Structure–activity relationships and docking studies of hydroxychavicol and its analogs as xanthine oxidase inhibitors

Article abstract of DOI:10.1248/cpb.c18-00197

Hydroxychavicol (HC), which is obtained from the leaves of Piper betle LINN. (Piperaceae), inhibits xanthine oxidase (XO) with an IC₅₀ value of 16.7μM, making it more potent than the clinically used allopurinol (IC₅₀=30.7μM). Herein, a structure–activity relationship analysis of the polar part analogs of HC was conducted and an inhibitor was discovered with a potency 13 times that of HC. Kinetic studies have revealed that HC and its active analog inhibit XO in an uncompetitive manner. The binding structure prediction of these inhibitor molecules to the XO complex with xanthine suggested that both compounds (HC and its analog) could simultaneously form hydrogen bonds with xanthine and XO.

Full text of DOI:10.1248/cpb.c18-00197

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c18-00197
Advance Publication
April 26, 2018
Chem. Pharm. Bull.
Regular Article
Structure–activity relationships and docking studies of hydroxychavicol
and its analogs as xanthine oxidase inhibitors
Keiji Nishiwaki*, Kanae Ohigashi, Takahiro Deguchi, Kazuya Murata, Shinya Nakamura,
†
Hideaki Matsuda , Isao Nakanishi
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, 3-4-1
Kowakae, Higashiosaka, Osaka 577-8502, Japan
†
Dedicate to Prof. H. M. who deceased on 26 April 2017.
*
Correspondence e-mail: k-nishi@phar.kindai.ac.jp
Ⓒ
2018 The Pharmaceutical Society of Japan

Summary
Hydroxychavicol (HC), which is obtained from the leaves of Piper betle Linn.
(
Piperaceae), inhibits xanthine oxidase (XO) with an IC50 value of 16.7 μM, making it
more potent than the clinically used allopurinol (IC50 = 30.7 μM). Herein, a
structure–activity relationship analysis of the polar part analogs of HC was conducted and
an inhibitor was discovered with a potency 13 times that of HC. Kinetic studies have
revealed that HC and its active analog inhibit XO in an uncompetitive manner. The binding
structure prediction of these inhibitor molecules to the XO complex with xanthine
suggested that both compounds (HC and its analog) could simultaneously form hydrogen
bonds with xanthine and XO.
Keywords hydroxychavicol; xanthine oxidase inhibitor; structure–activity relationship; docking study
Chemical and Pharmaceutical Bulletin Advance Publication

Introduction
Xanthine oxidase (XO) is a key enzyme in the purine metabolic pathway, catalyzing the
oxidation of hypoxanthine to uric acid. In addition, it is known as a target protein for the
1
, 2)
treatment of hyperuricemia and gout.
It also produces reactive oxygen species (ROS) in
parallel with the oxidation process. ROS have been associated with some pathological
3
)
conditions, such as the post-ischemic reperfusion injury, diabetes, and chronic heart failure.
Therefore, the selective inhibitor of XO may result in a broad-spectrum chemotherapeutic
4
)
agent applicable to gout, cancer, chronic inflammation, and oxidative damage.
Allopurinol (Fig. 1), a well-known XO inhibitor, has been widely prescribed for the
5)
treatment of gout for over 40 years. Since allopurinol is a purine analog, it could affect the
activities of an enzyme associated with purine and pyrimidine metabolism, thereby causing
life-threatening side effects, such as the hypersensitivity, i.e., the Stevens–Johnsons
6
)
syndrome. For this reason, there is a critical need for the discovery of novel XO inhibitors
4
)
independent of purine or pyrimidine scaffolds. In this decade, highly potent XO inhibitors,
such as febuxostat and topiroxostat (Fig. 1) with IC50 values of 4.4 and 69 nM, respectively,
Chemical and Pharmaceutical Bulletin Advance Publication

7
, 8)
have been developed.
Although these compounds are free from purine or pyrimidine
9
)
scaffolds, liver dysfunction has been reported as a serious side effect from their use.
10, 11)
Therefore, novel XO inhibitors with fewer and milder side effects are required.
From the extract of Piper betle Linn. (Piperaceae) leaves originating from South Asia and
Southeast Asia, in 2009, Matsuda et al. discovered hydroxychavicol (HC), a molecule that
1
2)
possesses an XO inhibitory activity that is twice that of allopurinol (Fig. 1). Although the
XO inhibitory activity of HC is relatively low compared with febxostat and topiroxostat, its
1
3)
ligand efficiency index value is as high as 0.60, indicating that HC is a good candidate as a
seed compound for a modification study. Recently, Honda and Masuda reported that
1
4)
pyrogallol (Fig. 1) in coffee also possessed XO inhibitory activity. It is interesting that such
polyphonols contained in natural plants indicated XO inhibiting potency. In this research,
therefore, the structure–activity relationship (SAR) of HC was investigated with a focus on
the polar component of the molecule. The number and position of the hydroxy groups on the
benzene ring were modified. In addition, the contribution of the allyl group was briefly
examined. On the other hand, the inhibition pattern of HC and its active analog were studied,
Chemical and Pharmaceutical Bulletin Advance Publication

and the binding structures of these compounds were predicted by a docking study.
Results and Discussion
Chemistry The designed HC analogs, including several known compounds, are listed in
Table 1 in addition to allopurinol and HC. These compounds were synthesized according to
1
5-23)
previously published methods.
Briefly, HC was obtained by removing a methylene group
of safrole. Chavibetol was synthesized via successive iodination and subsequent allylation by
Stille coupling, followed by the deacetylation of methoxyphenyl acetate. 3-Allylphenol (1)
was obtained from 1-bromo-3-methoxybenzene with an allyl Grignard reagent. 4-Allylphenol
(
2) was obtained by the demethylation of 4-allylanisole. 2-Allylhydroquinone (3) and
2
1
3
-allylresorcinol (4) were obtained from 2-bromo-1,4-dimethoxybenzene and
-bromo-3,5-dimethoxybenzene via the Grignard reaction and demethylation, respectively.
-Allylcatechol (5) was synthesized from the corresponding methyl ether via demethylation.
The monoallylation of resorcinol and subsequent Claisen rearrangement leads to
4
-allylresorcinol (6) and 2-allylresorcinol (7) at once. Finally, using the same procedure,
Chemical and Pharmaceutical Bulletin Advance Publication

5
-allylpyrogallol (8) was synthesized from pyrogallol. It should be noted that all reactions
were not optimized.
XO inhibition XO inhibition was measured according to the method developed by
2
4)
Chang. Allopurinol was used as a positive control, and all experiments were performed in
triplicate. The experimental data were tested for statistical significance using the
Bonferroni–Dunn multiple range test method. The IC50 values of HC and its analogs in vitro
are listed in Table 1.
Structure Activity Relationship First, we briefly examined the contribution of the alkyl
group. Catechol, formed by dropping the allyl group from HC, and 4-methylcatechol were
inactive. These results indicate that the allyl group may significantly contribute to the
inhibitory activity of HC. However, while phenol and catechol lost their inhibitory activity,
pyrogallol was as potent as allopurinol, with an IC value of 36.5 μM. The third hydroxy
5
0
group plays an effective role in enhancing any interaction. These results indicate that the allyl
group is not essential in XO inhibition; however, it is an important pharmacophoric moiety
for the HC analogs. Therefore, we examined the SAR of the polar site retaining the allyl
Chemical and Pharmaceutical Bulletin Advance Publication

group.
Because allylbenzene (Table 1) wherein two hydroxy groups on the benzene ring are
removed from HC was inactive, as the next step, the hydroxy groups were capped by methyl
group(s) to validate whether the hydroxy groups contribute as a hydrogen binding donor.
Both monomethylated analogs, i.e., eugenol and chavibetol, and the dimethylated analog, i.e.,
methyleugenol, were inactive. This suggests that the hydroxy groups would be involved in
hydrogen bond formation as proton donors. Subsequently, we examined whether the removal
of one hydroxy group could be tolerated for the inhibitory activity; thus, three positional
isomers, 2-, 3-, and 4-allylphenols, were tested. All three compounds were found to be
inactive, indicating that at least two hydroxy groups were mandatory for XO inhibition. For
this reason, all the dihydroxy derivatives of allylbenzene were tested. In total, there are six
isomers, compounds 3–7 (Table 1) and HC. Interestingly, all isomers except HC were found
to be inactive, indicating that the existence of at least two hydroxy groups at both the 3- and
4
-positions is required for the inhibitory activity. Finally, 5-allylpyrogallol (8) comprising
three hydroxy groups on allylbenzene at the 3-, 4-, and 5-positions was synthesized, and it
Chemical and Pharmaceutical Bulletin Advance Publication

indicated an IC50 value of 1.27 μM. This indicates that 8 is 13 times and 29 times more potent
than HC and pyrogallol, respectively.
Inhibition patterns To identify inhibition patterns of HC and 8, the inhibition kinetics of
both compounds were analyzed. The Lineweaver–Burk plots shown in Fig. 2 revealed that
both compounds inhibited XO by uncompetitive inhibition, indicating that these compounds
inhibit XO by binding to the XO complex with the substrate. It should be noted that the type
of inhibition of allopurinol and topiroxostat is competitive, and that of febuxostat is
2
5-27)
mixed.
Since the binding sites of HC and 8 were difficult to identify, it was assumed that
each compound bound just adjacent to the substrate in the enzyme–substrate complex. From
these observations, all hydroxy groups of catechol or pyrogallol units were found to form
hydrogen bonds with XO and/or the substrate.
Docking study Finally, a docking study was performed to investigate the binding
structures for HC and 8. The uncompetitive inhibition manner suggested docking both
compounds against XO under a complex state with a substrate. Therefore, the docking study
2
8)
of HC was performed using the crystal structure of XO with xanthine (PDB ID: 3EUB) and
Chemical and Pharmaceutical Bulletin Advance Publication

the docking site was set to around the area where xanthine was bound. Using the MOE
2
9)
dock, three binding structures were suggested for HC: A, B, and C (Fig. 3). In candidate A,
the catechol part of HC formed three hydrogen bonds, two of which were with xanthine and
one was with the carboxyl group of Glu802 sidechain of XO (Fig. 3a). Conversely, the
candidates B and C formed two hydrogen bonds with xanthine and XO (Figs. 3b and 3c). The
binding free energies ΔGbind for the three candidate structures were predicted using the
3
0)
molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method and are listed
in Table 2. Candidate A formed more hydrogen bonds than candidates B and C and thus had
the highest affinity. It should be noted that HC in candidates B and C could not maintain the
initial structures during MD simulations (Figs. 3e and 3f), whereas candidate A maintained
the docked structure keeping three hydrogen bonds (Fig. 3d). Consequently, candidate
structure A was adopted as the most appropriate binding structure of HC. This structure can
explain the active/inactive SAR of the monohydroxy and dihydroxy analogs.
Utilizing the same protocol, the binding structure and the binding free energy of 8 were
predicted. The most stable structure and its binding free energy are shown in Fig. 4 and Table
Chemical and Pharmaceutical Bulletin Advance Publication

2
, respectively. Compound 8 formed four hydrogen bonds through xanthine and XO,
increasing preferable electrostatic interactions compared with HC. Due to this effect, 8
obtained a higher affinity than HC by 2.8 kcal/mol. The difference of the predicted ΔGbind
values between HC and 8 did not reproduce the experimentally estimated value of 1.4
kcal/mol, with a twofold overestimation. This discrepancy might be caused by the
contribution of the binding entropy, which was being ignored by the prediction scheme.
Conclusion
Overall, a SAR study of HC and its analogs was performed and a novel inhibitor (8) was
discovered, with a potency 13 times that of HC (IC50 = 1.27 μM). These compounds inhibited
XO through an uncompetitive inhibitory mechanism. The docking study of HC and 8
suggested that HC and 8 formed three and four hydrogen bonds, respectively, with the
XO–xanthine complex and that the predicted binding affinity qualitatively reproduced
experimental values. To validate these predictions, hybrid compounds of HC (or 8) with
xanthine (or allopurinol) are currently under development. If our prediction is pertinent, we
Chemical and Pharmaceutical Bulletin Advance Publication

can obtain more potent compounds bound in a competitive manner.
Experimental Section
Chemistry
General Methods. Xanthine oxidase, methyleugenol and allopurinol were purchased
from Sigma-Aldrich (St. Louis, MO). Phenol and pyrogallol were purchased from Wako
pure chemical industries, Ltd (Osaka, Japan). Other chemicals and reagents were reagent
grade and were purchased unless otherwise noted. Melting points were taken on Yanaco
MP-S3 melting point apparatus and not corrected. NMR spectra were recorded on JEOL
1
13
1
13
JNM-AI400 ( H 400 MHz, C 100 MHz) or JEOL JNM-ECA500 ( H 500 MHz, C 125
3
MHz) spectrometers in CDCl with TMS as an internal standard. Mass spectra (EI) were
recorded on a JMS-HX100 spectrometer. Infrared spectra were recorded on a Jasco
FT/IR-460 Plus spectrophotometer. Microanalysis was performed on Yanaco MT-3. Unless
otherwise noted, all materials were obtained from commercial suppliers and used without
further purification. Thin-layer chromatography was carried out on a Merck precoated
Chemical and Pharmaceutical Bulletin Advance Publication

Silica gel 60 F254. Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. Flash column chromatography was
performed on Merck Silica gel 60 (230~400 mesh ASTM) according to Still's method. All
spectroscopic data for known compounds were in complete accord with literature values.
Registry numbers of synthesized compounds Hydroxychavicol: 1126-61-0, Chavibetol:
5
2
3
1
01-19-9,
3-Allylphenol
(1):
1446-24-8,
4-Allylphenol
(2):
501-92-8,
-Allylbenzene-1,4-diol (3): 5721-21-1, 5-Allylbenzene-1,3-diol (4): 142039-78-9,
-Allylcatechol (5): 1125-74-2, 4-Allylresorcinol (6): 1616-52-0, 2-allylresorcinnol (7):
746-89-0, 5-Allylpyrogallol (8): 850896-26-3
Syntheses of the test compounds
1
5)
Hydroxychavicol
To a solution of safrole 0.45 mL (3 mmol), BBr
3
1.22 g(3.3 mmol) in CH
2
Cl
2
(6.0 mL) was
o
added a solution of 1.0 M BBr
3
in CH
2
Cl
2
7.5 mL (7.5 mmol) dropwise at -78 C. The
o
solution was warmed up to 0 C for 1 h. The reaction mixture was added to sat. NaHCO
3
aq
2 2
and extracted with CH Cl and the subsequent purification by flash column chromatography
Chemical and Pharmaceutical Bulletin Advance Publication

(
2 3
hexane : Et O = 2 : 1 and then CHCl : EtOAc = 8 : 1) afforded hydroxychavicol (0.12 g,
2
7%).
1
6)
Chavibetol
2 2
To a solution of 2-methoxyphenyl acetate (0.75 mL, 5.1 mmol) in CH Cl (18 mL) iodine
o
monochloride (0.30 mL, 5.7 mmol) was added at 0 C under N
2
and stirred for 2 hs. The
reaction mixture was added to a cold sat. Na S O aq. and extracted with CH Cl and the
2
2
3
2
2
subsequent purification by flash column chromatography (hexane : CHCl = 4 : 1) afforded
3
5
-iodo-2-methoxyphenyl acetate (1.5 g, 99%). To a solution of 5-iodo-2-methoxyphenyl
3 4
acetate (0.84 g, 2.9 mmol), CsF (1.0 g, 7.2 mmol) and Pd(PPh ) (55 mg, 0.048 mmol) in
THF (30 mL) was added and stirred. To the mixture allyltributyltin (1.2 mL, 3.6 mmol) was
o
added and stirred for 10 hs at 45 C and then added water. The reaction mixture was shaken
vigorously and filtered off. The filtrate was extracted with ethyl acetate and the subsequent
2
purification by flash column chromatography (hexane : Et O = 3: 1) afforded
5
-allyl-2-methoxyphenyl acetate (0.42 mg, 71%). To a solution of 5-allyl-2-methoxyphenyl
acetate (0.42 mg, 2.0 mmol) in MeOH (1.7 mL), (THF 1.7 mL) and water (0.60 mL),
Chemical and Pharmaceutical Bulletin Advance Publication

2
LiOH･H O (0.337 mg, 8.0 mmol) was added and stirred for 3.5 hs. The reaction mixture
2
was added 1 M HCl aq and extracted with Et O. The extract afforded chavibetol (0.28 g,
8
4%) without further purification.
1
7)
3
-Allylphenol (1)
2
To magnesium (290 g, 12 mmol), 1-bromo-3-methoxybenzene (1 mL, 8 mmol) in Et O
(
40 mL) was added dropwise under nitrogen atmosphere. The mixture was refluxed until
o
magnesium was consumed and cooled to 0 C subsequently. To the solution allylbromide
(
2.4 mL, mmol) was added dropwise. After stirring for 2 hs at rt, the mixture was quenched
4
with sat. NH Cl aq. and extracted with ether and the subsequent purification by flash
column chromatography (hexane
:
ethyl acetate
=
19
:
1) afforded
1
-allyl-3-methoxybenzene
(667
mg,
56%).
To
a
stirred
solution
of
1
-allyl-3-methoxybenzene (667 mg, 4.5 mmol) in CH Cl (60 mL), an 1 M solution of
2 2
o
BBr
3
in CH
2
Cl
2
5.5 mL (5.5 mmol) was added dropwise at -78 C under nitrogen
o
atmosphere. The solution was allowed to warm up to 0 C and stirred for 2 hs at room
2 2
temperature. The reaction mixture was quenched with water and extracted with CH Cl .
Chemical and Pharmaceutical Bulletin Advance Publication

and the subsequent purification by flash column chromatography (hexane : diethyl ether =
3
: 1) afforded 3-allylphenol (302 mg, 50%).
1
8)
4
-Allylphenol (2)
To a stirred solution of 1-allyl-4-methoxybenzene (0.32 mL, 2 mmol) in CH
2
Cl
2
(30 mL),
o
an 1 M solution of BBr
3
in CH
2
Cl
2
7.8 mL (7.8 mmol) was added dropwise at -78 C under
o
nitrogen atmosphere. The solution was allowed to warm up to 0 C and stirred for 2hs at
room temperature. The reaction mixture was quenched with water and extracted with
CH Cl . and the subsequent purification by flash column chromatography (hexane : ethyl
2
2
acetate = 5 : 1) afforded 4-allylphenol (15 mg, 5.2%).
1
9)
2
-Allylhydroquinone (3)
To magnesium (290 g, 12 mmol), 2-bromo-1,4-dimethoxybenzene (1.2 mL, 8.0 mmol) in
Et O (40 mL) was added dropwise under nitrogen atmosphere. The mixture was refluxed
2
o
until magnesium was consumed and cooled to 0 C. Subsequently to the solution
allylbromide (2.1 mL, 8.4 mmol) was added dropwise. After stirring for 2 hs at rt the
4
mixture was quenched with sat. NH Cl aq. and extracted with ether and the subsequent
Chemical and Pharmaceutical Bulletin Advance Publication

purification by flash column chromatography (hexane : ethyl acetate = 19 : 1) afforded
1
2
-allyl-2,5- dimethoxybenzene (1.33g, 93%). The mixture of CS (70 mL), Al (2.0 g 74
mmol) and I (15 g 118 mmol) was refluxed for 2hs and cooled down to rt. To the stirred
2
2
solution, a solution of 1-allyl-2,5-dimethoxybenzene (1.33 g, 7.5 mmol) in CS (70 mL)
was added dropwise. The reaction mixture was stirred under reflux for 12hs. The reaction
mixture was quenched with water and extracted with Et O. The combined organic phase
2
washed with 1 M Na S O aqueous solution and dried over Na SO . The solvent was
2
2
3
2
4
removed and the resulting residue was dissolved in THF, subsequent purification by flash
3
column chromatography (CHCl : methanol = 5 : 1) afforded 2-allylhydroquinone (35 mg,
3
%).
2
0)
3
-Allylresorcinol (4)
To magnesium (290 g, 12 mmol), 1-bromo-3,5-dimethoxybenzene (0.86 g 4.0 mmol) in
THF (20 mL) was added dropwise under nitrogen atmosphere. The mixture was refluxed
o
until magnesium was consumed and cooled to 0 C subsequently. To the solution
allylbromide (1.2 mL, 15 mmol) was added dropwise. After stirring for 2 hs at rt the
Chemical and Pharmaceutical Bulletin Advance Publication

4
mixture was quenched with sat. NH Cl aq. was added and extracted with ether and the
subsequent purification by flash column chromatography (hexane : ethyl acetate = 19 : 1)
afforded 1-allyl-3,5-dimethoxybenzene (356 mg, 50%). The mixture of CS (35 mL), Al
2
(
2
0.40 g 74 mmol) and I (3 g 118 mmol) was refluxed for 2hs and cooled down to rt. To the
2
stirred solution, a solution of 1-allyl-3,5-dimethoxybenzene (0.75 g, 4.2 mmol) in CS (35
mL) was added dropwise. The reaction mixture was stirred under reflux for 12hs. The
reaction mixture was quenched with water and extracted with Et O. The combined organic
2
2 2 3 2 4
phase washed with 1 M Na S O aqueous solution and dried over Na SO . The solvent was
removed and the resulting residue was dissolved in THF. subsequent purification by flash
3
column chromatography (CHCl : methanol = 5 : 1) afforded 2-allylhydroquinone (34 mg,
6
%).
2
1)
3
-Allylcatechol (5)
To a stirred solution of 2-allyl-6-methoxyphenol (0.32 mL, 2 mmol) in CH
2
Cl
2
(30 mL),
o
an 1.0 M solution of BBr
3
in CH
2
Cl
2
7.8 mL (7.8 mmol) was added dropwise at -78 C
o
under nitrogen atmosphere. The solution was allowed to warm up to 0 C and stirred for 2hs
Chemical and Pharmaceutical Bulletin Advance Publication

at room temperature. The reaction mixture was quenched with water and extracted with
2 2
CH Cl . And the subsequent purification by flash column chromatography (hexane: ethyl
acetate = 5 : 1) afforded 3-allylcatechol (15 mg, 31%).
2
2)
4
-Allylresorcinol (6) and 2-allylresorcinol (7)
To a solution of resorcinol (0.77 g, 7.0 mmol) in dry acetone (7 mL), allylbromide (0.80
mL, 9.2 mmol) and K CO (1.1 g, 8.1 mmol) was added and stirred for 8hs. The reaction
2
3
mixture was filtered off. The filtrate was concentrated and purified by flash column
chromatography (hexane : Et O = 10: 1) afforded 3-(2-propenyloxy)phenol (0.43 g, 41%).
2
o
3
-(2-propenyloxy)phenol (0.43 g 0.28 mmol) was heated for 30 min. at 210 C. The
2
reaction mixture was purified with flash column chromatography (hexane : Et O = 10: 1)
afforded 4-allylresorcinol (6) (0.22 g, 51%) and 2-allylresorcinnol (7) (0.090 g, 21%).
2
3)
5
-Allylpyrogallol (8)
Suspension of allyl bromide (0.21 mL, 2.4 mmol) and sodium iodide (0.51 g, 3.4 mmol)
in acetone (5 mL) was stirred under nitrogen atmosphere at rt and prevented light by
aluminum foil. In other flask, suspension of pyllogarol (0.32 g, 2.5 mmol) and potassium
Chemical and Pharmaceutical Bulletin Advance Publication

bicarbonate (0.35 g, 2.5 mol) in acetone (5 mL) was stirred under nitrogen atmosphere at rt
and prevented light by aluminum foil. To a suspension of potassium salt of pyllogarol, allyl
iodide solution was added dropwise at rt. The mixture was refluxed for 6 days. The solvent
was evaporated and the residue was washed with sat. NH Cl solution. The aqueous phase
4
3
was extracted with CHCl and the subsequent purification by flash column chromatography
(
Ethyl acetate) afforded 5-allylpyrogallol (18 mg, 4.4%).
2
4)
XO inhibitory assay
Xanthine (3.0 mg) was dissolved in 100 ml of 0.1 M phosphate buffer (pH 7.8) with gentle
heating to make 200 μM of xanthine buffer. The test solution was prepared by dissolving
each sample into dimethyl sulfoxide. The assay mixture consisting of 10 μL of test solution
and 990 μL of xanthine buffer was prepared for the reaction. The reaction was initiated by
addition of 2 μL enzyme solution (20 units/mL). The mixture was vortexed and incubated at
room temperature for 4 min. The concentration of uric acid was measured by the optical
density (OD) at 295 nm by using a spectrophotometer (UV-2450PC, Shimadzu, Kyoto,
Japan).
Chemical and Pharmaceutical Bulletin Advance Publication

Computational studies
Docking studies were performed based on the crystal structure of the XO-xanthine complex
2
8)
(
PDB ID: 3EUB). Mammalian XO is formed with α, β-subunits. The α-subunit consists of
the FAD region and two 2Fe-2S clusters. The β-subunit contains a molybdopterin (Mo-pt)
cofactor. In this study, we used only Mo-pt containing region (Ala590-Thr1315) of the
β-subunit for the calculation to reduce computation time. Electrostatic potential (ESP) around
3
1)
Mo-pt was calculated using HF/6-31G*/LANL2DZ with Gaussian09 and restrained
3
2)
33)
electrostatics potential (RESP) charges for Mo-pt were assigned with Antechamber. The
3
4)
hydrogen atoms were added using SYBYL and all acidic and basic amino-acid residues
were set to charged state. To eliminate the distortion of hydrogen atoms, the structure
3
5)
optimization was done only to hydrogen atoms by the MMFF94x force field until the
maximum gradient was < 0.0001 kcal/(mol･Å). The structures of HC and 8 were optimized
using HF/6-31G*, and RESP charges for each atom were assigned with Antechamber.
Alpha spheres were generated around xanthine by Site Finder implemented in Molecular
2
9)
Operating Environment (MOE). Using MOE Dock, 100000 binding poses of HC were
Chemical and Pharmaceutical Bulletin Advance Publication

generated by a Triangle Matcher method and were evaluated using five scoring functions of
London dG, ASE, Affinity dG, Alpha HB and GBVI/WSA dG. Binding poses with the best
1
00 scores were subjected to structure optimization using the MMFF94x force field and then
scored again by the same evaluation functions. Out of five binding structures with the best
score for each scoring function, three structures which formed hydrogen bonds with both
xanthine and XO were selected as binding structure candidates.
3
6)
Water molecules with the TIP3P potential were spherically generated from the center of
3
7)
38)
the inhibitor molecule with the radius of 30 Å. The FF99SB and gaff force field were
assigned to XO and inhibitors, respectively. After structure optimization, molecular dynamics
3
9)
simulation was performed using Amber11 fixing the position of the Mo-pt moiety and
restraining the position of the protein atoms locating 20 Å far from inhibitor with 100
2
40)
kcal/(mol･Å ). All MD simulations were carried out at 300 K with the SHAKE procedure
to constrain hydrogen atoms from moving. An equilibration step of 500 ps, after a 50 ps
heating step, was followed by a production run of 2000 ps, with a time step of 1.0 fs, and
trajectories were collected every 5 ps. This simulation was repeated three times changing the
Chemical and Pharmaceutical Bulletin Advance Publication

initial velocity. Using total of 1200 snapshot structures, the binding free energy was
calculated by the mmpbsa_py_energy script of Amber11.
Acknowledgment
This study was supported by the MEXT-Supported Program for the Strategic Research
Foundation at Private Universities (S1411037, 2014–2018).
Conflict of Interest
The authors declare no conflict of interest.
Chemical and Pharmaceutical Bulletin Advance Publication

References and notes
1
) As a recent review for xanthine oxidase inhibitors: Šmelcerovič A, Tomovič K,
Šmelcerovič Z, Petronijevič Ž., Kocič G., Tomašič T., Jakopin Ž., Anderluh M., Eur. J.
Med. Chem., 135, 491-516. (2017).
2
) As a recent review for xanthine oxidase inhibitors: Ishikita H., Eger B. T., Okamoto K.,
Nishino T., Pai E. F., J. Am. Chem. Soc., 134, 999-1009 (2012).
3
4
5
) Nishino T., Okamoto K., J. Biol. Inorg. Chem., 20, 195-207 (2015).
) Chen C., Lü J. M., Yao Q., Med. Sci. Monit., 22, 2501-2512 (2016).
) Elion G. B., Callahan S., Nathan H., Bieber S., Rundles R. W., Hitchings G. H., Biochem.
Pharmacol., 12, 85-91 (1963).
6
7
) Pacher P., Nivorozhkin A., Szabó C., Pharmacol. Rev., 58, 87-114 (2006).
) Okamoto K., Kawaguchi Y., Eger B. T., Pai E. F., Nishino T., J. Am. Chem. Soc., 132,
1
7080-17083 (2010).
8
) Hosoya T., Ohno I., Nomura S., Hisatome I., Uchida S., Fujimori S., Yamamoto T, Hara
S., Clin. Exp. Nephrol., 18, 876-884 (2014).
Chemical and Pharmaceutical Bulletin Advance Publication

9
1
1
1
) Kikuchi H., Fujisaki H., Furuta T., Okamoto K., Leimkühler S., Nishino T., Sci. Rep., 2,
331(1)-331-(8) (2012).
0) Dhiman R., Sharma S., Singh G., Nepali K., Bedi P. M. S., Arch. Pharm. Chem. Life Sci.,
3
46, 7-16 (2013).
1) Fukunari A., Okamoto K., Nishino T., Eger B. T., Pai E. F., Kamezawa M., Yamada I.,
Kato N. J. Pharmacol. Exp. Ther., 311, 519-528 (2004).
2) Murata K., Nakao K., Hirata N., Namba K., Nomi T., Kitamura Y., Moriyama K.,
Shintani T., Iinuma M., Matsuda H., J. Nat. Med., 63, 355–359 (2009).
3) Hopkins A. L., Groom C. R., Alex A., Drug Discov. Today, 9, 430-431 (2004).
4) Honda S., Masuda T., J. Agric. Food Chem., 64, 7743−7749 (2016).
5) Brooks P. R., Wirtz M. C., Vetelino M. G., Rescek D. M., Woodworth G. F., Morgan B. P.,
Coe J. W., J. Org. Chem., 64, 9719-9721 (1999).
1
1
1
1
1
6) Qu M. N., Zhou L., Cao X. P., Chin. J. Chem., 24, 1625-1630 (2006).
7) Catarina B., Ingemar K., Susana A., Per-Ola J., Ismet D., Karolina E., Bertil S., Aasa R.,
PCT Int. Appl. WO 2008135488 (2008).
Chemical and Pharmaceutical Bulletin Advance Publication

1
1
2
2
2
2
8) Lin J. M., Prakasha G. A., S. Sharma A. K., Amin S., Bioorg. Med. Chem., 20, 3202-3211
(2012).
9) Yang H.-C., Yu J., Oh K.-B., Shin D.-S., Cho W.-J., Shin J., Kim S., Arch. Pharm. Res.,
3
0, 955-961 (2007).
0) Jo H., Choi M., Viji M., Lee Y. H., Kwak Y.-S., Lee, K., Choi N. S., Lee, Y.-J., Lee H.,
Hong J. T., Lee M. K., Jung J.-K., Molecules, 20, 15966-15975 (2015).
1) Coolen H. K. A. C., Meeuwis J. A. M., van Leeuwen P. W. M. N., Nolte R. J. M., J. Am.
Chem. Soc., 117, 11906-11913 (1995).
2) Gozzo F. C., Fernandes S. A., Rodrigues D. C., Eberlin M. N., Marsaioli A. J., J. Org.
Chem., 68, 5493-5499 (2003).
3) Liu Y, ZhaoY, Lu C, Zhang X., Zhengzhou Gongcheng Xueyuan Xuebao, 24, 25-28
(2003).
2
2
4) Chang W, Lee Y, Lu F, Chiang H., Anticancer Res., 13, 2165-2170 (1993).
5) Massey V., Komai H., Palmer G.,Elion G. B., J. Biol. Chem., 245, 2837-2844 (1970).
Chemical and Pharmaceutical Bulletin Advance Publication

2
2
6) Okamoto K., Eger B. T., Nishino T., Kondo S., Pai E. F. Nishimoto T., J. Biol Chem., 278,
1
848-1855 (2003).
7) Okamoto K., Matsumoto K., Hille R,. Eger B. T., Pai E. F., Nishino T., Proc. Natl. Acad.
Sci. USA, 101, 7931-7936 (2004).
2
2
3
8) Pauff J. M., Cao H., Hille R., J. Biol. Chem., 284, 8760-8767 (2009).
9) MOE Ver. 2011.10, Chemical Computing Group Inc., Montreal, Canada.
0) Kollman P. A., Massova I, Reyes C, Kuhn B., Huo S., Chong L., Lee M., Lee T., Duan Y.,
Wang W., Donini O., Cieplak P., Srinivasan J., Case D. A., Cheatham T. 3rd., Acc. Chem.
Res., 3., 889-897 (2000).
3
1) Frisch M. J., Trucks G. W., Schlegel H. B., Scuseria G. E., Robb M. A., Cheeseman J. R.,
Scalmani G., Barone V., Mennucci B., Petersson G. A., Nakatsuji H., Caricato M., Li X.,
Hratchian H. P., Izmaylov A. F., Bloino J., Zheng G., Sonnenberg J. L., Hada M., Ehara
M., Toyota K., Fukuda R., Hasegawa J., Ishida M., Nakajima T., Honda Y., Kitao O.,
Nakai H., Vreven T., Montgomery J. A., Peralta J. E., Ogliaro F., Bearpark M., Heyd J. J.,
Brothers E., Kudin K. N., Staroverov V. N., Keith T., Kobayashi R., Normand J.,
Chemical and Pharmaceutical Bulletin Advance Publication

Raghavachari K., Rendell A., Burant J. C., Iyengar S. S., Tomasi J., Cossi M., Rega N.,
Millam J. M., Klene M., Knox J. E., Cross J. B., Bakken V., Adamo C., Jaramillo J.,
Gomperts R., Stratmann R. E., Yazyev O., Austin A. J., Cammi R., Pomelli C., Ochterski
J. W., Martin R. L., Morokuma K., Zakrzewski V. G., Voth, G. A., Salvador P.,
Dannenberg J. J., Dapprich S., Daniels A. D., Farkas O., Foresman J. B., Ortiz J. V.,
Cioslowski J., Fox D. J. Gaussian 09 Revision C.01. Gaussian, Inc. 2010.
3
3
3
2) Christopher I. B., Piotr C., Wendy C., Peter, A. K., J. Phys. Chem., 97, 10269-10280
(1993).
3) Wang J. Wang W., Kollman P. A., Case, D. A. J. Mol. Graph. Model., 25, 247-260
(
2006).
4) SYBYL-X, Certara USA, Inc. 100 Overlook Center, Suite 101, Princeton, NJ 08540
USA.
3
3
5) Halgren T. A., Nachbar R. B., J. Comp. Chem., 17, 587-615 (1996).
6) Jorgensen W. L., Chandrasekhar J., Madura J. D., Impey R. W., Klein M. L., J. Chem.
Phys., 79, 926–935 (1983).
Chemical and Pharmaceutical Bulletin Advance Publication

3
3
3
4
7) Hornak V., Abel R. Okur A., Strockbine B., Roitberg A., Simmerling C., Proteins, 65,
7
12–725 (2006).
8) Wang J., Wolf R. M., Caldwell J. W., Kollman P. A., Case D. A. J. Comput. Phys., 25,
1157–1174 (2004).
9) Pearlman D. A., Case D. A., Caldwell J. W., Ross W. S., Cheatham T. E. III, DeBolt S.,
Ferguson D., Seibel G., Kollman P., Comp. Phys. Commun., 91, 1–41(1995).
0) Ryckaert J. P., Ciccotti G., Berendsen H. J. C., J. Comput. Phys., 23, 327–341 (1977).
Chemical and Pharmaceutical Bulletin Advance Publication

CN
O
N
NC
N
N
O
N
O
N
NH
N
S
OH
N
N
NH
H
Alloprinol
30.7)
Febxostat
(0.0044)
Topiroxostat
(0.069)
(
OH
OH
OH
OH
OH
Hydroxychavicol
16.7)
pyrogallol
(36.5)
(
Fig. 1. Structure of Alloprinol, Febxostat, Topiroxostat and HC. IC50 values (μM) are shown in parenthesis.
a)
b)
Fig. 2. Lineweaver−Burk plot analyses of XO by HC (a) and 8 (b) in the absence (control) and in the presence
of HC with xanthine as the substrate. (a) ◆: control, ▲: HC- 15 μM, ●: HC- 20 μM. (b) ◆: control, ▲: 8- 1
μM, ＊: 8- 2 μM, ×: 8- 3 μM, ●: 8- 5 μM, ■: and dashed line alloprinol 20 μM.
Chemical and Pharmaceutical Bulletin Advance Publication

a) Candidate sturcture A
d)
Asn768
Glu802
Glu802
xanthine
xanthine
Asn768
b) Candidate sturcture B
e)
Glu802
Glu802
xanthine
xanthine
c) Candidate sturcture C
f)
Asp872
xanthine
xanthine
Thr1010
Thr1010
Glu879
Fig. 3. The candidate binding structures for HC to the XO-xanthine complex.
a), b), and c) Candidate structures A, B, and C. d), e) and f) Structures after three MD simulations for candidate
A, B, and C (first: yellow, second: magenta, third: cyan). The blue dashed lines show hydrogen bonds.
Chemical and Pharmaceutical Bulletin Advance Publication