Synthesis and structure-activity relationship of dehydroxymethylepoxyquinomicin analogues as inhibitors of NF-κB functions

Article abstract of DOI:10.1016/S0968-0896(02)00315-2

We previously found dehydroxymethylepoxyquinomicin (DHMEQ) inhibited NF-κB activation and showed anti-inflammatory activity in vivo. Here we designed and synthesized analogues of DHMEQ and tested their biological activity as NF-κB inhibitors in human T cell leukemia Jurkat cells. The hydroxyl group at the 2-position of the benzamide moiety was found to be essential for the inhibitory activity. But etherification of this group did not diminish the activity completely. Thus, for further mechanistic studies the hydroxyl group at the 2-position may be useful for extension with a linker and biotin moiety.

Full text of DOI:10.1016/S0968-0896(02)00315-2

Bioorganic & Medicinal Chemistry 10 (2002) 3933–3939
Synthesis and Structure–Activity Relationship of
Dehydroxymethylepoxyquinomicin Analogues as
Inhibitors of NF-ꢀBFunctions
Chanya Chaicharoenpong,a Kuniki Katob and Kazuo Umezawaa,*
aDepartment of Applied Chemistry, Faculty of Science and Technology, Keio University,
3-14-1Hiyoshi, Kohoku-ku, Yokohama 223-0061, Japan
bResearch Laboratories, Pharmaceuticals Group, Nippon Kayaku Co. Ltd., 3-31 Shimo,
Kita-ku, Tokyo 115-0042, Japan
Received 31 May 2002;accepted 16 July 2002
Abstract—We previously found dehydroxymethylepoxyquinomicin (DHMEQ) inhibited NF-kB activation and showed anti-
inflammatory activity in vivo. Here we designed and synthesized analogues of DHMEQ and tested their biological activity as
NF-kB inhibitors in human T cell leukemia Jurkat cells. The hydroxyl group at the 2-position of the benzamide moiety was found
to be essential for the inhibitory activity. But etherification of this group did not diminish the activity completely. Thus, for further
mechanistic studies the hydroxyl group at the 2-position may be useful for extension with a linker and biotin moiety.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
from the culture broth of Amycolatopsis sp. MK
299-95F4, a microbial strain isolated from a soil sam-
ple collected in Sendai, Japan, as weak antibiotics.7
Epoxyquinomicin C 1 is the simplest among these 4
epoxyquinomicins, and it has a 4-hydroxy-5,6-epoxy-
cyclohexenone group that also exists in the structure of
panepoxydone 28 and cycloepoxydon 3.9 Compounds 2
and 3 were found to inhibit TNF-a-induced activation
of NF-kB by inhibiting the phosphorylation and degra-
dation of IkB.8 Epoxyquinomicin C 1 has an additional
hydroxymethyl group compared with panepoxydone 2,
and this compound did not inhibit NF-kB activation.
Therefore, we designed and synthesized the
5-dehydroxymethyl derivative (DHMEQ, 4) of 1.10
Compound 4 inhibited TNF-a-induced activation of
NF-kB in human T cell leukemia Jurkat cells. We
recently reported that compound 4 inhibited nuclear
translocation of NF-kB in Jurkat and African green
monkey COS-1 cells.11 It also inhibited type II collagen-
induced rheumatoid arthritis in a murine model.10 Like
epoxyquinomicin C 1, compound 4 showed no promi-
nent toxicity in mice. Thus we considered the possibility
of developing it as a chemotherapeutic agent. In the
present paper, we report the synthesis and the biological
activity of DHMEQ derivatives with potential to inhibit
NF-kB functions, and looked into their structure–
activity relationship.
Nuclear factor-kappa B (NF-kB) is an inducible and
ubiquitous transcription factor that elicits the expres-
sion of inflammatory cytokines such as IL-1, IL-6, IL-8,
and TNF-a and apoptosis inhibitory proteins (IAPs).
The most common form of NF-kB is a heterodimer
consisting of a 50-kD protein (p50) and a 65-kD protein
(p65 or RelA).1 NF-kB is a key mediator of TNF-a
responses and an attractive target for therapeutic inter-
vention against inflammatory diseases such as rheuma-
toid arthritis2 and neoplastic diseases.3 The number of
known roles of NF-kB is rapidly increasing. For
instance, erythropoietin was shown to inhibit apoptosis
of neuronal cells by increasing the tyrosine phosphoryl-
ation of IkB to activate NF-kB.4 On the other hand,
NF-kB was found to be often constitutively activated in
breast cancer cells and in human melanoma cells.5,6
Activation of NF-kB would provide apoptotic resis-
tance in these neoplastic tumors.
Recently, 4 novel 5,6-epoxycyclohexenone compounds,
that is, epoxyquinomicins A, B, C and D, were isolated
*Corresponding author. Tel.: +81-45-566-1558;fax: +81-45-566-
1558;e-mail: umezawa@applc.keio.ac.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00315-2

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C. Chaicharoenpong et al. / Bioorg. Med. Chem. 10 (2002) 3933–3939
Fortunately, however, treatment of 10c–j and 11a–b
with boron trifluoride etherate in CH2Cl2 at À20 ꢀC
furnished epoxyquinone compounds 14a–j in 57–85%
yields without any difficulty. Then, regioselective reduc-
tion of 14a–j was carried out by treatment with sodium
triacetoxyborohydride in methanol to provide the target
compounds 15a–j in 54–89% yields. No epimer was
detected in TLC after the reduction by sodium triace-
toxyborohydride for preparation of 15a–j. The relative
stereochemistry of 15a–j was determined based on the
physical data by comparison with those of DHMEQ,
whose stereochemistry was established unambiguously
by X-ray crystallography.10
Biological Activity
The newly synthesized analogues 15a–j were assayed for
their inhibitory activity toward TNF-a-induced NF-kB
activation in Jurkat cells, with DHMEQ 4 adopted as a
reference standard. The cells were transfected with kB-
luciferase DNA by the DEAE-dextran method. The
activity of NF-kB can be detected by transient trans-
fection of Jurkat cells with the reporter DNA having the
binding sequence for NF-kB and the luciferase gene.
Figure 1 shows inhibition of TNF-a-induced NF-kB
activation by all of the synthesized compounds. The
hydroxyl group at the 2-position of the benzamide ring
system was found to be essential for the inhibitory
activity, since the regional isomers 15a and 15b, posses-
sing a respective hydroxyl group at the 3- and 4-posi-
tions, and the deoxy compound 15c all completely lost
the activity. The methylated compound 15h and halo-
genated compounds such as 15i and 15j at the 2-position
also showed no activity. Interestingly, etherification of
the hydroxyl group at the 2-position did not diminish
the inhibitory activity. The presence of a hexyl group
(15f) or phenyl group (15g) weakened the activity, but
lower alkyl groups such as the methyl and ethyl of 15d
and 15e, respectively, did not affect the inhibitory
activity. For the detection of molecular target for
DHMEQ 4, the biotin-labeled derivative of 4 must be
useful. Thus, based on the results of the structure–activ-
ity relationship, we found that the hydroxyl functional
Chemistry
Synthesis of the target compounds 15a–j was accom-
plished by applying a methodology different from that
previously described for 4.10 Scheme 3 summarizes the
synthetic route for 15a–j. We selected racemic 3-amino-
4,4-dimethoxy-5,6-epoxycyclohex-2-en-1-on (8) as the
starting material, which could be readily obtained from
commercially available 2,5-dimethoxyaniline (5) as shown
in Scheme 1 according to the protocol of Wipf’s method.12
As lithium tert-butoxide was recommended as a base for
acylation of epoxyenamine 8 with various acyl chlor-
ides,13 compound 8 was coupled with the appropriate
acyl chloride, prepared in situ from carboxylic acids 9a–
j by oxalyl chloride in the presence of a catalytic amount
of DMF, by the aid of lithium tert-butoxide in THF to
afford amides 10a–j in 62–98% yields. Saponification of
ester group of compounds 10a and 10b with K2CO3 in
methanol gave 11a and 11b in 71 and 65% yields,
respectively. At first, regeneration of the ketone group
from acetal by hydrolysis was supposed to be trouble-
some. Taylar et al. reported that the direct deprotection
of the acetal group of 12 was unsuccessful due to aro-
matization, and therefore we converted 12 into the
corresponding epoxyquinone 13 via a 3-step reduction-
deprotection-oxidation sequence (Scheme 2).14
Scheme 1. Reagents and conditions: (i) Alloc-Cl, Et3N, THF;(ii) PhI(OAc) 2, MeOH;(iii) TBHP, DBU, CH 2Cl2.
Scheme 2. Reagents and conditions: (i) LiEt3BH, THF;(ii) montmorillonite K10, CH 2Cl2;(iii) PDC, CH 2Cl2.

C. Chaicharoenpong et al. / Bioorg. Med. Chem. 10 (2002) 3933–3939
3935
Scheme 3. Reagents and conditions: (i) (COCl)2, cat. DMF, CH2Cl2, rt, 0 ꢀC, 1.5 h;(ii) t-BuOLi, THF, À78 ꢀC, 30 min;(iii) K 2CO3, MeOH, rt, 30
ꢀ
ꢀ
.
min;(iv) BF 3 OEt2, CH2Cl2, À20 C, 30 min;(v) NaBH (OAc) 3, MeOH, 0 C, 30 min.
Analytical TLC was performed on Merck pre-coated
silica gel 60 F254 plates. Column chromatography was
performed by using silica gel 60 (particle size 0.063–
0.200 mm, Merck). Melting points were obtained with a
Yanaco micro melting point apparatus and left uncor-
1
rected. H NMR (300 MHz) and 13C NMR (75.4 MHz)
spectral data were obtained on a Jeol JNM-LA300
spectrometer, and chemical shifts were reported as parts
per million (ppm) downfield from tetramethylsilane as
an internal standard. High-resolution mass spectra were
recorded on a Jeol JMS-GCmate mass spectrometer.
General method for preparation of N-(2,2,-dimethoxy-
3,4-epoxy-5-oxo-cyclohex-6-enyl)benzamide derivatives
Dry DMF (10 mL) was added to a cold solution of
appropriate carboxylic acid (1 equiv) from 9a–j and
oxalyl chloride (2 equiv) in dry CH2Cl2. The clear solu-
tion was stirred at 0 ꢀC for 1.5 h and allowed to warm to
room temperature over 30 min. The solvent was
removed under reduced pressure;and after the addition
of dry toluene, the mixture was concentrated in vacuo.
Dry THF was added, and the solution was cannulated
under Ar to a flask containing epoxyenamine 8 (1 equiv).
To this stirred solution was added t-BuOLi (0.1 M THF
solution, 1 equiv) at À78 ꢀC. The reaction was allowed to
proceed with stirring at 0 ꢀC for 30 min. On completion,
the reaction mixture was extracted with ethyl acetate;and
the organic layers were washed with brine, dried over
anhydrous sodium sulphate, and evaporated to yield a
Figure 1. Effect of DHMEQ and its analogues on TNF-a-induced
activation of NF-kB in Jurkat cells. The cells were treated with each
compound with or without 20 ng/mL of TNF-a.
group at the 2-position of the benzamide ring is a suitable
candidate for extension with a linker and biotin moiety.
Experimental
All reactions requiring anhydrous and oxygen-free con-
ditions were conducted in an argon atmosphere. t-BuOLi
(0.1 M in THF) was obtained from Aldrich Chemical Co.

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C. Chaicharoenpong et al. / Bioorg. Med. Chem. 10 (2002) 3933–3939
residue. Purification of this residue by using column chro-
matography over silica gel with n-hexane/ethyl acetate
(2:1, v/v) as eluent yielded the benzamide derivatives 10a–j.
3.66 (s, 3H), 3.83 (d, 1H, J=4.5 Hz), 4.20 (t, 2H, J=7.2
Hz), 7.00 (d, 1H, J=8.4 Hz), 7.07 (t, 1H, J=7.2 Hz),
7.43 (d, 1H, J=1.8, Hz), 7.45 (td, 1H, J=8.1, 1.2 Hz),
8.15 (dd, 1H, J=7.8, 1.8 Hz), 10.30 (br s, 1H); 13C
NMR (CDCl3) d 13.9, 22.6, 25.5, 28.9, 31.5, 50.9, 51.4,
52.0, 69.8, 95.8, 110.0, 112.6, 121.0, 121.5, 132.8, 134.1,
146.1, 156.8, 164.6, 193.1. HRMS (EI) m/z calcd for
C21H27NO6, 389.1838;found, 389.1860.
3-Acetoxy-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-
6-enyl)benzamide (10a). (96%, mp 128–130 ꢀC): 1H
NMR (CDCl3) d 2.30 (s, 3H), 3.30 (s, 3H), 3.53 (dd, 1H,
J=4.2, 2.1 Hz), 3.67 (s, 3H), 3.84 (d, 1H, J=3.9 Hz),
7.23 (d, 1H, J=2.1 Hz), 7.30 (t, 1H, J=5.6 Hz), 7.45 (d,
1H, J=8.1 Hz), 7.51 (t, 1H, J=1.8 Hz), 7.55 (t, 1H,
J=8.1 Hz), 8.39 (br s, 1H); 13C NMR (CDCl3) d 21.0,
50.9, 51.3, 51.4, 52.1, 95.6, 109.1, 120.8, 124.0, 126.1,
130.0, 135.2, 145.1, 150.6, 164.8, 169.2, 192.8. HRMS
(EI) m/z calcd for C17H17NO7, 347.1005;found,
347.1011.
2-Phenoxy-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-
6-enyl)benzamide (10g). (64%, mp 69–71 ꢀC): H NMR
1
(CDCl3) d 3.21 (s, 3H), 3.25 (s, 3H), 3.48 (dd, 1H,
J=3.9, 2.1 Hz), 3.76 (d, 1H, J=4.4 Hz), 6.80 (d, 1H,
J=8.1 Hz), 7.12 (d, 2H, J=7.6 Hz), 7.20 (t, 1H, J=7.2
Hz), 7.26 (t, 1H, J=7.8 Hz), 7.34 (d, 1H, J=2.4 Hz),
7.44 (m, 3H), 8.24 (dd, 1H, J=6.0, 1.8 Hz), 10.54 (br s,
1H); 13C NMR (CDCl3) d 50.0, 51.0, 51.4, 52.1, 95.5,
109.0, 117.5, 120.2, 122.5, 123.7, 125.5, 130.3, 132.5,
134.1, 146.2, 154.6, 156.1, 163.4, 193.4. HRMS (EI) m/z
calcd for C21H19NO6, 381.1212;found, 381.1206.
4-Acetoxy-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-
6-enyl)benzamide (10b). (92%, mp 145–147 ꢀC): 1H
NMR (CDCl3) d 2.33 (s, 3H), 3.33 (s, 3H), 3.56 (dd, 1H,
J=3.6, 1.8 Hz), 3.70 (s, 3H), 3.87 (d, 1H, J=3.9 Hz),
7.26 (t, 3H, J=7.8 Hz), 7.80 (d, 2H, J=8.4 Hz), 8.42 (br
s, 1H); 13C NMR (CDCl3) d 21.1, 50.8, 51.3, 51.4, 52.1,
95.6, 108.9, 122.2, 128.6, 131.2, 145.3, 154.0, 164.9,
168.8, 171.1, 192.8. HRMS (EI) m/z calcd for
C17H17NO7, 347.1005;found, 347.1006.
2-Methyl-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-6-
ꢀ
1
enyl)benzamide (10h). (85%, mp 102–103 C): H NMR
(CDCl3) d 2.41 (s, 3H), 3.28 (s, 3H), 3.50 (dd, 1H,
J=4.2, 2.1 Hz), 3.59 (s, 3H), 3.80 (d, 1H, J=3.9 Hz),
7.21 (m, 3H), 7.34 (t, 2H, J=8.4 Hz), 8.00 (br s, 1H);
13C NMR (CDCl3) d 20.0, 50.7, 51.3, 51.5, 52.1, 95.5,
108.8, 126.1, 126.7, 131.1, 131.7, 134.8, 137.0, 145.3,
168.3, 192.8. HRMS (EI) m/z calcd for C16H17NO5,
303.1107;found, 303.1083.
N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-6-enyl)benz-
amide (10c). (95%, mp 117–118 ꢀC): H NMR (CDCl3)
1
d 3.35 (s, 3H), 3.56 (dd, 1H, J=4.2, 2.1 Hz), 3.72 (s,
3H), 3.88 (d, 1H, J=4.3 Hz), 7.31 (d, 1H, J=2.0 Hz),
7.54 (d, 2H, J=7.6 Hz), 7.57 (t, 1H, J=1.5 Hz), 7.77 (d,
2H, J=6.8 Hz), 8.48 (br s, 1H); 13C NMR (CDCl3) d
50.9, 51.3, 51.4, 52.2, 95.6, 108.8, 126.9, 128.9, 132.7,
133.5, 145.2, 165.6, 192.6. HRMS (EI) m/z calcd for
C15H15NO5, 289.0950;found, 289.0958.
2-Fluoro-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-6-
enyl)benzamide (10i). (72%, mp 126–128 ꢀC): H NMR
1
(CDCl3) d 3.32 (s, 3H), 3.52 (dd, 1H, J=3.9, 2.1 Hz),
3.66 (s, 3H), 3.85 (d, 1H, J=1.8 Hz), 7.16 (t, 1H, J=8.1
Hz), 7.28 (m, 2H), 7.53 (d, 1H, J=3.9 Hz), 8.05 (td, 1H,
J=7.8, 1.5 Hz), 9.20 (br s, 1H); 13C NMR (CDCl3) d
50.7, 51.2, 51.5, 52.1, 95.4, 109.5, 116.2, 116.6, 125.3,
132.4, 134.8, 145.6, 158.8, 162.0, 193.3. HRMS (EI) m/z
calcd for C15H14NO5F, 307.0856;found, 307.0853.
2-Methoxy-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-
6-enyl)benzamide (10d). (77%, mp 125–127 ꢀC): 1H
NMR (CDCl3) d 3.29 (s, 3H), 3.47 (dd, 1H, J=4.3, 2.1
Hz), 3.67 (s, 3H), 3.83 (d, 1H, J=4.4 Hz), 4.00 (s, 3H),
6.97 (d, 1H, J=8.4 Hz), 7.05 (t, 1H, J=7.4 Hz), 7.31 (d,
1H, J=2.2 Hz), 7.49 (td, 1H, J=7.8, 1.8 Hz), 8.10 (dd,
1H, J=8.1, 1.8 Hz), 10.58 (br s, 1H); 13C NMR
(CDCl3) d 50.6, 51.1, 51.5, 51.9, 56.0, 95.6, 108.8, 111.7,
120.6, 121.6, 132.4, 134.3, 146.3, 157.3, 164.0, 193.3.
HRMS (EI) m/z calcd for C16H17NO6, 319.1056;found,
319.1054.
2-Chloro-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-6-
enyl)benzamide (10j). (94%, mp 114–116 ꢀC): H NMR
1
(CDCl3) d 3.32 (s, 3H), 3.55 (dd, 1H, J=4.2, 1.8 Hz),
3.62 (s, 3H), 3.85 (d, 1H, J=3.9 Hz, 1H), 7.23 (d, 1H,
J=1.8 Hz), 7.35 (m, 1H), 7.45 (d, 2H, J=3.6 Hz, 2H),
7.72 (d, 1H, J=7.8 Hz), 8.66 (br s, 1H); 13C NMR
(CDCl3) d 50.6, 50.7, 51.4, 52.0, 95.5, 109.5, 126.7,
130.6, 131.4, 132.4, 133.5, 145.2, 165.0, 170.5, 193.2.
HRMS (EI) m/z calcd for C15H14NO5Cl, 323.0561;
found, 323.0566.
2-Ethoxy-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-6-
enyl)benzamide (10e). (98%, mp 157–159 ꢀC): H NMR
1
(CDCl3) d 1.55 (t, 3H, J=6.9 Hz), 3.32 (s, 3H), 3.51 (dd,
1H, J=4.3, 2.1 Hz), 3.66 (s, 3H), 3.83 (d, 1H, J=4.2
Hz), 4.29 (q, 2H, J=6.9 Hz), 7.00 (d, 1H, J=7.8 Hz),
7.06 (t, 1H, J=7.5 Hz), 7.47 (m, 2H), 8.17 (d, 1H,
J=7.8 Hz), 10.35 (br s, 1H); 13C NMR (CDCl3) d 14.7,
51.4, 52.0, 65.1, 95.9, 109.9, 112.5, 121.5, 122.1, 132.8,
134.2, 146.2, 156.7, 164.6, 193.1. HRMS (EI) m/z calcd
for C17H19NO6, 333.1212;found, 333.1206.
3-Hydroxy-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-
6-enyl)benzamide (11a). A solution of 10a (66.2 mg, 0.19
mmol) and K2CO3 (26.4 mg, 0.19 mmol) in MeOH (4
mL) and water (0.5 mL) was stirred for 30 min at room
temperature. The reaction mixture was diluted with
water (2 mL), acidified, and then extracted with EtOAc
(10 mL). The organic layer was washed with brine, dried
over anhydrous Na2SO4, and then concentrated under
reduced pressure. The resulting residue was chromato-
graphed on silica gel (n-hexane:EtOAc=2:1) to afford
11a (41.1 mg, 71%) as a white solid. Mp 152–154 ꢀC; 1H
2-Hexyloxy-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclo-
hex-6-enyl)benzamide (10f). (62%, mp 58–60 ꢀC): 1H
NMR (CDCl3) d 0.89 (t, 3H, J=6.9 Hz), 1.35 (m, 6H),
1.91 (m, 2H), 3.31 (s, 3H), 3.52 (dd, 1H, J=3.9, 1.8 Hz),

C. Chaicharoenpong et al. / Bioorg. Med. Chem. 10 (2002) 3933–3939
3937
NMR (CDCl3) d 3.30 (s, 3H), 3.52 (dd, 1H, J=3.9, 2.1
Hz), 3.66 (s, 3H), 3.85 (d, 1H, J=3.9 Hz), 7.04 (dd, 1H,
J=8.1, 1.8 Hz), 7.21 (t, 1H, J=7.8 Hz), 7.23 (d, 1H,
J=2.1 Hz), 7.28 (m, 2H), 7.54 (br s, 1H), 8.46 (br s,
1H); 13C NMR (CDCl3) d 50.8, 51.2, 51.4, 52.1, 95.6,
108.6, 114.3, 118.3, 120.2, 130.2, 134.9, 145.8, 157.0,
165.9, 193.4. HRMS (EI) m/z calcd for C15H15NO6,
305.0899;found, 305.0910.
2-Methoxy-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
amide (14d). (57%, mp 105–107 ꢀC): H NMR (CDCl3)
1
d 3.76 (dd, 1H, J=3.7, 2.2 Hz), 3.86 (d, 1H, J=3.7 Hz),
4.04 (s, 3H), 6.95 (d, 1H, J=8.4 Hz), 7.04 (t, 1H, J=7.3
Hz), 7.47 (td, 1H, J=8.1, 1.8 Hz), 7.64 (d, 1H, J=2.2 Hz),
8.08 (dd, 1H, J=7.7, 1.5 Hz), 10.74 (br s, 1H); 13C NMR
(CDCl3) d 52.6, 53.8, 56.2, 111.7, 115.5, 120.1, 121.7, 132.5,
134.7, 139.9, 157.6, 164.4, 188.3, 191.5. HRMS (EI) m/z
calcd for C14H11NO5, 273.0637;found, 273.0639.
4-Hydroxy-N-(2,2-dimethoxy-3,4-epoxy-5-oxo-cyclohex-
6-enyl)benzamide (11b). The same procedure as for 11a,
starting from 10b, was employed for the synthesis of
11b. Compound 11b was obtained as a white solid
(65%). Mp 170–172 ꢀC; 1H NMR (acetone-d6) d 3.36 (s,
3H), 3.46 (dd, 1H, J=4.2, 2.1 Hz), 3.66 (s, 3H), 4.08 (d,
1H, J=3.9 Hz), 6.91 (dd, 2H, J=6.9, 1.8 Hz), 7.10 (d,
1H, J=2.1 Hz), 7.75 (dd, 2H, J=6.9, 2.1 Hz), 8.71 (br s,
1H), 9.23 (br s, 1H); 13C NMR (acetone-d6) d 50.9, 51.5,
52.2, 52.7, 96.6, 108.0, 116.4, 125.8, 130.4, 147.6, 162.5,
166.1, 193.6. HRMS (EI) m/z calcd for C15H15NO6,
305.0899;found, 305.0898.
2-Ethoxy-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
amide (14e). (69%, mp 172–174 ꢀC): H NMR (CDCl3)
1
d1.63 (t, 3H, J=6.9 Hz), 3.78 (dd, 1H, J=3.6, 2.1 Hz),
3.86 (d, 1H, J=3.6 Hz), 4.22 (q, 2H, J=6.9 Hz), 6.95
(d, 1H, J=8.4 Hz), 7.03 (t, 1H, J=7.5 Hz), 7.45 (t, 1H,
J=6.9 Hz), 7.72 (d, 1H, J=1.8 Hz), 8.11 (dd, 1H,
J=6.3, 1.8 Hz), 10.66 (br s, 1H); 13C NMR (CDCl3) d
14.7, 52.6, 53.9, 65.4, 112.4, 115.8, 120.1, 121.5, 140.0,
157.2, 164.7, 188.4. HRMS (EI) m/z calcd for
C15H13NO5, 287.0794;found, 287.0797.
2-Hexyloxy-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
ꢀ
1
amide (14f). (71%, mp 65–67 C): H NMR (CDCl3) d
0.89 (t, 3H, J=7.2 Hz), 1.39 (m, 6H), 2.06 (m, 2H),
3.82 (dd, 1H, J=4.1, 1.8 Hz), 3.91 (d, 1H, J=3.9 Hz),
4.20 (td, 2H, J=6.9, 1.8 Hz), 7.00 (d, 1H, J=8.4 Hz),
7.07 (t, 1H, J=8.1 Hz), 7.49 (td, 1H, J=8.4, 1.8 Hz),
7.76 (d, 1H, J=1.8 Hz), 8.14 (dd, 1H, J=8.1, 1.8 Hz,
1H), 10.65 (br s, 1H); 13C NMR (CDCl3) d 14.0, 22.6,
25.8, 28.7, 31.4, 52.6, 53.8, 69.9, 112.4, 115.8, 120.1,
121.4, 132.7, 140.0, 157.3, 164.7, 188.3, 191.6. HRMS
(EI) m/z calcd for C19H21NO5, 343.1420;found,
343.1418.
General method for preparation of N-(2,5-dioxo-3,4-
epoxy-cyclohex-6-enyl)benzamide derivatives
To a stirred solution of appropriate compound (1 equiv)
from 10c–j and 11a–b in CH2Cl2 was added boron tri-
fluoride etherate (2.5 equiv) at À20 ꢀC. The reaction
mixture was stirred for 30 min and then warmed up to
room temperature. On completion the reaction mixture
was extracted with ethyl acetate;and the organic layers
were washed with brine, dried over anhydrous sodium
sulphate, and evaporated to yield a residue, which upon
purification by column chromatography over silica gel
using n-hexane/ethyl acetate (2:1, v/v) as eluent yielded
the epoxyquinone derivatives 14a–j.
2-Phenoxy-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
ꢀ
1
amide (14g). (77%, mp 128–130 C): H NMR (CDCl3)
d 3.75 (dd, 1H, J=3.6, 2.1 Hz), 3.80 (d, 1H, J=3.6 Hz),
6.86 (dd, 1H, J=8.4, 2.1 Hz), 7.11 (d, 2H, J=7.8 Hz),
7.17 (m, 2H), 7.38 (t, 3H, J=7.6 Hz), 7.68 (d, 1H,
J=2.4 Hz), 8.15 (dd, 1H, J=8.1, 1.8 Hz), 10.55 (br s,
1H); 13C NMR (CDCl3) d 52.5, 53.8, 115.8, 118.0,
119.7, 122.3, 123.8, 125.2, 130.3, 132.8, 134.4, 139.7,
154.6, 155.7, 164.0, 188.1, 192.4. HRMS (EI) m/z calcd
for C19H13NO5, 335.0794;found, 335.0807.
3-Hydroxy-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
ꢀ
1
amide (14a). (67%, mp 156–158 C) H NMR (acetone-
d6) d 3.91 (dd, 1H, J=4.0, 2.1 Hz), 4.12 (d, 1H, J=3.9
Hz), 7.10 (m, 1H), 7.37–7.40 (m, 3H), 7.50 (d, 1H,
J=2.1 Hz), 8.96 (br s, 1H); 13C NMR (acetone-d6) d
53.8, 54.8, 115.1, 115.2, 119.2, 120.8, 131.0, 135.6, 140.5,
158.7, 166.8, 189.0, 192.4. HRMS (EI) m/z calcd for
C13H9NO5, 259.0481;found, 259.0486.
3-Methyl-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
amide (14h). (85%, mp 134–136 ꢀC): H NMR (CDCl3)
1
4-Hydroxy-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
amide (14b). (75%, mp 193–194 ꢀC) H NMR (acetone-
1
d 2.46 (s, 3H), 3.84 (dd, 1H, J=3.8, 2.1 Hz), 3.92 (d, 1H,
J=3.6 Hz), 7.27 (d, 2H, J=5.7 Hz), 7.43 (m, 2H), 7.67
(d, 1H, J=2.1 Hz), 8.24 (br s, 1H); 13C NMR (CDCl3) d
20.1, 52.5, 53.8, 115.4, 126.2, 126.9, 131.6, 131.9, 133.9,
137.4, 138.3, 168.2, 188.1, 191.1. HRMS (EI) m/z calcd
for C14H11NO4, 257.0688;found, 257.0674.
d6) d 3.78 (dd, 1H, J=3.6, 1.8 Hz), 3.98 (d, 1H, J=3.6
Hz), 6.85 (d, 2H, J=7.8 Hz), 7.38 (d, 1H, J=2.1 Hz), 7.72
(d, 2H, J=9.0 Hz), 8.78 (br s, 1H), 9.18 (br s, 1H); 13C
NMR (acetone-d6) d 53.7, 54.8, 114.7, 116.4, 125.2, 130.7,
140.7, 156.6, 166.3, 189.1, 192.4. HRMS (EI) m/z calcd
for C13H9NO5, 259.0481;found, 259.0488.
2-Fluoro-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
amide (14i). (68%, mp 136–137 ꢀC): H NMR (CDCl3)
1
N - (2,5 - dioxo - 3,4 - epoxy - cyclohex - 6 - enyl)benzamide
(14c). (78%, mp 101–103 ꢀC): H NMR (CDCl3) d 3.87
d 3.85 (dd, 1H, J=3.6, 2.1 Hz), 3.95 (d, 1H, J=3.9 Hz),
7.21 (dd, 1H, J=8.4, 3.6 Hz), 7.30 (t, 1H, J=7.5 Hz),
7.56 (d, 1H, J=7.8 Hz), 7.69 (d, 1H, J=2.1 Hz), 8.06
(td, 1H, J=7.8, 1.8 Hz), 9.30 (br s, 1H); 13C NMR
(CDCl3) d 52.7, 54.0, 116.4, 116.8, 125.5, 132.4, 135.3,
135.5, 139.2, 159.1, 162.4, 188.0, 191.4. HRMS (EI) m/z
calcd for C13H8NO4F, 261.0437;found, 261.0436.
1
(dd, 1H, J=3.6, 2.3 Hz), 3.98 (d, 1H, J=3.8 Hz), 7.52
(d, 2H, J=7.7 Hz), 7.62 (t, 1H, J=7.2 Hz), 7.70 (d, 1H,
J=2.3 Hz), 7.83 (d, 2H, J=7.3 Hz), 8.65 (br s, 1H); 13C
NMR (CDCl3) d 52.5, 53.9, 115.5, 127.2, 128.9, 132.7,
133.1, 165.6, 188.0, 190.8. HRMS (EI) m/z calcd for
C13H9NO4, 243.0532;found, 243.0522.

3938
C. Chaicharoenpong et al. / Bioorg. Med. Chem. 10 (2002) 3933–3939
2-Chloro-N-(2,5-dioxo-3,4-epoxy-cyclohex-6-enyl)benz-
amide (14j). (62%, mp 125–127 ꢀC): H NMR (CDCl3)
2-Ethoxy-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-en-
yl)benzamide (15e). (64%, mp 187–189 ꢀC): 1H NMR
(CDCl3) d 1.52 (t, 3H, J=6.9 Hz), 3.49 (dd, 1H, J=3.9,
1.8 Hz), 3.85 (t, 1H, J=3.9 Hz), 4.20 (t, 2H, J=6.9 Hz),
4.67 (s, 1H), 6.87 (s, 1H), 6.95 (d, 2H, J=8.4 Hz), 7.03
(t, 1H, J=7.8 Hz), 7.44 (td, 1H, J=8.1, 1.8 Hz), 8.10
(dd, 1H, J=8.1, 1.5 Hz), 10.44 (br s, 1H); 13C NMR
(CDCl3) d 14.7, 53.6, 53.8, 65.5, 107.7, 112.3, 120.3,
121.6, 132.8, 134.4, 189.5. HRMS (EI) m/z calcd for
C15H15NO5, 289.0950;found, 289.0956.
1
d 3.84 (dd, 1H, J=3.6, 2.1 Hz), 3.93 (d, 1H, J=3.6 Hz),
7.35 (m, 1H), 7.44 (d, 2H, J=3.9 Hz), 7.66 (d, 1H, J=1.5
Hz), 7.73 (d, 1H, J=7.8 Hz), 8.84 (br s, 1H); 13C NMR
(CDCl3) d 52.6, 53.8, 116.0, 116.1, 127.5, 130.8, 130.9,
132.9, 133.0, 138.6, 165.1, 187.8, 191.1. HRMS (EI) m/z
calcd for C13H8NO4Cl, 277.0142;found, 277.0132.
General method for preparation of N-(2-hydroxy-3,4-
epoxy-5-oxo-cyclohex-6-enyl)benzamide derivatives
2-Hexyloxy-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-
enyl)benzamide (15f). (89%, mp 88–90 ꢀC): 1H NMR
(CDCl3) d 0.85 (t, 3H, J=7.2 Hz), 1.36 (m, 6H), 1.89
(m, 2H), 3.48 (dd, 1H, J=3.9, 2.1 Hz), 3.85 (t, 1H, J=3.9
Hz), 4.15 (m, 2H), 4.65 (s, 1H), 6.82 (d, 1H, J=1.9 Hz),
6.94 (d, 1H, J=8.0 Hz), 7.02 (t, 1H, J=7.8 Hz), 7.44 (td,
1H, J=7.5, 2.1 Hz), 8.10 (dd, 1H, J=8.0, 1.8 Hz), 10.39
(br s, 1H); 13C NMR (CDCl3) d 14.0, 22.6, 25.7, 28.7,
31.4, 53.6, 53.8, 65.5, 69.7, 107.7, 112.4, 120.3, 121.4,
132.7, 134.4, 149.7, 157.0, 164.9, 193.2. HRMS (EI) m/z
calcd for C19H23NO5, 345.1576;found, 345.1563.
To a stirred solution of the appropriate compound (1
equiv) from 14a–j in MeOH was added NaBH(OAc)3
(1 equiv) at 0 ꢀC. The reaction mixture was allowed to
warm to room temperature over 30 min. On comple-
tion the reaction mixture was diluted with ethyl ace-
tate;and the organic layers were washed with brine,
dried over anhydrous sodium sulphate, and evaporated
to yield a residue, which upon purification by column
chromatography over silica gel using chloroform/
MeOH (20:1, v/v) as eluent yielded the derivatives 15a–j
of DHMEQ.
2-Phenoxy-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-en-
yl)benzamide (15g). (75%, mp 138–140 ꢀC): H NMR
1
3-Hydroxy-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-
enyl)benzamide (15a). (60%, mp. 185–187 ꢀC): 1H
NMR (acetone-d6) d 3.76 (dd, 1H, J=3.9, 1.5 Hz), 3.84
(dd, 1H, J=4.2, 2.4 Hz), 4.94 (s, 1H), 6.79 (d, 1H,
J=1.8 Hz), 6.89 (t, 1H, J=1.8 Hz), 7.05 (m, 3H), 8.99
(br s, 1H); 13C NMR (acetone-d6) d 52.6, 56.0, 64.7,
108.7, 115.4, 119.5, 120.3, 130.6, 136.3, 152.6, 158.4,
167.9, 194.2. HRMS (EI) m/z calcd for C13H11NO5,
261.0637;found, 261.0647.
(CDCl3) d 3.44 (dd, 1H, J=4.2, 2.1 Hz), 3.76 (t, 1H,
J=4.2 Hz), 4.85 (s, 1H), 6.82 (d, 1H, J=8.1 Hz), 6.96
(s, 1H), 7.03 (d, 2H, J=8.1 Hz), 7.16 (m, 2H), 7.36 (t,
3H, J=7.8 Hz), 8.14 (dd, 1H, J=7.8, 1.8 Hz), 10.33 (br
s, 1H); 13C NMR (CDCl3) d 53.3, 53.8, 65.6, 107.6,
118.0, 119.7, 122.6, 123.8, 125.2, 130.3, 132.6, 134.2,
149.1, 154.7, 155.5, 164.1, 193.2. HRMS (EI) m/z calcd
for C19H15NO5, 337.0950;found, 337.0947.
4-Hydroxy-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-
enyl)benzamide (15b). (75%, mp. 197–199 ꢀC): 1H
NMR (acetone-d6) d 3.33 (dd, 1H, J=4.2, 2.1 Hz), 3.74
(dd, 1H, J=4.2, 2.4 Hz), 4.93 (s, 1H), 6.72 (d, 1H,
J=2.1 Hz), 6.90 (d, 2H, J=8.7 Hz), 7.80 (dd, 2H,
J=9.6, 2.4 Hz), 9.45 (br s, 1H); 13C NMR (acetone-d6)
d 52.7, 56.0, 108.2, 116.1, 125.8, 130.9, 151.1, 153.1,
162.4, 167.4, 194.1. HRMS (EI) m/z calcd for
C13H11NO5, 261.0637;found, 261.0630.
2-Methyl-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-en-
ꢀ
1
yl)benzamide (15h). (63%, mp 168–169 C): H NMR
(CDCl3) d 2.66 (s, 3H), 3.46 (dd, 1H, J=3.8, 2.1 Hz),
3.91 (t, 1H, J=4.2 Hz), 5.19 (s, 1H), 6.03 (d, 1H, J=1.8
Hz), 7.30 (d, 2H, J=7.8 Hz) 7.48 (t, 1H, J=6.6 Hz),
8.08 (d, 1H, J=7.8 Hz); 13C NMR (CDCl3) d 21.7, 50.9,
52.9, 66.9, 96.6, 125.7, 126.8, 130.8, 131.8, 133.0, 141.1,
155.8, 166.3, 189.9. HRMS (EI) m/z calcd for
C14H13NO4, 259.0845;found, 259.0870.
N-(2-hydroxy-3,4-epoxy-5-oxoꢀ-cyclohex-6-enyl)benz-
3-Fluoro-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-en-
yl)benzamide (15i). (61%, mp 141–142 ꢀC): 1H NMR
(acetone-d6) d 3.27 (dd, 1H, J=3.9, 2.1 Hz), 3.81 (t, 1H,
J=3.9 Hz), 4.82 (s, 1H), 6.86 (d, 1H, J=1.8 Hz), 7.17–
7.30 (m, 2H), 7.55 (d, 1H, J=6.3 Hz), 7.88 (td, 1H,
J=8.1, 1.8 Hz), 9.29 (br s, 1H); 13C NMR (acetone-d6)
d 54.0, 54.2, 65.8, 107.5, 117.1, 117.4, 126.1, 132.5,
135.6, 135.7, 150.3, 159.5, 162.8, 194.3. HRMS (EI) m/z
calcd for C13H10NO4F, 263.0594;found, 263.0610.
1
amide (15c). (78%, mp. 155–157 C): H NMR (CDCl3)
d 3.47 (dd, 1H, J=4.0, 2.0 Hz), 3.92 (t, 1H, J=3.5 Hz),
4.97 (s, 1H), 6.07 (d, 1H, J=2.5 Hz), 7.51 (t, 2H, J=7.6
Hz), 7.66 (t, 1H, J=7.4 Hz), 8.16 (d, 2H, J=7.4 Hz).
13C NMR (CDCl3) d 51.3, 53.3, 67.7, 96.9, 128.2, 128.7,
130.0, 134.2, 156.0, 166.0, 190.1. HRMS (EI) m/z calcd
for C13H11NO4, 245.0688;found, 245.0689.
2-Methoxy-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-
enyl)benzamide (15d). (54%, mp. 114–116 ꢀC): 1H
NMR (acetone-d6) d 3.25 (dd, 1H, J=4.0, 2.2 Hz), 3.80
(dd, 1H, J=4.0, 2.6 Hz), 3.96 (s, 3H), 4.79 (s, 1H), 6.91
(d, 1H, J=1.5 Hz), 7.01 (t, 1H, J=7.7 Hz), 7.11 (d, 1H,
J=8.4 Hz), 7.47 (td, 1H, J=7.7, 1.8 Hz), 7.96 (dd, 1H,
J=8.1, 1.8 Hz), 10.73 (br s, 1H); 13C NMR (acetone-d6)
d 54.0, 54.3, 56.8, 66.1, 106.9, 113.2, 121.6, 122.2, 132.8,
135.2, 151.1, 158.6, 165.1, 194.4. HRMS (EI) m/z calcd
for C14H13NO5, 275.0794;found, 275.0798.
3-Chloro-N-(2-hydroxy-3,4-epoxy-5-oxo-cyclohex-6-en-
yl)benzamide (15j). (70%, mp 139–141 ꢀC): 1H NMR
(acetone-d6) d 3.41 (dd, 1H, J=3.9, 2.1 Hz), 3.83 (t, 1H,
J=3.9 Hz), 4.66 (s, 1H), 6.92 (d, 1H, J=1.5 Hz), 7.33
(m, 1H), 7.39 (d, 2H, J=4.2 Hz), 7.60 (d, 1H, J=8.1
Hz), 8.72 (br s, 1H); 13C NMR (acetone-d6) d 53.6, 55.5,
65.2, 107.5, 109.1, 127.4, 130.6, 132.6, 133.5, 148.7,
151.1, 165.7, 193.8. HRMS (EI) m/z calcd for
C13H10NO4Cl, 279.0298;found, 279.0304.

C. Chaicharoenpong et al. / Bioorg. Med. Chem. 10 (2002) 3933–3939
3939
ꢀB/Luciferase reporter gene assay. Jurkat cells (2Â106
cells) were transfected with 2 mg of DNA consisting of 3
tandem kB repeats and the luciferase gene by the
DEAE-dextran method. The transfected cells were see-
ded into 12-well plates at 1Â106 cells/well. Chemicals
dissolved in DMSO and TNF-a were added at 14 and
16 h, respectively. Six hours after the TNF-a addition,
the cells were harvested and lysed, and the lysate was
used for the luciferase assay with luciferin substrate
buffer (20 mM Tricin-NaOH [pH 8.0], 1.07 mM mag-
nesium carbonate hydroxide, 2.67 mM MgSO4, 0.1 mM
EDTA, 33.3 mM DTT, 270 M CoA, 470 M luciferin,
and 530 mM ATP). Luminescence was measured with a
Lumat 9501 (Berthold). Each value was normalized by
the transfection efficiency obtained from the b-actin
promoter/b-galactosidase assay.
of Japan;and by funding from the Special Coordination
Funds for Promotion of Science and Technology of the
Science and Technology Agency of Japan.
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8. Erkel, G.;Anke, T.;Sterner, O.
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We wish to thank Miss Akiko Ariga of our Keio
laboratory for the evaluation of biological activity. This
work was financially supported in part by a grant from
Keio University Special Grants-in-Aid for Innovative
Collaborative Research Projects;by grants from the
programs Grants-in-Aid for Scientific Research on
Priority Areas (A) and Grants-in-Aid for Academic
Frontier Promotion Project of the Ministry of Educa-
tion, Science, Culture, and Sports of Japan;by grants
from the Science Research Promotion Fund of the Pro-
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