Development and biological evaluation of potent and selective c-KIT D816V inhibitors

Article abstract of DOI:10.1021/jm500413g

The c-KIT tyrosine kinase has emerged as a potential therapeutic target for an array of diseases. However, there exists a drug resistance that is caused by mutations in c-KIT; therefore, c-KIT remains as a clinical challenge due to limited effective treatment options for therapies. For example, the acquired activating point mutation D816V significantly impairs the efficacy of targeted cancer therapies. Understanding the mechanisms of drug resistance at the molecular level will aid in designing and developing particular inhibitors with the potential to overcome these resistance mutations. We undertake a structure-based de novo design of 7-azaindole as the molecular core using the modified scoring function. This approach led to an identification of new c-KIT inhibitors over 100-fold specific for the D816V mutant relative to the wild-type c-KIT with nanomolar inhibitory activity. More importantly, these compounds potently inhibit clinically relevant D816V mutations of c-KIT in biochemical and cellular studies.

Full text of DOI:10.1021/jm500413g

Article
pubs.acs.org/jmc
Development and Biological Evaluation of Potent and Selective
c‑KITD816V Inhibitors
Soyoung Lee,†,‡ Hyunseung Lee,§ Jinhee Kim,†,‡ Suhyun Lee,†,‡ Soo Jung Kim,§ Byong-Seok Choi,†
Soon-Sun Hong,*,§ and Sungwoo Hong*,‡,†
†Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, E6-4, Daejeon 305-701,
Korea
‡Center for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS), Daejeon 305-701, Korea
§Department of Biomedical Sciences, College of Medicine, Inha University, Incheon, 400-712, Korea
S
* Supporting Information
ABSTRACT: The c-KIT tyrosine kinase has emerged as a potential
therapeutic target for an array of diseases. However, there exists a drug
resistance that is caused by mutations in c-KIT; therefore, c-KIT remains as a
clinical challenge due to limited effective treatment options for therapies. For
example, the acquired activating point mutation D816V significantly impairs
the efficacy of targeted cancer therapies. Understanding the mechanisms of
drug resistance at the molecular level will aid in designing and developing
particular inhibitors with the potential to overcome these resistance
mutations. We undertake a structure-based de novo design of 7-azaindole
as the molecular core using the modified scoring function. This approach led
to an identification of new c-KIT inhibitors over 100-fold specific for the
D816V mutant relative to the wild-type c-KIT with nanomolar inhibitory
activity. More importantly, these compounds potently inhibit clinically
relevant D816V mutations of c-KIT in biochemical and cellular studies.
Chemotherapy of cancers with first-line drugs often leads to
resistance of the cancers.5,8 Gain-of-function point mutations in
the c-KIT kinase domain result in a ligand-independent
constitutive activation of c-KIT signaling, which leads to an
uncontrolled mast cell proliferation and apoptosis resist-
ance.9−11 Systemic mastocytosis patients have a gain-of-
function mutation (c-KITD816V) in many cases9,12 Introduction
of the D816V point mutation disrupts the inactive
conformation and impedes the binding process of first-line
drugs, including imatinib, to the ATP-binding site of c-KIT.13
To date, only a few multikinase inhibitors, such as dasatinib14
INTRODUCTION
■
The c-KIT tyrosine kinase belongs to a family of type III
receptor tyrosine kinase and is a transmembrane receptor that
mediates the pleiotropic biological effects through its ligand
stem cell factor (SCF, also known as mast cell growth factor).1
Once SCF binds to an extracellular immunoglobulin-like
domain of c-KIT, two monomeric c-KIT receptors become
dimerized, resulting in an activation of downstream intracellular
signal transduction, including phophatidylinositol-3-kinase
(PI3K)/Akt pathway, mitogen-activated protein kinase
(MAPK), and signal transducer and activator of transcription
(STAT), which are involved in cell proliferation, differentiation,
and survival2,3 Deregulated c-KIT kinase activity can be variably
detected in multiple different types of pathogenesis, including
small cell lung carcinoma, acute myeloid leukemia (AML),
neuroblastoma, malignant melanomas, colorectal cancer,
systemic mastocytosis (SM), and gastrointestinal stromal
tumors (GISTs).3,4 The activation loop of the wild-type c-
KIT exists in a dynamic equilibrium between the active and
inactive conformations. Impairing c-KIT activity with tyrosine
kinase inhibitors, such as imatinib, has yielded good responses
in some c-KIT-dependent cancers.5,6 Imatinib is effective
against the wild-type c-KIT by stabilizing the inactive
conformation of the c-KIT kinase domain and then preventing
the inactive form from switching to the active conformation of
c-KIT.5,7
and ponatinib,15 sufficiently block the activity of c-KITD816V
.
Simultaneously inhibiting off-targets can result in a target-
related toxicity because many individual biological targets are
involved in important cellular functions. In this way, many
multikinase inhibitors in clinical use might lead to possible side
effects because off-target kinase inhibition can cause toxicity to
normal cells.16 Recently, an interest has been growing in an
effort to discover an approach to only inhibit the mutant
therapeutic target, thereby minimizing the risk of potential side
effects associated with the inhibition of normal function. The
structural similarity has made the search for selective c-
KITD816V inhibitors a challenging process. In this regard, it
Received: March 17, 2014
Published: July 8, 2014
© 2014 American Chemical Society
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would be worthwhile to identify c-KITD816V inhibitors in a
specific manner over the wild-type c-KIT without normal SCF/
KIT signaling pathway disruption. This strategy would
potentially provide an effective treatment option as a special
type of cancer treatment. Herein we describe our efforts to
develop a new series of 7-azaindole-based inhibitors that may
be effective and specific against c-KITD816V and demonstrate the
biological activities of these compounds against imatinib-
resistant HMC-1.2 cell lines.
structural information regarding various kinase−inhibitor
complexes. The predicted hydrogen bonding network with
Tyr836, Asp810, and the inhibitor in PI3Kα (Figure 2A) was
based on previous studies using X-ray crystallography.19 Our
experience also confirmed the importance of a water-mediated
hydrogen bond with residues in the active site of PI3Kα.20
Although hydrogen bonds appeared to form between pyridyl
nitrogen at the 5 position of 1 and bound water molecule in
PI3K, this key hydrogen bonding interaction was not expected
in c-KITD816V (Figure 2B). We speculated that these structural
differences can possibly provide a guideline for improving the
selectivity via inhibitor modifications in this region of accessing
specific spaces of the gatekeeper. With this agenda, our initial
survey of the disruption of the key intermolecular hydrogen
bonding network in PI3Kα via C5 group modifications was
conducted, probing the structural differences affecting the
binding pockets of PI3Kα and c-KITD816V (Figure 2).
RESULTS AND DISCUSSION
■
Identification of New c-KITD816V Inhibitors. We began
our study by cross-screening our compound collection in search
of c-KITD816V inhibitors. This led to an identification of a 7-
azaindole-based derivative, combined with 3,4-dimethoxyben-
zene group at C3, and pyridylsulfonamide group at C5 as a c-
KITD816V inhibitor (1, IC50 = 346 nM) (Figure 1).17 The
Indeed, modification of the C5 group was essential for
enhancing the selectivity over PI3Kα, as shown in Table 1. Of
particular significance was the observation that the binding
affinity of 2 over PI3Kα was dramatically reduced (IC50 = 1.6
μM for PI3Kα) when the pyridyl group at the C5 position was
replaced with a phenyl group, probably due to the disruption of
the key hydrogen bond with a bound water molecule in PI3Kα.
We also noted that the sulfonyl group of aniline at the C5
position was allocated to generate further stabilizing
interactions through the formation of hydrogen bond with
Lys802 in PI3Kα. With the binding mode set in this way, the
sulfonyl group appeared to be unnecessary for producing an
inhibitory activity over c-KITD816V. To verify this assumption,
we then removed the sulfonyl group and assessed their
inhibitory potencies over PI3Kα and c-KITD816V. Subsequently,
compound 4 exhibited excellent selectivity over PI3Kα while
retaining the lowest POC number for c-KITD816V (IC50 = 6.3
nM for c-KITD816V, IC50 > 10000 nM for PI3Kα). Compound 3
was prepared as the control to investigate the role of the pyridyl
group. Next, a second set of data was obtained for compounds
in which C-5 aryl ring was substituted with a variety of patterns
of groups. From these results, it was evident that either a
removal of the sulfonamide group or a replacement of the
pyridyl group with a phenyl group at C5 led to a dramatic loss
in the inhibitory activity over PI3Kα. Remarkably, this strategy
has served well without compromising the c-KITD816V affinity,
and most of 3,4-dimethoxybenzene derivatized compounds are
Figure 1. Initial hit for c-KITD816V inhibitors. Compounds were tested
against c-KITD816V and PI3Kα.
synthesized inhibitor compound 1 may impair c-KITD816V
catalytic activity by specifically binding to the ATP binding
site as illustrated in Figure 2. Because 7-azaindole scaffold has
been used for PI3Kα inhibitors,18 we proceeded to screen this
series of compounds against PI3Kα. Indeed, the newly
identified c-KITD816V inhibitor 1 exhibited an inhibitory activity
against PI3Kα (IC50 = 12 nM). Because the initial hit 1 was
found to have a higher affinity for PI3Kα, our first design efforts
were focused on improving the selectivity against PI3Kα while
maintaining potent c-KITD816V inhibition.
The recent design of selective kinase inhibitors has been
facilitated by the availability of substantial three-dimensional
Figure 2. Design of new scaffolds as c-KITD816V inhibitors and opportunities for modification in the schematic active site of c-KITD816V. (A)
Predicted binding mode of 1 in PI3Kα. (B) Predicted binding mode of 1 in c-KITD816V
.
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Table 1. Exploration of Groups at the C5 Position of
a
Azaindole Core
Figure 3. Flowchart for the discovery process of potent and selective
inhibitors of D816V mutant of c-KIT through the structure-based de
novo design.
azaindole derivatives: one is the homology modeling for the
structure of c-KITD816V constructed by the MODELER
program on the basis of wild-type c-KIT in the active
conformation,21,22 and the other is the autoinhibited form of
the wild-type (PDB entry: 1T45)23 as for the wild-type c-KIT
model. First, a variety of 7-azaindole derivatives were generated
with the LigBuilder program24 based on the structure of the
D816V mutant−azaindole complex obtained from docking
simulations. To score the generated derivatives with consid-
eration of their binding affinity for c-KIT and c-KITD816V, the
program employed an empirical binding free energy function.25
As the starting point for generating 7-azaindole derivatives, we
analyzed the binding pocket in the ATP-binding site of the
D816V mutant using the POCKET module. The structure of c-
KITD816V in complex with 7-azaindole was used as the input to
identify key interacting residues in the ATP-binding site. From
this information, it could be possible to define a binding model
of 7-azaindole inhibitors at the ATP biding site. A variety of 7-
azaindole derivatives were then generated via an application of
genetic algorithm, whereby the structures evolved as the
chemical groups modified at the substitution positions of the 7-
azaindole core, and these derivatives were rescored with the
modified scoring function (see the Supporting Information).
The generated 7-azaindole derivatives were then selected
according to the calculated binding free energy to maximize
the affinity for the D816V mutant and minimize the affinity for
wild-type c-KIT.
a
Compounds were tested against c-KITD816V and PI3Kα.
found to have a greater binding affinity for c-KITD816V than
PI3Kα (Table 1). We also examined the significance of the
meta-substituents at the C5-aryl ring and observed that the
binding affinity for c-KITD816V was dramatically reduced when
the meta-amino group of compound 4 was moved to the ortho-
position (13).
De Novo Design. On the basis of the preliminary data,
compound 4 was selected as an initial hit compound to allow
for additional interactions to be identified. We explored the
space at 3- and 5- positions of 7-azaindole to identify the
derivatives with enhanced potency through the structure-based
de novo design, as summarized in Figure 3. Thus, we prepared
two receptor models to perform the de novo design of 7-
The de novo design studies identified a variety of functional
groups to introduce the 7-azaindole core at the 3- and 5-
positions. For the synthesized derivatives, IC50 (50% inhibitory
concentration) values were determined against the wild-type c-
KIT and D816V mutant.26 To determine whether or not the
3,4-dimethoxy phenyl group at the C3 position of initial hit 4
was optimal, the effects of its replacement with other groups
were explored, and a series of (hetero)aryl groups were
installed (Table 2). Because the 3-aniline group at the C5
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and dynamics simulation studies revealed that the activating
D816V mutation triggers spontaneous detachment of the
juxtamembrane domain from the c-KIT domain, which
stabilizes the activation loop (A-loop) in the extended
conformation.21 This conformational change in c-KITD816V
results in maintaining the active form, which enables
constitutive oncogenic activation. Figure 4 displays representa-
tive MD trajectory snapshots of the D816V mutant−4
complexes. The binding modes of 4 with the D816V mutant
shows that the 7-azaindole skeleton fully maintains the
bidentate hydrogen bonding pattern with the backbone groups
of Cys673, which reveal that the strengths and dynamic
stabilities of the bidentate hydrogen bonds with Cys673 serve
as critical factors that affect the potency and selectivity of c-
KITD816V inhibitors. Compound 4 appears to be stabilized
through the hydrophobic interactions with side chains Leu595,
Val603, Ala621, Tyr672, and Leu799. Another feature in the
binding modes of 4 is that the terminal 3,4-dimethoxybenzene
group appears to be exposed to the bulk solvent in the c-KIT−
4 complex and stabilized through interactions with the protein
Table 2. Exploration of Groups at the C3 Position of
Azaindole Core
a
groups at the entrance of the ATP-binding site of c-KITD816V
.
Having identified the significance of the 3,4-dimethoxyphenyl
group at the C3 position of 7-azaindole, we carried out a
broader exploration of analogues which incorporated this key
functional group (Table 3). In general, the synthesized
derivatives appear to maintain the bidentate hydrogen bonding
with the backbone groups of Cys673 in both c-KIT and c-
KITD816V. The aromatic ring system of these analogues is
further stabilized through hydrophobic interactions with
nonpolar residues, including Leu595, Val603, Ala621, Leu799,
Tyr672, and Cys674. The third hydrogen bond in the P-loop
appears to be established in a different fashion in complex of
wild-type c-KIT and the D816V mutant. The molecular
modeling study indicated that Asp-Phe-Gly (D810-F8111-
G812) motif in the A-loop and the glycine-rich phosphate-
binding loop (P-loop) form a deep hydrophobic pocket in c-
KITD816V. In contrast, the p-loop in the wild-type is away from
7-azaindole, as shown in Figure 4B. The 3-aminophenyl group
at C5 faced into the hydrophobic pocket constructed by
phosphate-binding loop and activation loop in the ATP-binding
sites of c-KITD816V. This suggests that there exists a possibility
of further elaboration of the substituents of C5 aryl ring (Figure
4).
With this agenda, a diverse set of substituents were
investigated to expand the structure−activity relationship, and
the analogues incorporated an additional substituent into the 3-
aminophenyl group in order to enhance the selectivity for c-
KITD816V. In this series of analogues, it appeared that the
presence of an additional substituent on the 3-aniline ring was
generally tolerant to its function on c-KITD816V. In contrast, the
potency on the wild-type c-KIT was tunable by modification of
the substitution patterns of the C5-aryl ring as seen in Table 3.
The relatively low potency for wild-type c-KIT of 7-azaindole
inhibitors with a larger group substituted 5-aryl ring may be
rationalized by the decreased volume of the ATP-binding
pocket in wild-type c-KIT due to the approach of JMR residues
to the gatekeeper site in the inactive conformation. The
substitution patterns of C5 aryl ring also proved to be an
important structural requirement for the design of c-KITD816V
selective inhibitors. Thus, introduction of an additional
methoxy group at the para-position of the 3-aniline ring (34)
resulted in highly amplified selectivity vs wild-type c-KIT (IC50
>10000 nM for c-KIT, IC50 = 25.6 nM for c-KITD816V). In the
a
IC50 (50% inhibitory concentration) values were determined against
the D816V mutant.
position of 4 proved to be effective for high inhibitory activity,
we tentatively fixed the 3-aniline group in this region, then
screened various groups at the C3 position. We observed that
the binding affinity and potency were reduced when the 3,4-
dimethoxy group was replaced with the 3,5-dimethoxy group
(27) or the monomethoxy group (25 and 26), indicating that
the orientation of the dimethoxy group is critical in sustaining
potency. The enzyme potency of the derivatives was also
decreased when the trimethoxy group was introduced (21),
demonstrating that the 3,4-dimethoxy phenyl group at the C3
position group is the maximum size that is suitable for this
position in the 7-azaindole core. A comparison of the IC50
values of 4 (IC50 = 6.3 nM) to that of 22 (IC50 = 104 nM)
clearly reveals that substitutions of other chemical groups for
the 3,4-dimethoxybenzene moiety lead to a large decrease in
the inhibitory activity against c-KITD816V. From the first round
of C3 array exploration, derivatives bearing 3,4-dimethoxyben-
zene appeared to be the superior inhibitors, suggesting that this
group is an effective substituent for the potent activity against c-
KITD816V
.
Enhancement of Selectivity for the D816V mutant.
The aniline 4 proved to be a potent c-KITD816V inhibitor (IC50
= 6.3 nM) and represented a potent hit in our c-KITD816V
program. To address the structural and mechanistic aspects that
are relevant to the selective inhibition of the D816V mutant by
these newly identified inhibitors, we carried out docking studies
for c-KITD816V in complex with 4. Previous molecular modeling
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Figure 4. Representative MD trajectory snapshots of the hydrophobic pocket constructed by phosphate-binding loop and activation loop in the
ATP-binding sites of (A) c-KITD816V and (B) wild-type c-KIT. (C) Predicted binding mode of 4 with the D816V mutant of c-KIT. Carbon atoms of
c-KIT and 4 are indicated in gray and green, respectively. Asp810 and Phe811 of DFG motif are indicated in magenta. Each navy dotted line
indicates a hydrogen bond.
D816V mutant, both substituents at the meta- and para-
position of C5 aryl ring of compound 34 are expected to extend
the P-loop and make favorable contacts with backbone groups
of residues in the pocket (Figure 5A). In addition, the methoxy
group at the para-position appeared to establish an additional
hydrogen bond with the side-chain amino group of Lys623. By
contrast, this binding mode cannot be formed in the c-KIT−34
complex because the P-loop is too distant from the ATP-
binding site, and accessing to the extended P-loop is prevented
by Phe811 in the wild-type c-KIT. Instead, it is observed that
the C5 aryl ring points toward a small hydrophobic pocket
formed by the side chains of Ile699, Thr670, Val622, Leu644,
and Lys623-Glu640 salt bridge in the wild-type (Figure 5B).
This hydrophobic region is too small to comfortably
accommodate the two substituents coming off the C5 aryl
ring, and unfavorable steric contacts would be present in the
wild-type c-KIT. The structural comparison of c-KIT and c-
KITD816V in the P-loop revealed that the interaction of 7-
azaindole with hydrophobic pocket in the P-loop could serve a
key structural determinant of the specificity of the D816V
mutant selective inhibitors.
Because these structural differences provided a guideline
toward favorable impact on selectivity improvement for the
D816V mutant, several other groups were designed to extend
the substituent at this region (Table 3). The preference of meta-
and para-substituents of C5-aryl ring for the enhancement of
selectivity for c-KITD816V is further illustrated by the potency of
analogue 31 bearing in the form of a morpholinoacetyl group
(IC50 = 9140 nM for c-KIT, IC50 = 8.5 nM for c-KITD816V).
The inhibitory activity was also increased over the D816V
mutant when the methoxy group was replaced with a 2-
morpholinoethoxy appendage (40, IC50 = 13.9 nM) while
retaining excellent selectivity over the wild-type c-KIT. The
3,4,5-trimethoxy aryl group was also tolerable to the D816V
mutant, and 32 (IC50 = 8.8 nM) was more potent than the
corresponding 4-methoxy derivative 18 (IC50 = 32.1 nM) and
3-methoxy derivative 41 (IC50 = 28.7 nM). Methyl substitution
(33) had no appreciable change on both the wild-type c-KIT
and the D816V mutant when compared with the corresponding
34. Intriguingly, replacement of the 3-NH2 group of 34 with a
3-OMe group (19) resulted in about 10-fold increase in
potency on the D816V mutant (IC50 = 2.1 nM). Attempts to
further increase potency by linking or repositioning substituents
of C5-aryl ring (35, 36, and 39) were unremarkable, and no
compound offered any advantage relative to the corresponding
compound 19. Cumulatively, these results indicate that
disubstitution at the meta- and para-position of C5 aryl ring
in this series was most effective for achieving high selectivity for
the D816V mutant.
Interestingly, moving the substituent from the meta- to the
para-position of C5 aryl ring led to increase in the inhibitory
activity against the wild-type c-KIT, thus reducing the
selectivity for the D816V mutant (18, 37, and 38). For
example, changing the substituent from 3-aminobenzene in 4 to
4-aminobenzene in 37 led to almost a 20-fold increase in the
inhibitory activity against the wild-type c-KIT (IC50 = 56.8 nM)
but only a slight change in the IC50 values with respect to the
D816V mutant (IC50 = 9.1 nM). Compound 18 bearing a 4-
methoxy group at the para-position also showed a decrease in
selectivity for the D816V mutant. When the para-amino group
in compound 37 was replaced by the methyl group in
compound 17, it resulted in a substantial loss of activity on
both the wild-type c-KIT (IC50 >10\000 nM) and the D816V
mutant (IC50 = 571.3 nM). Because moving the substituent
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Table 3. Influence of C5 Groups on Selectivity
from the meta- to the para-position of C5 aryl ring resulted in
an increased potency over the wild-type, the binding mode of
37 was further analyzed with MD simulations.
binding site and the P-loop are also observed in the wild-type−
37 complexes. For example, the inhibitor moves from the
activation loop to the ATP-binding site (residues 670−677) in
the c-KIT−37 complex, establishing a hydrogen bond between
the 4-aniline group of 37 and the backbone aminocarbonyl
oxygen of Asp810 (Figure 6B). This positional shift strengthens
the bidentate hydrogen bonds between 7-azaindole ring and
Cys673. The average N···H−N and N−H···O hydrogen bond
distances are 1.82 and 1.78 Å, respectively, in c-KIT−37
complex. The approach of 37 to the ATP-binding may also
have an effect in promoting van der Waals interactions between
the 7-azaindole group and the side chains of Val654, Tyr672,
and Leu799 (Figure 6B). Therefore, the strengthening of the
hydrogen bonds with the backbone groups of Asp810 and
The binding modes of 37 in the ATP-binding sites of the
wild-type c-KIT and the D816V mutant, obtained from energy
minimizations of the time-averaged structures of c-KIT−37 and
D816V−37 complexes, are compared in Figure 6. With regard
to the structure, 37 is stabilized through bidentate hydrogen
bonds between 7-azaindole ring and Cys673, hydrophobic
interactions of aromatic rings with nonpolar residues, and a
hydrogen bond between the NH2 group and the backbone
aminocarbonyl oxygen of Asp810. Despite the overall similarity
in the binding modes of 4 and 37 toward the wild-type c-KIT,
some differences in inhibitor positions with respect to the ATP-
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Figure 5. (A) Predicted binding mode of 34 with c-KITD816V and (B) with wild-type c-KIT. Each navy dotted line indicates a hydrogen bond.
Figure 6. Representative MD trajectory snapshots of 37 in the ATP-binding sites of (A) c-KITD816V and (B) wild-type. Carbon atoms of c-KIT and
37 are indicated in cyan and green, respectively. Each dotted line indicates a hydrogen bond.
Cys673, as well as hydrophobic interactions with nonpolar
residues around the ATP-binding site, may contribute to the
potent inhibitory activity of 37 with respect to the wild-type c-
KIT. The binding mode of 37 for the D816V mutant differs
from that of the wild-type in that the role of the hydrogen bond
acceptor with respect to the terminal aniline moiety is played
by a side chain carboxylate ion of Glu640 instead of the
backbone aminocarbonyl oxygen of Asp810 (Figure 6A). The
above modeling analysis similarly applies to compound 18,
leading to an increased potency over the wild-type c-KIT. The
loss of hydrogen bond interactions between derivative 17
bearing C5 p-tolyl group and the backbone aminocarbonyl
oxygen of Asp810 may explain the substantial loss of activity on
the wild-type c-KIT (IC50 >10000 nM).
Kinase Selectivity Profiling. To obtain the detailed
picture of selectivity profile of this series, potent inhibitors
were subjected to kinase selectivity profiling with a panel of 48
cancer related kinases, representing major branches (TK, TKL,
STE, CMGC, CAMK, AGC, atypical, and other) on the
kinome tree at 1 μM concentrations in a high-throughput
binding assay (Figure 7, KINOMEscan, Ambit Biosciences).25
Overall, this series of inhibitors showed moderate to good
specificity profiles relative to current multitargeted c-KITD816V
inhibitors such as dasatinib and ponatinib; however, these
inhibitors are less selective for the TK kinase family including
FLT3, JAK2, TRKA, and TRKB. Other kinases inhibited by this
series are DRAK1 (CAMK family), Aurora A, Aurora B, IKKa,
and IKKb as shown in Figure 7 (see Supporting Information
Table S1 for more details). Among this series of inhibitors,
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Figure 7. Selectivity profiles of compounds 19, 21, 31, 32, and 34. A panel of 48 kinases was tested at 1 μM concentrations in a high-throughput
binding assay (see Supporting Information for details).
trimethoxy derivatve 32 exhibited an improved selectivity
profile, and two kinases against which 32 demonstrated tight
off-target binding affinity (IC50 < 1 μM) were FLT3, and
DRAK1. On the other hand, compound 31 showed only
modest specificity profiles against other kinases and displayed
significant off-target activity over 10 kinases (ABLT315I, Aurora
A, DRAK1, FLT3, IKKa, IKKb, JAK2, ROCK1, TRKA, and
TRKB).
Antiproliferation Activity. This series of potent inhibitors
were further tested for cellular growth inhibition against human
mast cells (Table 4). We carried out an evaluation of their
antiproliferative activity on HMC-1.2 cell lines (V560G,
D816V), which are imatinib-resistant. To measure the
inhibitory effect of these compounds on cell growth, cell
viability was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay in HMC-1.2 at various
concentrations for 48 h. Notably, compounds 19, 21, 32, and
34 showed potent antiproliferative effects at submicromolar
concentration as shown in Table 4. This series of compounds
displayed significant off-target activity (IC50 < 10 μM) over
several kinases (DRAK1, FLT3, and IKK) which are believed to
be involved in cell proliferation. On the basis of the selectivity
data, off-target activity might be partially responsible for potent
antiproliferative effects. For example, DRAK1 and FLT3
displayed tight binding to this series of compounds (DRAK1
by 19, 31, 32, 34; FLT3 by 19, 31, and 32). Compound 31 also
exhibited significant actvities over IKKa and IKKb.
A couple of cases of discordances were observed between
IC50 values over c-KITD816V and cellular potencies, which may
be attributed to the inability of the enzymatic assay data to
accurately reflect the cell potencies. Because intracellular
activity is influenced by the physicochemical properties of
compounds, we further measured basic profiles of these
compounds (see Table S5 in Supporting Information). The
good ability of 21 and 34 to suppress the growth of HMC-1.2
cells might be attributable to their more desirable range of
properties, whereas more potent compound 31 in the
biochemical assay exhibited a lower AlogP value and higher
topological polar surface area (tPSA). Likewise, compounds 19
and 32 showed a higher AlogP value and lower tPSA than 21 or
34.
Table 4. Effect of C-KIT Inhibitors on Growth of HMC-1.2
HMC-1.2 (V560G,
D816V) IC50 (μM)
HMC-1.2 (V560G,
D816V) IC50 (μM)
compd
compd
imatinib
dasatinib
ponaitinib
4
17
21
31
32
33
34
0.81
2.01
0.91
1.43
0.87
0.82
0.35
1.68
0.76
19
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Cell-Signaling Effects. c-KIT signaling is transmitted
through several pathways, including signal transducer and
activator of transcription, and activation of the c-KIT pathway
leads to the phosphorylation of STAT3, Akt, and Erk, a
downstream effector of phosphatidylinositol 3-kinase. To
ensure that the new series of compounds was inhibiting c-
KIT-dependent downstream signaling pathways in HMC-1.2
cells, several potent inhibitors were further profiled for their
ability to suppress cellular biomarkers, using Western
immunoblotting. Consistent with the expectations for a potent
c-KIT inhibitor, 19, 21, 32, and 34 strongly suppressed the
phosphorylation of STAT3, Akt, and Erk. We also compared
the suppression of c-KIT signaling by these inhibitors with
imatinib, dasatinib, and ponatinib as shown in Figure 8.
Scheme 1. A two-step reaction sequence, starting from a
known-compound 45, was utilized to afford the desired
derivative 48. An initial Pd-catalyzed cross-coupling with C3
(hetero)aryl partners was carried out to afford a C3 aryl
intermediate, which were then subjected to benzenesulfonyl
group deprotection under basic conditions, resulting in
compound 46. An assortment of the C5 groups of 7-azaindole
was then explored through another Suzuki coupling reaction
with the appropriate arylboronic acids or esters, yielding
focused set of target compounds. For focused C5-aryl group
array, the key intermediate 47 was prepared via direct
borylation of 5-bromoazaindole 46. The corresponding desired
compounds 48 were then obtained by Pd-catalyzed cross-
coupling with the appropriate aryl boronic acids or esters.
CONCLUSION
■
The 7-azaindole-based hit 4 was modified by applying a
structure-based de novo design to obtain new potent and
selective inhibitors for the gain-of-function D816V mutant. The
7-azaindole derivatives with a 3,4-dimethoxybenzene moiety at
the 3-postion and a polar aromatic group at the 5-position were
found to have a nanomolar inhibitory activity for the D816V
mutant, with more than 100-fold greater potency compared to
the wild-type c-KIT. A decomposition analysis of the binding
free energies indicates that the potency of an inhibitor can be
optimized by reducing desolvation cost for enzyme−inhibitor
complexation as well as by strengthening the interactions
between the inhibitor and c-KIT in the ATP-binding site. Four
of the compounds suppress cell growth and proliferation
against imatinib-resistant HMC-1.2 cell lines in a submicromo-
lar concentration range. Compound 19 also induced apoptosis
through regulation of the c-KIT signaling pathway, which leads
to strong anticancer activity in HMC-1.2 cells. Future studies
may be necessary to fully identify the potential of this series as a
modality for cancer treatment.
Figure 8. Inhibition of cellular c-KIT pathway including STAT3, Akt,
and Erk in HMC-1.2 (imatinib-resistant human mast cell). HMC-1.2
cells were starved with serum-free IMEM for 2 h and treated with
imatinib, dasatinib, ponatinib, 19, 21, 32, and 34 for 1 h at 5 μM.
EXPERIMENTAL SECTION
■
I. General Methods and Materials. Unless stated otherwise,
reactions were performed in a flame-dried glassware under 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 via UV light (254
and 354 nm). Flash column chromatography was utilized on silica gel
(400−630 mesh). 1H NMR was recorded on 600 or 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 the 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). 13C NMR was recorded
on 100 or 150 MHz and was fully decoupled by broad band proton
decoupling. 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 Bruker
Daltonik HR-MS using the EI method or Agilent LR-MS. High-
performance liquid chromatography analyses for checking mass
spectral data (using EI method) and the purity (>95% area) and of
synthesized compounds were performed on a Agilent HPLC equipped
with an Agilent Poroshell 120 EC-C18 reverse phase column (4.6 mm
× 50 mm, 2.7 μm) and by quadrupole LC/MS. The mobile phase was
a mixture of MeOH (0.1% TFA) and H2O (0.1% TFA). Compound
purity was determined by integrating the peak areas of liquid
chromatogram, monitored at 254 nm. Flow rate (0.5 mL/min):
Gradient system, from MeOH (0.1% TFA) 10% and H2O (0.1% TFA)
90% (0 min) to MeOH (0.1% TFA) 90% and H2O (0.1% TFA) 10%
Cell-Cycle Distribution and Apoptotic Cell Death. In
general, cell apoptosis and growth correlate with the cell cycle
progression. Blocking the kinase pathway can change the cell
cycle distribution and promote apoptosis. Because compound
19 successfully demonstrated the ability to inhibit p-STAT3, p-
Akt, and pErk in HMC-1.2 cells, we further determined
whether the inhibition of c-KIT in HMC-1.2 cells by
compound 19 induces a cell cycle arrest. The cell cycle
distribution was evaluated by a flow cytometric analysis. The
treatment of 19 inhibited the cell cycle progression in HMC-1.2
cells, resulting in a significant increase in the percentage of cells
in the SubG1/G1 phase in a dose dependent manner (Figure
9).
We next analyzed the ability of 19 to induce apoptosis in
HMC-1.2 cells. When HMC-1.2 cells were exposed to various
doses of 19, the DNA fragmentation, one of the hallmarks of
apoptosis, was increased in a dose-dependent manner (Figure
10). A flow cytometric analysis of HMC-1.2 cells, which were
subjected to terminal deoxynucleotidyl transferase-mediated
nick end labeling (TUNEL) staining, revealed that the
percentage of TUNEL-positive cells increased after treatment
with 19, further supporting the conclusion that 19 induced
apoptosis.
Chemical Synthesis. The general synthesis of representa-
tive 3,5-disubstituted 7-azaindole derivatives 48 is shown in
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Figure 9. Effect of 19 on the cell cycle distribution. HMC-1.2 cells were exposed to increasing concentration of 19 and 5 μM imatinib for 24 h.
Compound 19 arrested the cell cycle at G1 phase in a dose-dependent manner.
(18 min), the solvent ratio was maintained for 23 min; the solvent
ratio was changed to MeOH (0.1% TFA) 10% and H2O (0.1% TFA)
90% (25 min). All final compounds were found to have >95% purity.
Commercial grade reagents and solvents were used without further
purification except as indicated below. Dichloromethane was distilled
from calcium hydride. THF was distilled from sodium.
7.13 (dd, J = 8.2, 2.0 Hz, 1H), 7.05 (d, J = 2.0 Hz, 1H), 6.97 (d, J = 8.2
Hz, 1H). 3.95 (s, 3H), 3.92 (s, 3H).
3-(3,4-Dimethoxyphenyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)-1H-pyrrolo[2,3-b]pyridine (47). A solution of 5-bromo-3-
(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (75 mg, 0.23
mmol), bis(pinacolato)diboron (74.6 mg, 0.29 mmol), KOAc (44.4
mg, 0.452 mmol), and PdCl2(dppf)·CH2Cl2 (37 mg, 0.045 mmol) in
anhydrous 1,4-dioxane was heated to 90 °C for 12 h under an
atmosphere of N2. The reaction mixture was cooled to rt 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 (15:1). The filtrate was
concentrated in vacuo then purified by flash column chromatography
(EtOAc/hexanes, gradient 1:3 to 1:1) to give 47 as brown solid (63.9
II. Preparation of Compounds. General Procedure (GP I) for
Suzuki Coupling. 5-Bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo-
[2,3-b]pyridine (46). A solution of 5-bromo-3-iodo-1-(phenylsulfon-
yl)-1H-pyrrolo[2,3-b]pyridine (600 mg, 1.3 mmol), 3,4-dimethox-
yphenylboronic acid (284 mg, 1.56 mmol), Cs2CO3 (1060 mg, 3.25
mmol), and PdCl2(dppf)·CH2Cl2 (212 mg, 0.26 mmol) in 1,4-
dioxane:H2O = 3:1 (7 mL) was heated to 80 °C for 2 h in sealed tube.
The resulting solution was cooled to rt, and 4N KOH solution (4 mL)
and MeOH was added. The reaction mixture was then heated to 60 °C
and stirred for 1 h (N-deprotection of benzenesulfonyl group). The
mixture was diluted with water and neutralized with 6 N HCl solution
in ice bath. 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
(EtOAc/hexanes, gradient 1:2 to 1:1) to give the product as yellow
1
mg, 74%). H NMR (300 MHz, chloroform-d) δ 12.14 (s, 1H), 8.82
(d, J = 1.4 Hz, 1H), 8.68 (d, J = 1.4 Hz, 1H), 7.54 (s, 1H), 7.28 (dd, J
= 8.3, 2.0 Hz, 1H), 7.17 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H),
3.99 (s, 3H), 3.96 (s, 3H), 1.41(s, 12H).
General Procedure (GP II) for Suzuki Coupling. 3-(3-(3,4-
Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline (4). A sol-
ution of 5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
(40 mg, 0.12 mmol), 3-aminophenylboronic acid (23 mg, 0.15 mmol),
Cs2CO3 (78 mg, 0.24 mmol), and PdCl2(dppf)·CH2Cl2 (20 mg, 0.024
1
solid (369 mg, 85%). H NMR (400 MHz, chloroform-d) δ 9.41 (s,
1H), 8.38 (d, J = 2.0 Hz, 1H), 8.28 (d, J = 2.0 Hz, 1H), 7.44 (s, 1H),
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1
solid (11 mg, 27%). H NMR (400 MHz, chloroform-d) δ 10.57 (s,
1H), 8.56 (d, J = 2.0 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 7.48 (s, 1H),
7.25 (t, J = 7.8 Hz, 1H), 7.20 (dd, J = 8.3, 2.0 Hz, 1H), 7.13 (d, J = 2.0
Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.92 (s,
1H), 6.69 (d, J = 7.8 Hz, 1H), 3.93 (s, 3H), 3.92 (s, 3H), 3.00 (br,
2H). 13C NMR (150 MHz, chloroform-d) δ 149.4, 148.6, 147.9, 146.9,
142.5, 140.8, 130.3, 129.9, 127.8, 126.6, 122.2, 119.6, 118. 6, 117.9,
116.7, 114.1, 113. 9, 111.9, 110.9, 56.1. HRMS (EI+) m/z calcd for
C21H19N3O2 [M + H]+, 346.1556; found, 346.1543.
N-(5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
pyridin-3-yl)benzenesulfonamide (1). Benzenesulfonyl chloride (10.7
μL, 0.084 mmol), pyridine (11.3 μL, 0.14 mmol), and dichloro-
methane (1 mL) was added to 5-(3-(3,4-dimethoxyphenyl)-1H-
pyrrolo[2,3-b]pyridin-5-yl)pyridin-3-amine (24.3 mg, 0.07 mmol).
The reaction mixture was stirred at rt for overnight and then diluted
with DCM and brine. Organic phase was extracted with DCM, dried
over MgSO4. The concentrated crude was purified with flash column
chromatography (CH2Cl2/MeOH, gradient 30:1 to 20:1) to give 1
1
(11.1 mg, 33%). H NMR (400 MHz, DMSO-d6) δ 12.05 (s, 1H),
10.83 (s, 1H), 8.67 (d, J = 2.0 Hz, 1H), 8.44 (d, J = 2.0 Hz, 1H), 8.30
(s, 2H), 7.88−7.77 (m, 4H), 7.64−7.54 (m, 3H), 7.29−7.26 (m, 2H),
7.03 (d, J = 8.9 Hz, 1H), 3.85 (s, 3H), 3.79 (s, 3H). MS (EI+) m/z
calcd for C26H22N4O4S [M + H]+, 487.1; found, 487.2.
5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
pyridin-3-amine (3). Compound 3 was prepared (42.6 mg, 40%)
according to GP II (CH2Cl2/MeOH, gradient 20:1 to 15:1) from 5-
bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (101 mg,
0.3 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-
1
3-amine (81 mg, 0.368 mmol) as white solid. H NMR (400 MHz,
DMSO-d6) δ 11.96 (s, 1H), 8.49 (s, 1H), 8.33 (s, 1H), 8.12 (s, 1H),
7.94 (s, 1H), 7.86 (s, 1H), 7.29 (d, J = 8.2 Hz, 1H), 7.23−7.30 (m,
2H), 7.02 (d, J = 8.2 Hz, 1H), 5.42 (s, 2H), 3.84 (s, 3H), 3.78 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 149.2, 148.7, 147.3, 144.9, 141.5,
135.4, 135.1, 134.6, 127.7, 126.3, 125.3, 124.1, 118.7, 117.9, 117.4,
114.8, 112.4, 110.7, 55.6. HRMS (EI+) m/z calcd for C20H18N4O2 [M
+ H]+, 347.1508; found, 347.1508.
Figure 10. Effect of 19 on apoptosis in HMC-1.2 cells. HMC-1.2 cells
were treated with various concentrations of 19 for 48 h, and apoptosis
was determined by flow cytometry using TUNEL staining.
3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
benzonitrile (5). Compound 5 was prepared (31.5 mg, 59%)
according to GP II (EtOAc/hexanes, gradient 1:1) from 5-bromo-3-
(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (50 mg, 0.15
mmol) and (3-cyanophenyl)boronic acid (26.4 mg, 0.18 mmol) as
white solid. 1H NMR (400 MHz, DMSO-d6) δ 11.98 (s, 1H), 8.61 (d,
J = 2.1 Hz, 1H), 8.49 (d, J = 2.1 Hz, 1H), 8.30 (s, 1H), 8.12 (d, J = 7.8
Hz, 1H), 7.86 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.67 (t, J = 7.8 Hz,
1H), 7.35 (dd, J = 8.2, 2.0 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H), 7.03 (d, J
= 8.3 Hz, 1H), 3.85 (s, 3H), 3.78 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 149.2, 148.8, 147.3, 141.9, 140.3, 131.8, 130.6, 130.4,
mmol) in 1,4-dioxane/H2O = 3:1 (1 mL) was charged in capped tube
and was heated to 100 °C for 3 h. The reaction mixture was cooled to
room temperature and concentrated in vacuo. The residue was diluted
with CH2Cl2, and organic phase was extracted with CH2Cl2. The
combined organic layer was dried with MgSO4 and concentrated in
vacuo. The mixture was purified with flash column chromatography
(CH2Cl2/MeOH, gradient 30:1 to 15:1) to give the product as white
Scheme 1. Synthesis of 7-Azaindole Derivatives
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130.1, 127.6, 126.5, 125.9, 124.2, 118.9, 118.8, 117.4, 115.1, 112.4,
112.1, 110.7, 55.6. HRMS (EI+) m/z calcd for C22H17N3O2 [M +
Na]+, 378.1218; found, 378.1217.
(400 MHz, DMSO-d6) δ 12.00 (s, 1H), 8.99 (s, 1H), 8.60 (d, J = 2.2
Hz, 1H), 8.56−8.57 (m, 1H), 8.47 (d, J = 2.2 Hz, 1H), 8.17 (d, J = 8.0
Hz, 1H), 7.87 (s, 1H), 7.48 (dd, J = 8.0, 4.8 Hz, 1H), 7.33 (dd, J = 8.2,
2.1 Hz, 1H), 7.29 (d, J = 2.1 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 3.85 (s,
3H), 3.78 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 149.2, 148.8,
147.9, 147.3, 141.8, 134.7, 134.5, 127.6, 125.8, 125.5, 124.2, 123.9,
118.8, 117.5, 115.0, 112.4, 110.7, 55.6, 55.6. HRMS (EI+) m/z calcd
for C20H17N3O2 [M + H]+, 332.1399; found, 332.1385.
3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-N,N-
dimethylaniline (6). Compound 6 was prepared (13.7 mg, 30%)
according to GP II (EtOAc/hexanes, gradient 1:1) from 5-bromo-3-
(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (40 mg, 0.122
mmol) and (3-(dimethylamino)phenyl) boronic acid (24.2 mg,
0.146 mmol) as white solid. 1H NMR (400 MHz, DMSO-d6) δ
11.85 (s, 1H), 8.51 (d, J = 2.1 Hz, 1H), 8.33 (d, J = 2.1 Hz, 1H), 7.81
(s, 1H), 7.29−7.21 (m, 3H), 7.03 (d, J = 7.5 Hz, 1H), 6.99−6.94 (m,
2H), 6.72 (d, J = 7.5 Hz, 1H), 3.84 (s, 3H), 3.77 (s, 3H) 2.95 (s, 6H).
13C NMR (100 MHz, DMSO-d6) δ 150.9, 149.1, 148.5, 147.2, 142.0,
3-(3,4-Dimethoxyphenyl)-5-o-tolyl-1H-pyrrolo[2,3-b]pyridine
(12). Compound 12 was prepared (13.3 mg, 68%) according to GP II
(CH2Cl2/MeOH, gradient 30:1 to 10:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (18.9 mg, 0.057 mmol)
1
and o-tolylboronic acid (9.3 mg, 0.068 mmol). H NMR (400 MHz,
DMSO-d6) δ 11.90 (s, 1H), 8.22 (d, J = 2.1 Hz, 1H), 8.12 (d, J = 2.1
Hz, 1H), 7.84 (s, 1H), 7.27−7.32 (m, 4H), 7.25−7.22 (m, 2H), 6.99
(d, J = 8.7 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 3H), 2.28 (s, 3H). MS (EI+)
m/z calcd for C22H20N2O2 [M + H]+, 345.2; found, 345.2.
139.9, 129.6, 129.4, 127.9, 125.3, 123.8, 118.5, 117.3, 115.2, 114.7,
112.5, 111.1, 111.1, 110.6, 55.6, 55.5, 40.2. HRMS (EI+) m/z calcd for
C23H23N3O2 [M + H]+, 374.1869; found, 374.1845.
3-(3,4-Dimethoxyphenyl)-5-(m-tolyl)-1H-pyrrolo[2,3-b]pfyridine
(7). Compound 7 was prepared (9.9 mg, 60%) according to GP II
(CH2Cl2/MeOH, gradient 60:1 to 30:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (16 mg, 0.048 mmol)
and m-tolylboronic acid (7.8 mg, 0.58 mmol) as white solid. 1H NMR
(400 MHz, DMSO-d6) δ 11.87 (s, 1H), 8.52 (d, J = 2.1 Hz, 1H), 8.34
(d, J = 2.1 Hz, 1H), 7.81 (s, 1H), 7.54 (s, 1H), 7.51 (d, J = 7.6 Hz,
1H), 7.36 (t, J = 7.6 Hz, 1H), 7.29 (dd, J = 8.2, 2.1 Hz, 1H), 7.25 (d, J
= 2.1 Hz, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 3.84
(s, 3H), 3.77 (s, 3H), 2.38 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
149.2, 148.5, 147.3, 141.8, 139.0, 138.1, 128.8, 128.7, 127.8, 127.7,
127.5, 125.2, 124.1, 123.9, 118.7, 117.4, 114.8, 112.5, 110.7, 55.6, 55.6,
21.1. HRMS (EI+) m/z calcd for C22H20N2O2 [M + Na]+, 367.1422;
found, 367.1423.
2-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
(13). Compound 13 was prepared (14.7 mg, 67%) according to GP II
(CH2Cl2/MeOH, gradient 30:1 to 1:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (21.4 mg, 0.064 mmol)
1
and 2-aminophenylboronic acid (10.5 mg, 0.0768 mmol). H NMR
(400 MHz, chloroform-d) δ 10.21 (s, 1H), 8.44 (d, J = 2.0 Hz, 1H),
8.30 (d, J = 2.0 Hz, 1H), 7.52 (s, 1H), 7.21−7.17 (m, 3H), 7.13 (d, J =
2.0 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 7.7 Hz, 1H), 6.80
(d, J = 7.7 Hz, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.75 (s, 2H). MS (EI+)
m/z calcd for C21H19N3O2 [M + H]+, 346.2; found, 346.2.
4-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
benzonitrile (14). Compound 14 was prepared (15.1 mg, 48%)
according to GP III (EtOAc/hexanes, gradient 1:1 to 2:1) from 3-(3,4-
dimethoxyphenyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrrolo[2,3-b]pyridine (34 mg, 0.0894 mmol) and 4-bromobenzoni-
3-(3,4-Dimethoxyphenyl)-5-(3-fluorophenyl)-1H-pyrrolo[2,3-b]-
pyridine (8). Compound 8 was prepared (18.2 mg, 65%) according to
GP II (EA/HX, gradient 1:3 to 1:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (26.5 mg, 0.08 mmol)
1
trile (19.5 mg, 0.107 mmol). H NMR (400 MHz, chloroform-d) δ
10.61 (s, 1H), 8.60 (d, J = 2.0 Hz, 1H), 8.35 (d, J = 2.0 Hz, 1H), 7.76−
7.71 (m, 4H), 7.54 (s, 1H), 7.20 (dd, J = 8.2, 1.9 Hz, 1H), 7.12 (d, J =
1.9 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H). MS
(EI+) m/z calcd for C22H17N3O2 [M + H]+, 356.2; found, 356.2.
3-(3,4-Dimethoxyphenyl)-5-(4-fluorophenyl)-1H-pyrrolo[2,3-b]-
pyridine (15). Compound 15 was prepared (8.4 mg, 31%) according
to GP II (CH2Cl2/MeOH, gradient 30:1 to 10:1) from 5-bromo-3-
(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (26 mg, 0.078
1
and 3-fluorophenylboronic acid (15.7 mg, 0.1 mmol). H NMR (400
MHz, chloroform-d) δ 10.80 (s, 1H), 8.59 (s, 1H), 8.34 (s, 1H), 7.53
(s, 1H), 7.45−7.39 (m, 2H), 7.31−7.33 (m, 1H), 7.21 (dd, J = 8.2, 2.0
Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.04−7.08 (m, 1H), 6.99 (d, J = 8.2
Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H). MS (EI+) m/z calcd for
C21H17FN2O2 [M + H]+, 349.1; found, 349.2.
3-(3,4-Dimethoxyphenyl)-5-phenyl-1H-pyrrolo[2,3-b]pyridine (9).
Compound 9 was prepared (42.4 mg, 43%) according to GP II
(EtOAc/CH2Cl2, gradient 1:5 to 1:3) from 5-bromo-3-(3,4-dimethox-
yphenyl)-1H-pyrrolo[2,3-b]pyridine (99 mg, 0.3 mmol) and phenyl-
1
mmol) and 4-fluorophenylboronic acid (13.2 mg, 0.094 mmol). H
NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H), 8.52 (d, J = 2.1 Hz,
1H), 8.35 (d, J = 2.1 Hz, 1H), 7.83 (s, 1H), 7.77−7.83 (m, 2H), 7.32−
7.28 (m, 3H), 7.26 (d, J = 2.0 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 3.84
(s, 3H), 3.78 (s, 3H). MS (EI+) m/z calcd for C21H17FN2O2 [M +
H]+ ,349.1; found, 349.1.
1
boronic acid (43.9 mg, 0.36 mmol) as white solid. H NMR (400
MHz, chloroform-d) δ 11.19 (s, 1H), 8.62 (d, J = 2.1 Hz, 1H), 8.37 (d,
J = 2.1 Hz, 1H), 7.67−7.61 (m, 2H), 7.53 (s, 1H), 7.48 (t, J = 7.5 Hz,
2H), 7.37 (t, J = 7.5 Hz, 1H), 7.22 (dd, J = 8.2, 2.0 Hz, 1H), 7.15 (d, J
= 2.0 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 149.27, 148.6, 147.3, 141.8, 139.1,
129.0, 128.6, 127.8, 127.1, 126.9, 125.3, 124.0, 118.7, 117.4, 114.9,
112.5, 110.6, 55.6. HRMS (EI+) m/z calcd for C21H18N2O2 [M +
Na]+, 353.1266; found, 353.1253.
3-(3,4-Dimethoxyphenyl)-5-(pyridin-4-yl)-1H-pyrrolo[2,3-b]-
pyridine (10). Compound 10 was prepared (19.9 mg, 72%) according
to GP II (CH2Cl2/MeOH, gradient 30:1 to 10:1) from 5-bromo-3-
(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (28 mg, 0.084
mmol) and 4-pyridinylboronic acid (12.4 mg, 0.101 mmol) as white
solid. 1H NMR (400 MHz, chloroform-d) δ 10.01 (s, 1H), 8.72−8.65
(m, 3H), 8.40 (d, J = 2.0 Hz, 1H), 7.56−7.58 (m, 2H), 7.52 (s, 1H),
7.20 (dd, J = 8.3, 2.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.00 (d, J = 8.3
Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 150.1, 149.3, 149.2, 147.4, 146.1, 141.8, 127.5, 125.7, 125.4, 124.4,
121.4, 118.8, 117.4, 115.2, 112.5, 110.7, 55.6. HRMS (EI+) m/z calcd
for C20H17N3O2 [M + H]+, 332.1399; found, 332.1372.
5-(4-Chlorophenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]-
pyridine (16). Compound 16 was prepared (20.3 mg, 76%) according
to GP II (EtOAc/hexanes, gradient 1:3 to 1:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (24.3 mg, 0.0735 mmol)
1
and 4-chlorophenylboronic acid (13.8 mg, 0.088 mmol). H NMR
(400 MHz, chloroform-d) δ 9.39 (s, 1H), 8.54 (d, J = 2.1 Hz, 1H),
8.29 (d, J = 2.1 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.47 (s, 1H), 7.43
(d, J = 8.5 Hz, 2H), 7.20 (dd, J = 8.2, 2.0 Hz, 1H), 7.12 (d, J = 2.0 Hz,
1H), 6.98 (d, J = 8.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H). MS (EI+)
m/z calcd for C21H17ClN2O2 [M + H]+, 365.1; found, 365.1.
3-(3,4-Dimethoxyphenyl)-5-p-tolyl-1H-pyrrolo[2,3-b]pyridine
(17). Compound 17 was prepared (34.4 mg, 57%) according to GP II
(CH2Cl2/MeOH, gradient 30:1 to 15:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (58 mg, 0.174 mmol)
and p-tolylboronic acid (28.5 mg, 0.209 mmol). 1H NMR (400 MHz,
chloroform-d) δ 11.34 (s, 1H), 8.60 (d, J = 2.1 Hz, 1H), 8.35 (d, J =
2.1 Hz, 1H), 7.51−7.54 (m, 3H), 7.28 (d, J = 7.7 Hz, 2H), 7.21 (dd, J
= 8.2, 2.0 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H),
3.93(s, 6H), 2.41 (s, 3H). MS (EI+) m/z calcd for C22H20N2O2 [M +
H]+, 345.2; found, 345.2.
3-(3,4-Dimethoxyphenyl)-5-(pyridin-3-yl)-1H-pyrrolo[2,3-b]-
pyridine (11). Compound 11 was prepared (31 mg, 62%) according to
GP II (CH2Cl2/MeOH, gradient 30:1 to 15:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (50 mg, 0.15 mmol) and
3-pyridinylboronic acid (22.1 mg, 0.18 mmol) as white solid. 1H NMR
3-(3,4-Dimethoxyphenyl)-5-(4-methoxyphenyl)-1H-pyrrolo[2,3-
b]pyridine (18). Compound 18 was prepared (22.2 mg, 68%)
according to GP II (EtOAc/hexanes, gradient 1:3 to 1:1) from 5-
6439
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Journal of Medicinal Chemistry
Article
bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (29.9 mg,
0.09 mmol) and 4-methoxyphenylboronic acid (16.4 mg, 0.108
mmol). 1H NMR (400 MHz, chloroform-d) δ 9.94 (s, 1H), 8.55 (d, J
= 2.1 Hz, 1H), 8.30 (d, J = 2.1 Hz, 1H), 7.55 (d, J = 8.7 Hz, 2H), 7.48
(s, 1H), 7.21 (dd, J = 8.2, 2.0 Hz, 1H), 7.15 (d, J = 2.0 Hz, 1H), 7.01
(d, J = 8.7 Hz, 2H), 6.98 (d, J = 8.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s,
3H), 3.85 (s, 3H). MS (EI+) m/z calcd for C22H20N2O3 [M + H]+,
361.2 ; found, 361.2.
2,3′-(1H-Pyrrolo[2,3-b]pyridine-3,5-diyl)dianiline (23). 2-(5-
Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline was prepared (32.5 mg,
50%) according to GP I (EtOAc/hexanes, gradient 1:3 to 1:1) from 5-
bromo-3-iodo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (105.2
mg, 0.2278 mmol) and 2-aminophenylboronic acid (37.4 mg, 0.2734
mmol). 1H NMR (300 MHz, chloroform-d) 11.42 (s, 1H), 8.64 (d, J =
2.0 Hz, 1H), 8.45 (d, J = 2.0 Hz, 1H), 7.80 (s, 1H), 7.56−7.47 (m,
2H), 7.18−7.13 (m, 2H). Compound 23 was prepared (8.9 mg,
26.3%) according to GP II (CH2Cl2/MeOH, gradient 30:1 to 15:1)
from 2-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (32.5 mg,
0.1128 mmol) and 3-aminophenylboronic acid (18.5 mg, 0.135
mmol). 1H NMR (400 MHz, chloroform-d) δ 9.52 (s, 1H), 8.58 (d, J
= 2.1 Hz, 1H), 8.11 (d, J = 2.1 Hz, 1H), 7.47 (s, 1H), 7.29 (d, J = 7.5
Hz, 1H), 7.23−7.14 (m, 2H), 7.00 (d, J = 7.5 Hz, 1H), 6.91 (s, 1H),
6.87−6.77 (m, 2H), 6.66 (d, J = 7.5 Hz, 1H), 3.83 (s, 2H), 3.74 (s,
2H). MS (EI+) m/z calcd for C19H16N4 [M + H]+, 301.1; found,
301.2.
3,5-Bis(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (19).
Compound 19 was prepared (27 mg, 58%) according to GP II
(EtOAc/hexanes, 2:1) from 5-bromo-3-(3,4-dimethoxyphenyl)-1H-
pyrrolo[2,3-b]pyridine (40 mg, 0.12 mmol) and 3,4-dimethoxyphe-
1
nylboronic acid (26.2 mg, 0.14 mmol) as white solid. H NMR (400
MHz, DMSO-d6) δ 11.86 (s, 1H), 8.54 (d, J = 2.1 Hz, 1H), 8.35 (d, J
= 2.1 Hz, 1H), 7.81 (s, 1H), 7.31−7.28 (m, 3H), 7.24 (dd, J = 8.3, 2.1
Hz, 1H), 7.01−7.05 (m, 2H), 3.86 (s, 3H), 3.86 (s, 3H), 3.79 (s, 3H),
3.78 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 149.2, 149.2, 148.3,
148.2, 147.2, 141.8, 131.9, 128.7, 127.9, 125.0, 123.8, 119.1, 118.6,
117.3, 114.7, 112.5, 112.4, 111.0, 110.6, 55.6, 55.5. HRMS (EI+) m/z
calcd for C23H22N2O4 [M + Na]+, 413.1477; found, 413.1482
5-(3,5-Difluorophenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-
b]pyridine (20). Compound 20 was prepared (24.1 mg, 82%)
according to GP II (EtOAc/hexanes, gradient 1:3 to 1:1) from 5-
bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (26.8 mg,
0.0804 mmol) and 3,5-difluorophenylboronic acid (15.2 mg, 0.0965
mmol). 1H NMR (400 MHz, chloroform-d) δ 10.23 (s, 1H), 8.48 (d, J
= 2.0 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 7.51 (s, 1H), 7.45 (td, J = 8.6,
6.4 Hz, 1H), 7.19 (dd, J = 8.1, 2.0 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H),
7.01−6.92 (m, 3H), 3.94 (s, 3H), 3.92 (s, 3H). MS (EI+) m/z calcd
for C21H16F2N2O2 [M + H]+, 367.1; found, 367.1.
5-Bromo-3-(p-tolyl)-1H-pyrrolo[2,3-b]pyridine (24). 5-Bromo-3-
(p-tolyl)-1H-pyrrolo[2,3-b]pyridine was prepared (22.4 mg, 36%)
according to GP I (EtOAc/hexanes, gradient 1:4 to 1:1) from 5-
bromo-3-iodo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (100 mg,
1
0.2168 mmol) and p-tolylboronic acid (37.4 mg, 0.2734 mmol). H
NMR (300 MHz, chloroform-d) 9.34 (s, 1H), 8.33 (s, 1H), 8.27 (s,
1H), 7.44−7.41 (m, 3H), 7.26−7.23 (m, 2H). Compound 24 was
prepared (10.2 mg, 45.2%) according to GP II (CH2Cl2/MeOH,
gradient 20:1 to 15:1) from 5-bromo-3-(p-tolyl)-1H-pyrrolo[2,3-
b]pyridine (22.4 mg, 0.078 mmol) and 3-aminophenylboronic acid
1
(14.6 mg, 0.094 mmol). H NMR (400 MHz, DMSO-d6) δ 11.88 (s,
1H), 8.45 (d, J = 2.1 Hz, 1H), 8.27 (d, J = 2.1 Hz, 1H), 7.82 (s, 1H),
7.63 (d, J = 7.9 Hz, 2H), 7.26 (d, J = 7.9 Hz, 2H), 7.11 (t, J = 7.7 Hz,
1H), 6.89 (s, 1H), 6.85 (d, J = 7.7 Hz, 1H), 6.57−6.54 (m, 1H), 5.15
(s, 2H), 2.33 (s, 3H). MS (EI+) m/z calcd for C20H17N3 [M + H]+,
300.2; found, 300.2.
3-(3-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
aniline (21). 5-Bromo-3-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[2,3-b]-
pyridine was prepared (91 mg, 77%) according to GP I from 5-bromo-
3-iodo-1-(phenyl- sulfonyl)-1H-pyrrolo[2,3-b]pyridine (150 mg, 0.325
mmol) and (3,4,5-trimethoxyphenyl)boronic acid (82.6 mg, 0.390
3-(3-(4-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
(25). 5-Bromo-3-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine was
prepared (118 mg, 60%) according to GP I from 5-bromo-3-iodo-1-
(phenyl-sulfonyl)-1H-pyrrolo[2,3-b]pyridine (300 mg, 0.649 mmol)
1
mmol). H NMR (400 MHz, chloroform-d) δ 9.62 (s, 1H), 8.39 (s,
1H), 8.27 (s, 1H), 7.46 (s, 1H), 6.75 (s, 2H), 3.93 (s, 6H), 3.89 (s,
3H). Compound 21 was prepared (42.3 mg, 47%) according to GP II
(EA/HX 2:1) 5-bromo-3-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[2,3-
b]pyridine (87 mg, 0.24 mmol) and 3-aminophenylboronic acid
(44.5 mg, 0.29 mmol) as white solid. 1H NMR (400 MHz, DMSO-d6)
δ 11.92 (s, 1H), 8.47 (d, J = 2.0 Hz, 1H), 8.29 (d, J = 2.0 Hz, 1H), 7.86
(s, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.95 (s, 2H), 6.91 (s, 1H), 6.87 (d, J
= 7.8 Hz, 1H), 6.56 (d, J = 7.8 Hz, 1H), 5.18 (s, 2H), 3.87 (s, 6H),
3.69 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 153.3, 149.2, 148.4,
141.7, 139.6, 135.9, 130.7, 129.5, 129.4, 124.7, 124.4, 117.3, 114.9,
114.5, 112.7, 112.4, 104.1, 60.15, 56.0. HRMS (EI+) m/z calcd for
C22H21N3O3 [M + Na]+, 398.1481; found, 398.1471.
1
and 4-methoxyphenylboronic acid (118.4 mg, 0.78 mmol). H NMR
(300 MHz, chloroform-d) δ 9.29 (s, 1H), 8.36 (dd, J = 2.1, 0.4 Hz,
1H), 8.28 (d, J = 2.1, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.41 (s, 1H), 7.00
(d, J = 8.8 Hz, 2H), 3.85 (s, 3H). Compound 25 was prepared (83.7
mg, 69%) according to GP II (EtOAc/hexanes, 2:1) from 5-bromo-3-
(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (117 mg, 0.39 mmol)
and 3-aminophenylboronic acid (71.8 mg, 0.46 mmol) as white solid.
1H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 8.45 (d, J = 2.0 Hz,
1H), 8.26 (d, J = 2.0 Hz, 1H), 7.76 (s, 1H), 7.66 (d, J = 8.7 Hz, 2H),
7.12 (t, J = 7.7 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H), 6.90 (s, 1H), 6.85 (d,
J = 7.7 Hz, 1H), 6.57 (d, J = 7.7 Hz, 1H), 5.15 (s, 2H), 3.78 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ 157.5, 149.1, 148.4, 141.6, 139.6,
3-(3-(3-Fluoro-4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
aniline (22). 5-Bromo-3-(3-fluoro-4-methoxyphenyl)-1H-pyrrolo[2,3-
b]pyridine was prepared (85.1 mg, 61%) according to GP I from 5-
bromo-3-iodo-1-(phenyl- sulfonyl)-1H-pyrrolo[2,3-b]pyridine (200
mg, 0.432 mmol) and 3-fluoro-4-methoxyphenylboronic acid (88.1
mg, 0.518 mmol). 1H NMR (400 MHz, chloroform-d) δ 9.79 (s, 1H),
8.38 (d, J = 2.1 Hz, 1H), 8.28 (d, J = 2.1 Hz, 1H), 7.44 (s, 1H), 7.32−
7.28 (m, 1H), 7.28−7.26 (m, 1H), 7.04 (t, J = 8.8 Hz, 1H), 3.93 (s,
3H). Compound 22 was prepared (37 mg, 46.1%) according to GP II
(EA/HX, gradient 2:1) from 5-bromo-3-(3-fluoro-4-methoxyphenyl)-
1H-pyrrolo[2,3-b]pyridine (77.3 mg, 0.24 mmol) and 3-amino-
129.4, 129.4 127.6 127.4, 124.8 123.5 117.3 114.7 114.4 112.7 112.4,
55.1 HRMS (EI+) m/z calcd for C20H17N3O [M + H]+, 316.1450;
found, 316.1424.
3-(3-(3-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
(26). 5-Bromo-3-(3-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine was
prepared (34.1 mg, 52%) according to GP I (EtOAc/hexanes,
gradient 1:5 to 1:1) from 5-bromo-3-iodo-1-(phenylsulfonyl)-1H-
pyrrolo[2,3-b]pyridine (99.7 mg, 0.216 mmol) and 3-methoxypheyl-
boronic acid (39.4 mg, 0.259 mmol). 1H NMR (400 MHz,
chloroform-d) δ 12.18 (s, 1H), 8.39 (d, J = 2.1 Hz, 1H), 8.32 (d, J
= 2.1 Hz, 1H), 7.97 (s, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.28 (d, J = 7.9
Hz, 1H), 7.20 (s, 1H), 6.84 (d, J = 7.9 Hz, 1H). Compound 26 was
prepared (6.5 mg, 36.8%) according to GP II (EtOAc/hexanes,
gradient 1:1 to 2:1) from 5-bromo-3-(3-methoxyphenyl)-1H-pyrrolo-
[2,3-b]pyridine (17 mg, 0.056 mmol) and 3-aminophenylboronic acid
1
phenylboronic acid (44.8 mg, 0.29 mmol) as white solid. H NMR
(400 MHz, DMSO-d6) δ 11.93 (s, 1H), 8.47 (d, J = 2.1 Hz, 1H), 8.28
(d, J = 2.1 Hz, 1H), 7.86 (s, 1H), 7.52−7.60 (m, 2H), 7.23 (t, J = 8.9
Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.91 (s, 1H), 6.87 (d, J = 7.8 Hz,
1H), 6.57 (d, J = 7.8 Hz, 1H), 5.15 (s, 2H), 3.86 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 151.9 (d, J = 243.2 Hz), 149.2, 148.4, 145.2
(d, J = 10.7 Hz), 141.8, 139.6, 129.6, 129.5, 128.3 (d, J = 7.0 Hz),
124.8, 124.3, 122.4 (d, J = 3.3 Hz), 117.1, 114.8, 114.4 (d, J = 2.3 Hz),
113.8 (d, J = 18.4 Hz), 113.3 (d, J = 1.9 Hz), 112.8, 112.5, 56.1.
HRMS (EI+) m/z calcd for C20H16FN3O [M + H]+, 334.1356; found,
334.1326.
1
(9.2 mg, 0.0672 mmol). H NMR (300 MHz, chloroform-d) δ 10.63
(s, 1H), 8.58 (s, 1H), 8.37 (s, 1H), 7.56 (s, 1H), 7.38 (s, 1H), 7.27−
7.20 (m, 3H), 7.03 (d, J = 8.1 Hz, 1H), 6.93 (s, 1H), 6.86 (d, J = 8.4
Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 3.86 (s, 3H), 3.79 (s, 2H). MS (EI
+) m/z calcd for C20H17N3O [M + H]+, 316.1; found, 316.2.
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Journal of Medicinal Chemistry
Article
3-(3-(3,5-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
(27). 5-Bromo-3-(3,5-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
was prepared (12.7 mg, 18%) according to GP I (EtOAc/hexanes,
gradient 1:4 to 1:1) from 5-bromo-3-iodo-1-(phenylsulfonyl)-1H-
pyrrolo[2,3-b]pyridine (100 mg, 0.216 mmol) and 3,5-dimethoxyphe-
nylboronic acid (47.2 mg, 0.259 mmol). 1H NMR (300 MHz,
methanol-d4) δ 8.31 (d, J = 2.1 Hz, 1H), 8.25 (d, J = 2.1 Hz, 1H), 7.61
(s, 1H), 7.16−7.14 (m, 2H), 7.02 (d, J = 8.9 Hz, 1H). Compound 27
was prepared (4.5 mg, 34%) according to GP II (EtOAc/hexanes,
gradient 1:1 to 2:1) from 5-bromo-3-(3,5-dimethoxyphenyl)-1H-
pyrrolo[2,3-b]pyridine (12.7 mg, 0.038 mmol) and 3-aminophenylbor-
(s, 3H) 3.61−3.60 (m, 4H), 4H. 13C NMR (100 MHz, DMSO-d6) δ
168.6, 149.2, 148.6, 147.3, 146.6, 141.6, 140.8, 128.8, 128.4, 127.8,
125.0, 124.0, 118.7, 117.6, 117.4, 114.9, 114.4, 113.8, 112.5, 110.7,
66.2, 55.6, 55.6. HRMS (EI+) m/z calcd for C26H26N4O4 [M + Na]+,
481.1852; found, 481.1866.
3-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrolo-
[2,3-b]pyridine (32). Compound 32 was prepared (37 mg, 59%)
according to GP II (EtOAc/hexanes, 2:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (50 mg, 0.15 mmol) and
(3,4,5-trimethoxyphenyl) boronic acid (38.2 mg, 0.18 mmol) as white
solid. 1H NMR (400 MHz, DMSO-d6) δ 11.89 (s, 1H), 8.57 (d, J = 2.1
Hz, 1H), 8.40 (d, J = 2.1 Hz, 1H), 7.82 (s, 1H), 7.32−7.29 (m, 2H),
7.02 (d, J = 8.1 Hz, 1H), 6.99 (s, 2H), 3.87 (s, 6H), 3.86 (s, 3H), 3.78
(s, 3H), 3.70 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 153.3, 149.2,
148.5, 147.2, 142.0, 136.8, 135.0, 128.9, 127.9, 125.5, 123.9, 118.6,
117.2, 114.8, 112.5, 110.6, 104.7, 60.0, 56.0, 55.6, 55.5. HRMS (EI+)
m/z calcd for C24H24N2O5 [M + Na]+, 443.1583; found, 443.1566.
5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-
methylaniline (33). Compound 33 was prepared (31.8 mg, 57%)
according to GP II (CH2Cl2/MeOH, gradient 60:1 to 30:1) from 5-
bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (50 mg,
0.15 mmol) and 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
1
onic acid (6.2 mg, 0.0456 mmol). H NMR (400 MHz, DMSO-d6) δ
11.97 (s, 1H), 8.46 (d, J = 2.1 Hz, 1H), 8.25 (d, J = 2.1 Hz, 1H), 7.91
(s, 1H), 7.12 (t, J = 7.9 Hz, 1H), 6.89 (s,, 1H), 6.85−6.83 (m, 3H),
6.56 (d, J = 7.9 Hz, 1H), 6.42 (s, 1H), 5.17 (s, 2H), 3.81 (s, 6H). MS
(EI+) m/z calcd for C21H19N3O2 [M + H]+, 346.2; found, 346.2.
3,3′-(1H-Pyrrolo[2,3-b]pyridine-3,5-diyl)dianiline (28). 3-(5-
Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline was prepared (27.8 mg,
45%) according to GP I (EtOAc/hexanes, gradient 1:2 to 1:1) from 5-
bromo-3-iodo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (100 mg,
0.216 mmol) and 3-aminophenylboronic acid (35.6 mg, 0.26 mmol).
1H NMR (300 MHz, methanol-d4) δ 8.35 (d, J = 2.1 Hz, 1H), 8.24 (d,
J = 2.1 Hz 1H), 7.57 (s, 1H), 7.16 (t, J = 7.9 Hz, 1H), 7.00 (s, 1H),
6.94−6.92 (m, 1H), 6.66 (dd, J = 7.9, 2.1 Hz, 1H). Compound 28 was
prepared (6.8 mg, 23.5%) according to GP II (CH2Cl2/MeOH,
gradient 30:1 to 15:1) from 3-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-
yl)aniline (27.8 mg, 0.0965 mmol) and 3-aminophenylboronic acid
(15.9 mg, 0.1158 mmol). 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s,
1H), 8.43 (d, J = 2.1 Hz, 1H), 8.29 (d, J = 2.1 Hz, 1H), 7.71 (s, 1H),
7.12 (t, J = 7.8 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.97 (s, 1H), 6.88 (s,
1H), 6.86−6.83 (m, 2H), 6.56 (d, J = 7.8 Hz, 1H), 6.46 (d, J = 7.6 Hz,
1H), 5.15 (s, 2H), 5.10 (s, 2H). MS (EI+) m/z calcd for C19H16N4 [M
+ H]+, 301.1; found, 301.2.
3-(3-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline (29).
5-Bromo-3-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine was prepared
(19.2 mg, 28%) according to GP I (EtOAc/hexanes, gradient 1:3 to
1:1) from 5-bromo-3-iodo-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]-
pyridine (101.4 mg, 0.2195 mmol) and 4-chlorophenylboronic acid
(41.2 mg, 0.2634 mmol). 1H NMR (300 MHz, DMSO-d6) δ 11.65 (s,
1H), 8.45 (d, J = 2.1 Hz, 1H), 8.33 (d, J = 2.1 Hz, 1H), 8.01 (s, 1H),
7.75 (d, J = 8.5 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H). Compound 29 was
prepared (2.4 mg, 12%) according to GP II (CH2Cl2/MeOH, 30:1)
from 5-bromo-3-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine (19.2
mg, 0.0624 mmol) and 3-aminophenylboronic acid (10.3 mg, 0.0749
mmol). 1H NMR (400 MHz, DMSO-d6) δ 12.02 (s, 1H), 8.46 (d, J =
2.1 Hz, 1H), 8.30 (d, J = 2.1 Hz, 1H), 7.94 (s, 1H), 7.79 (d, J = 8.5 Hz,
2H), 7.48 (d, J = 8.5 Hz, 2H), 7.12 (t, J = 7.8 Hz, 1H), 6.89 (s, 1H),
6.86 (d, J = 7.8 Hz, 1H), 6.57 (d, J = 7.8 Hz, 1H), 5.15 (s, 2H). MS
(EI+) m/z calcd for C19H14ClN3 [M + H]+, 320.1; found, 320.1.
N-(3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
phenyl)acetamide (30). Compound 30 was prepared (41.9 mg, 31%)
according to GP II (CH2Cl2/MeOH, gradient 30:1 to 15:1) from 5-
bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (117 mg,
0.351 mmol) and N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)acetamide (110 mg, 0.421 mmol). 1H NMR (400 MHz,
DMSO-d6) δ 11.91 (s, 1H), 10.02 (s, 1H), 8.50 (d, J = 2.0 Hz, 1H),
8.32 (d, J = 2.0 Hz, 1H), 7.91 (s, 1H), 7.84 (s, 1H), 7.59 (d, J = 7.1
Hz, 1H), 7.43−7.37 (m, 2H), 7.28−7.26 (m, 2H), 7.04 (d, J = 8.2 Hz,
1H), 3.85 (s, 3H), 3.78 (s, 3H), 2.06 (s, 3H). MS (EI+) m/z calcd for
C23H21N3O3 [M + H]+, 388.2; found, 388.2.
1
2-yl)aniline (42 mg, 0.18 mmol) as white solid. H NMR(400 MHz,
DMSO-d6) δ 11.82 (s, 1H), 8.44 (d, J = 2.1 Hz, 1H), 8.24 (d, J = 2.1
Hz, 1H), 7.79 (s, 1H), 7.26−7.23 (m, 2H), 7.04 (d, J = 8.0 Hz, 1H),
7.01 (d, J = 7.6 Hz, 1H), 6.95 (d, J = 1.9 Hz, 1H), 6.82 (dd, J = 7.6, 1.9
Hz, 1H), 4.91 (s, 2H), 3.84 (s, 3H), 3.78 (s, 3H), 2.08 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 149.2, 148.3, 147.2, 147.0, 141.6,
137.2, 130.5, 129.3, 127.9, 124.6, 123.7, 120.0, 118.5, 117.4, 114.7,
114.6, 112.5, 112.4, 110.6, 55.6, 55.6, 17.1. HRMS (EI+) m/z calcd for
C22H21N3O2 [M + H]+, 360.1712; found, 360.1691.
5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-me-
thoxyaniline (34). Compound 34 was prepared (44.2 mg, 39%)
according to GP II (CH2Cl2/MeOH, gradient 30:1) from 5-bromo-3-
(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.3
mmol) and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1
aniline (89.7 mg, 0.36 mmol) as white solid. H NMR (400 MHz,
chloroform-d) δ 10.98 (s, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.27 (d, J =
2.0 Hz, 1H), 7.46 (s, 1H), 7.19 (dd, J = 8.2, 2.0 Hz, 1H), 7.13 (d, J =
2.0 Hz, 1H), 6.97−6.95 (m, 3H), 6.87 (d, J = 8.7 Hz, 1H), 3.92 (s,
3H), 3.92 (s, 3H), 3.89 (s, 3H). 13C NMR (100 MHz, chloroform-d) δ
149.3, 148.3, 147.8, 146.8, 142.0, 136.6, 132.6, 130.1, 127.9, 126.3,
122.3, 119.6, 118.6, 117.4, 116.4, 114.0, 111.8, 110.8, 110.8, 56.0, 56.0,
55.6. HRMS (EI+) m/z calcd for C22H21N3O3 [M + H]+, 376.1661;
found, 376.1635.
5-(Benzo[d][1,3]dioxol-5-yl)-3-(3,4-dimethoxyphenyl)-1H-
pyrrolo[2,3-b]pyridine (35). Compound 35 was prepared (21.6 mg,
66%) according to GP II (EtOAc/hexanes, gradient 1:3 to 1:1) from
5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (29 mg,
0.087 mmol) and 3,4-(methylenedioxy)-phenyl boronic acid (17.3 mg,
1
0.1044 mmol). H NMR (400 MHz, chloroform-d) δ 10.49 (s, 1H),
8.53 (d, J = 2.0 Hz, 1H), 8.27 (d, J = 2.0 Hz, 1H), 7.50 (s, 1H), 7.20
(dd, J = 8.2, 2.0 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H), 7.10 (d, J = 1.8 Hz,
1H), 7.07 (dd, J = 7.9, 1.8 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 6.91 (d, J
= 7.9 Hz, 1H), 6.00 (s, 2H), 3.94 (s, 3H), 3.93 (s, 3H). MS (EI+) m/z
calcd for C22H18N2O4 [M + H]+, 375.1; found, 375.2.
5-(2,4-Dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrrolo-
[2,3-b]pyridine (36). Compound 36 was prepared (86.2 mg, 74%)
according to GP II (EtOAc/hexanes, gradient 1:3 to 1:1) from 5-
bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (100 mg,
0.3 mmol) and 2,4-dimethoxyphenyl boronic acid (65.5 mg, 0.36
(2-Amino-4-(3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-
5-yl)phenyl)(morpholino)-methanone (31). Compound 31 was
prepared (52.4 mg, 43%) according to GP II (CH2Cl2/MeOH,
gradient 20:1 to 15:1) from 5-bromo-3-(3,4-dimethoxyphenyl)-1H-
pyrrolo[2,3-b]pyridine (90 mg, 0.267 mmol) and (2-amino-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)(morpholino)methanone
1
mmol). H NMR (300 MHz, chloroform-d) δ 9.94 (s, 1H), 8.48 (s,
1H), 8.28 (s, 1H), 7.46 (s, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.19 (dd, J =
8.2, 2.0 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 6.95 (d, J = 8.2 Hz, 1H),
6.61−6.59 (m, 2H), 3.93 (s, 3H), 3.91 (s, 3H), 3.86 (s, 3H), 3.80 (s,
3H). MS (EI+) m/z calcd for C23H22N2O4 [M + H]+, 391.2; found,
391.2.
1
(107 mg, 0.321 mmol) as as pale-yellow solid. H NMR (400 MHz,
DMSO-d6) δ 11.90 (s, 1H), 8.51 (s, 1H), 8.33 (s, 1H), 7.82 (s, 1H),
7.28−7.25 (m, 2H), 7.12 (d, J = 7.8 Hz, 1H), 7.09 (s, 1H), 7.03 (d, J =
8.1 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 5.34 (s, 2H), 3.84 (s, 3H), 3.77
4-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
(37). Compound 37 was prepared (29.3 mg, 57%) according to GP II
(EtOAc/hexanes, gradient 1:1 to 3:1) from 5-bromo-3-(3,4-dimethox-
6441
dx.doi.org/10.1021/jm500413g | J. Med. Chem. 2014, 57, 6428−6443

Journal of Medicinal Chemistry
Article
yphenyl)-1H-pyrrolo[2,3-b]pyridine (50 mg, 0.15 mmol) and 4-
ASSOCIATED CONTENT
* Supporting Information
Spectra, rescoring of the generated 7-azaindole derivatives,
kinase selectivity profiling, and physicochemical properties
predicted by Accelrys DB. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (39.5 mg, 0.18
S
1
mmol) as yellow solid. H NMR (400 MHz, DMSO-d6) δ 11.76 (s,
1H), 8.44 (d, J = 2.1 Hz, 1H), 8.23 (d, J = 2.1 Hz, 1H), 7.77 (s, 1H),
7.41 (d, J = 8.5 Hz, 2H), 7.28 (dd, J = 8.2, 2.0 Hz, 1H), 7.25 (d, J = 2.0
Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 6.68 (d, J = 8.5 Hz, 2H), 5.16 (s,
2H), 3.85 (s, 3H), 3.78 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
149.2, 147.9, 147.9, 147.2, 141.3, 129.4, 128.0, 127.5, 126.4, 123.8,
123.5, 118.6, 117.4, 114.5, 114.4, 112.5, 110.6, 55.6, 55.6. HRMS (EI
+) m/z calcd for C21H19N3O2 [M + H]+, 346.1556; found, 346.1534.
5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
pyrimidin-2-amine (38). Compound 38 was prepared (30.2 mg, 29%)
according to GP II (EtOAc/hexanes, gradient 1:3 to 1:1) from 5-
bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (101.6
mg, 0.305 mmol) and (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
pyrimidin-2-amine (74.2 mg, 0.335 mmol). 1H NMR (400 MHz,
DMSO-d6) δ 11.90 (s, 1H), 8.63 (s, 2H), 8.46 (d, J = 2.0 Hz, 1H),
8.33 (d, J = 2.0 Hz, 1H), 7.82 (s, 1H), 7.31 (dd, J = 8.2, 2.1 Hz, 1H),
7.26 (d, J = 2.1 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.71 (s, 2H), 3.84
(s, 3H), 3.77 (s, 3H). MS (EI+) m/z calcd for C19H17N5O2 [M + H]+,
348.1; found, 348.2.
AUTHOR INFORMATION
Corresponding Authors
*For S.H.: phone, (+82) 42-350-2811; fax, (+82) 42-350-2810;
E-mail, hongorg@kaist.ac.kr.
*For S.-S.-H.: phone, (+82) 32-890-3683; fax, (+82) 32-890-
2462; E-mail, hongs@inha.ac.kr.
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-
0020322, Medical Research Center (2014009392)) and the
Institute for Basic Science (IBS-R010-G1).
3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-5-me-
thoxyaniline (39). Compound 39 was prepared (40.8 mg, 36%)
according to GP II (EtOAc/hexanes, 3:1) from 5-bromo-3-(3,4-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (100 mg, 0.3 mmol) and
3-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (89.7
ABBREVIATIONS USED
■
SCF, stem cell factor; c-Kit, receptor tyrosine kinase for the
stem cell factor; PI3K, phophatidylinositol-3-kinase; Akt,
known as protein kinase B (PKB); MAP kinase, mitogen-
activated protein kinase; JAK, Janus-activated kinase; STAT,
signal transducer and activator of transcription; Src, a
nonreceptor protein tyrosine kinase protein that in humans is
encoded by the SRC gene; SFK, Src family kinase; GISTs,
gastrointestinal stromal tumors; AML, acute myeloid leukemia;
SM, systemic mastocytosis; WT, wild-type; ATP, adenosine-5′-
triphosphate; A-loop, activation loop; SAR, structure−activity
relationship; DFG, Asp-Phe-Gly; ADP, adenosine diphosphate;
PDB, Protein Data Bank; POC, percent of control; IC50, half-
maximal inhibitory concentration; Erk, extracellular signal-
regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl
transferase-mediated nick end labeling; tPSA, topological polar
suface area
1
mg, 0.36 mmol) as white solid. H NMR(400 MHz, DMSO-d6) δ
11.86 (s, 1H), 8.47 (d, J = 2.1 Hz, 1H), 8.27 (d, J = 2.1 Hz, 1H), 7.81
(s, 1H), 7.28−7.26 (m, 2H), 7.04 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H),
6.44 (s, 1H), 6.16 (s, 1H), 5.19 (s, 2H), 3.85 (s, 3H), 3.78 (s, 3H),
3.73 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 160.8, 150.3, 149.2,
148.5, 147.3, 141.7, 140.6, 129.4, 127.8, 124.9, 123.8, 118.6, 117.3,
114.7, 112.5, 110.7, 105.6, 100.7, 98.2, 55.6, 55.6, 54.7. HRMS (EI+)
m/z calcd for C22H21N3O3 [M + H]+, 376.1661; found, 376.1643.
General Procedure (GP III) for Suzuki Coupling. 5-(3-(3,4-
Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-2-(2-
morpholinoethoxy)aniline (40). A solution of 47 (35 mg, 0.092
mmol), 5-bromo-2-(2-morpholinoethoxy)aniline (33 mg, 0.11 mmol),
Cs2CO3 (60 mg, 0.184 mmol), and PdCl2(dppf)·CH2Cl2 (18.8 mg,
0.023 mmol) in 1,4-dioxane/H2O = 3:1 (1.5 mL) was charged in
capped tube and was heated to 100 °C for 3 h. The reaction mixture
was cooled to room temperature and concentrated in vacuo. The
residue was diluted with CH2Cl2 and organic phase was extracted with
CH2Cl2. The combined organic layer was dried with MgSO4 and
concentrated in vacuo. The mixture was purified with flash column
chromatography (CH2Cl2/MeOH, gradient 30:1 to 15:1) to give the
product, compound 40 as white solid (12.9 mg, 30%). 1H NMR (400
MHz, chloroform-d) δ 9.48 (s, 1H), 8.52 (s, 1H), 8.27 (s, 1H), 7.44 (s,
1H), 7.20 (dd, J = 8.2, 2.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 6.98−
6.88 (m, 4H), 4.17 (t, J = 5.6 Hz, 2H), 3.94 (s, 3H), 3.92(s, 3H), 3.73
(t, J = 4.6 Hz, 4H), 2.82 (t, J = 5.6 Hz, 2H), 2.59 (t, J = 4.6 Hz, 4H).
13C NMR (150 MHz, chloroform-d) δ 149.4, 148.2, 148.0, 145.9,
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6.98 (d, J = 8.3 Hz, 1H), 6.92 (dd, J = 8.3, 2.5 Hz, 1H), 3.94 (s, 3H),
3.93 (s, 3H), 3.87 (s, 3H). MS (EI+) m/z calcd for C22H20N2O3 [M +
H]+, 361.2; found, 361.2.
6442
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Journal of Medicinal Chemistry
Article
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