Potent inhibition of checkpoint kinase activity by a hymenialdisine-derived indoloazepine

Article abstract of DOI:10.1016/j.bmcl.2004.05.079

The marine sponge metabolite hymenialdisine is a potent inhibitor of a variety of kinases including MEK-1, GSK-3β, and CK1. In addition, hymenialdisine and debromohymenialdisine exhibit inhibition of the G₂ cell cycle checkpoint at micromolar c

Full text of DOI:10.1016/j.bmcl.2004.05.079

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4319–4321
Potent inhibition of checkpoint kinase activity by a
hymenialdisine-derived indoloazepine
q
Vasudha Sharma and Jetze J. Tepe*
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA
Received 22 March 2004; revised 26 May 2004; accepted 27 May 2004
Available online 19 June 2004
Abstract—The marine sponge metabolite hymenialdisine is a potent inhibitor of a variety of kinases including MEK-1, GSK-3b, and
CK1. In addition, hymenialdisine and debromohymenialdisine exhibit inhibition of the G
concentrations. We report herein the potent inhibition of cell cycle kinase Chk2 by the indolic-hymenialdisine indoloazepine 1
IC50 ¼ 8 nM).
2004 Elsevier Ltd. All rights reserved.
2
cell cycle checkpoint at micromolar
(
ꢀ
1
. Introduction
The marine sponge metabolite hymenialdisine (Fig. 1)
was originally isolated from the sponges Axinella
verrucosa and Acantella aurantiaca and its structure was
DNA replication is a process that requires great accu-
racy and relies on surveillance mechanisms, which
monitor DNA damage and initiate DNA repair. The
inability to carry out DNA repair often leads to the
9
established on the basis of X-ray crystallography. Hy-
1
menialdisine and other oroidin compounds have been
established to be potent anti-proliferative, anti-neuro-
degenerative, and anti-inflammatory agents in various
2
transformation of normal cells into malignancies.
1
0–14
Upon DNA damage, cell cycle checkpoints get acti-
vated, which delay the cell cycle progression and
allow for DNA repair. A multi-faceted involvement of
cell lines and animal models.
found to be a potent kinase inhibitor of several related
Hymenialdisine was
10
CDKs. In addition, hymenialdisine was found to be a
potent inhibitor of the nuclear transcription factor
NF-jB and acts as a competitive nanomolar inhibitor
1
–3
these checkpoint pathways regulate DNA repair,
4
11
telomere length, and the induction of apoptotic cell
death.
1;5
of the cyclin-dependent kinases, GSK-3b and CK1 with
IC50 values of 10 and 35 nM, respectively. Crystallo-
graphic data portrayed the binding of the kinase inhib-
12
Protein kinases regulate a host of cellular processes such
as growth and differentiation, cell proliferation, and
apoptosis. DNA damage caused by radiation or che-
motherapy triggers the DNA damage-responsive protein
kinases ATM and ATR, which activate Chk1 and Chk2.
Chk1 and Chk2 in turn phosphorylate Cdc25 and pre-
vent Cdc2 activation, resulting in cell cycle arrest.
Hence, small molecules that can inhibit the checkpoints
may enhance the efficacy of DNA damaging chemo-
12
itor in the ATP binding pocket of the kinases. In
addition, Ireland and co-workers have identified hy-
menialdisine as a very potent mitogen-activated protein
kinase kinase-1 (MEK-1) inhibitor with low nanomolar
6
7
H
H
N
H
N
H N
N
2
H N
2
H N
2
8
O
therapeutics or radiation therapy.
O
O
N
N
N
NH
NH
NH
N
Br
N
N
H
O
Keywords: Hymenialdisine; Kinase; Checkpoint; Chk2; Apoptosis.
q
Supplementary data associated with this article can be found, in the
H
O
H
O
debromohymenialdisine
hymenialdisine
1
online version, at doi:10.1016/j.bmcl.2004.05.079
Corresponding author. Tel.: +1-517-355-9715; fax: +1-517-353-1322;
e-mail: tepe@cem.msu.edu
*
Figure 1. Structures of debromohymenialdisine, hymenialdisine, and
hymenialdisine derivative 1.
0
960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.079

4320
V. Sharma, J. J. Tepe / Bioorg. Med. Chem. Lett. 14 (2004) 4319–4321
HN
O
O
NH2CH2CH2COOEt
EDCI.HCl / CH2Cl2
LiOH / H O
2
COOH
N
H
N
H
OEt
9
8 %
93 %
O
Ph
O
P2O5,MeSO3H
10 0
5%
O
HN
O
O
N
NH
1
C
N
OH
N
TiCl / THF, Py
O
4
H
H
8
55%
H
O
N
O
SBn
NH.HCl
N
O
Ph
H N
2
H2N
N
NH
NH
LiH, EtOH
8%
N
H
O
N
2
O
H
Scheme 1. Synthesis of indolic-hymenialdisine derivative 1.
Table 1. IC50 values for kinase inhibition by compound 1, hymenialdisine, and debromohymenialdisine
Kinase
IC50 (nM) compound 1
Hymenialdisine
Debromohymenialdisine
1
1
1
1
1
0;12
0;12
3
CK1d(h)
CK2(h)
MEK1(h)
PKCa(h)
PKCbII(h)
Chk1
1352
>10,000
89
35
7000
6
NA
NA
13
824
0;12
0;12
2539
3381
237
700
1200
1950
42
NA
NA
725
183
Chk2
8
NA: value not available.
1
3
IC50 values (IC50 ¼ 6 nM). In these studies we describe
the synthesis and kinase activity of the indole-derivative
of hymenialdisine, 1.
Here, we present the potent inhibitory activity of the
indoloazepine 1 against several purified kinases includ-
15
ing the checkpoint kinases Chk1 and Chk2. Table 1
illustrates the comparison between the in vitro activity
of indoloazepine 1, hymenialdisine, and debromohy-
menialdisine. Compound 1 exhibits very potent inhibi-
tion of Chk2 activity in the low nanomolar range
2
. Chemistry and biology
Roberge and co-workers have shown the inhibition of
the G DNA damage checkpoint by debromohymeni-
aldisine as well as hymenialdisine in cells. Inhibition
of Chk1 and Chk2 by debromohymenialdisine was
reported to correspond with IC50 values of 3 and
.5 lM, respectively. Both debromohymenialdisine and
hymenialdisine showed comparable IC50 values for
inhibition of the G checkpoint at 8 and 6 lM, respec-
(
IC50 ¼ 8 nM). Interestingly, unlike the natural product
2
hymenialdisine, compound 1 exhibits an increased
selectivity for the checkpoint kinases. Compound 1
exhibited a significant increase in potency for Chk 1 and
Chk2 inhibition when compared to hymenialdisine.
However, a significant drop in activity was found for all
other kinases tested.
7
3
2
tively. This indicates that the bromide at the C2 position
These kinase inhibition studies suggest that analogs of
hymenialdisine may improve its overall kinase profile
and significantly affect its kinase selectivity. Further in
vivo studies would help in validating the therapeutic
potential of hymenialdisine analogs for cancer therapy
and neurodegenerative disorders.
of hymenialdisine did not significantly influence the
7
2
inhibition of the G checkpoint.
As a part of a program to develop analogs of the oroidin
family of natural products, we recently synthesized 1
from the indole carboxylic acid in five steps and overall
14
yield of 12% as shown in Scheme 1. The indole-based
hymenialdisine derivative 1 was found to contain potent
anti-inflammatory activity as well as anti-proliferative
14
activity. Concurrently with our studies, the inhibition
of purified GSK-3b, CDK5/p25, and CDK1/cyclin B by
several hymenialdisine derivatives was investigated.
Acknowledgements
10
The authors gratefully acknowledge the financial sup-
port by Research Scholar Grant RSG-04-018-01-CDD
from the American Cancer Society and Michigan State
University.
These studies indicated that the indole analogs exhibited
kinase inhibition profiles similar to those of the pyrrole-
based compounds.

V. Sharma, J. J. Tepe / Bioorg. Med. Chem. Lett. 14 (2004) 4319–4321
4321
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5. In vitro kinase assay: Compound 1, hymenialdisine and
debromohymenialdisine were tested in vitro against the
kinases indicated by Upstate, UK using a Kinase Profiler
Assay according to the manufacturer’s protocol. Briefly, in
a final volume of 25 lL, the kinase was incubated with the
desired buffer and the required polypeptide substrate, in
8
33
presence of 10 mM magnesium acetate and c- P-ATP
10 lM). After incubation for 40 min at room temperature,
the reaction was stopped by the addition of 3% H PO
5 lL). A 10 lL of the reaction was then spotted on a P30
PO and finally in
(
3
4
(
filtermat and washed 3· in 75 mM H
3
4
methanol. Samples were then dried and signals counted on
a scintillation counter.
2
002, 62, 5743–5748.