Design, synthesis and molecular modeling of aloe-emodin derivatives as potent xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.01.058

A series of aloe-emodin derivatives were synthesized and evaluated as xanthine oxidase inhibitors. Among them, four aloe-emodin derivatives showed significant inhibitory activities against xanthine oxidase. The compound 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbaldehyde (A1) possessed the best xanthine oxidase inhibitory activity with IC₅₀ of 2.79 μM. Lineweaver-Burk plot analysis revealed that A1 acted as a mixed-type inhibitor for xanthine oxidase. The docking study revealed that the molecule A1 had strong interactions with the active site of xanthine oxidase and this result was in agreement with kinetic study. Consequently, compound A1 is a new-type candidate for further development for the treatment of gout.

Full text of DOI:10.1016/j.ejmech.2014.01.058

Accepted Manuscript
Design, synthesis and molecular modeling of aloe-emodin derivatives as potent
xanthine oxidase inhibitors
Da-Hua Shi, Wei Huang, Chao Li, Yu-Wei Liu, Shi-Fan Wang
PII:
S0223-5234(14)00104-4
10.1016/j.ejmech.2014.01.058
EJMECH 6711
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2013
Revised Date: 23 January 2014
Accepted Date: 28 January 2014
Please cite this article as: D.-H. Shi, W. Huang, C. Li, Y.-W. Liu, S.-F. Wang, Design, synthesis and
molecular modeling of aloe-emodin derivatives as potent xanthine oxidase inhibitors, European Journal
of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.01.058.
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ACCEPTED MANUSCRIPT
GR APH IC AB S TR AC T
Design, synthesis and molecular modeling of aloe-emodin
derivatives as potent xanthine oxidase inhibitors
Da-Hua Shia,b, Wei Huanga, Chao Lia, Yu-Wei Liu c , Shi-Fan Wanga,
a School of Ocean, Hainan University, Haikou 570228, People’s Republic of China
b School of Chemical Engineering, Huaihai Institute of Technology, Lianyungang
222005, People’s Republic of China
c Marine Economic Research Center of Jiangsu, Huaihai Institute of Technology,
Lianyungang 222005, People’s Republic of China
The structure of compound A1 and the expected interaction between compound A1
and the binding site of XDH (PDB NO. 1VDV)
Address for Correspondence: School of Ocean, Hainan University, Haikou 570228,
People’s Republic of China, E-mail: wangsf777@163.com

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ꢀ We designed and synthesized a series of aloe-emodin derivatives.
ꢀ We evaluated the xanthine oxidase-inhibition activities of those compounds.
ꢀ The compound A1 possessed the best activity with IC50 of 2.79 µM.
ꢀ We identified A1 was a mixed-type xanthine oxidase inhibitor.
ꢀ Using the docking study we found A1 could interact with the catalyze site of
xanthine oxidase.

ACCEPTED MANUSCRIPT
Design, synthesis and molecular modeling of aloe-emodin
derivatives as potent xanthine oxidase inhibitors
Da-Hua Shia,b, Wei Huanga, Chao Lia, Yu-Wei Liuc, Shi-Fan Wanga,
a School of Ocean, Hainan University, Haikou 570228, People’s Republic of China
b School of Chemical Engineering, Huaihai Institute of Technology, Lianyungang
222005, People’s Republic of China
c Marine Economic Research Center of Jiangsu, Huaihai Institute of Technology,
Lianyungang 222005, People’s Republic of China
Address for Correspondence: School of Ocean, Hainan University, Haikou 570228,
People’s Republic of China, E-mail: wangsf777@163.com
1

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GR APH IC AB S TR AC T
Design, synthesis and molecular modeling of aloe-emodin
derivatives as potent xanthine oxidase inhibitors
Da-Hua Shia,b, Wei Huanga, Chao Lia, Yu-Wei Liuc , Shi-Fan Wanga,
a School of Ocean, Hainan University, Haikou 570228, People’s Republic of China
b School of Chemical Engineering, Huaihai Institute of Technology, Lianyungang
222005, People’s Republic of China
c Marine Economic Research Center of Jiangsu, Huaihai Institute of Technology,
Lianyungang 222005, People’s Republic of China
The structure of compound A1 and the expected interaction between compound A1
and the binding site of XDH (PDB NO. 1VDV)
Address for Correspondence: School of Ocean, Hainan University, Haikou 570228,
People’s Republic of China, E-mail: wangsf777@163.com
2

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ABSTRACT
A series of aloe-emodin derivatives were synthesized and evaluated as xanthine
oxidase inhibitors. Among them, four aloe-emodin derivatives showed significant
inhibitory
activities
against
xanthine
oxidase.
The
compound
4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carbaldehyde (A1) possessed the
best xanthine oxidase inhibitory activity with IC50 of 2.79 ꢀM. Lineweaver-Burk plot
analysis revealed that A1 acted as a mixed-type inhibitor for xanthine oxidase. The
docking study revealed that the molecule A1 had strong interactions with the active
site of xanthine oxidase and this result was in agreement with kinetic study.
Consequently, compound A1 is a new-type candidate for further development for the
treatment of gout.
Keywords: aloe-emodin; xanthine oxidase; inhibitor; gout
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1. Introduction
Xanthine oxidase (XO; EC 1.17.3.2) is a key enzyme in the purine metabolic
pathway[1]. There is an overwhelming acceptance that XO serum levels are increased
in various pathological states like hepatitis, inflammation, ischemia-reperfusion,
cancer and aging. Thus, the selective inhibition of XO may result in broad spectrum
therapeutics for gout, cancer, inflammation and oxidative damage[2]. Most XO
inhibitors such as allopurinol, 2-alkylhypoxanthines were purine analogs. The purine
analogs could effect on the activities of purine and pyrimidine metabolism enzyme
and induce life-threatening side effects[2]. Therefore, finding the non-purine
compounds with potent XO-inhibition activities and fewer side effects instead of the
purine analogs are in great demand[1]. As a matter of fact, some non-purine XO
inhibitors, such as Y-700, a 1-phenylpyrazole derivative[3], FYX-051, a
3,5-diaryltriazole
derivative[4],
2-(Pyridin-4-yl)-quinolin-4(1H)-one[5],
1-(3-(Furan-2-yl)-4,5-dihydro-5-(pyridin-4-yl)pyrazol-1-yl)ethanone[6], flavonoids[7]
and curcumin[8] have been reported (Fig 1.). Recently, febuxostat, a novel non-purine
XO inhibitor, has received approval for the treatment of hyperuricemia in gout
patients in USA and Europe (Fig 1.)[9]. Some natural products have been reported to
possess potent XO inhibitory activities [10, 11] and could be selected as the leading
compounds for developing novel XO inhibitors. For example, Wu et al. found that a
xanthone possessed good XO-inhibition activity, but the solubility of the xanthone
was very poor. So they modified the compound and got series of novel xanthone
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derivatives as XO inhibitors[12]. For this reason, screening and modifying of natural
products is an important way to find the novel non-purine XO inhibitors.
In our previous screening of XO inhibitors from natural products, we found that
the aloe-emodin (Fig. 2), an anthraquinone which is the major anthraquinone in aloe
plants not only possesses antibacterial, antiviral, hepatoprotective, anticancer, laxative
and anti-inflammation effects[13], but also showed the weak XO-inhibition activities
with the inhibitory rate of 22.47% at the concentration of 50 ꢀg/mL. In continuation
of our previous work on finding novel non-purine XO inhibitors[11, 14], we designed
and synthesized a series of aloe-emodin derivatives and tested their XO-inhibition
activities.
2. Results and Discussion
2.1. Chemistry
Four series of 18 aloe-emodin derivatives A1-A5, B1-B5, C1-C4 and D1-D4
(Scheme 1-2) were synthesized starting from aloe-emodin (1) according to the
methods we reported previously[15]. In series A (Scheme 1), the alcoholic hydroxyl
of aloe-emodin (1) was first oxidized into the formyl group in anhydrous CH2Cl2
under the catalytic action of pyridinium chlorochromate (PCC) to obtain the
compound A1. Then, the A1 was modified by the condensation or the acylation with
different amine to get compounds A2 and A3, respectively. The compound A1 was
further oxidized to acid A4 and A4 was amidated to obtain A5. In series B (Scheme 1),
aloe-emodin (1) was methylated to get B1. Then, B2 was synthesized similarly with
A1. Compounds B3-B5 were gotten by condensation of B2 with different amine in
high yields, respectively. In series C (Scheme 2), the alcoholic hydroxyl group of
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aloe-emodin (1) was initially replaced by a chlorine atom and then the quaternary
ammonium was introduced into the molecule of aloe-emodin to get three derivatives
of aloe-emodin C2-C4. In series D (Scheme 2), the alcoholic hydroxyl group of B1
was initially replaced by a chlorine atom to give D1. Then the quaternary ammonium
was introduced into the molecule of D1 to get three derivatives of aloe-emodin
D2-D4.
2.2. Biological activities
Anthraquinones, such as anthragallol, were reported to possess XO-inhibition
activities[10]. Aloe-emodin is the major anthraquinone in aloe plants and possesses
weak XO-inhibition activity. In this study, we synthesized a series of derivatives of
aloe-emodin. The in vitro bovine XO inhibitory activities of those aloe-emodin
derivatives were measured by spectrophotometry at 295 nm with the allopurinol as a
reference compound[11]. The results are shown in Table 1. Among the 18
aloe-emodin derivatives, the compound A1, A2, A3 and C3 showed the potent
XO-inhibition activities. Compared with the positive control of allopurinol (IC50 =
11.23 ꢀM), the IC50 values of A1, A2, A3 and C3 are 2.79 ꢀM, 3.87 ꢀM, 8.43 ꢀM and
18.67 ꢀM, respectively. The compound A1 that possessed the highest XO-inhibition
activity could inhibit the XO dose-dependently (Fig. 3). The Lineweaver-Burk plot
(Fig. 4) revealed that compound A1 was a mixed-type XO inhibitor. Compound
B1-B5 and compound D1-D4 with the methylation of 4,5-hydroxyl groups exhibited
significantly weak anti-XO activities compared with compounds A1-A5 and
compound C1-C4 (Table 1). These phenomena implied that the hydroxyl groups of
those aloe-emodin derivatives might play a significant role in XO-inhibition activities.
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These phenomena are consistent with the findings [16, 17]. According to the
XO-inhibition activities of the compound A1-A5, the side chains have also effects on
their inhibitory activities (Table 1). The XO-inhibition activities were decreased with
the increased volume of side chains. On the other hand, XO-inhibition activities of the
series C also revealed the importance of the side chains. The compound C3 possessed
the potent XO inhibitory activity with IC50 of 18.67 ꢀM, while the compound C2 and
C4 inhibited XO activities less than 10% inhibitory rate at the concentration of 50
ꢀM.
2.3. Molecular modeling
In order to understand the binding conformation of the most potent XO inhibitor
A1, its flexible molecular docking was carried out into the active site of XO using the
docking software AUTODOCK4.2 (University of California, San Francisco). The
xanthine-binding site of xanthine dehydrogenase (XDH) co-crystallized with Y-700
(PDB entry code 1VDV) was chosen as the template for docking because that both A1
and Y-700 are mixed-type XO inhibitors and that there is no difference between the
xanthine binding-sites of XO and XDH crystal structures[18]. The docking results
showed that A1 could bind to the xanthine-binding site of XDH in a way similar to
that for the Y-700 with the binding energy of -6.99. The Fig. 5 shows the docking
conformation of compound A1 at the binding site of XDH.
The major interactions of A1 with XDH included seven hydrogen bonds with
GLU802, SER876, ARG880, and THR1010. The 4-OH and 10-carbonyl of the
compound were involved in hydrogen bonding with GLU802 residue. The formyl
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group of the side chain interacted with the THR1010 residue with two H-bonds and
interacted with ARG880 with one H-bond. 9-carbonyl of the compound interacted
with SER876 and THR1010 by H-bonds at the same time (Fig. 5). The docking
results show again the free 4,5-hydroxyl groups and the formyl group in the molecule
A1 played a significant role in the XO-inhibition activities. One possible explanation
for these findings is that the phenolic moiety of the compounds via hydrogen bonding
and electrostatic force interacts with key amino acid residues such as ARG880,
GLU1261 and THR1010[16, 19].
3. Conclusions
A series of aloe-emodin derivatives were designed, synthesized and evaluated as
xanthine oxidase inhibitors. Among them, four compounds A1, A2, A3 and C3
showed the potent XO-inhibition activities and the IC50 values of A1, A2, A3 and C3
are 2.79 ꢀM, 3.87 ꢀM, 8.43 ꢀM and 18.67 ꢀM, respectively. Active-relationship
analysis and docking study revealed that A1 could interact with the xanthine-binding
site of XO. The free 4,5-hydroxyl groups and the formyl group of side chain of the
compound played significant roles for the XO-inhibition activity.
4. Experimental
4.1. Chemistry
Reaction and the resulted products were monitored by thin-layer chromatography
(TLC) on the self-made pre-coated silica gel 60 F254 plates. Spots were detected by a
UV lamp visualized at 254 nm and/or 365 nm. Melting points (˚C, uncorrected) were
determined on a XT4 MP apparatus (Taike Corp., Beijing, China). The IR spectra
1
were recorded on a Thermo iS10 IR spectrometer (KBr pellets) and HNMR spectra
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were recorded in CDCl3 or d6-DMSO on a Bruker DPX400 spectrometer with TMS
and solvent signals allotted as internal standards. Splitting patterns are as follows: s,
singlet; d, doublet; dd, double doublets; t, triplet; m, multiplet. Chemical shifts are
reported in δ (ppm) and coupling constants are given in Hertz. ESI mass spectra were
recorded on a LCMS-IT-TOF mass spectrometer. Aloe-emodin (1) were purchased
from Shanxi Sciphar Biotechnology Co. Ltd. (Shanxi, China) and recrystallized from
DMF, m.p. 225-227 ˚C; IRνmaxcm-1: 3131, 2367, 1672, 1628, 1572, 1400, 1288;
1HNMR (d6-DMSO): δ = 4.60 (s, 2H, CH2), 5.58 (s, 1H, HO-CH2), 7.21 (s, 1H,
H-C(2)), 7.31 (d, 1H, J = 8.0Hz, H-C(7)), 7.59 (s, 1H, H-C(4)), 7.62 (d, 1H, J =
7.5Hz, H-C(5), 7.74 (dd, 1H, J1 = 7.5Hz, J2 = 8.0Hz, H-C(6)), 11.90 (s, 1H, HO-C(1)),
11.97 (s, 1H, HO-C(8)); HRMS (ESI): calcd for [M+H]+ (C15H11O5) m/z 271.0606,
found 271.0611. XO from buttermilk, xanthine and allopurinol were purchased from
Sigma-Aldrich Co.. Other reagents, all being A.R. grade, were purchased from
Shanghai Chemical Reagent Company (Shanghai, China). All solvents were distilled
and dried before use.
4.1.1 Synthesis of 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-
carbaldehyde (A1)
0.33 g (1.50 mmol) of pyridinium chlorochromate (PCC) was added in 200 mL
of dichloromethane containing 0.27 g of compound 1 (1.0 mmol). The yielded
mixture was stirred at room temperature 6 h until the reaction was accomplished.
After the completion of reaction, the reaction mixture was washed with water in the
separatory funnel. The dichloromethane layer was separated and the aqueous layer
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was extracted twice with dichloromethane. The combined dichloromethane solutions
were dried for eight hours over anhydrous sodium sulfate and then the
dichloromethane was distilled off to afford a yellow solid. Recrystallization of the
yellow solid from ethanol gave compound A1 as yellow needle, yield 60 %; m.p.
208-210 ˚C; IRνmaxcm-1: 3131, 1703, 1672, 1626, 1401, 1267; 1HNMR (d6-DMSO): δ
= 7.44 (d, 1H, J = 8.2 Hz, 1H, H-C(6)), 7.78 (s, 1H, H-C(1)), 7.84(s, 1H, H-C(3)),
7.87 (dd, 1H, J1 = 8.2Hz, J2 = 7.5Hz, H-C(7)), 8.15 (d, 1H, J = 7.5Hz, H-C(8)), 10.13
(s, 1H, CHO), 11.94 (s, 2H, OH); HRMS (ESI): calcd for [M+H]+ (C15H9O5) m/z
269.0450, found 269.0453.
4.1.2. Synthesis of (E)-1,8-dihydroxy-3-((phenylimino)methyl)anthracene-9,10-dione
(A2)
0.093 g (1.0 mmol) of aniline was added into a solution of compound A1 (0.268
g, 1.0 mmol) in absolute toluene (100 mL) under stirring at 112 ˚C for 3 h, monitored
by TLC. After the completion of reaction, the toluene of the reaction mixture was
evaporated at reduced pressure to afford a yellow solid. Recrystallization of the
yellow solid from ethanol gave compound A2 as yellow needle, yield 45 %; m.p.
1
235-237 ˚C; IRνmaxcm-1: 3132, 3005, 2937, 1703, 1666, 1586, 1277; HNMR
(d6-DMSO): δ=7.08-7.70 (m, 5H, on the ring of phenyl), 7.69 (d, 1H; J=8.0Hz,
H-C(7)), 7.76 (dd, 1H, dd, J1 = 8.0 Hz, J2 = 7.8Hz, H-C(6)), 8.05 (d, 1H, J = 7.8Hz,
H-C(5)), 8.23 (s, 1H, H-C(2)), 8.35 (s, 1H, H-C(4)), 8.60 (s, 1H, CH=N), 11.95 (s,
1H, HO-C(1)), 12.01 (s, 1H, HO-C(8)); HRMS (ESI): calcd for [M+H]+ (C21H14NO4)
m/z 344.0923, found 344.0929.
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4.1.3. Synthesis of (3R,4S,5S,6R)-6-(acetoxymethyl)-3-((E)-((4,5-dihydroxy-
9,10-dioxo-9,10-dihydroanthracen-2-yl)methylene)amino)tetrahydro-2
H-pyran-2,4,5-triyl triacetate (A3)
As described for A2, compound A1 was treated with acetylated glucosamine to
give compound A3 as yellow needle from ethanol, yield 69 %; m.p. 231-233 ˚C;
1
IRνmaxcm-1: 3031, 2853, 1749, 1624, 1253, 1220, 761; HNMR (CDCl3): δ=
1.90-2.21(m, 12H, 4COCH3), 3.57-5.98 (m, 7H, H-C (acetyl glucosamine)), 7.33(d,
1H, J=7.7Hz, H-C(6)), 7.67(s, 1H, H-C(1)), 7.72(dd, 1H, J1=7.8Hz, J2=8.1Hz,
H-C(7)), 7.87(d, 1H, J=8.1Hz, H-C(8)), 8.12(s, 1H, H-C(3)), 8.30(s, 1H, CH=N),
12.01(s, 1H, OH-5), 12.04(s, 1H, OH-4); HRMS (ESI): calcd for [M+H]+
(C29H28NO13) m/z 598.1561, found 598.1555.
4.1.4. Synthesis of 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxylic
acid (A4)
A mixture of compound 1 (1 mmol, 0.27g), PCC (2 mmol, 0.45 g), DMF (100
ml) and 2mL of water were stirred at room temperature for 24h, monitored by TLC.
After the completion of reaction, 100 mL of ice water was added under stirring to
afford orange precipitate. The orange precipitate was filtered, washed and dried.
Recrystallization of the orange precipitate from ethanol gave compound A4 as brown
acicular crystals, yield 62 %; m.p. 310-313 ˚C; IRνmaxcm-1: 3432, 3059, 2930, 1695,
1628, 1270, 1192, 757; 1HNMR (d6-DMSO): δ=7.58 (d, 1H, J = 8.2 Hz, 1H, H-C(6)),
7.85 (s, 1H, H-C(1)), 7.97 (dd, 1H, J1 = 8.2Hz, J2 = 7.5Hz, H-C(7)), 8.01 (s, 1H,
H-C(3)), 8.45 (d, 1H, J = 7.5Hz, H-C(8)), 11.87 (s, 1H, HO-C(1)), 11.94 (s, 1H,
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HO-C(8)), 13.90 (s, 1H, COOH); HRMS (ESI): calcd for [M+H]+ (C15H9O6) m/z
285.0399, found 285.0403.
4.1.5. Synthesis of (3R,4S,5S,6R)-6-(acetoxymethyl)-3-(4,5-dihydroxy-
9,10-dioxo-9,10-dihydroanthracene-2-carboxamido)tetrahydro-2H-pyra
n-2,4,5-triyl triacetate (A5)
0.42 g (1.2 mmol) of acetylglucosamine and 0.25 g (1.2 mmol) of
dicyclohexylcarbodiimide (DCC) were added into a solution of compound A4
(0.284g, 1.0mmol) in anhydrous DMF (50 mL) under stirring at room temperature for
26 h, monitored by TLC. After the completion of reaction, the reaction mixture was
filtered to remove the precipitate. 2 mL of glacial acetic acid were slowly added into
the filtrate until no more precipitation was come out, standing at 4 ˚C for 12 h. The
precipitate was filtered and then 20 mL of water was added into the filtrate under
stirring to afford light yellow precipitate. The expected product was purified by
column chromatography with the acetic ether-cyclohexane mixture (v:v=4:5) to give
compound A5 as pale yellow needle, yield 53 %; m.p. 239-242 ˚C; IRνmaxcm-1: 3388,
1
2937, 1750, 1627, 1543, 1278, 1221, 753; HNMR (CDCl3): δ=2.08-2.14(m, 12H,
4COCH3), 3.96-5.84 (m, 7H, H-C (acetyl glucosamine)), 7.26 (d, 1H, J=8.3Hz,
H-C(6)), 7.30 (s, 1H, H-C(1)), 7.63 (dd, 1H, J1=8.0Hz, J2=8.5Hz, H-C(7)), 7.65(s,
1H, H-C(3)), 7.67 (d, 1H, J=8.2Hz, H-C(8)), 7.93(s, 1H, NH), 11.73(s, 1H, OH-5),
11.79(s, 1H, OH-4); HRMS (ESI): calcd for [M+H]+ (C29H28NO14) m/z 614.1510,
found 614.1504.
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4.1.6. Synthesis of
3-(hydroxymethyl)-1,8-dimethoxyanthracene-9,10-dione (B1)
Aloe-emodin (1 mmol, 0.27 g), dimethyl sulfate (10 mmol, 1 mL) and potassium
carbonate (10 mmol, 1.38 g) in 200 mL of dry acetone were refluxed for 12 h at 56 ˚C
till the reaction was accomplished. After the completion of reaction, the yielded
mixture was cooled to room temperature and filtered. The filtrate was distilled to
afford a yellow solid. Recrystallization of the yellow solid from acetone gave
compound B1 as yellow needle, yield 65 %; m.p. 223-225 ˚C; IR/cm-1: 3129, 2989,
1662, 1591, 1400, 752; 1HNMR (d6-DMSO): δ = 3.91 (s, 6H , 2CH3), 4.64 (d, J = 5.7
Hz, 2H, CH2O), 5.54 (t, J = 5.7Hz, 1H, OH), 7.21 (1H, s, H-C(2)), 7.30 (1H, d, J =
8.2 Hz, H-C(7)), 7.58 (1H, s, H-C(4)), 7.61 (1H, d, J = 7.5Hz, H-C(5)), 7.74 (1H, dd,
J1 = 8.2Hz, J2 = 7.5Hz, H-C(6)); HRMS (ESI): calcd for [M+H]+ (C17H15O5) m/z
299.0919, found 299.0924.
4.1.7. Synthesis of 4,5-dimethoxy-9,10-dioxo-9,10-dihydroanthracene-
2-carbaldehyde (B2)
0.33 g (1.50 mmol) of pyridinium chlorochromate (PCC) was added in 250 mL
of dichloromethane containing 0.30 g of compound B1 (1.0 mmol). The yielded
mixture was stirred at room temperature 3 h until the reaction was accomplished.
After the completion of reaction, the reaction mixture was washed with water in the
separatory funnel. The dichloromethane layer was separated and the aqueous layer
was extracted twice with dichloromethane. The combined dichloromethane solutions
were dried for eight hours over anhydrous sodium sulfate and then the
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dichloromethane was distilled off to afford a yellow solid. Recrystallization of the
yellow solid from acetone gave compound B2, yield 73 %; m.p. 195-197 ˚C;
IRνmaxcm-1: 3026, 2966, 2929, 2872, 2839, 1671, 1582, 1283; 1HNMR (d6-DMSO): δ
= 4.04 (s, 6H , 2CH3), 7.26 (s, 1H, H-C(1)), 7.34 (d, 1H, J = 8.2 Hz, H-C(6)), 7.68 (s,
1H, H-C(3)), 7.88 (d, 1H, J = 7.5Hz, H-C(8)), 8.32 (dd, 1H, J1 = 8.2Hz, J2 = 7.5Hz,
H-C(7)), 10.13 (s, 1H, CHO); HRMS (ESI): calcd for [M+H]+ (C17H13O5) m/z
297.0763, found 297.0755.
4.1.8. Synthesis of
(E)-4,5-dimethoxy-9,10-dioxo-9,10-dihydroanthracene- 2-carbaldehyde
oxime (B3)
0.11 g (1.5 mmol) of hydroxylamine hydrochloride and 0.28 g (2.0 mmol) of
anhydrous potassium carbonate were added into a solution of compound B2 (0.296 g,
1.0 mmol) in absolute ethyl alcohol (50 mL) under stirring at 65˚C for 0.5 h,
monitored by TLC. After the completion of reaction, the reaction mixture was filtered
to remove the solid of inorganic salt and the solvent was evaporated at reduced
pressure o afford a yellow solid. Recrystallization of the yellow solid from ethanol
gave compound B3 as yellow needle, yield 65 %; m.p. 247-249 ˚C; IRνmaxcm-1: 3315,
1
3059, 2929, 1659, 1587, 1283, 1237, 753; HNMR (d6-DMSO): δ=3.94 (s, 6H ,
2CH3), 7.53 (s, 1H, H-C(1)), 7.66 (d, 1H, J = 8.2 Hz, H-C(6)), 7.69 (s, 1H, H-C(3)),
7.74 (d, 1H, J = 7.5Hz, H-C(8)), 7.92 (dd, 1H, J1 = 8.2Hz, J2=7.5Hz, H-C(7)), 8.31 (s,
1H, CH), 11.78 (s, 1H, NOH) ; HRMS (ESI): calcd for [M+H]+ (C17H14NO5) m/z
312.0872, found 312.0879.
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4.1.9. Synthesis of
(E)-1,8-dimethoxy-3-((phenylimino)methyl)anthracene- 9,10-dione (B4)
As described for A2, compound B2 was treated with aniline to give compound
B4 as orange needle from ethanol, yield 70 %; m.p. 225-228 ˚C; IRνmaxcm-1: 3092,
1
2927, 1666, 1586, 1277, 1235, 788; HNMR (CDCl3): δ = 4.12 (s, 6H , 2CH3),
7.18-7.70(m, 5H, on the ring of phenyl), 7.65 (d, 1H; J = 8.1Hz, H-C(7)), 7.71 (dd,
1H, dd, J1 = 8.1Hz, J2 = 7.8Hz, H-C(6)), 8.03 (d, 1H, J = 7.5Hz, H-C(5)), 8.18 (s, 1H,
H-C(2)), 8.31 (s, 1H, H-C(4)), 8.57 (s, 1H, CH=N); HRMS (ESI): calcd for [M+H]+
(C23H18NO4) m/z 372.1236, found 372.1230.
4.1.10. Synthesis of
(E)-3-(hydrazonomethyl)-1,8-dimethoxyanthracene-9,10-dione (B5)
As described for B3, compound B2 was treated with hydrazine sulfate to give
compound B4 as bright yellow needle from ethanol, yield 74 %; m.p. 280 ˚C (dec.);
1
IRνmaxcm-1: 3062, 2958, 1668, 1590, 1286, 1235, 748; HNMR (CDCl3): δ = 3.54(s,
2H, NH2), 4.03 (s, 6H , 2CH3), 7.33 (d, 1H; J = 8.1Hz, H-C(7)), 7.67 (dd, 1H, dd, J1 =
8.1Hz, J2 = 7.8Hz, H-C(6)), 7.86 (d, 1H, J = 7.4Hz, H-C(5)), 7.95 (s, 1H, H-C(2)),
8.16 (s, 1H, H-C(4)), 8.73(s, 1H, CH=N); HRMS (ESI): calcd for [M+H]+
(C17H15N2O4) m/z 311.1032, found 311.1038.
4.1.11. Synthesis of
3-(chloromethyl)-1,8-dihydroxyanthracene-9,10-dione (C1)
2 mL of thionyl chloride was slowly added into a solution of compound A1 (0.27
g, 1.0 mmol) in anhydrous DMF (20 mL) under stirring at room temperature for 26 h,
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monitored by TLC. After the completion of reaction, 60 mL of ice water was added
under stirring to afford orange precipitate. The orange precipitate was filtered, washed
and dried. Recrystallization of the orange precipitate from ethanol gave compound C1
as yellow needle, yield 55 %; m.p. 177-178 ˚C; IRνmaxcm-1: 3426, 3041, 1672, 1627,
1569, 1478, 1454, 1422, 1381, 1276; 1HNMR (CDCl3): δ = 4.62 (s, 2H, CH2), 7.31 (s,
1H, H-C(4)), 7.35 (d, 1H, J = 8.0Hz, H-C(7)), 7.71 (dd, 1H, J1 = 7.5Hz, J2 = 8.0Hz,
H-C(6)), 7.85 (s, 1H, H-C(2)), 7.86 (d, 1H, J = 7.5Hz, H-C(5), 12.04 (s, 1H, HO),
12.07 (s, 1H, HO); HRMS (ESI): calcd for [M+H]+ (C15H10ClO4) m/z 289.0268,
found 289.0265.
4.1.12. Synthesis of
1-((4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-
2-yl)methyl)pyridi-1-ium chloride (C2)
1 mL of dry pyridine was slowly added into a solution of compound C1 (0.29 g,
1.0 mmol) in anhydrous CHCl3 (50 mL) under stirring at room temperature. The
reaction mixture was allowed to stand at room temperature for 2 weeks and orange
crystals were formed at the bottom of the vessel, yield 60 %; m.p. 235 ˚C (dec.);
IRνmaxcm-1: 3418, 3059, 2955, 1629, 1563, 1280, 1214, 755; 1HNMR (D2O): δ = 5.92
(s, 2H, CH2), 7.19 (d, 1H, J = 8.4Hz, H-C (6)), 7.24(s, 1H, H-C (1)), 7.43(d, 1H, J =
7.4Hz, H-C (8)), 7.46(s, 1H, H-C (3)), 7.62 (dd, 1H, J1 = 8.2Hz, J2 = 7.8Hz, H-C (7)),
8.20(d, 2H, J = 6.8Hz, H-C (pyridyl-3',5')), 8.70 (dd, 1H, J1 = 7.9Hz, J2 = 7.9Hz, H-C
(pyridyl-4')), 9.02ꢀd, 2H, J = 5.6Hz, H-C (pyridyl-2',6')), 11.81 (s, 2H, 2OH); HRMS
(ESI): calcd for [M-Cl]+ (C20H14NO4) m/z 332.0943, found 332.0941.
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4.1.13. Synthesis of
1-(4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-
2-yl)-N,N,N-trimethylmethanaminium chloride (C3)
As described for C2, compound C1 was treated with trimethylamine to give
compound C3 as yellow needle, yield 65 %; m.p. 230 ˚C (dec.); IRνmaxcm-1: 3413,
3022, 2957, 1629, 1477, 1285, 1200, 752; 1HNMR (D2O): δ = 3.20(s, 9H, CH3), 4.95
(s, 2H, CH2), 7.19(d, 1H, J = 8.4Hz, H-C (6)), 7.40(s, 1H, H-C (3)), 7.44(d, 1H, J =
7.5Hz, H-C (8)), 7.60(s, 1H, H-C (1)), 7.64(dd, 1H, J1 = 8.2Hz, J2 = 7.8Hz, H-C (7)),
12.05 (s, 2H, 2OH); HRMS (ESI): calcd for [M-Cl]+ (C18H18NO4) m/z 312.1256,
found 312.1257.
4.1.14. Synthesis of
N-((4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracen-2-
yl)methyl)-N,N-dimethylbenzenaminium chloride (C4)
As described for C2, compound C1 was treated with N,N-dimethylaniline to
give compound C4 as orange needle, yield 63 %; m.p. 182 ˚C (dec.); IRνmaxcm-1:
1
3414, 3008, 2928, 1636, 1489, 1271, 1215, 746; HNMR (D2O): δ = 3.52 (s, 6H,
2NCH3), 5.02 (s, 2H, CH2), 6.97 (s, 1H, H-C (1)), 7.05 (d, 1H, J = 8.4Hz, H-C (6)),
7.20 (s, 1H, H-C (3)), 7.28 (d, 1H, J = 7.4Hz, H-C (8)), 7.35 (dd, 1H, J1 = 8.2Hz, J2 =
7.8Hz, H-C (7)), 7.70-8.53 (m, 5H, H-C (benzene ring)), 12.05 (s, 2H, 2OH); HRMS
(ESI): calcd for [M-Cl]+ (C23H20NO4) m/z 374.1392, found 374.1396.
4.1.15. Synthesis of
3-(Chloromethyl)-1,8-dimethoxyanthracene-9,10-dione (D1)
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As described for C1, compound B1 was chloridized with thionyl chloride to give
compound D1 as yellow needle from acetone, yield 80%; m.p. 179-182 ˚C;
IRνmaxcm-1: 3004, 2932, 1666, 1586, 1284, 1237, 712; 1H NMR (CDCl3): d = 4.01 (s,
3H, CH3), 4.04 (s, 3H, CH3), 4.65 (s, 2H, CH2), 7.30 (d, 1H, J = 8.3 Hz, H-C(7)), 7.34
(s, 1H, HC(2)), 7.65 (dd, 1H, J1 = 7.8 Hz, J2 = 8.3 Hz, H-C(6)), 7.83 (s, 1H, HC(4)),
7.84 (d, 1H, J = 7.8 Hz, H-C(5)); HRMS (ESI): calcd for [M+H]+ (C17H14ClO4) m/z
317.0581, found 317.0585.
4.1.16. Synthesis of
1-((4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl)pyri
din-1-ium chloride (D2)
1 ml of dry pyridine was slowly added into a solution of compound D1 (0.32 g,
1.0 mmol) in anhydrous CHCl3 (50 mL) under stirring at room temperature. The
reaction mixture was allowed to stand at room temperature for 2 weeks and yellow
crystals were formed at the bottom of the vessel, yield 73%; m.p. 168 ˚C (dec); IRνmax
1
cm-1: 3219, 3047, 1663, 1587, 1277, 1239, 741; H NMR (D2O): d = 3.94 (s, 3H,
CH3-5), 3.96 (s, 3H, CH3-4), 5.93 (s, 2H, CH2), 7.47 (d, 1H, J = 8.6 Hz, H-C (6)),
7.50 (d, 1H, J = 6.7 Hz, H-C (8)), 7.52 (s, 1H, H-C (1)), 7.57 (s, 1H, H-C (3)), 7.68
(dd, 1H, J1 = 8.1 Hz, J2 = 8.0 Hz, H-C (7)), 8.16 (d, 2H, J = 6.8 Hz, H-C
(pyridyl-3′,5′)), 8.66 (dd, 1H, J1 = 7.9 Hz, J2 = 7.9 Hz, H-C (pyridyl-4′)), 8.99 (d, 2H,
J = 5.6 Hz, H-C (pyridyl-2′,6′)); HRMS (ESI): Calcd for [M-Cl]+(C22H18NO4) m/z
360.1236, found 360.1124.
4.1.17. Synthesis of
1-(4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)-N,N,N-trime
thylmethanaminium chloride (D3)
As described for D2, compound D1 was treated with trimethylamine to give
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compound D3 as yellow needle, yield 74%; m.p. 219 ˚C (dec); IR νmax cm-1: 3009,
1
2962, 1661, 1591, 1282, 1235, 746; H NMR (D2O): d = 3.20 (s, 9H, CH3), 3.90 (s,
6H, 2OCH3), 4.52 (s, 2H, CH2), 7.30 (d, 1H, J = 7.5 Hz, H-C (6)), 7.38 (d, 1H, J = 8.3
Hz, H-C 8)), 7.47 (s, 1H, H-C (3)), 7.57 (s, 1H, H-C (1)), 7.58 (dd, 1H, J1 = 6.8 Hz, J2
= 9.3 Hz, H-C (7)); HRMS (ESI): Calcd for [M-Cl]+ (C20H22NO4) m/z 340.1549,
found 340.1560.
4.1.18. Synthesis of
N-((4,5-Dimethoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl)-N,N
-dimethylbenzenaminium chloride (D4)
As described for D2, compound D1 was treated with N,N-dimethylaniline to give
compound D4 as yellow needle, yield 70%; m.p. 161 ˚C (dec); IRνmaxcm-1: 3005,
2963, 1674, 1585, 1285, 1241, 751; 1H NMR (D2O): d = 3.53 (s, 6H, 2NCH3), 3.74 (s,
6H, 2OCH3), 4.96 (s, 2H, CH2), 6.64 (s, 1H, H-C (1)), 6.81 (d, 1H, J = 7.5 Hz, H-C
(6)), 6.88 (s, 1H, H-C (3)), 7.12 (d, 1H, J = 8.1 Hz, HC (8)), 7.24 (dd, 1H, J1 = 8.3 Hz,
J2 = 7.7 Hz, H-C (7)), 7.64-7.73 (m, 5H, H-C (benzene ring)); HRMS (ESI): Calcd
for [M-Cl]+ (C25H24NO4) m/z 402.1705, found 402.1699.
4.2 Assay of the in vitro XO inhibitory activity
The XO activity with xanthine as the substrate was measured at 25 °C, according
to the protocol of Kong and others[20]. The reaction mixture contained 650 ꢀL of 50
mM K2HPO4 buffer (pH 7.8), 200 ꢀL of 84.8 ꢀg/mL xanthine in 50 mM K2HPO4
buffer, and 50 ꢀL of the various concentrations of tested compounds, which were
dissolved in DMSO. The reaction was started by addition of 100 ꢀL of XO (25
mU/mL) and was monitored for 6 min at 295 nm. The XO activity was expressed as
the change in absorbance per minute at 295nm.
4.2. Molecular Modeling Evaluations[21, 22]
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The pdb structure of xanthine dehydrogenase (XDH) co-crystallized with Y-700
(PDB entry code 1VDV) was obtained from the Protein Data Bank. The 3D structure
of compound A1 was constructed utilizing ChemBioOffice 2008 and optimized
according to the standard protocol of ChemBio 3D.
Docking studies were carried out using the AUTODOCK 4.2.3 program. Using
ADT[23], Polar hydrogen atoms were added to amino acid residues and Gasteiger
charges were assigned to all atoms of the enzyme. The resulting enzyme structure was
used as an input for the AUTOGRID program. AUTOGRID performed a
precalculated atomic affinity grid maps for each atom type in the ligand plus an
electrostatics map and a separate desolvation map present in the substrate molecule.
All maps were calculated with 0.375 Å spacing between grid points. The centre of the
grid box was placed at the centre of the Y-700. The dimensions of the active site box
were set at 50×50×50 Å.
Flexible ligand docking was performed for the compound. Docking calculations
were carried out using the Lamarckian genetic algorithm (LGA) and all parameters
were the same for each docking. A population of random individuals (population size:
150), a medium number of 2,500,000 energy evaluations, a maximum number of
generations of 27,000 was used. At the end of docking procedure (100 docking runs),
the resulting positions were clustered according to a root mean square criterion of 0.5
Å.
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Acknowledgment
The financial support of the Natural Science Foundation of P.R. China (grated
No: 21162006), Postdoctoral Science Foundation of China (grated No:
2012M521659) and Collegiate Natural Science Fund of Jiangsu Province
(12KJB350001) are gratefully acknowledged.
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Figure Captions
Figure 1. Some reported non-purine XO inhibitors.
Figure 2. Chemical structure of aloe-emodin.
Figure 3. The inhibition of compound A1 on the XO. Values are means ± SD, n=3.
Figure 4. Steady-state inhibition of XO by A1. Lineweaver-Burk plot of reciprocal of
initial velocities versus reciprocal of five fixed xanthine concentrations (20, 30, 40, 60
◆
and 80 ꢀg/mL) in the absence ( ) and presence of A1 at 1.4 ꢀM (■), 2.8 ꢀM (▲) and
5.6 ꢀM (●).
Figure 5. A close view of the potential interaction between compound A1 and the
binding site of XDH (PDB NO. 1VDV). A: Binding interactions of A1 in the catalytic
site of XDH. The blue molecular was Y-700; B: Compound A1 docked into the
binding site of XDH. The blue molecular was Y-700.
Scheme Captions
Scheme 1. Synthetic pathway for compounds A1-A5 and B1-B5
Scheme 2. Synthetic pathway for compounds C1-C4 and D1-D4
Table Caption
Table 1. The XO-inhibition activities of aloe-emodin derivatives
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Table 1. The XO-inhibition activities of aloe-emodin derivatives
Inhibition
Compound
Structures
Inhibition (%)a
IC50 (µM)
22.46
-
1
93.27
94.55
93.38
2.79 ± 0.64
3.87 ± 0.35
8.43 ± 1.81
A1
A2
A3
3.63
7.20
9.77
A4
A5
B1
-
-
-
-
-
O
O
O
36.47
n.a.b
17.47
n.a.
B2
B3
CH3O
OCH3
B4
B5
C1
C2
C3
18.45
n.a.
-
-
68.85
n.a.
18.67 ± 2.01
-
C4
D1
14.26
n.a.
D2
D3
n.a.

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D4
n.a.
Allopurinol
11.23 ± 0.11
aThe inhibition activities of the compounds at the concentration of 50 µM.
b n.a.:not active(less than 10% inhibition at 50 µM).

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