Structure-based de novo design and identification of D816V mutant-selective c-KIT inhibitors

Article abstract of DOI:10.1039/c4ob00053f

To identify potent and selective inhibitors of D816V, the most common gain-of-function c-KIT mutant, we carried out structure-based de novo design using 7-azaindole as the core and the scoring function improved by implementing an accurate solvation free energy term. This approach led to the identification of new c-KIT inhibitors specific for the D816V mutant. The 3-(3,4- dimethoxyphenyl)-7-azaindole scaffold was optimized and represents a lead structure for the design of the potent and specific inhibitors of the D816V mutant. The results of molecular dynamics simulations indicate that hydrogen bonding interactions between the 7-azadindole moiety and the backbone groups of Cys673 are the most significant determinant for the potency and selectivity of c-KIT inhibitors. This journal is

Full text of DOI:10.1039/c4ob00053f

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Structure-based de novo design and identification
of D816V mutant-selective c-KIT inhibitors†
Cite this: DOI: 10.1039/c4ob00053f
Hwangseo Park,*a Soyoung Lee,b Suhyun Leeb and Sungwoo Hong*b
To identify potent and selective inhibitors of D816V, the most common gain-of-function c-KIT mutant,
we carried out structure-based de novo design using 7-azaindole as the core and the scoring function
improved by implementing an accurate solvation free energy term. This approach led to the identification
of new c-KIT inhibitors specific for the D816V mutant. The 3-(3,4-dimethoxyphenyl)-7-azaindole scaffold
was optimized and represents a lead structure for the design of the potent and specific inhibitors of the
D816V mutant. The results of molecular dynamics simulations indicate that hydrogen bonding inter-
actions between the 7-azadindole moiety and the backbone groups of Cys673 are the most significant
determinant for the potency and selectivity of c-KIT inhibitors.
Received 8th January 2014,
Accepted 30th January 2014
DOI: 10.1039/c4ob00053f
www.rsc.org/obc
respect to this unusual activation, recent molecular dynamics
(MD) simulation studies have shown that the activating D816V
Introduction
The stem cell factor receptor (c-KIT) is a receptor tyrosine mutation promotes spontaneous detachment of the JMR from
kinase that is activated through dimerization upon binding the kinase domain, which is the triggering first step of the
the stem cell factor (SCF). Activated c-KIT phosphorylates intra- inactive-to-active state transition of c-KIT.7 Imatinib is effective
cellular substrates and thereby switches on downstream cell against wild-type c-KIT by stabilizing the inactive conformation
signaling pathways essential for cell proliferation, differen- of the kinase domain, but introduction of the D816V point
tiation, and survival.1 Deregulated c-KIT kinase activity is mutation disrupts the inactive conformation. Because the
responsible for the pathogenesis of various human cancers, active conformation of the c-KIT kinase domain is strongly
including acute myeloid leukemia, small cell lung carcinoma, favored in the D816V mutant through ligand independent con-
malignant melanomas, colorectal cancer, and gastrointestinal stitutive autophosphorylation, the D816V mutant is resistant
stromal tumors.2
to imatinib which targets the inactive conformation of c-KIT.8
Most cancers caused by c-KIT abnormalities stem from a The emergence of c-KIT mutants that are resistant to first-line
subset of mutations that make c-KIT kinase constitutively drugs has complicated clinical treatment, highlighting the
active.3 The activation loop (A-loop) of the wild-type c-KIT importance of identifying new c-KIT inhibitors with high
exists in dynamic equilibrium between the active and inactive potency against the D816V mutant for use as second-line
conformations. Mutations that induce tumorigenic effects treatments.
have been identified in a membrane-proximal immunoglobin-
Although the importance of constitutively active c-KIT
like domain, the auto-inhibitory juxtamembrane region (JMR), mutants in the pathogenesis of human cancers is well appreci-
and the protein tyrosine kinase domain.4 The mutation of ated, the discovery of inhibitors specific for such gain-of-func-
Asp816 in the A-loop to Val is the most frequently observed c- tion mutants lags behind biochemical and pharmaceutical
KIT mutation in human cancers.5 This D816V mutant of c-KIT studies. A few compounds, such as AP23464 and AP23848,
is capable of inducing downstream oncogenic signaling with a modest degree of selectivity (∼10-fold) toward D816V
regardless of ligand binding in the extracellular region.6 With relative to wild-type c-KIT have been described.9 The basis of
selectivity in this case could be attributed to the decrease in
binding affinity for the inactive conformation of wild-type c-
KIT. Because the normal activity of wild-type c-KIT is necessary
aDepartment of Bioscience and Biotechnology, Sejong University, Seoul, 143-747,
Korea. E-mail: hspark@sejong.ac.kr; Fax: (+82) 2-3408-3766; Tel: (+82) 2-3408-2811
bDepartment of Chemistry, Korea Advanced Institute of Science and Technology
for the survival, differentiation, and function of mast cells, the
selective inhibition of c-KIT gain-of-function mutants with
small-molecule inhibitors can be a novel strategy for the
development of anticancer agents that do not disrupt normal
SCF/KIT signaling, thereby minimizing the risk of potential
toxicity.
(KAIST), Daejeon, 305-701, Korea. E-mail: hongorg@kaist.ac.kr;
Fax: (+82) 42-350-2810; Tel: (+82) 42-350-2811
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ob00053f
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In this study, we seek to identify potent and selective inhibi-
tors for the most abundant gain-of-function c-KIT mutant
(D816V) based on the structure-based de novo design, chemical
synthesis, and biochemical evaluation of various derivatives of
7-azaindole. Because this chemical scaffold binds tightly at the
hinge region (Cys673), it is predicted to serve as a good start-
ing point from which potent and selective inhibitors can be
derived.10 To improve the correlation between computational
predictions and experimentally obtained biological data, we
used an accurate solvation model for calculating the binding
free energies between c-KIT and putative ligands (see the ESI†
for more details).11 By applying MD simulations of wild-type
c-KIT and the D816V mutant in complex with the newly identi-
fied inhibitors, we also address the structural and dynamic fea-
tures for the design of potent and selective inhibitors of the
D816V mutant.
Fig. 1 Docking poses of 7-azaindole in the ATP-binding sites of (a) the
wild-type c-KIT in the inactive conformation and (b) the active form of
the D816V mutant. The X-ray structure of the wild type (a) and the homo-
logy-modeled structure of the D816V mutant (b) were used in the
calculations. Each dotted line indicates a hydrogen bond.
Results and discussion
Because no X-ray crystal structure of the D816V mutant was
available, its atomic coordinates were obtained by homology
modeling using the structure of wild-type c-KIT in the active
conformation as the template (PDB entry: 1PKG).12 In this
structure, the JMR and A-loop were in open positions to allow
free access of bulk solvent to the active site. Homology model-
ing of the D816V mutant of c-KIT constructed by the MODEL-
LER program13 was useful because the coordinates of missing
residues (residues 694–762) in the original X-ray structure of
the template could be built in the target from a randomized
distorted structure that resides approximately between the two
anchoring regions. The calculated structure of the D816V
mutant appeared to be quite similar to that of wild-type in the
active conformation.14 As the receptor model for wild-type
c-KIT to be used in structure-based design, we utilized the X-ray
crystal structure of c-KIT in the autoinhibited conformation
(PDB entry: 1T45).15 The JMR segment was entered into the
active site in this inactive conformation, which led to the
obstruction of the active site by the A-loop.16 Using these two
receptor models—the D816V mutant and the autoinhibited
wild-type c-KIT—we perform de novo design of 7-azaindole
derivatives to identify potent and selective inhibitors of the
gain-of-function mutant.17
Fig. 2 Representative MD trajectory snapshots of 5 in the ATP-binding
sites of (a) wild-type and (b) D816V mutant of c-KIT. Carbon atoms of
c-KIT and 5 are indicated in cyan and green, respectively. Dotted lines
indicate a hydrogen bond.
elements between the wild-type and the mutant c-KIT. For
example, the glycine-rich phosphate-binding loop (P-loop)
stays quite distant from 7-azaindole and points to the solvent
area in wild-type c-KIT (Fig. 2a), whereas it forms a binding
pocket in the D816V mutant (Fig. 2b). In the calculated
binding modes shown in Fig. 2, we also note that the 6-mem-
bered ring of 7-azaindole stays closer to the P-loop than the
5-membered ring. Therefore, the introduction of a substituent at
the pyridine ring of 7-azaindole seems to increase the potency
for the D816V mutant only without significantly changing the
inhibitory activity against wild-type c-KIT. Actually, we have
been able to identify selective and potent inhibitors of the
D816V mutant via de novo design of 7-azaindole derivatives
To obtain the starting structure for de novo design, we
carried out docking simulations of 7-azaindole in the ATP-
binding site of c-KIT. The binding modes of 7-azaindole for
wild-type c-KIT and the D816V mutant were obtained with the
modified scoring function and compared in Fig. 1. In both
structures, 7-azaindole appears to be stabilized through biden-
tate hydrogen bonds with the backbone amide group of
Cys673 in the hinge region and hydrophobic interactions with
the side chain of Tyr672 in the ATP-binding site. Despite the
similarity in the interactions of 7-azaindole in the wild-type
c-KIT and the D816V mutant, 7-azaindole derivatives with large
substituents are expected to have different binding modes
owing to the differences in the configuration of key structural
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the modified scoring function, which takes into account the
effects of ligand solvation on protein–ligand association. Of
the 7-azaindole derivatives tested, 12 compounds are found to
have submicromolar inhibitory activity for the D816V mutant
and more than 100-fold greater potency relative to the wild-
type c-KIT. Notably, all these highly potent and selective
D816V inhibitors (5–16) contain
a 3,4-dimethoxybenzene
moiety in the 3-(Y) position of the 7-azaindole core. A compari-
son of the IC50 values of 14 with those of 19, 20, 22, and 23
clearly reveals that substitutions of other chemical groups for
the 3,4-dimethoxybenzene moiety in the Y position lead to a
large decrease in the inhibitory activity against the D816V
mutant and a relatively insignificant change in IC50 values for
wild-type c-KIT. The replacement of 3,4-dimethoxybenzene at
the Y-position in 14 with pyridine in 25 also leads to a
decrease in selectivity. Furthermore, the introduction of the
3,4-dimethoxybenzene moiety at the Y-position allows speci-
ficity for inhibition of the D816V mutant to be retained for
various chemical groups in the 5-(X) position of the 7-azain-
dole, as evidenced by the large differences between IC50 values
of 5–18 for the wild-type c-KIT and the D816V mutant. This
indicates that 3-(3,4-dimethoxyphenyl)-7-azaindole represents
Scheme 1 Synthetic route of azaindole derivatives.Reagents and con-
ditions: (a) aryl boronic acid, Pd(dppf)Cl2·CH2Cl2, Cs2CO3, 1,4-dioxane–
H2O = 3 : 1 , 80 °C, 2 h, then 4 N KOH, MeOH, 60 °C, 1 h; (b) aryl boronic
acid, Pd(dppf)Cl2·CH2Cl2, Cs2CO3, 1,4-dioxane–H2O = 3 : 1 , 100 °C, 4 h;
(c) 3,4-dimethoxyphenylboronic acid, Pd(dppf)Cl2·CH2Cl2, Cs2CO3, 1,4-
dioxane–H2O = 3 : 1, 80 °C, 2 h, then 4 N KOH, 60 °C, 1 h; (d) bis(pinico-
lato)diboron, Pd(dppf)Cl2·CH2Cl2, KOAc, 1,4-dioxane, 90 °C, 12 h; (e)
aryl bromide, Pd(dppf)Cl2·CH2Cl2, Cs2CO3, 1,4-dioxane–H2O = 3 : 1,
100 °C, 4 h.
based on the differences in calculated structures of c-KIT-
azaindole and D816V-azaindole complexes.
Next, structure-based de novo design of new c-KIT inhibitors a good starting point for the design of the specific inhibitors
specific for the D816V mutant was conducted, and a variety of
drug-like 7-azaindole derivatives were generated with the Lig-
of the D816V mutant.
Among the variety of substituents at the X position, a
Builder program (Fig. S1 in the ESI† for more details).18 The (2-aminophenyl)-morpholin-4-yl-methanone group in 5 is found
generated 7-azaindole derivatives were then scored according
to the calculated binding free energy with respect to the D816V
to be the most efficient in specifically inhibiting the D816V
mutant, yielding more than 1000-fold higher potency for the
mutant. The synthetic approach to the 7-azaindole analogs is mutant than for the wild-type c-KIT. The highly selective inhi-
shown in Scheme 1.19 First, Pd-catalyzed coupling was con-
bition of the D816V mutant by 5, 7, and 8 indicates that the
ducted at 80 °C with the C5 (hetero) aryl partners for
functionalization of the C3 position. The benzenesulfonyl chemical group in the X position can bind tightly in the ATP-
derivatives of 3-(3,4-dimethoxyphenyl)-7-azaindole with a large
group was removed under the basic reaction conditions to
provide intermediate 2. An assortment of C5 functional groups
binding site of the D816V mutant and show compromised
ability to be fully stabilized in the ATP-binding site of the inac-
was then explored by subjecting compound 2 to another Pd- tive form of wild-type c-KIT. The large substituents at the X
catalyzed Suzuki coupling with the appropriate boronic acids
or boronic esters at 100 °C. The enhanced potency produced
position seem to be stabilized in the internal region of the
ATP-binding site because the 6-membered ring of 7-azaindole
by introduction of the 3,4-dimethoxyphenyl group at the C3 points to the center of the N-terminal domain in docking
position of 7-azaindole suggested a broader exploration of
derivatives that incorporated this key functional group
simulations (Fig. 1). The relatively low potency for wild-type
c-KIT of 7-azaindole inhibitors with a large substituent is actu-
(Table 1). In this regard the synthesis of 3-(3,4-dimethoxyphe- ally not surprising because the volume of the ATP-binding
nyl)-7-azaindole derivatives was facilitated by converting 5-bromo-
pocket decreases in the inactive conformation due to the
azaindole into the corresponding boronic esters 3 via direct
approach of JMR residues (residues 547–581) to the gatekeeper
borylation reaction. Various (hetero)aryl groups were then site.
installed at the 5-position using Suzuki coupling with the
The preference for a large chemical group at the X position
for selective inhibition of the D816V mutant is further illus-
appropriate (hetero)aryl boronic acid. In the final step, IC50
(50% inhibitory concentration) values for the synthesized trated by the IC50 values of 9–26 for wild-type c-KIT and the
derivatives were then determined against both the wild-type
c-KIT and D816V mutant.20
D816V mutant. For example, inhibitors with aromatic rings
containing two or three substituents (9–12) exhibit greater
Table 1 lists the structures and IC50 values of 22 representa- selectivity than those with one or no substituent (13–26),
tive inhibitors. We note that most 7-azaindole derivatives
exhibit a higher potency against the D816V mutant than the
except for 17. However, the inhibitors with the same size may
also exhibit a different pattern of selectivity. For example,
wild-type c-KIT. Because all 7-azaindole derivatives were changing the NH2 group from the meta-position in 14 to the
selected to maximize the affinity for the D816V mutant and
minimize the affinity for the wild-type c-KIT, the potency and
para-position in 26 leads to a substantial decrease in the speci-
ficity for inhibition of the D816V mutant. Although the inhibi-
selectivity of the designed inhibitors exemplify the accuracy of tory activities of 14 and 26 with respect to the D816V mutant
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Table 1 Structures and inhibitory activities of representative 7-azaindole derivatives
IC50 (nM) for KIT
IC50 (nM) for KIT
Xa
Ya
WT
D816V
8.5
Xa
Ya
WT
D816V
261.4
5
9140
16
>10 000
6
7
>10 000
>10 000
29.2
15.8
17
18
142.0
3327
2.1
60.1
8
8540
13.9
24.6
25.6
26.2
19
20
21
22
1743
1822
2154
1208
47.8
104.1
134.6
94.7
9
>10 000
>10 000
>10 000
10
11
12
2754
9.8
23
4851
398.1
13
14
15 170
1064
63.8
6.3
24
25
311.6
95.1
32.1
13.1
15
1810
10.8
26
56.8
9.1
a Asterisks indicate the atom attached to the position of substitution.
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are similar, their IC50 values with respect to the wild-type c-KIT explain the greater inhibitory activity of 5 against the D816V
decrease significantly (14, IC50 = 1064 nM; 26, IC50 = 56.8 nM). mutant than the wild-type c-KIT.
This indicates that the large decrease in specificity for inhi-
Interestingly, compound 14 remains as a potent and
bition of the D816V mutant stems from an increase in the specific inhibitor of the D816V mutant despite the removal of
inhibitory activity of 26 against the wild-type c-KIT. Therefore, the morpholine-4-carbonyl group in 5. The binding modes of
the shift of the –NH2 group from the meta- to para-position is 14 with the wild-type c-KIT and the D816V mutant are similar
expected to cause the establishment of strong c-KIT-inhibitor to those of 5 in that the central 7-azaindole moiety forms the
interactions that enable the inhibitor to bind more tightly in bidentate hydrogen bonds with backbone groups of Cys673,
the ATP-binding site of the inactive conformation of wild-type with the aromatic rings being stabilized through the hydro-
c-KIT.
phobic interactions with the side chains of Leu595, Val603,
The representative MD trajectory snapshots for 5 bound in Ala621, Tyr672, and Leu799 (Fig. 3). The anilinic nitrogen also
the ATP-binding sites of the wild-type c-KIT and the D816V forms a hydrogen bond with the backbone aminocarbonyl
mutant are compared in Fig. 2. In both c-KIT-5 and D816V-5 oxygen of Asp810 in the c-KIT-14 complex and with that of
complexes, the pyridinyl nitrogen of the 7-azaindole ring Ala621 in the D816V-14 complex. As in the c-KIT-5 complex,
receives a hydrogen bond from the backbone amidic nitrogen the terminal 3,4-dimethoxy benzene group appears to be
and the neighboring –NH group donates a hydrogen bond to exposed to bulk solvent in the c-KIT-14 complex. On the other
the aminocarbonyl oxygen of Cys673. Another common struc- hand, it is directed toward the P-loop, where it forms a water-
tural feature of c-KIT-5 and D816V-5 complexes is that the car- mediated hydrogen bond with the backbone aminocarbonyl
bonyl group flanking the morpholine and phenyl rings of 5 oxygen of Ala597. Thus, the number of hydrogen bonds
forms a hydrogen bond with the side-chain ammonium ion of increases from three to four in going from the wild-type recep-
Lys623. However, the third hydrogen bond in the ATP-binding tor to the D816V mutant. Such strengthening of hydrogen
site appears to be established in a different fashion in c-KIT-5 bonding interactions may help to explain the greater inhibitory
and D816V-5 complexes. In the c-KIT-5 complex, the anilinic activity of 14 for the D816V mutant than for the wild-type
nitrogen of 5 plays the role of a hydrogen bond donor with c-KIT. Another common feature in the binding modes of 5 and
respect to the backbone aminocarbonyl oxygen of Asp810, 14 is that their terminal 3,4-dimethoxybenzene moiety is
which is a component of the highly conserved Asp-Phe-Gly stabilized by protein groups at the entrance of the ATP-binding
(D810-F811-G812) motif in the A-loop. In the D816V-5 site of the D816V mutant, whereas it points to bulk solvent in
complex, on the other hand, this anilinic nitrogen establishes
a solvent-mediated hydrogen bond with the side-chain carboxy-
late group of Asp677 at the entrance of the ATP-binding site.
Judging from the proximity to the 7-azaindole ring, this hydro-
gen bond is expected to play a role in orienting the 7-azaindole
moiety in a position to form the bidentate hydrogen bonds
with Cys673 in the ATP-binding site of the D816V mutant. In
both c-KIT-5 and D816V-5 complexes, three aromatic rings of 5
appear to be stabilized through the hydrophobic interactions
with the side chains of nonpolar residues including Leu595,
Val603, Ala621, Tyr672, and Leu799. In contrast, the terminal
morpholine ring exhibits different binding modes in c-KIT-5
and D816V-5 complexes. In the D816V mutant, it is stabilized
through the interactions with backbone groups of the P-loop
(residues 596–602, Fig. 2b). This binding mode cannot be
formed in the c-KIT-5 complex because the P-loop is too
distant from the ATP-binding site. Instead, the morpholine
group points toward a small hydrophobic pocket formed by
the side chains of Trp557, Leu644, Val654, and Thr670 in the
c-KIT-5 complex (Fig. 2a). The respective presence and absence
of the interactions with the P-loop in D816V-5 and c-KIT-5
complexes underscore the importance of the P-loop as a key
structural determinant of the binding specificity of c-KIT
inhibitors. The binding mode of the terminal 3,4-dimethoxy
benzene moiety also appears to be different in c-KIT-5 and
D816V-5 complexes. It is stabilized by the interactions with
Asn680 and Arg684 in the D816V-5 complex (Fig. 2b); in con-
Fig. 3 Representative MD trajectory snapshots of 14 in the ATP-binding
sites of (a) wild-type and (b) D816V mutant of c-KIT. Carbon atoms of
trast, it is exposed to bulk solvent in the c-KIT-5 complex
c-KIT and 14 are indicated in cyan and green, respectively. Each dotted
(Fig. 2a). This difference in binding modes can be invoked to line indicates a hydrogen bond.
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the ATP-binding site of the wild-type c-KIT. This difference in the bidentate hydrogen bonds between Cys673 and the
binding modes with respect to the wild-type c-KIT and the 7-azaindole ring are found to be dynamically unstable in c-KIT-5
D816V mutant confirms the effectiveness of the 3,4-dimethoxy- and c-KIT-14 complexes as shown in Fig. 4a and 4b. Although
benzene moiety in conferring high selectivity for the D816V the N⋯H–N hydrogen bond is maintained for more than
mutant.
86.3% of simulation time, the residence time for the N–H⋯O
Because the bidentate hydrogen bonding established hydrogen bond falls to 72.7% in c-KIT-5 and c-KIT-14 com-
between the 7-azaindole moiety and the backbone groups of plexes. These relative instabilities of the bidentate hydrogen
Cys673 is the most important interaction in the wild-type c-KIT bonds indicate that 5 and 14 would be bound weakly in the
and the D816V mutant, a comparison of the dynamic stabi- ATP-binding site of the wild-type c-KIT in the inactive confor-
lities of the two hydrogen bonds might shed light on the mation, which is consistent with the even weaker inhibitory
differences in inhibitory activities. A common feature of activities of 5 and 14 against the wild-type c-KIT than against
D816V-5, D816V-14, and D816V-26 complexes is that both the D816V mutant. Such weak binding of 5 and 14 to the wild-
N⋯H–N and N–H⋯O hydrogen bonds between the inhibitor type c-KIT might be related to the fact that the terminal 3,4-
7-azaindole ring and Cys673 are dynamically very stable, with an dimethoxy benzene group is exposed to the bulk solvent
associated time-averaged distance of 1.91–1.97 Å. All six hydro- because the P-loop remains distant from the ATP-binding site
gen bonds in the three D816V-inhibitor complexes are retained of c-KIT in the inactive conformation, making it possible for
for more than 99% of simulation time assuming a hydrogen the bidentate hydrogen bonds to be ruptured by solvent mole-
bond distance limit of 2.5 Å. This dynamic stability of biden- cules. As can be seen in Fig. 4c, however, the dynamic stability
tate hydrogen bonds is actually not surprising because 5, 14, of the bidentate hydrogen bonds between Cys673 and the
and 26 are found to be well-accommodated in the ATP-binding inhibitor increases significantly with a change in the substitu-
site of the D816V mutant owing to the presence of P-loop resi- ent at the X position from 3-aminobenzene in 14 to 4-amino-
dues near the entrance of the ATP-binding site. Thus, the benzene in 26. The N⋯H–N and N–H⋯O hydrogen bonds
maintenance of bidentate hydrogen bonds with dynamic between the inhibitor 7-azaindole ring and Cys673 exhibited
stability is consistent with tight binding of inhibitors in residence times of 99.5% and 98.6% in the c-KIT-26 complex.
the ATP-binding site of the D816V mutant, which helps to The resulting substantial increase in dynamic stability of
explain nanomolar inhibitory activities. On the other hand, the bidentate hydrogen bonds is consistent with the greater
Fig. 4 Time evolutions of the interatomic distances associated with the hydrogen bonds between the 7-azaindole core of (a) 5, (b) 14, and (c) 26
and backbone amide groups in the ATP-binding sites of the wild-type c-KIT and the D816V mutant.
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inhibitor potency of 26 compared with 5 and 14, confirming a 1,4-dioxane–H2O = 3 : 1 (7 mL) was heated to 80 °C for 2 h in a
close relation between the strength of bidentate hydrogen sealed tube.
bonds with Cys673 and inhibitory activity.
The resulting solution was cooled to rt and 4 N KOH solu-
tion (4 mL) was added. The reaction mixture was then heated
to 60 °C and stirred for 1 h (N-deprotection of the benzenesul-
fonyl group). The mixture was diluted with water and neutral-
ized with 6 N HCl solution in an 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
(EA–HX, gradient 1 : 2 to 1 : 1) to give the product as a yellow
Conclusions
We have identified new 7-azaindole-based c-KIT inhibitors
with nanomolar inhibitory activity and high selectivity for the
gain-of-function D816V mutant through the structure-based
de novo design. Owing to improvements in the scoring function,
achieved by implementing an accurate solvation free energy
term, the designed derivatives are potent and selective against
the D816V mutant. In particular, 7-azaindole derivatives with a
3,4-dimethoxybenzene moiety at the 3-postion and a polar aro-
matic group at the 5-position represent a lead structure for the
discovery of new anticancer agents that specifically inhibit the
D816V c-KIT mutant. The results of MD simulations confirm
that the strengths and dynamic stabilities of the bidentate
hydrogen bonds with Cys673 serve as the critical factors
affecting the potency and selectivity of c-KIT inhibitors.
1
solid (369 mg, 85%). H NMR (400 MHz, chloroform-d) δ 9.41
(s, 1H), 8.38 (d, J = 2.1 Hz, 1H), 8.28 (d, J = 1.8 Hz, 1H), 7.44 (d,
J = 2.5 Hz, 1H), 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).
General procedure (GP II) for Suzuki coupling
(2-Amino-4-(3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-
5-yl)phenyl)(morpholino)methanone (5). A solution of
5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
(90 mg, 0.267 mmol), (2-amino-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenyl)(morpholino)methanone (107 mg,
0.321 mmol), Cs2CO3 (174 mg, 0.534 mmol), and PdCl2-
(dppf)·CH2Cl2 (44 mg, 0.0534 mmol) in 1,4-dioxane–H2O =
3 : 1 (2 mL) was charged in a capped tube and was heated to
100 °C for 3 h. The reaction mixture was cooled to room temp-
erature and concentrated in vacuo. The residue was diluted
with CH2Cl2 and the organic phase was extracted with CH2Cl2.
The combined organic layer was dried with MgSO4 and con-
centrated in vacuo. The mixture was purified with flash
column chromatography (CH2Cl2–MeOH, gradient 20 : 1 to
15 : 1) to give the product as a pale yellow solid (52.4 mg, 43%).
Experimental
General methods and materials
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
1
silica gel (400–630 mesh). H NMR was recorded at 600 MHz,
1
mp 153–157 °C. H NMR (400 MHz, DMSO-d6) δ 11.90 (s, 1H),
400 MHz 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 appropri-
ate: 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 carried out at
100 MHz 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 chloro-
form-d. Mass spectral data were obtained using the EI method.
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.
8.51 (d, J = 2.2 Hz, 1H), 8.33 (d, J = 2.2 Hz, 1H), 7.82 (t, J =
2.1 Hz, 1H), 7.28–7.25 (m, 2H), 7.12 (d, J = 7.9 Hz, 1H), 7.09 (s,
1H), 7.03 (d, J = 8.1 Hz, 1H), 6.96 (dd, J = 7.9, 1.7 Hz, 1H), 5.34
(s, 2H), 3.84 (s, 3H), 3.77 (s, 3H) 3.61 (s, 4H). 13C NMR
(100 MHz, DMSO-d6) δ 168.62, 149.19, 148.64, 147.33, 146.45,
141.62, 140.81, 128.75, 128.42, 127.75, 125.00, 123.98, 118.66,
117.61, 117.41, 114.85, 114.43, 113.77, 112.45, 110.69, 66.19,
55.63, 55.60. HRMS (EI+) m/z calcd for C26H26N4O4 [M + Na]+,
481.1852; found 481.1866.
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 (EA–HX, 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 a white solid. mp 213–215 °C.
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), 7.80 (d, J = 2.5 Hz, 1H),
General procedure (GP I) for Suzuki coupling
5-Bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine 7.29–7.21 (m, 3H), 7.03 (d, J = 8.8 Hz, 1H), 6.99–6.94 (m, 2H),
(2). A solution of 5-bromo-3-iodo-1-(phenylsulfonyl)-1H- 6.72 (d, J = 7.5 Hz, 1H), 3.84 (s, 3H), 3.77 (s, 3H) 2.95 (s, 6H).
pyrrolo[2,3-b]pyridine (600 mg, 1.3 mmol), 3,4-dimethoxyphe- 13C NMR (100 MHz, DMSO-d6) δ 150.93, 149.13, 148.50,
nylboronic acid (284 mg, 1.56 mmol), Cs2CO3 (1059 mg, 147.20, 141.96, 139.85, 129.63, 129.44, 127.85, 125.33, 123.76,
3.25 mmol) and Pd(dppf)Cl2·CH2Cl2 (212 mg, 0.26 mmol) in 118.53, 117.28, 115.17, 114.70, 112.50, 111.12, 111.09, 110.55,
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55.58, 55.49, 40.21. HRMS (EI+) m/z calcd for C23H23N3O2 111.87, 110.92, 66.98, 66.83, 57.84, 56.08, 56.06, 54.04. HRMS
[M + H]+, 374.1869; found 374.1845.
(EI+) m/z calcd for C27H30N4O4 [M + H]+, 475.2345; found
475.2350.
5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
2-methylaniline (9). Compound 9 was prepared (31.8 mg,
General procedure (GP III) for Suzuki coupling
(3-Amino-5-(3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin- 57%) according to GP II (CH2Cl2–MeOH, gradient 60 : 1 to
5-yl)phenyl)(morpholino)methanone (7). A solution of 30 : 1) from 5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]-
5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine pyridine (50 mg, 0.15 mmol) and 2-methyl-5-(4,4,5,5-tetra-
(75 mg, 0.23 mmol), bis(pinacolato)diboron (74.6 mg, methyl-1,3,2-dioxaborolan-2-yl)aniline (42 mg, 0.18 mmol) as a
0.29 mmol), KOAc (44.4 mg, 0.452 mmol) and Pd(dppf) white solid. mp 170–174 °C. 1H NMR (400 MHz, DMSO-d6)
Cl2·CH2Cl2 (37 mg, 0.045 mmol) in anhydrous 1,4-dioxane was δ 11.82 (d, J = 2.6 Hz, 1H), 8.44 (d, J = 2.1 Hz, 1H), 8.24 (d, J =
heated to 90 °C for 12 h under an atmosphere of N2. The reac- 2.1 Hz, 1H), 7.79 (d, J = 2.6 Hz, 1H), 7.26–7.23 (m, 2H), 7.02
tion mixture was cooled to RT and concentrated in vacuo. The (dd, J = 10.24, 7.65 Hz, 2H), 6.95 (d, J = 1.9 Hz, 1H), 6.82 (dd,
residue was diluted with CH2Cl2 and MeOH and then filtered J = 7.6, 1.9 Hz, 1H), 4.91 (s, 2H), 3.84 (s, 3H), 3.78 (s, 3H), 2.08
through a short pad of silica (SiO2) and celite eluting with (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 149.15, 148.31,
CH2Cl2 and MeOH (15 : 1). The filtrate was concentrated 147.23, 147.04, 141.55, 137.22, 130.52, 129.32, 127.87, 124.56,
in vacuo and then purified by flash column chromatography 123.73, 120.04, 118.54, 117.36, 114.74, 114.62, 112.46, 112.36,
(EA–HX, gradient 1 : 3 to 1 : 1) to give 3 as a brown solid 110.60, 55.61, 55.58, 17.08. HRMS (EI+) m/z calcd for
(63.9 mg, 74%). 1H NMR (300 MHz, chloroform-d) δ 12.14 C22H21N3O2 [M + H]+, 360.1712; found 360.1691.
(s, 1H), 8.82 (d, J = 1.4 Hz, 1H), 8.68 (d, J = 1.4 Hz, 1H), 7.54
5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
(s, 1H), 7.28 (dd, J = 8.3, 2.0 Hz, 1H), 7.17 (t, J = 2.5 Hz, 1H), 2-methoxyaniline (10). Compound 10 was prepared (44.2 mg,
7.02 (d, J = 8.3 Hz, 1H), 3.99 (s, 3H), 3.96 (s, 3H), 1.41(s, 12H). 39%) according to GP II (CH2Cl2–MeOH, gradient 30 : 1) from
A solution of 3 (23 mg, 0.06 mmol), (3-amino-5-bromophenyl)- 5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
(morpholino)methanone (20.5 mg, 0.072 mmol), Cs2CO3 (100 mg, 0.3 mmol) and 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-
(39.1 mg, 0.12 mmol) and Pd(dppf)Cl2·CH2Cl2 (12.2 mg, dioxaborolan-2-yl)aniline (89.7 mg, 0.36 mmol) as a white
0.015 mmol) in 1,4-dioxane–H2O = 3 : 1 (1.5 mL) was charged solid. mp 193–195 °C. 1H NMR (400 MHz, Chloroform-d)
in a capped tube and was heated to 100 °C for 3 h. The reac- δ 10.98 (s, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.27 (d, J = 2.0 Hz, 1H),
tion mixture was cooled to room temperature and concen- 7.46 (s, 1H), 7.19 (dd, J = 8.2, 2.0 Hz, 1H), 7.13 (d, J = 2.0 Hz,
trated in vacuo. The residue was diluted with CH2Cl2 and the 1H), 6.97–6.95 (m, 3H), 6.87 (dd, J = 8.1, 1.2 Hz, 1H), 3.92
organic phase was extracted with CH2Cl2. The combined (s, 3H), 3.92 (s, 3H), 3.89 (s, 3H). 13C NMR (100 MHz, chloro-
organic layer was dried with MgSO4 and concentrated in vacuo. form-d) δ 149.32, 148.28, 147.80, 146.84, 142.04, 136.55,
The mixture was purified with flash column chromatography 132.61, 130.11, 127.94, 126.26, 122.31, 119.55, 118.59, 117.38,
(CH2Cl2–MeOH, gradient 30 : 1 to 15 : 1) to give the product as 116.37, 114.00, 111.82, 110.81, 110.77, 56.02, 55.99, 55.63.
a pale yellow solid (6 mg, 22%). mp 154–157 °C. 1H NMR HRMS (EI+) m/z calcd for C22H21N3O3 [M + H]+, 376.1661;
(400 MHz, DMSO-d6) δ 11.88 (s, 1H), 8.46 (d, J = 2.0 Hz, 1H), found 376.1635.
8.27 (d, J = 2.1 Hz, 1H), 7.82 (d, J = 2.3 Hz, 1H), 7.28–7.24 (m,
3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
2H), 7.04 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 2.1 Hz, 1H), 6.82 (d, J = 5-methoxyaniline (11). Compound 11 was prepared (40.8 mg,
1.9 Hz, 1H), 6.55 (s, 1H), 5.39 (s, 2H), 3.84 (s, 3H), 3.78 (s, 3H), 36%) according to GP II (EA–HX 3 : 1) from 5-bromo-3-
3.59 (br, 8H). 13C NMR (150 MHz, DMSO-d6) δ 169.59, 149.34, (3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (100 mg,
149.19, 148.55, 147.33, 141.65, 139.83, 137.08, 128.64, 127.76, 0.3 mmol) and 3-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-di-
125.05, 123.93, 118.66, 117.36, 114.81, 113.18, 112.67, 112.51, oxaborolan-2-yl)aniline (89.7 mg, 0.36 mmol) as a white solid.
1
110.85, 110.72, 66.17, 55.63, 40.04. HRMS (EI+) m/z calcd for mp 190–195 °C. H NMR (400 MHz, DMSO-d6) δ 11.86 (d, J =
C26H26N4O4 [M + Na]+, 481.1852; found 481.1860.
1.8 Hz, 1H), 8.47 (d, J = 2.1 Hz, 1H), 8.27 (d, J = 2.1 Hz, 1H),
5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)- 7.81 (d, J = 2.5 Hz, 1H), 7.28–7.26 (m, 2H), 7.04 (d, J = 8.8 Hz,
2-(2-morpholinoethoxy)aniline (8). Compound 8 was prepared 1H), 6.51 (t, J = 1.7 Hz, 1H), 6.44 (dd, J = 2.4, 1.5 Hz, 1H), 6.16
(12.9 mg, 30%) according to GP III (CH2Cl2–MeOH, gradient (t, J = 2.0 Hz, 1H), 5.19 (s, 2H), 3.85 (s, 3H), 3.78 (s, 3H), 3.73
30 : 1 to 15 : 1) from 3 (35 mg, 0.092 mmol) and 5-bromo-2- (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 160.78, 150.33,
(2-morpholinoethoxy)aniline as a white solid. mp 96–98 °C. 149.17, 148.52, 147.26, 141.69, 140.62, 129.36, 127.84, 124.90,
1H NMR (400 MHz, chloroform-d) δ 9.48 (s, 1H), 8.52 (s, 1H), 123.78, 118.57, 117.28, 114.70, 112.49, 110.65, 105.63, 100.66,
8.26 (d, J = 1.9 Hz, 1H), 7.44 (d, J = 2.2 Hz, 1H), 7.20 (dd, 98.17, 55.60, 55.57, 54.74. HRMS (EI+) m/z calcd for
J = 8.2, 2.0 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 6.98–6.88 (m, 4H), C22H21N3O3 [M + H]+, 376.1661; found 376.1643.
4.17 (t, J = 5.6 Hz, 2H), 3.94 (s, 3H), 3.92 (s, 3H), 3.73
3-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrolo-
(t, J = 4.6 Hz, 4H), 2.82 (t, J = 5.6 Hz, 2H), 2.59 (t, J = 4.3 Hz, [2,3-b]pyridine (12). Compound 12 was prepared (37 mg, 59%)
4H). 13C NMR (150 MHz, chloroform-d) δ 149.39, 148.17, according to GP II (EA–HX, 2 : 1) from 5-bromo-3-(3,4-
148.00, 145.87, 142.76, 137.38, 133.35, 130.35, 127.74, dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (50 mg, 0.15 mmol)
126.21, 121.79, 119.68, 118.43, 117.38, 116.91, 114.24, 113.31, and (3,4,5-trimethoxyphenyl)boronic acid (38.2 mg, 0.18 mmol)
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as a white solid. mp 202–204 °C. H NMR (400 MHz, DMSO-
3-(3,4-Dimethoxyphenyl)-5-(m-tolyl)-1H-pyrrolo[2,3-b]pyridine
d6) δ 11.89 (s, 1H), 8.57 (d, J = 2.1 Hz, 1H), 8.40 (d, J = 2.1 Hz, (16). Compound 16 was prepared (9.9 mg, 60%) according to
1H), 7.82 (d, J = 2.2 Hz, 1H), 7.32–7.29 (m, 2H), 7.02 (d, J = GP II (CH2Cl2–MeOH, gradient 60 : 1 to 30 : 1) from 5-bromo-
8.1 Hz, 1H), 6.99 (s, 2H), 3.87 (s, 6H), 3.86 (s, 3H), 3.78 (s, 3H), 3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (16 mg,
3.70 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 153.27, 149.15, 0.048 mmol) and m-tolylboronic acid (7.8 mg, 0.58 mmol) as a
148.51, 147.22, 142.03, 136.80, 134.98, 128.91, 127.85, 125.49, white solid. mp 184–190 °C. 1H NMR (400 MHz, DMSO-d6)
123.87, 118.59, 117.24, 114.78, 112.52, 110.59, 104.66, 60.04, δ 11.87 (s, 1H), 8.52 (d, J = 2.1 Hz, 1H), 8.34 (d, J = 2.1 Hz, 1H),
56.00, 55.61, 55.49. HRMS (EI+) m/z calcd for C24H24N2O5 7.81 (s, 1H), 7.54 (s, 1H), 7.51 (dd, J = 8.2, 1.6 Hz, 1H), 7.36
[M + Na]+, 443.1583; found 443.1566.
(t, J = 7.6 Hz, 1H), 7.29 (dd, J = 8.2, 2.1 Hz, 1H), 7.25 (d, J =
3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)- 2.0 Hz, 1H), 7.17 (ddt, J = 7.6, 1.8, 0.9 Hz, 1H), 7.03 (d, J =
benzonitrile (13). Compound 13 was prepared (31.5 mg, 59%) 8.3 Hz, 1H), 3.84 (s, 3H), 3.77 (s, 3H), 2.38 (s, 3H). 13C NMR
according to GP II (EA–HX, gradient 1 : 1) from 5-bromo-3-(3,4- (100 MHz, DMSO-d6) δ 149.15, 148.49, 147.26, 141.82, 139.01,
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (50 mg, 0.15 mmol) 138.11, 128.84, 128.68, 127.77, 127.69, 127.52, 125.21, 124.13,
and (3-cyanophenyl)boronic acid (26.4 mg, 0.18 mmol) as a 123.89, 118.65, 117.37, 114.79, 112.47, 110.65, 55.59, 55.56,
white solid. mp 198–206 °C. 1H NMR (400 MHz, DMSO-d6) 21.10. HRMS (EI+) m/z calcd for C22H20N2O2 [M + Na]+,
δ 11.98 (d, J = 2.7 Hz, 1H), 8.61 (d, J = 2.1 Hz, 1H), 8.49 (d, J = 367.1422; found 367.1423.
2.2 Hz, 1H), 8.30 (s, 1H), 8.12 (ddd, J = 7.9, 2.0, 1.1 Hz, 1H),
3,5-Bis(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (17).
7.86 (d, J = 2.6 Hz, 1H), 7.81 (dt, J = 7.8, 1.3 Hz, 1H), 7.67 (t, J = Compound 17 was prepared (27 mg, 58%) according to GP II
7.8 Hz, 1H), 7.35 (dd, J = 8.2, 2.0 Hz, 1H), 7.27 (d, J = 2.0 Hz, (EA–HX, 2 : 1) from 5-bromo-3-(3,4-dimethoxyphenyl)-1H-
1H), 7.03 (d, J = 8.3 Hz, 1H), 3.85 (s, 3H), 3.78 (s, 3H). 13C NMR pyrrolo[2,3-b]pyridine (40 mg, 0.12 mmol) and 3,4-dimethoxy-
(100 MHz, DMSO-d6) δ 149.15, 148.84, 147.34, 141.87, 140.31, phenylboronic acid (26.2 mg, 0.14 mmol) as a white solid. mp
131.82, 130.56, 130.44, 130.07, 127.57, 126.50, 125.89, 124.24, 218–223 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.86 (d, J =
118.89, 118.81, 117.39, 115.10, 112.43, 112.08, 110.70, 55.59. 2.6 Hz, 1H), 8.54 (d, J = 2.1 Hz, 1H), 8.35 (d, J = 2.1 Hz, 1H),
HRMS (EI+) m/z calcd for C22H17N3O2 [M + Na]+, 378.1218; 7.81 (d, J = 2.5 Hz, 1H), 7.31–7.28 (m, 3H), 7.24 (dd, J = 8.3, 2.1
found 378.1217.
Hz, 1H), 7.03 (dd, J = 8.6, 7.1 Hz, 2H), 3.86 (s, 3H), 3.86 (s, 3H),
3-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)- 3.79 (s, 3H), 3.78 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
aniline (14). Compound 14 was prepared (11 mg, 25%) accord- δ 149.18, 149.15, 148.32, 148.16, 147.21, 141.81, 131.94,
ing to GP II (CH2Cl2–MeOH, gradient 30 : 1) from 5-bromo- 128.71, 127.89, 125.01, 123.80, 119.13, 118.58, 117.33, 114.70,
3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (40 mg, 112.48, 112.38, 110.98, 110.57, 55.60, 55.52. HRMS (EI+) m/z
0.12 mmol) and 3-aminophenylboronic acid (20 mg, calcd for C23H22N2O4 [M + Na]+, 413.1477; found 413.1482.
0.15 mmol) as a white solid. mp 200–203 °C. 1H NMR
3-(3,4-Dimethoxyphenyl)-5-(pyridin-3-yl)-1H-pyrrolo[2,3-b]-
(400 MHz, chloroform-d) δ 10.57 (s, 1H), 8.56 (d, J = 2.0 Hz, pyridine (18). Compound 18 was prepared (31 mg, 62%)
1H), 8.32 (d, J = 2.0 Hz, 1H), 7.48 (s, 1H), 7.25 (t, J = 7.8 Hz, according to GP II (CH2Cl2–MeOH, gradient 30 : 1 to 15 : 1)
1H), 7.20 (dd, J = 8.3, 2.0 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.01 from 5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyri-
(dt, J = 7.8, 1.1 Hz, 1H), 6.97 (d, J = 8.2 Hz, 1H), 6.92 (t, J = dine (50 mg, 0.15 mmol) and 3-pyridinylboronic acid (22.1 mg,
2.1 Hz, 1H), 6.69 (dt, J = 7.9, 1.6 Hz, 1H), 3.93 (s, 3H), 3.92 (s, 0.18 mmol) as a white solid. mp 225–226 °C. 1H NMR
3H), 3.00 (br, 2H). 13C NMR (150 MHz, chloroform-d) δ 149.39, (400 MHz, DMSO-d6) δ 12.00 (s, 1H), 8.99 (d, J = 2.4 Hz, 1H),
148.61, 147.94, 146.93, 142.46, 140.76, 130.28, 129.90, 127.80, 8.60 (d, J = 2.2 Hz, 1H), 8.57 (dd, J = 4.9, 1.6 Hz, 1H), 8.47 (d,
126.61, 122.22, 119.64, 118.56, 117.86, 116.73, 114.07, 113.89, J = 2.1 Hz, 1H), 8.17 (dt, J = 7.9, 2.0 Hz, 1H), 7.87 (d, J = 2.4 Hz,
111.87, 110.85, 56.05. HRMS (EI+) m/z calcd for C21H19N3O2 1H), 7.48 (dd, J = 7.9, 4.8 Hz, 1H), 7.33 (dd, J = 8.1, 2.1 Hz, 1H),
[M + H]+, 346.1556; found 346.1543.
7.29 (d, J = 2.1 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 3.85 (s, 3H),
5-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)- 3.78 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 149.18, 148.80,
pyridin-3-amine (15). Compound 15 was prepared (42.6 mg, 147.94, 147.34, 141.78, 134.66, 134.45, 127.63, 125.79, 125.46,
40%) according to GP II (CH2Cl2–MeOH, gradient 20 : 1 to 124.19, 123.85, 118.77, 117.49, 115.00, 112.44, 110.67, 55.60,
15 : 1) from 5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]- 55.58. HRMS (EI+) m/z calcd for C20H17N3O2 [M + H]+,
pyridine (101 mg, 0.3 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2- 332.1399; found 332.1385.
dioxaborolan-2-yl)pyridin-3-amine (81 mg, 0.368 mmol) as a
3-(5-(3-Aminophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo-
white solid. mp 254–260 °C. 1H NMR (400 MHz, DMSO-d6) nitrile (19). 3-(5-Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile
δ 11.96 (s, 1H), 8.49 (s, 1H), 8.33 (s, 1H), 8.12 (s, 1H), 7.94 was prepared (54 mg, 84%) according to GP I from 5-bromo-
(s, 1H), 7.86 (s, 1H), 7.29 (d, J = 8.2 Hz, 1H), 7.24 (d, J = 3-iodo-1-(phenyl-sulfonyl)-1H-pyrrolo[2,3-b]pyridine (100 mg,
13.4 Hz, 2H), 7.02 (d, J = 8.2 Hz, 1H), 5.42 (s, 2H), 3.84 (s, 3H), 0.216 mmol) and 3-cyanophenylboronic acid (33 mg,
1
3.78 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 149.16, 148.67, 0.22 mmol). H NMR (300 MHz, DMSO-d6) δ 12.38 (s, 1H) 8.59
147.30, 144.92, 141.52, 135.42, 135.06, 134.61, 127.67, (d, J = 2.1 Hz, 1H), 8.35 (d, J = 2.1 Hz, 1H), 8.07–8.19 (m, 3H),
126.28, 125.25, 124.05, 118.66, 117.89, 117.44, 114.81, 112.44, 7.59–7.70 (m, 2H). Compound 19 was prepared (16 mg, 39%)
110.65, 55.59. HRMS (EI+) m/z calcd for C20H18N4O2 [M + H]+, according to GP II (CH2Cl2–MeOH, gradient 30 : 1 to 15 : 1)
347.1508; found 347.1508.
from 3-(5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (40 mg,
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0.13 mmol) and 3-aminophenylboronic acid (21 mg, pyridine (150 mg, 0.325 mmol) and (3,4,5-trimethoxyphenyl)
0.15 mmol) as a pale yellow solid. mp 221–223 °C. 1H NMR boronic acid (82.6 mg, 0.390 mmol). 1H NMR (400 MHz,
(400 MHz, DMSO-d6) δ 12.13 (s, 1H), 8.48 (d, J = 2.0 Hz, 1H), chloroform-d) δ 9.62 (s, 1H), 8.39 (s, 1H), 8.27 (d, J = 1.9 Hz,
8.38 (d, J = 2.1 Hz, 1H), 8.21 (d, J = 1.8 Hz, 1H), 8.14 (dd, J = 1H), 7.46 (d, J = 2.3 Hz, 1H), 6.75 (s, 2H), 3.93 (s, 6H), 3.89 (s,
7.7, 1.7 Hz, 1H), 8.09 (s, 1H), 7.70–7.62 (m, 2H), 7.13 (t, J = 3H). Compound 22 was prepared (42.3 mg, 47%) according to
7.8 Hz, 1H), 6.91 (s, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.58 (dd, J = GP II (EA–HX, 2 : 1) from 5-bromo-3-(3,4,5-trimethoxyphenyl)-
7.9, 2.3 Hz, 1H), 5.15 (s, 2H). 13C NMR (100 MHz, DMSO-d6) 1H-pyrrolo[2,3-b]pyridine (87 mg, 0.24 mmol) and 3-aminophe-
δ 149.13, 148.49, 142.16, 139.49, 136.37, 130.78, 130.09, nylboronic acid (44.5 mg, 0.29 mmol) as a white solid. mp
129.44, 129.35, 129.04, 125.89, 125.05, 119.04, 116.84, 114.89, 206–208 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.92 (d, J =
112.79, 112.61, 112.51, 112.06. HRMS (EI+) m/z calcd for 2.7 Hz, 1H), 8.47 (d, J = 2.0 Hz, 1H), 8.29 (d, J = 2.1 Hz, 1H),
C20H14N4 [M + Na]+, 333.1116; found, 333.1101.
7.86 (d, J = 2.6 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 6.95 (s, 2H),
3-(3-(3-Fluoro-4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)- 6.91 (t, J = 2.0 Hz, 1H), 6.87 (ddd, J = 7.5, 1.9, 1.0 Hz, 1H), 6.56
aniline (20). 5-Bromo-3-(3-fluoro-4-methoxyphenyl)-1H-pyrrolo- (ddd, J = 8.0, 2.2, 0.9 Hz, 1H), 5.18 (s, 2H), 3.87 (s, 6H), 3.69
[2,3-b]pyridine was prepared (85.1 mg, 61%) according to GP I (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 153.26, 149.16,
from 5-bromo-3-iodo-1-(phenyl-sulfonyl)-1H-pyrrolo[2,3-b]pyri- 148.41, 141.74, 139.56, 135.94, 130.66, 129.51, 129.41, 124.74,
dine (200 mg, 0.432 mmol) and 3-fluoro-4-methoxyphenyl- 124.42, 117.28, 114.92, 114.53, 112.69, 112.35, 104.07, 60.05,
boronic acid (88.1 mg, 0.518 mmol). 1H NMR (400 MHz, 55.97. HRMS (EI+) m/z calcd for C22H21N3O3 [M + Na]+,
chloroform-d) δ 9.79 (s, 1H), 8.38 (dd, J = 2.1, 0.4 Hz, 1H), 8.28 398.1481; found 398.1471.
(dd, J = 2.1, 0.7 Hz, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.32–7.28 (m,
3-(3-(4-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
1H), 7.28–7.26 (m, 1H), 7.04 (t, J = 8.8 Hz, 1H), 3.93 (s, 3H). (23). 5-Bromo-3-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
Compound 20 was prepared (37 mg, 46.1%) according to GP II was prepared (118 mg, 60%) according to GP I from 5-bromo-
(EA–HX, gradient 2 : 1) from 5-bromo-3-(3-fluoro-4-methoxy- 3-iodo-1-(phenyl-sulfonyl)-1H-pyrrolo[2,3-b]pyridine (300 mg,
phenyl)-1H-pyrrolo[2,3-b]pyridine (77.3 mg, 0.24 mmol) and 0.649 mmol) and 4-methoxyphenylboronic acid (118.4 mg,
1
3-aminophenylboronic acid (44.8 mg, 0.29 mmol) as a white 0.78 mmol). H NMR (300 MHz, chloroform-d) δ 9.29 (s, 3H),
solid. mp 199–201 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.93 (s, 8.36 (dd, J = 2.1, 0.4 Hz, 1H), 8.28 (dd, J = 2.1, 0.7 Hz, 1H), 7.49
1H), 8.47 (d, J = 2.1 Hz, 1H), 8.28 (d, J = 2.1 Hz, 1H), 7.86 (d, J = (d, J = 8.9 Hz, 2H), 7.41 (d, J = 2.6 Hz, 1H), 7.00 (d, J = 8.8 Hz,
2.0 Hz, 1H), 7.58 (dd, J = 12.9, 2.1 Hz, 1H), 7.53 (ddd, J = 8.5, 2H), 3.85 (s, 3H). Compound 23 was prepared (83.7 mg, 69%)
2.2, 1.0 Hz, 1H), 7.23 (t, J = 8.9 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), according to GP II (EA–HX, 2 : 1) from 5-bromo-3-(4-methoxy-
6.91 (t, J = 2.0 Hz, 1H), 6.87 (ddd, J = 7.6, 1.8, 0.9 Hz, 1H), 6.57 phenyl)-1H-pyrrolo[2,3-b]pyridine (117 mg, 0.39 mmol) and
(ddd, J = 7.9, 2.2, 0.9 Hz, 1H), 5.15 (s, 2H), 3.86 (s, 3H). 3-aminophenylboronic acid (71.8 mg, 0.46 mmol) as a white
13C NMR (100 MHz, DMSO-d6) δ 151.87 (d, J = 243.2 Hz), solid. mp 209–210 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.84 (s,
149.15, 148.42, 145.21 (d, J = 10.7 Hz), 141.83, 139.58, 129.62, 1H), 8.45 (d, J = 1.8 Hz, 1H), 8.26 (d, J = 2.1 Hz, 1H), 7.76 (d, J =
129.47, 128.26 (d, J = 7.0 Hz), 124.81, 124.33, 122.39 (d, J = 2.4 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.12 (t, J = 7.7 Hz, 1H),
3.3 Hz), 117.08, 114.75, 114.43 (d, J = 2.3 Hz), 113.84 (d, J = 7.02 (d, J = 8.7 Hz, 2H), 6.90 (s, 1H), 6.85 (d, J = 7.6 Hz, 1H),
18.4 Hz), 113.33 (d, J = 1.9 Hz), 112.75, 112.50, 56.05. HRMS 6.57 (dd, J = 8.1, 2.3 Hz, 1H), 5.15 (s, 2H), 3.78 (s, 3H).
(EI+) m/z calcd for C20H16FN3O [M + H]+, 334.1356; found, 13C NMR (100 MHz, DMSO-d6) δ 157.54, 149.14, 148.42,
334.1326.
141.63, 139.64, 129.44, 129.39, 127.58, 127.42, 124.79, 123.47,
3-(3,4-Dimethoxyphenyl)-5-phenyl-1H-pyrrolo[2,3-b]pyridine 117.28, 114.65, 114.38, 112.67, 112.43, 55.07. HRMS (EI+) m/z
(21). Compound 21 was prepared (42.4 mg, 43%) according to calcd for C20H17N3O [M + H]+, 316.1450; found 316.1424.
GP II (EA–CH2Cl2, gradient 1 : 5 to 1 : 3) from 5-bromo-3-(3,4-
3-(3,4-Dimethoxyphenyl)-5-(pyridin-4-yl)-1H-pyrrolo[2,3-b]-
dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (99 mg, 0.3 mmol) pyridine (24). Compound 24 was prepared (19.9 mg, 72%)
and phenylboronic acid (43.9 mg, 0.36 mmol) as a white solid. according to GP II (CH2Cl2–MeOH, gradient 30 : 1 to 10 : 1)
mp 192–194 °C. 1H NMR (400 MHz, chloroform-d) δ 11.19 from 5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
(s, 1H), 8.62 (d, J = 2.1 Hz, 1H), 8.37 (d, J = 2.0 Hz, 1H), (28 mg, 0.084 mmol) and 4-pyridinylboronic acid (12.4 mg,
7.67–7.61 (m, 2H), 7.53 (d, J = 1.9 Hz, 1H), 7.48 (t, J = 7.6 Hz, 0.101 mmol) as a white solid. mp 218–220 °C. 1H NMR
2H), 7.37 (t, J = 7.4 Hz, 1H), 7.22 (dd, J = 8.2, 2.0 Hz, 1H), 7.15 (400 MHz, chloroform-d) δ 10.01 (s, 1H), 8.72–8.65 (m, 3H),
(d, J = 2.0 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 8.40 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 5.5 Hz, 2H), 7.52 (d, J =
3H). 13C NMR (100 MHz, DMSO-d6) δ 149.17, 148.55, 147.29, 2.2 Hz, 1H), 7.20 (dd, J = 8.3, 2.0 Hz, 1H), 7.12 (d, J = 2.0 Hz,
141.83, 139.09, 128.98, 128.60, 127.77, 127.05, 126.89, 125.32, 1H), 7.00 (d, J = 8.2 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H). 13C NMR
123.96, 118.68, 117.42, 114.85, 112.47, 110.64, 55.59. HRMS (100 MHz, DMSO-d6) δ 150.09, 149.27, 149.17, 147.38, 146.11,
(EI+) m/z calcd for C21H18N2O2 [M + Na]+, 353.1266; found 141.75, 127.45, 125.74, 125.41, 124.36, 121.41, 118.79, 117.44,
353.1253.
3-(3-(3,4,5-Trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)- C20H17N3O2 [M + H]+, 332.1399; found 332.1372.
aniline (22). 5-Bromo-3-(3,4,5-trimethoxyphenyl)-1H-pyrrolo 3-(3-(Pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline
115.20, 112.47, 110.72, 55.60. HRMS (EI+) m/z calcd for
[2,3-b]pyridine was prepared (91 mg, 77%) according to (25). 5-Bromo-3-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine was pre-
GP I from 5-bromo-3-iodo-1-(phenyl-sulfonyl)-1H-pyrrolo[2,3-b]- pared (38.5 mg, 65%) according to GP I (EA–HX, gradient 1 : 1
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to 3 : 1) from 5-bromo-3-iodo-1-(phenyl-sulfonyl)-1H-pyrrolo-
[2,3-b]pyridine (100 mg, 0.216 mmol) and 3-pyridinyl boronic
acid (32 mg, 0.26 mmol). 1H NMR (300 MHz, CD3OD-d4) δ 8.78
(dd, J = 0.75, 2.2 Hz, 1H), 8.39 (dd, J = 1.6, 4.9 Hz, 1H), 8.36 (d,
J = 2.1 Hz, 1H), 8.28 (d, J = 2.1 Hz, 1H), 8.05–8.09 (m, 1H), 7.80
(s, 1H), 7.46 (dd, J = 4.9, 7.2 Hz, 1H). Compound 25 was pre-
pared (17.7 mg, 22%) according to GP II (CH2Cl2–MeOH, gradi-
ent 20 : 1 to 10 : 1) from 5-bromo-3-(pyridin-3-yl)-1H-pyrrolo
[2,3-b]pyridine (76 mg, 0.28 mmol) and 3-aminophenylboronic
acid (52 mg, 0.336 mmol) as a gray solid. mp 225–227 °C.
1H NMR (400 MHz, DMSO-d6) δ 12.13 (s, 1H), 9.00 (d, J =
2.3 Hz, 1H), 8.50 (d, J = 2.0 Hz, 1H), 8.46 (dd, J = 4.8, 1.6 Hz,
1H), 8.34 (d, J = 2.1 Hz, 1H), 8.17 (dt, J = 7.8, 2.0 Hz, 1H), 8.05
(s, 1H), 7.46 (dd, J = 7.9, 4.7 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H),
6.93 (s, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.57 (dd, J = 9.0, 1.7 Hz,
1H), 5.17 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 149.16,
148.52, 147.21, 146.64, 142.08, 139.41, 133.35, 130.90, 129.86,
129.46, 125.28, 124.88, 123.93, 117.09, 114.74, 112.78, 112.49,
111.10. HRMS (EI+) m/z calcd for C18H14N4 [M + Na]+,
309.1116; found, 309.1117.
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4-(3-(3,4-Dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-
aniline (26). Compound 26 was prepared (29.3 mg, 57%)
according to GP II (EA–HX, gradient 1 : 1 to 3 : 1) from
5-bromo-3-(3,4-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine
(50 mg, 0.15 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)aniline (39.5 mg, 0.18 mmol) as a yellow solid. mp
1
203–205 °C. H NMR (400 MHz, DMSO-d6) δ 11.76 (d, J = 2.7
Hz, 1H), 8.44 (d, J = 2.1 Hz, 1H), 8.23 (dd, J = 2.1, 0.6 Hz, 1H),
7.77 (d, J = 2.6 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.28 (dd, J =
8.1, 2.1 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 149.15, 147.91, 147.87,
147.18, 141.26, 129.35, 128.02, 127.52, 126.41, 123.79, 123.51,
118.56, 117.40, 114.49, 114.43, 112.49, 110.58, 55.59, 55.58.
HRMS (EI+) m/z calcd for C21H19N3O2 [M + H]+, 346.1 556;
found 346.1534.
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Acknowledgements
This research was supported by the National Research Foun-
dation of Korea (NRF) through general research grants
(NRF-2011-0022858, 0016436, 0020322) and the Institute for
Basic Science (IBS).
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