Synthesis and evaluation of a cyclopropane derivative of DHMEQ

Article abstract of DOI:10.1248/cpb.c13-00914

Dehydroxymethylepoxyquinomycin (DHMEQ, 1) is well known to inhibit nuclear factor-kappa B (NF-κB), which is closely associated with immune, inflammatory, and apoptotic processes as an inducible transcription factor. The inhibitory effect seems to be the r

Full text of DOI:10.1248/cpb.c13-00914

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Note
Chem. Pharm. Bull. 62(3) 304–307 (2014)
Vol. 62, No. 3
Synthesis and Evaluation of a Cyclopropane Derivative of DHMEQ
Eiko Yasui,* Kan Takayama, Takahiro Nakago, Nobuyuki Takeda, Yasutada Imamura, and
Shinji Nagumo*
Department of Applied Chemistry, Kogakuin University; 2665–1 Nakano, Hachioji, Tokyo 192–0015, Japan.
Received November 24, 2013; accepted December 19, 2013; advance publication released online December 26, 2013
Dehydroxymethylepoxyquinomycin (DHMEQ, 1) is well known to inhibit nuclear factor-kappa B (NF-
κB), which is closely associated with immune, inflammatory, and apoptotic processes as an inducible tran-
scription factor. The inhibitory effect seems to be the result of the ring opening of an epoxide of 1 with Cys38
of p65. We have synthesized an analog 4 containing a cyclopropanated quinol skeleton and examined its abil-
ity to inhibit NF-κB. Surprisingly, 4 showed no remarkable NF-κB inhibitory activity as determined through
expression of cyclooxygenase-2 (COX-2) in an RAW264.7 macrophage cell line.
Key words dehydroxymethylepoxyquinomycin (DHMEQ); cyclopropane; nuclear factor-kappa B (NF-κB);
cyclooxygenase-2 (COX-2); RAW264.7
The recent growth in molecular biology has generated NMR spectra. Although the regioselective ring opening of the
new drug targets causing a dramatic paradigm shift in the epoxide is essential for the additional inhibitory effect of 1
chemotherapeutics of cancer (e.g., molecularly targeted thera- against p65, it has not yet been completely confirmed whether
peutics). Recently, much attention has been concentrated on a similar chemical binding occurs in biological processes.
nuclear factor-kappa B (NF-κB), which belongs to a family In this paper, we describe the synthesis of a new DHMEQ
of transcription factors associated with immune and inflam- analog 4 with a cyclopropane ring instead of an epoxide and
matory processes. NF-κB is a homo- or heterodimer of Rel the evaluation of its NF-κB inhibitory activity to clarify the
family proteins including p65 (RelA), p50, RelB, c-Rel, significance of the epoxide in 1 using a structure–activity
and p52. Among them, p65 (RelA), c-Rel, and p52 have a relationship study.
transactivation domain. Continuous activation of NF-κB is
known to occur in various types of human tumors, and is
correlated with inactivation of apoptosis via transcriptional
Results and Discussion
Because 3b also inhibits NF-κB activation,4) synthesis of its
misregulation. NF-κB inhibitors are thus expected to be prom- analog 8 was initially conducted as shown in Chart 2. Dienone
inent candidates for anticancer drugs.1) Dehydroxymethyl- 5 was prepared from 2,5-dimethoxyaniline following a known
epoxyquinomycin (DHMEQ, 1), first synthesized by Umezawa procedure.5) Treatment of dienone 5 with trimethylsulfoxo-
and colleagues on the basis of a structural modification of ep- nium iodide in the presence of NaH resulted in selective cy-
oxyquinomycin C (Chart 1), shows a potent NF-κB inhibitory clopropanation at the C4,5 double bond to generate 6 in 77%
effect with little toxicity.2) The careful matrix assisted laser yield.6,7) Deacetalization of
6
with pyridinium p-
desorption ionization-time of flight (MALDI-TOF)-MS analy- toluenesulfonate (PPTS) afforded the diketone 7 in excellent
sis by Umezawa and colleagues of chymotrypsin-treated p65 yield. Finally, reduction of 7 with L-selectride® proceeded
peptide clarified that the bioactivity of 1 is a result of coupling stereoselectively to give 8 with cis-configuration. Next, we
with Cys38 of p65.3) To specify the covalent bond formed, they attempted the conversion of 8 into the second target 4. After
conducted a reaction of 1 with N-Boc-L-Cys methyl ester. The protection of the alcohol 8 with an acetyl group, the result-
resulting conjugate was identified as 2 based on its mass and ing acetate 9 was subjected to removal of an alloc group
Chart 1. Cyclopropane Derivative of DHMEQ
The authors declare no conflict of interest.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: bt13305@ns.kogakuin.ac.jp

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Chart 2. Synthesis of Quinol Derivative 4
Chart 3. Reaction of 4 with Nucleophiles
upon treatment with Pd(PPh3)4 and dimedone. Unfortunately,
although deprotection of 9 proceeded smoothly to give
amine 10, a partial O→N acetyl migration occurred to also
generate alcohol 11. We thus decided to synthesize 4 from
the dienone 12, which can be prepared in two steps from
2,5-dimethoxyaniline.8) Cyclopropanation of 12 with trimeth-
ylsulfoxonium iodide proceeded regioselectively to give 13
in good yield. Finally, 13 was successfully converted into the
target 4 by removal of dimethyl acetal and subsequent stere-
oselective reduction. The stereochemistry of the alcohol was
verified by nuclear Overhauser effect (NOE) measurement.
It is reported that DHMEQ reacts rapidly with nucleophiles
like benzylthiol and cysteine derivatives to give DHMEQ–
cysteine adducts in dimethyl sulfoxide (DMSO)/phosphine
buffer.3) Although compound 4 was submitted to the same
Cells were treated with or without chemicals at indicated concentrations for 1h
and stimulated with or without LPS at indicated concentrations for 8h. COX-2
expression was evaluated by Western blotting using anti-COX-2 antibody (BD, cat.
No. C22420) according to the manufacturer’s instructions.
Chart 4. COX-2 Expression in an RAW264.7 Macrophage Cell Line
reaction, no adduct formation was observed, even after a long inhibitory activity on NF-κB in RAW264.7 cells.
reaction time (Chart 3).
Next, its inhibitory activity on NF-κB was examined in
Conclusion
an RAW264.7 macrophage cell line. It is known that cyclo-
We have achieved the synthesis of 4, a cyclopropane
oxygenase-2 (COX-2) is expressed in the cell when NF-κB is derivative of DHMEQ (1), from commercially available
activated.9) Compounds 1 and 4 were dissolved in DMSO at 2,5-dimethoxyaniline in 5 steps (total yield 29%). We showed
1mg/mL, respectively, and 10µL was added to the cell cul- that 4 has no recognizable inhibitory activity on NF-κB in an
ture. After 1h, the medium was washed off. The cells were RAW264.7 macrophage cell line. These results strongly sup-
treated with lipopolysaccharide (LPS) and cultured for 8h. port the suggestion by Umezawa and colleagues, that is, the
Subsequently, the cells were lysed and the inhibitory activity covalent binding of the thiol group in Cys38 of p65 with the
on NF-κB was assesed by estimating the expression of COX-2. epoxide of DHMEQ is closely correlated with its inhibitory
The production level of COX-2 was inhibited in cells treated activity.
with DHMEQ (1), but cells treated with compound 4 produced
COX-2 at almost the same level as the control group (Chart
4). Therefore, we concluded that compound 4 has little or no
Experimental
1H- and 13C-NMR spectra were recorded on a JEOL JNM-

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Vol. 62, No. 3
1
ECX-400 spectrometer at 400 and 100MHz, respectively. Allyl Ester 8: mp 94–95°C; H-NMR (CDCl3, 400MHz) δ:
Chemical shifts were expressed in δ parts per million with 7.56 (s, 1H), 6.44 (t, J=1.2Hz, 1H), 5.92 (ddt, J=17.2, 10.8,
1
tetramethylsilane as internal standard (δ=0ppm) for H-NMR. 4.8Hz, 1H), 5.35 (dq, J=17.6, 1.2Hz, 1H), 5.27 (dq, J=10.4,
Chemical shifts of carbon signals were referenced to CDCl3 1.2Hz, 1H), 4.81 (dt, J=7.6, 1.2Hz, 1H), 4.63 (dt, J=5.6,
(δC=77.0ppm). The following abbreviations are used: s= 1.2Hz, 2H), 3.94 (d, J=7.6Hz, 1H), 1.95–2.09 (m, 2H), 1.25
singlet, d=doublet, t=triplet, q=quartet, m=multiplet, and (m, 1H), 1.06 (q, J=5.2Hz, 1H); 13C-NMR (CDCl3, 100MHz)
br=broad. IR spectra were recorded on a Shimadzu IR δ: 198.02, 152.26, 149.22, 131.60, 118.94, 104.24, 66.46,
Prestige-21. Mass spectra were recorded on a JEOL JMS- 64.41, 23.62, 17.56, 12.55; IR (KBr) 3225, 1744, 1620, 1551,
GCmate II. Melting points were determined on a Yanaco 1227cm−1; HR-MS (EI) Calcd for C11H13NO4 (M+) 223.0845,
MP-S3 micro melting point apparatus and were uncorrected.
Trimethylsulfoxonium iodide (12.9g, 57.4mmol) was dis-
Found 223.0844.
Trimethylsulfoxonium iodide (0.40g, 1.80mmol) was dis-
solved in DMSO (31.5mL) and stirred at room temperature solved in DMSO (1.6mL) and stirred at 0°C under an argon
under an argon atmosphere. To this solution was added atmosphere. To this solution was added sodium hydride
sodium hydride (50–72% in mineral oil, 2.25g). After stirring (50–72% in mineral oil, 86.0mg). After stirring for 20min at
for 30min, dienone 5 (0.73g, 2.87mmol) in DMSO (31.5mL) room temperature, dienone 12 (0.12g, 0.36mmol) in DMSO
was added and stirred for 17h. To this reaction mixture was (2mL) was added and stirred for 1.5h. To this reaction mix-
added aqueous saturated NH4Cl and extracted with ethyl ture was added aqueous saturated NH4Cl and extracted with
acetate for 3 times. The organic phase was dried over Na2SO4 ethyl acetate for 3 times. The organic phase was dried over
and concentrated in vacuo. The resulting residue was purified Na2SO4 and concentrated in vacuo. The resulting residue was
by silica gel column chromatography (hexane–ethyl acetate= purified by silica gel column chromatography (hexane–ethyl
1:1) to give 6 (0.59g, 77%).
acetate=1:1) to give 13 (0.10g, 92%).
(2,2-Dimethoxy-5-oxobicyclo[4.1.0]hept-3-en-3-yl)carbamic
N-(2,2-Dimethoxy-5-oxobicyclo[4.1.0]hept-3-en-3-yl)-2-
1
Acid Allyl Ester 6: H-NMR (CDCl3, 400MHz) δ: 7.28 (brs, hydroxy-benzamide 13: Amorphous 1H-NMR (CDCl3,
1H), 6.65 (d, J=1.2Hz, 1H), 5.94 (m, 1H), 5.36 (dq, J=17.2, 400MHz) δ: 11.47(brs, 1H), 8.79 (s, 1H), 7.45 (m, 2H), 7.11
1.6Hz, 1H), 5.28 (dd, J=10.4, 1.2Hz, 1H), 4.64 (q, J=1.6Hz, (d, J=1.2, 1H), 7.02 (dd, J=8.0, 0.8Hz, 1H), 6.94 (dt, J=8.0,
1H), 4.62 (q, J=1.6Hz, 1H), 3.56 (s, 3H), 3.27 (s, 3H), 2.08 0.8Hz, 1H), 3.66 (s, 3H), 3.31 (s, 3H), 2.16 (m, 1H), 2.06
(m, 1H), 2.00 (m, 1H), 1.29 (m, 1H), 0.96 (q, J=4.8Hz, 1H); (dt, J=7.6, 6.4Hz, 1H), 1.35 (dt, J=8.4, 5.2Hz, 1H), 1.05 (q,
13C-NMR (CDCl3, 100MHz) δ: 196.05, 152.01, 144.32, 131.54, J=5.2Hz, 1H); 13C-NMR (CDCl3, 100MHz) δ: 196.59, 168.93,
118.81, 107.01, 96.63, 66.26, 50.96, 49.42, 23.16, 18.15, 13.01; 161.58, 142.99, 135.31, 125.78, 119.28, 118.96, 114.65, 110.25,
IR (film) 1744, 1659, 1628, 1512, 1211cm−1; high resolution 97.10, 51.18, 49.97, 23.49, 18.32, 13.23; IR (film) 3248, 1659,
(HR)-MS (electron ionization (EI)) Calcd for C13H17NO5 (M+) 1636, 1605, 1520, 1211, 1065cm−1; HR-MS (EI) Calcd for
267.1107, Found 267.1122.
Dimethyl acetal 6 (0.28g, 1.06mmol) was dissolved in ace-
C15H13NO4 ([M−CH3OH]+) 271.0845, Found 271.0845.
Dimethyl acetal 13 (0.80g, 2.62mmol) was dissolved in
tone (10mL) and stirred at room temperature. To this solution acetone (27mL) and stirred at room temperature. To this solu-
was added H2O (1mL) and (PPTS, 0.27g, 1.06mmol). After tion was added H2O (2.6mL) and PPTS (0.66g, 2.62mmol).
stirring for 24h, aqueous saturated NaHCO3 was added and After stirring for 22h, aqueous saturated NaHCO3 was added
extracted with ethyl acetate for 3 times. The organic phase and extracted with ethyl acetate for 3 times. The organic
was dried over Na2SO4 and concentrated in vacuo. The result- phase was dried over Na2SO4 and concentrated in vacuo. The
ing residue was purified by silica gel column chromatography resulting residue was purified by silica gel column chroma-
(hexane–ethyl acetate=1:1) to give diketone 7 (0.23g, 97%).
tography (hexane–ethyl acetate=3:2) to give diketone (0.62g,
(2,5-Dioxobicyclo[4.1.0]hept-3-en-3-yl)carbamic Acid Allyl 92%). Diketone (0.62g, 2.42mmol) was dissolved in THF
1
Ester 7: H-NMR (CDCl3, 400MHz) δ: 7.57 (brs, 1H), 7.02 (d, (25mL) and stirred at −20°C under an argon atmosphere. To
J=2.0Hz, 1H), 5.92 (m, 1H), 5.93 (ddt, J=17.6, 10.8, 6.0Hz, this solution was added L-selectride® (1.0 M in THF solution,
1H), 5.37 (dq, J=17.6, 1.2Hz, 1H), 5.29 (dq, J=10.0, 1.2Hz, 4.9mL). After stirring for 1.5h, aqueous 10% HCl was added
1H), 4.66 (dt, J=6.0, 1.2Hz, 1H), 2.62–2.52 (m, 2H), 1.75 (dt, and extracted with ethyl acetate for 3 times. The organic
J=8.8, 5.2Hz, 1H), 1.66 (q, J=5.2Hz, 1H); 13C-NMR (CDCl3, phase was dried over Na2SO4 and concentrated in vacuo to
100 MHz) δ: 194.18, 190.52, 151.92, 138.67, 131.33, 119.14, give alcohol 4 (0.41g, 65%). The resulting solid was recrystal-
113.69, 66.71, 26.98, 25.40, 21.16; IR (film) 1744, 1658, 1512, lized from EtOH to give colourless crystal.
1203cm−1; HR-MS (EI) Calcd for C11H11NO4 (M+) 221.0688,
Found 221.0688.
2-Hydroxy-N-(2-hydroxy-5-oxobicyclo[4.1.0]hept-3-en-3-yl)-
benzamide 4: mp 212°C (dec.); 1H-NMR (DMSO-d6, 400MHz)
Diketone 7 (0.10g, 0.46mmol) was dissolved in tetrahydro- δ: 11.70 (s, 1H), 10.95 (s, 1H), 7.91 (dd, J=7.6, 2.0Hz, 1H),
furan (THF) (5mL) and stirred at −38°C under an argon 7.41 (dt, J=6.4, 2.0Hz, 1H), 6.97 (t, J=8.0Hz, 1H), 6.94 (dd,
atmosphere. To this solution was added L-selectride® (1.0 M in J=8.0, 0.8Hz, 1H), 6.74 (t, J=1.6Hz, 1H), 6.05 (d, J=8.0Hz,
THF solution, 0.47mL). After stirring for 3min, aqueous 10% 1H), 4.79 (dt, J=7.2, 1.6Hz, 1H), 1.93 (m, 1H), 1.79 (m, 1H),
HCl was added and extracted with ethyl acetate for 3 times. 1.16 (dt, J=8.0, 4.0Hz, 1H), 0.93 (q, J=4.8Hz, 1H); 13C-NMR
The organic phase was dried over Na2SO4 and concentrated in (DMSO-d6, 100MHz) δ: 197.04, 164.69, 156.03, 149.95,
vacuo. The resulting residue was purified by silica gel column 134.08, 131.10, 119.85, 118.47, 116.90, 105.80, 63.47, 23.24,
chromatography (hexane–acetone=2.5:1) to give alcohol 8 17.42, 12.07; IR (KBr) 3364, 1659, 1605, 1528cm−1; HR-MS
(92.0mg, 90%). The resulting solid was recrystallized from (EI) Calcd for C14H13NO4 (M+) 259.0845, Found 259.0845.
hexane–ethyl acetate to give colourless crystal.
(2-Hydroxy-5-oxobicyclo[4.1.0]hept-3-en-3-yl)carbamic Acid

March 2014
307
(cyclopropane ring and hydroxyl group are substituted as anti) and
their inhibitory activities against NF-κB would be reported in the
next paper.
References and Notes
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(2006).
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5821 (1981).
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(1965).
8) Suzuki Y., Sugiyama C., Ohno O., Umezawa K., Tetrahedron, 60,
7061–7066 (2004).
9) Vandoros G. P., Konstantinopoulos P. A., Sotiropoulou-Bonikou G.,
Kominea A., Papachristou G. I., Karamouzis M. V., Gkermpesi M.,
Varakis I., Papavassiliou A. G., J. Cancer Res. Clin. Oncol., 132,
76–84 (2006).
4) Compound 3a, a derivative of DHMEQ with the same stereochem-
istry, scarcely inhibited NF-κB activation, but it weakly binds to
p65. On the other hand, compound 3b, a derivative of DHMEQ
with the opposite stereochemistry, inhibited NF-κB activation dose-
dependently, but it does not bind to p65 (refer to ref. 5). Compound
8 was synthesized to examine structure–activity correlation of
cyclopropane derivatives. The synthesis of epimer of compound 8