Synthesis and evaluation of bioactive naphthoquinones from the Brazilian medicinal plant, Tabebuia avellanedae

Article abstract of DOI:10.1016/j.bmc.2009.07.039

A series of naphthoquinones based on the naphtho[2,3-b]furan-4,9-dione skeleton such as (-)-5-hydroxy-2-(1′-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (1) and its positional isomer, (-)-8-hydroxy-2-(1′-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (2), which are secondary metabolites found in the inner bark of Tabebuia avellanedae, were stereoselectively synthesized and their biological activities were evaluated in conjunction with those of their corresponding enantiomers. Compound 1 exhibited potent antiproliferative effect against several human tumor cell lines, but its effect against some human normal cell lines was much lower than that of mitomycin. On the other hand, its enantiomer (R)-1 was less active toward the above tumor cell lines than 1. The antiproliferative effect of 2 against all tumor cell lines was significantly reduced. These results indicated the presence of the phenolic hydroxy group at C-5 is of great important for increasing antiproliferative effect. In addition, 1 also showed higher cancer chemopreventive activity than 2, while there were no significant differences between 1 and 2 in antimicrobial activity. Both compounds displayed modest antifungal and antibacterial activity (Gram-positive bacteria), whereas they were inactive against Gram-negative bacteria.

Full text of DOI:10.1016/j.bmc.2009.07.039

Bioorganic & Medicinal Chemistry 17 (2009) 6286–6291
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of bioactive naphthoquinones from the Brazilian
medicinal plant, Tabebuia avellanedae
Mitsuaki Yamashita a, Masafumi Kaneko a, Harukuni Tokuda b, Katsumi Nishimura c,
Yuko Kumeda d, Akira Iida e,
*
a Faculty of Pharmacy, Takasaki University of Health and Welfare, Nakaorui-machi, Takasaki 370-0033, Japan
b Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan
c Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
d Osaka Prefectural Institute of Public Health, Nakamichi, Osaka 537-0025, Japan
e School of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of naphthoquinones based on the naphtho[2,3-b]furan-4,9-dione skeleton such as (ꢀ)-5-
hydroxy-2-(10-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (1) and its positional isomer, (ꢀ)-8-
hydroxy-2-(10-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (2), which are secondary metabolites found
in the inner bark of Tabebuia avellanedae, were stereoselectively synthesized and their biological activities
were evaluated in conjunction with those of their corresponding enantiomers. Compound 1 exhibited
potent antiproliferative effect against several human tumor cell lines, but its effect against some human
normal cell lines was much lower than that of mitomycin. On the other hand, its enantiomer (R)-1 was
less active toward the above tumor cell lines than 1. The antiproliferative effect of 2 against all tumor cell
lines was significantly reduced. These results indicated the presence of the phenolic hydroxy group at C-5
is of great important for increasing antiproliferative effect. In addition, 1 also showed higher cancer che-
mopreventive activity than 2, while there were no significant differences between 1 and 2 in antimicro-
bial activity. Both compounds displayed modest antifungal and antibacterial activity (Gram-positive
bacteria), whereas they were inactive against Gram-negative bacteria.
Received 6 June 2009
Revised 20 July 2009
Accepted 21 July 2009
Available online 23 July 2009
Keywords:
Stereoselective synthesis
Antiproliferative activity
Naphtho[2,3-b]furan-4,9-dione
Cancer chemopreventive activity
Tabebuia avellanedae
Antimicrobial activity
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
tumor cells as well as lapachol (3) and b-lapachone (4) that are
congeners of 1 in this plant ( Fig. 1).6 In addition, compound 1
Tabebuia avellanedae LORENTZ ex GRISEB1 (Bignoniaceae) (syn.
Tabebuia impetiginosa), which is widespread in South America
throughout Brazil to north Argentina, has been well known as a
traditional medicine since the Incan Era.2 The stem bark of T. avel-
lanedae has been used as a diuretic and as an astringent. In addi-
tion, the stem bark of this plant shows a wide array of biological
activities such as antitumor, antibacterial, antifungal and anti-
inflammatory activity.3 In particular, the discovery of its antitumor
activity has made T. avellanedae an important medicinal resource.4
A series of naphthoquinones and anthraquinones, a number of sim-
ple benzoic acid derivatives and iridoid glycosides has been iso-
lated as secondary metabolites of this plant.5 Among these
constituents, naphtho[2,3-b]furan-4,9-dione analogues such as
(ꢀ)-5-hydroxy-2-(10-hydoxyethyl)naphtho[2,3-b]furan-4,9-dione (1)
and its positional isomer, (ꢀ)–8-hydroxy-2-(10-hydoxyethyl)naph-
tho[2,3-b]furan-4,9-dione (2) are, in particular, worthy of attention
because of their potent antiproliferative activity against various
strongly inhibits Epstein-Barr virus early antigen (EBV-EA) activa-
tion induced by the tumor promoter, 12-O-tetradecanoylphorbol-
13-acetate (TPA) in Raji cell7 and TPA-induced tumor promotion
on mouse skin initiated with 7,12-dimethylbenz[a]anthracene
(DMBA) in two-stage carcinogenesis tests. Therefore, compound
1 is expected to be a potential cancer chemopreventer.8
OH
O
O
O
5
S
S
1'
O
O
OH
OH
8
O
OH
1
2
O
O
O
OH
3
O
4
O
* Corresponding author. Tel./fax: +81 742 43 7274.
E-mail address: iida@nara.kindai.ac.jp (A. Iida).
Figure 1.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.039

M. Yamashita et al. / Bioorg. Med. Chem. 17 (2009) 6286–6291
6287
In our precedent paper,9 we reported a concise and stereoselec-
tive synthesis of 1 and its antiproliferative effect toward several tu-
mor cell lines and in vitro cancer preventive activity. Through our
preliminary results, we clearly recognized that there were signifi-
cant differences between 1 and (R)-1 in their biological activities
mentioned above. This finding promoted us to synthesize both 2
and its enantiomer (R)-2 and to evaluate their biological activities
since it has been reported by Wagner et al. that compound 2 was
isolated in the state of a racemic mixture.5c In fact, we also could
obtain 2 only as a racemate through repeated HPLC of a CHCl3 ex-
tract of T. avellanedae. On the other hand, Fujimoto’s group, who
first obtained 1 and (R)-1 together with 2 and (R)-2 by optical res-
olution of the synthetic racemates 1 and 2, isolated 2 from the
same plant as a mixture in which the ratio of the (R)- and (S)-enan-
tiomer was 3: 1. Furthermore, they reported that there were no dif-
ferences between the two pairs of enantiomers in their
antiproliferative activity toward L1210 cells.6c These results also
encouraged us to advance our research program. Herein we report
the stereoselective synthesis of two pairs of enantiomers described
above via Noyori reduction as a key step and their antiproliferative
effect against some human tumor cell lines and their correspond-
ing human normal cell lines, in vitro cancer chemopreventive
activity and antimicrobial activity.10 In addition, in vivo cancer
chemopreventive activity of 1 and (R)-1 is also described.
one, which was synthesized from commercially available but-3-
en-2-one and bromine, in the presence of DBU in THF according
to the method reported by Hagiwara and others13 to give naph-
thodihydrofuran 9a. It should be noted that the gram-scale synthe-
sis afforded only 9a without the simultaneous formation of 10a
differently from the case of the milligram-scale synthesis.9a After
the crude product was washed with MeOH, naphthodihydrofuran
9a was treated with MnO2 in chloroform to provide the desired
naphthofuran 10a in 50% yield in two steps. Compound 10b was
synthesized from 8b via 9b in the same manner as that described
above. Subsequent Noyori reduction completed the stereoselective
synthesis of 1 and 2 (Scheme 2).14 Asymmetric transfer hydrogena-
tion of naphthofurans 10a and 10b, which was catalyzed by a
commercially available chiral Ru(II) complex, Ru [(S,S)-Tsdpen]
(p-cymene)15 in a formic acid–triethylamine mixture and CH2Cl2,
successfully afforded the corresponding secondary alcohols 1 and
2 in high chemical yield and enantiomeric excess (89–97% yield,
95–97% ee). The physico-chemical properties of all the compounds
synthesized in this study were identical in all respects to those
reported previously.13 The corresponding enantiomers (R)-1 and
(R)-2 were similarly prepared using Ru [(R,R)-Tsdpen] (p-cymene)
as a chiral catalyst.16
2.2. Biology
As reported so far, naphthoquinones isolated from the Tabebuia
plants are recognized as antiproliferative compounds.3b,6,7 Thus,
the two pairs of enantiomers synthesized were also evaluated for
their ability to suppress the growth of human tumor cell lines
including PC-3 (prostate), A549 (lung) and MCF-7 (breast) (Table
1). Compound 1 exhibited rather potent antiproliferative activity
2. Results and discussion
2.1. Chemistry
The gram-scale synthesis of 1 and 2 were accomplished simi-
larly according to our preliminary synthesis of 1.9 The synthetic
way to 1 and 2 is shown in Scheme 1. The synthesis of juglone
(6) was started from commercially available 1,5-hydroxynaphtha-
lene, which was oxidized with air in the presence of CuCl in the
dark to give 6 in 47% yield.11 Chemical transformations of 6 to 8
was carried out according to the reported methods12 with some
against all the three cell lines (0.14–0.78
equal to that of mitomycin (0.14–0.96 M) and much higher than
that of b-lapachone (1.13–9.96 M). However, its antiproliferative
lM), which was almost
l
l
effect against human normal cell lines including Hs888Lu (lung)
and SVCT-M12 (breast) was noticeably lower than that of mitomy-
cin (1: 3.36–54.5 lM, mitomycin: 0.56–1.46 lM) (Table 2).
modifications. Oxidative amination of
6 with dimethylamine
On the other hand, the antiproliferative activity of its enantio-
mer (R)-1 was discernibly reduced toward the tumor cell lines, in
particular against MCF-7 cells, compared with that of 1. These re-
sults suggested that the orientation of the hydroxy group at C-10
is crucial. Interestingly, ketone 10a was between 1 and (R)-1 in
antiproliferative activity. This finding suggests the superiority of
the a-oriented hydroxy group of 1. Regarding the four human nor-
mal cell lines tested (Table 2), the antiproliferative activity of (R)-1
was somewhat higher than 1 only against IE cells.
(2.0 M solution in THF) in toluene at ꢀ40 °C gave 2-dimethylami-
nojuglone (7a) and 3-dimethylaminojuglone (7b) in 42% and 11%
yields, respectively. As reported in the precedent paper,9 a THF
solution of dimethylamine is suitable reagent for practical use,
and moderate chemical yield and regioselectivity were observed.
Deamination of 2-dimethylaminojuglone (7a) with 10% aqueous
HCl gave 2,5-dihydroxy-1,4-naphthoquinone (8a) in quantitative
chemical yield.12b The naphtho[2,3-b]furan-4,9-dione skeleton
was constructed by the reaction of 8a with 3,4-dibromobutan-2-
The comparison between 1 and 2 showed that the position of a
hydroxy group attached to the aromatic ring also affects their abil-
ity to suppress the growth of the tumor cell lines. As shown in Ta-
ble 1, the antiproliferative effect of 2 that possesses a hydroxy
function at C-8 was considerably lower than that of 1 (1.57–
OH
OH
O
O
X
O
a
b
c
4.31 lM) although it was comparable with that of b-lapachone.
NMe2
Thus, the presence of a phenolic hydroxy group at C-5 seems to
play an important role in increasing antiproliferative effect. A sim-
ilar effect was also observed between 10a and 10b, supporting its
role described above.
5
6
OH
Y
O
7a: X = OH, Y = H
7b:
X = H, Y = OH
X
Y
O
X
Y
O
O
X
O
d
e
5 mol %
Ru [(S,S)-Tsdpen]
(p-cymene)
X
O
X
O
O
O
OH
O
O
(S)
O
Y
O
HCO2H–Et3N
(5:2)
O
O
OH
O
8a: X = OH, Y = H
8b:
9a: X = OH, Y = H
10a: X = OH, Y = H
10b:
9b:
Y
O
X = H, Y = OH
X = H, Y = OH
X = H, Y = OH
Y
10a:
O
CH2Cl2
rt, 24 h
89-97% yield, 95-97% ee
1:
X = OH, Y = H
X = OH, Y = H
10b: X = H, Y = OH
2: X = H, Y = OH
Scheme 1. Synthesis of 10. Reagents and conditions: (a) CuCl, CH3CN, air, rt, 47%;
(b) Me2NH, toluene, THF, ꢀ40 °C, 42% and 11% for 7a and 7b, respectively; (c) 10%
HCl, dioxane, reflux, 97%; (d) 3,4-dibromobutan-2-one, DBU, THF, room tempera-
ture; (e) MnO2, CHCl3, reflux, 50–60% in two steps.
Scheme 2. Chiral Ru catalyst mediated asymmetric hydrogenation of naphthofuran
10.

6288
M. Yamashita et al. / Bioorg. Med. Chem. 17 (2009) 6286–6291
Table 1
Table 3
Antiproliferative effects of 10a, 10b, 1, (R)-1, racemate 1, 2, b-lapachone, lapachol and
Inhibitory effect of 1, (R)-1, racemate 1, 2, (R)-2, racemate 2, b-lapachone and lapachol
on TPA-induced EBV-EA activation
mitomycin against human tumor cell linesa
Compound
EC50
(
lM)
Compound
EBV-EA-positive cells (% viability)a
Human tumor cell lines
Compound concentration (mol ratio/32 pmol TPA)
PC-3
A549
MCF-7
1000
500
100
10
IC50 (lM)
10a
10b
Racemate 1
(R)-1
1
0.84
3.00
0.56
0.93
0.14
1.57
1.13
1.97
0.14
2.0
0.78
2.61
8.5
10a
10b
Racemate 1
(R)-1
1
Racemate 2
(R)-2
2
b-Lapachone
Lapachol
Curcuminc
0 (60)
7.1
8.3
6.2 (70)
9.7
4.4 (60)
28.4
20.4
26.5
21.7
32.8 (60)
40.3
23.7
27.5
20.7
24.7
16.9
59.0
52.5
58.3
50.4
65.2
74.5
56.0
60.3
52.9
59.4
50.0
84.6
76.3
80.5
73.1
86.6
95.8
37.2
40.1
34.9
38.9
33.2
306.5
201.1
271.0
210.3
311.4
345
4.31
3.24
3.0
0.78
4.31
4.21
4.78
0.43
0 (70)
0 (60)b
0 (70)
9.3
0.51
2.61
9.96
10.15
0.96
0 (60)
2
8.9 (60)
4.2 (60)
7.4 (60)
4.7 (50)
8.9 (50)
8.9 (60)
b-Lapachone
Lapachol
Mitomycin
a
Human tumor cell lines: PC-3 = prostate, A549 = lung, MCF-7 = breast.
a
Values represent relative percentage to the positive control value (100%).
Values in parentheses represent viability percentages of Raji cells measured
b
Table 2
through trypan blue staining; unless otherwise stated, the viability percentage of
Raji cells were more than 80%.
Antiproliferative effects of 10a, 10b, 1, (R)-1, racemate 1, 2 and mitomycin against
human normal cell linesa
c
Positive control substance.
Compound
EC50
(lM)
Human normal cell lines
above. Moreover, the values of cell viability at 1000 mol ratio in Ta-
ble 3 indicate that the naphtho[2,3-b]furan-4,9-dione analogues
are less cytotoxic than b-lapachone and lapachol.
Fb
Hc
MPC-5
IE
Hs888Lu
SVCT-M12
10a
10b
Racemate 1
(R)-1
1
2
NTb
NT
45.4
39.7
11.1
NT
NT
NT
89.3
29.8
11.1
NT
NT
NT
89.3
65.9
29.7
NT
NT
NT
158
39.7
54.5
NT
7.7
14.6
NT
7.7
11.7
NT
In addition to in vitro cancer chemopreventive activity men-
tioned above, it has already been known that compound 1 exhibits
in vivo cancer chemopreventive activity.8a These findings inevita-
bly led to the idea that naphtho[2,3-b]furan-4,9-dione analogues
are likely to prove extremely useful for the development of cancer
chemopreventive agents. Therefore, we reexamined inhibitory ef-
fect of 1 that was the most active in vitro and compared with that
of (R)-1 on mouse skin tumor promotion in two-stage carcinogen-
esis experiments in which DMBA is used as an initiator and TPA as
a promoter. Figure 2a shows the incidence (%) of papilloma-bear-
ing mice during 20 weeks of promotion. In group 1, in which only
TPA was applied to mouse skin initiated by DMBA (positive con-
trol), the first tumors were observed at 6 weeks with about 7% inci-
dence. The rate of papilloma-bearing mice reached 100% in
additional 4 weeks. On the other hand, those in groups II–IV which
were treated with 1, (R)-1, racemate 1 before application of TPA
was approximately 7–20% at 10 weeks, 47–53% at 15 weeks and
67–80% even at 20 weeks, respectively. Twenty weeks of promo-
tion allowed group I to form 8.6 papillomas per mouse, while
groups II–IV possessed only 3.9–4.7 papillomas per mouse. The
inhibitory activities of 1, (R)-1 and racemate 1 were also observed
as the average numbers of papillomas per mouse during 20 weeks
of promotion (Fig. 2b). These results clearly demonstrate the po-
tential of naphtho[2,3-b]furan-4,9-dione analogues as cancer che-
mopreventive agents. It is noteworthy that (R)-1 was somewhat
more potent than 1 in this in vivo experiment model.
Antimicrobial activity of naphtho[2,3-b]furan-4,9-dione ana-
logues synthesized was also examined because some constituents
isolated from the Tabebuia plants including 3 and 4 are known to
be active toward bacteria and fungi (Table 4). All compounds
including a synthetic precursor 10a, which is also one of constitu-
ents of T. avellanedae, were found to have modest or potent
antibacterial activity against several Gram-positive bacteria
(0.78–6.25
lg/mL) and antifungal activity (1.56–100 lg/mL) in
comparison with penicillin G and amphotericin B. On the other
hand, all compounds were not active against common Gram-nega-
tive bacteria at concentrations up to 100 lg/mL. From these re-
NT
NT
5.51
14.6
0.56
3.36
14.6
0.96
Mitomycin
0.93
1.46
2.1
1.46
a
Human normal cell lines: Fb = skin, Hc = liver, MPC-5 = lung, IE = colon,
Hs888Lu = lung, SVCT-M12 = breast.
b
NT, not tested.
Unexpectedly, ketone 10a exhibited potent antiproliferative ef-
fect toward the tumor cell lines (Table 1). In particular, it strongly
suppressed the growth of MCF-7 cells, which was comparable to
that of 1. On the other hand, its antiproliferative effect against
Hs888Lu and SVCT-M12 was not as high as that of 1 (Table 2).
Therefore, 10a is also eminently suitable for the development of
anticancer drugs in addition to 1.
The mechanism of action of compound 1 for antiproliferative ef-
fect is still unknown and now being under investigation. However,
our preliminary results indicated that compound 1 affects E2F1, a
multifunctional transcription factor. On the other hand, b-lapach-
one, which is a congener of 1, has been shown to induce apoptosis
in a variety of cancer cell lines and to be involved in transcription
processes. Thus, we currently deduce that compound 1 also has a
similar mechanism to that of b-lapachone.6d
Compound 1 has already been known to act as a cancer chemo-
preventive agent and showed potent inhibition on EBV-EA activa-
tion without cytotoxicity against Raji cells dose-dependently.8 In
order to compare in vitro cancer chemopreventive activity of (R)-
1, 2 and (R)-2 with that of 1, they were evaluated for their ability
to inhibit EBV-EA activation induced by TPA in Raji cells as a pri-
mary screening test for antitumor promoters along with b-lapach-
one and lapachol (Table 3). Among them, both 1 and (R)-1 were
found to be potent inhibitors with IC50 values (mol ratio/32 pmol
TPA) of 33.2 and 38.9, respectively. In particular, compound 1
showed 10-fold higher inhibition than the positive control sub-
stance curcumin. Although (R)-2, which was unambiguously more
potent than 2, exhibited modest inhibitory effect that was compa-
rable with that of b-lapachone, it was much less potent than 1.
These results strongly suggest that the hydroxy group at C-5 plays
an important role in increasing the inhibitory effect as well as the
antiproliferative effect against the tumor cell lines described
sults, it is likely that the presence of the hydroxy group at C-5
makes a slight contribution toward increasing antimicrobial activ-
ity, while the other hydroxy group is not so important although de-
tailed structural requirements cannot be discussed.

M. Yamashita et al. / Bioorg. Med. Chem. 17 (2009) 6286–6291
6289
2 and (R)-2 were recognized at least in the in vitro cancer chemo-
preventive activity. Interestingly, (R)-1 showed somewhat higher
cancer chemopreventive activity in vivo. Further modification to-
ward structure–activity relationship studies is now in progress to
better understand structural requirements for increasing their bio-
logical activities.
100
90
80
70
60
50
40
30
20
10
0
1
(R)-1
racemate1
control
4. Experimental
1H and 13C NMR spectra were taken in CDC13 unless otherwise
noted. Chemical shift values are expressed in ppm relative to inter-
nal tetramethylsilane. Abbreviations are as follows: s, singlet; d,
doublet; t, triplet; m, multiplet. IR was expressed in cmꢀ1. The ex-
tract was dried over Na2SO4 unless otherwise noted. Purification
was carried out using silica gel column chromatography. All reac-
tions were carried out under an argon atmosphere unless other-
wise stated.
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
weeks
9
8
7
6
5
4
3
2
1
0
1
(R)-1
racemate1
control
5. Chemistry
5.1. 5-Hydroxy-1,4-naphthalenedione (6)11
This compound was prepared according to the reported proce-
dure in 47% yield as yellow needles of mp 163–165 °C. 1H NMR:
6.96 (1H, s), 7.29 (1H, dd, J = 2.0, 8.0 Hz), 7.62–7.67 (2H, m), 11.9
(1H, s). 13C NMR: 114.9, 119.2, 124.5, 131.7, 136.6, 138.6, 139.6,
161.4, 184.3, 190.3.
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
weeks
Figure 2. Inhibitory effect of 1 on mouse skin carcinogenesis induced by DMBA and
TPA: X: control DMBA (390 nmol) and TPA (1.7 nmol) (group I); s: DMBA
(390 nmol), TPA (1.7 nmol) and 1 (85 nmol) (group II), d: DMBA (390 nmol), TPA
(1.7 nmol) and (R)-1 (85 nmol) (group III), h: DMBA (390 nmol), TPA (1.7 nmol) and
racemate 1 (85 nmol) (group IV). (a) Percentage of mice with papillomas. (b)
Average number of papillomas per mouse. At 20 weeks of promotion, group II–IV
were significantly different from group I (p <0.05) on papillomas per mouse.
5.2. 2-(Dimethylamino)-5-hydroxy-1,4-naphthalenedione (7a)
and 2-(dimethylamino)-8-hydroxy-1,4-naphthalenedione (7b)12a
These compounds were prepared according to the reported pro-
cedure12 with some modifications. To the solution of 6 (4.28 g,
24.5 mmol) in toluene (125 mL) was added dimethyl amine
(18.4 mL, 2.0 M solution in THF, 36.8 mmol) at ꢀ40 °C. After the
mixture was stirred for 12 h, the solvent was removed under re-
duced pressure. The residue was purified by column chromatogra-
phy (hexane/EtOAc = 4/1) gave 7a (2.23 g, 42% yield) as yellow
needles of mp 145–146 °C and 7b (0.58 g, 11% yield) as yellow nee-
dles of mp 155–157 °C.
3. Conclusions
In conclusion, the concise stereoselective synthesis of 1 and 2
starting from 5 was accomplished by using Noyori reduction as a
key step, and the total yields of products 1 and 2 were 8.5% and
2.9%, respectively. Naphtho[2,3-b]furan-4,9-dione 1 synthesized
in this study showed potent antiproliferative effect and in vitro
and in vivo cancer chemopreventive activity. In addition, it dis-
played relatively strong antimicrobial activity against several
Gram-positive bacteria and fungi. On the other hand, its positional
isomer 2 was less active in antiproliferative effect and in vitro can-
cer chemopreventive activity, but exhibited similar antimicrobial
activity to that of 1. Furthermore, significant differences between
5.3. 2,5-Dihydroxy-1,4-naphthalenedione (8a)12b
This compound was prepared from 7a according to the reported
procedure in 97% yield as yellow needles of mp >210 °C (dec.). 1H
NMR: 6.31 (1H, s),7.35 (1H, dd, J = 1.2, 8.5 Hz), 7.44 (1H, s), 7.59
(1H, t, J = 8.5 Hz), 7.69 (1H, dd, J = 1.2, 8.5 Hz), 12.33 (1H, s). 13C
Table 4
Antimicrobial activitya of 1, (R)-1, 2, (R)-2 and 10a
1
(R)-1
2
(R)-2
10a
PenicillinGc
AmphotericinBc
Bacillus subtilis NBRC3134
0.78
3.13
0.78
>100
>100
>100
25
1.56
3.13
12.5
25
0.78
3.13
0.78
>100
>100
>100
25
1.56
3.13
12.5
25
1.56
6.25
1.56
>100
>100
>100
25
1.56
3.13
25
1.56
6.25
1.56
>100
>100
>100
25
1.56
3.13
25
1.56
1.56
0.78
>100
>100
>100
>100
1.56
1.56
100
0.39
0.39
1.56
NTb
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
1.56
1.56
0.78
1.56
1.56
0.78
Staphylococcus aureus NBRC13276
Bacillus mycoides ATCC 11778
Escherichia coli ATCC11775
Pseudomonas aeruginosa NBRC13275
Salmonella enteritidis OPS10
Candida albicans NBRC1060
Cryptococcus albidus NBRC0378
Saccharomyces cerevisiae NBRC10114
Aspergillus fumigatus NBRC4400
Penicillium expansum NBRC8800
Paecilomyces variotii NBRC4855
NT
NT
NT
50
50
50
50
50
100
25
25
a
Values represent minimum growth inhibitory concentration, MIC (
NT, not tested.
Positive control substance.
lg/mL).
b
c

6290
M. Yamashita et al. / Bioorg. Med. Chem. 17 (2009) 6286–6291
NMR: 110.3, 114.5, 119.8, 126.7, 129.3, 135.2, 156.9, 161.4, 181.2,
191.2.
(3H, s), 7.32 (1H, dd, J = 1.0, 8.5 Hz), 7.59 (1H, s), 7.67 (1H, dd,
J = 7.0, 8.5 Hz), 7.77 (1H, dd, J = 1.0, 7.0 Hz), 11.90 (1H, s). 13C
NMR: 26.7, 112.4, 115.3, 120.5, 125.5, 131.3, 133.2, 137.0, 152.3,
155.8, 163.0, 178.8, 178.8, 187.2.
5.4. 2,8-Dihydroxy-1,4-naphthalenedione (8b)12b
Compound 8b was prepared from 7b according to the reported
procedure in 97% yield as yellow needles of mp >215 °C (dec.). 1H
NMR: 6.31 (1H, s), 7.35 (1H, dd, J = 1.2, 8.5 Hz), 7.44 (1H, s), 7.59
(1H, t, J = 8.5 Hz), 7.69 (1H, dd, J = 1.2, 8.5 Hz), 12.33 (1H, s). 13C
NMR: 111.6, 113.1, 119.6, 123.4, 132.7, 138.2, 156.1, 161.6,
184.1, 185.1.
5.9. 5-Hydroxy-2-[(1S)-1-hydroxyethyl]-naphtho[2,3-b]furan-
4,9-dione (1)5c
To a flask was added ketone 10a (128 mg, 0.5 mmol), Noyori
asymmetric transfer hydrogenation catalyst Ru [(S,S)-Tsdpen](p-
cymene) (15 mg, 0.025 mmol, 5 mol %), CH2Cl2 (5.0 mL), formic
acid/Et3N (5:2, 1.3 ml). The resulting solution was stirred at room
temperature for 24 h. The reaction mixture was diluted by addition
of H2O and 10% HCl aq, and extracted with CHCl3. The organic ex-
tracts were washed with brine, and then dried over Na2SO4. Con-
centration and column chromatography (hexane/EtOAc = 2/1)
gave 1 (115 mg, 89% yield, 96% ee) as yellow needles of mp 171–
5.5. 2-Acetyl-2,3-dihydro-5-hydroxy-naphtho[2,3-b]furan-4,9-
dione (9a)
This compound was prepared by the method of Hagiwara. To a
solution of methyl vinyl ketone (215 g, 3.07 mol) in pentane
(700 mL) was slowly added a solution of bromine (500 g, 3.13 mol)
in pentane (600 mL) at ꢀ15 °C. After stirred for 10 min, concentra-
tion under reduced pressure gave 3,4-dibromobutan-2-one, which
was immediately added to the solution of 8a (97.3 g, 512 mmol) in
THF (2.5 mL). To the solution was slowly added DBU (551 mL,
3.69 mol) at 0 °C, and the mixture was stirred for overnight at room
temperature. The mixture was quenched with 10% HCl at 0 °C. The
mixture was extracted with CHCl3. The organic extracts were
washed with brine, and then dried. Concentration and crystalliza-
tion (CHCl3/benzene = 2/1), then washing with MeOH gave 9a
(92 g, 70% yield) as yellow needles of mp 186–188 °C. 1H NMR:
2.67 (3H, s), 7.32 (1H, dd, J = 1.0, 8.5 Hz), 7.59 (1H, s), 7.67 (1H, dd,
J = 7.0, 8.5 Hz), 7.77 (1H, dd, J = 1.0, 7.0 Hz), 7.77 (1H, dd, J = 1.0,
7.0 Hz), 11.90 (1H, s). 13C NMR: 26.6, 29.5, 87.4, 114.6, 119.7,
123.5, 126.0, 131.6, 135.4, 159.8, 161.3, 176.4, 187.5, 204.3. IR
(KBr): 3400, 1717, 1678, 1647, 1616, 1450, 1234, 760. HRMS (ESI)
m/z: [MꢀH]ꢀ calcd for [C14H9O5]ꢀ, 257.0450; found, 257.0452.
172 °C. ½a 2D4
ꢀ22.7 (c 0.58, CH3OH). 96% ee (HPLC, Daicel Chiralpak
ꢁ
AD-H; hexane/i-PrOH = 9/1, 1.0 mL/min; 254 nm, minor 37.9 min
and major 40.9 min). 1H NMR: 1.66 (3H, d, J = 6.8 Hz), 2.31 (1H,
br s), 5.05 (1H, m), 6.84 (1H, s), 7.27 (1H, dd, J = 1.0, 8.3 Hz), 7.62
(1H, dd, J = 8.0, 8.3 Hz), 7.75 (1H, dd, J = 0.9, 8.0 Hz), 12.18 (1H,
s). 13C NMR: 21.5, 63.8, 103.4, 115.2, 120.0, 125.3, 131.0, 132.6,
136.3, 152.0, 162.3, 165.4, 172.7, 186.5.
5.10. 5-Hydroxy-2-[(1R)-1-hydroxyethyl]-naphtho[2,3-b]furan-
4,9-dione ((R)-1)
½
a 2D4
+22.5 (c 0.42, CH3OH).
ꢁ
5.11. 8-Hydroxy-2-[(1S)-1-hydroxyethyl]-naphtho[2,3-b]furan-
4,9-dione (2)5c
Compound 2 was prepared from 10b in a manner similar to that
described for compound 10a. Concentration and column chroma-
tography (hexane/EtOAc = 2/1) gave 2 (97% yield, 97% ee) as yellow
5.6. 2-Acetyl-2,3-dihydro-8-hydroxy-naphtho[2,3-b]furan-4,9-
dione (9b)
needles of mp 181–182 °C. ½a D24
ꢁ ꢀ22.3 (c 0.16, CH3OH). 97% ee (HPLC,
Daicel Chiralpak AD-H; hexane/i-PrOH = 9/1, 1.0 mL/min; 254 nm,
minor 21.1 min and major 24.1 min). 1H NMR: 1.66 (3H, d,
J = 6.5 Hz), 2.61 (1H, br s), 5.05 (1H, q, J = 6.5 Hz), 6.85 (1H, s), 7.26
(1H, dd, J = 1.5, 8.0 Hz), 7.59 (1H, dd, J = 7.5, 8.0 Hz), 7.70 (1H, dd,
J = 1.5, 7.5 Hz), 12.00 (1H, s). 13C NMR: 21.5, 63.8, 104.7, 114.7,
120.1, 125.3, 131.9, 133.2, 136.3, 151.1, 162.6, 165.8, 178.5, 179.7.
Compound 9b was prepared from 8b using the same procedure
as described for compound 9a. Concentration and column chroma-
tography (chloroform only) gave 9b (72% yield) as yellow needles
of mp 131–133 °C. 1H NMR: 2.40 (3H, s), 3.40 (1H, d, J = 9.0 Hz),
3.40 (1H, d, J = 11.0 Hz), 5.29 (1H, dd, J = 9.0, 11.0 Hz), 7.23 (1H,
dd, J = 3.5, 6.0 Hz), 7.63–7.61 (2H, m), 11.60 (1H, s). 13C NMR:
26.4, 30.0, 87.2, 114.5, 119.4, 124.4, 124.6, 132.9, 137.1, 158.9,
162.1, 180.7, 181.8, 204.2. IR (KBr): 1736, 1650, 1620, 1439,
1246, 1177, 957, 756, 732. HRMS (ESI) m/z: [MꢀH]ꢀ calcd for
[C14H9O5]ꢀ, 257.0450; found, 257.0465.
5.12. 8-Hydroxy-2-[(1R)-1-hydroxyethyl]-naphtho[2,3-b]furan-
4,9-dione ((R)-2)
½
a 2D4
+21.7 (c 0.16, CH3OH).
ꢁ
5.7. 2-Acetyl-5-hydroxy-naphtho[2,3-b]furan-4,9-dione (10a)5c
5.13. Antiproliferative effect assay
Under Ar atmosphere,
78 mmol) in chloroform (1.0 L) was heated with MnO2 (90 g,
<5 m, activated, 1.04 mol) to reflux for 1 d. The mixture was
passed through a silica gel column chromatography. Concentration
gave 10a (13.6 g, 71% yield) as yellow needles of mp >247 °C (dec.).
1H NMR: 2.67 (3H, s), 7.32 (1H, dd, J = 1.0, 8.5 Hz), 7.60 (1H, s), 7.67
(1H, dd, J = 7.0, 8.5 Hz), 7.82 (1H, dd, J = 1.0, 7.0 Hz), 12.13 (1H, s).
13C NMR: 26.8, 111.9, 115.3, 120.5, 125.9, 130.5, 132.7, 136.7,
152.9, 155.7, 162.7, 173.2, 185.6, 187.5.
a
solution of compound 64 (20 g,
The antiproliferative effects of naphthoquinones were exam-
ined in cancer cell lines and normal cell lines derived from human
origin. These cells were maintained in usual 10% fetal serum Dul-
becco’s minimum essential medium (DMEM) through experiments
and exposed to four dose concentrations of naphthoquinones in a
humidified atmosphere (37 °C, 5% CO2) for 72 h. After reaction,
cells were further incubated with 0.25% trypan blue dye for
20 min and counted for viable cells under light microscopic appa-
ratus. IC50 values were calculated from separate experiments per-
formed in triplicate.
l
5.8. 2-Acetyl-8-hydroxy-naphtho[2,3-b]furan-4,9-dione (10b)5c
5.14. In vitro EBV-EA activation assay
Compound 10b was prepared from 9b using the same proce-
dure as described for compound 10a. Concentration gave 10b
(84% yield) as yellow needles of mp 220–221 °C. 1H NMR: 2.67
The inhibition of EBV-EA activation was assayed according to the
reported method.17 The cells were incubated for 48 h at 37 °C in a

M. Yamashita et al. / Bioorg. Med. Chem. 17 (2009) 6286–6291
6291
medium containing n-butyric acid (4 mM), TPA (32 pmol) and vari-
References and notes
ous amounts of the test compounds. Smears were made from the cell
suspension and the EBV-EA inducing cells were stained by means of
an indirect immunofluorescence technique.18 In each assay, at least
500 cells were counted and the number of stained cells (positive
cells) among them was recorded. Triplicate assays were performed
for each data point. The EBV-EA inhibitory activity of the test com-
pound was compared with that of the control experiment (100%)
with n-butyric acid plus TPA. In the experiments, the EBV-EA activ-
ities were ordinarily abound 40% and these values were taken as the
positive control (100%). The viability of cells was assayed against
treated cells by the trypan blue staining method. For the determina-
tion of cytotoxicity, the cell viability was required to be more than
60%.19 Student’s t-test was used for all statistical analyses.
1. Gentry, A. H. Ann. Missouri Bot. Garden 1973, 60, 945.
2. Hashimoto, G. Brazil Shokubutu-ki; Teikoku Shoin: Tokyo, 1962.
3. (a) Hartwell, J. L. Lloydia 1968, 31, 71; (b) Rao, M. M.; Kingston, D. G. I. J. Nat.
Prod. 1982, 45, 600; (c) Koyama, J.; Morita, I.; Tagahara, K.; Hirai, K.
Phytochemistry 2000, 53, 869; (d) Park, B. S.; Lee, K. G.; Shibamoto, T.; Lee, S.
E.; Takeoka, G. R. J. Agric. Food Chem. 2003, 51, 295; (e) Park, B. S.; Lee, H. K.; Lee,
S. E.; Piao, X. L.; Takeoka, G. R.; Wong, R. Y.; Ahn, Y. J.; Kim, J. H. J.
Ethnopharmacol. 2006, 105, 255; (f) Queiroz, M. L. S.; Valadares, M. C.; Torello,
C. O.; Ramos, A. L.; Oliveira, A. B.; Rocha, F. D.; Arruda, V. A.; Accorci, W. R. J.
Ethnopharmacol. 2008, 117, 228.
4. (a) Ueda, S.; Tokuda, H. Planta Med. 1990, 56, 669; (b) Zani, C. L.; Oliveira, A. B.;
O1iveira, G. G. Phytochemistry 1991, 30, 2379.
5. For quinones and benzene derivatives: (a) Burnett, A. R.; Thomson, R. H. J.
Chem. Soc. C 1967, 1261; (b) Burnett, A. R.; Thomson, R. H. J. Chem. Soc. C 1967,
2100; (c) Wagner, H.; Kreher, B.; Lotter, H.; Hamburger, M. O.; Cordell, G. A.
Helv. Chim. Acta 1989, 72, 659; For iridoids: (d) Nakano, K.; Maruyama, K.;
Murakami, K.; Takaishi, Y.; Tomimatsu, T. Phytochemistry 1993, 32, 37l; (e)
Warasina, T.; Nagatani, Y.; Noro, T. Phytochemistry 2004, 65, 2003; (f)
Warashina, T.; Nagatani, Y.; Noro, T. Phytochemistry 2005, 66, 589.
6. (a) Hussain, H.; Krohn, K.; Ahmad, V. U.; Miana, G. A.; Green, I. R. Arkivoc 2007,
145; (b) Pardee, A. B.; Li, Y. Z.; Li, C. J. Curr. Cancer Drug Targets 2002, 2, 227; (c)
Fujimoto, Y.; Eguchi, T.; Murasaki, C.; Ohashi, Y.; Kakinuma, K.; Takagi, H.; Abe,
M.; Inazawa, K.; Yamazaki, K.; Ikekawa, N.; Yoshikawa, O.; Ikekawa, T. J. Chem.
Soc., Perkin Trans. 1 1991, 2323; (d) Cragg, G. M.; Grothaus, P. G.; Newman, D. J.
Chem. Rev 2009, 109, 3012.
7. Ueda, S.; Umemura, T.; Dohguchi, K.; Matsuzaki, T.; Tokuda, H.; Nishino, H.;
Iwashima, A. Phytochemistry 1994, 36, 323.
8. Ueda, S. U.S. Patent 5663197, 2005; Chem. Abstr. 1994, 121, 50088.
9. (a) Yamashita, M.; Kaneko, M.; Iida, A.; Tokuda, H.; Nishimura, K. Bioorg. Med.
Chem. Lett. 2007, 17, 6417; (b) Iida, A.; Tokuda, H.; Yamashita, M. JP Patent
4077863, 2008.
5.15. In vitro antibacterial activity
MICs of (1, (R)-1, 2, (R)-2 and 10a) were determined by slightly
modified NCCLS M27-P broth dilution method.20 A 50
lL aliquot of
0.85% sodium chloride solution containing 106/mL bacterial cells
was added to 1 mL of Muller-Hinton broth (OXOID) containing
(1, (R)-1, 2, (R)-2 and 10a) at a concentration range of 0.1–
100
l
g/mL, followed by incubation for 24 h at 37 °C. Similarly, fun-
gal conidia suspended in 50
l
L of 0.05% Tween 80 solution (105/mL
conidia concentration) were added to 1 mL of potato dextrose
broth (DIFCO) containing (1, (R)-1, 2, (R)-2 and 10a) at a concentra-
tion range of 0.1–100 lg/mL and incubated for 3–5 days at 28 °C.
10. (a) Guiraud, P.; Steiman, R.; Campos-Takaki, G. M.; Seigle-Murandi, F.; Simeon
de Buochberg, M. Planta Med. 1994, 60, 373; (b) Nagata, K.; Hirai, K.; Koyama, J.;
Wada, Y.; Tamura, T. Antimicrob. Agents Chemother. 1998, 42, 700–702; (c)
Eyong, K. O.; Folefoc, G. N.; Kuete, V.; Beng, V. P.; Krohn, K.; Hussain, H.;
Nkengfack, A. E.; Saeftel, M.; Salem Ramadan Sarite, S. R.; Hoerauf, A.
Phytochemistry 2006, 67, 605.
5.16. In vivo two-stage carcinogenesis test on mouse skin
papillomas promoted by TPA
11. Tietze, L. F.; Güntner, C.; Gericke, K. M.; Schuberth, I.; Bunkoczi, G. Eur. J. Org.
Chem. 2005, 2459.
12. (a) Chaker, L.; Pautet, F.; Fillion, H. Chem. Pharm. Bull. 1994, 42, 2238; (b)
Oliveira, A. B.; Ferreira, D. T.; Raslan, D. S. Tetrahedron Lett. 1988, 29,
155.
Mice were divided into groups, with 15 females in each group and
housed in a controlled environment. All mice were shaved with elec-
tric clippers 1 day before application of the agents to backs of the
mice. 7,12-Dimethylbenz[a]anthracene (DMBA, 390 nmol) in
0.1 mL acetone were applied topically with pipet. After one week,
the same skin portion was painted with 1, (R)-1, racemate 1
(85 nmol) in acetone or acetone alone, 60 min before TPA (1.7 nmol).
Results of tumor response are present as the average number of mice
with papillomas and the percentage of mice with papillomas.
13. Hagiwara, H.; Sato, K.; Nishino, D.; Hoshi, T.; Suzuki, T.; Ando, M. J. Chem. Soc.,
Perkin Trans. I 2001, 2946.
14. (a) Fujii, A.; Hashiguchi, S.; Uematsu, N.; Ikariya, T.; Noyori, R. J. Am. Chem. Soc.
1996, 118, 2521; (b) Noyori, R.; Hashiguchi, S. Acc. Chem. Res. 1997, 30, 97.
15. Tsdpen = N-(p-toluenesulfonyl)-1,2-diphenylethanediamine.
16. The purity of synthesized 1 and (R)-1, 2, (R)-2 used for biological evaluation is
95–97% ee.
17. Ito, Y.; Yanase, S.; Fujita, J.; Harayama, T.; Takashima, M.; Imanaka, H. Cancer
Lett. 1981, 13, 29.
18. Henle, G.; Henle, W. J. Bacteriol. 1966, 91, 1248.
19. Ohigashi, H.; Takamura, H.; Koshimizu, K.; Tokuda, H.; Ito, Y. Cancer Lett. 1986,
30, 143.
Acknowledgments
20. National Committee for Clinical Laboratory Standards. Reference Method for
Broth Dilution Antifungal Susceptibility Testing of Yeast (Proposal Standard M27-
P); NCCLS: Villanova, PA, 1992.
The authors are grateful to Taheebo Japan Co., Ltd for gener-
ously providing the powdered inner bark of Tabebuia avellanedae
and SANSHIN METAL WORKING CO., LTD for generous financial
support of this project.