The synthesis and biological activity of pyranonaphthoquione derivatives from Streptomyces sp. and their related substances

Article abstract of DOI:10.1246/bcsj.77.1925

The total synthesis of a novel pyranonaphthoquinone (2) having Cdc25A phosphatase inhibitory activity is achieved. The key step in this synthetic pathway is the intramolecular Michael addition for construction of the pyran ring system. The inhibitory acti

Full text of DOI:10.1246/bcsj.77.1925

ꢀ 2004 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 77, 1925–1930 (2004) 1925
The Synthesis and Biological Activity of Pyranonaphthoquione
Derivatives from Streptomyces sp. and Their Related Substances
ꢀ
Akiko Shimbashi, Ayako Tsuchiya,1 Masaya Imoto,1 and Shigeru Nishiyama
Department of Chemistry, Faculty of Science and Technology, Keio University,
Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522
1Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University,
Hiyoshi 3-14-1, Kohoku-ku, Yokohama 223-8522
Received May 6, 2004; E-mail: nisiyama@chem.keio.ac.jp
The total synthesis of a novel pyranonaphthoquinone (2) having Cdc25A phosphatase inhibitory activity is achiev-
ed. The key step in this synthetic pathway is the intramolecular Michael addition for construction of the pyran ring sys-
tem. The inhibitory activity of several derivatives, readily obtained from synthetic intermediates of 2, is assessed. The
resulting data indicates that the pyran ring moiety is not a crucial factor for the activity.
Four pyranonaphthoquinones (1–4, Fig. 1), isolated from the
fermentation broth of Streptomyces sp. by the Eli Lilly group,1
exhibited weak inhibitory activity against Cdc25A phospha-
tase which plays a pivotal role in the regulation of the G1/S
transition for activating several cell cycle-dependent kinases
(CDKs) by dephosphorylating an inhibitory phosphotyrosine
and phosphothreonine,2 and are closely related to the oncogen-
ic properties in human tumors, such as in breast cancer.3 Be-
cause of its significant role in cell proliferation and its correla-
tion with a variety of cancers, Cdc25A is a potential and attrac-
tive target for anticancer chemotherapeutic agents. Structural-
ly, the benzoquinone moiety, including the amino substituents,
is located at the edge of the tricyclic molecule, whereas those
of nanaomycins,4 eleutherin,5 and freolicins6 are inserted be-
tween the aromatic and pyran rings. Against such a back-
ground, we undertook the assembly of pyranonaphthoquinones
1–4 and their related substances, as well as the evaluation of
their inhibitory activity against the dual-specificity of the pro-
tein phosphatase Cdc25A. We describe herein the synthesis of
(ꢁ)-2, with relatively simple substitutions, and the evaluation
of the related derivatives in the Cdc25A enzyme inhibition
assay.7
O
O
OH
O
CO H
2
MeHN
1
O
O
OH
O
CO H
2
MeHN
2
O
CO H
2
O
OH
S
Me
N
H
26
O
CO H
2
MeHN
O
3,4: isomers at the C-26 position
Results and Discussion
Fig. 1. Structures of Cdc25A phosphatase inhibitors.
Synthesis of Pyranonaphthoquinone (2). Based on the
retrosynthetic analysis of 2 (Scheme 1), the tricyclic com-
pound 13 might be easily converted to the natural substance
2. The pyran ring moiety of 13 can be constructed from the
functionalized derivative 12 by using the intramolecular
Michael addition. The naphthalene 6, which can be obtained
from 5-bromoveratraldehyde (5), can provide 12.
sulfonium methylide10 gave an epoxide. After isomerization of
the epoxide under ZnBr2/PhH conditions,11 the resultant alde-
hyde was subjected to the Horner–Wadsworth–Emmons olefi-
nation to furnish the ꢀ,ꢁ-unsaturated ester 9 in 78% yield from
8. A reduction with DIBAL produced the corresponding allyl
alcohol 10, which underwent silylation to give 11 in good
yield. The coupling reaction of 11 with valeraldehyde via bro-
mine–lithium exchange reaction, and then desilylation, provid-
ed 12. Oxidation to the naphthoquinone, followed by treatment
with MnO2, gave the ꢀ,ꢁ-unsaturated aldehyde, which was un-
expectedly converted into a cyclic aldehyde. The cyclic alde-
hyde was automatically transformed into a methyl ester, due
In our previous paper,7 5-bromoveratraldehyde (5) was ef-
fectively converted to the intermediate 6 (Scheme 2). Naphtha-
lene 6 was exposed to basic conditions8 and the successive
reduction to give 7. The regioselective bromination of 7 with
Pyr HBr39 was followed by oxidation and methylation to give
ꢂ
the aldehyde 8 in 76% yield. The reaction of 8 with dimethyl-
Published on the web October 9, 2004; DOI 10.1246/bcsj.77.1925

1926 Bull. Chem. Soc. Jpn., 77, No. 10 (2004)
Synthesis of Pyranonaphthoquinone
OBn OMe OH
O
OH
O
CO H
2
MeO
OH
MeHN
OMe
O
O
2
12
OBn OAc
CHO
OMe
O
MeO
CO Et
2
MeO
Br
CO Me
2
MeO
OMe
OMe
O
6
13
5
Scheme 1. Retrosynthetic analysis of 2.
CHO
OBn OAc
OBn OH
a, b
ref.7
OH
MeO
Br
MeO
CO Et
MeO
Br
2
OMe
OMe
OMe
5
6
7
OBn OMe
OBn OMe
Br
i
f - h
c - e
COOEt
MeO
CHO
MeO
OMe
OMe
8
9
OBn OMe
OBn OMe
Br
Br
j
k, l
MeO
OH
MeO
OTBS
OMe
OMe
10
11
OBn OMe OH
O
O
OH
m - q
O
MeO
OH
CO Me
2
OMe
MeO
12
13 (cis / trans 2:1)
O
O
OH
r
s
O
2 (cis / trans 4:1)
CO Me
2
MeHN
14 (cis / trans 1:3)
Scheme 2. Reagents and conditions: (a) K2CO3, EtOH, rt; (b) LiAlH4, THF, rt, 79% in 2 steps; (c) Pyr HBr3, THF, 0 ꢃC; (d)
ꢂ
ꢃ
SO3 Pyr., TEA, DMSO, CH2Cl2, rt; (e) MeI, K2CO3, DMF, rt, 76% in 3 steps; (f) Me3SI, NaH, DMSO, THF, 0 C; (g) ZnBr2,
ꢂ
ꢃ
ꢃ
PhH, reflux; (h) (EtO)2P(O)CH2CO2Et, NaH, THF, ꢄ78 C, 78% in 3 steps; (i) DIBAL, THF, ꢄ78 C, 93%; (j) TBSCl, Imid.,
DMF, rt, 96%; (k) valeraldehyde, n-BuLi, THF, ꢄ78 ꢃC; (l) TBAF, THF, 0 ꢃC, 80% in 2 steps; (m) DDQ, t-BuOH, aq. CH2Cl2, rt;
ꢃ
ꢃ
ꢃ
(n) MnO2, CH2Cl2, 40 C; (o) PDC, DMF, rt; (p) TMSCHN2, MeOH, rt; (q) BBr3, CH2Cl2, ꢄ78 C to 0 C, 13% from 12; (r)
ꢃ
MeNH2, MeOH, rt; (s) KOH, H2O, MeOH, 0 C to rt, 87%.
13
to the labile character of the ꢀ,ꢁ-unsaturated aldehyde. Expo-
sure of the methyl ester to BBr3 resulted in the selective
demethylation at C-6 in 13% yield from 12.12 Introduction
of an N-methyl group with MeNH2 provided the methyl-
amine 14,14 and the successive hydrolysis with KOH in MeOH
quantitatively afforded pyranonaphthoquinone (2), contami-

A. Shimbashi et al.
Bull. Chem. Soc. Jpn., 77, No. 10 (2004) 1927
active derivatives and other pyranonaphthoquinones such as
chloroquinocin.17 In addition, it was revealed that the pyran
ring system was not crucial for the expression of Cdc25A in-
hibition. The syntheses of related substances and more detailed
biological investigations are in progress.
O
O
OMe OH
a - c
12
MeO
OAc
15
Experimental
O
O
OH OH
General. IR spectra were recorded on a JASCO Model A-202
1
d, e
spectrophotometer. H NMR and 13C NMR spectra were obtained
MeHN
OH
on JEOL JNM EX-270 and JEOL JNM GX-400 spectrometers in
CDCl3 using tetramethylsilane as an internal standard, unless oth-
erwise stated. High-resolution mass spectra were obtained on a
Hitachi M-80 B GC–MS spectrometer operating at an ionization
energy of 70 eV. Melting points were measured on a Yanaco
MP-S3 and were uncorrected. Silica-gel column chromatography
was carried out using Kanto Chemical silica 60N (sherical, neu-
tral, 63–210 mm). Preparative and analytical thin-layer chromatog-
raphy (TLC) were carried out on silica-gel plates (Kieselgel 60
F254, E. Merck AG, Germany). The reaction was monitored by
UV (254 nm) light and/or stained with 5% phosphomolybdic acid
in ethanol as a developing agent, followed in the latter case by
heating on an electric plate. Work-up procedure: a mixture was
partitioned between EtOAc or CHCl3 and H2O. The organic layer
was washed with brine, dried (Na2SO4), and then evaporated.
8-Benzyloxy-3-hydroxymethyl-5,6-dimethoxy-1-naphthol
(7): A mixture of 6 (3.76 g, 8.9 mmol) and K2CO3 (12.7 g, 92
mmol) in EtOH (80 mL) was stirred at room temperature for 18
h. After evaporation, the work-up afforded a crude naphthol. A
mixture of the naphthol and LiAlH4 (0.70 g, 19 mmol) in THF
(40 mL) was stirred at room temperature for 30 min. After the ad-
dition of saturated aq. NH4Cl at 0 ꢃC, the mixture was worked up
and recrystallized from hexane–EtOAc to afford 7 (2.39 g, 79%)
as a yellow crystal: mp 131–132 ꢃC; IR (disk) 3386, 2941,
16
f, g
2
Scheme 3. Reagents and conditions: (a) Ac2O, Pyr.,
CH2Cl2, rt; (b) DDQ, t-BuOH, aq. CH2Cl2, 0 ꢃC; (c)
BBr3, CH2Cl2, ꢄ78 ꢃC, 16% from 12; (d) MeNH2,
THF, 0 ꢃC; (e) K2CO3, MeOH, rt, 87% in 2 steps; (f)
ꢃ
MnO2, CH2Cl2, 40 C; (g) PꢃDC, DMF, rt, 19% from 16;
Jones reagent, acetone, ꢄ20 C, 43% from 16.
Table 1. Inhibitory Activity of Cdc25A Phosphatase
Compound
Pyranonaphthoquinone (2)a)
IC50/mM
>50
>50
>50
13
13
14
16
b)
Na3VO4
0.005
2844, 1635, 1616 cmꢄ1 1H NMR ꢂ 3.89 (s, 3H), 3.94 (s, 3H),
;
a) 15.5% Inhibition at 10 mM concentration was reported in
Ref. 1. b) Positive control.
4.74 (s, 2H), 5.24 (s, 2H), 6.66 (s, 1H), 6.72 (s, 1H), 7.39–7.50
(complex, 6H), 9.25 (s, 1H); 13C NMR ꢂ 57.1, 61.0, 65.5, 72.1,
72.3, 96.5, 107.4, 109.6, 110.6, 127.9, 128.8, 129.0, 131.5,
134.9, 137.6, 137.7, 141.1, 148.0, 151.8, 154.7. HRMS calcd for
C20H20O5 (Mþ) 340.1311, found m=z 340.1305. Anal. Calcd for
C20H20O5: C, 70.57; H, 5.92%. Found: C, 70.48; H, 5.79%.
5-Benzyloxy-3-bromo-4,7,8-trimethoxynaphthalene-2-carb-
nated by the corresponding trans-isomer of substituents in the
benzopyran moiety (cis:trans = 4/1).15
To accomplish the efficient synthesis of 2, we planned to
construct the pyran ring moiety in the final stage, as shown
in Scheme 3. At first, the primary allyl alcohol of 12 was se-
lectively acetylated, followed by the oxidation and selective
demethylation to give p-quinone 15 in 16% yield from 12. In-
troduction of a methylamino group and then basic treatment
provided 16, which upon oxidation in two steps gave 2. Their
spectroscopic data was superimposable with those of the re-
ported data, which indicated the cis substitution in the pyran
moiety based on the NOE experiments.1,16
Biological Evaluation. The synthetic intermediates 13, 14,
and 16 were evaluated as inhibitors of the Cdc25A phospha-
tase, using 3-O-methylfluorescein phosphate (OMFP) as the
substrate. Their inhibitory activities are shown in Table 1.
Compound 16 showed a stronger activity than the natural sub-
stance 2 and its derivatives 13, 14. This result suggested that
the pyran ring system is not necessary for the inhibitory activ-
ity.
aldehyde (8): A mixture of 7 (2.42 g, 7.1 mmol) and Pyr HBr3
ꢂ
ꢃ
(2.52 g, 7.9 mmol) in THF (50 mL) was stirred at 0 C for 1 h.
After the addition of saturated aq. Na2S2O3, the work-up gave a
crude bromodiol. A mixture of the crude product, Et3N (5.9
mL, 43 mmol), and SO3 Pyr (3.38 g, 21 mmol) in CH2Cl2 (20
ꢂ
mL)–DMSO (18 mL) was stirred at room temperature for 20
min. After the addition of saturated aq. NH4Cl at 0 ꢃC, the
work-up afforded a crude aldehyde. A mixture of the aldehyde,
K2CO3 (3.93 g, 28 mmol), and MeI (0.88 mL, 14 mmol) in
DMF (30 mL) was stirred at room temperature for 1 h. The
work-up and purification by silica-gel column chromatography
(hexane–EtOAc = 1:1) afforded 8 (2.32 g, 76%) as a yellow
plate: mp 129–130 ꢃC (hexane/EtOAc); IR (disk) 2943, 2848,
1687, 1612, 1577 cmꢄ1 1H NMR ꢂ 3.80 (s, 3H), 3.95 (s, 3H),
;
3.96 (s, 3H), 5.20 (s, 2H), 6.91 (s, 1H), 7.38 (d, 1H, J ¼ 7:2
Hz), 7.43 (t, 2H, J ¼ 7:2 Hz), 7.56 (d, 2H, J ¼ 7:2 Hz), 8.50 (s,
1H), 10.50 (s, 1H); 13C NMR ꢂ 56.9, 61.5, 61.9, 62.0, 62.1,
72.8, 100.5, 103.3, 113.8, 119.6, 121.4, 127.7, 128.1, 128.6,
129.9, 131.5, 136.3, 138.9, 149.0, 151.0, 154.1, 192.2. HRMS
calcd for C21H1979BrO5 (Mþ) 430.0416, found m=z 430.0345.
In conclusion, we have accomplished a total synthesis of
(ꢁ)-pyranonaphthoquinone (2) using the intramolecular Mi-
chael addition for construction of the pyran ring system. This
synthetic route can be utilized for the synthesis of optically

1928 Bull. Chem. Soc. Jpn., 77, No. 10 (2004)
Synthesis of Pyranonaphthoquinone
Anal. Calcd for C21H19BrO5 0.2H2O: C, 58.00; H, 4.50%. Found:
C, 57.78; H, 4.44%.
2H, J ¼ 7:2 Hz), 7.56 (d, 2H, J ¼ 7:2 Hz), 7.73 (s, 1H); 13C NMR
ꢂ ꢄ5:0, 18.5, 25.7, 26.0, 39.6, 56.9, 61.3, 61.7, 63.7, 72.6, 99.9,
115.9, 116.8, 117.7, 127.6, 127.7, 127.9, 128.5, 130.6, 131.6,
135.3, 136.8, 137.0, 139.0, 148.2, 151.1. HRMS calcd for
C30H3979BrO5Si (Mþ) 586.1750, found m=z 586.1758. Anal.
Calcd for C30H39BrO5Si: C, 61.32; H, 6.69%. Found: C, 61.21;
H, 6.60%.
ꢂ
Ethyl (2E)-4-(5-Benzyloxy-3-bromo-4,7,8-trimethoxy-2-
naphthyl)but-2-enoate (9): A suspension of NaH (517 mg,
60% dispersion in mineral oil, 13 mmol) in DMSO (5 mL) was
stirred at room temperature for 2 h, then diluted with THF (50
mL). A solution of trimethylsulfonium iodide (2.75 g, 13 mmol)
in DMSO (10 mL) was slowly added at 0 ꢃC, and the mixture
was stirred below 5 ꢃC. After 40 min, a solution of 8 (1.89 g,
4.4 mmol) in DMSO (25 mL) was added at 0 ꢃC over 10 min. Af-
ter 45 min, saturated aq. NH4Cl was slowly added. The work-up
afforded a crude oxirane. ZnBr2 (2.68 g, 12 mmol) in PhH (60
mL) was refluxed for 40 min. After the addition of a solution of
the crude oxirane in PhH (40 mL), the mixture was heated at
the same temperature for 20 min. The work-up gave a crude prod-
uct. To a solution of triethyl phosphonoacetate (4.1 mL, 21 mmol)
in THF (40 mL) was added NaH (816 mg, 60% dispersion in min-
eral oil, 20 mmol) at 0 ꢃC. After 1.5 h, a solution of the above
mixture in THF (40 mL) was added at ꢄ78 ꢃC. After 25 min,
the reaction was quenched by the addition of saturated aq. NH4Cl
at 0 ꢃC and worked up. Purification by silica-gel column chroma-
tography (hexane–EtOAc = 3:1) afforded 9 (1.77 g, 78%) as a
pale yellow oil: IR (film) 2937, 2846, 1716, 1614, 1591, 1573
(2E)-4-(5-Benzyloxy-3-(1-hydroxypentyl)-4,7,8-trimethoxy-
2-naphthyl)but-2-en-1-ol (12): To a solution of 11 (1.06 g, 1.8
mmol) in THF (30 mL) were added n-BuLi (3.4 mL of a 1.57 M
solution in hexane, 5.3 mmol) and valeraldehyde (1.0 mL, 9.4
ꢃ
mmol) at ꢄ78 C all at once. After the addition of saturated aq.
NaHCO3, the work-up gave a crude product. To a solution of
the crude product in THF (20 mL) was added TBAF (9 mL of a
1 M solution in THF, 9 mmol) at 0 ꢃC. Stirring was continued
at room temperature for 1.5 h. After the addition of H2O at 0
ꢃC, the work-up and purification by silica-gel column chromatog-
raphy (hexane–EtOAc = 3:1) gave 12 (693 mg, 80%) as a yellow
oil: IR (film) 3408, 2933, 2858, 1620, 1574 cmꢄ1; 1H NMR ꢂ 0.92
(t, 3H, J ¼ 6:2 Hz), 1.37–1.39 (complex, 2H), 1.63–1.80 (com-
plex, 2H), 1.92–2.04 (complex, 2H), 3.57 (d, 2H, J ¼ 5:4 Hz),
3.80 (s, 3H), 3.90 (s, 3H), 3.93 (s, 3H), 4.09 (d, 2H, J ¼ 5:6
Hz), 4.99 (m, 1H), 5.11 (d, 1H, J ¼ 11 Hz), 5.19 (d, 1H, J ¼
11 Hz), 5.64 (dt, 1H, J ¼ 5:6, 15 Hz), 5.91 (dt, 1H, J ¼ 5:4, 15
Hz), 6.73 (s, 1H), 7.36 (d, 1H, J ¼ 7:2 Hz), 7.41 (t, 2H, J ¼ 7:2
Hz), 7.53 (d, 2H, J ¼ 7:2 Hz), 7.65 (s, 1H); 13C NMR ꢂ 14.2,
22.8, 29.2, 36.6, 39.0, 50.3, 56.9, 61.1, 63.4, 64.0, 71.1, 72.9,
100.3, 113.7, 115.1, 118.2, 127.4, 127.8, 128.4, 130.7, 131.0,
131.2, 136.8, 136.9, 137.0, 147.9, 151.1, 154.8, 163.6. HRMS
calcd for C29H36O6 (Mþ) 480.2512, found m=z 480.2493. Anal.
cmꢄ1
;
1H NMR ꢂ 1.27 (t, 3H, J ¼ 7:0 Hz), 3.77 (s, 3H), 3.82
(d, 2H, J ¼ 6:2 Hz), 3.90 (s, 3H), 3.95 (s, 3H), 4.18 (q, 2H, J ¼
7:0 Hz), 5.18 (s, 2H), 5.81 (d, 1H, J ¼ 16 Hz), 7.18 (dt, 1H,
J ¼ 6:2, 16 Hz), 7.36 (d, 1H, J ¼ 7:2 Hz), 7.42 (t, 2H, J ¼ 7:2
Hz), 7.56 (d, 2H, J ¼ 7:2 Hz), 7.74 (s, 1H); 13C NMR ꢂ 14.3,
39.5, 56.8, 60.2, 61.1, 61.8, 72.5, 100.1, 116.1, 116.3, 118.4,
122.7, 127.6, 127.9, 128.4, 130.3, 130.5, 136.4, 136.5, 136.7,
140.1, 145.7, 148.4, 151.0, 153.7, 166.2. HRMS calcd for
C26H2779BrO6 (Mþ) 514.0991, found m=z 514.0966. Anal. Calcd
for C26H27BrO6: C, 60.59; H, 5.28%. Found: C, 60.63; H, 5.33%.
(2E)-4-(5-Benzyloxy-3-bromo-4,7,8-trimethoxy-2-naphthyl)-
but-2-en-1-ol (10): To a solution of 9 (147 mg, 0.29 mmol) in
THF (3 mL) was added DIBAL (1.5 mL of a 1 M solution in tol-
Calcd for C29H36O6 0.2H2O: C, 71.94; H, 7.58%. Found: C,
71.78; H, 7.60%.
ꢂ
Ã
Ã
Methyl 2-((1R ,3R )-1-Butyl-3,4-dihydro-10-hydroxy-7-
methoxy-6,9-dioxobenzo[g]-1H-isochromen-3-yl)acetate (13):
To a solution of 12 (120 mg, 0.25 mmol) in CH2Cl2 (1 mL)
was added a suspension of DDQ (171 mg, 0.75 mmol) in t-BuOH
(1 mL)–H2O (1 mL) at 0 ꢃC. After 15 min, saturated aq. NaHCO3
was added, and the work-up afforded a crude naphthoquinone. A
mixture of the crude product and MnO2 (872 mg, 10 mmol) in
CH2Cl2 (5 mL) was heated at 40 ꢃC. After 3.5 h, precipitates were
filtered through a Celite pad and washed with CHCl3–MeOH, then
the filtrate and washings were then evaporated to give a residue,
ꢃ
uene, 1.5 mmol) at ꢄ78 C. After 35 min, 4 M HCl was slowly
added, the work-up and purification by silica-gel column chroma-
tography (hexane–EtOAc = 1:1) afforded 10 (125 mg, 93%) as a
pale yellow plate: mp 104–105 ꢃC (hexane/EtOAc); IR (disk)
1
3521, 2902, 2860, 1614, 1589, 1574 cmꢄ1; H NMR ꢂ 1.82 (brs,
1H), 3.66 (d, 2H, J ¼ 6:2 Hz), 3.77 (s, 3H), 3.90 (s, 3H), 3.94
(s, 3H), 4.13 (q, 2H, J ¼ 5:4 Hz), 5.17 (s, 2H), 5.75 (dt, 1H,
J ¼ 5:4, 16 Hz), 5.95 (dt, 1H, J ¼ 6:2, 16 Hz), 6.74 (s, 1H),
7.37 (d, 1H, J ¼ 7:2 Hz), 7.42 (t, 2H, J ¼ 7:2 Hz), 7.56 (d, 2H,
J ¼ 7:2 Hz), 7.73 (s, 1H); 13C NMR ꢂ 39.6, 56.8, 61.1, 61.7,
63.4, 63.5, 72.5, 99.8, 115.8, 116.6, 117.7, 127.6, 127.8, 128.3,
129.5, 129.6, 130.5, 131.0, 131.1, 136.7, 138.5, 148.2, 151.0,
153.3. HRMS calcd for C24H2579BrO5 (Mþ) 472.0885, found
m=z 472.0893. Anal. Calcd for C24H25BrO5: C, 60.90; H,
5.32%. Found: C, 60.95; H, 5.63%.
8-Benzyloxy-2-bromo-3-[(2E)-4-(t-butyldimethylsiloxy)-2-
butenyl]-1,5,6-trimethoxynaphthalene (11): A mixture of 10
(125 mg, 0.26 mmol), imidazole (180 mg, 2.6 mmol), and TBSCl
(215 mg, 1.4 mmol) in DMF (3 mL) was stirred at 0 ꢃC for 15
min. The work-up and purification by silica-gel column chroma-
tography (hexane–EtOAc = 3:1) afforded 11 (149 mg, 96%) as
a yellow oil: IR (film) 2931, 2894, 2854, 1614, 1591, 1576
which was passed through
a silica-gel column (hexane–
EtOAc = 1:1) to give a crude product. A mixture of the crude al-
dehyde and PDC (1.19 g, 3.2 mmol) in DMF (5 mL) was stirred at
room temperature for 21 h. The work-up gave a crude product. A
mixture of the crude product and TMSCHN2 (0.4 mL of a 2 M so-
lution in hexane, 0.8 mmol) in MeOH (1 mL) was stirred at room
temperature for 2 h. The work-up afforded a crude naphthopyran.
A mixture of the naphthopyran and BBr3 (0.1 mL of a 1 M solu-
tion in CH2Cl2, 0.1 mmol) in CH2Cl2 (1 mL) was stirred at ꢄ78
ꢃC for 20 min. The work-up and purification by preparative TLC
(hexane–EtOAc = 1:1) afforded 13 (12.6 mg, 13%) as a yellow
oil: 1H NMR ꢂ 0.92 (t, 3H, J ¼ 6:8 Hz), 1.25–1.58 (complex,
4H), 1.74–1.76 (m, 1H), 1.92–1.98 (m, 1H), 2.58–2.78 (complex,
4H), 3.74 (s, 3H), 3.91 (s, 3H), 3.91–3.97 (m, 1H), 4.38 (m, 1H),
6.06 (s, 1H), 7.41 (s, 1H), 12.6 (s, 1H). HRMS calcd for C21H24O7
(Mþ) 388.1522, found m=z 388.1532.
cmꢄ1
;
1H NMR ꢂ 0.07 (s, 6H), 0.90 (s, 9H), 3.65 (d, 2H, J ¼
Methyl 2-((1R ,3R )-1-Butyl-3,4-dihydro-10-hydroxy-7-
(methylamino)-6,9-dioxobenzo[g]-1H-isochromen-3-yl)acetate
(14): To a solution of 13 (3.9 mg, 10 mmol) in THF (1 mL) was
Ã
Ã
6:8 Hz), 3.76 (s, 3H), 3.89 (s, 3H), 3.94 (s, 3H), 4.19 (q, 2H, J ¼
5:2 Hz), 5.18 (s, 2H), 5.67 (dt, 1H, J ¼ 5:2, 15 Hz), 5.93 (dt, 1H,
J ¼ 6:8, 15 Hz), 6.74 (s, 1H), 7.36 (d, 1H, J ¼ 7:2 Hz), 7.42 (t,
ꢃ
added MeNH2 (20 mL, 40% MeOH solution, 0.3 mmol) at 0 C.

A. Shimbashi et al.
Bull. Chem. Soc. Jpn., 77, No. 10 (2004) 1929
After 30 min, the solvent was evaporated to afford crude 14:
1H NMR (C3D6O) ꢂ 0.82 (t, 3H, J ¼ 6:8 Hz), 1.18–1.82 (com-
plex, 5H), 2.16 (m, 1H), 2.47–2.93 (complex, 4H), 2.86 (d, 3H,
J ¼ 5:4 Hz), 3.55 (s, 3H), 3.83 (m, 1H), 4.26 (m, 1H), 5.43 (s,
1H), 7.17 (s, 1H), 7.17 (m, 1H), 13.8 (s, 1H). HRMS calcd for
C21H25NO6 (Mþ) 387.1682, found m=z 387.1658.
J ¼ 5:4 Hz), 3.43 (complex, 2H), 4.11 (d, 2H, J ¼ 5:2 Hz),
4.84 (m, 1H), 5.57 (s, 1H), 5.65 (dt, 1H, J ¼ 5:2, 15 Hz), 5.81
(dt, 1H, J ¼ 5:9, 15 Hz), 6.17 (s, 1H), 7.38 (s, 1H), 14.07 (s,
1H); 13C NMR ꢂ 14.2, 21.5, 22.7, 28.6, 29.3, 36.2, 36.3, 49.3,
49.4, 63.2, 70.9, 99.2, 121.0, 128.6, 131.8, 139.0, 149.6, 159.7,
188.8, 208.0. HRMS calcd for C20H25NO5 (Mþ + H) 360.1811,
found m=z 360.1800.
2-[(1R ,3R )-1-Butyl-3,4-dihydro-10-hydroxy-7-(methyl-
amino)-6,9-dioxobenzo[g]-1H-isochromen-3-yl]acetic Acid (2):
PDC oxidation. A mixture of 16 (9.9 mg, 28 mmol) and ꢃMnO2
(106 mg, 1.2 mmol) in CH2Cl2 (3 mL) was heated at 40 C for
2.5 h. The work-up gave a crude product. A mixture of the crude
product (4.4 mg, 12 mmol) and PDC (215 mg, 0.57 mmol) in DMF
(2 mL) was stirred at room temperature for 3 h. The work-up af-
forded a crude pyranonaphthoquinone 2 (1.9 mg, 19% from 16).
Jones oxidation. A mixture of the crude aldehyde and the
Jones reagent (ca. 1 M, excess amount) in acetone (0.7 mL) was
stirred at ꢄ20 ꢃC for 15 min. The work-up and purification by
preparative TLC (chloroform–methanol = 5:1) afforded pyrano-
naphthoquinone 2 (2.3 mg, 43% from 16).
Ã
Ã
2-[(1R ,3R )-1-Butyl-3,4-dihydro-10-hydroxy-7-(methyl-
amino)-6,9-dioxobenzo[g]-1H-isochromen-3-yl]acetic Acid (2):
A mixture of 14 (1.3 mg, 3.4 mmol) and KOH (1 mg, 17 mmol) in
H2O (0.3 mL) in MeOH (1 mL) was kept for 24 h. A solution of 1
M HCl was slowly added, and the work-up afforded 2 (1.9 mg,
Ã
Ã
87%): IR (film) 3355, 2924, 1710, 1612 cmꢄ1 1H NMR ꢂ 0.87
;
(t, 3H, J ¼ 6:8 Hz), 1.25–1.39 (complex, 4H), 2.15 (m, 1H),
2.78 (complex, 4H), 2.95 (d, 3H, J ¼ 5:2 Hz), 3.98 (m, 1H),
5.14 (m, 1H), 5.59 (s, 1H), 6.10 (m, 1H), 7.34 (s, 1H), 13.64 (s,
1H); 13C NMR (CD3COCD3) ꢂ 14.3, 23.3, 27.8, 34.4, 36.0,
40.8, 70.6, 75.1, 99.0, 113.3, 119.5, 129.3, 135.7, 142.7, 151.4,
159.1, 172.1, 181.6, 189.7. HRMS calcd for C20H23NO6 (Mþ)
1
373.1525, found m=z 373.1549. trans-isomer: H NMR ꢂ 0.94 (t,
3H, J ¼ 6:8 Hz), 1.25–1.39 (complex, 4H), 2.33 (m, 1H), 2.82
(complex, 4H), 2.95 (d, 3H, J ¼ 5:2 Hz), 4.40 (m, 1H), 5.05
(m, 1H), 5.59 (s, 1H), 6.10 (m, 1H), 7.34 (s, 1H), 13.50 (s, 1H);
13C NMR (CD3COCD3) ꢂ 14.3, 23.0, 27.4, 28.9, 32.6, 34.1,
41.1, 72.8, 79.2, 99.0, 114.8, 119.7, 129.1, 136.8, 140.4, 155.7,
164.7, 172.1, 182.7, 189.7.
Enzymatic Assay of Cdc25A Phosphatase Inhibition. Pro-
tein purification. Esherichia coli BL21 (DE-3) cells were trans-
formed with a plasmid encoding GST fusion of cdc25A. Cultures
ꢃ
were grown at 37 C to an A600 of 0.8, and isopropyl 1-thio-ꢁ-D-
galactopyranoside (IPTG) was added to a final concentration of
ꢃ
(2E)-4-(3-(1-Hydroxypentyl)-4,7-dimethoxy-5,8-dioxo(2-
naphthyl)(but-2-enyl Acetate (15): A mixture of 12 (111 mg,
0.23 mmol), pyridine (36 mL, 0.46 mmol), and Ac2O (15 mL,
0.16 mmol) in CH2Cl2 (5 mL) was stirred at room temperature
for 2 days. The work-up afforded a crude secondary alcohol. A
mixture of the crude secondary alcohol in CH2Cl2 (5 mL) and
DDQ (109 mg, 0.48 mmol) in t-BuOH (2 mL)–H2O (2 mL) was
stirred at 0 ꢃC for 10 min. After the addition of saturated aq.
NaHCO3, the mixture was worked up to afford a crude naphtho-
quinone. To a solution of BBr3 (0.20 mL of a 1 M solution in
CH2Cl2, 0.20 mmol) in CH2Cl2 (3 mL) was added a solution of
the crude naphthoquinone in CH2Cl2 (5 mL) at ꢄ78 ꢃC. After
20 min, the work-up and purification by preparative TLC (hex-
ane–EtOAc = 1:2) afforded 15 (13.8 mg, 16%) as a yellow oil
and the naphthoquinone (25.8 mg, 29%). 15: IR (film) 3534,
0.1 mM. After growing for an additional 4 h at 37 C, the cells
were collected by centrifugation. Cell pellets were suspended in
lysis buffer (MT-PBS (150 mM NaCl, 20 mM Na2HPO4, 4 mM
NaH2PO4), 0.1% tritonX-100, 10 mg/mL leupeptin, 1 mM phenyl-
methanesulfonyl fluoride) and lysed by sonication. GST-cdc25A
was precipitated with GSH-agarose beads (sigma, St. Louis,
MO.), washed four times with MT-PBS, and eluted by elution buf-
fer (50 mM Tris–HCl (pH 8.0), 20 mM reduced glutathione). The
amount of GST-cdc25A was estimated by comparison with a BSA
standard after SDS-PAGE and Coomasie blue staining.
Cdc25A assay in vitro. The activity of the GST-cdc25A was
measured in a 96-well microtiter plate using the substrate OMFP
(sigma), which is readily metabolized to the fluorescent O-methyl
fluorescein. The inhibitors were resuspended in methanol, and all
reactions including controls were performed at a final concentra-
tion of 1% methanol. Approximately 30 mg of purified GST-
2956, 1739, 1683, 1625 cmꢄ1
;
1H NMR ꢂ 0.90 (t, 3H, J ¼ 6:8
ꢃ
Hz), 1.33–1.42 (complex, 4H), 1.50–1.76 (complex, 2H), 2.05
(s, 3H), 3.50 (d, 2H, J ¼ 5:8 Hz), 3.92 (s, 3H), 4.52 (d, 2H, J ¼
6:2 Hz), 4.86 (m, 1H), 5.59 (dt, 1H, J ¼ 5:8, 15 Hz), 5.87 (dt,
1H, J ¼ 6:2, 15 Hz), 6.07 (s, 1H), 7.48 (s, 1H), 13.11 (s, 1H);
13C NMR ꢂ 14.1, 21.0, 22.7, 28.6, 36.3, 36.4, 49.2, 49.3, 56.7,
64.4, 70.6, 109.3, 121.7, 126.8, 129.0, 131.6, 138.4, 144.4,
cdc25A was preincubated at 25 C for 15 min in reaction buffer
consisting of 50 mM Tris–HCl (pH 7.5) and various concentra-
tions of inhibitors, the reaction was started by the addition of 25
mM OMFP. The fluorescence emission from the product was
measured over a 30 min reaction period with a maltiwell plate
reader (Fluoroskan Ascent FL: Labsystem). The excitation wave-
length was 355 nm, and emission was selected using a 538 nm fil-
ter. All experiments were done in triplicate. The reaction was lin-
ear over the time used in the experiments and was directly propor-
tional to both the enzyme and substrate concentration.
159.5, 161.0, 190.7, 206.5. HRMS calcd for C22H26O7 (Mþ
+
H) 403.1757, found m=z 403.1732. Anal. Calcd for
C22H26O7 0.2H2O: C, 65.08; H, 6.55%. Found: C, 65.10; H,
6.45%.
ꢂ
7-[(2E)-4-Hydroxybut-2-enyl]-5-hydroxy-6-(1-hydroxypen-
tyl)-2-(methylamino)-1,4-naphthoquinone (16): A mixture of
15 (3.9 mg, 10 mmol) and MeNH2 (20 mL, 40% MeOH solution,
This work was supported by a Grant-in-Aid for the 21st
Century COE program ‘‘Keio Life Conjugated Chemistry’’,
as well as Scientific Research C from the Ministry of Educa-
tion, Culture, Sports, Science and Technology.
ꢃ
0.28 mmol) in THF (1 mL) was stirred at 0 C for 30 min. The
solvents were then evaporated to afford a crude (aminomethyl)-
quinone. A mixture of the crude product and K2CO3 (20 mg,
0.14 mmol) in MeOH (1 mL) was stirred at room temperature
for 40 min. The work-up and purification by preparative TLC
(EtOAc) afforded 16 (3.1 mg, 87%) as a yellow oil: IR (film)
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;
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1H NMR ꢂ 0.90 (t, 3H, J ¼ 6:8 Hz),
2

1930 Bull. Chem. Soc. Jpn., 77, No. 10 (2004)
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8
The combined yield in two steps (K2CO3/EtOH, then
LiAlH4/THF) was better than a one-step procedure using only