New xanthine oxidase inhibitors from the fruiting bodies of Tyromyces fissilis

Article abstract of DOI:10.1080/09168451.2019.1576501

Excessive uric acid production, which causes gout and hyperuricemia, can be blocked by inhibiting xanthine oxidase (XO). However, some agents to block on XO often cause side effects, thereby necessitating the identification of new inhibitors. During the s

Full text of DOI:10.1080/09168451.2019.1576501

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20
New xanthine oxidase inhibitors from the fruiting
bodies of Tyromyces fissilis
Shunsuke Mitomo, Mitsuru Hirota & Tomoyuki Fujita
To cite this article: Shunsuke Mitomo, Mitsuru Hirota & Tomoyuki Fujita (2019): New xanthine
oxidase inhibitors from the fruiting bodies of Tyromycesꢀfissilis, Bioscience, Biotechnology, and
Biochemistry, DOI: 10.1080/09168451.2019.1576501
To link to this article: https://doi.org/10.1080/09168451.2019.1576501
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1576501
New xanthine oxidase inhibitors from the fruiting bodies of Tyromyces fissilis
Shunsuke Mitomo, Mitsuru Hirota and Tomoyuki Fujita
Graduate School of Science and Technology, Department of Agriculture, Shinshu University, Nagano, Japan
ABSTRACT
ARTICLE HISTORY
Received 11 December 2018
Accepted 21 January 2019
Excessive uric acid production, which causes gout and hyperuricemia, can be blocked by
inhibiting xanthine oxidase (XO). However, some agents to block on XO often cause side
effects, thereby necessitating the identification of new inhibitors. During the screening of XO
inhibitors from various mushroom extracts, we found that a methanolic extract of the fruiting
bodies of Tyromyces fissilis, an inedible and non-toxic fungus, showed inhibitory activity. Both
n-hexane and ethyl acetate layers, obtained by partitioning this extract exhibited XO inhibi-
tory activity. Subsequently, using an activity-guided separation method, eight active com-
pounds (1–8) were isolated. The structures of five of the new compounds, 2–4, 6, and 7, were
elucidated by spectral analysis and chemical derivatization. All compounds had a salicylic acid
moiety with an aliphatic group at the C-6 position. Notably, 2-hydroxy-6-pentadecylbenzoic
acid (1) showed the highest level of XO noncompetitive inhibition (58.9 2.2% at 25 µM).
KEYWORDS
Tyromyces fissilis; xanthine
oxidase inhibitor; anacardic
acid analogues; structure-
activity relationship
Uric acid, the causative agent of gout, is the final
product of purine (a nucleic acid) metabolism,
which is excreted in urine. It is produced by the
successive oxidation of hypoxanthine and xanthine
by the catalytic enzyme xanthine oxidase (XO) [1].
When excess uric acid accumulates in blood, the
urate crystals tend to precipitate and aggregate at
relatively low temperatures. A painful inflammation
occurs when white blood cells attack the deposited
crystals, leading to the symptoms collectively known
as gout [2]. The onset of gout is determined by the
plasma uric acid level. When this level reaches
7.0 mg/dL or more, the patient is diagnosed with
hyperuricemia [3], which has been correlated with
cardiovascular diseases [4], hypertension [5], and
renal diseases [6]. To treat both gout and hyperur-
icemia, the XO inhibitors, allopurinol and febuxo-
stat, which inhibit uric acid production, were
developed [7]. However, treatment using these inhi-
bitors has been associated with various side-effects,
such as the development of hepatic disorders.
Therefore, the discovery of new uric acid production
inhibitors that have a low risk of causing side-effects
is desired [8] and has led to increased interest in
natural products, particularly XO-inhibiting phyto-
chemicals, including flavonoids, phenylpropanoids,
anacardic acids [9], hispidin derivatives [10], and
berchemiosides [11].
for their activities. For example, several lanosterol
derivatives, whose physiological activities have yet to
be clarified [13,14], and 2-hydroxybenzoic acid analo-
gues, which appear to have anti-inflammatory activ-
ities [15], have been isolated from this species of
fungus. Furthermore, a screening of mushroom
extracts for XO enzymatic inhibition demonstrated
that a methanolic extract of T. fissilis exhibited the
highest inhibitory activity (data not shown).
In the present study, we analyzed possible XO
inhibitors that were isolated from T. fissilis fruiting
bodies by methanol extraction. To our knowledge,
this is the first study that has conducted the isolation,
structural determination, and inhibitory activity of
the XO inhibitors derived from T. fissilis.
Materials and methods
Fungal material
Fruiting bodies of T. fissilis were collected and pro-
vided by Mr. Mizuki Nomura and Dr. Mitsuru Hirota
of the Ina Campus, Shinshu University, Japan in
October 2014. The fungal material was identified by
Dr. Akiyoshi Yamada of the Faculty of Agriculture,
Shinshu University. A voucher specimen (Mito-01)
was deposited in a laboratory in the Faculty of
Agriculture, Shinshu University.
Tyromyces fissilis is a white-rot fungus of the
Polyporaceae family that grows on fallen broad-
leaved trees from summer to autumn [12]. While the
fruiting bodies are hard and unsuitable for consump-
tion, various components have been isolated and tested
Chemicals
XO (EC 1.17.3.2, from bovine milk), xanthine,
1-methyl-3-nitro-1-nitrosoguanidine, Dess-Martin
periodinane, and 4-(dimethylamino) pyridine
CONTACT Tomoyuki Fujita
tfujita@shinshu-u.ac.jp
© 2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
S. MITOMO ET AL.
(DMAP) were obtained from Sigma Chemical
Company. Ethylenediaminetetraacetic acid
successively partitioned with n-hexane (500 mL,
thrice), EtOAc (500 mL, thrice), and n-BuOH
(375 mL, thrice). The dried n-hexane extract
(9.29 g), EtOAc extract (39.4 g), and n-BuOH extract
(7.37 g) were then evaluated for XO inhibition. The
n-BuOH extract showed low XO inhibition (4.5% at
100 µg/mL), while the n-hexane and EtOAc extracts
showed relatively high inhibitory activity against XO
with inhibitory rates of 28.5% and 37.8%, respec-
tively, at 100 µg/mL (The XO inhibitory activities of
these extracts are shown in Supplementary Data).
The n-hexane extract was further subjected to silica
gel column chromatography. Elution was performed
using a stepwise mixture of n-hexane/EtOAc (100:0,
90:10, 80:20, 70:30, and 0:100, v/v; 100 mL each) and
MeOH, resulting in 27 fractions (TFH1–TFH27) based
on TLC analysis. Fraction TFH4 (a part of the 90%
n-hexane/EtOAc eluate, 302.5 mg) was subsequently
subjected to additional silica gel column chromatography
with a stepwise mixture of n-hexane/EtOAc (100:0, 99:1,
98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, and 90:10, v/v;
30 mL each) and MeOH as the eluent, resulting in 12
additional fractions (TFH4-1 to TFH4-12). The MeOH
eluate, TFH4-12 (34.7 mg), was further separated using
the ODS column with MeCN/H2O (90:10, 91:9, 92:8, and
93:7, v/v; 5 mL each) as the eluent to give compound 1
(19.7 mg). The mixture of the TFH14 and TFH15 frac-
tions (parts of the 80% n-hexane/EtOAc eluate)
(60.4 mg) was fractionated using the ODS column with
MeCN/H2O (60:40, 65:35, 70:30, 75:25, and 80:20, v/v;
30 mL each) as the eluent, resulting in 15 subfractions
[TFH(14,15)-1 to TFH(14,15)-15)]. The 80% MeCN/H2
O eluate (subfractions 13–15, 17.5 mg) was mixed and
separated by reverse-phase HPLC (80:20, v/v; MeCN/H2
O) to give compound 2 (5.0 mg). Three parts of the 80%
n-hexane/EtOAc eluate, TFH16–18 (58.8 mg), were sub-
jected to ODS flash column elution with MeCN/H2
O (60:40, 65:35, 70:30, 75:25, and 80:20, v/v; 15 mL
each) as the eluent to give compound 3 (9.0 mg). Part
of the 80% n-hexane/EtOAc eluate, TFH21 (32.8 mg),
was similarly separated to obtain compound 4 (20.5 mg).
Part of the 70% n-hexane/EtOAc eluate, TFH25
(152.4 mg), was fractionated using the ODS column with
MeCN/H2O (60:40, 65:35, 70:30, 75:25, 80:20, and 85:15,
v/v; 60 mL each) as the eluent to produce 11 subfractions
(1–11). Part of the 75% MeCN/H2O eluate TFH25-6
(26.0 mg) was separated by recrystallization from
MeCN to give compound 5 (12.9 mg).
(EDTA) was purchased from Nacalai Tesque Inc.
Deuterochloroform (CDCl3) was obtained from
Merck. Methanol (MeOH), n-hexane, n-butanol
(n-BuOH), acetonitrile (MeCN), chloroform
(CHCl3), and ethyl acetate (EtOAc) were obtained
from
Kanto
Chemical
Company.
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDC∙HCl), (1R,2R)-2-(anthracene-
2,3-dicarboximido)cyclohexanecarboxylic acid, and
(1S,2S)-2-(anthracene-2,3-dicarboximido)cyclohex-
anecarboxylic acid were obtained from the Tokyo
Chemical Industry Corporation.
General experimental methods
One and two dimentional nuclear magnetic resonance
1
(NMR) spectra, including the H-1H correlation spec-
troscopy (COSY), heteronuclear multiple quantum cor-
relation (HMQC), and heteronuclear multiple bond
correlation (HMBC) spectra, were recorded on the
Bruker DRX500 spectrometer operated at 500 MHz
1
1
for H and 125 MHz for 13C. H chemical shifts were
determined relative to the internal standard tetra-
methylsilane (TMS). 13C chemical shifts were deter-
mined using the CDCl3 solvent peak (δC 77.0) as the
reference. High-resolution fast atom bombardment
mass spectra (HR-FAB-MS) were obtained on the
JEOL JMS-700MStation spectrometer with glycerol as
the matrix. Optical rotations were measured using the
JASCO DIP-100 digital polarimeter (L = 1 cm). Melting
points were measured using a Yanaco MP-S3 measur-
ing device. IR spectra were recorded on the JASCO FT/
IR-480 Plus spectrometer, while UV spectra were mea-
sured on the Shimadzu UV-1800 spectrometer. The
silica gels, silica gel 60 F254 and 60 RP-18 F254S
(Merck), were used for analytical thin-layer chromato-
graphy (TLC). Column chromatography was per-
formed using a normal-phase silica gel (Fuji Silysia
BW-350) and reverse-phase octadecyl functionalized
silica gel (ODS; Fuji Silysia BM1020T). High-
performance liquid chromatography (HPLC) was per-
formed on the Shimadzu system (Kyoto, Japan) con-
sisting of the LC-10AD pump, CTO-10A column oven,
and SPD-10A UV-VIS detector, which were connected
to the C-R7A plus Chromatopac integrator. A reverse-
phase column (10ϕ × 250 cm, 5 µm, Inertsil ODS-3),
flow rate of 3 mL/min, and wavelength of 254 nm were
used for the analysis. All solvents were HPLC-grade.
2-Hydroxy-6-pentadecylbenzoic acid (1): Colorless
amorphous powder; mp 86–89°C; UV (EtOH) λmax
(log ε) 209 (4.47), 243 (3.77), and 310 (3.58) nm; IR
(NaCl) νmax 3500–2500, 2916, 2853, 1657, 1605, 1448,
1308, 1249, 882, 815, 730, 708, 647, and 596 cm−1;
HR-FAB-MS m/z 347.2584 [M−H]− (calcd. for C22
H35O3, 347.2586); 1H NMR (500 MHz, CDCl3) δ 0.88
(3H, t, J = 7.1 Hz), 1.20–1.38 (24H, m), 1.57 (2H, m),
2.94 (2H, dd, J = 7.6, 7.9 Hz), 6.75 (1H, d, J = 7.5 Hz),
Isolation of compounds 1–8
Fruiting bodies of T. fissilis (2.1 kg) were soaked in
MeOH (4 L) for 4 months. After filtration and eva-
poration of MeOH in vacuo, the resulting crude
extract was dissolved in H2O (250 mL) and then

XANTHINE OXIDASE INHIBITORS FROM TYROMYCES FISSILIS
3
6.84 (1H, d, J = 8.2 Hz), and 7.31 (1H, dd, J = 7.5,
8.2 Hz); 13C NMR (125 MHz, CDCl3) δ 14.1, 22.7,
29.4, 29.5, 29.7, 29.7, 29.8, 31.9, 32.0, 36.3, 111.2,
115.7, 122.7, 135.0, 147.6, 163.0, and 176.1.
(1H, d, J = 7.5 Hz), 6.85 (1H, d, J = 8.3 Hz), and 7.33
(1H, dd, J = 7.5, 8.3 Hz); 13C NMR (125 MHz, CDCl3) δ
14.1, 18.8, 25.2, 28.7, 28.9, 28.9, 29.0, 29.0, 29.1, 29.6,
32.1, 36.5, 37.0, 39.5, 72.5, 110.9, 115.6, 122.5, 134.9,
147.5, 163.5, and 174.6.
2-Hydroxy-6-(12-oxoheptadecyl)benzoic acid (2):
Colorless amorphous powder; mp 93–94°C; UV
(EtOH) λmax (log ε) 209 (4.48), 243 (3.79), and 311
(3.60) nm; IR (NaCl) νmax 3500–2500, 2916, 2850,
1705, 1652, 1607, 1449, 1299, 1222, 907, 813, and
720 cm−1; HR-FAB-MS m/z 389.2686 [M−H]−
The EtOAc extract was subjected to silica gel column
chromatography with the n-hexane/EtOAc mixture
(100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70,
20:80, 10:90, 0:100, v/v; 400 mL each) and MeOH as
the eluent, producing 13 fractions (TFE1–TFE13) based
on solvent composition and TLC analyses. Part of the
70% n-hexane/EtOAc eluate, TFE4 (2764.1 mg), was
further subjected to silica gel column chromatography
with an n-hexane/EtOAc mixture (100:0, 90:10, 80:20,
70:30, 60:40, 50:50, and 40:60, v/v; 240 mL each) as the
eluent, affording 25 subfractions (TFE4-1 to TFE4-25).
Two parts of the 70–80% n-hexane/EtOAc eluate, TFE4-
9 and TFE4-10 (599.1 mg), were mixed and dissolved in
1
(calcd. for C24H37O4, 389.2692); H and 13C NMR,
see Tables 1 and 2.
2-Hydroxy-6-(12-oxopentadecyl)benzoic acid (3):
Colorless amorphous powder; mp 82–84°C; UV
(EtOH) λmax (log ε) 209 (4.40), 243 (3.70), and 310
(3.50) nm; IR (NaCl) νmax 3500–2500, 2922, 2850,
1706, 1654, 1608, 1449, 1301, 1218, 903, 814, and
721 cm−1; HR-FAB-MS m/z 361.2377 [M−H]−
1
(calcd. for C22H33O4, 361.2379); H and 13C NMR,
Table 1. 13C NMR spectral data for 2–4, 6, and 7 (125 MHz, CDCl3).
see Tables 1 and 2.
(R)-2-Hydroxy-6-(12-hydroxyheptadecyl)benzoic
acid (4): Colorless amorphous powder; mp 75–76°C;
1
2
3
4
5
6
7
8
1
2
3
4
5
6
111.2 110.6 110.6 111.2 110.9 111.0 110.8 110.7
163.0 163.5 163.5 163.3 163.5 163.5 163.5 163.5
115.7 115.7 115.7 115.6 115.6 115.6 115.7 115.7
135.0 135.1 135.1 134.7 134.9 134.7 135.0 135.0
122.7 122.6 122.6 122.4 122.5 122.4 122.6 122.5
147.6 147.6 147.6 147.4 147.5 147.3 147.5 147.5
36.3
32.0
31.9
29.4
29.5
29.7
29.7
29.7
29.7
29.7
29.7
[α]18.7 −0.5° (c 0.42, MeOH); UV (EtOH) λmax (log
D
ε) 209 (4.44), 243 (3.75), and 310 (3.55) nm; IR
(NaCl) νmax 3500–2500, 3446, 2926, 2854, 1653,
1605, 1452, 1311, 1246, 1214, 1166, 824, and
709 cm−1; HR-FAB-MS m/z 391.2838 [M−H]−
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
11′
12′
13′
14′
15′
16′
17′
36.5
32.0
29.6
29.3
29.2
29.2
29.2
29.1
29.0
23.6
42.8
36.5
32.0
29.6
29.3
29.2
29.2
29.2
29.1
29.0
23.7
42.8
36.4
32.1
31.8
29.5
29.1
29.0
28.9
28.9
28.8
25.3
37.0
72.7
37.3
25.2
28.6
22.6
14.0
36.5
32.1
29.6
29.1
29.0
29.0
28.9
28.9 130.6
28.7 127.4
25.2
37.0 132.0
72.5 124.7
36.7
32.2
29.8
29.0
28.5
28.5
26.8
36.5
32.1
29.8
29.5
29.5
29.4
29.4
29.4
29.4
29.4
29.4
25.6
39.0
68.7
23.2
36.5
32.1
29.9
29.5
29.5
29.4
29.4
29.3
29.3
29.3
29.3
29.3
29.2
25.6
39.1
68.7
23.3
1
(calcd. for C24H39O4, 391.2848); H and 13C NMR,
see Tables 1 and 2.
(R)-2-Hydroxy-6-(12-hydroxypentadecyl)benzoic
acid (5): Colorless oil; [α]19.9 −1.0° (c 0.19, MeOH);
D
UV (EtOH) λmax (log ε) 209 (4.41), 243 (3.71), and 310
(3.52) nm; IR (NaCl) νmax 3500–2500, 3451, 2925, 2853,
1647, 1605, 1451, 1310, 1246, 1213, 1166, 823, and
742 cm−1; HR-FAB-MS m/z 363.2546 [M−H]− (calcd.
for C22H35O4, 363.2535); 1H NMR (500 MHz, CDCl3) δ
0.94 (3H, t, J = 6.6 Hz), 1.23–1.40 (14H, m), 1.40–1.54
(8H, m), 1.58 (2H, m), 2.96 (2H, m), 3.70 (1H, m), 6.75
25.8
29.7 213.0 212.9
29.8
22.7
14.1
42.8
23.8
31.4
22.4
13.9
44.8
17.3
13.8
39.3
18.8
14.7
34.9
72.0
38.4
18.8
14.0
COOH 175.0 175.0 175.0 174.6 174.6 174.1 175.0 174.7
1
Table 2. H NMR spectral data for 2–4, 6, and 7 (500 MHz, CDCl3).
2
3
4
6
7
1
2
3
4
5
6.86, d (8.3)
7.35, dd (7.5, 8.3)
6.77, d (7.5)
6.86, d (8.3)
7.35, dd (7.4, 8.3)
6.77, d (7.4)
6.84, d (8.2)
7.32, dd (7.5, 8.2)
6.75, d (7.5)
6.84, d (8.2)
7.32, dd (7.5, 8.2)
6.74, d (7.5)
6.85, d (8.3)
7.33, dd (7.5, 8.3)
6.76, d (7.5)
6
1′
2′
3′6′
7′
8′
9′
10′
11′
12′
13′
14′
15′
16′
17′
COOH
2.97, dd (7.7, 8.0)
1.59, m
2.97, dd (7.7, 8.0)
1.58, m
2.95, m
1.58, m
2.86–3.02, m
1.59, m
2.96, dd (7.7, 8.0)
1.59, m
1.23–1.40, m
1.23–1.40, m
1.23–1.40, m
1.23–1.40, m
1.57, m
1.23–1.41, m
1.23–1.41, m
1.23–1.41, m
1.23–1.41, m
1.56, m
1.23–1.40, m
1.23–1.40, m
1.23–1.40, m
1.23–1.40, m
1.47, m
1.47, m
3.69, m
1.47, m
1.47, m
1.23–1.40, m
1.31, m
0.89, t (6.7)
1.28–1.45, m
2.06, q (6.8)
5.38, dt (6.8, 11.0)
5.37, dt (6.5, 11.0)
2.83, t (6.5)
5.60, dt (6.5, 10.6)
5.43, dt (6.5, 10.6)
2.33, m
3.81, m
1.53, t (6.3)
1.41, m, 1.53, m
0.95, t (7.0)
1.24–1.55, m
1.24–1.55, m
1.24–1.55, m
1.24–1.55, m
1.24–1.55, m
1.24–1.55, m
1.24–1.55, m
1.36, m
2.41, t (7.5)
2.41, t (7.3)
2.41, t (7.5)
1.57, m
1.26, m
1.31, m
0.89, t (7.2)
2.40, t (7.4)
1.60, m
0.91, t (7.4)
3.88, m
1.23, d (6.2)

4
S. MITOMO ET AL.
MeCN and then, the supernatant (178.3 mg) was further
fractionated using the ODS column with MeCN/H2
O (60:40, 65:45, 70:30, 75:25, 80:20, and 85:15, v/v;
70 mL each) as the eluent to give 36 subfractions
(1–36). Of these, subfractions 6 and 7 (14.8 mg), which
are parts of the 70–75% MeCN/H2O eluate, were sub-
jected to silica gel column chromatography with a CHCl3
/MeOH (100:0, 98:2, and 96:4, v/v; 4.8 mL each) as the
eluent to obtain compound 6 (7.7 mg). Part of the 60%
n-hexane/EtOAc eluate, TFE5 (393.2 mg), was subjected
to additional silica gel column chromatography with an
n-hexane/EtOAc mixture (100:0, 90:10, 80:20, 70:30,
60:40, and 50:50, v/v; 30 mL each) as the eluent, resulting
in 22 additional fractions (TFE5-1 to TFE5-22). Part of
the 70% n-hexane/EtOAc eluate, TFE5-11 (111.2 mg),
was further fractionated using the ODS column with
MeCN/H2O (60:40, 65:35, 70:30, 75:25, and 80:20, v/v;
45 mL each) as the eluent to afford 22 subfractions
(1–22). Two parts of the 75% MeCN/H2O eluate, sub-
fractions 7 and 8 (25.4 mg) were further separated by
recrystallization from MeCN to give compound 7
(20.1 mg). The MeCN eluate, TFE5-11–22 (5.3 mg),
was similarly separated and recrystallized from MeCN
to give compound 8 (5.2 mg).
Methylation of compounds 1, 2, and 4–8 by
diazomethane
The method used to obtain the methyl esters of 1, 2, and
4–8 was based on that described by Nomura et al. [15].
For example, compound 1 (1.44 mg) was dissolved in
MeOH (3 mL). An excess amount of diazomethane (in
Et2O) was then added, and the reaction mixture was
stirred at room temperature for 15 h. Subsequently,
methyl ester 9 (1.33 mg) was obtained from 1 by remov-
ing the solvents in vacuo from the reaction mixture.
Compounds 2, and 4–8 (2.00, 4.68, 4.55, 0.54, 5.56, and
1.20 mg, respectively) were also treated similarly to
obtain methyl ester derivatives 10 (2.98mg), 11
(5.21mg), 12 (4.62mg), 13 (0.45mg), 14 (6.16mg), and
15 (1.48 mg), respectively.
Methyl 2-methoxy-6-pentadecylbenzoate (9): Colorless
amorphous powder; 1H NMR (500 MHz, CDCl3) δ 0.88
(3H, t, J = 7.0 Hz), 1.20–1.35 (24H, m), 1.52–1.62
(2H, m), 2.53 (2H, dd, J = 7.8, 8.0 Hz), 3.82 (3H, s),
3.91 (3H, s), 6.76 (1H, d, J = 8.3 Hz), 6.82 (1H, d,
J = 7.7 Hz), and 7.27 (1H, dd, J = 7.7, 8.3 Hz).
Methyl 2-methoxy-6-(12-oxoheptadecyl)benzoate
(10): Colorless amorphous powder; 1H NMR
(500 MHz, CDCl3) δ 0.89 (3H, t, J = 7.3 Hz),
1.20–1.34 (18H, m), 1.56 (6H, m), 2.38 (4H, t,
J = 7.5 Hz), 2.53 (2H, dd, J = 7.7, 8.1 Hz), 3.82
(3H, s), 3.91 (3H, s), 6.76 (1H, d, J = 8.3 Hz),
6.82 (1H, d, J = 7.7 Hz), and 7.27 (1H, dd,
J = 7.7, 8.3 Hz); 13C NMR (125 MHz, CDCl3) δ
13.9, 22.5, 23.6, 23.9, 29.3, 29.4, 29.5, 29.5, 29.6,
31.2, 31.4, 33.5, 42.8, 42.8, 52.2, 55.8, 108.3, 121.5,
123.4, 130.2, 141.3, 156.2, 169.0, and 211.8.
2-Hydroxy-6-((R,8Z,11Z)-14-hydroxyheptadeca-8,11-
dien-1-yl)benzoic acid (6): Colorless oil; [α]18.7D +2.6° (c
0.17, methanol); UV (EtOH) λmax (log ε) 204 (4.39), 240
(3.62), and 309 (3.39) nm; IR (NaCl) νmax 3500–2500,
3436, 3008, 2926, 2854, 1653, 1605, 1451, 1308, 1247,
1214, 1166, 1123, 1014, 823, and 708 cm−1; HR-FAB-MS
m/z 387.2538 [M−H]− (calcd. for C24H35O4, 387.2535);
1H and 13C NMR, see Tables 1 and 2.
(R)-2-Hydroxy-6-(14-hydroxypentadecyl)benzoic
acid (7): Colorless amorphous powder; mp 74–76°
Methyl
(R)-2-(12-hydroxyheptadecyl)-6-methoxy-
benzoate (11): Colorless oil; 1H NMR (500 MHz,
CDCl3) δ 0.89 (3H, t, J = 6.9 Hz), 1.21–1.36 (18H, m),
1.35–1.49 (8H, m), 1.53–1.61 (2H, m), 2.53 (2H, dd,
J = 7.8, 8.1 Hz), 3.55–3.62 (1H, m), 3.82 (3H, s), 3.91
(3H, s), 6.76 (1H, d, J = 8.3 Hz), 6.82 (1H, d, J = 7.7 Hz),
and 7.27 (1H, dd, J = 7.7, 8.3 Hz).
C; [α]18.7 −3.8° (c 0.40, methanol); UV (EtOH)
D
λmax (log ε) 209 (4.43), 243 (3.74), and 311 (3.55)
nm; IR (NaCl) νmax 3500–2500, 3451, 2925, 2853,
1653, 1605, 1451, 1310, 1246, 1214, 1166, 823, and
708 cm−1; HR-FAB-MS m/z 363.2540 [M−H]−
(calcd. for C22H35O4, 363.2535); 1H and
13C NMR, see Tables 1 and 2.
Methyl
(R)-2-(12-hydroxypentadecyl)-6-methoxy-
benzoate (12): Colorless oil; 1H NMR (500 MHz,
CDCl3) δ 0.93 (3H, t, J = 6.7 Hz), 1.20–1.36 (14H, m),
1.36–1.50 (8H, m), 1.52–1.61 (2H, m), 2.53 (2H, dd,
J = 7.8, 8.0 Hz), 3.57–3.64 (1H, m), 3.82 (3H, s), 3.91
(3H, s), 6.76 (1H, d, J = 8.3 Hz), 6.82 (1H, d, J = 7.7 Hz),
(R)-2-Hydroxy-6-(16-hydroxyheptadecyl)benzoic
acid (8): Colorless amorphous powder; mp 83–84°C;
[α]22.0 −4.2° (c 0.11, methanol); UV (EtOH) λmax
D
(log ε) 209 (4.36), 243 (3.66), and 310 (3.47) nm; IR
(NaCl) νmax 3500–2500, 3470, 2916, 2849, 1683, 1575,
1470, 1314, 1248, 1104, 823, 772, 716, and 701 cm−1;
HR-FAB-MS m/z 391.2858 [M−H]− (calcd. for C24
H39O4, 391.2848); 1H NMR (500 MHz, CDCl3) δ 1.22
(3H, d, J = 6.2 Hz), 1.24–1.55 (26H, m), 1.59 (2H, m),
2.96 (2H, dd, J = 7.1, 8.7 Hz), 3.87 (1H, m), 6.76 (1H,
d, J = 7.5 Hz), 6.86 (1H, d, J = 8.3 Hz), and 7.34 (1H,
dd, J = 7.5, 8.3 Hz); 13C NMR (125 MHz, CDCl3) δ
23.3, 25.6, 29.2, 29.3, 29.3, 29.4, 29.4, 29.5, 29.9, 32.1,
36.5, 39.1, 68.7, 110.7, 115.7, 122.6, 135.0, 147.5,
163.5, and 174.7.
and 7.27 (1H, dd, J = 7.7, 8.3 Hz).
Methyl 2-((R,8Z,11Z)-14-hydroxyheptadeca-8,11-dien
1
-1-yl)-6-methoxybenzoate (13): Colorless oil; H NMR
(500 MHz, CDCl3) δ 0.93 (3H, t, J = 6.4 Hz), 1.20–1.40
(10H, m), 1.40–1.52 (2H, m), 1.52–1.62 (2H, m), 2.04
(2H, q, J = 7.0 Hz), 2.24 (2H, t, J = 6.7 Hz), 2.53 (2H, dd,
J = 7.8, 8.0 Hz), 2.81 (2H, t, J = 6.8 Hz), 3.62–3.68
(1H, m), 3.82 (3H, s), 3.91 (3H, s), 5.29–5.35 (1H, m),
5.36–5.42 (1H, m), 5.44 (1H, dt, J = 7.5, 10.6 Hz), 5.55
(1H, dt, J = 7.3, 10.7 Hz), 6.76 (1H, d, J = 8.3 Hz), 6.82
(1H, d, J = 7.7 Hz), and 7.27 (1H, dd, J = 7.7, 8.3 Hz).

XANTHINE OXIDASE INHIBITORS FROM TYROMYCES FISSILIS
5
Methyl
(R)-2-(14-hydroxypentadecyl)-6-methoxy-
2-(anthracene-2,3-dicarboximido)cyclohexanecar-
benzoate (14): Colorless amorphous powder; 1H NMR
(500 MHz, CDCl3) δ 1.18 (3H, d, J = 6.2 Hz), 1.23–1.35
(20H, m), 1.35–1.50 (2H, m), 1.57 (2H, m), 2.53 (2H,
dd, J = 7.8, 8.0 Hz), 3.79 (1H, m), 3.82 (3H, s), 3.91 (3H,
s), 6.76 (1H, d, J = 8.3 Hz), 6.82 (1H, d, J = 7.7 Hz), and
7.27 (1H, dd, J = 7.7, 8.3 Hz).
boxylic acid and then purified by chromatography to
afford the corresponding (1S,2S)-esters, 18 (0.23 mg),
20 (0.34 mg), 22 (0.1 mg), 24 (0.51 mg), and 26
(0.17 mg).
(R: 1R,2R)-Ester (17) formed from derivative 11:
1
Yellow powder; H NMR (500 MHz, CDCl3) δ 0.70
Methyl
(R)-2-(16-hydroxyheptadecyl)-6-methoxy-
(3H, t, J = 6.9 Hz), 0.91–0.95 (8H, m), 0.91–1.03
(5H, m), 1.03–1.13 (6H, m), 1.13–1.23 (3H, m),
1.23–1.35 (5H, m), 1.42–1.61 (4H, m), 1.85–1.91
(3H, m), 2.15–2.25 (2H, m), 2.51 (2H, dd, J = 7.8,
8.0 Hz), 3.56 (1H, dt, J = 3.4, 11.8 Hz), 3.82 (3H, s),
3.91 (3H, s), 4.49 (1H, dt, J = 3.6, 11.9 Hz), 4.74 (1H, m),
6.77 (1H, d, J = 8.3 Hz), 6.82 (1H, d, J = 7.7 Hz), 7.28
(1H, dd, J = 7.7, 8.3 Hz), 7.60 (2H, m), 8.06 (2H, m), 8.47
(2H, s), and 8.61 (2H, s).
benzoate (15): Colorless amorphous powder; 1H NMR
(500 MHz, CDCl3) δ 1.19 (3H, d, J = 6.2 Hz), 1.22–1.35
(24H, m), 1.35–1.50 (2H, m), 1.52–1.62 (2H, m), 2.53
(2H, dd, J = 7.8, 8.0 Hz), 3.79 (1H, m), 3.82 (3H, s), 3.91
(3H, s), 6.76 (1H, d, J = 8.3 Hz), 6.82 (1H, d, J = 7.7 Hz),
and 7.27 (1H, dd, J = 7.7, 8.3 Hz).
Oxidation of methylated derivative 11 by
Dess-Martin periodinane
(R: 1S,2S)-Ester (18) formed from derivative 11:
1
Yellow powder; H NMR (500 MHz, CDCl3) δ 0.55
(3H, t, J = 7.0 Hz), 0.85–1.09 (12H, m), 1.09–1.18
(3H, m), 1.18–1.35 (12H, m), 1.35–1.66 (4H, m),
1.81–1.92 (3H, m), 2.13–2.23 (2H, m), 2.52 (2H, dd,
J = 7.6, 8.1 Hz), 3.56 (1H, dt, J = 3.2, 12.4 Hz), 3.82
(3H, s), 3.91 (3H, s), 4.49 (1H, dt, J = 3.4, 12.2 Hz),
4.73 (1H, m), 6.76 (1H, d, J = 8.4 Hz), 6.82 (1H, d,
J = 7.6 Hz), 7.29 (1H, dd, J = 7.6, 8.4 Hz), 7.61
(2H, m), 8.07 (2H, m), 8.48 (2H, s), and 8.62 (2H, s).
(R: 1R,2R)-Ester (19) formed from derivative 12:
Compound 11 was oxidized via the method described by
Nomura et al. [15]. In CH2Cl2 (2 mL) of 11 (2.61 mg), an
excessive amount of Dess-Martin periodinane was
added. The resulting solution was stirred at room tem-
perature for 2 h and extracted twice with EtOAc
(5.0 mL). The combined EtOAc extracts were then eva-
porated to dryness and fractionated by silica gel column
chromatography with n-hexane/EtOAc (80:20, v/v) as
the eluent to obtain oxidation derivative 16 (2.41 mg).
Methyl 2-methoxy-6-(12-oxoheptadecyl)benzoate (16):
Colorless amorphous powder; HR-FAB-MS m/z
417.2979 [M−H]− (calcd. for C26H41O4, 417.3005);
1H NMR (500 MHz, CDCl3) δ 0.89 (3H, t, J = 7.1 Hz),
1.20–1.36 (18H, m), 1.56 (6H, m), 2.38 (4H, t, J = 7.5 Hz),
2.53 (2H, dd, J = 7.8, 8.0 Hz), 3.82 (3H, s), 3.91 (3H, s),
6.76 (1H, d, J = 8.3 Hz), 6.82 (1H, d, J = 7.7 Hz), and 7.27
(1H, dd, J = 7.7, 8.3 Hz); 13C NMR (125 MHz, CDCl3) δ
13.9, 22.5, 23.6, 23.9, 29.3, 29.4, 29.5, 29.5, 29.6, 31.1, 31.4,
33.5, 42.8, 42.8, 52.2, 55.8, 108.3, 121.5, 123.4, 130.2,
141.3, 156.2, 169.0, and 211.8.
1
Yellow oil; H NMR (500 MHz, CDCl3) δ 0.71 (3H, t,
J = 7.3 Hz), 0.75–0.90 (8H, m), 0.90–1.03 (4H, m),
1.03–1.23 (8H, m), 1.23–1.38 (3H, m), 1.38–1.63
(4H, m), 1.83–1.93 (3H, m), 2.13–2.27 (2H, m), 2.51
(2H, dd, J = 6.0, 7.8 Hz), 3.56 (1H, dt, J = 3.5, 11.8 Hz),
3.82 (3H, s), 3.91 (3H, s), 4.49 (1H, dt, J = 3.7, 12.0 Hz),
4.75 (1H, m), 6.77 (1H, d, J = 8.3 Hz), 6.82 (1H, d,
J = 7.7 Hz), 7.29 (1H, dd, J = 7.7, 8.3 Hz), 7.60 (2H, m),
8.06 (2H, m), 8.48 (2H, s), and 8.61 (2H, s).
(R: 1S,2S)-Ester (20) formed from derivative 12: Yellow
oil; 1H NMR (500 MHz, CDCl3) δ 0.51 (3H, t, J = 7.3 Hz),
0.85–1.08 (10H, m), 1.08–1.20 (3H, m), 1.20–1.35
(10H, m), 1.40–1.65 (4H, m), 1.81–1.93 (3H, m),
2.13–2.25 (2H, m), 2.52 (2H, dd, J = 7.5, 8.2 Hz), 3.57
(1H, dt, J = 3.8, 11.5 Hz), 3.82 (3H, s), 3.91 (3H, s), 4.49
(1H, dt, J = 3.5, 12.3 Hz), 4.75 (1H, m), 6.76 (1H, d,
J = 8.2 Hz), 6.82 (1H, d, J = 7.7 Hz), 7.29 (1H, dd,
J = 7.7, 8.2 Hz), 7.61 (2H, m), 8.08 (2H, m), 8.48 (2H,
s), and 8.63 (2H, s).
Esterification of compounds 11–15 with (1R,2R)-
or (1S,2S)-reagent
The method described by Ohtaki et al. [16] was used
for the esterification of derivatives 11–15. (1R,2R)-
2-(Anthracene-2,3-dicarboximido)cyclohexanecar-
boxylic acid (1.2 eq.) was dissolved in a mixture of
toluene and acetonitrile (1:1,v/v; 200 µL). Derivatives
11–15 (1.0, 1.0, 0.2, 0.8, and 0.4 mg, respectively) were
added to the solution along with EDC∙HCl (10 eq.)
and DMAP (10 mg). The mixture was stirred over-
night at room temperature, and then evaporated in
vacuo. The mixture was purified by silica gel column
chromatography to give the corresponding (1R,2R)-
esters, 17 (0.98 mg), 19 (2.1 mg), 21 (0.1 mg), 23
(0.84 mg), and 25 (0.24 mg). In the same manner,
derivatives 11–15 (1.0, 1.0, 0.2, 0.49, and 0.4 mg,
(R: 1R,2R)-Ester (21) formed from derivative 13:
1
Yellow oil; H NMR (500 MHz, CDCl3) δ 0.69 (3H, t,
J = 7.3 Hz), 0.83–0.98 (2H, m), 1.08–1.35 (13H, m),
1.35–1.69 (4H, m), 1.79–1.93 (5H, m), 1.93–2.28
(4H, m), 2.47 (2H, m), 2.53 (2H, dd, J = 7.2, 8.4 Hz),
3.55 (1H, m), 3.81 (3H, s), 3.90 (3H, s), 4.47 (1H, dt,
J = 4.8, 11.3 Hz), 4.77 (1H, t, J = 6.4 Hz), 4.97–5.12
(3H, m), 5.12–5.20 (1H, m), 6.76 (1H, d, J = 8.5 Hz), 6.82
(1H, d, J = 8.0 Hz), 7.23–7.30 (1H, m, overlapped with
CHCl3), 7.62 (2H, m), 8.07 (2H, m), 8.48 (2H, s), and
8.62 (2H, s).
respectively)
were
esterified
with
(1S,2S)-

6
S. MITOMO ET AL.
(R: 1S,2S)-Ester (22) formed from derivative 13: Yellow
2.76 mL of 40 mM sodium carbonate buffer containing
0.1 mM EDTA (pH 10.0), 0.06 mL of 10 mM xanthine,
and 0.06 mL of the sample solution (dissolved in
DMSO). The reaction was started by the adding
0.12 mL of XO solution (0.04 U), and the absorbance
at 293 nm was recorded for 90 s. Control experiments
were carried out by replacing the sample solution with
the same amount of DMSO and/or by omitting
xanthine in the reaction mixture. The XO inhibitory
activity, in terms of the rate of uric acid production
inhibition (%), was calculated as follows:
oil; 1H NMR (500 MHz, CDCl3) δ 0.45 (3H, t,
J = 7.2 Hz), 0.80–1.08 (5H, m), 1.08–1.40 (10H, m),
1.38–1.70 (4H, m), 1.80–1.97 (5H, m), 2.12–2.23
(4H, m), 2.53 (2H, dd, J = 7.9, 8.0 Hz), 3.51 (1H, m),
3.81 (3H, s), 3.90 (3H, s), 4.47 (1H, m), 4.76 (1H, m),
5.12–5.23 (2H, m), 5.23–5.32 (2H, m), 6.75 (1H, d,
J = 8.8 Hz), 6.81 (1H, d, J = 7.6 Hz), 7.23–7.30 (1H, m,
overlapped with CHCl3), 7.62 (2H, m), 8.09 (2H, m),
8.48 (2H, s), and 8.63 (2H, s).
(R: 1R,2R)-Ester (23) formed from derivative 14:
Yellow powder; 1H NMR (500 MHz, CDCl3) δ
0.80–0.90 (6H, m), 0.90–0.98 (2H, m), 1.00 (3H, d,
J = 6.2 Hz), 1.05–1.13 (2H, m), 1.13–1.20 (4H, m),
1.20–1.35 (6H, m), 1.35–1.65 (4H, m), 1.80–1.93
(3H, m), 2.10–2.30 (2H, m), 2.54 (2H, dd, J = 7.8,
8.0 Hz), 3.50 (1H, dt, J = 3.8, 8.3 Hz), 3.82 (3H, s),
3.91 (3H, s), 4.47 (1H, dt, J = 3.8, 13.5 Hz), 4.74 (1H, m),
6.76 (1H, d, J = 8.2 Hz), 6.83 (1H, d, J = 7.7 Hz), 7.27
(1H, dd, J = 7.7, 8.2 Hz), 7.61 (2H, m), 8.07 (2H, m), 8.48
(2H, s), and 8.62 (2H, s).
XO inhibition (%) = {1 – (Asample Asample
–
(no
xanthine))/(Acontrol – Acontrol (no xanthine))} × 100
Results and discussion
Using the XO inhibitory activity-guided separation
method described by Nguyen et al. [17] (see
Supplementary Data), we isolated 1–8 from the fruit-
ing bodies of T. fissilis through methanol extraction,
followed by various types of chromatographic separa-
tion (Figure 1). Compounds 1, 5, and 8 were identi-
fied as 2-hydroxy-6-pentadecylbenzoic acid [18], (R)-
(R: 1S,2S)-Ester (24) formed from derivative 14:
1
Yellow powder; H NMR (500 MHz, CDCl3) δ 0.88
(3H, d, J = 6.2 Hz), 0.92–1.15 (12H, m), 1.15–1.35
(11H, m), 1.40–1.66 (4H, m), 1.80–1.93 (3H, m),
2.13–2.29 (2H, m), 2.53 (2H, dd, J = 7.8, 8.0 Hz), 3.52
(1H, dt, J = 3.1, 11.9 Hz), 3.82 (3H, s), 3.91 (3H, s), 4.44
(1H, dt, J = 3.5, 12.3 Hz), 4.74 (1H, m), 6.76 (1H, d,
J = 8.3 Hz), 6.82 (1H, d, J = 7.7 Hz), 7.26–7.28 (1H, m,
overlapped with CHCl3), 7.62 (2H, m), 8.08 (2H, m),
8.49 (2H, s), and 8.63 (2H, s).
(R: 1R,2R)-Ester (25) formed from derivative 15:
Yellow powder; 1H NMR (500 MHz, CDCl3) δ
0.82–0.89 (10H, m), 0.89–0.98 (3H, m), 1.00 (3H, d,
J = 6.2 Hz), 1.04–1.37 (13H, m), 1.37–1.72 (5H, m),
1.82–1.95 (3H, m), 2.10–2.27 (2H, m), 2.54 (2H, dd,
J = 7.6, 8.1 Hz), 3.50 (1H, m), 3.82 (3H, s), 3.91 (3H, s),
4.46 (1H, m), 4.73 (1H, m), 6.76 (1H, d, J = 8.0 Hz), 6.82
(1H, d, J = 7.7 Hz), 7.25–7.30 (1H, m, overlapped with
CHCl3), 7.61 (2H, m), 8.08 (2H, m), 8.48 (2H, s), and
8.63 (2H, s).
(R: 1S,2S)-Ester (26) formed from derivative 15: Yellow
powder; 1H NMR (500 MHz, CDCl3) δ 0.83–0.92
(2H, m), 0.88 (3H, d, J = 6.1 Hz), 0.92–1.09 (10H, m),
1.09–1.36 (14H, m), 1.36–1.73 (5H, m), 1.80–1.93
(3H, m), 2.13–2.27 (2H, m), 2.53 (2H, dd, J = 8.0,
8.1 Hz), 3.52 (1H, m), 3.82 (3H, s), 3.91 (3H, s), 4.45
(1H, m), 4.75 (1H, m), 6.76 (1H, d, J = 8.6 Hz), 6.82 (1H,
d, J = 7.9 Hz), 7.25–7.30 (1H, m, overlapped with
CHCl3), 7.62 (2H, m), 8.08 (2H, m), 8.49 (2H, s), and
8.63 (2H, s).
2-hydroxy-6-(12-hydroxypentadecyl)benzoic
acid
[15], and (R)-2-hydroxy-6-(16-hydroxyheptadecyl)
benzoic acid [19], respectively, by comparing their
physicochemical properties and spectral data to
those in the literature.
The negative HR-FAB-MS of
2
showed
a deprotonated molecular ion at m/z 389.2686 [M−H] –
(calcd. 389.2692), indicating that the molecular formula
is C24H38O4 with five unsaturations. The IR spectrum
suggested the presence of a carboxy (3500–2500 and
1652 cm−1), isolated carbonyl (1705 cm−1), and aromatic
(907, 813, and 720 cm−1) groups. The 13C NMR spec-
trum indicates the presence of one carbonyl (δC 213.0),
one carboxy (δC 175.0), one benzene (δC 110.6, 115.7,
122.6, 135.1, 147.6, and 163.5), one methyl (δC 13.9), and
15 methylene (δC 22.4, 23.6, 23.8, 29.0, 29.1, 29.2, 29.2,
29.2, 29.3, 29.6, 31.4, 32.0, 36.5, 42.8, and 42.8) groups
(Table 1). The deduced substituted form of the aromatic
ring is 1,2,3-trisubstituted benzene based on the coupling
constants (J) of the aromatic proton signals at δH 6.77
(1H, d, J = 7.5 Hz), 6.86 (1H, d, J = 8.3 Hz), and 7.35 (1H,
dd, J = 7.5, 8.3 Hz) in the 1H NMR spectrum (Table 2).
These data are similar to those of 1 except for the signal
owing to a ketone, and the aromatic group is presumed
to be salicylic acid with an alkyl side chain at the C-6
position. This is confirmed by the presence of long-range
correlations between H-1′ and C-1, C-5 and C-6, and
H-2′ and C-6 in the HMBC spectrum.
The methylene protons adjacent to the ketone are
observed at δH 2.41 (4H, t, J = 7.5 Hz) (Table 2,
Figure 2). The correlation with the four methylene
protons is observed in order from the terminal methyl
proton δH 0.89 (3H, t, J = 7.2 Hz) to the fourth
XO inhibitory activity of the isolated compounds
The XO inhibitory activity was evaluated by measuring
the uric acid level according to the method described by
Masuoka et al. [9]. The reaction mixture consisted of

XANTHINE OXIDASE INHIBITORS FROM TYROMYCES FISSILIS
7
Figure 1. Chemical structures of 1–8.
methylene proton at δH 2.41 in the 1H-1H COSY spec-
trum (Figure 2), indicating that the length of the alkyl
side chain is C17. The HMBC spectrum also exhibited
C–H correlations between the three methylene protons
(δH 2.41 and 1.57) and carbonyl carbon (δC 213.0). This
indicates that the position of the carbonyl carbon is
C-12′. Thus, 2 was determined as 2-hydroxy-6-(12-
oxoheptadecyl)benzoic acid, which is a new compound.
The NMR and IR profiles of 3 were similar to
those of 2, indicating that 3 also has C6-substituted
salicylic acid with alkyl side chain. The negative HR-
FAB-MS confirmed that molecular formula of 3 is
C22H34O4 (found: m/z 361.2377 [M−H]−). The
13C NMR spectrum also indicates the presence of 22
carbons, as shown in Table 1. These data directly
show that 3 has a C15 alkyl side chain with one
carbonyl group. The 1H-1H COSY spectrum sug-
gested the presence of a partial structure based on
the correlations between a terminal methyl proton
(H-15′, δH 0.91) and methylene protons (H-14′, δH
1.60) and between two methylene protons (H-14′, δH
1.60 and H-13′, δH 2.40). Furthermore, the HMBC
spectrum showed correlations between the carbonyl
carbon (δC 212.9) and two methylene protons (H-13′,
δH 2.40 and H-14′, δH 1.60). Thus, the carbonyl
group was assigned to the C-12′ position in the
alkyl side chain. These spectral data indicated that 3
is another new compound, specifically 2-hydroxy-
6-(12-oxopentadecyl)benzoic acid.
The negative HR-FAB-MS confirmed that 4 has
the molecular formula, C24H40O4 (found: m/z
391.2838 [M−H]–), indicating that its mass number
is 2H more than that of 2. Its 13C NMR spectrum is
similar to that of 2, suggesting the presence of the C6-
substituted salicylic acid moiety with C15 alkyl side
chain. The difference between 2 and 4 is the absence
of the carbonyl signal at δC 213.0 for the latter; a new
signal (δC 72.7) for oxymethine appears instead
(Table 1). Based on detailed analysis of the HMQC
and HMBC spectra, this oxymethine group is
Figure 2. Important COSY (bold) and HMBC (→) correlations for 2 and 6.

8
S. MITOMO ET AL.
expected to be at C-12′ position in the alkyl side
chain.
Figure 2. Thus, the position of oxymethine and the
two double bonds are C-14′, C-12′, C-11′, C-9′, and
C-8′, respectively. The HMBC data also supported
this assignment.
Based on the J values of the olefinic protons at δH 5.37
to 5.38 (11.0 Hz) and 5.43 to 5.60 (10.6 Hz), the config-
uration of the double bonds is Z. Compound 6 was
determined as the new compound, 2-hydroxy-
6-((8Z,11Z)-14-hydroxyheptadeca-8,11-dien-1-yl)ben-
zoic acid.
The methylation of 4 by diazomethane afforded
methyl ester 11, which was then treated with Dess-
Martin periodinane to oxidize the hydroxy group
and, form derivative 16 (Scheme 1). The spectral
data of 16 were completely identical to those of 10;
comparison with the methylated 2 indicates that the
hydroxy group of 4 is positioned at C-12′. Thus,
compound 4 was identified as the new compound,
2-hydroxy-6-(12-hydroxyheptadecyl)benzoic acid.
Compound 7 has the same molecular formula, C22H36
O4, as that of 5. Because its 1H and 13C NMR spectra are
also similar to that of 5, it was suspected to be a positional
isomer of 5 at the hydroxy group. Detailed assignment of
the NMR signals based on the 1H-1H COSY and HMBC
spectra indicated a hydroxy group at the C-14′. Thus,
compound 7 was determined as 2-hydroxy-6-(14-
hydroxypentadecyl)benzoic acid as a new compound.
Because compounds 4–8 have an asymmetric carbon
in the alkyl side chain, their absolute configurations were
determined using the method described by Ohrui et al.
[16,20]. Compounds 4–8 have several overlapping sig-
nals in their 1H NMR spectra owing to the long alkyl side
chain; thus, the data was difficult to analyze using the
modified Mosher’s method as it requires many hydrogen
assignments [21]. Therefore, we used this method, whose
empirical rule between the absolute configuration and
chemical shift at the terminal methyl of the alkyl side
chain of the secondary alcohol is known. First, the phe-
nolic hydroxy and carboxy groups of 4–8 were methy-
lated with diazomethane to produce the corresponding
methyl esters, 9–15. Each methyl ester was then deriva-
tized using (1R,2R)- or (1S,2S)-2-(anthracene-2,3-dicar-
boximido)cyclohexanecarboxylic acid to form the
(1R,2R)- or (1S,2S)-ester, respectively. The (R)- and (S)-
configurations of the asymmetric carbon atoms were
judged by comparing the chemical shifts of the terminal
The negative HR-FAB-MS of
a deprotonated molecular ion at m/z 387.2538 [M−H]
(calcd. 387.2535), indicating that the molecular for-
6
showed
–
mula is C24H36O4 with six unsaturations. The 13C NMR
spectrum indicates the presence of one carboxy (δC
174.1), four olefinic (δC 124.7, 127.4, 130.6, and
132.0), one benzene (δC 111.0, 115.6, 122.4, 134.7,
147.3, and 163.5), one oxymethine (δC 72.0), one methyl
(δC 14.0), and 11 methylene (δC 18.8, 25.8, 26.8, 28.5,
28.5, 29.0, 29.8, 32.2, 34.9, 36.7, and 38.4) groups (Table
1). The 1H NMR signals at δH 6.74 (1H, d, J = 7.5 Hz),
6.84 (1H, d, J = 8.2 Hz), and 7.32 (1H, dd, J = 7.5,
8.2 Hz) indicate the presence of a 1,2,3-trisubstituted
benzene ring moiety (Table 2). Thus, 6 is also assumed
to contain a derivative of the C6-substituted salicylic
acid, which has a C17 side chain with C–C double bonds
and an oxymethine group.
From the H–H correlations in the COSY spectrum
was deduced the sequence from the terminal methyl
group (δH 0.95) to the four olefinic protons, that is
between methyl: H-17′, (δH 0.95), methylenes: H-16′
(δH 1.41) and H-15′ (δH 1.53), oxymethine: H-14′ (δH
3.81) and H-13′ (δH 2.33), olefinic proton: H-12′ (δH
5.43), olefinic proton: H-11′ (δH 5.60), olefinic pro-
ton: H-10′ (δH 2.83), olefinic proton: H-9 (δH 5.37),
and olefinic proton: H-8 (δH 5.38), as shown in
Scheme 1. Chemical derivatization of 2 and 4.

XANTHINE OXIDASE INHIBITORS FROM TYROMYCES FISSILIS
9
Figure 3. Structural representation of the chemical shifts observed for the terminal methyl group of the side chain.
methyl group of the alkyl side chain of the (1R,2R)- and
(1S,2S)-esters for each compound.
cooperative inhibition, while the alkenyl side chain
enhanced this effect by interacting with the hydropho-
bic site of XO [9].
The ester derivatives undergo a change in the
1,3-syn configuration of the carbonyl oxygen and
the hydrogen on the α-carbon of the alcohol, as well
as in the s-trans configuration of the carbonyl oxygen
and the α-hydrogen at the C2 position of the cyclo-
hexane moiety as shown in Figure 3.
For the (R)-ester, the change in the chemical
shift of the terminal methyl, {Δδ [δ (1R,2R) − δ
(1S,2S)] ppm}, is positive, whereas that of the (S)-
ester is negative. Therefore, because the differences
between the chemical shifts of the (1R,2R)-esters
and (1S,2S)-esters of 4–8 are positive (Table 3),
their hydroxy groups have the (R)-configuration.
Compounds 2–4, 6, and 7 are newly isolated com-
pounds, while the (S)-isomers of 5 and 8 have not
been reported thus far.
The compounds that have a salicylic acid moiety
with an aliphatic group at the C-6 position are known
as bioactive phytochemicals [22]. For example, com-
pound 1 has been shown to have inhibitory activity
against glyceraldehyde-3-phosphate dehydrogenase
[18] and antitumor activity [23]. The known com-
pounds, 5 and 8, exhibit inflammation suppressive
[15] and tyrosinase inhibitory activities [24].
Because a few anacardic acid derivatives isolated
from edible plants, such as cashew nuts [25], cashew
apple [26], mango peel [27], the newly isolated com-
pounds from T. fissilis are expected to show various
biological activities, such as enzyme inhibition and
antitumor activity, without causing a side effect.
Finally, we evaluated the XO inhibitory activities of
all isolated compounds and their derivatives following
the method described by Masuoka et al. [9] (Table 4).
Allopurinol was chosen as the positive control. Among
the tested compounds, compound 1 exhibits the highest
level of XO inhibition at a concentration of 25 μM
(inhibitory activity = 58.9 2.2%), which is comparable
to that of the positive control (inhibitory activ-
ity = 64.0 0.6%). Furthermore, XO inhibition by 1 is
noncompetitive, as shown in Figure 4. Compound 6,
which has two cis- double bonds in its alkyl side chain,
shows the next highest level of inhibition, 30.6 0.7% at
a concentration of 100 μM. Interestingly, methylated
derivative 9, exhibits a remarkably low activity com-
pared with its parent compound (1), suggesting that the
carboxy and hydroxy groups of the salicylic acid moiety
are essential for XO inhibition. Although further inves-
tigation is necessary, the existence of the salicylic acid
moiety and level of the side chain hydrophobicity seems
to play a significant role on XO inhibitory activity, based
on the correlation between the latter and the structure.
This supports previous experimental results showing
that the salicylic acid moiety of anacardic acid caused
Table 3. Differences in the chemical shifts for the terminal
methyl of the esters.
Chemical shift (ppm)
Compounds (1R,2R)-ester (1S,2S)-ester Δδ [δ (1R,2R) − δ (1S,2S)]
4
5
6
7
8
0.70
0.71
0.69
1.00
1.00
0.55
0.51
0.45
0.88
0.88
+0.15
+0.20
+0.24
+0.12
+0.12
Table 4. Xanthine oxidase inhibitory activity of 1–9 (n = 3).
Inhibition (%)
Compounds
25 µM
100 µM
1
2
3
4
5
6
7
8
58.9 2.2
7.8 1.9
11.0 2.3
9.1 1.4
0.9 2.0
13.2 0.6
8.4 1.5
8.1 1.2
7.9 1.5
64.0 0.6
63.4 1.4
16.9 0.5
18.6 0.3
15.9 2.6
10.3 1.8
30.6 0.7
7.4 1.2
11.1 1.9
3.4 4.3
98.3 0.6
9
Allopurinol

10
S. MITOMO ET AL.
Figure 4. Lineweaver-Burk plots of uric acid formation by XO. Multiple xanthine concentrations (0–50 μM) were oxidized by XO
in the presence of varying concentrations of 1 (●, 0 µM; ■, 25 µM; and ▲, 50 µM). Each data point represents the average of
three tests.
peroxide and superoxide formation. J Biol Chem.
1981;256:9096–9103.
Conclusions
In this study, eight XO-inhibiting compounds (1–8),
including five new compounds (2–4, 6, and 7), were
isolated from the fruiting bodies of T. fissilis, and their
chemical structures were elucidated by instrumental
analysis and chemical derivatization. In summary,
these results indicated a significant structure-activity
relationship, whereby the presence of a salicylic acid
moiety and hydrophobicity of the alkyl side chain
were found to be essential for XO inhibition.
Although additional work is required to evaluate the
biological activity of these compounds from fungus, this
study provides a new framework for further investiga-
tion on the structure-related activity of XO inhibitors.
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Author contributions
Conceived the work: T. F. and M. H. Designed the work:
T. F. Isolation and structure determination of compounds:
S. M. and T. F. Instrumental analysis and derivatization of
compounds: S. M. Wrote the manuscript: S. M. and
T. F. Reviewed the manuscript: M. H.
[7] Borghi C, Desideri G. Urate-lowering drugs and
prevention of cardiovascular disease: the emerging
role of xanthine oxidase inhibition. Hypertension.
2016;67:496–498.
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Febuxostat compared with allopurinol in patients
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2005;353:2450–2461.
Acknowledgments
[9] Masuoka N, Kubo I. Characterization of xanthine
oxidase inhibition by anacardic acids. Biochim
Biophys Acta. 2004;1688:245–249.
[10] Kemami Wangun HV, Hertweck C. Squarrosidine
and Pinillidine: 3,3′-Fused bis(styrylpyrones) from
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[11] Kang KB, Park EJ, Kim J, et al. Berchemiosides A-C,
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J Nat Prod. 2017;80:2778–2786.
We are grateful to Dr. Yasuko Okamoto (Tokushima Bunri
University) for performing the HR-FAB-MS measurements.
We also thank Mr. M. Nomura and Prof. M. Hirota (Shinshu
University) for collecting and providing the mushroom. This
work was supported by the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) KAKENHI under
Grant Number JP15H01751.
Disclosure statement
[12] Imazeki R, Ohtani K, Hongou T. Nihon no kinoko
[Japanese mushrooms]. Tokyo: Yama-kei Publishers;
1988. Japanese.
No potential conflict of interest was reported by the authors.
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Tyromycic acids B–E, new lanostane triterpenoids
from the mushroom Tyromyces fissilis. J Nat Prod.
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