Synthesis of ustalic acid, an inhibitor of Na+,K+-ATPase

Article abstract of DOI:10.1016/j.tet.2008.04.051

Ustalic acid, an inhibitor of Na⁺,K⁺-ATPase isolated from a poisonous mushroom, was synthesized in 8?steps using the Suzuki-Miyaura coupling and oxidation of methylene acetal as key steps.

Full text of DOI:10.1016/j.tet.2008.04.051

Tetrahedron 64 (2008) 5873–5877
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Synthesis of ustalic acid, an inhibitor of Naþ,Kþ-ATPase
*
Ichiro Hayakawa, Hidekazu Watanabe, Hideo Kigoshi
Department of Chemistry, Graduate School of Pure and Applied Sciences, and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tennodai,
Tsukuba 305-8571, Japan
a r t i c l e i n f o
a b s t r a c t
Ustalic acid, an inhibitor of Naþ,Kþ-ATPase isolated from a poisonous mushroom, was synthesized in
8 steps using the Suzuki–Miyaura coupling and oxidation of methylene acetal as key steps.
Ó 2008 Elsevier Ltd. All rights reserved.
Article history:
Received 5 March 2008
Received in revised form 11 April 2008
Accepted 14 April 2008
Available online 3 May 2008
1. Introduction
Synthesis of the ustalic acid (1) started from commercially
available sesamol (Scheme 2). Sesamol was transformed into cat-
Ustalic acid (1) was isolated from a poisonous mushroom,
Tricholoma ustale (Kakishimeji in Japanese), by Kawagishi et al. in
2002 (Fig. 1).1 Ustalic acid (1) inhibited Naþ,Kþ-ATPase; IC50 values
of ustalic acid (1) against the commercially available enzyme pu-
rified from porcine cerebral cortex and the crude enzyme from
mouse intestinal mucosal cells were 5.2 and 0.77 mM, respectively.
In 2006, Nishikawa et al. first achieved the total synthesis of ustalic
acid dimethyl ester (2).2 We planned an efficient synthesis of ustalic
acid (1), which will provide a practical supply for further biological
studies. We report here the first synthesis of ustalic acid (1) in 8
steps using Suzuki–Miyaura coupling3 as a key step. Recently,
Takahashi et al. have reported the total synthesis of a similar
compound vialinin A, by a similar cross-coupling strategy.4
echol 7,5 and the hydroxyl group of 7 was then protected as an
acetonide. The introduction of two boronic acid moieties into 8 was
accomplished through a double lithiation using n-BuLi and TMEDA
at ꢀ78 ꢁC, and trapping with B(OMe)3 to give diboronic acid 9 and
monoboronic acid 10.
We tried the Suzuki–Miyaura coupling3 with diboronic acid 9
and iodobenzene (Scheme 3). The coupling reaction with Pd(PPh3)4
and Na2CO3 in dioxane afforded diphenyl compound 11 in one step,
but the yield was low (25%). In contrast, the cross-coupling reaction
of monoboronic acid 10 with iodobenzene proceeded by the same
conditions to give monophenyl compound 12 in 71% yield.
O
O
O
O
2. Results and discussions
HO2C CO2H
O
O
Our synthetic plan of ustalic acid (1) is shown in Scheme 1. Our
synthetic route to ustalic acid (1) involved the Suzuki–Miyaura
coupling3 at C-2–C-10. We therefore synthesized organoboron
compound 5.
phlebiarubrone (3)
ustalic acid (1)
O
O
O
R1O
O
O
R2O
OR2
OR2
2
1'
OR1
O
O
B
B
R2O
2
4'
R1O
OR1
1'
4
RO2C CO2R
1
R = H; ustalic acid (1)
R = Me (2)
5
O
O
O
Figure 1. Structures of ustalic acid (1) and its derivative.
R1O
OR1
OH
sesamol
Scheme 1. Retrosynthetic analysis of ustalic acid (1).
6
* Corresponding author. Tel.: þ81 29 853 4313.
E-mail address: kigoshi@chem.tsukuba.ac.jp (H. Kigoshi).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.04.051

5874
I. Hayakawa et al. / Tetrahedron 64 (2008) 5873–5877
Table 1
O
O
O
O
O
O
O
O
Study of the removal of the acetonide group in 11
ref. 5
a
O
O
O
O
conditions
HO
OH
HO
7
sesamol
O
8
O
O
O
O
O
O
O
11
HO
OH
OH
HO
HO
b
O
O
O
O
B
B
B
HO
O
O
O
O
9
10
phlebiarubrone (3)
Scheme 2. Synthesis of organoboronic acids. Reagents and conditions: (a) PPTS, iso-
propenyl methyl ether, benzene, reflux, 96%, (b) n-BuLi, TMEDA, B(OMe)3, THF, Et2O,
ꢀ78 ꢁC to rt, 9: 33%, 10: 24%.
15
O
O
Compound 12 was converted into boronic acid 13 by lithiation
followed by treatment with B(OMe)3. The coupling reaction of 13
with iodobenzene was subjected to the same conditions to give
diphenyl compound 11.
HO
OH
14
Entry
Conditions
Yield (%)
The investigation of removal of the acetonide group in 11 is
summarized in Table 1. Acidic hydrolysis of 11 gave phlebiarubrone
(3), an oxidative compound of catechol 14 (entry 1), which was
isolated from the culture of the fungus Phlebia strigozonata.6 Syn-
thetic phlebiarubrone (3) gave spectral data (1H NMR, 13C NMR, and
HRMS) in full agreement with those of the natural one.7 Because
catechol 14 readily accepted air oxidation, 14 was not isolated.
Because of the irreproducible yield, we tried to synthesize ortho-
quinone 3 by selective oxidation (entries 2–5). The reaction of 11
with DDQ gave only the undesired ortho-quinone 15 (entry 2). The
oxidation by ammonium cerium(IV) nitrate (CAN) afforded the
desired ortho-quinone 3 (12% yield) and the undesired ortho-qui-
none 15 (35% yield) (entry 3). The reactions at low temperature
increased the selectivity of ortho-quinone 3 (entries 4 and 5).
However, we could not satisfy the yield and selectivity in this
transformation; therefore, we next tried oxidation of protected
catechol by two methylene acetal groups.
14
3
15
11
1
2
3
4
5
6 M HCl, THF–MeOH, 100 ꢁC
DDQ, CH2Cl2–H2O, rt, 2 days
CAN, MeCN–H2O, 0 ꢁC, 5 min
CAN, MeCN–H2O, ꢀ20 ꢁC, 5 min
CAN, MeCN–H2O, ꢀ40 ꢁC, 10 min
0
w59
0
0
37
35
47
25
0
50
0
0
0
d
d
d
d
12
31
27
Next, we attempted a Suzuki–Miyaura coupling,3 as depicted in
Table 2. A cross-coupling reaction between diboronic acid 17 and
iodobenzene with Pd(PPh3)4 or PdCl2(dppf) afforded monophenyl
compound 22 and bis-methylene acetal 16 (entries 1 and 2). An
attempt at a cross-coupling reaction of the diboronic acid 17 with
PdCl2(PPh3)2 and Cs2CO3 in DMF at room temperature gave the
desired diphenyl compound 21, but the yield was low (8%) (entry 3).
O
O
O
O
a
b
The hydroxyl groups of catechol 7 were protected by the second
methylene acetal (Scheme 4).8 The bis-methylene acetal 16 was
converted to diboronic acid 17 by double lithiation followed by
treatment with B(O-iPr)3. Another borate reagent, B(OMe)3, was
less effective in this case. Furthermore, the bis-methylene acetal 16
was converted to boronate 19 and pinacolborane 20 by sequential
boronation and esterification.
HO
OH
O
O
7
16
O
O
O
O
O
O
HO
HO
OH
HO
HO
B
B
B
OH
O
O
O
17
18
O
O
O
O
O
O
O
O
HO
HO
OH
OH
a
O
B
B
O
O
O
c
B
B
d
O
O
O
O
O
9
11
19
O
O
O
O
O
O
O
O
O
O
O
HO
OH
OH
b
c
O
O
O
O
B
B
d
B
B
HO
O
O
O
10
12
13
20
Scheme 3. Study of Suzuki–Miyaura coupling. Reagents and conditions: (a) PhI,
Pd(PPh3)4, Na2CO3, dioxane, rt, 25%; (b) PhI, Pd(PPh3)4, Na2CO3, dioxane, rt, 71%; (c)
n-BuLi, TMEDA, B(OMe)3, THF, Et2O, ꢀ78 ꢁC to rt, 49%; (d) PhI, Pd(PPh3)4, Na2CO3,
dioxane, rt, 40%.
Scheme 4. Synthesis of boronate compounds. Reagents and conditions: (a) CH2Br2,
Cs2CO3, DMF, 90 ꢁC, 46% (from sesamol); (b) n-BuLi, TMEDA, B(O-iPr)3, Et2O, ꢀ78 ꢁC to
rt, 17: 55%, 18: 12%, recovered 16: 21%; (c) trimethylene glycol, toluene, reflux; (d)
pinacol, MgSO4, CH2Cl2, rt.

I. Hayakawa et al. / Tetrahedron 64 (2008) 5873–5877
5875
Table 2
Study of Suzuki–Miyaura coupling
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
PhI
conditions
RO
RO
OR
OR
B
B
21
22
16
17: R = H
19: R, R =
20: R, R =
Me2C CMe2
Entry
Substrate
Conditions
Catalyst
Yield (%)
Solvent
Base
Temp (ꢁC)
21
22
16
1
2
3
17
17
17
17
19
20
Pd(PPh3)4
1,4-Dioxane
DME
DMF
DMF
DMF
Na2CO3 aq
K3PO4 aq
Cs2CO3 aq
Cs2CO3 aq
Cs2CO3
rt
60
rt
90
rt
90
0
0
8
30
43
62
16
43
13
18
16
31
8
11
4
32
0
PdCl2(dppf)
PdCl2(PPh3)2
PdCl2(PPh3)2
PdCl2(PPh3)2
PdCl2(PPh3)2
4
5a
6a
DMF
Cs2CO3
3.2
a
Isolated yields calculated from 16 (in 3 steps).
The reaction at 90 ꢁC afforded the desired diphenyl compound 21 in
30% yield along with monophenyl compound 22 (18%) and bis-
methylene acetal 16 (4%) (entry 4). The cross-coupling reaction of
the boronate 19 with PdCl2(PPh3)2 and Cs2CO3 in DMF afforded the
desired diphenyl compound 21 in 43% yield (entry 5). Treatment of
pinacolborane 20 under the same conditions gave the desired
diphenyl compound 21 in 62% yield (entry 6). In entries 5 and 6, the
crude boronic esters 19 and 20 were employed, and the yields were
calculated from 16 in 3 steps. Thus, this cross-coupling was most
efficiently effected by using pinacolborane 20. Also, monophenyl
compound 22 was transformed into the desired diphenyl com-
pound 21 by the same reactions (Scheme 5).
O
O
O
O
b
a
O
O
O
O
O
O
phlebiarubrone (3)
21
O
O
c
MeO2C CO2Me
2
HO2C CO2Me
ustalic acid monomethyl ester (23)
Since the diphenyl compound 21 has been synthesized in an
available yield, we attempted oxidation of 21. Oxidation of 21 with
CAN gave phlebiarubrone (3) in quantitative yield (Scheme 6). To
convert phlebiarubrone (3) to ustalic acid dimethyl ester (2), we
followed the procedure reported by Nishikawa et al.2 with a mod-
ification. Phlebiarubrone (3) was treated with Pb(OAc)4 (20 equiv)
in MeOH and toluene in the presence of K2CO3 (2.1 equiv) to give
ustalic acid dimethyl ester (2). This modification increased the yield
of the ustalic acid dimethyl ester (2) to 11%. Hydrolysis of the ustalic
acid dimethyl ester (2) with 3 M KOH aq in DMSO at room tem-
perature for 1 day afforded the ustalic acid monomethyl ester (23).
The monomethyl ester 23 was treated under the same conditions
for 4 days to give ustalic acid (1) in 38% yield. The reaction at 70 ꢁC
for 39 h gave ustalic acid (1) in 23% yield. Synthetic ustalic acid (1)
gave spectral data (1H NMR, 13C NMR, IR, and HRMS) in full
agreement with those of the natural one,1 thus completing the total
synthesis.
e
O
O
d
HO2C CO2H
ustalic acid (1)
Scheme 6. Synthesis of ustalic acid (1). Reagents and conditions: (a) CAN, MeCN–H2O,
0 ꢁC, quant.; (b) Pb(OAc)4, K2CO3, toluene, MeOH, rt, 11%; (c) 3 M KOH aq, DMSO, rt; (d)
3 M KOH aq, DMSO, rt, 38% in 2 steps; (e) 3 M KOH aq, DMSO, 70 ꢁC, 23%.
3. Conclusion
In summary, we achieved the first synthesis of ustalic acid (1) by
using the Suzuki–Miyaura coupling as a key step. Further struc-
ture–activity relationship studies are now in progress.
4. Experimental
O
O
O
O
O
O
O
O
4.1. General methods
a-c
1H NMR spectra were recorded on a JEOL JNM-EX270 (270 MHz)
or a Bruker AVANCE 500 (500 MHz) spectrometer. Chemical shifts
for 1H NMR are reported in parts per million (ppm) downfield
from tetramethylsilane as the internal standard and coupling con-
stants are in hertz (Hz). The following abbreviations are used
for spin multiplicity: s¼singlet, d¼doublet, t¼triplet, q¼quartet,
m¼multiplet, and br¼broad. 13C NMR spectra were recorded on
22
21
Scheme 5. Synthesis of diphenyl compound 21 from monophenyl compound 22. Re-
agents and conditions: (a) n-BuLi, TMEDA, B(O-iPr)3, Et2O, ꢀ78 ꢁC to rt; (b) pinacol,
MgSO4, CH2Cl2, rt; (c) PhI, PdCl2(PPh3)2, Cs2CO3, DMF, 90 ꢁC, 27% in 3 steps (recovered
16: 27%).

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I. Hayakawa et al. / Tetrahedron 64 (2008) 5873–5877
a JEOL JNM-EX270 (67.8 MHz) or a Bruker AVANCE 500 (125 MHz)
spectrometer. Chemical shifts for 13C NMR are reported in parts
per million relative to the central line of a triplet at 77.0 ppm for
deuteriochloroform. IR spectra were recorded on a JASCO FT/IR-300
instrument and are reported in wavenumbers (cmꢀ1). ESI mass
spectra were recorded on a Applied Biosystems QStar/Pulsar i
spectrometer. Elemental analyses were recorded on a Yanaco CHN
CORDER MT-6. TLC analysis was conducted on E. Merck precoated
silica gel 60 F254 (0.25 mm layer thickness). Fuji Silysia silica gel
BW-820 MH was used for column chromatography unless other-
wise noted. Organic solvents for moisture-sensitive reactions were
distilled from the drying agents: THF, Et2O, DME, and 1,4-dioxane
(Na-benzophenone ketyl), benzene and toluene (Na). Anhydrous
acetone, MeOH, CH2Cl2, and DMF were purchased from Kanto
Chemical Co., Inc. or Wako Pure Chemical Industries, Ltd., and used
without further drying. All moisture-sensitive reactions were per-
formed under an atmosphere of argon or nitrogen and the starting
materials were azeotropically dried with benzene before use. All
new compounds were determined to be >95% pure by 1H NMR
unless otherwise noted.
under nitrogen flow and diluted with H2O (10 mL) at room tem-
perature. The resultant mixture was filtrated with Celite, and this
Celite was rinsed with Et2O (3ꢂ10 mL). The layers were separated
and the aqueous layer was extracted with Et2O (3ꢂ15 mL). The
combined Et2O layers were washed with 1 M HCl (ꢂ2), H2O (ꢂ1),
and brine (ꢂ1), dried (Na2SO4), and concentrated. The residual solid
was purified by recrystallization from n-hexane–CH2Cl2 to give
diphenyl compound 21 (451 mg, 59% in 3 steps) as colorless crystals.
The mother liquid was concentrated, and the residual solid was
purified by recycle HPLC [JAIGEL-1H-40 (600ꢂ20 mm) and JAIGEL-
2H-40 (600ꢂ20 mm); flow rate 3.8 mL/min; detection UV 254 nm;
solvent CHCl3] to give diphenyl compound 21 (21.7 mg, 2.8% in 3
steps; total 473 mg, 62% in 3 steps) and monophenyl compound 22
(18.7 mg, 3.2% in 3 steps) as colorless crystals, respectively. Diphenyl
compound 21. Mp 204–205 ꢁC (n-hexane–CH2Cl2); 1H NMR
(270 MHz, CDCl3)
d
7.82–7.86 (m, 4H), 7.42–7.48 (m, 4H), 7.31–7.37
139.0,131.2,128.8,
(m, 2H), 6.00 (s, 4H); 13C NMR (67.8 MHz, CDCl3)
d
128.2, 127.8, 107.8, 100.8. Anal. Calcd for C20H14O4: C, 75.46; H, 4.43;
O, 20.10%. Found: C, 75.19; H, 4.57; O, 20.24%. Monophenyl com-
pound 22. Mp 138–139 ꢁC (n-hexane–CH2Cl2); 1H NMR (270 MHz,
CDCl3)
d
7.78–7.83 (m, 2H), 7.41–7.47 (m, 2H), 7.31–7.37 (m,1H), 6.49
141.4, 138.7, 131.2,
4.2. Bis-methylene acetal 16
(s, 1H), 5.93 (s, 4H); 13C NMR (67.8 MHz, CDCl3)
d
128.7, 128.2, 127.8, 108.9, 101.0, 92.1. Anal. Calcd for C14H10O4: C,
69.42; H, 4.16; O, 26.42%. Found: C, 69.25; H, 4.26; O, 26.49%.
To a stirred solution of catechol 7 (1.02 g, 6.62 mmol) and
Cs2CO3 (2.67 g, 8.19 mmol) in DMF (10 mL) was added CH2Br2
(0.71 mL, 9.93 mmol) at room temperature and the mixture was
stirred at 90 ꢁC for 19 h. After cooling to room temperature, the
mixture was diluted with Et2O (30 mL), filtrated with Celite, and
this Celite was rinsed with Et2O (3ꢂ6 mL). The filtrate and rinse
were washed with H2O (3ꢂ10 mL) and the aqueous layer was
extracted with Et2O (3ꢂ6 mL). The combined organic layers were
washed with 1 M NaOH aq (ꢂ3), H2O (ꢂ2), and brine (ꢂ1), dried
(Na2SO4), and concentrated. The residual oil was purified
by column chromatography on silica gel (15 g, n-hexane–EtOAc
30:1/10:1) to give bis-methylene acetal 16 (489 mg, 46%) as
a white solid: colorless crystals. Mp 139–140 ꢁC (n-hexane–
4.4. Phlebiarubrone (3)
To a stirred solution of diphenyl compound 21 (410 mg,
1.28 mmol) in acetonitrile (97 mL) was added CAN (1.0 M solution
in H2O, 3.9 mL, 3.90 mmol) at 0 ꢁC and the mixture was stirred at
0 ꢁC for 3 min. The mixture was diluted with H2O (80 mL) and
extracted with CHCl3 (3ꢂ15 mL). The combined extracts were dried
(Na2SO4) and concentrated. The residual oil was purified by column
chromatography on silica gel (10 g, n-hexane–EtOAc 10:1/4:1) to
give red solid. The red solid was purified by recrystallization from
n-hexane–CH2Cl2 to give phlebiarubrone (3) (389 mg, quant.) as
red crystals. Mp 248–250 ꢁC (n-hexane–CH2Cl2); 1H NMR
CH2Cl2); 1H NMR (270 MHz, CDCl3)
d
6.48 (s, 2H), 5.86 (s, 4H); 13C
NMR (67.8 MHz, CDCl3) 141.1, 101.0, 93.0. Anal. Calcd for
C8H6O4: C, 57.84; H, 3.64; O, 38.52%. Found: C, 57.76; H, 3.76; O,
38.57%.
d
(270 MHz, CDCl3)
d
7.60–7.65 (m, 4H), 7.34–7.48 (m, 6H), 6.13 (s,
175.9, 156.1, 129.4, 128.7, 128.6,
2H); 13C NMR (67.8 MHz, CDCl3)
d
128.1, 113.7, 102.7; HRESIMS m/z 327.0636, calcd for C19H12NaO4
[MþNa]þ 327.0633.
4.3. Diphenyl compound 21
4.5. Ustalic acid dimethyl ester (2)
To a stirred solution of bis-methylene acetal 16 (400 mg,
2.41 mmol) in Et2O (10 mL) at 0 ꢁC were added TMEDA (1.0 mL,
6.71 mmol) and n-BuLi (1.61 M solution in n-hexane, 4.5 mL,
7.25 mmol) under nitrogen flow, and the resultant mixture was
stirred at 0 ꢁC for 30 min. After cooling to ꢀ78 ꢁC, B(O-iPr)3 (2.8 mL,
12.2 mmol) in Et2O (4.2 mL) was added and the resultant mixture
was stirred at ꢀ78 ꢁC for 1 h. The mixture was stirred at room
temperature for 17 h, diluted with 1 M HCl to pH 1, and extracted
with CHCl3 (4ꢂ15 mL). The combined extracts were dried (Na2SO4)
and concentrated to afford crude diboronic acid 17 (670 mg), which
was used for the next reaction without further purification.
The crude diboronic acid 17 (670 mg), pinacol (2.26 g,
19.1 mmol), and MgSO4 (1.30 g,10.8 mmol) were dissolved in CH2Cl2
(16 mL), and the resultant mixture was stirred at room temperature
for 17.5 h. The mixture was filtrated with Celite, and this Celite was
rinsed with CH2Cl2 (3ꢂ5 mL). The filtrate and rinse were combined
and concentrated to afford crude diboronic pinacol ester 20 (1.28 g),
which was used for the next reaction without further purification.
All solvents were degassed by freeze-thawing. To a stirred so-
lution of crude diboronic pinacol ester 20 (1.28 g) and iodobenzene
(0.8 mL, 7.18 mmol) in DMF (10 mL) were added PdCl2(PPh3)2
(190 mg, 0.24 mmol) and Cs2CO3 (2.34 g, 7.18 mmol) at room tem-
perature in a glove box. The mixture was stirred at 90 ꢁC for 14 h
Phlebiarubrone (3) (100 mg, 0.329 mmol), Pb(OAc)4 (2.92 g,
6.59 mmol), and K2CO3 (96.2 mg, 0.696 mmol) were dissolved in
toluene (8.2 mL) and MeOH (5.8 mL). The resultant mixture was
stirred at room temperature for 47 h, diluted with H2O (15 mL) and
ethylene glycol (a few drops), and filtrated with Celite. The filtrate
was extracted with Et2O (4ꢂ15 mL). The combined extracts were
washed with H2O, saturated aqueous NaHCO3, and H2O, dried
(Na2SO4), and concentrated. The residual oil was purified by col-
umn chromatography on silica gel (8.0 g, n-hexane–EtOAc 5:1/
2:1), preparative TLC (CH2Cl2), and preparative TLC (n-hexane–
EtOAc 2:1) to give ustalic acid dimethyl ester (2) (13 mg, 11%) as
a yellow solid. IR (film) 1718, 1637 cmꢀ1; 1H NMR (270 MHz, CDCl3)
d
7.31–7.42 (m, 10H), 5.41 (s, 2H), 3.73 (s, 6H); 13C NMR (67.8 MHz,
CDCl3)
d 167.5, 148.6, 134.4, 129.8, 127.9, 127.8, 115.2, 95.9, 52.2;
HRESIMS m/z 389.0998, calcd for C21H18NaO6 [MþNa]þ 389.1001.
4.6. Ustalic acid (1)
The ustalic acid dimethyl ester (2) (2.6 mg, 7.10 mmol) was
treated with 3 M KOH aq–DMSO (1:1, 0.45 mL) at room tempera-
ture for 24 h. The mixture was diluted with saturated NaH2PO4 to
pH 3 and extracted with CHCl3 (ꢂ4). The combined extracts were

I. Hayakawa et al. / Tetrahedron 64 (2008) 5873–5877
5877
dried (Na2SO4) and concentrated to afford crude ustalic acid
monomethyl ester (23) (2.3 mg), which was used for the next re-
action without further purification.
Functional Molecules’) and by the 21st COE program from the
Ministry of Education, Culture, Sports, Science, and Technology
(MEXT), Japan. We would like to thank Mr. Ikuo Iida (University of
Tsukuba) for the elemental analyses.
The crude ustalic acid monomethyl ester (23) (2.3 mg) was
treated with 3 M KOH aq–DMSO (1:1, 0.45 mL) at room temperature
for 4 days. The mixture was diluted with saturated NaH2PO4 to pH 3
and extracted with CHCl3 (ꢂ4). The combined extracts were dried
(Na2SO4) and concentrated to afford crude ustalic acid (1). The re-
sidual oil was purified by HPLC (Develosil ODS-HG-5 (250ꢂ20 mm),
flow rate 5 mL/min; detection UV 254 nm; solvent 50% MeOH/0.1%
TFA) to give ustalic acid (1) (0.9 mg, 38%, retention time 91.2 min) as
a white solid. IR (film) 1698, 1630 cmꢀ1; 1H NMR (270 MHz, CDCl3)
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d
7.29–7.41 (m,10H), 5.46 (s, 2H); 13C NMR (67.8 MHz, CDCl3)
d 174.2,
149.2, 133.4, 129.9, 128.2, 127.9, 115.0, 96.3; HRESIMS m/z 361.0686,
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This work was supported in part by Grants-in-Aid for Scientific
Research on Priority Area (No. 16073204, ‘Creation of Biologically