Angiogenesis inhibitor epoxyquinol a: total synthesis and inhibition of transcription factor NF-kappaB.

Article abstract of DOI:10.1021/ol026513n

[reaction: see text] The asymmetric synthesis of the natural product (+)-epoxyquinol A (1) and related epoxyquinoid dimers, employing a cascade oxidation/electrocyclization/Diels-Alder dimerization sequence, is reported. In addition, we show that 1 and related molecules inhibit activation of the transcription factor NF-kappaB.

Full text of DOI:10.1021/ol026513n

ORGANIC
LETTERS
2002
Vol. 4, No. 19
3267-3270
Angiogenesis Inhibitor Epoxyquinol A:
Total Synthesis and Inhibition of
Transcription Factor NF-KB
Chaomin Li,† Sujata Bardhan,† Emily A. Pace,‡ Mei-Chih Liang,‡
Thomas D. Gilmore,‡ and John A. Porco, Jr.*
,†
Department of Chemistry and Center for Streamlined Synthesis and Department of
Biology, Boston UniVersity, Boston, Massachusetts 02215
porco@chem.bu.edu.
Received July 13, 2002
ABSTRACT
The asymmetric synthesis of the natural product (+)-epoxyquinol A (1) and related epoxyquinoid dimers, employing a cascade oxidation/
electrocyclization/Diels−Alder dimerization sequence, is reported. In addition, we show that 1 and related molecules inhibit activation of the
transcription factor NF-KB.
Angiogenesis, new blood vessel formation, involves a
number of distinct processes, including endothelial cell
migration, proliferation, and capillary tube formation, and
is believed to be a key requirement for tumor growth and
metastasis.1 Angiogenesis inhibition thus represents an
important approach to cancer chemotherapy, and several
agents, including those derived from natural product leads,
are now entering clinical development.2 The pentaketide
don (inset, Figure 1)5 and jesterone).6 Thus far, chemical
dimer (+)-epoxyquinol A (1) was recently isolated from an
uncharacterized fungus by Osada and co-workers3 and was
shown to have potent antiangiogenic activity. The relative
stereochemistry of 1 was determined by X-ray crystal-
lography. We have previously reported syntheses of the
dimeric epoxyquinone natural product torreyanic acid4 and
two monomeric epoxyquinol natural products (cycloepoxy-
synthesis of an epoxyquinol dimer has not been reported. In
this Letter, we report the synthesis and absolute stereochem-
ical assignment of (+)-epoxyquinol A and four related
epoxyquinoid dimers. In addition, we show that 1 and related
molecules inhibit activation of transcription factor NF-κB.
A retrosynthetic analysis for (+)-epoxyquinol A is de-
picted in Figure 1. Compound 1 may be prepared by an
† Department of Chemistry and Center for Streamlined Synthesis.
‡ Department of Biology.
(1) (a) Ryan, C. J.; Wilding, G. Drugs Aging 2000, 17, 249. (b) Folkman,
J.; Browder, T.; Palmblad, J. Thromb. Haemostasis 2001, 86, 23.
(2) For a review of natural products as angiogenesis inhibitors, see:
Paper, D. H. Planta Med. 1998, 64, 686.
(3) Kakeya, H.; Onose, R.; Koshino, H.; Yoshida, A.; Kobayashi, K.;
Kageyama, S.-I.; Osada, H. J. Am. Chem. Soc. 2002, 124, 3496.
(4) Li, C.; Lobkovsky, E.; Porco, J. A., Jr. J. Am. Chem. Soc. 2000,
122, 10484.
(5) Li, C.; Pace, E. A.; Liang, M.-C.; Lobkovsky, E.; Gilmore, T. D.;
Porco, J. A., Jr. J. Am. Chem. Soc. 2001, 123, 11308.
(6) Hu, Y.; Li, C.; Kulkarni, B.; Strobel, G.; Lobkovsky, E.; Torczynski,
R. M.; Porco, J. A., Jr. Org. Lett. 2001, 3, 1649.
10.1021/ol026513n CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/17/2002

Scheme 1a
Figure 1. Retrosynthetic analysis for epoxyquinol A.
a Reagents and conditions: (a) (E)-tributyl-1-propenyl-stannane,
Pd2dba3, AsPh3, PhCH3, 110 °C, 2 h (98%); (b) 48% HF, CH3CN,
rt, 1 h (84%); (c) DIBAL-H, THF, -78 °C, 10 min (72%); (d) O2
(1 atm), TEMPO, CuCl, DMF, 3 h; (e) 40% MeOH-H2O, 10 h, 1
(55%), 10 (14%); (f) Dess-Martin periodinane, CH2Cl2, 1.5 h; SiO2,
2 h, 67%. DIBAL-H ) diisobutylaluminum hydride, TEMPO )
2,2,6,6-tetramethyl-1-piperidinyloxy.
oxidation/electrocyclization/Diels-Alder dimerization of di-
astereomeric 2H-pyran monomers 2/2′.4,6 In this biomimetic,
endo-selective7 Diels-Alder heterodimerization, the methyl
groups of the pyran are anti to one another and the dienophile
approaches the diene anti to the epoxide moiety. Monomers
2/2′ may be derived from selective primary oxidation of
epoxyquinol 3 (RK-302, isolated from the same fermentation
broth as 13) followed by 6π-electrocyclic ring closure of the
resulting dienal 4. Compound 3 may be prepared from readily
available chiral epoxide 5, which is a known intermediate
in our previous synthesis of cycloepoxydon.5
The synthesis was initiated by Stille coupling of 5 with
(E)-tributyl-1-propenyl-stannane to afford R-propenyl enone
6 in near quantitative yield (Scheme 1).8 Sequential hydroly-
sis of the cyclic acetal and silyl ether protecting groups
(aqueous HF) afforded the quinone monoepoxide 7. Regio-
and stereoselective reduction of 7 using Kiyooka’s condi-
tions9 (2 equiv of DIBAL-H, THF, -78 °C) afforded 310
(72%) as the major diastereomer.
In line with our previous studies,4,6 treatment of 3 with
Dess-Martin periodinane afforded a crude product mixture,
which was subjected to column chromatography to provide
two major fractions. NMR analysis indicated that the major
fraction contained 2H-pyrans 2/2′ and aldehyde 4 (cf. Figure
1), as well as the desired dimer 1. The minor fraction
comprised quinone epoxide 7, the related quinone 2H-pyran
monomers 8/8′, and quinone dimer 9 (Scheme 1).11 This
result indicates that undesired oxidation of the secondary
allylic alcohol was occurring under these conditions.12 To
promote the desired electrocyclization/Diels-Alder dimer-
ization, the major fraction was treated with silica gel in
CHCl3 for 20 h.4 Epoxyquinol A was obtained in 26% yield
after silica gel chromatography. Synthetic (+)-epoxyquinol
A was confirmed to be identical to natural (+)-epoxyquinol
1
A by H and 13C NMR, mass spectrum, [R]D (+66° (c )
0.23, CHCl3)), and TLC Rf values in three different solvent
systems.
Alternative oxidation methods were next evaluated in order
to obtain higher selectivity for oxidation of the primary vs
secondary allylic alcohol of 3 (Scheme 1). We found that
selective oxidation could be achieved using conditions
reported by Semmelhack (CuCl (cat.), O2, TEMPO, DMF).13
Under these conditions, no oxidation of the secondary allylic
alcohol or overoxidation to the carboxylic acid was detected.
After oxidation, the crude product mixture14 was dissolved
1
in CDCl3, and H NMR analysis was used to monitor the
disappearance of aldehyde and 2H-pyran. Use of this slightly
acidic condition for dimerization afforded endo dimer 1
(36%) along with a related dimer 10 (30%) after column
chromatography. In contrast, Diels-Alder dimerization in
40% aqueous MeOH (10 h) afforded epoxyquinol A and 10
in 55% and 14% yield, respectively.15 The structure of 10
was determined to be an exo-dimer of two identical 2H-pyran
monomers16 by X-ray crystal structure analysis (Figure 2).
(7) In refs 3 and 4, epoxyquinol and epoxyquinone dimers were referred
to as “exo.” However, it is more appropriate to refer to the dimers as “endo”
with respect to the carbonyl substituent on the 2H-pyran dienophile.
(8) Attempted Stille coupling using Pd(PPh3)4 as catalyst afforded the
undesired 1,3-diketone as the main product; cf.: Suzuki, M.; Watanabe,
A.; Noyori, R. J. Am. Chem. Soc. 1980, 102, 2095.
(12) It is also conceivable that internal hydride transfer of dienal 4 to a
quinone epoxide may occur. For a recent example of an internal hydride
transfer, see: Szendi, Z.; Forgo´, P.; Tasi, G.; Bo¨cskel, Z.; Nyerges, L.;
Sweet, F. Steroids 2002, 67, 31.
(9) Kiyooka, S.; Kuroda, H.; Shimasaki, Y. Tetrahedron Lett. 1986, 27,
3009.
(10) Synthetic 3 has the same HPLC retention time and the same Rf by
TLC with the natural compound RK-302 (see Supporting Information).
(11) Endo quinone dimer 9 was also produced in 67% yield by Dess-
Martin periodinane oxidation of 7, followed by treatment with silica gel
(Scheme 1). See Supporting Information for details.
(13) Semmelhack, M. F.; Schmid, C. R.; Cortes, D. A.; Chou, C. S. J.
Am. Chem. Soc. 1984, 106, 3374.
(14) 1H NMR (CDCl3) analysis of the crude reaction mixture indicates
a ratio of dienal 4:2H-pyran 2:dimer 1:dimer 10 of 1:4.9:7.9:7.7.
(15) For enhancement of endo/exo selectivity by water in Diels-Alder
reactions, see: Otto, S.; Engberts, J. B. F. N. Pure Appl. Chem. 2000, 72,
1365.
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Org. Lett., Vol. 4, No. 19, 2002

diene syn to the epoxide. Dimer 12 was similarly determined
to be an exo dimer in which the dienophile approaches the
diene syn to the epoxide. Interestingly, dimer 11 is derived
from Diels-Alder reaction of diastereomeric 2H-pyran
monomers (cf. 1), and 12 from identical 2H-pyran monomers
(cf. 10). Evidently, 11 and 12 are not kinetically favored
products and may be obtained under microwave irradiation
as a result of the higher activation energy provided under
these conditions. On the basis of these results, three under-
lying rules for Diels-Alder dimerizations of 2H-pyran
epoxyquinol monomers are thus apparent: (1) two identical
or diastereomeric 2H-pyran monomers may undergo endo-
or exo-Diels-Alder cycloaddition; (2) the methyl groups of
the diene and dienophile tend to orient themselves away from
one another, presumably to avoid steric interactions; and (3)
kinetically, the dienophile approaches the diene anti to the
epoxide moiety (cf. 1 and 10),19 whereas thermodynamically
(by higher activation energy with microwave irradiation),
the dienophile can approach the diene syn to the epoxide
moiety.
Figure 2. X-ray structure of dimer 10.
With diastereomeric epoxyquinol dimers 1 and 10 in hand,
we next investigated their reactivity to thermolysis. Surpris-
ingly, both dimers were found to be stable at 80 °C in CDCl3.
Microwave irradiation was next used as a method to establish
rapid thermodynamic equilibration of the compounds.17
Treatment of 1 in the CEM Discover microwave system
(chlorobenzene, 180 °C, 5 min) afforded exo dimer 10 (14%),
dimer 11 (43%) (Figure 3), and 12 (10%). Extended
Because other epoxyquinoids, including both natural5,20
and synthetic21 molecules, have been shown to inhibit
activation of transcription factor NF-κB,22 we determined
whether epoxyquinol A and three synthetic derivatives also
had this ability. As shown in Table 1 and Figure 4, dimers
Table 1. Inhibition of NF-κB DNA Binding by Epoxyquinol A
(1) and Related Compounds
compound
IC50 (µM)
epoxyquinol A (1)
quinone dimer (9)
quinol monomer (3)
quinone monomer (7)
11a
10a
20b
2.3b
a Average of four experiments. b Average of two experiments.
Figure 3. Microwave irradiation of epoxyquinol A and AM1
equilibrium geometries of 11 and 12 (PC Spartan Pro, v. 1.0.7).
1 and 9 and monomers 3 and 7 all inhibit tumor necrosis
factor (TNF)-induced activation of NF-κB DNA binding in
mouse 3T3 cells. In these assays, monomer 7 was ap-
proximately 5-10 times more effective than the others, with
an IC50 of 2.3 µM. Of note, transcription of the gene encoding
vascular endothelial growth factor (VEGF), one of the
primary mediators of angiogenesis, is known to be controlled,
at least in part, by NF-κB.23 Indeed, the concentrations of 1
microwave irradiation of 1 (180 °C, 2 × 5 min) led to the
production of exo dimer 10 in higher yield (36%) along with
11 (25%) and 12 (10%). However, when dimer 10 was
irradiated using the same conditions, starting material was
recovered, indicating higher stability of 10, possibly derived
from intramolecular hydrogen bonding.18 The structure of
11 was assigned by NOE difference NMR spectroscopy to
be an endo dimer in which the dienophile approaches the
(19) For an example of Diels-Alder cycloaddition with π-facial selectiv-
ity anti to the diene epoxide moiety, see: Gillard, J. R.; Newlands, M. J.;
Bridson, J. N.; Burnell, D. J. Can. J. Chem. 1991, 69, 1337.
(20) (a) Cycloepoxydon: Gehrt, A.; Erkel, G.; Anke, T.; Sterner, O. J.
Antibiot. 1998, 51, 455. (b) Panepoxydon: Erkel, G.; Anke, T.; Sterner, O.
Biochem. Biophys. Res. Commun. 1996, 226, 214.
(21) Umezawa, K.; Ariga, A.; Matsumoto, N. Anticancer Drug Des. 2000,
15, 239.
(22) For a review of natural products as modulators of the NF-κB
signaling pathway, see: Bremner, P.; Heinrich, M. J. Pharm. Pharmacol.
2002, 54, 453.
(23) For recent reports on suppression of angiogenesis by inhibition of
NF-κB in cancer cells, see: (a) Huang, S.; Robinson, J. B.; DeGuzman,
A.; Bucana, C. D.; Fidler, I. J. Cancer Res. 2000, 60, 5334. (b) Huang, S.;
Pettaway, C. A.; Uehara, H.; Bucana, C. D.; Fidler, I. J. Oncogene 2001,
20, 4188.
(16) For endo Diels-Alder dimerization of two identical 2H-pyran
monomers, see ref 4.
(17) For select examples of microwave irradiation of natural products,
see: (a) Salmoria, G. V.; Dall’Oglio, E. L.; Zucco, C. Synth. Commun.
1997, 29, 4335. (b) Das, B.; Venkataiah, B. Synth. Commun. 1999, 29,
863. (c) Das, B.; Madhusudhan, P.; Venkataiah, B. Synth. Commun. 2000,
30, 4001. (d) Das, B.; Venkataiah, B.; Kashinatham, A. Tetrahedron 1999,
55, 6585.
(18) Dimer 10 shows a distance of 2.7-2.8 Å between the two hydroxyl
oxygens (X-ray, two conformers), as well as an anomalous downfield shift
for one hydroxyl proton (1H NMR). For a recent example of intramolecular
OH-OH hydrogen bonding, see: Vasquez, T. E., Jr.; Bergset, J. M.;
Fierman, M. B.; Nelson, A.; Roth, J.; Khan, S. I.; O’Leary, D. J. J. Am.
Chem. Soc. 2002, 124, 2931.
Org. Lett., Vol. 4, No. 19, 2002
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epoxyquinol 2H-pyran monomers. Additional epoxyquinone
and epoxyquinol dimers have been produced as part of these
studies, which further clarifies the underlying rules for
Diels-Alder dimerizations of epoxide-containing 2H-pyran
monomers. Notably, microwave irradiation of epoxyquinol
A has been used to produce novel stereoisomers of 1 that
are not accessible at ambient temperature. Future biological
experiments will be aimed at identifying the molecular
target(s) for epoxyquinol A mediated inhibition of NF-κB
activation and angiogenesis. Similarly, it will be of interest
to determine whether epoxyquinol A and related compounds
also inhibit other biological processes, including inflamma-
tion and the growth of certain tumor cells, that are dependent
on NF-κB.
Figure 4. Compound 7 inhibits tumor necrosis factor-induced
activation of NF-κB. Mouse 3T3 cells were untreated (lane 1) or
were treated with TNF-R (lanes 2-6), and NF-κB DNA-binding
activity was analyzed in an electrophoretic mobility shift assay. In
lanes 3-6, cells were preincubated with 0.675, 1.25, 2.5, and 5
µM, respectively, 7 prior to treatment with TNF-R; in lanes 1 and
2, cells were preincubated with solvent (CH3OH). The arrow
indicates the induced NF-κB (p50-RelA) complex.
Acknowledgment. Financial support from the American
Cancer Society (RSG-01-135-01, J.A.P, Jr.), the NIH
(CA47763, T.D.G.), and the Ministry of Education, Taiwan
(M.-C.L.) is gratefully acknowledged. We thank Prof.
Hiroyuki Osada and Dr. Hideaki Kakeya (RIKEN Institute)
for providing authentic epoxyquinol A and for HPLC
comparison of synthetic and natural RK-302, CEM Corpora-
tion (Matthews, NC) for providing the Discover microwave
system, and Profs. J. Panek and S. E. Schaus (Boston
University) for helpful comments.
reported to inhibit cell migration induced by VEGF3 (ED100
) 7 µM) and activation of NF-κB (Table 1) are similar. Thus,
our demonstration that epoxyquinol A and related compounds
inhibit induction of NF-κB may explain the molecular basis
for inhibition of angiogenesis by epoxyquinol A. If so, one
would predict that 7, the most effective inhibitor of TNF-
induced activation of NF-κB, would also be the most
effective blocker of angiogenesis.
Supporting Information Available: Experimental pro-
cedures and characterization data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
In summary, we have completed an asymmetric synthesis
of epoxyquinol A employing a [4 + 2] dimerization of
OL026513N
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