Discovery of wrightiadione as a novel template for the TrkA kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.09.070

Enzymatic kinase assays and docking simulation studies have shown that the natural product wrightiadione displays inhibitory activity toward TrkA and PLK3. In this study, the template of wrightiadione served as a starting point for Trk inhibitor developme

Full text of DOI:10.1016/j.bmcl.2015.09.070

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of wrightiadione as a novel template for the TrkA kinase
inhibitors
a,b
a
a,b,
⇑
Yujeong Jeong , Sang Min Lim , Sungwoo Hong
a
Center for Catalytic Hydrocarbon Functionalization, Institute for Basic Science (IBS), Daejeon 305-701, Republic of Korea
Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Enzymatic kinase assays and docking simulation studies have shown that the natural product wrightia-
dione displays inhibitory activity toward TrkA and PLK3. In this study, the template of wrightiadione
served as a starting point for Trk inhibitor development campaigns. Molecular simulation provided
structural insights for the design of derivatives that were efficiently generated by our recently developed
Received 31 July 2015
Revised 24 September 2015
Accepted 29 September 2015
Available online xxxx
3-step tandem synthetic approach, resulting in the discovery of compound 2h with biochemical potency
at the single-digit micromolar level.
Keywords:
Kinase
Ó 2015 Published by Elsevier Ltd.
Trk inhibitor
Wrightiadione
Modeling
Anticancer
Since the approval of imatinib by the FDA for the treatment of
chronic myelogenous leukemia in 2001, the development of kinase
inhibitors has been a central topic in the field of drug discovery for
We recently reported a highly efficient one-pot synthetic route
consisting of 3-step tandem dehydrogenation/oxidation/oxidative
cyclization reactions using a Pd/Cu catalytic system, which enabled
the straightforward preparation of wrightiadione (1) and its
1
a variety of therapeutic applications. As a result, 26 kinase inhibi-
6
tors have been approved as drugs, and 266 human kinases have
been targeted using small molecule inhibitors.2 However, kinase
inhibitors are unevenly distributed across the human kinome:
approximately 50% of human kinases have largely been unexplored
using small molecules.2b Thus, the development of small-molecule
new chemical entities (NCEs) remains a high priority because
structurally new inhibitors spanning diverse structures and
properties can provide ample opportunities to expand chemical
spaces and to develop selective molecular probes.3 Moreover,
novel molecular frames of inhibitors may present different and
derivatives from easily accessible starting materials. With the goal
7
of identifying a new class of potent Trk inhibitors, we investigated
the binding modes of wrightiadione in the active site of Trk and
extensively modified the structure of wrightiadione. Herein we
report that wrightiadione revealed high affinity toward tropomyo-
sin receptor kinase A (TrkA) as assessed using enzymatic kinase
assays. Subsequent synthesis of wrightiadione derivatives guided
by structure-based drug design demonstrated that wrightiadione
served as a promising starting point for the development of TrkA
inhibitors, resulting in the discovery of compound 2h with sin-
gle-digit micromolar potency.
4
improved profiles of potency and selectivity. Natural products or
synthetic compounds based on natural-product pharmacophores
broaden and diversify the current chemical space and provide priv-
ileged molecular templates from which a variety of biologically
active compounds can be generated. Recognition and utilization
of the potential of natural products as novel inhibitor platforms
has been one of the most powerful methods used to produce
new classes of inhibitors in terms of molecular targets, potency,
selectivity profile, and physiochemical properties.5
Wrightiadione, isolated from the dried bark of Wrightia tomen-
tosa, is a natural product that exhibits a wide range of biological
activities, including cytotoxicity against leukemia cell lines
8
(Fig. 1). However, the molecular mechanisms underlying its anti-
cancer activities are unknown. Our docking simulation studies
indicated that the tetracyclic isoflavone moiety of wrightiadione
could fit into the ATP binding sites of kinases, and provide a new
molecular platform for the development of potential kinase inhibi-
tors (vide infra). These observations led us to hypothesize that the
anticancer activity of wrightiadione may be attributed in part to
9
the inhibition of several different kinases. Thus, wrightiadione
⇑
Corresponding author. Tel.: +82 42 350 2811; fax: +82 42 350 2812.
E-mail address: hongorg@kaist.ac.kr (S. Hong).
was subjected to enzymatic kinase assays to test the hypothesis
http://dx.doi.org/10.1016/j.bmcl.2015.09.070
960-894X/Ó 2015 Published by Elsevier Ltd.
0
Please cite this article in press as: Jeong, Y.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.070

2
Y. Jeong et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
9
O
O
10
O
O
N
1
8
B
2
3
A
7
O
4
O
O
O
Wrightiadione (1)
wrightiadione (1)
PLK3 IC50 = 8.97 µM
TrkA IC50 = 55.8 µM
2a
PLK3 IC50 > 200 µM
TrkA IC50 = 28.9 µM
Figure 1. Structure of naturally occurring wrightiadione.
Figure 3. Kinase activities of wrightiadione (1) and its derivative 2a.
by performing a high-throughput binding assay at 10 lM against a
1
0
panel of representative panel of 97 cancer-related kinases. As
shown in Figure 2, these preliminary data indicated that wrightia-
dione inhibits TrkA and PLK3 with remarkable selectivity com-
pared to other kinases (Fig. 2 and Supporting information). The
activity of wrightiadione was further confirmed using radiometric
kinase assays where wrightiadione exhibited good potency against
TrkA (IC50 = 55.8 lM) and PLK3 (IC50 = 9.0 lM). To the best of our
knowledge, the tetracyclic isoflavone scaffold of wrightiadione has
not been reported as kinase inhibitors thus far.
As a strategy to further increase potency over TrkA employing
the wrightiadione scaffold, we envisioned that the enaminone
derivatives of wrightiadione would potentially provide additional
handle to manipulate potency by modifying the N-substituents. A
representative compound 2a was obtained by employing our effi-
cient 3-step tandem synthetic method (vide infra)6 and subse-
quently subjected to IC50 measurement. Switching to the
enaminone scaffold resulted in an enhancement of the potency
against TrkA: compound 2a was found to be more potent (IC50 of
new scaffolds is highly desired to identify improved selectivity pro-
files and physiochemical properties.
To obtain structural insight into the inhibitory mechanisms of
compound 2a, its binding mode was investigated by employing
the TrkA crystal structure (PDB ID: 4AOJ) as a simulation tem-
15f
plate.
Figure 4 shows the lowest energy conformation of
11
compound 2a as calculated using Discovery Studio software.
Docking simulations indicated that the carbonyl group at C11
forms key hydrogen bonding with the backbone N–H of Met592
in the hinge region of the ATP binding site. The binding of
compound 2a can be further stabilized via hydrophobic
interactions with residues Leu516, Val524, Ala542, and Leu657.
We also observed that the ring B of compound 2a is located
adjacent to the polar residues Arg593 and Arg599. Based on
these observations, we envisaged that installation of hydrophobic
or hydrophilic groups on appropriate positions of ring B would
further strengthen the binding of the resulting derivatives of
compound 2a.
2
8.9
the replacement of an enolone system with an enaminone had
resulted in drastic loss of activity for PLK3 (e.g., 2a, IC50 > 200 M).
lM) than wrightiadione (Fig. 3). Importantly, we observed that
Based on the structural analysis, we planned to install a variety
of substituents around the wrightiadione scaffold to expand the
structure–activity relationship (SAR) profiles. Our synthetic strat-
egy for wrightiadione derivatives is illustrated in Scheme 1.
l
Considering its low molecular weight (ꢀ260), compound 2a is antic-
ipated to serve as a new Trk inhibitor scaffold from which more
potent inhibitors can be derived. Tropomyosin receptor kinases
Recently, our group reported an efficient route for the three-step
tandem reaction process employing a Pd/Cu catalytic system.6
(
Trks), which are mainly expressed in neuronal tissues, serve as
Thus, the required starting materials, 2-benzyl substituted
receptors for neurotrophins and play an important role in the devel-
opment and maintenance of the central and peripheral nervous sys-
chromanones or enaminones 3 were conveniently prepared by 1,4-
16
addition using appropriate benzyl cuprate reagents. Next, a
tems. In addition to their role in chronic pain and inflammation,13
numerous reports have indicated that Trks are involved in malig-
nant transformation, metastasis, survival, migration and invasion
signaling in a variety of human cancers, including prostate, colorec-
12
tandem dehydrogenation/oxidation/oxidative cyclization process
from 2-benzyl dihydroquinolinones successfully provided the
desired wrightiadione derivatives in moderate to good yields.
The IC50 values of the synthesized derivatives were then deter-
mined for TrkA (Table 1). Our early investigation on the effects of
the substituents at the C3 position of ring A suggested that sub-
stituents on the Ring A might not be beneficial to enhance the
potency (2b and 2c), which prompted us to study the effect of sub-
stituents on the Ring B. Again, installing a methyl group on various
1
4
tal, pancreatic, breast and lung cancers and neuroblastoma. Thus,
the development of Trk kinase inhibitors has received considerable
attention from the field of drug discovery.15 However, most Trk
kinase inhibitors have been derived from relatively well-known
kinase scaffolds, and the development of Trk inhibitors based on
Figure 2. Selected results of the KINOMEscan profile of wrightiadione (1). Panel of 97 kinases were tested at 10 lM in a high-throughput binding assay (KINOMEscan).
POC = percent of control; values are the average of duplicate measurement; lower values indicate stronger hits (see Supporting information for details).
Please cite this article in press as: Jeong, Y.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.09.070

Y. Jeong et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Figure 5. Calculated binding mode of compound 2h in the ATP binding pocket of
TrkA (PDB ID: 4AOJ).
Figure 4. Calculated binding mode of compound 2a in the ATP binding pocket of
TrkA (PDB ID: 4AOJ).
active site of TrkA via a key hydrogen bond with the backbone
N–H of Met592 and hydrophobic interactions with residues
Leu516, Val524, Ala542 and Leu657.
In conclusion, we discovered that the natural product wrightia-
dione can serve as a new template for the development of kinase
inhibitors. The wrightiadione scaffold could be easily equipped
with an additional substituent using the newly developed syn-
thetic approach and we successfully identified compound 2h with
positions of the Ring B was detrimental to the overall activity
(2d–2f). We then speculated that the introduction of substituents
on Ring B that can form molecular interactions with polar residues,
such as Arg593 and Arg599, might be helpful to enhance the
potency against TrkA. Importantly, as presented in compounds
2
h and 2i, a methoxy substituent at C8 or C9 of Ring B was advan-
tageous in improving TrkA activity: compound 2h showed an IC50
of 6.6 M, featuring our most potent TrkA inhibitor at this stage.
l
potent activity (IC50 = 6.6 lM), thereby expanding the chemical
Placing substituents at proper locations appeared to be important,
as compounds 2g and 2j were not active at all. We also tested dif-
ferent groups on the nitrogen atom. Interestingly, replacement of
the methyl group with either the benzyl group or ethyl ester was
well tolerated, and as a result, compounds 2k and 2l was approx-
imately two-fold more potent than compound 2a. However, when
we evaluated compound 2m, which was obtained by hybridizing
compounds 2h and 2k, only a modest increase was observed.
The most potent derivative 2h exhibited similar configurations
with comparable interactions with the amino acid residues in the
active site (Fig. 5). Moreover, docking simulations clearly indicated
that the methoxy group at C8 of 2h appeared to form two addi-
tional hydrogen bonds with the backbone carbonyl oxygen of
Arg593 and guanidine group of Arg599 (Fig. 5). Presumably, these
additional molecular interactions of 2h might be attributed to the
improved potency, which was approximately 4 times greater than
that of 2a. Compound 2h could also be further stabilized in the
space of TrkA inhibitors. The molecular simulation results provided
insights into the generation of potent TrkA inhibitors. Overexpres-
sion or activating mutation of TrkA has been implicated in human
1
7,18
acute myeloid leukemia (AML) cells.
As wrightiadione dis-
played cytotoxicity against murine leukemia cell lines,8 we envi-
sion that our wrightiadione-based TrkA inhibitors may show
promising anticancer effects for human AML. Further studies
Table 1
Structure and activity relationship (SAR) of wrightiadione derivatives
1
0
9
2
O
R
8
1
2
B
R1
A
7
3
N
R
4
3
O
2
O
O
X
Compd
R1
R2
R3
TrkA IC50a
(lM)
a
b
R1
R1
R2
2a
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Bn
28.9
117
88.5
73.3
47.2
—
X
X = O or NR
3
2b
2c
3-CF
3-Cl
H
H
H
H
H
H
H
3
2
2
2
2
2
2
d
e
f
g
h
i
7-Me
8-Me
9-Me
7-OH
8-OMe
9-OMe
10-OMe
H
H
4
O
O
X
R2
141
6.6
R1
R2
R1
14.4
209
10.3
16.5
16.4
X
2j
2k
O
H
H
O
1
, 2a-2m
2l
CO
Bn
2
Et
2m
H
8-OMe
Scheme 1. Synthetic route of wrightiadione derivatives. Reagents and conditions:
a) R MgX (X = Cl, Br), CuI, TMSCl, THF/DCM, À78 °C, 41–89%; (b) Pd(OAc)
OAc) , Ag O, NaOAc, PivOH, O , 120 °C, 50–76%.
2
a
(
(
2
, Cu
The IC50 measurements were performed at the Reaction Biology Corp (Malvern,
PA, USA).
2
2
2
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Y. Jeong et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
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0. POC determinations were performed by the KINOMEscan Corp. (San Diego, CA,
1
USA).
Acknowledgment
11. The IC50 determinations were performed using radiometric kinase assays
33
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