An improved method for the synthesis of butein using SOCl2/ETOH as catalyst and deciphering its inhibition mechanism on xanthine oxidase

Article abstract of DOI:10.3390/molecules24101948

Butein (3,4,2⁰,4⁰-tetrahydroxychalcone) belongs to the chalcone family of flavonoids and possesses various biological activities. In this study, butein was synthesized through aldol condensation catalyzed by thionyl chloride (SOCl₂)/ethyl alcohol (EtOH) for the first time. The optimal reaction conditions including the molar ratio of reactants, the dosage of catalyst, and the reaction time on the yield of product were investigated, and the straightforward strategy assembles the yield of butein up to 88%. Butein has been found to inhibit xanthine oxidase (XO) activity. Herein, the inhibitory mechanism of butein against XO was discussed in aspects of inhibition kinetic, fluorescence titration, synchronous fluorescence spectroscopy, and molecular docking. The inhibition kinetic analysis showed that butein possessed a stronger inhibition on XO in an irreversible competitive manner with IC₅₀ value of 2.93 × 10^?6 mol L^?1. The results of fluorescence titrations and synchronous fluorescence spectroscopy indicated that butein was able to interact with XO at one binding site, and the fluorophores of XO were placed in a more hydrophobic environment with the addition of butein. Subsequently, the result of molecular docking between butein and XO protein revealed that butein formed hydrogen bonding with the amino acid residues located in the hydrophobic cavity of XO. All the results suggested that the inhibitory mechanism of butein on XO may be the insertion of butein into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and inducing conformational changes in XO.

Full text of DOI:10.3390/molecules24101948

molecules
Article
An Improved Method for the Synthesis of Butein
Using SOCl /EtOH as Catalyst and Deciphering Its
2
Inhibition Mechanism on Xanthine Oxidase
Yu-Xue Hou, Shi-Wei Sun, Yang Liu, Yan Li, Xiao-Hong Liu, Wei Wang, Shuang Zhang and
Wei Wang *
Department of Natural Medicine and Pharmacognosy, School of Pharmacy, Qingdao University,
Qingdao 260021, China; hyx19931008@163.com (Y.-X.H.); sunsw@qdu.edu.cn (S.-W.S.);
buckuper@163.com (Y.L.); liyanyaohua@126.com (Y.L.); liuxiaohong1043@163.com (X.-H.L.);
justwangwade@126.com (W.W.); qdeduzhangshuang@163.com (S.Z.)
*
Correspondence: w.w.wangwei@263.net; Tel./Fax: +86-532-8699-1172
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Atanas G. Atanasov, Karel Šmejkal and Elke Heiss
Received: 30 April 2019; Accepted: 19 May 2019; Published: 21 May 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
0
0
Abstract: Butein (3,4,2 ,4 -tetrahydroxychalcone) belongs to the chalcone family of flavonoids
and possesses various biological activities. In this study, butein was synthesized through aldol
condensation catalyzed by thionyl chloride (SOCl )/ethyl alcohol (EtOH) for the first time. The optimal
2
reaction conditions including the molar ratio of reactants, the dosage of catalyst, and the reaction
time on the yield of product were investigated, and the straightforward strategy assembles the yield
of butein up to 88%. Butein has been found to inhibit xanthine oxidase (XO) activity. Herein, the
inhibitory mechanism of butein against XO was discussed in aspects of inhibition kinetic, fluorescence
titration, synchronous fluorescence spectroscopy, and molecular docking. The inhibition kinetic
analysis showed that butein possessed a stronger inhibition on XO in an irreversible competitive
− −1
6
manner with IC50 value of 2.93
×
10 mol L . The results of fluorescence titrations and synchronous
fluorescence spectroscopy indicated that butein was able to interact with XO at one binding site, and
the fluorophores of XO were placed in a more hydrophobic environment with the addition of butein.
Subsequently, the result of molecular docking between butein and XO protein revealed that butein
formed hydrogen bonding with the amino acid residues located in the hydrophobic cavity of XO.
All the results suggested that the inhibitory mechanism of butein on XO may be the insertion of
butein into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and
inducing conformational changes in XO.
Keywords: butein; SOCl /EtOH; xanthine oxidase; inhibitory mechanism; kinetic analysis; florescence
2
spectroscopy; molecular docking
1
. Introduction
Hyperuricemia characterized by an elevated level of uric acid in human blood is a critical cause
of gout [
1
–
3
]. It can result in the deposition of monosodium urate crystals in joint and soft tissue,
]. Currently, the increasing
with accompanying inflammation and degenerative consequences [4,5
clinical reports have demonstrated that hyperuricemia is associated with some other diseases such as
cardiovascular disorders, renal dysfunction, obesity, diabetes, hyperlipidemia, cancer, hypertension,
and metabolic syndrome [6]. Thus, maintaining uric acid at a normal level becomes an important therapy
for prevention of gout and related disorders. Xanthine oxidase (XO), with a function of catalyzing
hypoxanthine and xanthine to uric acid, is a pivotal enzyme in purine metabolism. Structurally, XO is a
homodimer with each monomer consisted of the molybdopterin (Mo-pt), the flavin adenine dinucleotide
Molecules 2019, 24, 1948; doi:10.3390/molecules24101948
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1948
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(FAD) and two distinct [2Fe–2S] centers [7]. Although a series of promising alternative treatments
of hyperuricemia including the utilization of interleukin-1 inhibitors, recombinant uricase therapy,
or the targeting of renal urate transporters have been respectively investigated [ 10], XO inhibitors
8–
remain a primary therapy owing to the fundamental inhibition of uric acid production. For decades,
allopurinol, a purine analogue, has been widely applied to manage hyperuricemia as a potent XO
inhibitor. While clearly effective, only about 40% of patients are able to meet treatment goals via
allopurinol, and it occasionally causes Stevens Johnson syndrome, which may be fatal [11]. Febuxostat,
a non-purine XO inhibitor, is efficacious as a second-line therapy in lowering serum uric acid levels in
patients with gout [12]. Unfortunately, mild adverse reactions of febuxostat including musculoskeletal
symptoms, abnormal liver-function values, diarrhea, and headache are exhibited, causing the Food
and Drug Administration to require a cautionary statement on the drug insert [13
,14]. Accordingly, the
search for XO inhibitors with fewer side effects than allopurinol and febuxostat is highly warranted.
0
0
Early studies have reported the XO inhibitory ability of butein (3,4,2 ,4 -tetrahydroxychalcone)
belonging to the chalcone family of flavonoids, however, the inhibitory mechanism of butein against
XO was unclear until now [15,16]. Therefore, clarifying the inhibitory mechanism of butein on
XO activity may provide new insights into the application of butein as a XO inhibitor. Although
butein has been isolated from several plants, such as Butea monosperma, Dalbergia odorifera, Semecarpus
anacardium, and Toxicodendron vernicifluum [17], the preparation of butein from natural plants suffers
from drawbacks such as tedious extraction and time-consuming isolation procedures, consumption of
large amounts of organic solvents, and poor yield. The main method for the synthesis of butein involves
Claisen-Schmidt condensation between 2,4-dihydroxyacetophenone and 3,4-dihydroxybenzaldehyde
in the present of basic catalyst [18]. Nonetheless, the issues of complex process and long reaction
time are exposed due to the protection and deprotection of hydroxyl groups [16]. Comparing with
the basic catalyst, the problem of low productivity was revealed via the adoption of the typical acidic
catalyst [19]. Fortunately, thionyl chloride (SOCl )/ethyl alcohol (EtOH) as a novel acidic catalyst for the
2
synthesis of butein was first developed to compensate the shortcoming in this paper, and the optimized
reaction condition was established. Subsequently, the inhibitory mechanism of butein against XO was
further explored using inhibition kinetic, fluorescence titration, synchronous fluorescence spectroscopy,
and molecular docking.
2
. Results and Discussion
2
.1. Synthesis of Butein
As outlined in Scheme 1, butein (c) was prepared via aldol condensation catalyzed by SOCl in
2
ethyl alcohol between 2,4-dihydroxyacetophenone (
a) and 3,4-dihydroxybenzaldehyde (b). Although
the SOCl /EtOH employed to synthesis chalcones has already been described [20
,21], butein was
2
synthesized in this study using SOCl /EtOH as a catalyst in one step for the first time and the protection
2
of the hydroxyl groups were avoided. The reaction mixture was extracted by ethyl acetate to obtain
the crude product. Pure butein was purified by reversed-phase preparative HPLC and structural
elucidation was identified by MS and NMR spectra. In order to further improve the synthetic reaction
efficiency of butein, we studied the influence of various parameters of the reaction such as the
molar ratio of reactants, the dosage of catalyst, and reaction time. The overall yield of butein was
then measured by HPLC. Firstly, the concentration of SOCl in ethyl alcohol was critical in aldol
2
condensation. The reactants
a
and
b (1:1, molar ratio) with different concentrations of SOCl (0.25, 0.5,
2
−
1
1
, 1.5, 2.0 mol L ) were evaluated for the synthetic reaction capacity. As depicted in Table 1, the yields
−1
−1
increased quickly among the concentrations of SOCl from 0.25 mol L to 1 mol L (entry 1–3), and
declined suddenly when the concentrations of SOCl exceeded 1 mol L (entry 4–5). The yields of
high catalyst concentration (2.0 mol L ) and low catalyst concentration (0.25 mol L ) were only 9%
2
−
1
2
−1
−1
and 2%, respectively. The highest yield of butein (85%) was afforded when the concentration of catalyst
−1
was 1 mol L , and thus was chosen for further study. Secondly, the molar ratio of two reactants was

Molecules 2019, 24, 1948
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also investigated. As can be seen in Table 1, the molar ratio of
a
and
b
increasing from 1:0.6 to 1:1
would lead to the increased of yields (entry 6–7). However, the yields decreased with the molar ratio
increasing from 1:1 to 1:1.4 (entry 8–9). Therefore, the molar ratio of two reactants of 1:1 (entry 3) was
recommended in our case. Besides, the yield of butein was not significantly increased at the longer
reaction time. To our delight, the yields could reach 88% within 1 h (entry 10–12), which extremely
saved the preparation time. On the basis of above results, a sets of parameters to synthesize butein
−1
was proposed: Reaction was carried out at SOCl 1 mol L , under room temperature for 1 h, and the
2
molar ratio of a and b is 1:1 (entry 11).
OH
O
O
OH O
OH SOCl /EtOH
OH
OH
2
CH3
H
HO
RT
HO
OH
a
b
c
Scheme 1. Synthesis of butein.
Table 1. Optimization of parameters for the synthesis of butein.
SOCl (mol L 1)
−
Entry
Molar Ratio (a/b)
Time (h)
Yield (%)
2
1
2
3
4
5
6
7
8
9
0.25
0.5
1
1.5
2
1
1
1
1
1:1
1:1
1:1
1:1
1:1
1:0.6
1:0.8
1:1.2
1:1.4
1:1
2
2
2
2
2
2
2
2
2
2
74
85
37
9
50
60
75
76
84
88
87
1
1
1
0
1
2
1
1
1
0.5
1
1.5
1:1
1:1
2
.2. Inhibition of Butein on XO Activity
The inhibitory effect of butein on XO activity was evaluated by the formation of uric acid using
xanthine as the substrate. As shown in Figure 1, the activity of XO was effectively inhibited by butein;
the inhibition rate increased significantly with increasing concentration of the inhibitor. The IC value
5
0
−
6
−1
of butein was determined to be 2.93
×
10 mol L , which was slightly higher than that of allopurinol
−6
−1
(
1.10
×
10 mol L ). This IC50 value of butein was in keeping with the result of previous report on
XO inhibitory activity [16]. The results indicated that butein was an effective XO inhibitor compared to
positive control allopurinol, as a result of the presence of carbonyl group and four hydroxyl groups
0
0
on two phenyl rings [22]. In the previous research, chalcones 3,4,2 ,5 -tetrahydroxychalcone and
0 0
3
,4,2 ,6 -tetrahydroxychalcone with a similar structure as butein have shown weaker inhibition on XO
−
6
−1
−6 −1
10 mol L , respectively [16]. The presence
with IC50 values of (17
±
8)
×
10 mol L and (35
±
10)
×
0
0
of hydroxyl groups at C (2 ) and C (4 ) on ring B showed stronger activity than those with the equivalent
substitutes but located at para- position on ring B at C (2 ) and C (5 ) or meta- position at C (2 ) and
C (6 ) may be contributes to an increment in the stabilization of the aromatic ring due to induce
0
0
0
0
0
0
effect [22
,
23]. Similarly, compound 2,4,2 ,5 -tetrahydroxychalcone with two hydroxyl groups located at
−6
−1
the meta- position on ring A at C (2) and C (4) showed weaker inhibition activity (16.3 × 10 mol L )
than butein [22]. That may be because the higher polarizability enhances the attractive dispersion
interactions with an aromatic residue of enzyme binding site through
π-π stacking interactions [24,25].
Thus, the potential inhibitory mechanism of butein on XO is worthy of further investigation.

Molecules 2019, 24, 1948
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Figure 1. Inhibitory effect of butein on xanthine oxidase (XO) activity (pH = 7.5, T = 298 K). The final
− −1 −1
6
concentrations of xanthine and XO were 300
×
10 mol L and 0.024 U mL , respectively. Each value
represents means (±) standard deviation (n = 3).
2
.3. Irreversibility
The inhibition of XO by diverse concentrations of butein was illustrated in Figure 2. The plots of
the velocity versus at various concentrations of XO were constructed to confirm the irreversibility of
butein mediated inhibition. The plots offered a series of parallel straight lines and the slopes of the
lines were unchanged with the increasing concentrations of butein, which indicated that butein was
an irreversible inhibitor on XO. To the best of our knowledge, most of flavonoids were reported as a
reversible inhibitor on XO [26–28]. The different inhibition type of butein on XO prompts us to further
understand the potential mechanism between butein and XO.
−
6
−1
−6
−1
Figure 2. Plots of
ν
versus [XO], c (xanthine) = 300
×
10 mol L , c(butein) = 0, 1, 2, 3 and 4
×
10 mol L
for curves a → e, respectively. Each value represents means (±) standard deviation (n = 3).
2
.4. Inhibition Kinetic Analysis
Enzyme inhibition kinetic experiments were carried out to further characterize the binding type
of butein against XO by analyzing the data from Lineweaver–Burk plots. As shown in Figure 3a,
the vertical axis intercept (1/Vmax) of the double-reciprocal Lineweaver–Burk plots remained the
same and horizontal axis intercept (
suggesting that butein is a competitive inhibitor. Figure 3b exhibits good linearity between K
−
1/K
m
) increased with accumulating concentrations of butein,
app
m
and the relative concentration of butein, signifying that butein bound in a single class of inhibition
sites on XO. The inhibition kinetic analysis can be inferred that butein bound to the same active site
of XO as xanthine and competed with xanthine during catalysis, just as the allopurinol under the
uniform experimental conditions [29]. The value of K (inhibition constant) was determined to be
i
.46 × 10− mol L .
7
−1
0

Molecules 2019, 24, 1948
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(
a)
(b)
−
−6
−1
Figure 3.
for curves d
deviation (n = 3).
(
a
) Lineweaver–Burk plots. c(XO)=0.08 U mL 1, c(butein) = 0.8, 0.4, 0.2
×
10 mol L and 0
) standard
app
→
a, respectively; (
b
) the K
m
versus [butein]. Each value represents means (
±
2
.5. Fluorescence Quenching of XO upon Butein
Fluorescence spectroscopy analysis was further used to research the binding properties of butein
with XO on account of its ability to provide information on the molecular level of the binding mode,
binding constant, and intermolecular distance. As shown in Figure 4a, under 280 nm excitation
wavelength, the fluorescence emission spectra of XO inhibited by different concentrations of butein
were excited. XO displayed two strong fluorescence emissions at 340 nm and 405 nm wavelengths
due to the presence of three intrinsic fluorophores on XO, including tryptophan, tyrosine, and
phenylalanine [30]. Nevertheless, butein has no intrinsic fluorescence under the same experiment
conditions. Besides, fluorescence intensity of XO is mainly attributed to tryptophan residues due to
the low quantum yield of phenylalanine. The results showed that XO emission intensities decreased
dramatically with increasing of butein concentrations, which indicated that butein directly interacted
with XO and quenched its intrinsic fluorescence.
(
a)
(b)
−
1
Figure 4.
3.03, 36.36
butein only, c(butein) = 36.36
XO by butein at different temperatures.
(a) c(XO) = 0.1 U mL and c(butein) = 0, 3.96, 7.84, 11.65, 15.39, 19.05, 22.64, 26.17, 29.63,
−6
−1
3
×
10 mol L for curves a
→
k, respectively; curve m shows the emission spectrum of
−
6
−1
×
10 mol L ; ( ) Stern–Volmer plots for the fluorescence quenching of
b
Generally, the ground state complex caused the static quenching, while the collision between the
fluorophore and the quencher lead to the dynamic quenching. Their excited-state lifetime was used to
distinguish two modes of mechanism [31]. The Stern–Volmer equation was applied to calculate the

Molecules 2019, 24, 1948
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KSV and K
q
values with maximal emission at 340 nm in order to ascertain the mechanism of butein
binding to XO, as follows [30]:
F0
F
=
1 + KSV[Q] = 1 + Kqτ [Q]
(1)
0
F0 and F are steady-state fluorescence intensities of fluorophore in the absence and presence
of butein, respectively. KSV and [Q] are Stern–Volmer dynamic quenching constant and quencher
q q
0
concentration, respectively. K is the quenching rate constant of biomolecule (K = KSV/τ0). τ is the
−8
average biomolecule lifetime, and its value is 10 s [32]. KSV can be obtained from linear regression
plot slope of F /F versus [Q].
0
As shown in Figure 4b, the plots of F /F versus [Q] at three temperatures (298, 304 and 310 K), and
0
the relevant K
q
values were given in Table 2. A high linearity suggested that only one type of quenching
process occurred, either static or dynamic quenching. When the fluorophore is a biomacromolecule,
1
0
−1 −1
its maximum scatter collision quenching constant is 2.0
×
10 L mol
s
[
q
33]. The K values
12
12
1012 L mol
−1 −1
(at 298, 304 and 310 K, respectively) were 2.09
×
10 , 2.55
×
10 , 3.10
×
s , which were
considerably greater than the maximum scatter collision quenching constant. The results implied that
the fluorescence quenching mechanism of XO by butein is static quenching, and resulted from the
formation of a butein–XO complex [34].
Afterwards, the fluorescence titration data were further analyzed to acquire the association
constant (Ka) applying the modified Stern–Volmer equation [35]:
F0
F0 − F
1
1
fa
=
+
(2)
f K [Q]
a
a
where f
gave the K
a
is the fraction of accessible fluorescence. The linear regression of F /(F0
−
F) versus 1/[Q]
values were in
0
a
4
values for the butein-XO complex at 298, 301, and 310 K (Table 2). The K
a
−1
order of 10 L mol and the high linear correlation coefficient R indicated a moderate affinity for the
butein–XO interaction [26].
For the static quenching reaction, it was assumed that small molecules bind independently into a
class of equivalent sites on a macromolecule. The apparent binding constant K and the number of
b
binding sites n can be calculated from the following equation [36]:
F − F
0
lg
= nlg K + nlg[Q]
(3)
b
F
where [Q] is the total concentrations of butein. As shown in Table 2, the values of n were found to be
nearly equal to 1, implying the existence of one binding site for butein on XO, which was consistent
with the results of inhibition-mode studies.
2
.6. Thermodynamic Analysis
Generally, hydrogen bonds, van der Waals forces, electrostatic forces and hydrophobic interactions
are regarded as the main forces acting small ligands bind to biomolecules. The acting force of the
butein–XO complexation could be illustrated by calculating the thermodynamic parameters. The values
of ∆H and ∆S were determined from the van’t Hoff equation:
∆
H
∆S
lgKa = −
+
(4)
(5)
2
.303RT 2.303R
∆
G = ∆H − T∆S
and R represent the binding constant and the gas constant (8.314 J mol
The temperatures were 298, 304 and 310 K. The values of H and
logK versus 1/T, and then the free energy change ( G) was obtained from Equation (5). As shown
−1
−1
where K
a
K ).
∆
∆S were determined by plotting
a
∆

Molecules 2019, 24, 1948
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in Table 2, the negative signal for
The positive values of ∆H (103.45 kJ mol ) and ∆S (427.94 J mol K ) suggested that the binding was
an exothermic process, and the complexation was predominately driven by hydrophobic forces [37].
∆
G meant that the interaction of butein with XO was spontaneous.
− −1 −1
1
Table 2. Quenching rate constant K
q a
, modified Stern–Volmer association constant K , and relative
thermodynamic parameters for the interaction between butein and XO at different temperatures.
Kq
Ka
∆H
(kJ mol
∆G
(kJ mol
∆S
(J mol
T (K)
Ra
Rb
n
× 1012 L mol
−1
S
−1
)
(× 10 L mol
4
−1
)
−1
)
−1
)
−1 −1
K )
(
298
304
310
2.09
2.55
3.10
0.9953
0.9878
0.9944
2.96
3.86
10.51
0.9921
0.9931
0.9991
0.91
0.75
1.00
−24.09
−26.66
−29.23
103.45
427.94
2
.7. Synchronous Fluorescence
The synchronous fluorescence spectroscopy has been performed to explore the molecular
environment around the chromophore groups (tyrosine and tryptophan) of XO [38]. Both of the
synchronous fluorescence intensities of tyrosine (∆λ = 15 nm) and tryptophan (∆λ = 60 nm) residues
declined obviously with the addition of butein to XO solution. As shown in Figure 5, tyrosine had
a visible blue-shift (from 290 nm to 286 nm) and tryptophan had a slight blue-shift (from 276 nm to
275 nm), suggesting that the polarity decreased and the hydrophobicity increased in the vicinity of
tyrosine and tryptophan, specifically, that the fluorophores of XO were exposed in a less hydrophilic
environment, and more exposed to the solutions with the addition of butein, which was consistent
with the result of thermodynamic analysis.
(
a)
(b)
Figure 5. Synchronous fluorescence spectra of XO in the absence and presence of butein c(XO) =
.1 U mL 1
−
,
and c(butein) = 0, 3.96, 7.84, 11.65, 15.39, 19.05, 22.64, 26.17, 29.63, 33.03, 36.36 × 10−6 mol L
−1
0
for curves a → k, respectively. (a) ∆λ = 15 nm; (b) ∆λ = 60 nm.
2
.8. Computational Docking of the Butein–XO Complex
Molecular docking was carried out to improve the understanding of interactions between butein
and XO protein. The crystal structure of XO enzyme (PDB ID, 3EUB) in complex with xanthine (Xan)
from bovine milk was used for the docking calculations [39]. For validation of the credible docking
method, the natural ligand Xan was docked back into the 3EUB protein as a control until the root
mean square deviation (RMSD) of best docked ligand conformation was less than 2.0 Å. As shown in
Figure 6A, a total of six conformational clusters were obtained from 100 docking runs at an RMSD
−1
tolerance of 2.0 Å, and the lowest binding energy with 7.77 kcal mol ,were the most optimal among
−
all the clusters (38 out of 100, red histogram in Figure 6A). In particular, as shown in Figure 6B,C,
ligand Xan and potent inhibitor butein were located at the same hydrophobic pocket of XO, which is
the active site with molybdenum atomic (Mo) domain, indicating that butein is a competitive xanthine
oxidase inhibitor against xanthine with occupying the catalytic center of XO to avoid the entrance of

Molecules 2019, 24, 1948
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xanthine and inducing conformational changes in XO. The docking results for butein suggest that
three hydrogen bonds were formed between the OH group of butein and the oxygen or hydrogen
atom of key amino acids (Figure 6C,D). The carboxy group of Glu802 was found to form H-bonds
0
with the hydrogen atom of 3-OH and 2 -OH in butein respectively. The carbonyl group in butein
was connected with the hydrogen atom of β-OH in Ser876. In addition, key hydrophobic contacts of
residues Ala1079, Phe914, Phe1009, Ala1078, Arg880, Val1011, Leu1014, Leu873 and Leu648 were also
found in the hydrophobic region of XO. All of these suggest that hydrogen bonds and hydrophobic
interaction play an important role in the binding between butein and XO.
Figure 6. Molecular docking results of butein to XO. (
runs of butein with 3EUB. ( ) The binding active positions of xanthine (Xan) and butein with 3EUB.
D) Analysis of docked butein bound to 3EUB showing the key interactions in the binding pocket.
A) Cluster analyses of the AutoDock docking
B,C
(
3
. Experimental Section
3
.1. Materials
Mass spectra were obtained with a Bruk micro-TOFQ mass spectrometer. NMR spectra were
acquired on a Bruker AV-500 FT-NMR spectrometer (Bruker Daltonics, Bremen, Germany) operating
1
13
◦
at 500.1 MHz for H and at 125.8 MHz for C at 25 C. The chemical shifts were referenced to the
residual solvent signals 2.05 and 206.2, 29.8 for (CD ) CO. 2,4-dihydroxyacetophenone and
δ
H
δ
C
3 2
3
,4-dihydroxybenzaldehyde were purchased from Aladdin Chemical Reagent Company (Shanghai,
China). Xanthine oxidase from bovine milk (0.2 units/mg protein), whereas xanthine, sodium phosphate,
allopurinol were purchased from Sigma and Aldrich Chemical Co. (St. Louis, MO, USA).

Molecules 2019, 24, 1948
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3
.2. General Procedure for the Synthesis of Butein
SOCl (0.4 mL) was added dropwise to a stirred solution of the 2,4-dihydroxyacetophenone
2
(1 mmol) and 3,4-dihydroxybenzaldehyde (1 mmol) in ethanol (5.1 mL). The reaction mixture was
stirred at room temperature for 2 h. At the end of reaction, the mixture was extracted with ethyl acetate
by the addition of water. The ethyl acetate layer was washed with water and saturated brine, dried
over anhydrous Na SO . After concentration under reduced pressure, the crude product was further
2
4
purified by reversed-phase preparative HPLC (Agilent Prepstar SD-1 pump connected to a Prostar
UV-Vis detector, column Megress ODS-C , 20 250 nm, i.d., 10 m) to give butein. The elution was
×
µ
18
MeOH-H O (55:45, v/v) at a flow rate 3.0 mL/min, and the sample injection volume was 200
µL. The UV
2
detector was set at 254 and 365 nm.
1
[
1-(2,4-Dihydroxyphenyl)-3-(3,4-dihydroxyphenyl) prop-2-en-1-one] (
c
): Yellow power, H NMR
0
0
(
(
6
acetone-d6)
δ: 13.62 (1H, s, 2 -OH), 8.11 (1H, d, J = 8.8 Hz, H-6 ), 7.77 (1H, d, J = 15.2 Hz, H-
β), 7.68
1H, d, J = 15.2 Hz, H-
α
), 7.35 (1H, br s, H-2), 7.22 (1H, d, J = 8.1 Hz, H-6), 6.91 (1H, d, J = 8.1 Hz, H-5),
0
0
13
.47 (1H, dd, J = 8.8, 1.8 Hz, H-5 ), 6.37 (1H, d, J = 1.8 Hz, H-3 ). C NMR (acetone-d ) : 192.8 (C=O),
0 0
67.6 (C-4 ), 165.6 (C-2 ), 149.3(C-4), 146.5 (C-3), 145.5 (C-β), 133.3 (C-6 ), 128.2 (C-1), 123.4 (C-6), 118.3
δ
6
0
1
0 0 0
+
(C-α), 116.4 (C-2), 116.0 (C-5), 114.5 (C-1 ), 108.7 (C-5 ), 103.8 (C-3 ). ESI-MS m/z 273.08, [M + H] .
3
.3. Enzyme Activity Assay
The XO inhibitory activity assay was performed according to the method modified by our
group [29]. The butein was dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted
−1
with phosphate buffer (0.07 mol L , pH = 7.5) to a final concentration containing less than 1%
DMSO (v/v). In the 0.8 mL reaction system, a series of assay solutions prepared in phosphate
−
1
−1
buffer (0.07 mol L , pH = 7.5) consisting of a fixed concentration of XO (0.024 U mL ) and various
◦
concentrations of inhibitor solutions were incubated at 25 C for 30 min, and the reaction was initiated
by adding the substrate xanthine (300
was incubated at 25 C for 30 min. The reaction was stopped by the addition of HCl (1 mol L ).
The absorbance of uric acid level was determined by an HPLC method using a Diamonsil ODS-C18
−6
−1
×
10 mol L ) to the complex solutions. The assay mixture
◦
−1
column (250
×
4.6 mm i.d., 5 µm, Dikma Technologies, Beijing, China) on an Agilent 1260 system
equipped with a G1311C quaternary pump, a G1329B autosampler, a G1316A thermostatted column
compartment, and a G1314F variable wavelength detector coupled with an analytical workstation
(Agilent Technologies, Inc., Santa Clara, CA, USA). Averages of three replicates are presented and the
allopurinol was used as a positive control.
3
.4. Kinetic Analysis for Inhibitory
The assay was investigated applying the XO activity assay methodology in the absence and
−
6
presence of butein with a series of concentrations of substrate xanthine (25, 37.5, 50, 75, and 100
×
10
−
1
−6 −1
mol L ), where butein was at diverse concentrations (0, 0.2, 0.4, 0.8
×
10 mol L ) and xanthine at a
fixed concentration. The involved inhibition mode of the butein on xanthine oxidase was analyzed from
Lineweaver–Burk plots, and K value was identified by the software GraphPad Prism 5.0 (GraphPad
i
Software Inc., La Jolla, CA, USA). For competitive inhibition, the Lineweaver–Burk equation can be
written in double reciprocal form as follows:
!
1
v
Km
Vmax
[I] 1
K [S]
i
1
=
1 +
+
(6)
(7)
Vmax
A secondary plot can be plotted from:
Km[I]
Kmapp
=
+ Km
Ki

Molecules 2019, 24, 1948
10 of 13
m
where K and K represent the inhibition constant and Michaelis–Menten constant, respectively, and
i
their values can be calculated from Equations (6) and (7). v denotes enzyme reaction rate in the absence
and presence of butein. [I] and [S] are the concentrations of the inhibitor and substrate, respectively.
app
app
K
m
presents the apparent Michaelis-Menten constant. The secondary plot of K
m
versus [I] is
linearly fitted, indicating a single inhibition site or a single class of inhibition site [40].
3
.5. Fluorescence Titration
Fluorescence titration assay was conducted by a spectrofluorimeter (Model F-4600, Hitachi, Japan)
equipped with a thermostat bath and a 150 W xenon lamp to characterize the interaction between butein
−1
and XO. Briefly, 2.0 mL sample of XO solution (0.1 U mL ) was titrated with different concentrations
of butein from 0 to 36.36
− −1
6
×
10 mol L and then the solutions were incubated 5 min to equilibrate.
Afterwards, fluorescence emission spectra of the solutions were measured using a spectrofluorometer
at different temperatures (298, 304 and 310 K) in 300–500 nm wavelength range upon excitation 280 nm.
The widths of both the excitation and emission slits were set at 5 nm. The background fluorescence of
the buffer (0.07 mol L PBS solution, pH = 7.5) was subtracted from butein–XO complexes.
Synchronous fluorescence spectra were performed by setting the excitation and emission
wavelength interval (∆λ) at 15 and 60 nm over a wavelength range of 260–330 nm and 250–330 nm,
respectively.
−1
All the fluorescence-quenching data were corrected for absorption of excitation light and
reabsorption of emitted light by the following relationship [41]:
F = Fme(A1 + A2)/2
(8)
where F
c m
1 2
and F represent the corrected and measured florescence. A and A are the absorbance of
butein at excitation and emission wavelengths, respectively.
3
.6. Molecular Docking
The potential binding site of XO-butein was researched on AutoDock (version 4.2, The Scripps
Research Institute, La Jolla, CA, USA). The X-ray crystal structure (PDB ID, 3EUB) of XO used for the
docking studies [42] was download from the Protein Data Bank (http://www.rcsb.org/pdb). All water
molecules and ligand in XO were removed prior to the docking program running, after which the
polar hydrogen atoms and Gasteiger charges were added to the macromolecule file. The 3D structures
of butein were obtained in Chem 3D Ultra 8.0. The active site with a fixed grid spacing (0.375 points)
was enclosed by a rigid dimension of docking center (100
×
100
×
100 points). The best scoring docked
model of the ligand was chosen to represent its most desirable binding mode predicted by AutoDock.
The PyMol molecular graphic system was used to visualize the conformations and interactions between
the butein and the target proteins.
4
. Conclusions
In summary, a simple and effective method for the synthesis of butein by aldol condensation with
the catalytic system SOCl /EtOH was conducted for the first time in the present study. The optimal
2
reaction conditions including the molar ratio of reactants, the dosage of catalyst, and the reaction time
were investigated, and its potential inhibition of XO was discussed in aspect of inhibition kinetics,
fluorescence titrations, synchronous fluorescence spectroscopy, and molecular docking. The butein
0
0
can be prepared in good yield from 2 ,4 -dihydroxyacetophenone and 3,4-dihydroxybenzaldehyde
based on the catalytic system SOCl /EtOH, without protecting the hydroxyl groups. In addition,
2
the inhibitory activity on XO showed that butein could significantly reduce uric acid concentration
− −1
6
and the IC50 value of restraining XO was found as 2.93
×
10 mol L in vitro. Additionally, an
attractive inhibition mechanism of irreversible competition is worth further exploration, as it might
be helpful in treating gout. The intrinsic fluorescence of XO was quenched statically by butein, and

Molecules 2019, 24, 1948
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butein bound spontaneously into the active cavity of XO to form the butein–XO complex with one
high-affinity binding sites. Besides, hydrophobic interaction is the main driving force of butein to
binding with XO. The molecule docking revealed butein is a competitive XO inhibitor and has a high
affinity binding site on XO. The effectiveness of butein in reducing uric acid concentration in the blood
is required to evaluate by the further in vivo studies. This study has supplied significant insights into
the inhibitory mechanism of butein on XO, which will promote butein to be a leading compound by
structural modification on the treatment of gout.
Author Contributions: The contributions of the respective authors are as follows: Y.-X.H. performed the synthesis
of butein. Y.L. (Yang Liu) engaged in the bioassay of butein in vitro. S.Z. and W.W. (the 6th author) carried out
the kinetic analysis and fluorescence titration assay. X.-H.L. performed the computational docking. S.-W.S. and
Y.L. (Yan Li) contributed to checking and confirming all of the procedures of the experiments. This study was
performed based on the design of W.W. (the 8th author), the corresponding author. All the authors read and
approved the final manuscript.
Funding: This project was founded by Shandong Provincial Natural Science Foundation, China, grant numbers
[ZR2018BD030 and ZR2016HB22].
Conflicts of Interest: The authors declare no conflict of interest.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).