Xanthouroleuconaphin: A yellowish pigment from the aphid Uroleucon nigrotuberculatum and its total synthesis

Article abstract of DOI:10.1016/j.tet.2012.12.070

A novel yellowish pigment, xanthouroleuconaphin, was isolated from the aphid Uroleucon nigrotuberculatum. Its structure was established by detailed analyses of 1D and 2D NMR spectra and EI-HRMS. Furthermore, the total synthesis of (±)-xanthouroleuconaphin was accomplished via oxidative phenolic coupling of 6-hydroxymusizin, which was prepared by the Fries rearrangement using BF₃·2AcOH complex.

Full text of DOI:10.1016/j.tet.2012.12.070

Tetrahedron 69 (2013) 1808e1814
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Xanthouroleuconaphin: a yellowish pigment from the aphid
Uroleucon nigrotuberculatum and its total synthesis
,
a
a
a
Taichi Nishimura a, Mitsuyo Horikawa a , Kouji Yamada , Ayaka Sogabe , Takeshi Nishii ,
*
Hiroto Kaku a, Makoto Inai a, Masami Tanaka a, Shigeru Takahashi b, Tetsuto Tsunoda a
,
*
a Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
b Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A
novel yellowish pigment, xanthouroleuconaphin, was isolated from the aphid Uroleucon ni-
Received 27 September 2012
Received in revised form 26 December 2012
Accepted 28 December 2012
Available online 4 January 2013
grotuberculatum. Its structure was established by detailed analyses of 1D and 2D NMR spectra and EI-
HRMS. Furthermore, the total synthesis of (ꢀ)-xanthouroleuconaphin was accomplished via oxidative
phenolic coupling of 6-hydroxymusizin, which was prepared by the Fries rearrangement using
BF3$2AcOH complex.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Structure determination
Aphid pigment
Xanthouroleuconaphin
Uroleucon nigrotuberculatum
Total synthesis
Phenolic coupling
1. Introduction
established as C26H22O8 by EI-HRMS (m/z 462.1317, [M]þ). The IR
spectrum of
1 indicated the presence of hydroxy groups
Aphids produce novel pigments, such as protoaphins,1 fur-
anaphin,2 uroleuconaphins,3,4 viridaphin A1 glucoside,5 and
megouraphin glucosides,6 which may possess various biological
activities, including cytotoxicity.2,3,5,7 In our previous papers, we
reported that the red aphid Uroleucon nigrotuberculatum that feeds
on Solidago altissima produced mainly two red pigments, uroleu-
conaphins A1 and B1,3 and four yellowish pigments, uroleucona-
phins A2a, A2b, B2a, and B2b (Fig. 1).4 As part of our continuing efforts
toward determination of chemical structures of pigments in aphids,
we reinvestigated the aphid U. nigrotuberculatum and isolated
a yellow pigment, named xanthouroleuconaphin (1), as a minor
component of the aphid (Fig. 1). Herein we describe the structural
elucidation and the total synthesis of (ꢀ)-1 in detail.
(3340 cmꢂ1, br) and intramolecularly hydrogen-bonded carbonyl
groups (1624 cmꢂ1). The 13C NMR spectrum of 1 clearly exhibited
only 13 carbon resonances, which were classified as 2 methyls, 2
methines, and 9 quaternary carbon atoms by using distortionless
enhancement by polarization transfer (DEPT) 13C NMR analysis. In
the 1H NMR spectrum, three hydroxy protons (15.34 in acetone-d6,
10.52 and 8.34 ppm in diethyl ether-d10) were observed and the
proton resonating at 15.34 ppm was hydrogen-bonded presumably
with a carbonyl group. The heteronuclear multiple quantum co-
herence (HMQC) spectra of 1 (Table 1) revealed the presence of two
methyl carbons [C 20.1/H 2.13 (6H, s) ppm] and [C 32.1/H 2.66 (6H,
s) ppm] and two aromatic carbons [C 101.8/H 5.84 (2H, d) ppm] and
[C 102.4/H 6.30 (2H, d) ppm]. The 1H NMR spectrum of compound 1
was very similar to that of 6-hydroxymusizin (2) (Fig. 2), which was
isolated from the aphid Aphis spiraecola.2,8
2. Results and discussion
From the mass spectrum of 1 and its peracetate (C38H34O14, m/z
714 [M]þ), the structure of 1 was suggested as a dimer of 2. In fact,
the presence of six acetyl groups in the peracetate was confirmed
by observing six MS-fragments at m/z 462 (Mþꢂ42ꢃ6), 504
(Mþꢂ42ꢃ5), 546 (Mþꢂ42ꢃ4), 588 (Mþꢂ42ꢃ3), 630 (Mþ-42ꢃ2),
and 672 (Mþꢂ42). To reveal the position of dimerization of 2, 2D
NMR (NOESY and HMBC) experiments of 1 were carried out in
diethyl ether-d10 as a solvent (Fig. 3).9 In the NOESY experiments,
Compound 1 was isolated from the ether extract of U. nigrotu-
berculatum and purified by silica gel column chromatography as
a yellow-hued solid, mp 268 ꢁC (dec). Its molecular formula was
* Corresponding authors. Tel.: þ81 88 602 8452; fax: þ81 88 655 3051; e-mail
addresses: horikawa@ph.bunri-u.ac.jp (M. Horikawa), tsunoda@ph.bunri-u.ac.jp
(T. Tsunoda).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.12.070

T. Nishimura et al. / Tetrahedron 69 (2013) 1808e1814
1809
O
OH
O
OH
O
OH OH
O
5
4
10
O
R
6
3
11
O
O
OH
9
2
O
HO
HO
HO
HO
HO 7
1
8
8'
1'
4'
2'
3'
R
R
OH
9'
O
O
O
O
7'
6'
HO
HO
11'
O OH
O
HO
5'
10'
HO
O
O
O
OH OH
H
H
OH
O
OH O
H
O
OH
1
uroleuconaphin A1: R = H
uroleuconaphin B1: R = OH
uroleuconaphin A2a: R = H
uroleuconaphin B2a: R = OH
uroleuconaphin A2b: R = H
uroleuconaphin B2b: R = OH
Fig. 1. Structures of uroleuconaphins and xanthouroleuconaphin (1).
a correlation between H-8 and H-9 was observed in compound 1,
but not in 2. Finally, since HMBC measurements suggested that the
carbon at the C-1 position bore no proton, we determined that
two molecules of 2 should bind each other at C-1 to form dimeric
1, whose specific rotation was determined to be a plus value,
Table 1
13C (150 MHz) and 1H (600 MHz) NMR data of 1 in diethyl ether-d10
Position
dC, mult.
dH (J in Hz)
1, 10
127.8, C
133.6, C
115.8, C
166.3, C
108.5, C
161.1, C
102.4, CH
162.5, C
101.8, CH
140.1, C
20.1, CH3
203.5, C
32.1, CH3
2, 20
[a
]
22 þ18.5 (c 0.98, MeOH). Although 1 should be an atropisomeric
3, 30
D
4, 40
isomer, the absolute configuration of it could not be determined.
To validate the structural determination of 1 and continue our
investigations into the biological activities of 1, including antiviral,
antibacterial, and anti-fungal activities, we conducted a total syn-
thesis of 1. Our retrosynthetic analysis of 1 is illustrated in
Scheme 1. A phenolic coupling of protected 6-hydroxymusizin 3
would be a key step in the synthesis of 1. Fries rearrangement of
acetate 4, which could be afforded by Michael addition of sulfone 5
to ethyl crotonate (6) followed by intramolecular cyclization, would
be used to prepare methyl ketone 3.
4a, 40a
5, 50
6, 60
6.30, d (2.5)
5.84, d (2.5)
7, 70
8, 80
8a, 80a
9, 90
2.13,a
s
10, 100
11, 110
4, 40 eOH
5, 50 eOH
7, 70 eOH
2.66, s
15.34,b brs
10.52, s
8.34, s
For the preparation of rearrangement precursor 4 (Scheme 1),
we chose sulfone 710 as a starting material, which was obtained by
the reaction of 3,5-dimethoxybenzylbromide with PhSO3Na in DMF
in 95% yield. The sulfone 7 was subjected to Michael addition11 with
ethyl crotonate (6) under basic conditions followed by hydrolysis to
give carboxylic acid 8 in 88% yield (two steps, Scheme 2). The
conversion of 8 to the naphthalene skeleton was accomplished by
treatment with trifluoroacetic acid anhydride (TFAA) in 1,2-
dichloroethane (1,2-DCE) under reflux conditions followed by
a
This signal was overlapped with acetone-d6.
This signal was observed in acetone-d6 by 500 MHz NMR.
b
OH
OH
O
aromatization by b-elimination of sulfonyl group with t-BuOK to
HO
afford 1012 in 86% yield (two steps). The acetylation of 10 with
acetic anhydride in pyridine gave rearrangement precursor 1113 in
satisfactory yield.
2
Fig. 2. Structure of 6-hydroxymusizin (2).
With the desired precursor 11 in hand, we undertook the Fries
rearrangement of acetate 11 using 10 equiv of BF3$2AcOH in CH2Cl2
heated to reflux for 1 h to obtain rearranged product 1214e17 in 89%
yield. The cleavage of methyl ethers on 12 was achieved with BBr3
to furnish 6-hydroxymusizin (2) (87%),18e20 which was acetylated
selectively with acetic acid anhydride in CH2Cl2 at ꢂ40 ꢁC to give
diacetate 13.
OH
OH
O
CH3
We made several attempts to accomplish phenolic coupling of 13
using oxidizing agents such as FeCl3, K3Fe(CN)6, Mn(acac)3, and ceric
ammonium nitrate (CAN), and we found that CAN in MeCN gave the
best results in the regioselective oxidative dimerization of 13 at the
C-4 position to afford the desired product 14 along with keto-type
tautomer 15 (14/15z1:1).21 Previous reports in the literature in-
dicated that keto-type tautomers were generated in the phenolic
coupling22 and they were converted easily to the phenol-type tau-
tomer in a polar solvent such as EtOH.22b,c,e,23 So, after the coupling
reaction using CAN, we added water and EtOH into the reaction
mixture and obtained 14 in 63% yield by chromatographic purifi-
cation. Finally, the acetyl groups were removed with a 6 M aqueous
HO
CH3
H
8
H
H3C9
OH
H
H3C11
O
OH
OH
Fig. 3. HMBC (dotted line) and NOESY (solid line) correlations of 1 in diethyl ether-d10
.

1810
T. Nishimura et al. / Tetrahedron 69 (2013) 1808e1814
O
PGO HO
O
PGO
O
1
PGO
PGO
3
4
PGO
O
+
OEt
PGO
SO2Ph
5
6
Scheme 1. Retrosynthesis of xanthouroleuconaphin (1).
1) n-BuLi
MeO
MeO
O
ethyl crototate (6)
THF, –78 °C
OH
2) 2 M KOH
MeOH
MeO
MeO
SO2Ph
SO2Ph
88% (2 steps)
8
7
MeO
O
MeO
OH
TFAA
t-BuOK
1,2-DCE
THF, reflux
92%
MeO
MeO
reflux, 94%
SO2Ph
9
10
O
MeO
O
MeO
OH
O
BF3·2AcOH
Ac2O
CH2Cl2, reflux
89%
Pyridine
98%
MeO
MeO
11
12
OH OH
O
AcO
OH
O
Ac2O, Pyridine
BBr3
CH2Cl2, –40 °C
70 %
CH2Cl2
87%
HO
AcO
4
13
6-hydroxymusizin (2)
Scheme 2. Synthesis of 6-hydroxymusizin (2) and coupling precursor 13.

T. Nishimura et al. / Tetrahedron 69 (2013) 1808e1814
1811
NaOH solution in THF to give (ꢀ)-xanthouroleuconaphin (1)
(Scheme 3). All spectroscopic data (1H, 13C NMR, IR, and HRMS) for
the synthetic 1 agreed with the isolated pigment 1 in this study.
Furthermore, the LC analysis of hexamethyl ether of natural and
UV-1650pc spectrophotometer. IR spectra were measured on
a JASCO FT/IR-410 spectrophotometer. 1H NMR spectra were
recorded on Varian Unity-600 (600 MHz), Unity-500 (500 MHz),
Unity-400 (400 MHz), and Mercury-300 (300 MHz) NMR spec-
AcO
OH
O
AcO
O
O
CAN
AcO
AcO
H
+
13
H
OAc
OAc
MeCN
O
HO
OAc
O
O
OAc
14
15
H2O/EtOH/MeCN
OH OH
O
6 M NaOH
HO
14
OH
THF
quant.
O
HO HO
( )-xanthouroleuconaphin (1)
Scheme 3. Total synthesis of (ꢀ)-xanthouroleuconaphin (1).
synthetic 1 using a chiral column (CHIRALPAKÒ IB & IC) revealed that
the natural 1 was a racemic mixture (62:38). The comparison of CD
spectra of natural 1 with that of singucanol24 suggested that the
absolute configuration of major enantiomer of natural 1 should be S.
trometers with TMS as an internal standard in solvent. 13C NMR
spectra were recorded with Varian Unity-600 (150 MHz), Unity-
500 (125 MHz), and Varian Unity-400 (100 MHz) NMR spectrom-
eters; chemical shifts were referenced to the residual solvent signal
(diethyl ether-d10: dC 14.5, CDCl3: dC 77.0, acetone-d6: dC 29.8).
3. Conclusion
Signal multiplicities were established with DEPT experiments. Mass
spectra including HRMS were recorded on a JEOL JMX AX-500, JMS-
700 spectrophotometers. For column chromatography, stationary
The structure of xanthouroleuconaphin (1) isolated from the
aphid U. nigrotuberculatum was determined by detailed analyses of
1D and 2D NMR spectra and EI-HRMS. Furthermore, we have
achieved the total syntheses of (ꢀ)-1, 6-hydroxymusizin (2) and its
dimethyl ether 12 by utilizing the Fries rearrangement, followed by
phenolic coupling. Further work on the biological activities of
compounds 1, 2, and 12 are under investigation in our laboratory
and will be reported in due course.
phases of silica gel (Kanto Chemical Co., Inc., 60N 63e210 mm, Fuji
Silysia Chemical LTD., BW-127ZH and BW-300) were used. For the
TLC analysis, Merck pre-coated silica gel plates (60F) were used.
Acetyl chloride, 4-(N,N-dimethylamino)pyridine (DMAP), N,N-dii-
sopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), ethyl
crotonate, 1,2-dichloroethane, pyridine, acetonitrile (MeCN), and
ceric ammonium nitrate (IV) (CAN) were purchased from Nacalai
Tesque Inc. n-BuLi 1.5 M Hex soln, t-BuOK, tetrahydrofuran (THF,
dehydrated stabilizer free: super plus), and CH2Cl2 (dichloro-
methane, dehydrated: super) were purchased from Kanto
Chemical Co., Inc. Trifluoroacetic acid (TFA) and acetic anhydride
(Ac2O) were purchased from Wako Pure Chemical Industries, Ltd.
BBr3 (1.0 M CH2Cl2 soln) was purchased from Tokyo Chemical
Industry Co., Ltd. BF3$2AcOH was purchased from SigmaeAldrich
Co. All of these solvents and reagents were used without any
purification.
4. Experimental section
4.1. General information
Melting points were determined on a Yanaco MP-3 apparatus
€
and Buchi Melting Point B-545 device and are uncorrected.
Optical rotations were obtained on JASCO DIP-1000 and P-1030
polarimeters. UVevisible spectra were measured on a Shimadzu

1812
T. Nishimura et al. / Tetrahedron 69 (2013) 1808e1814
4.2. Material
129.9, 128.8, 128.7, 108.6, 101.1, 63.0, 55.3; IR lmax (neat, cmꢂ1):
1594, 1474, 1446, 1429; EIMS m/z (%): 292 [M]þ, 228, 151 (100), 77;
EI-HRMS m/z: 292.0771 (calcd for C15H16O4S 292.0769).
The aphid U. nigrotuberculatum feeding on S. altissima was col-
lected in Tokushima Prefecture, Japan in June 1999.
4.4.4. Preparation of 8. n-BuLi (1.5 M in hexane, 5.3 mL, 7.9 mmol)
was added dropwise to a solution of 7 (1.92 g, 6.57 mmol) in THF
(30 mL) at ꢂ78 ꢁC under Ar atmosphere, and the reaction mixture
was stirred for 30 min at the same temperature. After dropwise
4.3. Extraction and isolation
The red aphid U. nigrotuberculatum was removed from the plant
with a soft paintbrush and collected into a plastic Erlenmeyer flask
equipped a plastic funnel in June in Tokushima Prefecture, Japan.
The aphids (200 g) were crushed in diethyl ether (with a mortar),
and then the ethereal supernatant solution was separated by de-
cantation. The residue was washed with several portions of fresh
ether (total: 3 L). The combined ethereal solutions were dried over
Na2SO4 and were evaporated to give a crude extract (21.9 g). The
reddish residue was subjected to silica gel column chromatography
(600 g, n-hexane/EtOAc¼3:1 to 1:3) to give four fractions. The
fraction 2 (3.2 g) was applied to silica gel column chromatography
(150 g, n-hexane/EtOAc¼4:1 to 0:1) as an eluent to afford the red
pigment uroleuconaphin A1 (1.2 g) and a mixture of yellow pig-
ments (780 mg). These yellow pigments were subjected to repeated
chromatographic purification over silica gel (n-hexane/EtOAc¼4:1
to 0:1) to afford 1 (82 mg) and a mixture of uroleuconaphin A2a and
A2b (210 mg).
addition of ethyl crotonate (6) (815 mL, 6.57 mmol), the resulting
mixture was stirred for 30 min at ꢂ78 ꢁC and quenched with sat-
urated aqueous NH4Cl (30 mL) at the same temperature. The mix-
ture was extracted with diethyl ether (30 mLꢃ3), and the combined
organic extracts were washed with brine (60 mL), dried over
Na2SO4, filtered, and concentrated.25 The crude residue (2.92 g) was
dissolved in MeOH (10 mL) and treated with a 2 M KOH aqueous
solution (6 mL) at ambient temperature. After hydrolysis of ester
was completed, solvents were removed under reduced pressure,
and then diethyl ether (40 mL) was added to the residue, which was
extracted with H2O (30 mLꢃ3). The combined aqueous extracts
were acidified with 6 M HCl aqueous solution and extracted with
diethyl ether (30 mLꢃ3), and then the combined organic extracts
were washed with brine (60 mL), dried over Na2SO4, filtered, and
concentrated to give analytically pure pale yellow amorphous
solids of 8 (mixture of two diastereomers, major/minor¼64:36) in
88% yield (two steps). Major diastereomer of 8:25 1H NMR
4.4. Spectroscopic data of 1 and synthesis
(400 MHz, CDCl3):
d
7.57 (2H, dd, J¼8.4, 1.2 Hz), 7.47 (1H, dddd,
J¼7.6, 7.6, 1.2, 1.2 Hz), 7.33 (2H, dd, J¼7.6, 7.6 Hz), 6.28 (3H, s), 4.05
(1H, d, J¼8.4 Hz), 3.66 (6H, s), 3.18e3.10 (1H, m), 2.66 (1H, dd,
J¼16.0, 3.2 Hz), 2.14 (1H, dd, J¼16.0, 8.8 Hz), 1.43 (3H, d, J¼6.8 Hz).
4.4.1. Xanthouroleuconaphin (1). Yellow solid; mp 268 ꢁC (dec); Rf
22
0.34 (SiO2, n-hexane/EtOAc¼1:2); [
a]
þ18.5 (c 0.98, MeOH); UV
D
(CH3CN) lmax (log
3
): 234 (4.60), 275 (4.68), and 398 (4.11); CD
13C NMR (100 MHz, CDCl3):
d 177.5, 160.6, 138.7, 134.4, 133.1, 128.5,
D
3
þ19.4 and
D
3
ꢂ21.1 (c 3.11ꢃ10ꢂ5, CH3CN); 1H NMR
128.4, 108.1, 100.7, 75.2, 55.3, 38.7, 30.9, 19.0. Minor diastereomer of
267nm
235nm
(600 MHz, diethyl ether-d10) and 13C NMR (150 MHz, diethyl ether-
d10) data (Table 1); IR lmax (ATR, cmꢂ1): 3340 and 1624; EIMS m/z:
462 [M]þ; EI-HRMS m/z: 462.1317 (calcd for C26H22O8, 462.1315).
8:25 1H NMR (400 MHz, CDCl3):
d
7.57 (2H, dd, J¼8.4, 1.2 Hz), 7.48
(1H, dddd, J¼7.6, 7.6, 1.2, 1.2 Hz), 7.34 (2H, dd, J¼7.6, 7.6 Hz), 6.31
(3H, s), 4.22 (1H, d, J¼8.0 Hz), 3.68 (6H, s), 3.18e3.08 (1H, m), 2.91
(1H, dd, J¼17.2, 5.6 Hz), 2.72 (1H, dd, J¼16.8, 6.8 Hz), 1.10 (3H, d,
4.4.2. Hexaacetate of 1. To a suspension of 1 (11 mg) in CH2Cl2
J¼6.8 Hz); 13C NMR (100 MHz, CDCl3):
d 177.2, 160.5, 138.4, 133.9,
(17 mL) with acetyl chloride (400
(w20 mgꢃ8) and DIPEA (10 L) with stirring at ambient temper-
ature. After 15 min, the resulting mixture was quenched with water
(20 L). The mixture was separated and the organic layer was dried
mL) were added DMAP
133.2, 128.6, 128.5, 108.3, 100.7, 73.2, 55.3, 38.9, 30.0, 18.1. Mixture
of 8; IR lmax (ATR, cmꢂ1): 1707, 1594, 1460, 1447, 1429;1 EIMS m/z
(%): 378 [M]þ, 277, 237 (100), 219, 191, 151; EI-HRMS m/z: 378.1131
(calcd for C19H22O6S 378.1137).
m
m
over Na2SO4. After evaporation of the solvent, the crude residue
was purified by silica gel column chromatography (3.5 g, n-hexane/
EtOAc¼3:1 to 1:1) to give 5.2 mg of hexaacetate of 1 as a colorless
4.4.5. Preparation of 9. To a solution of a diastereomeric mixture of
8 (9.8 g, 25.9 mmol) in 1,2-dicholoroethane was added trifluoro-
acetic anhydride (4 mL, 28.5 mmol) at ambient temperature, and
the reaction mixture was refluxed for 1 h. After neutralization with
a saturated aqueous solution of NaHCO3 (70 mL) at 0 ꢁC, the
resulting mixture was extracted with CH2Cl2 (70 mLꢃ3). The
combined organic extracts were dried over Na2SO4, filtered, and
concentrated. The residue was purified by silica gel column chro-
matography (n-hexane/EtOAc¼1:1) to give a mixture of two di-
astereomers of 9 in 94% yield (major/minor¼61:39) as pale yellow
amorphous solids. Major diastereomer of 9:25 1H NMR (400 MHz,
solid; mp 124e130 ꢁC (n-hexane/EtOAc). UV (MeOH) lmax (log
3
):
7.08 (2H, d,
J¼2.2 Hz), 6.74 (2H, d, J¼2.2 Hz), 2.56 (6H, s), 2.42 (6H, s), 2.39
(6H, s), 2.22 (6H, s), 1.89 (6H, s); 13C NMR (150 MHz, CDCl3):
203.3,
232 (4.88), 302 (4.14) nm. 1H NMR (600 MHz, CDCl3):
d
d
169.0, 168.8, 168.6, 149.2, 146.3, 141.2, 135.0, 134.6, 133.3, 132.1,
118.0, 117.2, 115.0, 32.3, 21.2, 21.2, 20.9, 17.2; IR lmax (neat, cmꢂ1):
1772, 1707, 1628 (C]O); EIMS m/z (%): 714 [M]þ, 672, 630, 588,
546(100), 504, 462; EI-HRMS m/z: 714.1932 (calcd for C38H34O14
,
714.1949).
CDCl3):
d
7.71 (2H, dd, J¼8.4, 1.2 Hz), 7.66 (1H, dddd, J¼7.6, 7.6, 1.2,
4.4.3. Preparation of 7. To
a
solution of 3,5-dimethoxyben-
1.2 Hz), 7.51 (2H, dd, J¼7.6, 7.6 Hz), 6.48 (1H, d, J¼2.4 Hz), 5.88 (1H,
d, J¼2.4 Hz), 4.10 (1H, s), 3.89 (3H, s), 3.62 (3H, s), 3.28 (1H, dd,
J¼17.8, 6.4 Hz), 3.15e3.08 (1H, m), 2.31 (1H, ddd, J¼17.2, 1.6, 1.6 Hz),
zylbromide (496.3 mg, 2.15 mmol) in DMF (3 mL) was added
benzene sulfinic acid sodium salt (388.5 mg, 2.37 mmol) with
stirring at ambient temperature. After 2 h, 30 mL of water was
added at 0 ꢁC and the resulting mixture was extracted with CH2Cl2
(20 mLꢃ2). The combined organic extracts were washed with brine
(30 mL), dried over Na2SO4, filtered, and concentrated. The residue
was purified by silica gel column chromatography (n-hexane/
EtOAc¼2:1) to give sulfone 7 in 95% yield as colorless needles; mp
1.08 (3H, d, J¼7.2 Hz), 13C NMR (100 MHz, CDCl3):
d 192.8, 163.2,
162.2, 137.4, 135.8, 134.0, 129.2, 129.0, 116.4, 108.4, 100.1, 72.0, 56.1,
55.3, 42.7, 27.5, 20.9. Minor diastereomer of 9:25 1H NMR (400 MHz,
CDCl3):
d
7.61e7.55 (3H, m), 7,42 (2H, dd, J¼8.0, 8.0 Hz), 6.39 (1H, d,
J¼2.0 Hz), 5.52 (1H, d, J¼2.0 Hz), 4.25 (1H, d, J¼3.6 Hz), 3.87 (3H, s),
3.47 (3H, s), 3.12 (1H, dd, J¼18.8, 13.2 Hz), 2.85e2.74 (1H, m), 2.63
(1H, ddd, J¼18.6, 6.4, 1.2 Hz), 1.59 (3H, d, J¼7.2 Hz); 13C NMR
108.8 ꢁC (n-hexane/EtOAc). 1H NMR (400 MHz, CDCl3):
d 7.68 (2H,
dd, J¼8.0, 1.2 Hz), 7.61 (1H, dddd, J¼7.6, 7.6, 1.2, 1.2 Hz), 7.47 (2H, dd,
(100 MHz, CDCl3): d 194.1, 162.7, 161.9, 140.1, 138.9, 133.7, 129.0,
J¼7.6, 7.6 Hz), 6.39 (1H, dd, J¼2.2, 2.2 Hz), 6.19 (2H, d, J¼2.4 Hz),
128.7, 116.7, 106.8, 99.9, 71,3, 56.1, 55.2, 43.1, 33.1, 19.1. Mixture of 9;
4.24 (2H, s), 3.67 (6H, s); 13C NMR (100 MHz, CDCl3):
d 137.8, 133.6,
IR lmax (ATR, cmꢂ1): 1667, 1598, 1456; EIMS m/z (%): 360 [M]þ, 219

T. Nishimura et al. / Tetrahedron 69 (2013) 1808e1814
1813
(100), 191, 77; EI-HRMS m/z: 360.1029 (calcd for C19H20O5S
195.0e200.0 ꢁC (dec) (n-hexane/EtOAc). 1H NMR (400 MHz, ace-
360.1031).
tone-d6): d 17.47 (1H, s), 10.46 (1H, s), 9.13 (1H, s), 6.82 (1H, s), 6.53
(1H, d, J¼2.4 Hz), 6.37 (1H, d, J¼2.0 Hz), 2.73 (3H, s), 2.58 (3H, s); 13C
4.4.6. Preparation of 10. To a solution of diastereomeric mixture of
9 (8.8 g, 24.4 mmol) in THF (150 mL) was added t-BuOK (13.7 g,
122 mmol) at ambient temperature under Ar atmosphere, and the
mixture was refluxed for 3 h. After acidification with a 2 M HCl
aqueous solution at 0 ꢁC, the resulting mixture was extracted with
diethyl ether (100 mLꢃ3), and combined organic extracts were
dried over Na2SO4, filtered, and concentrated. The residue was
purified by silica gel column chromatography (n-hexane/
EtOAc¼5:1) to give 10 in 92% yield as colorless needles; mp
NMR (100 MHz, acetone-d6): d 204.8, 169.8, 162.4, 161.0, 141.1, 135.7,
121.0, 113.3, 108.0, 102.5, 101.9, 31.7, 24.8; IR lmax (neat, cmꢂ1):
3369, 1632, 1575, 1402; EIMS m/z (%): 232 [M]þ, 217 (100), 115;
EI-HRMS m/z: 232.0735 (calcd for C13H12O4 232.0736).
4.4.10. Preparation of 13. To a suspension of 6-hydroxymusizin (2)
(49.9 mg, 0.214 mmol) in CH2Cl2 (1 mL) were added pyridine (35 mL,
0.434 mmol) and acetic anhydride (44
m
L, 0.466 mmol) at ꢂ40 ꢁC
under Ar atmosphere. After stirring for 24 h at the same temper-
ature, the reaction mixture was treated with a saturated aqueous
NH4Cl solution (3 mL) at ꢂ40 ꢁC and extracted with EtOAc
(15 mLꢃ3). The combined organic extracts were washed with brine
(40 mL), dried over MgSO4, filtered, and concentrated. The residue
was purified by silica gel column chromatography (n-hexane/
EtOAc¼5:1 to 2:1) to give 13 in 70% yield as yellow needles; mp
85.8e86.0 ꢁC (n-hexane/CH2Cl2); 1H NMR (400 MHz, CDCl3):
d 9.02
(1H, s), 6.98 (1H, dddd, J¼0.4, 0.4, 0.4, 0.4 Hz), 6.92 (1H, d, J¼2.0 Hz),
6.57 (1H, d, J¼1.6 Hz), 6.37 (1H, d, J¼2.0 Hz), 3.99 (3H, s), 3.87 (3H,
s), 2.38 (3H, s); 13C NMR (100 MHz, CDCl3):
d 157.7, 157.1, 154.2,
138.4, 137.5, 117.5, 110.3, 108.9, 98.8, 96.7, 56.0, 55.2, 21.6; IR lmax
(neat, cmꢂ1): 3402, 1634, 1577, 1443; MS (EI): m/z (%): 218 ([M]þ,
100), 175, 132; EI-HRMS: m/z: 218.0937 (calcd for C13H14O3
218.0943).
129.1 ꢁC (EtOAc). 1H NMR (400 MHz, CDCl3):
d 15.02 (1H, s), 7.29
(1H, d, J¼2.0 Hz), 7.02 (1H, s), 6.86 (1H, d, J¼2.0 Hz), 2.70 (3H, s),
2.65 (3H, s), 2.37 (3H, s), 2.33 (3H, s); 13C NMR (100 MHz, CDCl3):
4.4.7. Preparation of 11. To a solution of 10 (4.9 g, 22.5 mmol) in
pyridine (45 mL) was added acetic anhydride (22 mL) at ambient
temperature. After stirring for 9 h at ambient temperature, the
reaction mixture was treated with H2O (130 mL) at 0 ꢁC and
extracted with diethyl ether (130 mLꢃ3). The combined organic
d 205.7, 169.9, 168.6, 163.7, 151.2, 149.9, 138.6, 135.2, 121.1, 116.2,
115.9, 115.5, 114.7, 33.0, 25.0, 21.24, 21.20; IR lmax (neat, cmꢂ1):
1769, 1628, 1579, 1469; EIMS m/z (%): 316 [M]þ, 274, 232 (100), 217,
83, 43; EI-HRMS m/z: 316.0952 (calcd for C17H16O6 316.0949).
extracts were washed with
a
0.5
M
HCl aqueous solution
4.4.11. Preparation of 14. To a solution of 13 (49.0 mg, 0.154 mmol)
in CH3CN (1 mL) was added CAN (174.3 mg, 0.318 mmol) at ambient
temperature. After stirring for 5 min, the reaction mixture was
treated with saturated aqueous Na2S2O3 (3 mL) at ambient
temperature and extracted with EtOAc (5 mLꢃ3). The combined
organic extracts were washed with H2O (5 mLꢃ2) and brine
(10 mL), dried over MgSO4, filtered, and concentrated. The residue
(54.7 mg) was dissolved in a mixture of CH3CN, EtOH, and H2O
(1.0 mL, 1/0.5/0.5), and the resulting mixture was stirred for 16 h at
ambient temperature. After concentration and addition of brine
(10 mL), the resulting mixture was extracted with EtOAc (5 mLꢃ3).
The combined organic extracts were dried over MgSO4, filtered, and
concentrated. The residue was purified by silica gel column chro-
matography (CHCl3/EtOAc¼15:1) to give dimer 14 in 63% yield (two
steps) as yellow solids; mp 231.8 ꢁC (dec). 1H NMR (400 MHz,
(260 mLꢃ2), a saturated aqueous NaHCO3 (260 mL), and brine
(260 mL), dried over MgSO4, filtered, and concentrated. The residue
was purified by silica gel column chromatography (n-hexane/
EtOAc¼3:1) to give 11 in 98% yield as colorless needles; mp 102.6 ꢁC
(n-hexane/EtOAc). 1H NMR (400 MHz, CDCl3):
d 7.34 (1H, dddd,
J¼1.2, 0.4, 0.4, 0.4 Hz), 6.75 (1H, dd, J¼1.2, 0.4 Hz), 6.66 (1H, d,
J¼2.4 Hz), 6.43 (1H, d, J¼2.4 Hz), 3.87 (6H, s), 2.42 (3H, d, J¼0.4),
2.34 (3H, s); 13C NMR (100 MHz, CDCl3):
d 170.3, 158.2, 156.2, 146.3,
137.8, 136.7, 124.6, 119.0, 113.0, 98.5, 98.3, 55.9, 55.2, 21.2, 21.0; IR
lmax (neat, cmꢂ1): 1753, 1613, 1584; EIMS m/z (%): 260 [M]þ, 218
(100), 175; EI-HRMS m/z: 260.1041 (calcd for C15H16O4 260.1049).
4.4.8. Preparation of 12. To a solution of acetate 11 (816.4 mg,
3.14 mmol) in CH2Cl2 (5 mL) was added BF3$2AcOH (4.4 mL,
31.6 mmol) at ambient temperature under Ar atmosphere. After
refluxing for 1 h, the reaction mixture was treated with H2O
(20 mL) at 0 ꢁC and extracted with EtOAc (15 mLꢃ3). The combined
organic extracts were washed with a saturated aqueous NaHCO3
(20 mL) and brine (20 mL), dried over MgSO4, filtered, and con-
centrated. The residue was purified by silica gel column chroma-
tography (n-hexane/EtOAc¼10:1) to give 12 in 89% yield as pale
yellow needles; mp 103.8e104.0 ꢁC (n-hexane/EtOAc). 1H NMR
CDCl3):
2.67 (6H, s), 2.42 (6H, s), 2.20 (6H, s), 2.11 (6H, s); 13C NMR
(100 MHz, CDCl3): 206.4, 170.0, 168.4, 160.8, 151.4, 149.9, 138.0,
d
14.02 (2H, s), 6.94 (2H, d, J¼2.0 Hz), 6.69 (2H, d, J¼2.0 Hz),
d
134.4, 126.8, 118.2, 115.7, 115.3, 114.9, 33.0, 21.2, 21.1, 20.9; IR lmax
(neat, cmꢂ1): 1765, 1616, 1578, 1391; FABMS m/z (%) 653 [MþNa]þ,
546, 463, 154 (100), 136, 43; FAB-HRMS m/z 653.1644 (calcd for
C34H30O12Na 653.1634).
Keto-type tautomer 15: 1H NMR (300 MHz, CDCl3):
d 6.76 (2H, d,
(400 MHz, CDCl3):
d
9.78 (1H, s), 6.96 (1H, d, J¼0.8 Hz), 6.59 (1H, d,
J¼2.1 Hz), 6.71 (2H, d, J¼2.1 Hz), 3.92 (2H, s), 2.31 (6H, s), 2.30
(6H, s), 2.26 (6H, s), 2.23 (6H, s); CIMS m/z (%): 630 ([M]þ, 100);
CI-HRMS m/z: 630.1758 (calcd for C34H30O12 630.1737).
J¼2.0 Hz), 6.41 (1H, d, J¼2.4 Hz), 4.01 (3H, s), 3.87 (3H, s), 2.61 (3H,
s), 2.35 (3H, d, J¼1.2 Hz); 13C NMR (100 MHz, CDCl3):
d 205.1, 158.9,
157.7, 153.4, 137.6, 135.1, 122.4, 119.1, 108.7, 98.8, 97.5, 56.2, 55.3,
32.3, 20.4; IR lmax (neat, cmꢂ1): 3338, 1669, 1622, 1588, 1450; EIMS
m/z (%): 260 [M]þ, 245 (100), 230; EI-HRMS m/z: 260.1055 (calcd for
C15H16O4 260.1049).
4.4.12. Preparation of (ꢀ)-xanthouroleuconaphin (1). To a solution
of 14 (30.9 mg, 0.049 mmol) in THF (1 mL) was added a 6 M NaOH
aqueous solution (0.5 mL) at 0 ꢁC. After stirring for 5 min at the
same temperature, the reaction mixture was poured into a 6 M HCl
aqueous solution (2 mL) with ice and extracted with diethyl ether
(10 mLꢃ3). The combined organic extracts were washed with H2O
(10 mLꢃ3) and brine (20 mL), dried over MgSO4, filtered, and
concentrated. The residue was analytically pure yellow solids of
(ꢀ)-xanthouroleuconaphin (1) (quantitative yield); mp 260.0 ꢁC
4.4.9. Preparation of 6-hydroxymusizin (2). To a solution of 12
(260 mg, 1.00 mmol) in CH2Cl2 (5 mL) was added dropwise BBr3
1.0 M CH2Cl2 soln (3 mL) at 0 ꢁC under Ar atmosphere. After stirring
for 24 h at ambient temperature, the reaction mixture was treated
with H2O (20 mL) at 0 ꢁC and extracted with EtOAc (50 mLꢃ3). The
combined organic extracts were washed with H2O (60 mL) and
brine (60 mL), dried over MgSO4, filtered, and concentrated. The
residue was purified by silica gel column chromatography (n-hex-
ane/EtOAc¼3:1) to give 12 in 97% yield as yellow platelets; mp
(dec). 1H NMR (500 MHz, diethyl ether-d10):
d 16.18 (2H, br s), 10.46
(2H, br s), 8.32 (2H, br s), 6.30 (2H, d, J¼2.0 Hz), 5.83 (2H, d,
J¼2.0 Hz), 2.66 (6H, s), 2.14 (6H, s); 13C NMR (125 MHz, diethyl
ether-d10):
d 203.5, 166.7, 162.5, 161.2, 140.1, 133.6, 127.8, 115.5,

1814
T. Nishimura et al. / Tetrahedron 69 (2013) 1808e1814
108.4, 102.4, 101.7, 32.0, 20.2; IR lmax (ATR, cmꢂ1): 3338, 1622; EIMS
m/z (%): 462 ([M]þ, 100), 447, 420, 405, 216; EI-HRMS m/z: 462.1316
(calcd for C26H22O8 462.1315).
14. Nishimura, T.; Iwata, T.; Maegawa, H.; Nishii, T.; Matsugasako, M.; Kaku, H.;
Horikawa, M.; Inai, M.; Tsunoda, T. Synlett 2012, 1789e1792.
15. The dimethyl ether 12 was a natural compound isolated from Cortinarius rufo-
olivaceus and Karwinskia humboldtiana (Rhamnaceae). See: (a) Gao, J.-M.; Qin,
J.-C.; Pescitelli, G.; Pietro, D. S.; Ma, Y.-T.; Zhang, A.-L. Org. Biomol. Chem. 2010, 8,
3543e3551; (b) Mitscher, L. A.; Gollapudi, S. R.; Oburn, D. S.; Drake, S. Phyto-
chemistry 1985, 24, 1681e1683.
Acknowledgements
16. Several syntheses of 12 had been reported. See Ref. 12e and (a) Chevenier, E.;
Lucatelli, C.; Pandya, U.; Wang, W.; Gimbert, Y.; Greene, A. E. Synlett 2004,
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1053e1059; (c) Dodd, J. H.; Garigipati, R. S.; Weinreb, S. M. J. Org. Chem. 1982,
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This work was supported partially by a Grant-in-Aid for Scien-
tific Research (C) from MEXT (the Ministry of Education, Culture,
Sports, Science and Technology of Japan). We are also thankful to
the MEXT-Supported Program for the Strategic Research Founda-
tion at Private Universities, 2008e2012.
17. All spectroscopic data (1H NMR, IR, and HRMS) for the synthetic 12 agreed with
those reported previously in the literature. See Refs. 15b and 16e.
Supplementary data
18. A total synthesis of 2 through compound 13 using a biomimetic strategy had
been reported. See: Harris, T. M.; Wittek, P. J. J. Am. Chem. Soc. 1975, 97,
3270e3271.
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2012.12.070.
19. All spectroscopic data (1H, 13C NMR, IR, and HRMS) for the synthetic 2 agreed
with those reported previously in the literature. See Refs. 2 and 20q.
20. As natural products, several 6-hydroxymusizin derivatives were isolated. See
Ref. 16e and (a) Wang, Z.-W.; Wang, J.-S.; Luo, J.; Wei, D.-D.; Kong, L.-Y. Chem.
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€
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