A new NF-κB inhibitor based on the amino-epoxyquinol core of DHMEQ

Article abstract of DOI:10.1016/j.bmcl.2010.08.036

The amino-epoxyquinols 6a and 6b were synthesized as soluble derivatives of an NF-κB inhibitor DHMEQ (1). In spite of the opposite configuration from 1, 6b rather than 6a affected the deactivation of NF-κB, based on NO secretion and MALDI-TOF MS analysis.

Full text of DOI:10.1016/j.bmcl.2010.08.036

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5638–5642
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A new NF-
jB inhibitor based on the amino-epoxyquinol core of DHMEQ
Tsuyoshi Saitoh a, Chika Shimada b, Masatoshi Takeiri b, Mitsuhiro Shiino b, Shigeru Ohba c, 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
c Department of Chemistry, Faculty of Letters, Keio University, Hiyoshi 4-1-1, Kohoku-ku, Yokohama 223-8521, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The amino-epoxyquinols 6a and 6b were synthesized as soluble derivatives of an NF-
(1). In spite of the opposite configuration from 1, 6b rather than 6a affected the deactivation of NF-
based on NO secretion and MALDI-TOF MS analysis. It was indicated that 6b inhibited the activation
by different manner from that of 1.
j
B inhibitor DHMEQ
B,
Received 12 July 2010
Revised 4 August 2010
Accepted 6 August 2010
Available online 11 August 2010
j
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
NF-jB inhibitor
Epoxyquinol derivatives
DHMEQ
The NF-
inflammation but also in the development of cancer. Therefore,
NF- B inhibitors are expected to be novel candidates as chemo-
therapeutic agents for inflammatory and cancer diseases, as well
as bioprobes for the characterization of intracellular biological re-
sponses and cell function.1 Over the past decade, a number of
j
B signaling pathway plays a central role not only in
structure. In fact, although isolation of MM14201 (Fig. 1), which
is a regioisomer of 5, has been reported, its biological properties
have not been discussed due to its instability.6 Therefore, we pre-
pared the N-protected epoxyquinol 6 and studied their inhibitory
activities.
j
Wipf et al. reported an efficient route for obtaining amino-substi-
structurally diverse small molecules that block the NF-
pathway, have been identified. Especially, the epoxyquinol class
NF-
B inhibitors, such as DHMEQ (1),2 cycloepoxydon (2),3 and
panepoxydon (3),4 were found to exhibit remarkable inhibitory
activity against NF- B activation (Fig. 1). In our previous study,
jB signaling
tuted epoxyquinols in the synthesis of LL-C10037a
(Scheme 1).7 We
used their intermediate 11 for synthesis of our desired epoxyquinol
derivatives with several improvements. As shown in Scheme 1, the
synthesis commenced with protection of 2,5-dimethoxyanilline
(7) to afford the protected 8. Oxidation by iodobenzene diacetate
under neutral conditions afforded the acetal 9, which was regiose-
lectively hydrolyzed in AcOH–acetone, yielding the dimethoxyqui-
none 10 (80% in three steps). Epoxidation of 10 with H2O2 and
j
j
we synthesized parasitenone (4) carrying the same epoxyquinol
moiety as DHMEQ (1), and the quinol moiety was shown to be cru-
cial for its inhibitory activity against NF-
their structural similarity (Fig. 1), all of them showed completely
different modes of action in the inhibition of NF- B activation.
Whereas DHMEQ (1) exhibits inhibitory activity by covalently
binding to the NF- B components, cycloepoxydon (2) and panep-
oxydon (3) show inhibition by interfering with the degradation
of I B- and activation of I
B kinase (IKK).3,4 In this study, we syn-
j
B activation.5 Despite
j
OH
O
OH OH
OH OH
O
OH
O
H
N
j
O
O
O
O
O
O
OH
OH
O
j
a
j
DHMEQ (1)
Panepoxydon (2)
Cycloepoxydon (3) Parasitenone (4)
thesized an epoxyquinol derivative carrying the same amide moi-
ety as 1 to examine the structure–activity relationship.
Compound 5, with a free amine instead of the salicylamide of
DHMEQ (1), was considered the simplest analog of 1. However,
several attempts to obtain 5 in the pure state have been unsuccess-
ful, probably due to the extremely unstable b-keto enamine
OH
H
OH
H
O
N
N
NH2
R
O
O
O
O
OH
O
O
OH
DHMEQ (1)
5: R = H
6: R = Alloc
MM14201
⇑
Corresponding author. Tel./fax: +81 45 566 1717.
E-mail address: nisiyama@chem.keio.ac.jp (S. Nishiyama).
Figure 1. NF-jB inhibitors possessing epoxyquinol moiety.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.036

T. Saitoh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5638–5642
5639
Wipf's total synthesis of LL-C10037a
O
O
OMe
OMe
NHAlloc
OMe
NH2
O
MeO
NHAc
O
HO
2,5-dimethoxyanilline (7)
LL-C10037α
11
OMe
OMe
NHAlloc
OMe
NH2
OMe
a
b
MeO
MeO
MeO
MeO
NHAlloc
8
9
2,5-dimethoxyanilline (7)
OMe
OMe
NHAlloc
O
OMe
OMe
NHAlloc
11
c
d, e
f
O
O
10
O
O
O
O
g
OH
NHAlloc
6a (syn)
OH
O
NHAlloc
O
O
NHAlloc
6b (anti)
12
Scheme 1. Reagents and conditions: (a) AllocCl, TEA, THF, rt; (b) PhI(OAc)2, NaHCO3, MeOH, rt; (c) AcOH, acetone, rt, 80% in three steps; (d) 30% H2O2 aq, 1 M K2CO3 aq, THF;
(e) TsOHꢀH2O, acetone, 60% in two steps; (f) TFA; (g) NaBH(OAc)3, MeOH, 60% in two steps, 6a:6b = 4:1.
A
B LPS (1 μg/mL)
-
-
+
6 h
6b
10 30
6a
1
-
1
3
1
3 10 30 10
(μg/mL)
iNOS
α-tubulin
Figure 2. Inhibition of NO secretion and iNOS expression by 6a and 6b. (A) Effects on NO secretion in RAW264.7 cells and cell viability. Cells were treated with or without
chemicals at indicated concentrations for 1 h, and stimulated with or without 1 g/mL LPS for 24 h. NO secretion was assessed by Griess reaction. The cell viability was
l
assessed by MTT exclusion. (B) Effects on iNOS expression. Cells were treated with or without 6a and 6b at indicated concentrations for 1 h, then stimulated with 1 lg/mL LPS
for 6 h. Total cell lysates were subjected to SDS-PAGE and immunoblotted with anti-iNOS antibody. Tubulin was used as a control.
K2CO3 in THF gave a 3:2 mixture of 11 and 10, which on treatment
with TsOHꢀH2O in acetone gave 11 (60% in two steps). During this
reaction, the unreacted acetal 10 was smoothly deprotected into
the corresponding benzoquinone, whereas 11 remained intact

5640
T. Saitoh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5638–5642
of a ketone to afford a mixture of the epoxyquinols8 (60% in two
steps, 6a (syn):6b (anti) = 4:1).
DHMEQ (1) suppressed the lipopolysaccharide (LPS)-induced
expression of inflammatory mediators and cytokines, such as iNOS,
-
-
+
LPS (1 μg/mL)
30 min
6b
1
ss
1
3
10 30 10
(μg/mL)
-
COX-2, IL-6, and TNF-a, in the mouse macrophage cell line
RAW264.7.9 We evaluated the inhibitory activities of syn- and
anti-derivatives (6a and 6b) carrying the amino-substituted epox-
yquinol moiety similar to 1, against secretion of inflammatory
mediators.
NF-κB
Compound 6a, which has the same configuration as that of 1,
scarcely inhibited NO production at even 30
hand, 6b dose-dependently reduced NO production; at 30
l
g/mL. On the other
g/mL,
l
and showed cellar toxicity (Fig. 2A). Inducible NO synthase (iNOS)
is also a major inflammatory mediator contributing to the patho-
genesis of cancer and inflammation. As shown in Fig. 2B, 6b inhib-
ited LPS-induced iNOS expression again more strongly than 6a.
Inhibition of NF-
ity shift assay (EMSA), as shown in Figure 3. Since 1 is known to
bind to NF-
B components, including p65,10 the binding property
jB by 6b was confirmed by electrophoretic mobil-
Free probe
j
of 6a and 6b with p65 was evaluated by MALDI-TOF MS analysis
(Fig. 4). Compound 6a showed a mass-shift at 4 equiv mol concen-
tration (1 effected the mass-shift by addition of 2 equiv mol),
whereas no shift was observed in 6b. It was suggested that 6a
binds weakly and nonspecifically to p65, and 6b did not bind to
p65. Therefore, the mechanism of inhibition should be different
from that of 1.
Figure 3. Effects of 6b on NF-
treated with or without 6b, then stimulated or not 1
thereafter, total nuclear extracts containing equal amounts of protein (5
then analyzed by electrophoretic mobility shift assay (EMSA) for DNA binding
j
B activation in RAW264.7 cells. RAW264.7 cells were
g/mL LPS for 30 min, and
g) were
l
l
activity of NF-j
B using a 32P-labeled oligonucleotide with the high affinity site
50-AGTTGAGGGGACTTTCCCAGGC-30. The inducible NF-
section of the fluorogram from a native gel is shown.
jB complex is indicated. A
To examine the effects of chirality, optical resolution of 6b was
attempted. The optical resolution was accomplished with a chiral
stationary phase column (Daicel Chiralpak AS) with MeOH as the
under the reaction conditions. Finally, the dimethoxy acetal of 11
was removed by trifluoroacetic acid, followed by selective reduction
p65(325)
wild type
2 eq.
4 eq.
6a
6b
control (DMSO only)
6a or 6b
Figure 4. MALDI-TOF MS analysis of p65(1–325) with 6a and 6b. Recombinant p65(1–325) was treated with or without 2 or 4 equiv ( )-6a and 6b. The p65(1–325) protein
(20 M) was treated with indicated equivalent of ( )-6a or 6b for 1 h at 4 °C. After incubation, the proteins were used for MALDI-TOF MS analysis.
l

T. Saitoh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5638–5642
5641
sampl - CH2
1000000
(-)-6b
(+)-6b
500000
0
5.0
10.0
15.0
20.0
0.0
Retention Time [min]
Figure 5. Chiral HPLC profile of 6b. Column: Daicel Chiralpak AS, column size: 10 mm I.D. 250 mm; eluent: methanol; flow rate: 2.0 mL/min; detection: 274 mm.
Figure 6. Inhibition of NO secretion by (+)-6b and (ꢁ)-6b. Effects on NO secretion in RAW264.7 cells and cell viability. Cells were treated with or without chemicals at
indicated concentrations for 1 h, and stimulated with or without 1
exclusion.
lg/mL LPS for 24 h. NO secretion was assessed by Griess reaction. The cell viability was assessed by MTT
eluent. Figure 5 shows the HPLC profile of 6b. Aliquots of 30 mg of
racemic 6b were added to the chiral column to give 11.8 and
8.5 mg of the corresponding (ꢁ)- and (+)-enantiomers. The former
bility of 6b in such solvents as MeOH, EtOH, and MeCN will
provide further biological information.
showed optical rotation of ½a D25
ꢁ103.3 (c 1.00, MeCN), while the
ꢂ
Acknowledgments
latter showed optical rotation of ½a D25
ꢂ
+96.5 (c 1.00, MeCN). Their
enantiomeric purities were >98% ee based on the results of HPLC
analyzes. In NO secretion assay of DHMEQ enantiomers, we ob-
served that (ꢁ)-1 was 10-times more effective than (+)-1.11 How-
ever, as can be seen in Figure 6, approximately same activity was
observed in the case of (+)-6b and (–)-6b.
This work was supported by the High-Tech Research Center
Project for Private Universities Matching Fund (2006–2011) from
MEXT, The Science Research Promotion Fund from The Promotion
and Mutual Aid Corporation for Private Schools of Japan, and the
program Promotion of Fundamental Studies in Health Sciences of
the National Institute of Biomedical Innovation (NIBIO), 2006–
2011. T.S. was indebted to Global COE Program ‘Center for Human
Metabolomic Systems Biology’ from MEXT.
In conclusion, we synthesized the N-protected amino-epoxyqu-
inol derivatives 6a and 6b, and assessed their inhibitory activities
against NF-jB activation. The results of the biological investigation
of 6a, which has the same configuration of the epoxyquinol moiety
as that of 1, indicated that the syn relationship of the epoxy-alcohol
References and notes
is crucial for binding to the cysteine residue of NF-
6b inhibited the NF- B activation by a different mode of action
from 1, which may inhibit the IKK activity. The syn- and anti-ste-
reochemistry controlled binding to NF- B. Furthermore, although
jB. Compound
j
1. Kataoka, T. J. Antibiot. 2009, 62, 655.
2. Ariga, A.; Namekawa, J.; Matsumoto, N.; Inoue, J.; Umezawa, K. J. Biol. Chem.
2002, 277, 24625.
3. Gehrt, A.; Erkel, G.; Anke, T.; Sterner, O. J. Antibiot. 1998, 51, 455.
4. Erkel, G.; Anke, T.; Sterner, O. Biochem. Biophys. Res. Commun. 1996, 226, 214.
5. Saitoh, T.; Suzuki, E.; Takasugi, A.; Obata, R.; Ishikawa, Y.; Umezawa, K.;
Nishiyama, S. Bioorg. Med. Chem. Lett. 2009, 19, 5383.
j
DHMEQ (1) exhibited remarkable activity, 1 has been applied only
in DMSO solution. To inspect biological activity, it is generally nec-
essary to dissolve test samples in various solvents. The high solu-

5642
T. Saitoh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5638–5642
6. Box, S. J.; Gilpin, M. L.; Gwynn, M.; Hanscomb, G. J. Antibiot. 1983, 36, 1631.
7. Wipf, P.; Kim, Y.; Jahn, H. Synthesis 1995, 1549.
Since the crystals of ( )-6b were very thin needles, the p-nitrobenzoate
derivative 6c has been prepared and the crystal structure determined by X-ray
diffraction method to confirm the anti configuration. For 6c, monoclinic, P21,
a = 17.065(3), b = 14.071(3), c = 7.1425(14) Å, b = 100.352(16)°, V = 1687.2(6)
Å3, and Z = 4. The structure of 6c has been deposited at the Cambridge
Crystallographic Data Center and allocated the deposition number CCDC
782800.
8. (a) Selected data. Compound 6a: HRMS (ESI-MS) calcd for C10H12NO5 (M+H)+
226.0715, obsd m/z 226.0708. 1H NMR (CDCl3): d 3.18 (br s, 1H), 3.52 (m, 1H),
3.86 (t, 1H, J = 2.0 Hz), 4.58 (br s, 1H), 4.64 (d, 2H, J = 3.7 Hz), 5.30 (m, 2H), 5.91
(m, 1H), 6.58 (s, 1H), 7.33 (br s, 1H). 13C NMR (CDCl3): d 53.5, 54.0, 65.4, 66.9,
105.1, 119.4, 131.5, 149.3, 152.2, 192.7. Compound 6b: HRMS (ESI-MS) calcd for
C
10H12NO5 (M+H)+ 226.0715, obsd m/z 226.0703. 1H NMR (CDCl3): d 3.46 (d,
9. Suzuki, E.; Umezawa, K. Biomed. Pharmacother. 2006, 60, 578.
10. Yamamoto, M.; Horie, R.; Takeiri, M.; Kozawa, I.; Umezawa, K. J. Med. Chem.
2008, 51, 5780.
1H, J = 2.3 Hz), 3.81 (dd, 1H, J = 0.9, 2.3 Hz), 4.41 (br s, 1H), 4.66 (d, 2H,
J = 3.7 Hz), 4.99 (s, 1H), 5.30–5.38 (m, 2H), 5.74 (s, 1H), 5.91 (m, 1H), 7.09 (s,
1H). 13C NMR (CDCl3): d 52.3, 53.3, 63.1, 67.4, 108.9, 119.8, 131.1, 151.3, 152.8,
11. Suzuki, Y.; Sugiyama, C.; Ohno, O.; Umezawa, K. Tetrahedron 2004, 60, 7061.
192.7.
(b) Relative configuration of 6b.
O
OH
O
O
O
H
N
H
N
p-nitrobenzoyl chloride
DMAP, CH2Cl2
80%
O
O
O2N
O
O
O
O
6b
6c