Discovery of new azaindole-based PI3Kα inhibitors: Apoptotic and antiangiogenic effect on cancer cells

Article abstract of DOI:10.1016/j.bmcl.2010.10.108

Phosphatidylinositol-3-kinase alpha (PI3Kα) is an important target in cancer due to the deregulation of the PI3K/AKT signaling pathway in many tumors. In this study, we designed [3,5-d]-7-azaindole analogs as PI3Kα inhibitors through the fragment-growing strategy. By varying groups at the 3,5-positions of azaindole, we developed the SAR (Structure-activity relationship) and identified a series of potent PI3Kα inhibitors. Representative azaindole derivatives showed activity in a cellular proliferation and apoptosis assays. Moreover, B3 exhibited strong antiangiogenic effects on cancer cells.

Full text of DOI:10.1016/j.bmcl.2010.10.108

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7212–7215
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new azaindole-based PI3K
a inhibitors: Apoptotic
and antiangiogenic effect on cancer cells
Seunghee Hong a, Soyoung Lee a, Bomi Kim a, Hyunseung Lee b, Soon-Sun Hong b, , Sungwoo Hong a,
⇑
⇑
a Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
b Department of Biomedical Sciences, College of Medicine, Inha University, Incheon 400-712, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphatidylinositol-3-kinase alpha (PI3K
a
) is an important target in cancer due to the deregulation of
Received 28 September 2010
Revised 20 October 2010
Accepted 21 October 2010
Available online 26 October 2010
the PI3K/AKT signaling pathway in many tumors. In this study, we designed [3,5-d]-7-azaindole analogs
as PI3K inhibitors through the fragment-growing strategy. By varying groups at the 3,5-positions of aza-
indole, we developed the SAR (Structure–activity relationship) and identified a series of potent PI3K
a
a
inhibitors. Representative azaindole derivatives showed activity in a cellular proliferation and apoptosis
assays. Moreover, B3 exhibited strong antiangiogenic effects on cancer cells.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Phosphatidylinositol-3-kinase
Azaindole
Fragment-growing strategy
Antiangiogenesis
Solid tumors contain hypoxic regions that have considerably
lower oxygen tension than the normal tissues. The transcription
factor hypoxia-inducible factor 1 (HIF-1) is a major regulator of
tumor cell adaptation to hypoxic stress. In addition to HIF-1
association in tumor progression, HIF-1 has been implicated in
the upregulation of genes involved in angiogenesis including
vascular endothelial growth factor (VEGF), inducible nitric oxide
synthase (NOS) and heme oxygenase-1. Angiogenesis is an essen-
tial process for tumor growth and metastasis, and VEGF is a
chemical signal stimulates the growth of new blood vessels from
pre-existing vessels. Recently, numerous studies have shown that
PI3K/Akt pathway plays critical roles in angiogenesis and expres-
Together, these observations strongly suggest that PI3K
pathway is among the most commonly activated signaling
pathway in cancer and the inhibition of the PI3K/Akt pathway
(PI3K/Akt, HIF-1a, VEGF) may lead to greater therapeutic potential.
Herein, we report the identification of new azaindole-based PI3K
inhibitors that demonstrate apoptotic and antiangiogenic effects
on cancer cell lines.
Fragment-based growing strategy,5 which creates novel
structures by adding interacting moieties to a starting scaffold, is
an alternative approach to high-throughput screening for the iden-
tification of active compounds for therapeutic targets. For inhibit-
ing kinases, the most exemplified approach targets the kinase
domain by competitive inhibition of the ATP binding site. Some
heterocyclic fragments mimicking the pyrimidine portion of ATP
can bind to the hinge region of the kinase via hydrogen bonds with
the backbone and represent start points for a fragment-growing
approach. As substantial structural and biochemical information
is available, PI3 kinases may represent one of good targets for
structure-based drug design to facilitate the discovery of new
potent inhibitors. With the goal of discovery of a new structural
class of inhibitors PI3K, we initiated a pharmacophore-directed
design. Among many hetero-aromatics, we selected 7-azaindole
as a scaffold for a PI3K hinge region binding moiety. 7-Azaindole
possesses both H-donor and H-acceptor for interaction with the
hinge region of the kinase6 and also has a strong advantage in that
it can be easily diversified in 3 and 5-positions via simple organic
reactions. To assess back pocket (DFG-motif, gate keeper and cata-
lytic lysine), we thought to take advantage of the information
known in the literature7 and combine a suitable pharmacophore
sion of HIF-1a
and VEGF.1
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that
catalyze phosphorylation of the 3-hydroxyl position of PIP2 (phos-
phatidylinositol 4,5-diphosphate) to PIP3 (phosphatidylinositol
3,4,5-triphosphate). The resulting second messenger, PIP3 can
regulate multiple physiological processes, including cell growth,
differentiation, survival, and motility.2 Deregulation of PI3K path-
way leads to elevated PIP3 levels and downstream activation of
Akt, which might be involved in the pathology of cancer, inflamma-
tion, immune disorders, and cardiovascular diseases.3 In particular,
the PIK3CA gene that encodes p110a catalytic subunit is frequently
mutated and overexpressed in a range of primary cancers.4
⇑
Corresponding authors. Tel.: +82 42 350 2811; fax: +82 42 350 2810 (S.H.);
tel.: +82 32 890 3683; fax: +82 32 890 2462 (S.-S.H).
E-mail addresses: hongs@inha.ac.kr (S.-S. Hong), hongorg@kaist.ac.kr (S. Hong).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.108

S. Hong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7212–7215
7213
to yield novel molecules with potent activity. In fact, a number of
inhibitors show various moieties which can interact in this back
pocket and the pyridyl sulfonamide group profoundly influenced
the activity of PI3K inhibitors by binding this region.7f Thus, our de-
sign strategy prioritized the incorporation of the pyridyl sulfon-
amide pharmacophore into azaindole at C5 to generate novel
hybrid molecules with the expectation of combining high affinity
for PI3 kinases (Fig. 1).
The general synthetic route for the preparation of substituted
azaindole derivatives is shown in Scheme 1. In all cases, the C3 po-
sition of the azaindole was functionalized prior to performing a
Susuki coupling with the C5 aryl bromide. The starting material
bromo-azaindole (1) was treated with N-iodosuccinimide in
acetone room temperature for 2 h to give the corresponding 5-bro-
mo-3-iodo-azaindole. After protection of NH group with benze-
nesulfonylcholoride, various aryl groups were attached to the
3-position of the azaindole core using palladium-mediated cross-
coupling, and benzenesulfonyl group was then deprotected to yield
the desired product 3. To facilitate the exploration of C5 position,
compound 3 underwent another palladium catalyzed Suzuki cou-
pling with different boronic acid derivatives. Finally, the various
sulfonyl groups were introduced to build the target products, by
treatment with arylsulfonylcholoride and pyridine at room tem-
perature. To build acetyl group at C3 position, Friedel–Crafts acet-
ylation of 1 with aluminium chloride and acetyl chloride provided
intermediate 5 as a white solid in 98% yield. The aryl group at C5
was then installed by Suzuki coupling in a similar manner.
Figure 2. Putative binding mode of B3 with the PI3K
PI3K
hydrogen bond with the backbone of Val851, and the sulfonamide bind to the
catalytic lysine (Lys802). Selected residues (A810 and Y836) are shown for possible
interaction with pyridyl sulfonamide subunit.
a
homology model, based on
c
crystal structures. In this model, the azaindole forms a key hinge region
In the resulting compound A1, an pyridyl sulfonamide was
grown from the C5 position of the azaindole to enalble into a
hydrophobic region in the back pocket of the enzyme. Compound
Table 1
Inhibition of PI3Ka of selected azaindole derivatives: structure–activity relationship
with various Ar and R groups
R
5
Ar
3
N
N
H
Compd
R1
R2
H
PI3K
IC50
a
(lM)
A1
A2
5.2
0.62
SO2Ph
HN
Acetyl
A3
A4
A5
3-Cyanophenyl
3-Pyridyl
3,4-Dimethoxyphenyl
0.031
0.012
0.012
N
Figure 1. Design of an azaindole scaffold as PI3K inhibitor and opportunities for
modification.
H
N
A6
0.95
SO2M e
B1
B2
B3
Acetyl
3-Cyanophenyl
3,4-Dimethoxyphenyl
0.73
0.13
0.016
O
R1
I
Br
Br
Br
N
c
a,b
O2S
H2N
N
H
N
N
N
H
N
N
SO2Ph
N
1
3
2
C1
C2
3-Cyanophenyl
3,4-Dimethoxyphenyl
0.027
0.003
F
O
O
d,e
R1
S
f
HN
F
O
N
N
Ar
d,e
Br
NH2
N
H
N
N
H
N
D1
E1
3,4-Dimethoxyphenyl
3,4-Dimethoxyphenyl
0.46
1.6
4
5
SO Ph
2
HN
Scheme 1. Reagents and conditions: (a) NIS, acetone, rt, 2 h; (b)
benzenesulfonylchloride, 50% NaOH, Bu4NHSO4, DCM, rt, 12 h; (c) aryl boronic
acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane/H2O = 3:1, 80 °C, 3 h, then 4 N KOH, MeOH, rt,
1 h; (d) aryl boronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane/H2O = 3:1, 100 °C, 5 h;
(e) arylsulfonylchloride, pyridine, rt, 12 h; (f) AcCl, AlCl3, DCM, rt, 6 h.

7214
S. Hong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7212–7215
Table 2
A1 had an IC50 of 5.2 lM for inhibition of PI3Ka. Analysis of the
docking model8 reveals that the azaindole anlogues maintains
the hydrogen-bonding pattern the hinge (Val851), while the
pyridyl sulfonamide group is lined by Y836 and A810 (Fig. 2).
Building upon the success observed with pyridyl sulfonamide
group, an investigation of the effect of the appendage at C3 posi-
tion was conducted. Profound effects on potency were achieved
with compounds incorporating the C3-aryl group, and A3 was
the earliest example in the azaindole series to display potency
Cancer cell proliferation of selected azaindole derivatives
Compd
Growth IC50
SK-BR3
(lM)
T47D
MCF7
A4
A5
B3
3.6
8.5
0.7
12
22
3.7
6.8
—
—
over PI3Ka enzyme at double-digit nanomolar concentration
(IC50 = 31 nM). The significant increase in activity afforded by
C3-aryl group prompted further investigation through synthesis
of a series of related analogs. Some heteroaromatic groups were
well tolerated, and in pyridyl analog A4 modest increase in activity
was observed. The presence of dimethoxyl groups resulted in
compounds which were generally of equivalent or better enzyme
potency than the corresponding unsubstituted derivatives, and
possesses good activity in a cellular proliferation assay. In the next
design cycle, we examined the structure–activity relationship
(SAR) of the sulfonamide moiety, and the highest PI3K
a potency
was obtained with 2,4-diF substituted phenyl groups (IC50 of
C2 = 3 nM). After mixing and matching, reversal of the sulfonamide
connectivity and addition of morpholine led to B3 which showed
increased solubility and provided an advantage in terms of cellular
activity. The pyridyl moiety at the 5 position was found to be
essential for enhanced potency, and replacement of pyridyl group
with phenyl group resulted in a significant loss of activity suggest-
ing that it was deemed important to preserve the intermolecular
hydrogen bonds that the pyridyl nitrogen makes with a conserved
active site water molecule7f (Table 1).
Given the impressive enzyme activity profile, several com-
pounds from this series were further tested for cellular proliferation
activity against cancer cell lines. To measure the inhibitory effect
of compounds on cell growth, cell viability was tested by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Figure 3. B3 promotes early-apoptosis of T47D breast cancer cells. T47D cells were
treated with various concentrations of B3 for 24 h and apoptosis was determined by
flow cytometric analysis using Annexin V-FITC and PI staining.
Figure 4. Representative images depicting the formation of capillary-like tubular network formation of HUVECs by treatment with DMSO (control) and B3 (0.1
10 M).
lM, 1 lM and
l

S. Hong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7212–7215
7215
Figure 5. Inhibition of HUVECs migration by B3 in a wound-induced migration assay. Migration was quantified by counting the number of cells that moved beyond the
reference line.
assay in T47D, SK-BR3, and MCF-7 human breast cancer cell
Acknowledgments
cultures. Notably, B3 showed potent antiproliferative effects at
submicromolar concentration (IC50 = 0.7 lM) as shown in Table 2.
This research was supported by National Research Foundation
of Korea (NRF) through general research Grants (NRF-2010-
001540 and 2010-0022179).
Activation of the PI3K pathway leads to phosphorylation of
Thr308 and Ser473 of AKT and subsequently a number of down-
stream substrates, such as IKK, GSK and Caspase-9. To ensure that
new series of compounds were inhibiting PI3K signaling in cells,
the most active compound based on growth assays, B3 was further
profiled for its ability to suppress cellular biomarker. B3 demon-
strated the ability to inhibit p-AKT (at Ser473) in T47D cancer cells
and promotes apoptosis of cancer cells, inducing cleaved-PARP
(cPARP) as a marker of apoptosis (data not shown). The percentage
of early-apoptotic cells was measured by the percentage of Annex-
in V-positive/PI-negative cells after incubation with various con-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.108.
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