Synthesis and structure-activity relationships of dehydroaltenusin derivatives as selective DNA polymerase α inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.08.051

Herein, we describe the synthesis and structure-activity relationships of dehydroaltenusin derivatives as inhibitors of a mammalian DNA polymerase α. We have newly synthesized nine dehydroaltenusin derivatives modified at the side chains or benzoquinone m

Full text of DOI:10.1016/j.bmc.2009.08.051

Bioorganic & Medicinal Chemistry 17 (2009) 7227–7238
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of dehydroaltenusin
derivatives as selective DNA polymerase
a inhibitors
b
c
b
b
Kouji Kuramochi a, , Keishi Fukudome , Isoko Kuriyama , Toshifumi Takeuchi , Yoshihiro Sato ,
*
Shinji Kamisuki b, Kazunori Tsubaki a, Fumio Sugawara b, , Hiromi Yoshida c,d, Yoshiyuki Mizushina c,d,
*
*
a Graduate School of Life and Environmental Science, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan
b Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan
c Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
d Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we describe the synthesis and structure–activity relationships of dehydroaltenusin derivatives as
inhibitors of a mammalian DNA polymerase . We have newly synthesized nine dehydroaltenusin deriv-
atives modified at the side chains or benzoquinone moiety. We also achieved the first synthesis of des-
methylaltenusin and desmethyldehydroaltenusin, metabolites of Alternaria sp. or Talaromyces flavus,
respectively. Among all synthesized derivatives, demethoxydehydroaltenusin was the most selective
Received 24 July 2009
Revised 26 August 2009
Accepted 26 August 2009
Available online 31 August 2009
a
inhibitor of DNA polymerase a. The o-hydroxy-p-benzoquinone (2-hydroxycyclohexa-2,5-dienone) moi-
Keywords:
ety is essential for the inhibition of DNA polymerases. Substitution at the 5-position of dehydroaltenusin
is important for the inhibitory potency. Because dehydroaltenusin is conjugated with N-acetylcysteine
methyl ester at the o-hydroxy-p-benzoquinone moiety, one or more cysteine residues of DNA polymerase
Structure–activity relationships
DNA polymerase inhibitor
Natural product synthesis
Dehydroaltenusin
a
may act as a target for this compound.
Ó 2009 Elsevier Ltd. All rights reserved.
Chemical biology
1. Introduction
except for the fragment ions having 14 mass units less. The 1H
NMR of desmethyldehydroaltenusin triacetate was similar to that
of dehydroaltenusin diacetate, which was prepared by acetylation
of 1 with acetic anhydride in pyridine. The structure of dehydroal-
tenusin diacetate was originally suggested to be a d-lactone (4) by
Development of highly potent and selective enzyme inhibitors
is important for both drug development and chemical genetic stud-
ies.1,2 Using this approach, several molecular target drugs have
been developed that are effective and show relatively low toxicity
in clinical use.3 Furthermore, many of these enzyme inhibitors
have been used as tools to characterize the biological function of
the proteins of interest.4
Dehydroaltenusin (1) was first isolated from mycelium extracts
of Alternaria tennuis and Alternaria kikuchiana by Rosett et al. in
19575 and its structure was subsequently determined by X-ray
crystallography (Fig. 1).6 Desmethyldehydroaltenusin (2), a related
natural product, was isolated from the organic soluble metabolites
of Talaromyces flavus by Ayer and Racok.7 The structure of 2 was
determined in the form of its acetate. After treatment of 2 with
acetic anhydride in pyridine, the product was assigned as 3
(Fig. 2). The mass fragmentation pattern of desmethyldehydroal-
tenusin triacetate was similar to that of dehydroaltenusin diacetate
Ayer and Racok,7 but was later revised to be a
c-lactone (5) follow-
ing our NMR analyses (COSY, HMQC, HMBC and INADEQUATE).8
We found that compound 5 was a major product of acetylation
of 1 with acetic anhydride in pyridine. Compound 4 was a minor
product of the acetylation with acetyl chloride in pyridine and
dichloromethane.
Nakanishi et al. reported that dehydroaltenusin (1) inhibits the
calmodulin-dependent activity of myosin light chain kinase
* Corresponding authors. Tel./fax: +81 75 703 5603 (K.K.); tel.: +81 4 7124
1501x3400; fax: +81 4 7123 9757 (F.S.); tel.: +81 78 974 1551x3232; fax: +81 78
974 5689 (Y.M.).
E-mail addresses: kuramoch@kpu.ac.jp (K. Kuramochi), sugawara@rs.noda.
tus.ac.jp (F. Sugawara), mizushin@nutr.kobegakuin.ac.jp (Y. Mizushina).
Figure 1. Structures of dehydroaltenusin (1) and desmethyldehydroaltenusin (2).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.051

7228
K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
Figure 2. Proposed structure of desmethyldehydroaltenusin triacetate (3) prepared from 2 with acetic anhydride in pyridine, and structures of dehydroaltenusin diacetate (4
and 5, respectively).
(MLCK) with an IC50 value of 0.69
tenusin (1), isolated from a fungus A. tennuis, selectively inhibited
the activity of a mammalian DNA polymerase . Compound 1
inhibited DNA polymerase with an IC50 value of 0.68 M and
had no effect on other replicative DNA polymerases such as DNA
polymerase d and e 10–16
Interestingly, related natural products,
l
M.9 We found that dehydroal-
onates (Scheme 2).18–20 This convergent approach will be applica-
ble to the synthesis of various dehydroaltenusin derivatives.
First, we decided to prepare desmethyldehydroaltenusin (2)
(Scheme 3). Coupling of aryl triflate 621 with aryl boronate 718 in
the presence of PdCl2(dppf) (0.04 equiv) and K2CO3 (2.4 equiv)
gave a biaryl 8 in 87% yield. Deprotection of all protecting groups
in 8 by BCl3 yielded desmethylaltenusin 9.22 Oxidation of 9 with
FeCl3 afforded desmethyldehydroaltenusin (2), which was con-
verted into its triacetate by the procedure reported by Ayer and Ra-
cok.7 The 1H and IR spectra of desmethyldehydroaltenusin
triacetate derived from synthetic 2 are identical to those reported.
Ayer and Racok proposed the structure for desmethyldehydroal-
tenusin triacetate as 3, which possessed the d-lactone ring. How-
ever, the NMR and IR data of our desmethyldehydroaltenusin
a
a
l
.
such as altenuene, alternariol and alternariol 9-methyl ether, did
not inhibit the activity of DNA polymerases.11
Our group has reported the total synthesis of 1 with a palla-
dium-catalyzed Suzuki coupling as a key reaction.17,18 The struc-
ture–activity relationships of dehydroaltenusin acetates suggest
that the o-hydroxy-p-benzoquinone (2-hydroxycyclohexa-2,5-die-
none) moiety is essential for inhibition of DNA polymerase
found that compound 1 existed in an equilibrium of two tautomers
possessing -lactone and d-lactone in polar solvents (Scheme 1).
a.
8 We
c
support the structure of c-lactone 10. In a previous paper we de-
Thus, we proposed that the equilibrium between 1 and 1a or plau-
sible active o-quinone species (1b) might be important for the inhi-
bition of DNA polymerase a.
In this paper, we prepared several dehydroaltenusin derivatives
including desmethyldehydroaltenusin and investigated the struc-
scribed the preparation and characterization of dehydroaltenusin
diacetates (4 and 5) (Fig. 3).8 The IR absorption at 1724 cmꢀ1 and
three singlet peaks derived from H-30, H-50 and CH3 in 4 are char-
acteristic of a d-lactone. On the other hand, the IR absorption at
1778 cmꢀ1 and the long-range coupling between H-30 and CH3 in
ture–activity relationships as DNA polymerase
tail. We also examined the mechanism of inhibition of DNA
a
inhibitors in de-
5
are characteristic of
1778 cmꢀ1 and the long-range coupling between H-30 and CH3 in
10 indicates that the compound possesses a -lactone ring.
a c-lactone. The IR absorption at
polymerase
a
caused by 1.
c
We next synthesized dehydroaltenusin derivatives by modify-
ing the methoxy group at the C-5 position. The biaryl 8 serves as
a potential starting material for the synthesis of various substi-
tuted dehydroaltenusin derivatives (Scheme 4). Deprotection of
the benzyl group with Pd(OH)2 on carbon under an atmosphere
of H2 gave a common intermediate 11. Mitsunobu reaction of 11
with various alcohols gave ethers 12(a–d). Deprotection of the ace-
tonide and methylene acetal in 12(a–d), followed by oxidation
2. Results
2.1. Synthesis of dehydroaltenusin derivatives
We planned to synthesize dehydroaltenusin derivatives by
palladium-catalyzed Suzuki coupling of aryl triflates with aryl bor-
Scheme 1. Tautomerization of dehydroaltenusin (1) in polar solvents.

K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
7229
Scheme 2. Synthetic approach for dehydroaltenusin derivatives.
Scheme 3. Synthesis of desmethyldehydroaltenusin (2) and its acetate (10). Reagents and conditions: (a) PdCl2(dppf), K2CO3, DME, 85 °C (87%); (b) BCl3, CH2Cl2–heptane, 0 °C
(90%); (c) FeCl3, EtOH–H2O (50%); (d) Ac2O, pyridine (64%).
Figure 3. Assignment of desmethyldehydroaltenusin triacetate by comparison with that of dehydroaltenusin diacetates (4 and 5, respectively).

7230
K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
Scheme 4. Synthesis of dehydroaltenuisin (1) and its derivatives (14–16). Reagents and conditions: (a) H2, Pd(OH)2, THF–MeOH (87%); (b) alcohol, DIAD, PPh3, THF (92% for
12a, 79% for 12b, 75% for 12c, 73% for 12d); (c) BCl3, CH2Cl2–heptane, 0 °C (97% for 13a, 77% for 13b, 75% for 13c, 82% for 13d); (d) FeCl3, EtOH–H2O (71% for 1, 63% for 14, 63%
for 15, 57% for 16).
with FeCl3 gave dehydroaltenusin (1) and its derivatives (14–16).
We prepared demethoxydehydroaltenusin (20) by the same proce-
dure in three steps starting from 1723 and 7 via the intermedi-
ates18 and 19 (Scheme 5).
Although compounds 1, 2, 14–16 and 20 predominantly form
the d-lactone in CDCl3, they exist in an equilibrium mixture of
neous lactone formation gave 25 in 77% yield. Deprotection of ben-
zyl groups with Pd(OH)2 on carbon under an atmosphere of H2
afforded isocoumarin 26 in quantitative yield.
We also examined the importance of the hydroxyl group at C-40
in dehydroaltenusin (1) in terms of its inhibitory activity against
DNA polymerase
a. Thus, we prepared dehydroaltenusin deriva-
two tautomers, d-lactone and
of the tautomers in DMSO-d6 is summarized in Table 1. The
c
-lactone, in polar solvents. The ratio
tives that lack the hydroxyl group at C-40. 3-Iodo-4-methylphenol
(27)25 was converted into the corresponding boronate 28. The pal-
ladium-catalyzed Suzuki coupling of 28 with 23 or 17 yielded 29 or
30, respectively. Hydrolysis of 29 and 30 and oxidation of the
resultant phenols with PhI(OAc)2 gave 31 and 32 in 66% and 77%
yield, respectively.26 No reaction occurred when FeCl3 was used
as an oxidant in the oxidation of the phenol. Moreover, oxidation
of the phenol with CAN resulted in decomposition of the phenol
(Scheme 7).
c
-lac-
tone form of 1, 2, 14–16 and 20 is the major tautomer in DMSO-d6.
Next, we modified at the quinone moiety in dehydroaltenusin.
An isocoumarin derivative was prepared according to a similar
strategy (Scheme 6). Treatment of 2124 with bis(pinacolato)dibo-
ron in the presence of PdCl2(dppf) and KOAc in DMF gave boronate
22 in 53% yield. Coupling of 22 with 2318 under Suzuki coupling
conditions afforded 24. Methanolysis of 24 followed by simulta-
Scheme 5. Synthesis of desmethyldehydroaltenusin 20. Reagents and conditions: (a) PdCl2(dppf), K2CO3, DME, 85 °C (89%); (b) BCl3, CH2Cl2–heptane, 0 °C (77%); (c) FeCl3,
EtOH–H2O (80%).

K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
7231
Table 1
2.2. Effect of synthetic dehydroaltenusin derivatives on
mammalian DNA polymerases
Ratio of c-lactone and d-lactone tautomers of dehydroaltenusin and its derivatives in
DMSO-d6
The inhibitory effect of chemically synthesized dehydroaltenu-
sin (1) and its derivatives (2, 14–16, 20, 25, 26, 31 and 32) on
two different DNA polymerases were investigated. Specifically,
we used calf DNA polymerase a, which is a representative replica-
tive DNA polymerase, and rat DNA polymerase b, which is a major
repair-related DNA polymerase. Dehydroaltenusin (1) dose-depen-
dently inhibited the activity of DNA polymerase
bition observed at a concentration of 0.68 M (Table 2) and
almost complete inhibition at 4
M.17 Although we previously re-
a, with 50% inhi-
l
l
ported that the naturally purified dehydroaltenusin from a fungus
(Acremonium sp.) was a specific inhibitor of mammalian DNA poly-
merase
polymerase b with an IC50 value of 64
dehydroaltenusin derivatives, compounds 25, 26, 31 and 32 did
not influence the activities of DNA polymerases and b. The struc-
tural difference between dehydroaltenusin (1) and compound 2 is
the group at 5-position of these compounds. Thus, five derivatives
modified at this position were synthesized (2, 14–16 and 20) and
their inhibitory activities compared. All five compounds dose-
a,
10 this compound was also effective at inhibiting rat DNA
lM. From the prepared
Compound
Ratio of c-lactone/d-lactone
a
1
2
14
15
16
20
1a/1 = 5
2a/2 = 7
14a/14 = 6
15a/15 = 6
16a/16 = 6
20a/20 = 13
dependently inhibited the activity of calf DNA polymerase a. Com-
pound 16 had the strongest inhibitory effect among the com-
pounds tested. The relative strength of the inhibitory effect of
these derivatives could be ranked as follows: 16 > 15 > 14 >
The ratio of
c
-lactone and d-lactone tautomers was determined on the basis of the
1H NMR integration.
Scheme 6. Synthesis of isocoumarin derivative 26. Reagents and conditions: (a) PdCl2(dppf), bis(pinacolato)diboron, KOAc, DMF, 80 °C (53%); (b) PdCl2(dppf), K2CO3, DME,
80 °C (61% and 21% of recovered 23); (c) K2CO3, MeOH (77%); (d) H2, Pd(OH)2, THF (quant.).
Scheme 7. Synthesis of dehydroaltenusin derivatives 31 and 32. Reagents and conditions: (a) PdCl2(dppf), bis(pinacolato)diboron, KOAc, DMF, 70 °C (86%); (b) PdCl2(dppf),
K2CO3, DME, 55 °C (76% for 29, 67% for 30); (c) LiOH, THF–H2O, then PhI(OAc)2, MeOH (77% for 31, 64% for 32 in two steps).

7232
K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
Table 2
MeOH–H2O (1:1) for 30 min gave an inseparable 1:1 mixture of
34 and 35 in 91% yield (Scheme 8). Other amino acid derivatives,
such as N-acetyllysine methyl ester, N-acetylarginine methyl ester,
N-acetylhistidine methyl ester, N-acetyltyrosine ethyl ester and N-
Boc-serine, did not react with 1. These results suggested that 1
IC50 values of dehydroaltenusin (1) and its derivatives on the activities of mammalian
DNA polymerases
a and b
Compound
IC50 value of DNA polymerase (lM) IC50 value ratio
pol b/pol
a
a
b
might bind to a cysteine residue of DNA polymerase
the activity of the enzyme.
a and inhibit
Dehydroaltenusin (1) 0.68
64
126
35
24
11
94.1
142
194
267
183
371
—
—
—
—
2
0.89
0.18
0.09
0.06
0.24
>200
>200
>200
>200
14
15
16
20
25
26
31
32
3. Discussion
89
In our previous report, we described the total synthesis of
dehydroaltenusin using Suzuki coupling for construction of the
biaryl moiety as a key transformation.18 Here, we have successfully
applied our synthetic method with slight modifications to the syn-
thesis of dehydroaltenusin derivatives. We have accomplished a
first synthesis of two natural products, desmethylaltenusin (9)
and desmethyldehydroaltenusin (2). Our method is also applicable
to the synthesis of related Alternaria toxins. Indeed, a similar strat-
egy was used in the synthesis of alternariol, alternariol methyl
ether, altenuene and isoaltenuene by Podlech et al.19,20
>200
>200
>200
>200
These compounds were incubated with calf DNA polymerase
merase b (0.05 units each). Enzyme activity in the absence of the compound was
taken as 100%.
a or rat DNA poly-
20 > 1 > 2 (Table 2). However, in each case the effect of these com-
pounds on rat DNA polymerase b activity was significantly weaker
than on calf DNA polymerase a. The inhibitory effect of the dehyd-
roaltenusin derivatives on rat DNA polymerase b varied markedly
in the following order: 16 > 15 > 14 > 1 > 20 > 2 (Table 2). The
Using the newly synthesized derivatives of dehydroaltenusin (2,
14–16, 20, 25, 26, 31 and 32) we investigated the structure–activ-
ity relationships for inhibition of DNA polymerase
a and b. Com-
pounds 2, 14–16 and 20, which have the o-hydroxy-p-
benzoquinone unit, exhibited inhibitory activity against DNA poly-
IC50 value ratio against DNA polymerase b/DNA polymerase
a
can be ranked as follows: 20 [371] > 15 [267] > 14 [194] > 16
[183] > 2 [142] > 1 [94.1] (Table 2). These results suggested that
demethoxydehydroaltenusin (20) was the most specific mamma-
merases
DNA polymerases
16 > 15 > 14 > 1 > 20 > 2, respectively. These compounds were
more selective DNA polymerase inhibitors than dehydroaltenu-
sin (1). Among the derivatives, demethoxydehydroaltenusin (20)
was the most selective inhibitor of DNA polymerase . On the
a
and b. The rank orders of the inhibitory effect against
a
and b were 16 > 15 > 14 > 20 > 1 > 2 and
lian DNA polymerase
a inhibitor of the synthesized derivatives,
a
and the specificity of this compound was approximately 4-fold
stronger than that of dehydroaltenusin (1).
a
other hand, isocoumarins (25 and 26) and p-benzoquinones (31
and 32) did not significantly influence on the activity of DNA poly-
merases. These results indicate that the o-hydroxy-p-benzoqui-
none moiety is essential for inhibiting DNA polymerases and that
substitution of the alkoxy group at C-5 greatly influences the inhib-
itory potency. The long alkoxy side chains might interact with the
hydrophobic region of DNA polymerases, resulting in the enhance-
ment of the inhibitory activity.
2.3. Model studies for determining the binding site of
dehydroaltenusin in DNA polymerase
a
Dehydroaltenusin exists as an equilibrium of tautomers, 1 and
1a via the plausible intermediate 1b in polar solvents. Because o-
quinone is a highly reactive species, which reacts with thiols or
amines,27–30 compound 1b might conjugate to a sulfhydryl or ami-
no group present in DNA polymerase
a, and thereby inhibit the
Compounds 1, 2, 14–16 and 20, which display inhibitory activ-
DNA polymerase activity. To clarify the type of residue that forms
a dehydroaltenusin-adduct, 1 was reacted with various amino
acids. Treatment of 1 with N-acetylcysteine methyl ester (33) in
ity against DNA polymerase
two tautomers, d-lactone and
-lactone/d-lactone does not correlate with either the strength or
a
, exist as an equilibrium mixture of
c-lactone, in DMSO-d6. The ratio of
c
Scheme 8. Reaction of dehydroaltenusin (1) with N-acetylcysteine methyl ester (33).

K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
7233
selectivity of the inhibitory activity. Our previous report shows
that the inhibitory activities of dehydroaltenusin diacetates 4 and
Melting points were determined with Yanaco MP-3S and were
uncorrected. Infrared spectra (IR) were recorded on a Jasco FT/IR-
410 spectrometer using NaCl (neat) or KBr pellets (solid), and were
reported on wavenumbers (cmꢀ1). Mass spectra (MS) were ob-
tained on a Hitachi M-80 spectrometer or an Applied Biosystems
mass spectrometer (APIQSTAR pulsar i) under conditions as High
resolution, using poly (ethylene glycol) as internal standard.
5, which form d-lactone and
c-lactone, respectively, are much
weaker than that of dehydroaltenusin.18 These observations sug-
gest that the o-quinone species of dehydroaltenusin derivatives
such as compound 1b, which possesses a plausible intermediate
between d-lactone and
c-lactone, are in fact the active species in
the inhibition of DNA polymerase
a. Actually, inactive compounds
25, 26, 31 and 32 did not form the o-quinone species, since these
compounds do not tautomerize.
5.1.1. 7-Benzyloxy-2,2-dimethyl-5-(5-methyl-1,3-benzodioxol-
6-yl)-4H-1,3-benzodioxin-4-one (8)
Generally, o-quinones are highly reactive species that react
covalently with cellular nucleophiles.27 Indeed, o-quinones can re-
act with sulfhydryl, amine, amide, indole and imidazole groups of
proteins or amino acids. The results obtained from the reaction of
dehydroaltenusin (1) with various amino acid derivatives indicate
that dehydroaltenusin might specifically conjugate with one or
To a degassed stirred solution of 6 (445.5 mg, 1.30 mmol), 7
(355.7 mg, 1.36 mmol) and K2CO3 (429.3 mg, 3.11 mmol) in DME
(10.0 mL) was added PdCl2 (dppf) (37.7 mg, 0.052 mmol), and the
mixture was stirred at 85 °C for 17 h. After addition of Celite, the
mixture was stirred for 30 min and filtered through a pad of Celite.
The filtrate was diluted with CHCl3, and this organic layer was
washed with water and brine, dried and concentrated. Chromatog-
more cysteines of DNA polymerase
a
. We previously demonstrated
by dehydroaltenusin
that the inhibition of calf DNA polymerase
a
raphy on silica gel with hexane–EtOAc (5:1–1:1) yielded 8
was competitive with the DNA template-primer and noncompeti-
tive with the deoxyribonucleotide triphosphates (dNTPs).10 We
also showed that dehydroaltenusin binds to the core domain of
(376.2 mg, 87%) as white solids. Mp = 200.5–201.5 °C. IR mmax
(KBr): 2994, 2917, 1730, 1604, 1575, 1486, 1425, 1381, 1342,
1281, 1231, 1200, 1166, 1129, 1034, 929, 856 cmꢀ1 1H NMR
.
the largest subunit of mouse DNA polymerase
a
, p180, but not to
(600 MHz, CDCl3): d 7.40 (4H, m), 7.36 (1H, m), 6.70 (1H, s), 6.58
(1H, s), 6.52 (1H, d, J = 2.5 Hz), 6.51 (1H, d, J = 2.5 Hz), 5.96 (1H,
d, J = 1.5 Hz), 5.94 (1H, d, J = 1.5 Hz), 5.09 (2H, s), 2.00 (3H, s),
1.73 (3H, s), 1.73 (3H, s). 13C NMR (150 MHz, CDCl3): d 163.7,
158.8, 158.5, 147.0, 146.9, 145.3, 135.6, 133.0, 128.7, 128.7,
128.4, 128.2, 127.5, 127.5, 113.7, 109.9, 108.6, 106.0, 105.1,
101.5, 100.9, 70.4, 26.0, 25.3, 19.7. HRMS (ESI) calcd for
C25H22O6Na ([M+Na]+) 441.1308, found 441.1304. Anal. Calcd for
C25H22O6: C, 71.76; H, 5.30. Found: C, 71.52; H, 5.02.
the smallest subunit or the DNA primase p46. The p180 domain
contains many cysteine residues in the DNA binding domain of
the enzyme.31,32 These data suggest that dehydroaltenusin may di-
rectly conjugate with a cysteine residue of the template-primer
DNA-binding site of DNA polymerase
a, resulting in the inhibition
of DNA polymerase . Studies to identify the binding site of dehyd-
a
roaltenusin in DNA polymerase
a are currently underway.
4. Conclusion
5.1.2. Desmethylaltenusin (3,40,5,50-tetrahydroxy-20-methylbi-
phenyl-2-carboxylic acid) (9)
We synthesized dehydroaltenusin derivatives and investigated
their inhibitory activity against DNA polymerases and b. Our re-
sults demonstrate that the o-hydroxy-p-benzoquinone (2-
hydroxycyclohexa-2,5-dienone) moiety is essential for inhibition
of DNA polymerases
dehydroaltenusin strongly influences the inhibitory potency. Of
all the synthesized derivatives, demethoxydehydroaltenusin (20)
was the most selective DNA polymerase
tained from the reaction of dehydroaltenusin with N-acetylcyste-
To a stirred solution of 8 (313.8 mg, 0.75 mmol) in CH2Cl2
(8.0 mL) was added BCl3 (1.0 M solution in heptane, 4.50 mL,
4.5 mmol) at 0 °C and the mixture was stirred at room temperature
for 19.5 h. Then the mixture was diluted with EtOAc. The organic
layer was washed with water and brine, dried and concentrated.
Chromatography on silica gel with hexane–EtOAc–AcOH
(4000:1000:1–2000:1000:1) yielded 9 (186.4 mg, 90%) as yellow
foam. IR mmax (KBr): 3381, 2928, 2850, 1650, 1614, 1516, 1454,
a
a and b. Substitution at the 5-position of
a inhibitor. Results ob-
1171, 1066, 1011, 852, 790 cmꢀ1 1H NMR (600 MHz, CD3OD): d
.
ine methyl ester indicates that dehydroaltenusin might
specifically conjugate with a cysteine of DNA polymerase a.
6.58 (1H, s), 6.49 (1H, s), 6.29 (1H, d, J = 2.5 Hz), 6.08 (1H, d,
J = 2.5 Hz), 1.91 (3H, s); 13C NMR (150 MHz, CD3OD): d 174.3,
165.7, 163.2, 148.3, 144.7, 143.1, 135.6, 127.4, 117.3, 116.6,
112.3, 105.9, 102.4, 19.2; HRMS (ESI) calcd for C14H12O6Na
([M+Na]+) 299.0526, found 299.0519.
5. Experimental
5.1. Analyses of structure determination
5.1.3. Desmethyldehydroaltenusin (2)
All non-aqueous reactions were carried out by using freshly dis-
tilled solvents under argon atmosphere. THF was distilled from so-
dium/benzophenone prior to use. Dichloromethane was distilled
from P2O5 prior to use. N,N-Dimethylformamide was distilled from
calcium hydride prior to use. All other reagents were commercially
available and used without further purification.
To a stirred solution of 9 (186.4 mg, 0.675 mmol) in EtOH–H2O
(1:1, 4.0 mL) was added a 0.2 M solution of FeCl3 in H2O (10.1 mL)
and the mixture was stirred at room temperature for 15 min and
diluted with CH2Cl2. The organic layer was washed with water
and brine, dried and concentrated. Chromatography on silica gel
with hexane–EtOAc (1:1) and recrystallization from EtOAc–hexane
gave 2 (92.3 mg, 50%) as yellow crystals. Mp = 185.0–185.5 °C (de-
comp.). IR mmax (KBr): 3368, 3053, 2980, 2928, 1678, 1640, 1490,
All reactions were monitored by TLC, which was carried out on
Silica Gel 60 F254 plates (Merck, Germany). Flash chromatography
separations were performed on PSQ 100B (Fuji Silysia Co., Ltd,
Japan).
1395, 1263, 1173, 1107, 1073, 1025, 856, 802 cmꢀ1 1H NMR
.
(600 MHz, CDCl3–CD3OD): d 6.69 (1H, d, J = 2.0 Hz), 6.66 (1H, s),
6.55 (1H, d, J = 2.0 Hz), 6.26 (1H, s), 1.72 (3H, s). 1H NMR
(600 MHz, DMSO-d6): 2a: d 10.90 (1H, s), 10.59 (1H, s), 9.54 (1H,
s), 6.35 (1H, d, J = 1.4 Hz), 6.28 (1H, br q, J = 1.1 Hz), 5.98 (1H, d,
J = 1.4 Hz), 1.58 (3H, d, J = 1.4 Hz). Compound 2: d 11.10 (1H, s),
11.10 (1H, s), 9.58 (1H, s), 6.84 (1H, d, J = 2.0 Hz), 6.75 (1H, s),
6.50 (1H, d, J = 2.0 Hz), 6.16 (1H, s), 1.64 (3H, s). 13C NMR
(150 MHz, CDCl3–CD3OD): d 181.1, 167.4, 164.9, 163.8, 151.6,
1H and 13C NMR spectra were recorded on a Bruker 600 MHz or
400 MHz spectrometer (Avance DRX-600, Avance DRX-400) or a
JEOL 270 MHz spectrometer (EX-270W), using CDCl3 (with TMS
for 1H NMR and chloroform-d for 13C NMR as the internal refer-
ence) solution, unless otherwise noted. Chemical shifts were ex-
pressed in
d (ppm) relative to Me4Si or residual solvent
resonance, and coupling constants (J) were expressed in hertz.

7234
K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
146.9, 135.1, 121.2, 116.4, 105.2, 104.2, 98.3, 78.8, 28.6. HRMS (ESI)
calcd for C14H11O6Na ([M+H]+) 275.0550, found 275.0543. Anal.
Calcd for C14H11O6: C, 61.32; H, 3.68. Found: C, 61.06; H, 3.58.
10.4. HRMS (ESI) calcd for C21H22O6Na ([M+Na]+) 393.1314, found
393.1299. Anal. Calcd for C21H22O6: C, 68.10; H, 5.99. Found: C,
68.04; H, 5.84.
5.1.4. Desmethyldehydroaltenusin triacetate (10)
5.2.3. 7-Hexyloxy-2,2-dimethyl-5-(5-methyl-1,3-benzodioxol-
6-yl)-4H-1,3-benzodioxin-4-one (12c)
A solution of 2 (5.4 mg, 19.5 lmol) in Ac2O and pyridine (1:1,
1.0 mL) was stirred at room temperature for 25 h and then concen-
trated. Chromatography on silica gel with hexane–EtOAc (3:2) and
yielded 10 (5.0 mg, 64%) as amorphous solids. IR (KBr) 3537, 3026,
2928, 2850, 1778, 1687, 1665, 1613, 1472, 1431, 1369, 1302, 1188,
The title compound was prepared by the coupling of 11 and 1-
hexanol in 75% yield as white acicular crystals. Mp = 43.0–43.7 °C.
IR mmax (KBr): 2995, 2956, 2928, 2860, 1732, 1608, 1576, 1481,
1430, 1384, 1344, 1283, 1241, 1189, 1132, 1058, 1033, 969, 933,
1152, 1123, 1090, 1055, 1013, 979, 913, 756 cmꢀ1
.
1H NMR
912, 868 cmꢀ1 1H NMR (600 MHz, CDCl3): d 6.70 (1H, s), 6.59
.
(600 MHz, CDCl3):
d 7.10 (1H, d, J = 1.7 Hz), 6.97 (1H, d,
(1H, s), 6.42 (1H, d, J = 2.4 Hz), 6.41 (1H, d, J = 2.4 Hz), 5.96 (1H,
d, J = 1.4 Hz), 5.93 (1H, d, J = 1.4 Hz), 3.98 (2H, t, J = 6.5 Hz), 2.02
(3H, s), 1.79 (2H, quin, J = 6.5 Hz), 1.73 (3H, s), 1.72 (3H, s), 1.45
(2H, quin, J = 7.3 Hz), 1.34 (4H, m), 0.90 (3H, t, J = 6.8 Hz). 13C
NMR (150 MHz, CDCl3): d 164.3, 158.9, 158.6, 146.9, 146.9, 145.3,
133.2, 128.3, 113.5, 109.9, 108.7, 105.6, 105.0, 101.0, 100.9, 68.6,
31.5, 28.9, 26.1, 25.6, 25.3, 22.5, 19.7, 13.9. HRMS (ESI) calcd for
C24H28O6Na ([M+Na]+) 435.1778, found 435.1796. Anal. Calcd for
C24H28O6: C, 69.88; H, 6.84. Found: C, 69.69; H, 7.12.
J = 1.7 Hz), 6.37 (1H, s), 6.31 (1H, d, J = 1.4 Hz), 2.43 (3H, s), 2.32
(3H, s), 2.29 (3H, s), 1.72 (3H, d, J = 1.4 Hz). 13C NMR (150 MHz,
CDCl3): d 178.0, 168.2, 168.2, 167.8, 165.3, 157.1, 154.4, 149.7,
149.6, 145.6, 129.9, 127.8, 118.4, 115.2, 113.0, 82.8, 21.1, 20.6,
20.4, 17.0. HRMS calcd for C20H16O9Na ([M+Na]+) 423.0686, found
423.0702.
5.1.5. 7-Hydroxy-2,2-dimethyl-5-(5-methyl-1,3-benzodioxol-6-
yl)-4H-1,3-benzodioxin-4-one (11)
To a stirred solution of 8 (747.4 mg, 1.81 mmol) in THF–MeOH
(1:1, 400 mL) was added 10% Pd(OH)2 on carbon (74.7 mg,
0.53 mmol), and the mixture was stirred under an atmosphere of
H2 at room temperature for 3 h. After addition of Celite, the mix-
ture was stirred for 30 min and filtered through a pad of Celite.
The filtrate was concentrated, and the crude product was recrystal-
lized from EtOAc to afford 11 (530.0 mg, 87%) as crystals.
Mp = 247.0–247.5 °C (decomp.). IR mmax (KBr): 3755, 3382, 3002,
2892, 1703, 1610, 1479, 1392, 1282, 1189, 1035, 921, 859,
5.2.4. 7-Dodecyloxy-2,2-dimethyl-5-(5-methyl-1,3-benzodioxol-
6-yl)-4H-1,3-benzodioxin-4-one (12d)
The title compound was prepared by the coupling of 11 and 1-
dodecanol in 73% yield as white solids. Mp = 111.9–112.2 °C. IR
mmax (neat): 2919, 2851, 1738, 1604, 1580, 1476, 1430, 1389,
1343, 1279, 1237, 1182, 1132, 1039, 937, 912, 869 cmꢀ1 1H NMR
.
(600 MHz, CDCl3): d 6.70 (1H, s), 6.59 (1H, s), 6.42 (1H, d,
J = 2.5 Hz), 6.41 (1H, d, J = 2.5 Hz), 5.96 (1H, d, J = 1.3 Hz), 5.94
(1H, d, J = 1.3 Hz), 3.98 (2H, t, J = 6.5 Hz), 2.02 (3H, s), 1.81–1.76
(2H, m), 1.73 (3H, s), 1.72 (3H, s), 1.46–1.41 (2H, m), 1.21–1.38
(16H, br m), 0.88 (3H, t, J = 7.0 Hz). 13C NMR (100 MHz, CDCl3): d
164.3, 158.9, 158.5, 146.8, 146.8, 145.3, 133.1, 128.2, 113.5,
109.9, 108.6, 105.5, 105.0, 101.0, 100.9, 68.6, 31.9, 29.6, 29.6,
29.5, 29.5, 29.3, 29.3, 28.9, 26.0, 25.9, 25.3, 22.7, 19.7, 14.1. HRMS
(ESI) calcd for C30H40O6Na ([M+Na]+) 519.2717, found 519.2750.
Anal. Calcd for C30H40O6: C, 72.55; H, 8.12. Found: C, 72.31; H, 8.42.
727 cmꢀ1 1H NMR (600 MHz, DMSO-d6): d 10.9 (1H, s), 6.76 (1H,
.
s), 6.61 (1H, s), 6.39 (1H, d, J = 2.3 Hz), 6.26 (1H, d, J = 2.3 Hz),
5.99 (1H, s), 5.97 (1H, s), 1.90 (3H, s), 1.66 (3H, s), 1.65 (3H, s).
13C NMR (150 MHz, DMSO-d6): d 163.7, 158.3, 158.3, 146.4,
146.3, 144.9, 133.4, 127.7, 114.1, 109.6, 108.6, 104.9, 104.0,
102.2, 100.8, 25.6, 24.9, 19.4. HRMS (ESI) calcd for C18H16O6Na
([M+Na]+) 351.0839, found 351.0842. Anal. Calcd for C18H16O6: C,
65.85; H, 4.91. Found: C, 65.78; H, 4.67.
5.3. General method for the preparation of 13
5.2. General method for the preparation of 12
To a stirred solution of 12 (1.0 equiv) in CH2Cl2 was added BCl3
(5.0 equiv) at 0 °C and the mixture was stirred at room tempera-
ture for 18–20 h. Then the mixture was diluted with EtOAc. The or-
ganic layer was washed with water and brine, dried and
concentrated. Chromatography on silica gel with hexane–EtOAc–
AcOH yielded 13.
To a stirred solution of 11 (1.0 equiv), alcohol (1.5 equiv) and
PPh3 (1.5 equiv) in THF was added DIAD (1.5 equiv) at 0 °C, and
then the mixture was stirred at room temperature for 20 min. After
concentration of the mixture, chromatography on silica gel with
hexane–EtOAc yielded 12.
5.2.1. 7-Methoxy-2,2-dimethyl-5-(5-methyl-1,3-benzodioxol-6-
yl)-4H-1,3-benzodioxin-4-one (12a)
5.3.1. Altenusin (13a)
The title compound was prepared from 12a in 97% yield as yel-
low amorphous solids. The spectral data of 13a were identical to
those of our authentic sample.18
The title compound was prepared by the coupling of 11 and
methanol in 92% yield as white acicular crystals. The spectral data
of 12a were identical to those of our authentic sample.18
5.3.2. 3,40,50-Trihydroxy-20-methyl-5-propoxybiphenyl-2-carb-
oxylic acid (13b)
5.2.2. 7-Propoxy-2,2-dimethyl-5-(5-methyl-1,3-benzodioxol-6-
yl)-4H-1,3-benzodioxin-4-one (12b)
The title compound was prepared from 12b in 77% yield as yel-
low amorphous solids. IR mmax (KBr): 3536, 3397, 2972, 2873, 2556,
The title compound was prepared by the coupling of 11 and 1-
pronanol in 79% yield as white acicular crystals. Mp = 133.5–
134.0 °C. IR mmax (KBr): 2979, 2939, 2883, 1733, 1606, 1576,
1481, 1428, 1382, 1342, 1282, 1233, 1186, 1130, 1034, 970,
1640, 1613, 1519, 1456, 1367, 1340, 1284, 1255, 1219, 1184, 1065,
955, 868 cmꢀ1. 1H NMR (600 MHz, CD3OD): d 6.49 (1H, s), 6.40 (1H,
s), 6.30 (1H, d, J = 3.0 Hz), 6.06 (1H, d, J = 3.0 Hz), 3.81 (2H, t,
J = 7.0 Hz), 1.80 (3H, s), 1.65 (2H, quin, J = 7.0 Hz), 0.90 (3H, t,
J = 7.0 Hz). 13C NMR (150 MHz, CD3OD): d 174.3, 165.7, 164.5,
148.0, 145.0, 143.3, 135.5, 127.5, 117.5, 116.8, 112.0, 107.0,
101.2, 70.8, 23.5, 19.3, 10.7. HRMS (ESI) calcd for C17H18O6Na
([M+Na]+) 341.0995, found 341.0996. Anal. Calcd for C17H18O6: C,
64.14; H, 5.70. Found: C, 64.16; H, 5.93.
932, 914, 854 cmꢀ1 1H NMR (600 MHz, CDCl3): d 6.70 (1H, s),
.
6.59 (1H, s), 6.43 (1H, d, J = 2.5 Hz), 6.42 (1H, d, J = 2.5 Hz), 5.96
(1H, d, J = 1.3 Hz), 5.94 (1H, d, J = 1.3 Hz), 3.96 (2H, m), 2.02
(3H, s), 1.82 (2H, sextet, J = 7.6 Hz), 1.73 (3H, s), 1.72 (3H, s),
1.04 (3H, t, J = 7.6 Hz). 13C NMR (150 MHz, CDCl3): d 164.3,
158.9, 158.6, 146.9, 146.9, 145.3, 133.1, 128.3, 113.5, 109.8,
108.7, 105.6, 105.0, 101.0, 100.9, 70.1, 26.1, 25.3, 22.3, 19.7,

K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
7235
5.3.3. 5-Hexyloxy-3,40,50-trihydroxy-20-methylbiphenyl-2-carb-
oxylic acid (13c)
1259, 1212, 1184, 1110, 1080, 1028, 972, 891, 857 cmꢀ1
.
1H NMR
(600 MHz, CDCl3): d 11.26 (1H, s), 6.73 (1H, d, J = 2.1 Hz), 6.68
(1H, s), 6.61 (1H, d, J = 2.1 Hz), 6.39 (1H, s), 6.26 (1H, s), 4.05 (2H,
t, J = 6.6 Hz), 1.82 (2H, quin, J = 6.6 Hz), 1.73 (3H, s), 1.48 (2H, quin,
J = 6.6 Hz), 1.35 (4H, m), 0.92 (3H, t, J = 6.5 Hz). 1H NMR (600 MHz,
DMSO-d6): 15a: d 11.02 (1H, s), 9.53 (1H, s), 6.42 (1H, d, J = 1.9 Hz),
6.31 (1H, d, J = 1.9 Hz), 6.29 (1H, br q, J = 1.4 Hz), 5.71 (1H, s), 3.95
(2H, m), 1.66 (2H, m), 1.57 (3H, d, J = 1.4 Hz), 1.37 (2H, m), 1.31–
1.26 (4H, m), 0.86 (3H, t, J = 6.9 Hz). Compound 15: d 11.14 (1H,
s), 9.59 (1H, s), 7.09 (1H, d, J = 2.2 Hz), 6.96 (1H, s), 6.70 (1H, d,
J = 2.2 Hz), 6.18 (1H, s), 4.14 (2H, m), 1.72 (2H, m), 1.64 (3H, s),
1.42 (2H, m), 1.31–1.26 (4H, m), 0.89 (3H, t, J = 6.9 Hz).13C NMR
(150 MHz, CDCl3): d 180.8, 167.3, 166.0, 164.7, 153.3, 146.1,
134.9, 120.7, 116.1, 104.7, 104.1, 99.6, 79.0, 69.0, 31.4, 29.5, 28.8,
25.5, 22.5, 13.9. HRMS (ESI) calcd for C20H22O6Na ([M+Na]+)
381.1308, found 381.1316. Anal. Calcd for C20H22O6: C, 67.03; H,
6.19. Found: C, 67.03; H, 6.19.
The title compound was prepared from 12c in 75% yield as yel-
low foam. IR
m
max (KBr): 3400, 2928, 1645, 1611, 1516, 1457, 1364,
1255, 1177, 1068, 1028, 854 cmꢀ1
.
1H NMR (600 MHz, CDCl3): d
6.71 (1H, s), 6.61 (1H, s), 6.45 (1H, d, J = 2.5 Hz), 6.21 (1H, d,
J = 2.5 Hz), 3.96 (2H, t, J = 6.9 Hz), 1.93 (3H, s), 1.76 (2H, quin,
J = 6.9 Hz), 1.42 (2H, quin, J = 6.9 Hz), 1.32 (4H, m), 0.88 (3H, t,
J = 6.6 Hz). 13C NMR (150 MHz, CDCl3): d 173.4, 165.5, 145.5,
164.2, 143.5, 141.3, 132.6, 128.4, 117.1, 115.9, 111.8, 103.6,
100.9, 68.5, 31.5, 28.9, 25.6, 22.5, 19.0, 13.9. HRMS (ESI) calcd for
C20H24O6Na ([M+Na]+) 383.1465, found 383.1491.
5.3.4. 5-Dodecyloxy-3,40,50-trihydroxy-20-methylbiphenyl-2-
carboxylic acid (13d)
The title compound was prepared from 12d in 82% yield as yel-
low foam. IR mmax (neat): 3334, 3021, 2926, 2855, 1650, 1609,
1517, 1461, 1217, 1071, 1038, 853 cmꢀ1 1H NMR (600 MHz,
.
CDCl3): d 6.70 (1H, s), 6.61 (1H, s), 6.44 (1H, d, J = 2.3 Hz), 6.20
(1H, d, J = 2.3 Hz), 3.95 (2H, t, J = 6.5 Hz), 1.93 (3H, s), 1.76 (2H,
m), 1.42 (2H, m), 1.38–1.20 (16H, br m), 0.87 (3H, t, J = 7.0 Hz).
13C NMR (100 MHz, CDCl3): d 173.2, 165.4, 164.1, 145.4, 143.5,
141.3, 132.6, 128.4, 117.1, 115.9, 111.8, 103.5, 100.8, 68.5, 31.9,
29.6, 29.6, 29.6, 29.5, 29.3, 29.3, 28.9, 25.9, 22.7, 19.0, 14.1. HRMS
(ESI) calcd for C26H36O6Na ([M+Na]+) 467.2404, found 467.2412.
5.4.4. 9-Dodecyloxy-3,7-dihidroxy-4a-metyl-4aH-benzo[c]chro-
mene-2,6-dione (16)
The title compound was prepared from 13d in 57% yield as yel-
low acicular crystal. Mp = 119.2–119.8 °C. IR (KBr): 3281, 3054,
2921, 2851, 1681, 1645, 1572, 1508, 1462, 1384, 1356, 1304,
1257, 1180, 1111, 1080, 1029, 893, 855 cmꢀ1 1H NMR (600 MHz,
.
CDCl3): d 11.30 (1H, s), 6.73 (1H, d, J = 2.2 Hz), 6.69 (1H, s), 6.61
(1H, d, J = 2.2 Hz), 6.40 (1H, s), 6.28 (1H, s), 4.04 (2H, m), 1.82
(2H, m), 1.73 (3H, s), 1.47 (2H, m), 1.39–1.22 (16H, br m), 0.88
(3H, t, J = 7.0 Hz). 1H NMR (600 MHz, DMSO-d6): 16a: d 11.02
(1H, s), 9.53 (1H, s), 6.41 (1H, d, J = 1.7 Hz), 6.30 (1H, d,
J = 1.7 Hz), 6.28 (1H, br q, J = 1.2 Hz), 5.70 (1H, s), 3.95 (2H, m),
1.65 (2H, m), 1.57 (3H, d, J = 1.2 Hz), 1.36 (2H, m), 1.23 (16H, br
m), 0.85 (3H, t, J = 7.1 Hz). Compound 16: d 11.14 (1H, s), 9.59
(1H, s), 7.09 (1H, d, J = 2.1 Hz), 6.96 (1H, s), 6.70 (1H, d,
J = 2.1 Hz), 6.18 (1H, s), 4.13 (2H, m), 1.73 (2H, m), 1.65 (3H, s),
1.41 (2H, m), 1.23 (16H, br m), 0.85 (3H, t, J = 7.1 Hz). 13C NMR
(100 MHz, CDCl3): d 180.8, 167.4, 166.0, 164.7, 153.3, 146.1,
134.9, 120.7, 116.2, 104.7, 104.1, 99.6, 79.1, 69.0, 31.9, 29.6, 29.6,
29.6, 29.5, 29.5, 29.3, 29.3, 28.9, 25.9, 22.7, 14.1. HRMS (ESI) calcd
for C26H34O6Na ([M+Na]+) 465.2247, found 465.2249. Anal. Calcd
for C26H34O6: C, 70.56; H, 7.74. Found: C, 70.77; H, 8.00.
5.4. General method for the preparation of 1, 14, 15 and 16
To a solution of 13 (1.0 equiv) in EtOH–H2O (1:1, 25 mL) was
added a 0.2 M solution of FeCl3 (3.0 equiv) in H2O, and the mixture
was stirred at room temperature for 10–35 min. The mixture was
diluted with CH2Cl2. The organic layer was washed with water
and brine, dried and concentrated. Chromatography on silica gel
with hexane–EtOAc (3:2) yielded 1 (0.84 g, 71%).
5.4.1. Dehydroaltenusin (1)
The title compound was prepared from 13a in 71% yield as yel-
low acicular crystals. The spectral data of 1 were identical to those
of our authentic sample.18
5.4.2. 3,7-Dihidroxy-4a-metyl-9-propoxy-4aH-
benzo[c]chromene-2,6-dione (14)
5.4.5. 2,2-Dimethyl-5-(5-methyl-1,3-benzodioxol-6-yl)-4H-1,3-
benzodioxin-4-one (18)
The title compound was prepared from 13b in 63% yield as yel-
low acicular crystals. Mp = 124.5–125.0 °C. IR mmax (KBr): 3284,
3048, 2968, 2938, 2878, 1684, 1644, 1573, 1496, 1455, 1387,
To a stirred solution of 17 (365.2 mg, 0.95 mmol), 7 (352.4. mg,
1.08 mmol) and K2CO3 (448.0 mg, 3.24 mmol) in DME (10 mL) was
added PdCl2 (dppf) (39.2 mg, 0.054 mmol), and the mixture was
stirred at 85 °C for 17 h. After addition of Celite, the mixture was
stirred for 30 min and filtered through a pad of Celite. The filtrate
was diluted with chloroform, and the organic layer was washed
successively with water and brine, dried and concentrated. Chro-
matography on silica gel with hexane–EtOAc (8:1) yielded 18
(299.0 mg, 89%) as a white solid. Mp = 187.8–188.4 °C. IR mmax
(KBr): 3003, 2895, 1738, 1582, 1474, 1385, 1311, 1277, 1225,
1357, 1108, 1077, 1027, 952, 891 851 cmꢀ1 1H NMR (600 MHz,
.
CDCl3): d 11.27 (1H, s), 6.73 (1H, d, J = 2.4 Hz), 6.70 (1H, s), 6.61
(1H, d, J = 2.4 Hz), 6.40 (1H, s), 6.28 (1H, s), 4.05 (2H, m), 1.86
(2H, quin, J = 7.3 Hz), 1.73 (3H, s), 1.07 (3H, t, J = 7.3 Hz). 1H NMR
(600 MHz, DMSO-d6): 14a: d 11.03 (1H, s), 9.53 (1H, s), 6.42 (1H,
d, J = 1.8 Hz), 6.31 (1H, d, J = 1.8 Hz), 6.29 (1H, br q, J = 1.3 Hz),
5.71 (1H, s), 3.92 (2H, m), 1.68 (2H, sextet, J = 7.2 Hz), 1.57 (3H,
d, J = 1.3 Hz), 0.94 (3H, t, J = 7.2 Hz). Compound 14: d 11.15 (1H,
s), 9.59 (1H, s), 7.10 (1H, d, J = 2.2 Hz), 6.96 (1H, s), 6.70 (1H, d,
J = 2.2 Hz), 6.18 (1H, s), 4.11 (2H, m), 1.76 (2H, sextet, J = 7.4 Hz),
1.64 (3H, s), 0.99 (3H, t, J = 7.4 Hz).13C NMR (150 MHz, CDCl3): d
180.8, 167.3, 165.9, 164.7, 153.2, 146.1, 134.9, 120.7, 116.1,
104.7, 104.1, 99.6, 79.1, 70.4, 29.5, 22.3, 10.4. HRMS (ESI) calcd
for C17H16O6Na [M+Na]+ 339.0839, found 339.0844. Anal. Calcd
for C17H16O6: C, 64.55; H, 5.10. Found: C, 64.68; H, 5.01.
1119, 1074, 1035, 930, 871, 829 cmꢀ1 1H NMR (600 MHz, CDCl3):
.
d 7.52 (1H, t, J = 7.8 Hz), 6.97 (1H, dd, J = 1.07 Hz, 7.8 Hz), 6.88 (1H,
dd, J = 1.07), 6.71 (1H, s), 6.58 (1H, s), 5.97 (1H, d, J = 1.5 Hz), 5.95
(1H, d, J = 1.5 Hz), 3.96 (2H, m), 2.00 (3H, s), 1.75 (3H, s), 1.74 (3H,
s). 13C NMR (150 MHz, CDCl3): d 159.0, 156.7, 146.9, 145.3, 145.1,
135.0, 132.9, 128.2, 125.9, 116.4, 112.9, 109.9, 108.7, 105.2,
100.8, 26.0, 25.3, 19.7. HRMS (ESI) calcd for C18H16O5Na
([M+Na]+) 335.0889, found 335.0909. Anal. Calcd for C18H16O5: C,
69.22; H, 5.16. Found: C, 69.42; H, 5.06.
5.4.3. 9-Hexyloxy-3,7-dihidroxy-4a-metyl-4aH-benzo[c]chro-
mene-2,6-dione (15)
The title compound was prepared from 13c in 63% yield as yel-
low solids. Mp = 136.3–136.7 °C. IR mmax (KBr): 3277, 3053, 2940,
2866, 1681, 1644, 1576, 1514, 1463, 1412, 1386, 1356, 1307,
5.4.6. 3,40,50-Trihydroxy-20-methyl-biphenyl-2-carboxylic acid (19)
To a stirred solution of 18 (268.5 mg, 0.86 mmol) in CH2Cl2
(9.0 mL) was added BCl3 (1.0 M solution in heptane, 4.3 mL,

7236
K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
4.3 mmol) at 0 °C and the mixture was stirred at room temperature
for 18 h. Then the mixture was diluted with EtOAc. The organic
layer was washed with water and brine, dried and concentrated.
Chromatography on silica gel with hexane–EtOAc–AcOH
(1333:667:1) yielded 19 (172.8 mg, 77%) as a yellow foam. IR mmax
(KBr): 3462, 3366, 3044, 1662, 1601, 1514, 1442, 1340, 1281, 1249,
centrated. The residue was chromatographed over silica gel.
Elution with hexane–ethyl acetate (6:1) gave 24 (73.2 mg, 61%)
amorphous solids and recovered 23 (15.7 mg, 21%). IR mmax (KBr):
3001, 2937, 1739, 1610, 1516, 1458, 1383, 1277, 1203, 1093,
1016, 914, 850, 741, 698, 636 cmꢀ1 1H NMR (270 MHz, CDCl3): d
;
7.27–7.47 (10H, m), 6.83 (1H, s), 6.77 (1H, s), 6.44 (1H, d,
J = 2.7 Hz), 6.42 (1H, d, J = 2.7 Hz), 5.13 (4H, s), 3.80 (3H, s), 2.01
(3H, s), 1.74 (6H, s). 13C NMR (68 MHz, CDCl3): d 169.0, 164.4,
158.6, 158.1, 149.2, 146.4, 141.9, 141.3, 137.1, 136.7, 128.4 (ꢁ2),
128.3 (ꢁ2), 127.8, 127.6, 127.4 (ꢁ2), 127.3 (ꢁ2), 125.3, 116.5,
113.0, 108.7, 105.5, 104.9, 101.1, 72.0, 71.3, 55.7, 26.5, 24.9, 20.9.
HRMS (ESI) calcd for C33H30O8Na ([M+Na]+) 577.1832, found
577.1822.
1224, 1171, 1045, 951, 871, 813 cmꢀ1 1H NMR (600 MHz, MeOD):
.
d 7.32 (1H, t, J = 7.8 Hz), 6.87 (1H, d, J = 7.8 Hz), 6.61 (1H, d,
J = 7.8 Hz), 6.59 (1H, s), 6.50 (1H, s), 1.73 (3H, s). 13C NMR
(150 MHz, MeOD): d 173.7, 161.2, 145.3, 144.9, 143.1, 134.7,
133.7, 127.7, 123.5, 117.4, 117.0, 116.4, 19.4. HRMS (ESI) calcd
for C14H12O5Na [M+Na]+ 283.0576, found 283.572. Anal. Calcd for
C14H12O5: C, 64.61; H, 4.65. Found: C, 64.52; H, 4.64.
5.4.7. 3,7-Dihidroxy-4a-methyl-4aH-benzo[c]chromene-2,6-
dione (20)
5.4.10. 2,3-Dibenzyloxy-7-trihydroxy-9-methoxy-6H-benzo[c]-
chromen-6-one (25)
To a solution of 19 (134.7 mg, 0.66 mmol) in EtOH–H2O (1:1,
3.0 mL) was added a 0.2 M solution of FeCl3 in H2O (6.0 mL) and
the mixture was stirred at room temperature for 10 min. The mix-
ture was diluted with CH2Cl2. The organic layer was washed with
water and brine, dried and concentrated. Chromatography on silica
gel with hexane–EtOAc (2:3) yielded 20 (134.8 mg, 80%) as a yel-
low acicular crystal. Mp = 180.0–181.2 °C. IR mmax (KBr): 3388,
3103, 3053, 2980, 2927, 1682, 1647, 1578, 1494, 1458, 1419,
1379, 1341, 1318, 1271, 1233, 1207, 1170, 1102, 1051, 1024,
To a solution of 24 (73.2 mg, 0.132 mmol) and K2CO3 (92.3 mg,
0.139 mmol) in MeOH (5 mL) was stirred at room temperature for
5 h. The mixture was diluted with CHCl3, and the reaction was
quenched by the addition of 1 M HCl solution. The layers were sep-
arated. The organic layer was washed with water, brine, dried
(Na2SO4) and concentrated. The residue was chromatographed
over silica gel. Elution with CHCl3 gave 25 (46.3 mg, 77%) as white
solids. Mp = 199–200 °C. IR mmax (KBr): 3444, 3068, 2941, 2875,
1668, 1626, 1568, 1512, 1456, 1410, 1371, 1315, 1275, 1188,
943, 887, 843, 815 cmꢀ1 1H NMR (600 MHz, CDCl3): d 11.13 (1H,
.
1157, 1095, 1034, 993, 924, 868, 833, 750, 696 cmꢀ1 1H NMR
.
s), 7.64 (1H, t, J = 8.01 Hz), 7.22 (1H, dd, J = 0.89, 8.02 Hz), 7.19
(1H, dd, J = 0.89, 8.02 Hz), 6.74 (1H, s), 6.73 (1H, s) 6.70 (1H, s),
6.47 (1H, s), 6.29 (1H, s), 1.75 (3H, s). 1H NMR (600 MHz, DMSO-
d6): 20a: d 11.10 (1H, s), 9.58 (1H, s), 7.54 (1H, t, J = 7.9 Hz), 6.96
(1H, d, J = 7.9 Hz), 6.71 (1H, d, J = 7.9 Hz), 6.32 (1H, br s), 5.77
(1H, s), 1.55 (3H, br s). Compound 20: d 10.99 (1H, s), 9.61 (1H,
s), 7.37 (1H, t, J = 8.4 Hz), 7.51 (1H, d, J = 8.4 Hz), 7.20 (1H, d,
J = 8.4 Hz), 6.88 (1H, s), 6.19 (1H, s), 1.67 (3H, s). 13C NMR
(150 MHz, CDCl3): d 180.7, 167.5, 162.3, 152.9, 146.3, 137.0,
133.8, 121.1, 120.9, 115.9, 115.7, 106.5, 79.6, 29.7. HRMS (ESI)
calcd for C14H10O5Na ([M+Na]+) 281.0420, found 281.0425. Anal.
Calcd for C14H10O5: C, 65.12; H, 3.90. Found: C, 65.42; H, 3.76.
(270 MHz, CDCl3): d 11.52 (1H, s), 7.50–7.32 (11H, m), 6.50 (1H,
d, J = 2.4 Hz), 5.22 (4H, s), 3.91 (3H, s). 13C NMR (68 MHz, CDCl3):
d 166.7, 165.2, 164.6, 151.6, 146.0, 146.0, 136.7, 136.6, 135.8,
128.6 (ꢁ2), 128.5 (ꢁ2), 128.1, 128.0, 127.4 (ꢁ2), 127.1 (ꢁ2),
110.5, 108.9, 102.9, 99.8, 99.2, 98.7, 72.3, 71.1, 55.8. HRMS (ESI)
calcd for C28H22O6Na ([M+Na]+) 477.1308, found 477.1327. Anal.
Calcd for C28H22O6: C, 74.00; H, 4.88. Found: C, 74.13; H, 4.77.
5.4.11. 2,3,7-Trihydroxy-9-methoxy-6H-benzo[c]chromen-6-
one (26)
A solution of 25 (34.3 mg, 75.5 lmol) and Pd(OH)2 on carbon
powder (20% Pd, 5.8 mg) in THF (6 mL) was stirred under an atmo-
sphere of H2 at room temperature for 9 h. The mixture was filtrated
through a pad of Celite and washed with CHCl3–MeOH (10:1). The
filtrate was concentrated. The residue was chromatographed over
silica gel. Elution with CHCl3–MeOH (20:1?10:1) gave 26
(21.1 mg, quant.) as white solids. Mp = 272 °C (decomp.). IR mmax
(KBr): 3531, 1645, 1516, 1454, 1277, 1161, 1032, 976, 914, 837,
5.4.8. 4,4,5,5,-Tetramethyl-2-(2-acetoxyl-4,5-dibenzyloxy-
phenyl)-1,3,2-dioxaborolane (22)
To a degassed solution of 21 (1.33 g, 2.81 mmol), bis(pinacola-
to)diboron (1.02 g, 4.02 mmol) and KOAc (0.83 g, 8.46 mmol) in
DMF (20 mL) was added PdCl2(dppf) (204 mg, 0.28 mmol). The
mixture was stirred at 80 °C for 6 h, then cooled to room tempera-
ture and filtrated through a pad of Celite and washed with EtOAc.
The organic layer was washed with water, brine, dried (Na2SO4)
and concentrated. The residue was chromatographed over silica
gel. Elution with hexane–ethyl acetate (6:1) gave 22 (711 mg,
53%) as white solids. Mp = 113–115 °C. IR mmax (KBr): 2983, 1757,
785 cmꢀ1 1H NMR (270 MHz, CD3OD): d 7.36 (1H, s), 6.88 (1H, d,
.
J = 2.2 Hz), 6.82 (1H, s), 6.49 (1H, d, J = 2.2 Hz), 3.94 (3H, s). 13C
NMR (68 MHz, DMSO-d6): d 166.6 (ꢁ2), 164.9, 163.6, 149.3,
144.2, 143.7, 137.5, 108.9, 103.4, 100.0, 98.6, 97.9, 56.1. HRMS
(ESI) calcd for C14H10O6Na ([M+Na]+) 297.0369, found 297.0359.
1622, 1514, 1001, 912, 849, 744, 696, 584, 499 cmꢀ1 1H NMR
.
5.4.12. 4,4,5,5-Tetramethyl-2-(2-methyl-4-hydroxyphenyl)-
1,3,2-dioxaborolane (28)
To a degassed solution of 27 (180 mg, 0.770 mmol), bis(pinaco-
lato)diboron (256 mg, 1.00 mmol) and KOAc (246 mg, 2.51 mmol)
(270 MHz, CDCl3): d 7.27–7.45 (11H, m), 6.63 (1H, d, J = 1.6 Hz),
5.10 (4H, s), 2.27 (3H, s), 1.29 (12H, s). 13C NMR (68 MHz, CDCl3):
d 170.4, 152.2, 150.6, 150.6, 146.1, 137.0, 136.3, 128.3 (ꢁ2),
128.1 (ꢁ2), 127.6, 127.5, 127.3 (ꢁ2), 126.9 (ꢁ2), 121.4, 108.2,
83.4 (ꢁ2), 71.7, 70.7, 24.8 (ꢁ4), 21.1. HRMS (ESI) calcd for
C28H31BO6Na ([M+Na]+) 497.2105, found 497.2103.
in DMF (5 mL) was added PdCl2(dppf) (56.3 mg, 76.4 lmol). The
mixture was stirred at 70 °C for 11 h, cooled to room temperature
and then filtrated through a pad of Celite and washed with EtOAc.
The organic layer was washed with water, brine, dried (Na2SO4)
and concentrated. The residue was chromatographed over silica
gel. Elution with hexane–ethyl acetate (7:1) gave 28 (155 mg,
86%) as white solids. Mp = 113–114 °C. IR mmax (KBr): 3394, 2976,
2929, 1599, 1506, 1433, 1356, 1290, 1217, 1142, 1063, 962, 914,
5.4.9. 7-Methoxy-2,2-dimethyl-5-(2-acetoxy-4,4-dibenzyloxy-
phenyl)-4H-1,3-benzodioxin-4-one (24)
To
(76.3 mg, 0.214 mmol) and K2CO3 (92.3 mg, 0.668 mmol) in DME
(3 mL) was added PdCl2(dppf) (8.2 mg, 11.2 mol). The mixture
a degassed solution of 22 (126 mg, 0.265 mmol), 23
l
856, 820, 752, 727 cmꢀ1 1H NMR (270 MHz, CDCl3): d 11.52 (1H,
;
was stirred at 80 °C for 6.5 h, cooled to room temperature and then
filtrated through a pad of Celite and washed with EtOAc. The or-
ganic layer was washed with water, brine, dried (Na2SO4) and con-
s), 7.50–7.32 (11H, m), 6.50 (1H, d, J = 2.4 Hz), 5.22 (4H, s), 3.91
(3H, s). 13C NMR (68 MHz, CDCl3): d 152.6, 136.5, 131.0, 121.9,

K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
7237
117.8, 117.8, 83.6 (ꢁ2), 24.9 (ꢁ4), 21.9. HRMS (EI) calcd for
5.4.16. 7-Hydroxy-4a-methyl-4aH-benzo[c]chromene-2,6-
dione (32)
C13H19BO3 ([M]+) 234.1427, found 234.1426.
A solution of 30 (55.2 mg, 0.194 mmol) and LiOHꢂH2O (164 mg,
3.90 mmol) was stirred at 50 °C for 18 h. The mixture was diluted
with EtOAc. The reaction was quenched by the addition of 1 M HCl
solution. The layers were separated. The organic layer was washed
with water, brine, dried (Na2SO4) and concentrated.
5.4.13. 5-(5-Hydroxy-2-methylphenyl)-7-methoxy-2,2-dim-
ethyl-4H-1,3-benzodioxin-4-one (29)
To
(49.6 mg, 0.212 mmol) and K2CO3 (89.2 mg, 0.645 mmol) in
DME (4 mL) was added PdCl2(dppf) (19.2 mg, 26.2 mol). The
a degassed solution of 23 (90.6 mg, 0.254 mmol), 28
To a solution of the crude carboxylic acid (33.1 mg) in MeOH
(3 mL) was added PhI(OAc)2 (75.6 mg, 0.235 mmol) at 0 °C, and
the mixture was stirred at room temperature for 1.5 h. The mixture
was concentrated. The residue was chromatographed over silica
gel. Elution with CHCl3 gave 32 (30.2 mg, 64% in two steps) as yel-
low solids. Mp = 124–129 °C; IR mmax (KBr): 3348, 3055, 2925,
2925, 2854, 1674, 1635, 1577, 1493, 1460, 1367, 1281, 1213,
l
mixture was stirred at 55 °C for 9 h, cooled to room temperature
and then filtrated through a pad of Celite and washed with EtOAc.
The organic layer was washed with water, brine, dried (Na2SO4)
and concentrated. The residue was chromatographed over silica
gel. Elution with hexane–ethyl acetate (3:1) gave 29 (44.1 mg,
76%) as amorphous solids. IR mmax (KBr): 3437, 3074, 2925,
2854, 1738, 1616, 1579, 1423, 1281, 1209, 1144, 1049, 939,
1149, 1067, 1032, 930, 906, 822, 730, 698 cmꢀ1
.
1H NMR
868, 768, 704, 671 cmꢀ1 1H NMR (270 MHz, CDCl3): d 7.03 (1H,
;
d, J = 8.4 Hz), 6.68 (1H, dd, J = 8.4 Hz, 2.7 Hz), 6.56 (1H, d, J
=2.7 Hz), 6.45 (1H, d, J = 2.7 Hz), 6.43 (1H, d, J = 2.7 Hz), 5.45
(1H, br s), 3.84 (3H, s), 2.00 (3H, s), 1.73 (6H, s). 13C NMR
(270 MHz, CDCl3): d 7.64 (1H, br t, J = 8.4 Hz), 7.23 (1H, dd,
J = 7.6 Hz, 0.8 Hz), 7.17 (1H, dd, J = 8.4 Hz, 0.8 Hz), 7.11 (1H, d,
J = 10.0 Hz), 6.66 (1H, d, J = 1.6 Hz), 6.35 (1H, dd, J = 10.0 Hz,
1.6 Hz), 1.72 (3H, s). 13C NMR (68 MHz, CDCl3): d 184.3, 167.1,
162.1, 149.1, 146.7, 137.0, 133.8, 127.9, 123.4, 120.6, 115.5,
106.2, 28.7. HRMS (EI) calcd for C14H10O4 ([M]+) 242.0579, found
242.0581.
(68 MHz, CDCl3):
d 164.6, 159.2, 158.4, 153.4, 146.8, 141.0,
130.4, 126.5, 115.2, 114.6, 112.5, 105.3, 105.2, 100.7, 55.8, 26.2,
25.2, 19.0. HRMS (ESI) calcd for C18H18O5Na ([M+Na]+)
337.1046, found 337.1040.
5.4.17. 30-{[2-(Acetylamino)-3-methoxy-3-oxopropyl]sulfanyl}-
3,40,50-trihydroxy-20-methyl-5-methoxybiphenyl-2-carboxylic
acid (34) and 20-{[2-(acetylamino)-3-methoxy-3-oxopropyl]-
sulfanyl}-3,30,40-trihydroxy-60-methyl-5-methoxybiphenyl-2-
carboxylic acid (35)
5.4.14. 5-(5-Hydroxy-2-methylphenyl)-2,2-dimethyl-4H-1,3-
benzodioxin-4-one (30)
To a degassed solution of 17 (258 mg, 0.792 mmol), 28 (144 mg,
0.614 mmol) and potassium carbonate (256 mg, 1.85 mmol) in
A
solution of 1 (21.2 mg, 0.074 mmol) and 33 (21.8,
DME (7 mL) was added PdCl2(dppf) (86.4 mg, 185 lmol). The mix-
0.123 mmol) in MeOH–H2O (1:1, 2 mL) was stirred at room tem-
perature for 30 min and then concentrated. The residue was puri-
fied by silica gel chromatography (CHCl3–MeOH = 9:1 with 1%
AcOH) to give the crude product (35.3 mg). This crude products
was further purified by silica gel chromatography (EtOAc–
MeOH = 10:1 with 0.5% AcOH) to give a 1:1 inseparable mixture
of 34 and 35 (30.2 mg, 91%) as amorphous solids.33 IR mmax (KBr):
3367, 3089, 3010, 2954, 2850, 1739, 1653, 1608, 1423, 1375,
1281, 1255, 1205, 1157, 1072, 1039, 983, 910, 842, 800, 756,
ture was stirred at 55 °C for 23 h, cooled to room temperature and
then filtrated through a pad of Celite and washed with EtOAc. The
organic layer was washed with water, brine, dried (Na2SO4) and
concentrated. The residue was chromatographed over silica gel.
Elution with hexane–ethyl acetate (3:1) gave 30 (118 mg, 67%) as
amorphous solids. IR mmax (KBr): 3377, 2997, 2924, 2860, 1718,
1581, 1477, 1385, 1319, 1205, 1045, 926, 845, 814, 754,
698 cmꢀ1 1H NMR (270 MHz, CDCl3): d 7.51 (1H, br t, J = 7.6 Hz),
.
7.05–6.94 (2H, m), 6.86 (1H, br d, J = 7.6 Hz), 6.54 (1H, d,
J = 2.7 Hz), 6.63 (1H, m), 5.93 (1H, br s), 1.97 (3H, s), 1.73 (6H, s).
13C NMR (68 MHz, CDCl3): d 159.6, 156.4, 153.5, 145.1, 140.1,
135.2, 130.3, 126.2, 125.5, 116.3, 115.3, 114.7, 112.5, 105.2, 26.1,
25.2, 19.0. HRMS (EI) calcd for C17H16O4 ([M]+) 284.1049, found
284.1049.
723, 667 cmꢀ1 1H NMR (270 MHz, CDCl3 with 1% TFA at 40 °C).
.
Compound 34: d 6.84 (1H, s), 6.54 (1H, d, J = 2.7 Hz), 6.22 (1H, d,
J = 2.7 Hz), 4.65 (1H, m), 3.83 (3H, s), 3.74 (3H, s), 2.76 (2H, m),
2.18 (3H, s), 1.98 (3H, s). Compound 35: d 6.84 (1H, s), 6.54 (1H,
d, J = 2.7 Hz), 6.14 (1H, d, J = 2.7 Hz), 4.75 (1H, m), 3.85 (3H, s),
3.73 (3H, s), 2.88 (2H, m), 2.18 (3H, s), 1.93 (3H, s). HRMS (ESI)
calcd for C21H23NO9NaS ([M+Na]+) 488.0985, found 488.0979.
5.4.15. 7-Hydroxy-9-methoxy-4a-methyl-4aH-benzo[c]chro-
mene-2,6-dione (31)
5.5. DNA polymerase assay
A solution of 29 (44.1 mg, 0.140 mmol) and LiOHꢂH2O (130 mg,
3.09 mmol) was stirred at 50 °C for 18 h. The mixture was diluted
with EtOAc. The reaction was quenched by the addition of 1 M HCl
solution. The layers were separated. The organic layer was washed
with water, brine, dried (Na2SO4) and concentrated.
In mammalian DNA polymerases, DNA polymerase
a was puri-
fied from calf thymus by immuno-affinity column chromatography
as described previously.34 DNA polymerase b was purified from a
recombinant plasmid expressing rat DNA polymerase b.35 The
To a solution of the crude carboxylic acid (39.7 mg) in MeOH
(3 mL) was added PhI(OAc)2 (67.6 mg, 0.209 mmol) at 0 °C, and
the mixture was stirred at room temperature for 2 h. The mixture
was concentrated. The residue was chromatographed over silica
gel. Elution with CHCl3 gave 31 (33.7 mg, 77% in two steps) as pale
yellow solids. Mp = 225–230 °C. IR mmax (KBr): 3450, 3209, 3060,
2925, 2852, 1676, 1614, 1570, 1487, 1433, 1389, 1358, 1290,
reaction mixtures for DNA polymerases
a and b were described
previously.36,37 For the DNA polymerases, poly(dA)/oligo(dT)12–18
(A/T = 2/1) and [3H]-dTTP were used as the DNA template-primer
and nucleotide (i.e., 20-deoxyribonucleotide 50-triphosphate, dNTP)
substrate, respectively. The dehydroaltenusin derivative was dis-
solved in dimethyl sulfoxide (DMSO) at various concentrations
and sonicated for 30 s. Four microlitres of each sonicated sample
1196, 1167, 1113, 1070, 1036, 978, 839, 644 cmꢀ1 1H NMR
;
were mixed with 16 lL of each enzyme (final 0.05 units) in
(270 MHz, CDCl3): d 11.3 (1H, s), 7.10 (1H, d, J = 10.0 Hz), 6.74
(1H, d, J = 2.4 Hz), 6.62 (1H, d, J = 2.4 Hz), 6.62 (1H, d, J = 1.9 Hz),
6.33 (1H, dd, J = 10.0 Hz, 1.9 Hz), 3.91 (3H, s), 1.70 (3H, s). 13C
NMR (68 MHz, CDCl3): d 184.4, 166.9, 166.3, 164.6, 149.3, 147.0,
135.1, 127.8, 123.3, 104.0, 103.4, 99.7, 56.0, 28.5. HRMS (EI) calcd
for C15H12O5 ([M]+) 272.0685, found 272.0692.
50 mM Tris–HCl (pH 7.5) containing 1 mM dithiothreitol, 50% glyc-
erol and 0.1 mM ethylenediamine tetraacetic acid (EDTA), and kept
at 0 °C for 10 min. These inhibitor–enzyme mixtures (8
lL) were
added to 16 L of each enzyme standard reaction mixture, and
l
incubated at 37 °C for 60 min. Activity without the inhibitor was
considered 100%, and the remaining activity at each concentration

7238
K. Kuramochi et al. / Bioorg. Med. Chem. 17 (2009) 7227–7238
13. Nakai, C. M.; Maeda, N.; Yonezawa, Y.; Kuriyama, I.; Kamisuki, S.; Takahashi, S.;
Sugawara, F.; Yoshida, H.; Sakaguchi, K.; Mizushina, Y. Biochim. Biophys. Acta
2004, 1674, 193.
14. Maeda, N.; Kokai, Y.; Ohtani, S.; Sahara, H.; Kuriyama, I.; Kamisuki, S.;
Takahashi, S.; Sakaguchi, K.; Sugawara, F.; Yoshida, H.; Sato, N.; Mizushina, Y.
Biochem. Biophys. Res. Commun. 2007, 352, 390.
15. Kuriyama, I.; Fukudome, K.; Kamisuki, S.; Kuramochi, K.; Tsubaki, K.;
Sakaguchi, K.; Sugawara, F.; Yoshida, H.; Mizushina, Y. Int. J. Mol. Med. 2008,
22, 793.
16. Kuriyama, I.; Mizuno, T.; Fukudome, K.; Kuramochi, K.; Tsubaki, K.; Usui, T.;
Imamoto, N.; Sakaguchi, K.; Sugawara, F.; Yoshida, H.; Mizushina, Y. Molecules
2008, 13, 2948.
of the inhibitor was determined relative to this value. One unit of
pol activity was defined as the amount of enzyme that catalyzed
the incorporation of 1 nmol of dNTP (i.e., dTTP) into synthetic
DNA template-primer in 60 min at 37 °C under the normal reaction
conditions for each enzyme.36,37
Acknowledgements
We gratefully acknowledge Professor Kobayashi and Professor
Nakazaki of Tokyo University of Science for giving us helpful ad-
vice. This work was supported in part by a Grant-in-Aid for
Kobe-Gakuin University Joint Research (A), and the ‘Academic
Frontier’ Project for Private Universities: matching fund subsidy
from the Ministry of Education, Science, Sports, and Culture of Ja-
pan (MEXT), 2006–2010, (H.Y. and Y.M.). Y.M. acknowledges a
Grant-in-Aid for Young Scientists (A) (No. 19680031) from MEXT,
and The Salt Science Research Foundation, No. 09S3 (Japan).
17. Takahashi, S.; Kamisuki, S.; Mizushina, Y.; Sakaguchi, K.; Sugawara, F.; Nakata,
T. Tetrahedron Lett. 2003, 44, 1875.
18. Kamisuki, S.; Takahashi, S.; Mizushina, Y.; Hanashima, S.; Kuramochi, K.;
Kobayashi, S.; Sakaguchi, K.; Nakata, T.; Sugawara, F. Tetrahedron 2004, 60,
5695.
19. Koch, K.; Podlech, J.; Pfeiffer, E.; Metzler, M. J. Org. Chem. 2005, 70, 3275.
20. Altemöller, M.; Podlech, J.; Fenske, D. Eur. J. Org. Chem. 2006, 1678.
21. Dushin, R. G.; Danishefsky, S. J. J. Am. Chem. Soc. 1992, 114, 655.
22. Aly, A. H.; Edrada-Ebel, R.; Indriani, I. D.; Wray, V.; Müller, W. E.; Totzke, F.;
Zirrgiebel, U.; Schächtele, C.; Kubbutat, M. H.; Lin, W. H.; Proksch, P.; Ebel, R. J.
Nat. Prod. 2008, 71, 972.
23. Hadfield, A.; Schweitzer, H.; Trova, M. P.; Green, K. Synth. Commun. 1994, 24,
1025.
24. Li, C. C.; Xie, Z. X.; Zhang, Y. D.; Chen, J. H.; Yang, Z. J. Org. Chem. 2003, 68, 8500.
25. Pache, S.; Lautens, M. Org. Lett. 2003, 5, 4827.
References and notes
26. Felpin, F. X. Tetrahedron Lett. 2007, 48, 409.
27. Bitter, S. Amino Acids 2006, 30, 205.
1. Crews, C. M.; Mohan, R. Curr. Opin. Chem. Biol. 2000, 4, 47.
2. Stockwell, B. R. Nature 2004, 432, 846.
28. Kutyrev, A. A. Tetrahedron 1991, 47, 8043.
3. Noble, M. E.; Endicott, J. A.; Johnson, L. N. Science 2004, 303, 1800.
4. Stockwell, B. R. Nat. Rev. Genet. 2000, 1, 116.
5. Rosett, T.; Sankhala, R. H.; Stickings, C. E.; Talyor, M. E.; Thomas, R. Biochem. J.
1957, 67, 390.
29. Kalyanaraman, B.; Felix, C. C.; Sealy, R. C. Environ. Health Perspect. 1985, 64, 185.
30. Cao, K.; Stack, D. E.; Ramanathan, R.; Gross, M. L.; Rogan, E. G.; Cavalieri, E. L.
Chem. Res. Toxicol. 1998, 11, 909.
31. Duman, R. K.; Heath, R. T.; Bose, R. N. FEBS Lett. 1999, 455, 49.
32. Wang, T. S.; Wong, S. W.; Korn, D. FASEB J. 1989, 3, 14.
33. The 13C NMR spectra for 34 and 35 could not be obtained with a sufficient
resolution because of the low solubility in NMR solvents.
34. Tamai, K.; Kojima, K.; Hanaichi, T.; Masaki, S.; Suzuki, M.; Umekawa, H.;
Yoshida, S. Biochim. Biophys. Acta 1988, 950, 263.
6. Rogers, D.; Williams, D. J.; Thomas, R. J. Chem. Soc. Chem. D 1971, 393.
7. Ayer, W. A.; Racok, J. S. Can. J. Chem. 1990, 68, 2085.
8. Kamisuki, S.; Takahashi, S.; Mizushina, Y.; Sakaguchi, K.; Nakata, T.; Sugawara,
F. Bioorg. Med. Chem. 2004, 12, 5355.
9. Nakanishi, S.; Toki, S.; Saitoh, Y.; Tsukuda, E.; Kawahara, K.; Ando, K.; Matsuda,
Y. Biosci., Biotechnol., Biochem. 1995, 59, 1333.
35. Date, T.; Yamaguchi, M.; Hirose, F.; Nishimoto, Y.; Tanihara, K.; Matsukage, A.
Biochemistry 1988, 27, 2983.
36. Mizushina, Y.; Tanaka, N.; Yagi, H.; Kurosawa, T.; Onoue, M.; Seto, H.; Horie, T.;
Aoyagi, N.; Yamaoka, M.; Matsukage, A.; Yoshida, S.; Sakaguchi, K. Biochim.
Biophys. Acta 1996, 1308, 256.
10. Mizushina, Y.; Kamisuki, S.; Mizuno, Y.; Takemura, M.; Asahara, H.; Linn, S.;
Yamaguchi, T.; Matsukage, A.; Hanaoka, F.; Yoshida, S.; Saneyoshi, M.;
Sugawara, F.; Sakaguchi, K. J. Biol. Chem. 2000, 275, 33957.
11. Kamisuki, S.; Murakami, C.; Ohta, K.; Yoshida, H.; Sugawara, F.; Sakaguchi, K.;
Mizushina, Y. Biochem. Pharmacol. 2002, 63, 421.
37. Mizushina, Y.; Yoshida, S.; Matsukage, A.; Sakaguchi, K. Biochim. Biophys. Acta
1997, 1336, 509.
12. Maeda, N.; Kamisuki, S.; Takahashi, S.; Yoshida, H.; Sakaguchi, K.; Sugawara, F.;
Mizushina, Y. Curr. Bioactive Comp. 2006, 2, 3.