Efficient synthesis of (±)-parasitenone, a novel inhibitor of NF-κB

Article abstract of DOI:10.1016/j.bmcl.2009.07.120

Dehydroxymethylepoxyquinomicin (DHMEQ, 1) is a novel nuclear factor-κB (NF-κB) inhibitor that inhibits DNA binding of NF-κB components including p65. To inspect its biological activity of 1, we synthesized parasitenone (3), possessing the common epoxycyclohexenone moiety of 1. Assessment of the inhibitory activity against NF-κB indicated that the epoxycyclohexenone moiety is the most essential element for the NF-κB inhibitory activity and the salicylic acid moiety may contribute the binding efficiency and specificity.

Full text of DOI:10.1016/j.bmcl.2009.07.120

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5383–5386
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of ( )-parasitenone, a novel inhibitor of NF-
jB
Tsuyoshi Saitoh a, Eriko Suzuki b, Arisa Takasugi b, Rika Obata a, Yuichi Ishikawa a,
Kazuo Umezawa b, Shigeru Nishiyama a,
*
a Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan
b Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Dehydroxymethylepoxyquinomicin (DHMEQ, 1) is a novel nuclear factor-
j
B (NF-jB) inhibitor that inhib-
Received 12 May 2009
Revised 24 July 2009
Accepted 27 July 2009
Available online 30 July 2009
its DNA binding of NF-
sized parasitenone (3), possessing the common epoxycyclohexenone moiety of 1. Assessment of the
inhibitory activity against NF- B indicated that the epoxycyclohexenone moiety is the most essential ele-
jB components including p65. To inspect its biological activity of 1, we synthe-
j
ment for the NF-
j
B inhibitory activity and the salicylic acid moiety may contribute the binding efficiency
and specificity.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
NF-jB inhibitor
DHMEQ
Parasitenone
Anodic oxidation
Epoxycyclohexenone
Binding of nuclear factor-
j
B (NF-
j
B) to the
j
B sequence on tar-
same epoxycyclohexenone structure as 1 prompted us to initiate
its synthesis and assess the NF- B inhibitory activity.
get genes induces the transcription of several inflammatory cyto-
kines, such as interleukins, TNF- , and antiapoptotic proteins.
Therefore, NF- B inhibitors were expected to be novel candidates
j
a
Selective reduction of phillostin (6),6 assisted with the neigh-
boring primary hydroxyl group, was expected to be a good ap-
proach to the target molecule 3 (Scheme 1). The asymmetric
structure of 6 was accessible by selective epoxidation to the ste-
rically less hindered site of 7 or 8, which was readily produced
by anodic oxidation of 9, followed by selective hydrolysis.
According to the synthetic plan mentioned above, the synthesis
was commenced by NaBH4 reduction of commercially available
2,5-dimethoxybenzaldehyde (10), followed by protection of the
benzyl alcohol to afford the siloxy ether 9 (98% in two steps).
j
as anti-inflammatory and anticancer agents.
DHMEQ (1), designed based on the structure of epoxyquinomi-
cin C (2) and synthesized by Umezawa and co-workers,1 exhibited
a remarkable inhibitory effect (Fig. 1). The known NF-
such as panepoxydon (4) and cycloepoxydon (5), inhibit the activa-
tion of NF- B by interference with the degradation process of I B-
and activation of IKK kinase.2,3 Despite the structural similarity
to 4 and 5, 1 showed a completely different mode of action: 1 spe-
jB inhibitors,
j
j
a
cifically deactivates NF-
jB by inhibiting the DNA binding of NF-jB
components.4
In the previous study, it was observed that the epoxycyclohex-
enone moiety of 1 covalently binds the 38th cysteine residue of
OH
O
p65 to appear the inhibitory activity against NF-j
B.4 To under-
OH
O
H
H
N
N
stand the detailed mode of action, and develop more effective
inhibitor, biological activities of the epoxycyclohexanone were
investigated. The appropriate substrate carrying the same cis
relationship of the corresponding OH and epoxide groups as that
of 1 was parasitenone (3), isolated from the marine algicolous
fungus Aspergillus parasiticus.5 It showed mild scavenging activity
of free radicals, such as 1,1-diphenyl-2-picrylhydrazyl (DPPH,
O
O
O
OH
O
OH
HO
Epoxyquinomicin C (2)
DHMEQ (1)
OH
OH OH
OH OH
O
O
O
O
IC50 = 57.0 l lM). The
M) and peroxynitrite (ONOOÀ) (IC50 = 52.6
O
OH
O
O
Parasitenone (3) Panepoxydon (4) Cycloepoxydon (5)
* Corresponding author. Tel./fax: +81 45 566 1717.
E-mail address: nisiyama@chem.keio.ac.jp (S. Nishiyama).
Figure 1. Bioactive compounds with the epoxycyclohexenone moiety.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.120

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T. Saitoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5383–5386
OMe
CHO
OMe
OMe
MeO OMe
OTBDPS
a), b)
c), d)
e)
OTBDPS
OTBDPS
O
OMe
10
O
9
7
8
O
OH
OH OH
OTBDPS
f)
g)
h)
O
O
O
O
O
O
racemic-
11
6
3
racemic-
racemic-
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH, 0 °C; (b) TBDPSCl, Imid., DMF, rt, 98% in two steps; (c) C.P.E. (1.15 V vs SCE.), 1% KOH/ MeOH, platinum wire
(cathode)—platinum net (anode), 0 °C; (d) 5% AcOH, acetone, quant. in two steps; (e) propane-1,3-diol, PPTS, benzene, reflux, 90%; (f) TBHP, tBuOK, THF, À78 to À35 °C, 57%,
(8, 39%), (g) HF-pyr., CH3CN; (h) NaBH(OAc)3, MeOH, 0 °C, 43% in two steps.
Anodic oxidation of 9 in 1% KOH–MeOH7 provided the corre-
sponding bisdimethylacetal as the two-electron oxidation prod-
uct, which on regioselective hydrolysis8 yielded the vinylogous
dimethylacetal 7 (100% in two steps). A number of efforts for di-
rect epoxidation were unsuccessful. Thus, the substrate of the
epoxidation reaction was changed to 8 (90%) by the Porco proto-
col.9 Epoxidation of 8 with tBuOOH and tBuOK provided the de-
sired epoxide 11 (57%, conversion yield: 98%), which on
treatment with HF-pyr. provided phillostin (6). Finally, NaB-
H(OAc)3 reduction of 6 effected the hydride-attack only from
the desired side to produce ( )-parasitenone (3) as a single dia-
stereomer (43% in two steps). The spectral data of synthetic 3
was identical to the reported data.10
DHMEQ (1) suppressed the LPS (lipopolysaccharide)-induced
secretion of inflammatory mediators and cytokines, such as iNOS,
COX-2, IL-6, and TNF-a, in the mouse macrophage cell line
RAW264.7.11 We evaluated the inhibitory activities of parasite-
none (3) against secretion of inflammatory mediators and cyto-
kines. Thus, 3 was subjected to the assay of NO production,
which is an inflammatory mediator in LPS-stimulated RAW264.7
cells.
A: Cell viability
100
80
60
As shown in Figure 2, 3 lowered cell viability at 30
while at 10 g/mL it only inhibited cell growth. Compound 3
clearly suppressed NO production in a dose-dependent manner
(Fig. 3A). In contrast to salicylic acid, at 10 g/mL, 3 decreased
lg/mL,
l
40
20
0
24 h
48 h
l
NO production to the control level. NO is produced by inducible
NO synthase (iNOS), the expression of which is mediated by
NF-
dependent manner (Fig. 3B). Accordingly, we assessed the effects
of racemic 1 and 3 on the transcriptional activity of NF- B in
RAW264.7 cell line. In Figure 4, compound 3 inhibited LPS-in-
jB. Compound 3 also diminished iNOS protein in a dose-
0
1
10
100
j
Racemic parasitenone (3) (μg/mL)
duced transcription of NF-jB, although it did so more weakly
than 1.
B: Cell growth
Parasitenone (3) was shown to covalently bind to the NF-jB
120
component p65 in MALDI-TOF MS analysis (Fig. 5), although
the binding was weak and non-specific compared with DHMEQ
(1).4
0 μg/mL
1 μg/mL
3 μg/mL
10 μg/mL
30 μg/mL
100
80
60
40
20
0
In conclusion, effective synthesis of racemic parasitenone
(3) was accomplished. Assessment of
iNOS induction, and NF- activation, indicated that the
epoxycyclohexenone core is the crucial factor for the inhibi-
tory activity against NF- B, while salicylic acid showed no
inhibition of NF- (Fig. 3A). Despite lower activities than
3 by NO production,
jB
j
jB
those of 1, the dose-dependent inhibition of 3 was consistent
with our previous observation that the epoxycyclohexenone
core may react with the 38th cysteine residue.4 However,
the biological effect and the p65 binding ability were lower
than DHMEQ. Therefore, the salicylic acid moiety is likely to
be required for more efficient and specific covalent-binding
to the cysteine residue.
0
24
48
Time (h)
Figure 2. Effect of parasitenone (3) on cell viability (A) and cell growth (B) in
RAW264.7 cells. The cell viability was assessed by trypan blue dye exclusion.

T. Saitoh et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5383–5386
5385
20
10
0
A
24h
DHMEQ (1)
Parasiten one (3)
salicylic acid
(μg/mL)
control
0
1
3
10
1 μg/mL LPS
1 μg/mL LPS
3 (μg/mL)
B
control
0
1
3
10
1 (10 μg/mL)
iNOS
135 kDa
55 kDa
tubulin
Figure 3. Inhibition of NO production and iNOS expression by parasitenone (3). (A) Effect on NO secretion in RAW264.7 cells. Cells were incubated without or with chemicals
at various concentrations for 1 h, stimulated or not 1 g/mL LPS for 24 h. NO secretion was assessed by Griess reaction. (B) Effect on iNOS expression. Cells were incubated
l
with or without parasitenone (3) at various concentration for 1 h, then stimulated with 1
lg/mL LPS for 24 h. Total cell lysates were subjected to SDS–PAGE and
immunoblotted with anti-iNOS anti-body. (Tublin was used as a control.)
37780
7000
6000
5000
4000
3000
37550 37720
37880
control
DHMEQ (1) 2 eq
parasitenone (3)10 eq
100 eq
Figure 5. MALDI-TOF MS analysis of p65(1-325) with parasitenone (3) and DHMEQ
(1). The p65(1-325) protein (20 M) was treated with several equiv of racemic
parasitenone (3) and racemic DHMEQ (1) for 1 h. After incubation, the proteins
2000
1000
0
l
were used for the MALDI-TOF MS analysis.
Acknowledgments
control
0
1
3
10
1
3
10
This work was supported by High-Tech Research Center Project
for subsidy from MEXT, 2006–2011, Japan. T.S. was indebted to
Global COE Program ‘Center for Human Metabolomic Systems Biol-
ogy’ from MEXT.
1 (μg/mL)
3 (μg/mL)
1 μg/mL LPS
Figure 4. Effect of parasitenone (3) on transcription activity of NF-
jB in RAW264.7
cells. Cells were transfected by diethylaminoethyl–dextran method and incubated
for 24 h. Transfected cells were treated with the indicated concentrations of
References and notes
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prepared and assayed for luciferase activity. Each value is the mean SD of
triplicate determinations.
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