell adaptation to hypoxic stress. HIF-
1 has been implicated in the up-regulation of angiogenic factors
including vascular endothelial growth factor (VEGF), heme
oxygenase 1, and inducible nitric oxide synthase (iNOS). Our
research was stimulated by the observation that inhibition of
RESULTS AND DISCUSSION
■
Identification of 7-Azaindole-Based Trk Inhibitors.
The availability to control the 3D-structure of therapeutic
targets has enhanced opportunities for the rapid identification
of biologically active compounds utilizing structure-based drug
design.17−19 Although the structures of the Trk extracellular
ligand-binding domains have been published, the structure of
the kinase domain has not yet been reported. The absence of
structural information describing the nature of Trk−inhibitor
interactions has made it difficult to design potent Trk
inhibitors. Through a concurrent program supporting the
development of PI3K inhibitors, we synthesized a set of kinase-
targeted compound library based on an azaindole scaffold.20
Extensive kinase cross-screening of our compound collection
revealed that a 7-azaindole scaffold combined with a
pyridylsulfonamide group at C5 and phenylsulfonamide group
at C3 exhibited moderate activity against TrKA (POC = 9.6).
To our knowledge, the 7-azaindole scaffold is not present in
any of the Trk inhibitors reported so far. The newly identified
Received: March 2, 2012
Published: May 10, 2012
© 2012 American Chemical Society
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Figure 1. (A) Azaindole 1 guides the design of the Trk kinase inhibitor. (B) Calculated binding mode of compound 1 in the ATP-binding site of the
TrkA homology model. Carbon atoms of compound 1 are indicated in pink.
inhibitor compound 1 may impair TrkA catalytic activity by
specifically binding to the ATP binding site (Figure 1B).
Considering the tolerance within the TrkA active site,
compound 1 was expected to serve as a good scaffold from
which more potent inhibitors could be derivatized. In addition,
the series was attractive because a 7-azaindole scaffold can be
easily diversified in the 3 and 5 positions via a convergent
synthetic approach for the rapid elucidation of structure−
activity relationship (SAR). For these reasons, compound 1 was
selected for further optimization by in-depth structural
modification. Structure-based mechanisms that lead to the
high affinity of compound 1 to TrkA were gained through
comparative investigations of the mode of binding to the ATP
binding site. Figure 1B describes the lowest-energy conforma-
tion of compound 1 in the ATP binding site of TrkA, as
calculated using the modified AutoDock program (see
Experimental Section).21 The docking simulation started with
the calculation of 3D grids of interaction energy for all possible
atom types. These uniquely defined potential grids for TrkA
were then used in common for docking simulations. For the
center of the common grids, we used the center of mass
coordinates of the IGF1R inhibitor22 whose binding mode in
the ATP binding site of TrkA had also been predicted in the
homology modeling together with the structure of TrkA itself.
The calculated grid maps had dimensions of 61 × 61 × 61
points with a spacing of 0.375 Å, which yielded a receptor
model that includes the atoms within 22.9 Å of the grid center.
These grid maps are actually sufficient to cope with the entire
part of the kinase domain of TrkA. Hydrogen bonding groups
on the 7-azaindole moiety appeared to point toward the hinge
region with the hydrophobic groups situated in proximity to the
amino acid residues in the ATP binding site. In addition, this
structure suggested that the phenyl ring of compound 1 would
be π-stacking with the Phe589 gatekeeper residue.
SAR efforts were focused on improving selectivity against PI3K
while maintaining potent Trk inhibition. There are structural
differences in PI3K and Trk around the moiety at the 5 position
of compound 1. Therefore, we hypothesized that the proper
orientation and modification of the C5 pyridyl sulfonamide
group are essential for creating selective Trk inhibitors. In
particular, we were interested in the one conserved water
molecule that establishes the hydrogen bonding network with
Tyr867, Asp841, and the ligand in ligand-bound PI3K. This
ligand binding interaction plays a significant role in complex
structure stabilization. In the case of compound 1, the pyridyl
nitrogen at the 5 position seemed to form hydrogen bonds with
the bound water molecule in PI3K, and this interaction was not
expected in Trk compounds. Our initial survey of the C5 group
disturbing this key intermolecular hydrogen bond was
conducted, probing the structural differences affecting the
binding pockets of PI3K and Trks.
The 7-azaindole scaffold can be easily equipped with
functional groups at the C5 position by standard synthetic
chemistry, allowing substitutions for rapid exploration of the
SAR profile. Scheme 1 illustrates the general synthetic route for
the preparation of substituted azaindole derivatives. In all cases,
the C3 position of the azaindole was functionalized prior to
performing a Suzuki coupling with the C5 (hetero)aryl
partners. Various aryl groups were attached to the 3-position
of the azaindole core of 2 using palladium-mediated cross-
coupling. The benzenesulfonyl group was then deprotected to
yield the desired product 3. To facilitate the exploration of an
assortment of C5 functional groups, compound 3 underwent
another palladium catalyzed Suzuki coupling with different
boronic acid derivatives. In some cases, 3-position function-
alized 5-bromoazaindole was borylated via Miyaura borylation
in order to introduce various aryl groups to the 5-position.
Then the borylated azaindole 4 was reacted with an aryl group
under Suzuki coupling reaction conditions in order to afford
the desired final compound 5.
Improving Selectivity over PI3K. Since the initial hit 1
was found to be moderately potent against TrkA (POC = 9.6)
and was more potent against PI3Kα (POC = 0.9), our initial
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a
data, the 3-aminophenyl group at the azaindole C5 faced into
the solvent front, and there was available space for amino acid
residues utilizing an additional inhibitor design. We expanded
the structure−activity relationship of a diverse set of
substituents while aiming to modify the 3-aniline at C5 in the
new analogues (Table 2). The lead 5f was tolerant to
substitution with methyl, dimethyl, or acyl group at the aniline
amino group. The orientation of the amino group was not
critical, as 5f and 6f were found to be nearly equipotent. The
next round of analogues incorporated an additional substituent
into the 3-aniline ring in order to identify the most suitable
moiety. In this series of analogues, it appeared that the
substitution around the 3-aniline ring was relatively important
to its function. In general, having a substituent at the 4 or 5
position of aniline enhanced its potency, whereas a substitution
at the 6 position was detrimental to Trk inhibitory activity. The
substituent at the 4 position of the aniline ring was varied to
include methyl, methoxy, and chloro groups, and these
derivatives were all tolerated (6g, 6h, 6i). The morpholine
moiety was employed to enhance the physicochemical
properties, but replacement of the mothoxy group with
morpholinomethanone (6j) affected a slightly decreased
potency. This trend was similar to the one found for the
substituent at the 5 position of the aniline ring when comparing
compounds 6l and 6m. We further attempted to explore the
SAR in the side chain linked to the C5 position of the aniline
ring. Introducing an oxyethaneamine group into the C5
position of the aniline yielded an increase in Trk inhibitory
activity (6n and 6o). In this series, benzenesulfonamide
substitution of the aniline also provided a boost in potency
(6q IC50 = 9.4 nM). This prompted us to further investigate the
effect of various sulfonyl groups on Trk binding affinity.
Notably, m-CF3-containing analogue 6t was an exceptionally
potent Trk inhibitor with an IC50 of 1.67 nM. Next, we
attempted to replace the sulfonamide group with amide (6u),
which also showed nanomolar level inhibition. However, the
amide analogue 6u was almost inactive in the cell-based assay,
which may be attributed to poor cellular permeability and water
solubility. Separately, while a substitution at the 4- or 5-position
of the 3-aniline ring was relatively well tolerated, a substitution
at the 6-position was deleterious to inhibitory activity (6v, 6w).
Espectially, when the 5-sulfonamide group was moved to the 6-
position, it resulted in a drastic loss of activity (6w). This result
demonstrated that the orientation of the 5-substituent in the
aniline ring plays an important role in determining activity.
With the impressive Trk enzyme activity profile, cell potency of
these TrkA inhibitors was further determined in a more cancer-
related setting. For this purpose, compounds from this series
were tested for cellular proliferation activity against MCF-7
human breast cancer cells. To measure the inhibitory effect of
compounds on cell growth, cell viability was tested by 3-(4,5-
dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT)
assay in MCF-7 human breast cancer cell cultures as shown in
Table 2.
Scheme 1. Synthetic Route of Azaindole Derivatives
a
Reagents and conditions: (a) arylboronic acid, Pd(dppf)Cl2, K2CO3,
1,4-dioxane/H2O = 3:1, 80 °C, 3 h, then 6 N KOH, 80 °C, 30 min;
(b) arylboronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane/H2O = 3:1,
100 °C, 5 h; (c) 4-pyridinylboronic acid, Pd(dppf)Cl2, K2CO3, 1,4-
dioxane/H2O = 3:1, 80 °C, 3 h (d) bis(pinacolato)diboron,
Pd(dppf)Cl2, KOAc, 1,4-dioxane, 100 °C, 12 h; (e) aryl bromide,
Pd(dppf)Cl2, K2CO3, 1,4-dioxane/H2O = 3:1, 100 °C, 5 h, then 6 N
KOH, 80 °C, 30 min.
The resulting compounds were tested over PI3Kα and TrkA
at 10 μM in a high-throughput binding assay (KINOMEscan,
Ambit Biosciences). Of particular significance was the
observation that the binding affinity over PI3Kα was
dramatically reduced (5b: POC = 31, Kd = 2300 nM for
PI3Kα) (Table 1) when the pyridyl group at the C5 position
was replaced with a phenyl group, probably because of
disruption of the key intermolecular hydrogen bond with a
bound water molecule in PI3K. Interestingly, this replacement
resulted in a slightly increased potency against TrkA. The
sulfonyl group was not required for producing the Trk
inhibitory activity. We also examined the significance of the
anilinic group at C5 and observed that the binding affinity was
reduced when the 3-amino group was replaced with a CN
group (5e). Next, in order to determine whether or not the 3,4-
dimethoxy phenyl group at the C3 was optimal, the effects of its
replacement with other groups were explored. Of these, the 4-
pyidyl group was found to be the most effective moiety in the
C3 position (5f: POC = 0, Kd = 64 nM for TrkA) while
retaining excellent selectivity over PI3Kα (POC = 31, Kd =
2900 nM). The 4-pyidyl group seemed to π-stack efficiently
with the Phe589, and 4-nitrogen may be an essential hydrogen
bonding donor for Lys544. Considering its simple structure and
low molecular weight (<300), 5f was expected to serve as a
good inhibitor scaffold from which more potent inhibitors
could be derivatized. Interestingly, replacement of the 7-
azaindole scaffold with the indole scaffold resulted in a
complete loss of activity (POC = 72), thus revealing the
indispensible nature of the 7-azaindole scaffold in maintaining
activity in this series. On the basis of these findings, lead 5f was
selected for further structural modification to investigate SAR in
order to optimize both the kinase and cell-based activity.
Lead Optimization. On the basis of the structure of
compound 5f, the enhanced potency of the 4-pyridyl moiety at
C3 and the 3-aniline at position C5 suggested a need to more
broadly explore analogues incorporating these key functional
groups. Thus, we fixed a 4-pyridyl group at the C3 position of
7-azaindole and then screened various groups at the C5
position, expecting to find improved enzyme interaction and
physicochemical properties. On the basis of the docking model
Binding Mode. On the basis of the docking results and
SAR, we identified the binding mode and the detailed
interactions responsible for stabilizing compound 6t in the
TrkA homology model. Figure 2 shows the lowest-energy
conformations of 6t in the TrkA ATP binding site, calculated
using the modified AutoDock program. The results of the
docking simulations were self-consistent in the sense that
compounds 1 and 6t were positioned in similar configurations
with comparable interactions with the amino acid residues in
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Table 1. Azaindole Scaffold as Trk Inhibitors
a
Compounds were tested against TrkA and PI3Kα at 10 μM. Lower numbers of POC (percent of control) indicate stronger hits. Values show an
average of duplicate measurements.
the ATP binding site. Hydrogen bonding groups on the
inhibitors appeared to point toward the backbone groups of the
ATP binding site, with their hydrophobic groups situated in
proximity to the Gly loop. However, 6t is more closely directed
toward the pocket around Lys544 and Phe589, while
compound 1 may not reach this region because of unfavorable
steric interactions of the bulky sulfonamide group on the C3
phenyl ring. Therefore, nitrogen at the 4-position of the pyridyl
group of 6t can form a hydrogen bond with Lys544, and the
NH group of the 7-azaindole donates a hydrogen bond to the
backbone aminocarbonyl oxygen of Glu590. In addition, the
nitrogen at the 7-position of the azaindole scaffold appeared to
form a hydrogen bond with the backbone aminocarbonyl
nitrogen of Met592. The terminal anilinic group of 6t donated
a hydrogen bond to the backbone aminocarbonyl oxygen of
Arg593. The sulfonamide group appeared to form a hydrogen
bond with the backbone aminocarbonyl oxygen of Leu516.
These five hydrogen bonds appeared to be the most significant
binding forces stabilizing 6t in the ATP binding site.
Compound 6t could be further stabilized in the TrkA ATP
binding site via π-stacking interaction with the Phe589
gatekeeper residue, and hydrophobic interactions with the
side chains of Met592, Phe589, Lys544, Tyr591, Gly595,
Met671, Asp596, L657, and Ile675. The overall structural
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Table 2. Exploration of the Groups at C5
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compared the cell growth in three human breast cell lines
(MDA231, MCF-7, and SKBr3) after treatment by 6t using a 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. We observed that 6t clearly induced cell death
in MCF-7 and SKBr3 human breast cancer cells in a dose-
dependent manner (Figure 3A). In particular, 6t induced strong
cell growth reduction in MCF-7 human breast cancer cells in a
range between 40% and 70%. Trk pathway stimulates the
PI3K/Akt pathway, resulting in a cascade of downstream
signaling events.23 Since the activation of the Trk pathway leads
to phosphorylation of Akt, we investigated the effects of 6t on
the Trk/Akt pathway in MCF-7 human breast cancer cells.
When MCF-7 human cancer cells were treated with various
concentrations of 6t, phosphorylations of TrkA and Akt were
effectively suppressed (Figure 3B). The most potent com-
pound, 6t in enzyme assay, was moderately active in cell-based
assay and Western blotting, which may be attributed to poor
cellular permeability and water solubility.
Effect on Apoptotic Cell Death. The occurrence of
apoptotic nuclei condensation and TUNEL reaction were
investigated to determine whether or not MCF-7 cell death was
a result of apoptosis. In order to characterize the cell death
induced by 6t, we examined the nuclear morphology of dying
cells with a fluorescent DNA-binding agent, DAPI. MCF-7 cells
treated with 6t displayed the typical morphological features of
apoptotic cells including condensed and fragmented nuclei,
while homogeneous nuclear chromatin was evident in the
control cells. The induction of apoptosis by 6t was confirmed
using the TUNEL assay (Figure 4A). The TUNEL assay, based
on the labeling of DNA strand breaks generated during
apoptosis, revealed that 6t induced apoptosis in MCF-7 cells. In
order to further explore the mechanisms of this induced
apoptosis, we examined the expression of apoptotic regulatory
proteins such as cytochrome C, Bcl-2, Bax and cleaved caspase-
3 using immunofluorescence staining.24 As expected, 6t
increased the expression of cytochrome C, Bax, cleaved
caspase-3, as well as decreased the expression of Bcl-2 in
MCF-7 breast cancer cells (Figure 4B). These results showed
that 6t induces cell apoptosis in MCF-7 breast cancer cells.
Effect on the Expression of HIF-1α and VEGF. HIF-1α
is an important target modulator of angiogenic factors along
with VEGF, an immediate downstream target gene of HIF-1α.
Thus, it was appropriate to investigate the effect of 6t on the
expression of hypoxia-induced HIF-1α and VEGF. MCF-7 cells
were treated with various concentrations of 6t under hypoxia in
order to mimic the conditions induced by 100 μM CoCl2 for 6
h. As shown in Figure 5, HIF-1α expression increased under
hypoxic conditions, whereas 6t inhibited the hypoxia-induced
HIF-1α expression in a dose-dependent manner. In addition,
we identified the effects of 6t (0.1−10 μM) on the production
of the hypoxia-induced VEGF. When the cells were treated with
6t under hypoxic conditions, the VEGF protein level reduced
dramatically.
Figure 2. Calculated binding mode of 6t in the ATP-binding site of
Trk. Carbon atoms of the protein and the ligand are indicated in gray
and green, respectively. Each dotted line indicates a hydrogen bond.
features derived from the docking simulations suggested that
the nanomolar inhibitory activity of 6t could be attributed to
the establishment of multiple hydrogen bonds and hydrophobic
interactions in a simultaneous fashion in the TrkA ATP binding
site.
The most potent inhibitor 6t was subjected to kinase
selectivity profiling over a panel of 30 cancer related kinases at
1 μM in a high-throughput binding assay (KINOMEscan,
Ambit Biosciences). Three kinases KIT, TrkA, and TrkB
displayed tight binding to 6t (kinases with POC < 10) as shown
in Table 3. Subsequently the measured IC50 demonstrated that
6t is about 100-fold selective for TrkA over KIT (TrkA IC50
=
1.67 nM and KIT IC50 = 145 nM).
a
Table 3. KINOMEscan Profile of Compound 6t
b
b
b
kinase
POC
kinase
POC
kinase
POC
ABL1
81
FGFR2
GSK3b
JAK2
61
81
34
3
PDPK1
PKAC-a
PLK1
90
AKT1
AKT2
AURKA
AURKB
BRAF
CDK11
EGFR
ERK1
FAK
94
88
100
100
85
100
64
KIT
PI3KCA
PIM1
MEK1
MEK2
MET
100
100
96
100
79
69
97
100
26
PLK1
100
75
PLK3
100
100
88
P38α
ROCK2
TRKA
TRKB
50
PAK1
PDGFRA
0.8
1.2
a
Panel of 30 kinases were tested at 1 μM in a high-throughput binding
b
assay (Ambit Bioscience). Lower numbers of POC (percent of
control) indicate stronger hits. Values show an average of duplicate
measurements.
Effect on Cell Migration. The antiangiogenic activity of 6t
was further evaluated using assays including wound induced
migration of human umbilical vein endothelial cells (HUVEC).
Since cell migration is the one of the major processes for
endothelial cells in the formation of blood vessels during
angiogenesis and metastasis, we examined the effects of 6t on
the migration of HUVECs. Endothelial cells were wounded and
then incubated in the presence of 6t (1−10 μM) for 16 h. As
shown in Figure 6, compound 6t reduced the migration of
HUVECs in a dose-dependent manner.
MECHANISM STUDIES
■
The Trk pathway plays a critical role in cell progression by
promoting cell proliferation and inhibiting apoptosis. For a
selected Trk inhibitor, 6t displaying both potent enzymatic and
cellular activity and the mechanism of cancer cell death was
assessed, followed by an antiangiogenic evaluation.
Effect on Cell Proliferation and Cell Signaling Down-
stream. In order to identify the anticancer effect of 6t, we
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Figure 3. Effect of 6t on the cell growth/proliferation and cell signaling pathways in human breast cancer cells. Effect of 6t on growth/proliferation
was measured in human breast cancer cells (MDA231, MCF-7, and SKBr3) by MTT assay (A). Results are expressed as a percent of relative cell
proliferation compared to control. The inhibition of Trk/Akt signaling pathway by 6t in MCF-7 cells was analyzed by Western blotting (p-Trk and
p-Akt). Data are represented as the mean SD from the triplicate wells (B).
Figure 4. Effect of 6t on the apoptosis of MCF-7 breast cancer cells. (A) MCF-7 breast cancer cells treated with 6t (10 μM) for 24 h. The induction
of apoptosis by 6t was conducted by DAPI and TUNEL staining and photographed at ×400 magnification. (B) The expressions of cytochrome C,
Bcl-2, Bax, and cleaved caspase-3 were observed by immunofluorescence staining in cells treated with 6t (10 μM) for 24 h at ×800 magnification.
Effect on Tubular Network Formation. The antiangio-
genic effect on tube formation was analyzed by treating
HUVEC with compound 6t. HUVECs were plated on Matrigel
in order to mimic in vivo HUVEC associated angiogenesis and
then treated with various concentrations of 6t (1−10 μM).
While the HUVECs were plated, they aligned with one another
and formed tubes resembling a capillary plexus. The 6t
produced significant inhibition of the VEGF-induced formation
of vessel-like structures, resulting in the elongation and
alignment of the HUVECs in a dose-dependent manner
(Figure 7). Considering that endothelial migration and tube
formation are all highly relevant properties in the process of
angiogenesis, these results showed that TrkA inhibitor 6t has
the ability to block VEGF-induced angiogenesis by suppressing
hypoxia-induced VEGF expression and preventing tube
formation and endothelial cell migration.
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Figure 5. Effect of 6t on the angiogenesis of MCF-7 breast cancer cells. Effect on the expression of HIF-1α and production of VEGF by 6t in
hypoxia-induced MCF-7 breast cancer cells (CoCl2, 100 μM). Data compared to CoCl2 from three independent experiments are represented as the
mean SD: (∗∗) P < 0.01.
Figure 6. Effects of 6t on cell migration. HUVECs were plated at 90% confluence and then scratched with a razor blade. Inhibition of HUVEC
migration by 6t in the wound-induced migration assay.
Figure 7. Effect of 6t on tube formation. Representative images depicting the formation of a HUVEC capillary-like tubular network by treatment
with compound 6t (1, 5, and 10 μM) and DMSO (control).
CONCLUSION
EXPERIMENTAL SECTION
■
■
General Chemistry. Unless stated otherwise, reactions were
performed in flame-dried glassware under a positive pressure of
nitrogen using freshly distilled solvents. Analytical thin layer
chromatography (TLC) was performed on precoated silica gel 60
F254 plates, and visualization on TLC was achieved by UV light (254
and 354 nm). Flash column chromatography was undertaken on silica
gel (400−630 mesh). 1H NMR was recorded on 400 or 300 MHz, and
chemical shifts were quoted in parts per million (ppm) referenced to
the appropriate solvent peak or 0.0 ppm for tetramethylsilane. The
following abbreviations were used to describe peak splitting patterns
when appropriate: br = broad, s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, dd = doublet of doublet. Coupling constants, J,
were reported in hertz unit (Hz). Chemical shifts were reported in
ppm referenced to the center line of a triplet at 77.0 ppm of
chloroform-d. Mass spectral data were obtained from the KAIST Basic
Science Institute by using the EI method. High performance liquid
chromatography analyses for checking purity (>95% area) of
synthesized compounds were performed on Waters HPLC instrument
equipped with an Agilent Prep-C18 reverse phase column (21.2 mm ×
Utilizing the structural-based design strategy, we designed and
synthesized a series of new 7-azaindole derivatives to be used as
potent Trk inhibitors. Through the systematic exploration of
the appropriate functional groups to place in the C3 and C5
positions of 7-azaindole scaffolds, we successfully developed a
SAR profile around the series and found that a 4-pyridyl group
at the C3 position and a selection of moieties (e.g., 5-
sulfonamide, amide, and oxyethaneamine on aniline ring) at the
C5 position of the 7-azaindole scaffold profoundly influenced
Trk binding affinity and resulted in nanomolar inhibitors with
good antiproliferative cellular activity. Moreover, compound 6t
exhibited strong apototic and antiangiogenic effects by
inhibiting HIF-1α and VEGF expression. These results suggest
that compound 6t represses the angiogenic process by
inhibiting endothelial cell migration and tube formation.
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150 mm, 10 μm) and by HRMS. The mobile phase was a mixture of
MeOH (0.1% TFA) and H2O (0.1% TFA). Compound purity was
determined by integrating peak areas of the liquid chromatogram,
monitored at 254 nm. Parameters were as follows: flow rate of 8 mL/
min; gradient system, from 10% MeOH (0.1% TFA) and 90% H2O
(0.1% TFA) (0 min) to 90% MeOH (0.1% TFA) and 10% H2O (0.1%
TFA) (15 min). The solvent ratio was maintained for 18 min. The
solvent ratio was then changed to 10% MeOH (0.1% TFA) and 90%
H2O (0.1% TFA) (20 min). All final compounds were found to have
>95% purity. Commercial grade reagents and solvents were used
without further purification except as indicated below. Dichloro-
methane was distilled from calcium hydride. THF was distilled from
sodium. Unless otherwise stated, all commercial reagents and solvents
were used without additional purification.
General Procedure (GP I) for Suzuki Coupling. 5-(3-(3,4-
Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridin-3-amine
(5c). A solution of 5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-
b]pyridine (129 mg, 0.387 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)pyridin-3-amine (121 mg, 0.548 mmol), K2CO3
(158 mg, 1.14 mmol), and PdCl2(dppf)·CH2Cl2 (64 mg, 0.04
mmol) in 1,4-dioxane/H2O = 3:1 (2 mL) was heated to 100 °C for
5 h under an atmosphere of N2. The reaction mixture was cooled to
room temperature and concentrated in vacuo. The residue was diluted
with CH2Cl2 and MeOH (10:1, 2 mL) and then filtered through a
short plug of silica (SiO2) and Celite, eluting with CH2Cl2 and MeOH
(20:1). The filtrate was purified with flash column chromatography
(CH2Cl2/MeOH, gradient 40:1 to 10:1) to give the product as a white
solid (71 mg, 53% yield). 1H NMR (300 MHz, CDCl3) δ 3.78 (s, 2H),
3.93 (s, 3H), 3.94 (s, 3H), 6.98 (d, J = 7.9 Hz, 1H), 7.13 (s, 1H), 7.19
(s, 1H), 7.21−7.23 (m, 1H), 7.47 (d, J = 2.1 Hz, 1H), 8.10 (d, J = 2.3
Hz, 1H), 8.30 (s, 2H), 8.54 (s, 1H), 9.20 (s, 1H). HRMS (EI+) m/z
calcd for C20H18N4O2 [M + H]+, 347.1508; found 347.1508.
N-(3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)benzenesulfonamide (5a). Benzenesulfonyl chloride (32 μL,
0.25 mmol) and pyridine (44 μL, 0.54 mmol) were added to 5c (65
mg, 0.18 mmol) in CH2Cl2 (1.5 mL) under an atmosphere of N2. The
reaction mixture was stirred for 5 h at room temperature, and then the
solvent was evaporated. The residue was purified with flash column
chromatography (CH2Cl2/MeOH = 15:1) to give the product as a
pale yellow solid (24 mg, 28% yield). 1H NMR (300 MHz, DMSO-d6)
δ 3.85 (s, 3H), 3.79 (s, 3H), 7.04 (d, J = 8.9 Hz, 1H), 7.26−7.28 (m,
2H), 7.55−7.59 (m, 3H), 7.72 (s, 1H), 7.81 (d, J = 7.5 Hz, 2H), 7.86
(d, J = 2.5 Hz, 1H), 8.25 (d, J = 2.3 Hz, 1H), 8.29 (d, J = 2.0 Hz, 1H),
8.43 (d, J = 2.0 Hz, 1H), 8.63 (s, 1H), 10.69 (s, 1H), 11.99 (s, 1H).
HRMS (EI+) m/z calcd for C26H22N4O4S [M + Na]+, 509.1259;
found, 509.1267.
Hz, 1H), 7.22 (dd, J = 8.2, 2.0 Hz, 1H), 7.54 (d, J = 2.4 Hz, 1H), 7.59
(d, J = 7.7 Hz, 1H), 7.63−7.64 (m, 1H), 7.85−7.91 (m, 2H), 8.32 (d, J
= 2.0 Hz, 1H), 8.57 (d, J = 2.0 Hz, 1H), 10.12 (s, 1H). HRMS (EI+)
m/z calcd for C22H17N3O2 [M + Na]+, 378.1218; found, 378.1217.
General Procedure (GP II) for Suzuki Coupling. 5-Bromo-3-
(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine. A solution of 5-
bromo-3-iodo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (315 mg,
0.680 mmol), dimethoxyboronic acid (124 mg, 0.680 mmol), K2CO3
(282 mg, 2.04 mmol), and PdCl2(dppf)·CH2Cl2 (111 mg, 0.136
mmol) in 1,4-dioxane/H2O = 3:1 (4 mL) was heated to 80 °C for 3 h
under an atmosphere of N2. The resulting solution was cooled to room
temperature, and 6 N KOH solution (2 mL) was added. The reaction
mixture was then heated to 80 °C and stirred for 30 min (N-
deprotection of benzenesulfonyl group). The mixture was diluted with
water and neutralized with 6 N HCl solution. The two-phase mixture
was extracted with CH2Cl2. The combined organic layer was dried
(MgSO4) and concentrated in vacuo. The residue was purified with
flash column chromatography (CH2Cl2/MeOH, gradient 40:1 to
1
10:1) to give the product as a yellow solid (178 mg, 78.6% yield). H
NMR (300 MHz, CDCl3) δ 3.91 (s, 3H), 3.93 (s, 3H), 6.91−6.95 (m,
1H), 7.03−7.08 (m, 2H), 7.41 (s, 1H), 8.23 (s, 1H), 8.32 (s, 1H),
11.25 (s, 1H).
3-(3-(Pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline (5f). 5-
Bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine was prepared (243
mg, 53.3% yield) according to GP II from 4-pyridinylboronic acid (398
mg, 3.24 mmol) and 5-bromo-3-iodo-1-(phenylsulfonyl)-1H-pyrrolo-
[2,3-b]pyridine (1.50 g, 3.24 mmol). 1H NMR (300 MHz, DMSO-d6)
δ 7.97 (d, J = 5.9 Hz, 2H), 8.41 (d, J = 2.2 Hz, 1H), 8.47 (d, J = 2.7
Hz, 1H), 8.60 (d, J = 6.0 Hz, 2H), 8.70 (d, J = 2.1 Hz, 1H), 12.69 (s,
1H). Compound 5f was prepared (11 mg, 42% yield) according to GP
I from 3-aminophenylboronic acid (23 mg, 0.17 mmol) and 5-bromo-
1
3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (39 mg, 0.14 mmol). H
NMR (300 MHz, DMSO-d6) δ 5.18 (s, 2H), 6.59 (d, J = 7.8 Hz, 1H),
6.90 (d, J = 7.7 Hz, 1H), 6.93 (s, 1H), 7.14 (t, J = 7.7 Hz, 1H), 7.81 (d,
J = 6.0 Hz, 2H), 8.25 (s, 1H), 8.45 (d, J = 1.8 Hz, 1H), 8.50 (d, J = 1.7
Hz, 1H), 8.56 (d, J = 5.9 Hz, 2H), 12.28 (s, 1H). 13C NMR (100
MHz, DMSO-d6) δ 111.6, 112.6, 112.9, 114.9, 116.9, 120.4, 125.3,
127.0, 129.5, 130.3, 139.4, 142.3, 142.4, 148.7, 149.2, 145.0. HRMS
(EI+) m/z calcd for C18H14N4 [M + H]+, 287.1297; found, 287.1289.
3-(3-(Pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline (5g). 5-
Bromo-3-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine was prepared (35
mg, 30% yield) according to GP II from 3-pyridinylboronic acid (53
mg, 0.43 mmol) and 5-bromo-3-iodo-1-(phenylsulfonyl)-1H-pyrrolo-
[2,3-b]pyridine (0.20 g, 0.43 mmol). 1H NMR (300 MHz, CD3OD) δ
7.46 (dd, J = 4.9, 7.2 Hz, 1H), 7.80 (s, 1H), 8.05−8.09 (m, 1H), 8.28
(d, J = 2.1 Hz, 1H), 8.36 (d, J = 2.1 Hz, 1H), 8.39 (dd, J = 1.6, 4.9 Hz,
1H), 8.78 (dd, J = 0.75, 2.2 Hz, 1H). Compound 5g was prepared (11
mg, 30% yield) according to GP I from 3-aminophenylboronic acid
(35 mg, 0.15 mmol) and 5-bromo-3-(pyridin-3-yl)-1H-pyrrolo[2,3-
b]pyridine (35 mg, 0.13 mmol). 1H NMR (300 MHz, CD3OD) δ 6.78
(d, J = 7.5 Hz, 1H), 7.03 (d, J = 7.6, 1H), 7.08 (d, J = 1.8 Hz, 1H),
7.25 (t, J = 7.8 Hz, 1H), 7.54−7.57 (m, 1H), 7.87 (s, 1H), 8.22−8.25
(m, 1H), 8.42−8.54 (m, 3H), 8.94 (d, J = 1.7 Hz, 1H). HRMS (EI+)
m/z calcd for C18H14N4 [M + Na]+, 309.1116; found, 309.1117.
3-(3-Phenyl-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline (5h). 5-Bromo-
3-phenyl-1H-pyrrolo[2,3-b]pyridine was prepared (41 mg, 58% yield)
according to GP II from phenylboronic acid (30 mg, 0.24 mmol) and
5-bromo-3-iodo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (113
N-(3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)benzenesulfonamide (5b). Compound 5b was prepared (2.4
mg, 28% yield) according to the procedure of benzenesulfonylation
(the same as 5a) from 5d (6.0 mg, 0.02 mmol). 1H NMR (300 MHz,
CD3OD) δ 3.82 (s, 3H), 3.85 (s, 3H), 6.96−6.99 (m, 1H), 7.01−7.16
(m, 3H), 7.28−7.32 (m, 3H), 7.41−7.46 (m, 3H), 7.53 (s, 1H), 7.78
(dd, J = 8.2, 1.3 Hz, 2H), 8.18 (d, J = 2.1 Hz, 1H), 8.29 (d, J = 2.0 Hz,
1H). HRMS (EI+) m/z calcd for C27H23N3O4S [M + Na]+, 508.1307;
found, 508.1292.
3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
(5d). Compound 5d was prepared (11 mg, 25% yield) according to
GP I from 3-aminophenylboronic acid (20 mg, 0.15 mmol) and 5-(3-
(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridin-3-
amine (40 mg, 0.12 mmol). 1H NMR (300 MHz, CD3OD) δ 3.87 (s,
3H), 3.90 (s, 3H), 6.68−6.75 (m, 1H), 6.93−7.06 (m, 3H), 7.20−7.26
(m, 3H), 7.59 (s, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.43 (d, J = 2.0 Hz,
1H). HRMS (EI+) m/z calcd for C21H19N3O2 [M + H]+, 346.1556;
found, 346.1543.
1
mg, 0.24 mmol). H NMR (300 MHz, DMSO-d6) δ 7.26 (t, J = 7.4
Hz, 1H), 7.41−7.46 (m, 2H), 7.69−7.72 (m, 2H), 7.95 (s, 1H), 8.32
(d, J = 2.1 Hz, 1H), 8.43 (d, J = 2.1 Hz, 1H), 12.17 (s, 1H).
Compound 5h was prepared (24 mg, 56% yield) according to GP I
from 3-aminophenylboronic acid (24 mg, 0.16 mmol) and 5-bromo-3-
phenyl-1H-pyrrolo[2,3-b]pyridine (41 mg, 0.15 mmol). 1H NMR (300
MHz, DMSO-d6) δ 5.15 (s, 2H), 6.56 (d, J = 7.9 Hz, 1H), 6.85 (d, J =
7.2 Hz, 1H), 6.90 (s, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.23−7.28 (m,
1H), 7.42−7.47 (m, 2H), 7.75 (d, J = 7.2 Hz, 2H), 7.88 (d, J = 2.5 Hz,
1H), 8.30 (d, J = 2.0 Hz, 1H), 8.46 (d, J = 2.0 Hz, 1H), 11.95 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ 112.5, 112.7, 114.5, 114.7, 117.2,
124.4, 124.9, 125.7, 126.4, 128.9, 129.5, 129.6, 135.0, 139.6, 141.8,
3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
benzonitrile (5e). Compound 5e was prepared (6.4 mg, 15% yield)
according to GP I from 3-cyanophenylboronic acid (20 mg, 0.14
mmol) and 5-(3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-
yl)pyridin-3-amine (40 mg, 0.12 mmol). 1H NMR (300 MHz, CDCl3)
δ 3.94 (s, 3H), 3.95 (s, 3H), 7.00 (d, J = 8.3 Hz, 1H), 7.11 (d, J = 1.9
5345
dx.doi.org/10.1021/jm3002982 | J. Med. Chem. 2012, 55, 5337−5349

Journal of Medicinal Chemistry
Article
148.5, 149.2. HRMS (EI+) m/z calcd for C19H15N3 [M + H]+,
286.1344; found, 286.1346.
added. The reaction mixture was then heated to about 80 °C and
stirred for 30 min (N-deprotection of benzenesulfonyl group). The
mixture was diluted with water and neutralized with 6 N HCl solution.
All solvent was removed in vacuum. The residue was dissolved into
CH2Cl2/MeOH = 5:1, and an insoluble salt was removed by filtration.
The filtrate was concentrated and purified with flash column
chromatography (CH2Cl2/MeOH, gradient 30:1 to 10:1) to give the
3-(5-(3-Aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile
(5i). 3-(5-Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile was pre-
pared (54 mg, 84% yield) according to GP II from 3-cyanophenylbor-
onic acid (33 mg, 0.22 mmol) and 5-bromo-3-iodo-1-(phenyl-
sulfonyl)-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.216 mmol). 1H
NMR (300 MHz, DMSO-d6) δ 7.59−7.70 (m, 2H), 8.07−8.19 (m,
3H), 8.35 (d, J = 2.1 Hz, 1H), 8.59 (d, J = 2.1 Hz, 1H), 12.38 (s, 1H).
Compound 5i was prepared (16 mg, 39% yield) according to GP I
from 3-aminophenylboronic acid (21 mg, 0.15 mmol) and 3-(5-
bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (40 mg, 0.13
1
product as a yellow solid (13 mg, 45% yield). H NMR (300 MHz,
DMSO-d6) δ 2.75 (d, J = 5.0 Hz, 3H), 5.72−5.75 (m, 1H), 6.57 (d, J =
7.8 Hz, 1H), 6.86 (s, 1H), 6.92 (d, J = 7.7 Hz, 1H), 7.20 (t, J = 7.8 Hz,
1H), 7.81 (d, J = 6.0, 2H), 8.24 (s, 1H), 8.47 (d, J = 1.9 Hz, 1H), 8.53
(d, J = 1.8 Hz, 1H), 8.56 (d, J = 6.0 Hz, 2H), 12.27 (s, 1H). 13C NMR
(100 MHz, DMSO-d6) δ 29.8, 110.5, 110.5, 111.7, 114.8, 116.9, 120.4,
125.5, 127.02, 129.5, 130.4, 139.5, 142.3, 142.4, 148.7, 150.0, 150.4.
HRMS (EI+) m/z calcd for C19H16N4 [M + Na]+, 323.1273; found,
323.1252.
1
mmol). H NMR (300 MHz, CD3OD) δ 6.73 (dd, J = 8.0, 1.3 Hz,
1H), 6.96−7.03 (m, 2H), 7.20 (t, J = 7.8 Hz, 1H), 7.58−7.60 (m, 2H),
7.78 (s, 1H), 7.99−8.01 (m, 2H), 8.35 (d, J = 2.0 Hz, 1H), 8.46 (d, J =
1.9 Hz, 1H). HRMS (EI+) m/z calcd for C20H14N4 [M + Na]+,
333.1116; found, 333.1101.
N,N-Dimethyl-3-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
aniline (6c). Compound 6c was prepared (7.2 mg, 18% yield)
according to GP I from 3-(dimethylamino)phenylboronic acid (26 mg,
0.16 mmol) and 5-bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine
3-(3-(3-Aminophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzonitrile
(5j). 3-(5-Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline was prepared
(158 mg, 50.9% yield) according to GP II from 3-aminophenylboronic
acid (237 mg, 1.08 mmol) and 5-bromo-3-iodo-1-(phenylsulfonyl)-
1H-pyrrolo[2,3-b]pyridine (500 mg, 1.08 mmol). 1H NMR (300
MHz, CD3OD) δ 6.65−6.68 (m, 1H), 6.90−7.00 (m, 2H), 7.23−7.48
(m, 1H), 7.48 (d, J = 2.5 Hz, 1H), 8.35 (d, J = 2.0 Hz, 1H), 8.39 (d, J
= 2.1 Hz, 1H), 9.71 (s, 1H). Compound 5j was prepared (25 mg, 26%
yield) according to GP I from 3-cyanophenylboronic acid (51 mg, 0.35
mmol) and 3-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (91 mg,
0.32 mmol). 1H NMR (300 MHz, DMSO-d6) δ 5.09 (s, 2H), 6.49 (d,
J = 7.9, Hz, 1H), 6.92 (d, J = 7.7 Hz, 1H), 7.02 (s, 1H), 7.09 (t, J = 7.7
Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.78 (s, 1H), 7.83 (d, J = 7.8 Hz,
1H), 8.14 (d, J = 8.0 Hz, 1H), 8.28 (s, 1H), 8.48 (d, J = 2.2 Hz, 1H),
8.61 (d, J = 2.1 Hz, 1H), 11.97 (s, 1H). 13C NMR (100 MHz, DMSO-
d6) δ 111.8, 112.1, 112.1, 114.4, 115.7, 117.4, 118.9, 124.3, 126.0,
126.5, 129.4, 130.1, 130.5, 130.5, 131.9, 135.1, 140.4, 141.9, 148.9,
149.0. HRMS (EI+) m/z calcd for C20H14N4 [M + Na]+, 333.1116;
found, 333.1098.
1
(35 mg, 0.13 mmol). H NMR (300 MHz, CDCl3) δ 3.03 (s, 6H),
6.77−6.94 (m, 1H), 6.94−6.99 (m, 2H), 7.36 (t, J = 7.7 Hz, 1H), 7.59
(dd, J = 4.6, 1.6 Hz, 2H), 7.78 (s, 1H), 8.44 (d, J = 1.9 Hz, 1H), 8.63−
8.65 (m, 3H), 11.30 (s, 1H). HRMS (EI+) m/z calcd for C20H18N4
[M + H]+, 315.1610; found, 315.1613.
5-(3-Methoxyphenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine
(6d). Compound 6d was prepared (10 mg, 35% yield) according to
GP I from 3-methoxyphenylboronic acid (22 mg, 0.14 mmol) and 5-
bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (26 mg, 0.09
1
mmol). H NMR (300 MHz, CD3OD) δ 3.89 (s, 3H), 6.96 (dd, J
= 7.8, 1.8 Hz, 1H), 7.22−7.27 (m, 2H), 7.41 (t, J = 7.9 Hz, 1H), 7.80
(dd, J = 4.7, 1.5 Hz, 2H), 8.02 (s, 1H), 8.52−8.54 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) δ 55.2, 111.8, 112.6, 112.9, 117.0, 119.6, 120.4,
125.8, 127.2, 129.3, 130.0, 140.3, 142.3, 142.5, 148.9, 150.0, 159.8.
HRMS (EI+) m/z calcd for C19H15N3O [M + Na]+, 324.1113; found,
324.1113.
5-(3-(Pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)pyridin-3-amine
(6a). Compound 6a was prepared (10 mg, 28% yield) according to GP
I from 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-amine
(42 mg, 0.19 mmol) and 5-bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-
N-(3-(3-(Pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)-
acetamide (6e). Compound 6e was prepared (5.7 mg, 12% yield)
according to GP I from N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)acetamide (50 mg, 0.19 mmol) and 5-bromo-3-(pyridin-
1
1
b]pyridine (35 mg, 0.13 mmol). H NMR (300 MHz, DMSO-d6) δ
4-yl)-1H-pyrrolo[2,3-b]pyridine (40 mg, 0.15 mmol). H NMR (300
5.40 (s, 2H), 7.25 (t, J = 7.25, 1H), 7.83 (dd, J = 4.6, 1.6 Hz, 2H), 7.95
(d, J = 4.3 Hz, 1H), 8.15 (d, J = 2.0 Hz, 1H), 8.26 (s, 1H), 8.52 (s,
2H), 8.55 (dd, J = 4.6, 1.5 Hz, 2H), 12.38 (s, 1H). HRMS (EI+) m/z
calcd for C17H13N5 [M + Na]+, 310.1069; found, 310 0.1065.
MHz, DMSO-d6) δ 2.07 (s, 3H), 7.38−7.50 (m, 2H), 7.66 (d, J = 7.5
Hz, 1H), 7.82 (d, J = 5.7 Hz, 2H), 7.88 (s, 1H), 8.27 (d, J = 2.7 Hz,
1H), 8.51−8.57 (m, 4H), 10.06 (s, 1H), 12.33 (s, 1H). 13C NMR (100
MHz, DMSO-d6) δ 24.1, 111.7, 117.0, 117.7, 117.8, 120.5, 122.0,
125.6, 127.4, 129.3, 129.5, 139.2, 139.9, 142.3, 142.5, 148.9, 149.8,
168.4. HRMS (EI+) m/z calcd for C20H16N4O [M + H]+, 329.1402;
found, 329.1399.
General Procedure (GP III) for Suzuki Coupling. N-Methyl-3-
(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline (6b). 5-
Bromo-1-(phenylsulfonyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine
was prepared from 4-pyridinylboronic acid and 5-bromo-3-iodo-1-
(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (73% yield) according to
the procedure of GP II without N-deprotection step of benzene-
sulfonyl group. 1H NMR (300 MHz, DMSO-d6) δ 7.62−7.67 (m, 2H),
7.73−7.78 (m, 1H), 7.85−7.87 (m, 2H), 8.15−8.18 (m, 2H), 8.56 (d, J
= 2.1 Hz, 1H), 8.62−8.65 (m, 3H), 8.67 (d, J = 2.2 Hz, 1H). A
solution of 5-bromo-1-(phenylsulfonyl)-3-(pyridin-4-yl)-1H-pyrrolo-
[2,3-b]pyridine (1.0 g, 2.24 mmol), bis(pinacolato)diboron (685 mg,
2.69 mmol), KOAc (660 mg, 6.72 mmol), and PdCl2(dppf)·CH2Cl2
(183 mg, 0.224 mmol) in anhydrous 1,4-dioxane was heated to 100 °C
for 12 h under an atmosphere of N2. The reaction mixture was cooled
to room temperature and concentrated in vacuo. The residue was
diluted with CH2Cl2 and MeOH and then filtered through a short pad
of silica (SiO2) and Celite, eluting with CH2Cl2 and MeOH (30:1).
The filtrate was concentrated in vacuo to give the crude compound 4
as a brown solid (590 mg, 57.1% yield). The crude product was used
for the next Suzuki coupling without further purification. A solution of
3-bromo-N-methylaniline (25 mg, 0.13 mmol), compound 4 (78 mg,
0.17 mmol), K2CO3 (54 mg, 0.39 mmol), and PdCl2(dppf)·CH2Cl2
(10 mg, 0.012 mmol) in 1,4-dioxane/H2O = 3:1 (2 mL) was heated to
100 °C for 5 h under an atmosphere of N2. The resulting solution was
cooled to room temperature, and 6 N KOH solution (2 mL) was
4-(3-(Pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline (6f).
Compound 6f was prepared (8.3 mg, 27% yield) according to GP I
from 4-aminophenylboronic acid pinacol ester (30 mg, 0.14 mmol)
and 5-bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (29 mg, 0.11
1
mmol). H NMR (300 MHz, DMSO-d6) δ 5.20 (s, 2H), 6.68(d, J =
8.4 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.81 (d, J = 6.2 Hz, 2H), 8.19
(d, J = 2.8 Hz, 1H), 8.40 (s, 1H), 8.48 (s, 1H), 8.54 (d, J = 6.0 Hz,
2H), 12.17 (s, 1H). HRMS (EI+) m/z calcd for C18H14N4 [M + H]+,
287.1297; found, 287.1295.
2-Methoxy-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
aniline (6g). Compound 6g was prepared (5.9 mg, 11% yield)
according to GP III from 5-bromo-2-methoxyaniline (35 mg, 0.17
mmol). 1H NMR (300 MHz, DMSO-d6) δ 3.80 (s, 3H), 4.81 (s, 2H),
6.88−6.95 (m, 2H) 7.02 (d, J = 1.7 Hz, 1H), 7.79 (d, J = 6.1 Hz, 2H),
8.21 (s, 1H), 8.40 (d, J = 1.9 Hz, 1H), 8.47 (d, J = 1.9 Hz, 1H), 8.55
(d, J = 6.0 Hz, 2H), 12.22 (s, 1H). HRMS (EI+) m/z calcd for
C19H16N4O [M + H]+, 317.1402; found, 317.1384.
2-Chloro-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
(6h). Compound 6h was prepared (5.6 mg, 15% yield) according to
1
GP III from 5-bromo-2-chloroaniline (24 mg, 0.12 mmol). H NMR
(300 MHz, DMSO-d6) δ 5.24 (s, 2H), 6.96 (dd, J = 8.1, 1.7 Hz, 1H),
7.17 (d, J = 1.7 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.80 (d, J = 5.6 Hz,
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2H), 8.24 (s, 1H), 8.46 (s, 1H), 8.49 (s, 1H), 8.55 (d, J = 5.6 Hz, 2H),
12.30 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 111.7, 113.9, 115.9,
116.4, 116.9, 120.4, 125.4, 127.2, 129.1, 129.4, 138.3, 142.1, 142.3,
145.0, 148.8, 150.0. HRMS (EI+) m/z calcd for C18H13ClN4 [M +
H]+, 321.0907; found, 321.0924.
2-Methyl-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
(6i). Compound 6i was prepared (12 mg, 28% yield) according to GP
I from 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
(51 mg, 0.22 mmol) and 5-bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-
b]pyridine (40 mg, 0.15 mmol). 1H NMR (300 MHz, CD3OD) δ 2.17
(s, 3H), 6.91 (dd, J = 7.7, 1.8 Hz, 1H), 7.03 (d, J = 1.7 Hz, 1H), 7.07
(d, J = 7.7 Hz, 1H), 7.75 (d, J = 4.8, 1.4 Hz, 2H), 7.96 (s, 1H), 8.45−
8.48 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 17.1, 111.6, 112.6,
115.0, 117.0, 120.3, 120.4, 125.0, 127.0, 130.2, 130.5, 136.9, 142.2,
142.4, 147.0, 148.6, 150.0. HRMS (EI+) m/z calcd for C19H16N4 [M +
Na]+, 323.1273; found, 323.1267.
(2-Amino-4-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)(morpholino)methanone (6j). Compound 6j was prepared
(10 mg, 13% yield) according to GP III from (2-amino-4-
bromophenyl)(morpholino)methanone (34 mg, 0.12 mmol). 1H
NMR (300 MHz, CDCl3) δ 3.70−3.72 (m, 8H), 4.53 (s, 2H),
6.97−6.99 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.58 (d, J = 5.5 Hz, 2H),
7.72 (s, 1H), 8.38 (s, 1H), 8.59 (s, 1H), 8.65 (d, J = 5.6 Hz, 2H), 9.55
(s, 1H). HRMS (EI+) m/z calcd for C23H21N5O2 [M + H]+, 400.1773;
found, 400.1808.
N-(3-Amino-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)methanesulfonamide (6p). Compound 6p was prepared (10
mg, 18% yield) according to GP I from N-(3-amino-5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanesulfonamide (65
mg, 0.20 mmol) and 5-bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]-
1
pyridine (42 mg, 0.16 mmol). H NMR (300 MHz, DMSO-d6) δ
2.99 (s, 3H), 5.34 (s, 2H), 6.51 (s, 1H), 6.66 (s, 2H), 7.77 (d, J = 6.1
Hz, 2H), 8.24 (s, 1H), 8.39 (s, 1H), 8.44 (s, 1H), 8.55 (d, J = 6.0 Hz,
2H), 12.30 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 48.6, 103.8,
106.5, 108.6, 111.6, 116.9, 120.4, 125.3, 127.2, 130.0 139.7, 140.2,
142.1, 142.4, 148.8, 150.0, 150.1. HRMS (EI+) m/z calcd for
C19H17N5O2S [M + H]+, 380.1181; found, 380.1179.
N-(3-Amino-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)benzenesulfonamide (6q). Compound 6q was prepared (65
mg, 81% yield) according to GP I from N-(3-amino-5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)benzenesulfonamide (103
mg, 0.274 mmol) and 5-bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]-
pyridine (50 mg, 0.18 mmol). 1H NMR (300 MHz, DMSO-d6) δ 5.26
(s, 2H), 6.43 (s, 1H), 6.52 (s, 1H), 6.56 (s, 1H), 7.53−7.60 (m, 3H),
7.74 (d, J = 5.9 Hz, 2H), 7.80 (s, 1H), 7.82 (s, 1H), 8.23 (s, 1H), 8.26
(s, 1H), 8.30 (s, 1H), 8.56 (d, J = 5.7 Hz, 2H), 10.13 (s, 1H), 12.25 (s,
1H). 13C NMR (100 MHz, DMSO-d6) δ 104.1, 106.5, 108.8, 111.6,
116.9, 120.4, 125.1, 126.7, 127.2, 129.1, 129.8, 132.7, 138.9, 139.8,
139.9, 141.9, 142.3, 148.8, 149.9, 150.0. HRMS (EI+) m/z calcd for
C24H19N5O2S [M + H]+, 442.1338; found 442.1349.
2-(2-(Dimethylamino)ethoxy)-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-
b]pyridin-5-yl)aniline (6k). Compound 6k was prepared (2.4 mg,
8.3% yield) according to GP III from 5-bromo-2-(2-(dimethylamino)-
N-(3-Amino-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)-4-methylbenzenesulfonamide (6r). Compound 6r was
prepared (21 mg, 49% yield) according to GP I from N-(3-amino-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-4-methylbenze-
nesulfonamide (46 mg, 0.12 mmol) and 5-bromo-3-(pyridin-4-yl)-1H-
1
ethoxy)aniline (20 mg, 0.079 mmol). H NMR (300 MHz, CDCl3) δ
2.35 (s, 6H), 2.77 (t, J = 6.0 Hz, 2H), 4.07 (s, 2H), 4.15 (t, J = 6.0 Hz,
2H), 6.91−6.96 (m, 2H), 6.96 (s, 1H), 7.56 (d, J = 5.0 Hz, 2H), 7.70
(s, 1H), 8.34 (s, 1H), 8.56 (s, 1H), 8.62 (d, J = 5.1 Hz, 2H), 10.73 (s,
1H). HRMS (EI+) m/z calcd for C22H23N5O [M + H]+, 374.1981;
found, 374.1982.
3-Methoxy-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
aniline (6l). Compound 6l was prepared (5.0 mg, 26% yield)
according to GP III from 3-bromo-5-methoxyaniline (12 mg, 0.060
mmol). 1H NMR (300 MHz, CDCl3) δ 3.84 (m, 5H), 6.28 (t, J = 2.0
Hz, 1H), 6.55 (d, J = 1.7 Hz, 1H), 6.57 (d, J = 1.9 Hz, 1H), 7.57 (d, J
= 6.1 Hz, 2H), 7.72 (d, J = 1.7 Hz, 1H), 8.39 (d, J = 1.9 Hz, 1H), 8.59
(d, J = 2.0 Hz, 1H), 8.64 (d, J = 6.1 Hz, 2H), 10.02 (s, 1H). HRMS
(EI+) m/z calcd for C19H16N4O [M + H]+, 317.1402; found,
317.1390.
(3-Amino-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)(morpholino)methanone (6m). Compound 6m was prepared
(11 mg, 19% yield) according to GP III from (3-amino-5-
bromophenyl)(morpholino)methanone (39 mg, 0.14 mmol). 1H
NMR (400 MHz, CDCl3, 60 °C) δ 3.64−3.67 (m, 8H), 3.85 (s,
2H), 6.70 (dd, J = 2.2, 1.4 Hz, 1H), 6.95 (t, J = 1.8 Hz, 1H), 7.00 (t, J
= 1.4 Hz, 1H), 7.54 (d, J = 4.6 Hz, 2H), 7.66 (s, 1H), 8.35 (d, J = 2.0
Hz, 1H), 8.56 (d, J = 2.0 Hz, 1H), 8.65 (d, J = 4.6 Hz, 2H), 9.03 (s,
1H). HRMS (EI+) m/z calcd for C23H21N5O2 [M + H]+, 400.1773;
found, 400.1752.
3-(2-(Dimethylamino)ethoxy)-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-
b]pyridin-5-yl)aniline (6n). Compound 6n was prepared (12 mg, 25%
yield) according to GP III from 3-bromo-5-((dimethylamino)-
methoxy)aniline (26 mg, 0.10 mmol). 1H NMR (300 MHz,
CD3OD) δ 3.01 (s, 6H), 3.64 (t, J = 5.0 Hz, 2H), 4.46 (t, J = 4.9
Hz, 2H), 6.76 (t, J = 1.9 Hz, 1H), 7.09 (s, 2H), 8.41 (s, 1H), 8.44 (s,
1H), 8.57 (s, 1H), 8.65−8.67 (m, 3H), 8.71(d, J = 1.9 Hz, 1H).
HRMS (EI+) m/z calcd for C22H23N5O [M + H]+, 374.1981; found,
374.2013.
1
pyrrolo[2,3-b]pyridine (26 mg, 0.095 mmol). H NMR (300 MHz,
DMSO-d6) δ 2.31 (s, 3H), 5.26 (s, 2H), 6.42 (s, 1H), 6.53 (s, 1H),
6.55 (s, 1H), 7.34 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.75
(d, J = 6.1 Hz, 2H), 8.23 (s, 1H), 8.28 (d, J = 2.0 Hz, 1H), 8.31 (d, J =
1.9 Hz, 1H), 8.56 (d, J = 6.0 Hz, 2H), 10.00 (s, 1H), 12.27 (s, 1H).
13C NMR (100 MHz, DMSO-d6) δ 20.9, 103.8, 106.3, 108.6, 111.6,
116.9, 120.4, 125.1, 126.8, 127.2, 129.6, 129.8, 136.9, 139.1, 139.8,
141.9, 142.3, 143.0, 148.8, 149.9, 150.0. HRMS (EI+) m/z calcd for
C25H21N5O2S [M + H]+, 456.1494; found, 456.1476.
N-(3-Amino-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)-2,4-difluorobenzenesulfonamide (6s). Compound 6s was
prepared (36 mg, 45% yield) according to GP I from N-(3-amino-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2,4-difluoroben-
zenesulfonamide (104 mg, 0.254 mmol) and 5-bromo-3-(pyridin-4-
1
yl)-1H-pyrrolo[2,3-b]pyridine (46 mg, 0.17 mmol). H NMR (300
MHz, CD3OD) δ 6.57 (t, J = 1.9 Hz, 1H), 6.71 (d, J = 1.7 Hz, 1H),
6.73 (d, J = 1.7 Hz, 1H), 7.04−7.18 (m, 2H), 7.78 (d, J = 6.3 Hz, 2H),
7.90−7.98 (m, 1H), 8.02 (s, 1H), 8.38 (d, J = 1.9 Hz, 1H), 8.39 (s,
1H), 8.53 (d, J = 6.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ
103.9, 105.8, 106.0, 106.2, 106.3, 109.0, 111.6, 112.1, 112.2, 112.4,
112.4, 116.9, 120.4, 124.0, 124.0, 124.1, 124.2, 125.2, 127.3, 129.8,
132.5, 132.6, 138.2, 140.0, 142.0, 142.3, 148.8, 149.9, 150.0. HRMS
(EI+) m/z calcd for C24H17F2N5O2S [M + H]+, 478.1149; found,
478.1120.
N-(3-Amino-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)-3-(trifluoromethyl)benzenesulfonamide (6t). Compound 6t
was prepared (37 mg, 49% yield) according to GP I from N-(3-amino-
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-
(trifluoromethyl)benzenesulfonamide (98 mg, 0.22 mmol) and 5-
bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (41 mg, 0.15
1
mmol). H NMR (DMSO-d6, 300 MHz) δ 5.33 (s, 2H), 6.44 (s,
1H), 6.50 (s, 1H), 6.60 (s, 1H), 7.74 (d, J = 6.1 Hz, 2H), 7.80−7.85
(m, 1H), 8.00 (d, J = 7.5 Hz, 1H), 8.07−8.09 (m, 2H), 8.24 (d, J = 2.6
Hz, 1H), 8.28 (s, 1H), 8.29 (s, 1H), 8.55 (d, J = 6.0 Hz, 2H), 10.24 (s,
1H), 12.30 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 104.6, 106.9,
109.3, 111.6, 116.9, 120.4, 123.3 (q, J = 271 Hz), 123.4, 125.1, 127.3,
129.5, 129.7, 129.8 (q, J = 33 Hz), 130.8, 130.9, 138.4, 140.1, 140.8,
141.9, 142.3, 148.8, 150.0, 150.1. HRMS (EI+) m/z calcd for
C25H18F3N5O2S [M + H]+, 510.1212; found, 510.1183.
3-(2-Morpholinoethoxy)-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]-
pyridin-5-yl)aniline (6o). Compound 6o was prepared (12 mg, 28%
yield) according to GP III from 3-bromo-5-(morpholinomethoxy)-
aniline (31 mg, 0.10 mmol). 1H NMR (300 MHz, CD3OD) δ 2.63 (t,
J = 4.6 Hz, 4H), 2.83 (t, J = 5.4 Hz, 2H), 3.72 (t, J = 4.7 Hz, 4H), 4.17
(t, J = 5.4 Hz, 2H), 6.35 (s, 1H), 6.58 (s, 1H), 6.65 (s, 1H), 7.79 (d, J
= 6.4 Hz, 2H), 8.01 (s, 1H), 8.48−8.52 (m, 4H). HRMS (EI+) m/z
calcd for C24H25N5O2 [M + H]+, 416.2087; found, 416.2086.
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Article
N-(3-Amino-5-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)acetamide (6u). Compound 6u was prepared (30 mg, 48%
yield) according to GP I from N-(3-amino-5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)acetamide (65 mg, 0.24 mmol) and 5-
bromo-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (50 mg, 0.18
energy minimization with the AMBER program to remove the bad
steric contacts in the structure of TrkA.
Biological Assays and Methods. Trk Enzyme Assay. The IC50
determinations were performed using radiometric kinase assays
([γ-33P]ATP) at the Reaction Biology Corp. (Malvern, PA, U.S.).
Compounds were tested in a 10-dose IC50 mode with 3-fold serial
dilution starting at 1 μM. POC and Kd determinations were performed
at the Ambit Bioscience Corp (San Diego, CA, U.S.). Compounds
were profiled at 1 μM against a panel of 96 kinases in a high-
throughput binding assay (Ambit Bioscience).
Cells and Materials. The human breast cell lines MDA-MB-231,
MCF-7, and SKBR3 breast cancer cells were purchased from ATCC
(Manassas, VA). FBS, medium, penicillin−streptomycin, and all other
agents used in cell culture studies were purchased from Invitrogen
(Carlsbad, CA). Cultures were maintained at 37 °C in a CO2
incubator with a controlled humidified atmosphere composed of
95% air and 5% CO2. HUVECs were grown in a gelatin coated 75 cm2
flask in an M199 medium containing 3 ng/mL basic fibroblast growth
factor (bFGF), 5 U/mL heparin, and 20% FBS at 37 °C in a
humidified atmosphere of 5% CO2/95% air.
Cell Growth/Proliferation. Cell viability was performed by MTT.
Briefly, MDA-MB-231, MCF-7, and SKBR3 breast cancer cells were
plated at a density of (5−10) × 103 cells/well in 96-well plates for 24
h. Then the medium was removed, and cells were treated with either
DMSO as a control or various concentrations of 6t. The final
concentration of DMSO in the medium was ≤0.1% (v/v). After the
cells were incubated for 48 h, supernatants were removed from the
wells and 10 μL MTT solutions (2 mg/mL) were added to each well
for another 4 h at 37 °C. The formazan crystals that formed were
dissolved in DMSO (100 μL/well) by constant shaking for 5 min. The
plate was then read on a microplate reader at 540 nm. Six replicate
wells were used for each analysis.
DAPI Staining and TUNEL Assay. MCF-7 cells were plated onto an
18 mm cover glass in RPMI-1640 medium at ∼70% confluence for 24
h. The cells were then treated with 6t at 10 μM for 24 h. They were
fixed in ice-cold 2% paraformaldehyde (PFA), washed with PBS, and
then stained with 2 μg/mL 4,6-diamidino-2-phenylindole (DAPI) for
20 min at 37 °C. The stained cells were examined under a fluorescence
of nuclear fragmentation. Terminal deoxynucleotidyl transferase-
mediated nick end labeling (TUNEL) was performed following the
manufacturer’s protocol for TUNEL kit (Chemicon, Temecula, CA).
Wound Migration Assay. HUVECs plated on 60 mm diameter
culture dishes at 90% confluence were wounded with a razor blade
score 2 mm in width and marked at the injury line. After wounding,
the peeled off cells were removed with a serum-free medium and
further incubated in M199 with 2% FBS, 1 mM thymidine (Sigma-
Aldrich), and 6t (1−10 μM). HUVECs were allowed to migrate for 16
h and were rinsed with a serum-free medium, followed by fixing with
absolute alcohol.
1
mmol). H NMR (300 MHz, DMSO-d6) δ 2.02 (s, 3H), 5.22 (s,
2H), 6.60 (s, 1H), 6.96 (s, 1H), 7.03 (s, 1H), 7.77 (d, J = 6.1 Hz, 2H),
8.24 (d, J = 2.6 Hz, 1H), 8.39 (s, 1H), 8.44 (d, J = 1.8 Hz, 1H), 8.56
(d, J = 6.1 Hz, 2H), 9.74 (s, 1H), 12.28 (s, 1H). 13C NMR (100 MHz,
DMSO-d6) δ 24.1, 103.6, 106.0, 107.9, 111.6, 116.9, 120.3, 125.1,
127.1, 130.3, 139.4, 140.5, 142.1, 142.4, 148.7, 149.5, 150.0, 168.1.
HRMS (EI+) m/z calcd for C20H17N5O [M + Na]+, 366.1331; found,
344.1328.
4-Methoxy-3-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
aniline (6v). Compound 6v was prepared (5.5 mg, 15% yield)
according to GP III from 3-bromo-4-methoxyaniline (27 mg, 0.12
1
mmol). H NMR (300 MHz, CD3OD) δ 2.91 (s, 3H), 5.97 (dd, J =
8.6, 2.8 Hz, 1H), 6.06 (d, J = 2.7 Hz, 1H), 6.12 (d, J = 8.6 Hz, 1H),
6.97 (dd, J = 4.7, 1.6 Hz, 2H), 7.20 (s, 1H), 7.59 (d, J = 1.9 Hz, 1H),
7.65 (d, J = 2.0 Hz, 1H), 7.70 (dd, J = 4.7, 1.6 Hz, 2H). HRMS (EI+)
m/z calcd for C19H16N4O [M + H]+, 317.1402; found, 317.1409.
N-(4-Amino-2-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)benzenesulfonamide (6w). Compound 6w was prepared (8
mg, 22% yield) according to GP III from N-(4-amino-2-
bromophenyl)benzenesulfonamide (27 mg, 0.08 mmol). 1H NMR
(300 MHz, CD3OD) δ 6.61−6.75 (m, 2H), 6.96 (d, J = 8.5 Hz, 1H),
7.12 (t, J = 7.5 Hz, 2H), 7.21−7.27 (m, 1H), 7.30−7.42 (m, 2H),
7.75−7.84 (m, 2H), 8.05 (s, 1H), 8.11 (s, 2H), 8.54 (d, J = 5.5 Hz,
2H). HRMS (EI+) m/z calcd for C24H19N5O2S [M + H]+, 442.1338;
found, 442.1320.
Homology Modeling of TrkA. Because the 3D structure of TrkA
has not been reported so far, we carried out homology modeling using
the X-ray crystal structure of insulin-like growth factor 1 receptor
(IGF1R, PDB entry 3I81)22 as the template to obtain a high-quality
structure of TrkA suitable docking simulations. Sequence alignment
between the kinase domains of TrkA and IGF1R was derived with the
ClustalW program using the BLOSUM matrices for scoring the
alignments. The parameters of GAP OPEN, GAP EXTENTION, and
GAP DISTANCE were set equal to 10, 0.05, 8, respectively. Opening
and extension gap penalties were thus changed systematically, and the
obtained alignment was inspected for violation of structural integrity in
the structurally conserved regions. On the basis of the best-scored
sequence alignment, the 3D structure of the catalytic domain of TrkA
was constructed using the latest version of the MODELLER program.
In this model building, we employed an optimization method
involving conjugate gradients and molecular dynamics to minimize
the violations of the spatial restraints. With respect to the structure of
gap regions, the coordinates were built from a randomized distorted
structure that resides approximately between the two anchoring
regions as implemented in the MODELLER program. To increase the
accuracy of the calculated structures, loop modeling was also
performed with the enumeration algorithm. Then we calculated the
conformational energies of the predicted structures of TrkA with the
ProSa 2003 program2 for the purpose of evaluation.
Docking. We used the AutoDock program because the out-
performance of its scoring function over those of the others had been
shown in several target proteins. The atomic coordinates of TrkA
obtained from the homology modeling were used as the receptor
model with docking simulations. Special attention was paid to assign
the protonation states of the ionizable Asp, Glu, His, and Lys residues
in the homology-modeled structure of TrkA. The side chains of Asp
and Glu residues were assumed to be neutral if one of their carboxylate
oxygens pointed toward a hydrogen-bond-accepting group including
the backbone aminocarbonyl oxygen at a distance within 3.5 Å, a
generally accepted distance limit for a hydrogen bond of moderate
strength. Similarly, the lysine side chains were protonated unless the
NZ atom was in proximity to a hydrogen-bond donating group. The
same procedure was also applied to determine the protonation states
of ND and NE atoms in His residues. After determining the
protonation state of each protein atom, we carried out 200 cycles of
Tube Formation Assay. Matrigel (200 μL) (10 mg/mL) (BD
Biosciences, NJ) was polymerized for 30 min at 37 °C. HUVECs were
suspended in M199 (5% FBS) medium at a density of 1.0 × 105 cells/
mL, and 0.2 mL of cell suspension was added to each well coated with
Matrigel, together with or without the indicated concentrations of 6t
for 10 h. The morphological changes of the cells and HUVEC tubes
formation were observed under a phase-contrast microscope and
photographed at ×200 magnification.
ASSOCIATED CONTENT
■
S
* Supporting Information
Effect of 61 on apoptosis of cancer cells. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*For S.-S.H.: phone, (+82) 32-890-3683; fax, (+82) 32-890-
2462; e-mail, hongs@inha.ac.kr. For S.H.: phone, (+82) 42-
350-2811; fax, (+82) 42-350-2810; e-mail, hongorg@kaist.ac.kr.
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Journal of Medicinal Chemistry
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Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by National Research Foundation
of Korea (NRF) through General Research Grants NRF-2011-
0016436 and 2011-0020322. S.H. is the recipient of a Global
Ph.D. Fellowship (Grant NRF-2011-0007581).
ABBREVIATIONS USED
■
RTK, receptor tyrosine kinase; TrkA, tropomyosin-related
kinase A; HIF-1, hypoxia-inducible factor; VEGF, vascular
endothelial growth factor; PI3K, phosphoinositide 3-kinase;
iNOS, inducible nitric oxide synthase; HUVEC, human
umbilical vein endothelial cell; ATP, adenosine 5′-triphosphate;
SAR, structure−activity relationship; NIC, N-iodosuccinimide;
DCM, dichloromethane; DMF, N,N-dimethylformamide;
DIPE, diisopropyl ether; DPPE, 1,1′-bis(diphenylphosphino)-
ferrocene; NBS, N-bromosuccinimide; MTT, 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
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