Polyphenols based on isoflavones as inhibitors of Helicobacter pylori urease

Article abstract of DOI:10.1016/j.bmc.2007.03.045

Twenty polyphenols were synthesized and evaluated for their effect on Helicobacter pylori urease. Among these compounds, 4-(p-hydroxyphenethyl)pyrogallol (15) (IC₅₀ = 0.03 mM) and 7,8,4′-trihydroxyisoflavone (19) (IC₅₀ = 0.14 mM) showed potent inhibitory activities, and inhibited Helicobacter pylori urease in a time-dependent manner. The structure-activity relationship of these polyphenols revealed: the two ortho hydroxyl groups were essential for inhibitory activity of polyphenol. When the C-ring of isoflavone was broken, the inhibitory activity markedly decreased. As for deoxybenzoin, the carboxyl group was clearly detrimental.

Full text of DOI:10.1016/j.bmc.2007.03.045

Bioorganic & Medicinal Chemistry 15 (2007) 3703–3710
Polyphenols based on isoflavones as inhibitors of
Helicobacter pylori urease
Zhu-Ping Xiao,a,b Da-Hua Shi,a Huan-Qiu Li,a Li-Na Zhang,a
Chen Xua and Hai-Liang Zhua,*
aInstitute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, PR China
bDepartment of Chemistry, Shangrao Normal College, Shangrao 334000, PR China
Received 20 January 2007; accepted 14 March 2007
Available online 18 March 2007
Abstract—Twenty polyphenols were synthesized and evaluated for their effect on Helicobacter pylori urease. Among these com-
pounds, 4-(p-hydroxyphenethyl)pyrogallol (15) (IC50 = 0.03 mM) and 7,8,40-trihydroxyisoflavone (19) (IC50 = 0.14 mM) showed
potent inhibitory activities, and inhibited Helicobacter pylori urease in a time-dependent manner. The structure–activity relationship
of these polyphenols revealed: the two ortho hydroxyl groups were essential for inhibitory activity of polyphenol. When the C-ring
of isoflavone was broken, the inhibitory activity markedly decreased. As for deoxybenzoin, the carboxyl group was clearly
detrimental.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Polyphenols, such as flavones and isoflavones, constitute
one of the most represented classes of compounds in
higher plants including medicinal and edible plants.
Extensive epidemiological and animal studies and
in vitro experiments with polyphenols have indicated
their broad variety of biological activities, including
anticancer,8 anti-inflammatory,9 antibacterial,10 cardio-
protective,11 anti-osteoporotic12, and enzyme-inhibi-
tory13 activities. More attention has been focused on
exploring novel biological properties of polyphenols.
As an example, tea polyphenols and flavones as urease
inhibitors were reported by Wotherspoon et al.14 and
Tamura,15 respectively. In general, isoflavones exhibited
similar biological activities as flavones,10,12,16–19 which
inspired us to screen isoflavones and their bioisosteres
for urease inhibitors. Twenty compounds were designed
to test for urease inhibitory activities against H. pylori
urease. To our knowledge, this is the first report on
the screening of isoflavone-based compounds for their
urease inhibitory activities.
Urease (urea amidohydrolase; E.C.3.5.1.5) is widely dis-
tributed in a variety of bacteria, fungi, algae, and plants.
People infected by these bacteria characterized by urease
activity such as Helicobacter pylori (H. pylori)and Pro-
teus mirabilis are exposed to a high risk for chronic
atrophic gastritis, peptic ulcer1, and urolithiasis.2 Struc-
tural studies of the enzymes from Klebsiella aerogenes,
Bacillus pasteurii, and H. pylori have revealed a dinucle-
ar Ni active site with a carbamylated lysine residue that
bridges the deeply buried metal atoms.3–5 Urease cata-
lyzes the hydrolysis of urea to produce ammonia and
carbon dioxide, and to protect the bacteria in the acidic
environment through the elevation in pH.6 Many urease
inhibitors have been described in the past decades, like
fluorofamide, hydroxyureas, and hydroxamic acids,
but part of them were prevented from using in vivo be-
cause of their toxicity or instability. For instance, aceto-
hydroxamic acid was demonstrated to be teratogenic in
rats.7 Thus, current efforts are focused on seeking novel
urease inhibitors with good bioavailability and low
toxicity.
2. Results and discussion
2.1. Chemistry
Keywords:
Polyphenols.
*
Urease
inhibitors;
Isoflavones;
Deoxybenzoins;
A series of isoflavone-based polyphenols with structural
diversity were designed for urease inhibitors. Inspection
Corresponding author. Tel.: +86 25 8359 2572; fax: +86 25
83592672; e-mail: zhuhl@nju.edu.cn
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.045

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Z.-P. Xiao et al. / Bioorg. Med. Chem. 15 (2007) 3703–3710
of the chemical structure of isoflavone suggested that the
compound could be divided into three subunits: A-, B-,
and C-rings (Fig. 3). Initial structure–activity relation-
ship (SAR) studies were performed by modification of
the parent compound to determine if any of the subunits
displayed urease inhibitory activity. Based on this con-
sideration, we further designed some deoxybenzoins by
breaking C-ring of the corresponding isoflavones. The
deoxybenzoins (4, 5, 6, 7, 8, and 9) were successfully syn-
thesized by the method of Wa¨ha¨la¨,20 while compounds
13 and 14 were produced in the presence of gas HCl
by treating phloroglucinol and 1,3,5-trihydroxy-2-meth-
oxybenzene with p-hydroxyphenylacetonitrile in dry
ether, respectively.21 Compounds 1 and 14 were trans-
formed into 17, 18, and 19 by CH3SO2Cl in anhydrous
DMF in the presence of BF3ÆEt2O. All compounds were
fully characterized by spectroscopic methods and ele-
mental analysis. Out of the compounds, four (com-
pounds 11, 12, 15, and 16) were reported for the first
time.
2.2. Biological activity
Twenty polyphenols 1–20 (Figs. 1–3) were evaluated
against H. pylori urease. Percent inhibition at a
400 lg/mL concentration of compounds 1–20 was ini-
tially determined, and the results are reported in Table
1. Compounds 1, 3, 4, 5, 9, 15, and 17 (with catechol skel-
eton) exhibited potent inhibitory activities, especially 15
(IC50 = 0.03 mM) and 17 (IC50 = 0.14 mM) which dem-
onstrated excellent in vivo activities. However, replacing
one or all of the hydroxyl groups on the catechol skeleton
by methoxy produced a partial or complete loss in activity
(10, 11, 12, 14, 18, and 19). So the two ortho hydroxyl
groups presented on aromatic ring of polyphenol mole-
cule may be responsible for inhibitory activity and coordi-
nate with the nickel (active site) of enzyme. Any
compounds (2, 6, 7, 8, and 13) with resorcinol skeleton
showed no or extremely weak inhibitory activity. Proba-
bly, the long distance between the two hydroxyl groups
at 1 and 3 positions on benzene ring counted for less inter-
Figure 1. Chemical structures of compounds (1–14).
Figure 2. Chemical structures of compounds (15–17).

Z.-P. Xiao et al. / Bioorg. Med. Chem. 15 (2007) 3703–3710
3705
and 17 were dependent on the length of preincubation
with the urease. Three hours of preincubation with
compounds 15 and 17 was indispensable for the
maximum inhibition (Fig. 4), which indicated that
compounds 15 and 17 were the irreversible inhibitors.
These results agreed with published observations that
the inhibitory actions of a,b-unsaturated ketones were
dependent on the length of preincubation with
urease.24
It was reported that some urease inhibitors depressed
H. pylori urease activities by interacting with the sulfhy-
dryl groups, especially the Cystein-592 residue, which is
a key residue in the active sites.25 The interaction of an
isoflavone with the sulfhydryl group may be related to
its inhibitory influence on urease, and the keto group
may be responsible for the interaction. In fact, dithio-
threitol (DTT) and 2-mercaptoethanol (2-ME) dimin-
ished the activity of 17 dose-dependently and even at
low concentrations (Table 3, Figs. 5 and 6). These re-
sults, together with the SAR, suggested that the keto
group and the two ortho hydroxyl groups in 17 hooked
the active site of H. pylori urease concurrently. On the
other hand, 2-ME and DTT showed weak effect on
the inhibitory activity of 15 and AHA, in which no keto
Figure 3. Chemical structures of compounds (18–20).
Table 1. Percent inhibition of compounds 1–20 against H. pylori
urease at the concentration of 400 lg/mL
Compound
Percent
inhibition (%)
Compound
Percent
inhibition (%)
1
2
38
14
41
51
44
16
14
—
39
—
11
12
13
14
15
16
17
18
19
20
—
—
3
—
17
4
5
94.5
42
6
7
81.7
36
8
100
9
10
23
—
80
action with the nickel of the enzyme. Deoxybenzoins (4,
14, and 13) were regarded as products by breaking the
C-rings of isoflavones (17, 18 (19), and 20), respectively
(Figs. 1 and 3). A comparison of the inhibitory activities
of isoflavones with the corresponding deoxybenzoins,
that is, comparing 4 and 17, 14 and 18 (19), 13 and 20,
showed clear difference in their activities. This indicated
that the C-ring of isoflavone was critical to its inhibitory
effect. Changing the carbonyl group of 4 for a methylene
(15) significant increased activity. Meanwhile replacing
the carbonyl group of 12 by hydroxyl group (16) also pro-
vided significantly increase in activity. These result
showed the carbonyl group was clearly detrimental to
polyphenolic activity.
60
40
15 (200 µg/mL)
17 (200 µg/mL)
AHA (40 µM)
20
0
0
1
2
3
4
5
Preincubation time (h)
Figure 4. Time course of inhibition of H. pylori urease activity by
acetohydroxamic acid (AHA) and compounds 15 and 17. Urease
activity was assayed as described in Section 4. Data are the average of
three experiments and error bars indicate the standard deviations.
Generally, reversible inhibition is characterized by a
definite degree of inhibition which is usually attained
rapidly.22 The depression by acetohydroxamic acid
(AHA), a reversible urease inhibitor,23 rapidly attained
the maximal constant degree. Under the assay condi-
tions, compounds 15 and 17 showed potent inhibitory
activities with IC50 = 0.03 and 0.14 mM, respectively
(Table 2). The inhibitory activities of compounds 15
Table 3. Effects of additives on the inhibitory activity of compounds
15 and 17 against H. pylori urease
a
IC50 (mM)
Compound
Additives
15
None
+2-ME (0.4 mM)
+DTT (0.4 mM)
0.030
0.071
0.053
Table 2. Inhibition of H. pylori urease by compounds 15 and 17
17
None
+2-ME (0.4 mM)
+DTT (0.4 mM)
0.14
>1.48
>1.48
a
IC50 (mM)
Compound
15
17
0.030 0.0008
0.140 0.003
0.017 0.002
Acetohydroxamic acid
None
+2-ME (0.4 mM)
+DTT (0.4 mM)
0.017
0.019
0.018
Acetohydroxamic acid
a Urease activity was assayed as described in Section 4 with 3 h pre-
incubation. Each value is represented as means SD from triplicate
measurements.
a Urease activity was assayed as described in Section 4 with 3 h
preincubation.

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Z.-P. Xiao et al. / Bioorg. Med. Chem. 15 (2007) 3703–3710
100
100
90
80
70
60
50
40
30
20
10
0
0.0 µM
0.0 µM
90
80
70
60
50
40
30
20
10
0
12.5
50.0
µ
µ
M
M
12.5
25.0
50.0
µ
µ
µ
M
M
M
100.0 µM
100.0
400.0
µM
µ
M
10
100
1000
10
100
1000
15(µg/mL)
17 (µg/mL)
Figure 5. Effect of DTT on the inhibition of urease activity by compounds 15 and 17. Urease activity was assayed as described in Section 4 with 3 h
preincubation.
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
0.0 µM
0.0 µM
12.5 µM
25.0 µM
50.0 µM
400.0 µM
12.5 µM
25.0 µM
50.0 µM
100.0 µM
200.0 µM
400.0 µM
10
100
1000
10
1000
100
15 (µg/mL)
17 (µg/mL)
Figure 6. Effect of 2-ME on the inhibition of urease activity by compounds 15 and 17. Urease activity was assayed as described in Section 4 with 3 h
preincubation.
groups existed (Fig. 6, Table 3). This implied that the
Cystein-592 residue of the active sites was unlikely to
be involved in binding to 15. Further studies are re-
quired to elucidate the mechanism by which polyphenols
exhibit the urease inhibitory activity.
polyphenols disclosed: the two ortho hydroxyl groups
were essential for inhibitory activity of polyphenol.
When the C-ring of isoflavone was broken, the inhibi-
tory activity markedly decreased. As for deoxybenzoin,
the carboxyl group was clearly detrimental. Further
efforts to modify the potent urease inhibitors (15 and
17) are underway and will be reported in due course.
Toxicity and rapid metabolic degradation prevented
some efficient urease inhibitors in vitro from developing
into therapeutic agents such as AHA. Heinonen et al.
reported that compound 17 was one of the metabolites
of daidzein.26 Therefore, this result attracted our interest
in further structural modifications of compound 17 as a
leading compound which provided new template struc-
tures for the design of new inhibitors with an optimized
pharmacological profile.
4. Experiments
4.1. Materials
Protease inhibitors (Complete mini EDTA-free) were
purchased from Roche Diagnostics GmbH (Mannheim,
Germany) and brucella broth was from Becton–Dickin-
son (Cockeysville, MD). Horse serum was from Hyclone
(Utah, America).
3. Conclusion
Twenty polyphenols were prepared and evaluated for
their activity against H. pylori urease. Among these com-
pounds, 15 (IC50 = 0.03 mM) and 17 (IC50 = 0.14 mM)
showed potent inhibitory activities. The SAR of these
4.2. Bacteria
Helicobacter pylori (ATCC 43504; American Type Cul-
ture Collection, Manassas, VA) was grown in brucella

Z.-P. Xiao et al. / Bioorg. Med. Chem. 15 (2007) 3703–3710
3707
broth supplemented with 10% heat-inactivated horse
serum for 24 h at 37 ꢁC under microaerobic conditions
(5% O2, 10% CO2, and 85% N2), as previously
described.27
(begin to decompose above 150 ꢁC) (lit., 169–172 ꢁC).
1H NMR d: 2.52 (s, 3H, CH3), 6.39 (d, 1H, J = 9 Hz,
H-5), 7.30 (d, 1H, J = 9 Hz, H-6), 8.59 (s, 1H, 3-
OH), 10.06 (s, 1H, 4-OH), 12.59 (s, 1H, 2-OH). ESI-
MS C8H9O4 [M+H]+ 169. Anal. Calcd for C8H8O4:
C, 57.14; H, 4.80. Found: C, 57.21; H, 4.77.
4.3. Preparation of H. pylori urease
30
For urease inhibition assays, 50 mL broth cultures
(2.0 · 108 CFU/mL) were centrifuged (5000g, 4 ꢁC) to
collect the bacteria, and after washing twice with phos-
phate-buffered saline (pH 7.4), the H. pylori precipita-
tion was stored at ꢀ80 ꢁC. H. pylori was returned to
room temperature, and after addition of 3 mL of dis-
tilled water and protease inhibitors, sonication was per-
formed for 60 s. Following centrifugation (15,000g,
4 ꢁC), the supernatant was desalted through Sephadex
G-25 column (PD-10 columns, Amersham–Pharmacia
Biotech, Uppsala, Sweden). The resultant crude urease
solution was added to an equal volume of glycerol and
stored at 4 ꢁC until use in the experiment.
4.5.2. 2,4-Dihydroxyacetophenone (2).. To the solution
of resorcinol (2.2 g; 20 mmol) in 6.3 mL of acetic acid
was added 2.8 g (20 mmol) of anhydrous ZnCl2. The
mixture was heated under reflux on the oil-bath for
5 h. After cooling, the product was poured into 150 mL
of ice-water. The precipitate was sucked and crystallized
from water to give compound 2 (2.8 g; 92%) as dark red
1
needles. Mp 145–146 ꢁC (lit., 141–144 ꢁC). H NMR d:
2.52 (s, 3H, CH3), 6.24 (d, 1H, J = 2 Hz, H-3), 6.37
(d · d, 1H, J = 9 Hz, J = 2 Hz, H-5), 7.76 (d, 1H,
J = 9 Hz, H-6), 10.60 (s, 1H, 4-OH), 12.60 (s, 1H, 2-OH).
ESI-MS C8H9O3 [M+H]+ 153. Anal. Calcd for
C8H8O3: C, 63.15; H, 5.30. Found: C, 63.27; H, 5.26.
4.4. Measurement of urease activity
4.5.3. 3,4-Dihydroxyacetophenone (3.). 31,32 A similar
treatment of catechol (2.2 g; 20 mmol) in 6.3 mL of ace-
tic acid with 2.8 g (20 mmol) of anhydrous ZnCl2 gave
the product as dark gummy material which was purified
by chromatography (petroleum ether/EtOAc = 2:1) to
yield compound 3 (2.2 g; 72%) as orange red powder.
The assay mixture, containing 25 lL (4 U) of H. pylori
urease and 25 lL of the test compound, was preincu-
bated for 3 h at room temperature in a 96-well assay
plate. Urease activity was determined by measuring
ammonia production using the indophenol method as
described by Weatherburn.28
1
Mp 117–119 ꢁC (lit., 116, 91, 184, 180 ꢁC). H NMR
d: 2.43 (s, 3H, CH3), 6.80 (d, 1H, J = 8 Hz, H-5), 7.33
(s, 1H, H-2), 7.34 (d, 1H, J = 8 Hz, H-6), 9.32 (s, 1H,
3-OH), 9.83 (s, 1H, 4-OH). ESI-MS C8H9O3 [M+H]+
153. Anal. Calcd for C8H8O3: C, 63.15; H, 5.30. Found:
C, 63.30; H, 5.21.
4.5. Chemistry
The compounds previously reported were synthesized
according to the methods of the references with appro-
priate modifications. Separation of the compounds by
column chromatography was carried out with silica gel
60 (200–300 mesh ASTM, E. Merck). The quantity of
silica gel used was 50–100 times the weight charged on
the column. Then, the eluates were monitored using
TLC. Melting points (uncorrected) were determined on
a XT4 MP apparatus (Taike Corp., Beijing, China).
ESI-mass spectra were obtained on a Mariner System
4.5.4. 2,3,4,40-Tetrahydroxydeoxybenzoin (4.). 33 0.63 g
(5 mmol) of pyrogallol and 0.76 g (5 mmol) of
p-hydroxyphenylacetic acid were dissolved into 5 mL
of fresh distilled BF3ÆEt2O. The mixture was stirred
and heated on the oil-bath at 80–85 ꢁC for about 3 h.
After cooling, the contents were poured into 150 mL
of ice-cold aqueous sodium acetate (wt% = 10%) with
stirring. Then, the precipitate was filtered and washed
thrice with water. Crystallization from methanol to
water gave compound 4 (1.15 g; 89%) as pale yellow
1
5304 mass spectrometer, and H NMR spectra were re-
corded at DPX500 or DPX300 in DMSO-d6 on a Bruker
spectrometer. Elemental analyses were performed on a
CHN-O-Rapid instrument and were within 0.4% of
the theoretical values.
1
crystals. Mp 206–208 ꢁC (lit., 190–192 ꢁC). H NMR d:
4.12 (s, 2H, ArCH2), 6.40 (d, 1H, J = 9 Hz, H-5), 6.69
(d, 2H, J = 8 Hz, H-30, 50), 7.07 (d, 2H, J = 8 Hz, H-20,
60), 7.48 (d, 1H, J = 9Hz, H-6), 8.64 (s, 1H, 3-OH), 9.30
(s, 1H, 40-OH), 10.12 (s, 1H, 4-OH), 12.59 (s, 1H,
2-OH). ESI-MS C14H13O5 [M+H]+ 261. Anal. Calcd for
C14H12O5: C, 64.61; H, 4.65. Found: C, 64.68; H, 4.58.
29
4.5.1. Gallacetophenone (1).. Equimolar quantities of
pyrogallol (2.5 g; 19.8 mmol) and Ac2O (1.9 mL;
20.1 mmol) were refluxed with a drop of concentrated
H2SO4 in a current of nitrogen on the oil-bath for
about 3 h. The contents were cooled and 10 mL of
water containing 2 mL of ethanol was added. Then
two drops of concentrated HCl were added, and the
solution was refluxed for 2 h to decompose the excess
Ac2O. The reaction mixture was cooled which was ex-
tracted thrice with EtOAc. The EtOAc solution was
washed with aqueous saturated NaHCO3 and brine,
dried, and concentrated under reduced pressure to give
the product as a dark gummy material which on crys-
tallization from methanol to water gave compound 1
as pale yellow leaflets (3.2 g; 96%). Mp 169–171 ꢁC
4.5.5. 2,3,4-Trihydroxy-40-methoxydeoxybenzoin (5). A
similar treatment described for 4 of pyrogallol (0.63 g;
5 mmol) with p-methoxyphenylacetic acid (0.83 g;
5 mmol) afforded compound 5 (1.03 g; 76%) as nearly
colorless crystals. An analytical sample was prepared
by crystallization from methanol to water. Mp 145–
1
147 ꢁC. H NMR d: 3.72 (s, 3H, OCH3), 4.20 (s, 2H,
ArCH2), 6.40 (d, 1H, J = 9 Hz, H-5), 6.87 (d, 2H,
J = 9 Hz, H-30, 50), 7.19 (d, 2H, J = 9 Hz, H-20, 60),
7.49 (d, 1H, J = 9 Hz, H-6), 8.63 (s, 1H, 3-OH), 10.11
(s, 1H, 4-OH), 12.54 (s, 1H, 2-OH). ESI-MS C15H15O5

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Z.-P. Xiao et al. / Bioorg. Med. Chem. 15 (2007) 3703–3710
[M+H]+ 275. Anal. Calcd for C15H14O5: C, 65.69; H,
5.14. Found: C, 65.75; H, 5.17.
4.5.10. 2,3,4,40-Tetramethoxydeoxybenzoin (10) and 2-(4-
methoxyphenyl)-1-(2,3,4-trimethoxyphenyl)-propan-1-one
(11). 0.78 g (3 mmol) of compound 4 and 0.90 mL
(15 mmol) of CH3I were dissolved into 15 mL of dry
acetone. To the solution, 2.1 g (15 mmol) of potassium
carbonate was added. The mixture was stirred and
heated under reflux for 24 h. After cooling, the contents
were poured into 80 mL of water with stirring. Then, the
mixture was extracted with EtOAc and dried. After re-
moval of solvent, the residue was purified by a flash
chromatography with EtOAc–petroleum ether (1:10)
to afford two fractions. The first fraction gave com-
4.5.6. 2,4,40-Terthydroxydeoxybenzoin (6.).34 A similar
treatment described for 4 of resorcinol (0.55 g; 5 mmol)
with p-hydroxyphenylacetic acid (0.76 g; 5 mmol) affor-
ded compound 6 (1.0 g; 82%) as colorless crystals. An
analytical sample was prepared by crystallization from
methanol to water. Mp 188–190 ꢁC (lit., 189–191 ꢁC).
1H NMR d: 4.13 (s, 2H, ArCH2), 6.24 (s, 1H, H-3),
6.37 (d, 1H, J = 9 Hz, H-5), 6.90 (d, 2H, J = 8 Hz, H-
30, 50), 7.07 (d, 2H, J = 8 Hz, H-20, 60), 7.92 (d, 1H,
J = 9 Hz, H-6), 9.29 (s, 1H, 40-OH), 10.67 (s, 1H, 4-
OH), 12.60 (s, 1H, 2-OH). ESI-MS C14H13O4 [M+H]+
245. Anal. Calcd for C14H12O4: C, 68.85; H, 4.95.
Found: C, 68.89; H, 4.93.
1
pound 11 as colorless oil (0.53 g). H NMR d: 1.34 (d,
3H, J = 7 Hz, ArCHCH3), 3.68 (s, 3H, 3-OCH3), 3.71
(s, 3H, 40-OCH3), 3.76 (s, 3H, 2-OCH3), 3.84 (s, 3H,
4-OCH3), 4.63 (q, 1H, J = 7 Hz, ArCH), 6.80 (d, 1H,
J = 9 Hz, H-5), 6.82 (d, 2H, J = 9 Hz, 30, 50-H), 7.10
(d, 2H, J = 9 Hz, 20, 60-H), 7.24 (d, 1H, J = 9 Hz, H-
6). ESI-MS C19H23O5 [M+H]+ 331. Anal. Calcd for
C19H22O5: C, 69.07; H, 6.71. Found: C, 68. 69.11; H,
6.68. The second fraction gave compound 10 as yellow-
4.5.7. 2,4-Dihydroxy-40-methoxydeoxybenzoin (7)..35
A
similar treatment described for 4 of resorcinol (0.55 g;
5 mmol) with p-methoxyphenylacetic acid (0.83 g;
5 mmol) afforded compound 7 (1.1 g; 85%) as pale yel-
low crystals. An analytical sample was prepared by
crystallization from methanol to water. Mp 160–
1
ish solid (0.36). Mp 51–53 ꢁC. H NMR d: 3.71 (s, 3H,
40-OCH3), 3.76 (s, 3H, 3-OCH3), 3.85 (s, 3H, 2-
OCH3), 3.87 (s, 3H, 4-OCH3), 4.16 (s, 2H, ArCH2),
6.85 (d, 2H, J = 8 Hz, H-30, 50), 6.89 (d, 1H, J = 9 Hz,
H-5), 7.11 (d, 2H, J = 8 Hz, H-20,60), 7.39 (d, 1H,
J = 9 Hz, J = 9Hz, H-6). ESI-MS C18H20O5Na
[M+Na]+ 339. Anal. Calcd for C18H20O5: C, 68.34; H,
6.37. Found: C, 68.38; H, 6.34.
1
162 ꢁC. H NMR d: 3.71 (s, 3H, OCH3), 4.20 (s, 2H,
ArCH2), 6.24 (d, 1H, J = 2 Hz, H-3), 6.39 (d · d, 1H,
J = 2 Hz, J = 9 Hz, H-5), 6.87 (d, 2H, J = 9 Hz, H-30,
50), 7.19 (d, 2H, J = 9 Hz, H-20, 60), 7.94 (d, 1H,
J = 9 Hz, H-6), 10.65 (s, 1H, 4-OH), 12.55 (s, 1H, 2-
OH). ESI-MS C15H15O4 [M+H]+ 259. Anal. Calcd for
C15H14O4: C, 69.76; H, 5.46. Found: C, 69.71; H, 5.41.
36
4.5.11. 2-Hydroxy-3,4,40-trimethoxydeoxybenzoin (12)..
4.5.8. 2,4-Dihydroxy-40-nitrodeoxybenzoin (8). A similar
treatment described for 4 of resorcinol (0.55 g; 5 mmol)
with p-methoxyphenylacetic acid (0.90 g; 5 mmol) affor-
ded the crude product of compound 8 as dark oil which
was extracted with EtOAc and dried. After removal of
solvent, the residue was purified by a flash chromatogra-
phy with EtOAc–petroleum ether (1:3) to afford brown
oil which crystallized from methanol to water to give
4-Methoxyphenacetyl chloride was prepared by treating
4-methoxyphenacetic acid with thionyl chloride at room
temperature for 24 h. The solvent was removed in vacuo
and further purification was not necessary. To a mixture
of pyrogallol trimethoxyl ether (2 g; 11.9 mmol) and 4-
methoxyphenacetyl chloride (2.3 g; 12.2 mmol) in dried
carbon disulfide (4 mL) was added anhydrous aluminum
chloride (2.8 g) with stirring at room temperature over-
night. Then the mixture was heated under reflux for
0.5 h. After removal of the solvent by decantation, the
residue was poured into ice-cold diluted hydrochloric
acid. The precipitate was sucked and crystallized from
methanol to give compound 12 as yellow crystals
(3.3 g; 92%). Mp 122–123 ꢁC (lit., 121–122 ꢁC). 1H
NMR d: 3.68 (s, 3H, 3-OCH3), 3.72 (s, 3H, 40-OCH3),
3.88 (s, 3H, 4-OCH3), 4.27 (s, 2H, ArCH2), 6.70 (d,
1H, J = 9 Hz, H-5), 6.87 (d, 2H, J = 8 Hz, H-30, 50),
7.20 (d, 2H, J = 8 Hz, H-20, 60), 7.87 (d, 1H, J = 9 Hz,
H-6). 12.32 (s, 3H, 2-OH). ESI-MS C17H19O5 [M+H]+
303. Anal. Calcd for C17H18O5: C, 67.54; H, 6.00.
Found: C, 67.50; H, 6.03.
1
pale yellow crystals (0.80 g; 59%). Mp 206–208 ꢁC. H
NMR d: 4.54 (s, 2H, ArCH2), 6.28 (s, 1H, H-3), 6.41
(d, 1H, J = 8 Hz, H-5), 7.56 (d, 2H, J = 8 Hz, H-20,
60), 7.94 (d, 1H, J = 8 Hz, H-6), 9.19 (d, 2H, J = 8 Hz,
H-30, 50), 10.72 (s, 1H, 4-OH), 12.25 (s, 1H, 2-OH).
ESI-MS C14H12NO5 [M+H]+ 274. Anal. Calcd for
C14H11NO5: C, 61.54; H, 4.06; N, 5.13. Found: C,
61.57; H, 4.01; N, 5.15.
20
4.5.9. 3,4,40-Terthydroxydeoxybenzoin (9).. A similar
treatment described for 4 of catechol (0.55 g; 5 mmol)
with p-hydroxyphenylacetic acid (0.76 g; 5 mmol) affor-
ded the crude product of compound 9 as dark oil which
was extracted with EtOAc and dried. After removal of
solvent, the residue was purified by a flash chromatogra-
phy with EtOAc–petroleum ether (1:1) to afford pale
brown solid (0.74 g; 62%). Mp 207–209 ꢁC (lit.,
4.5.12. 2,4,40,6-Tetrahydroxydeoxybenzoin (13).2.1 A solu-
tion of p-hydroxyphenylacetonitrile (1 g; 7.5 mmol) and
anhydrous phloroglucinol (0.94 g; 7.5 mmol) in dry ether
(8 mL) was saturated with hydrogen chloride at 0 ꢁC and
set aside for 24 h. The solvent was removed by decanta-
tion. Fifteen microliters of water was added and heated
under reflux for 1 h with stirring. After cooling, yellow
needles were separated and washed with water. Further
purification was not necessary (1.4 g; 72%). Mp 258–
1
211 ꢁC). H NMR d: 4.05 (s, 2H, ArCH2), 6.67 (d, 2H,
J = 8 Hz, H-30, 50), 6.80 (d, 1H, J = 8 Hz, H-5), 7.02
(d, 2H, J = 8 Hz, H-20, 60), 7.37 (s, 1H, H-2), 7.44 (d,
1H, J = 8 Hz, H-6), 9.22 (s, 1H, 3-OH), 9.30 (s, 1H,
40-OH), 9.82 (s, 1H, 4-OH). ESI-MS C14H13O4
[M+H]+ 245. Anal. Calcd for C14H12O4: C, 68.85; H,
4.95. Found: C, 68.79; H, 4.92.
1
260 ꢁC with decomposition (lit., 259 ꢁC). H NMR d:

Z.-P. Xiao et al. / Bioorg. Med. Chem. 15 (2007) 3703–3710
3709
4.21 (s, 2H, ArCH2), 5.81 (s, 2H, H-3,5), 6.67 (d, 2H,
J = 8 Hz, H-30, 50), 7.00 (d, 2H, J = 8 Hz, H-20, 60),
9.16 (s, 1H, 40-OH), 10.34 (s, 1H, 4-OH), 12.21 (s, 2H,
2,6-OH). ESI-MS C14H13O5 [M+H]+ 260. Anal. Calcd
for C14H12O5: C, 64.61; H, 4.65. Found: C, 64.67; H,
4.62.
4.5.16. 7,8,40-Trihydroxyisoflavone (17). Compound 5
(260 mg, 1 mmol) in anhydrous DMF (1.6 mL) was
treated cautiously with BF3ÆEt2O (0.4 mL, 3.3 mmol)
on the ice-water bath. To this mixture was added a solu-
tion of CH3SO2Cl (0.3 mL, 3 mmol) in anhydrous DMF
(0.7 mL) at 50 ꢁC. The mixture was heated at 80 ꢁC for
3 h. The cooling product was poured into ice-cold aque-
ous NaOAc (50 mL, wt% = 10%). The mixture was ex-
tracted with EtOAc. After removal of the solvent, the
residue was purified via crystallization from aqueous
methanol (v% = 85%) to give compound 17 as colorless
needles (190 mg, 70%), which decomposed over 180 ꢁC.
1H NMR d: 6.80 (d, 2H, J = 7 Hz, H-30, 50), 6.95 (d, 1H,
J = 8 Hz, H-6), 7.38 (d, 2H, J = 7 Hz, H-20, 60), 7.46 (d,
1H, J = 8 Hz, H-5), 8.33 (s, 1H, H-2), 9.44 (s, 1H, 40-
OH), 9.52 (s, 1H, 8-OH), 10.31 (s, H, 7-OH). ESI-MS
C15H11O5 [M+H]+ 271. Anal. Calcd for C15H10O5: C,
66.67; H, 3.73. Found: C, 66.61; H, 3.75.
4.5.13.
2,4,40,6-Tetrahydroxy-3-methoxyphenyldeoxy-
37
benzoin (14).. A similar treatment described for 13 of
p-hydroxyphenylacetonitrile (266 mg, 2 mmol) with
anhydrous 1,3,5-trihydroxy-2-methoxybenzene (312 mg,
2 mmol) afforded compound 14 as yellow needles. Fur-
ther purification was not necessary (380 mg, 66%). Mp
224–225 ꢁC (lit., 228 ꢁC). 1H NMR d: 3.59 (s, 3H,
3-OH), 4.22 (s, 2H, ArCH2), 5.91 (s, 1H, H-5), 6.67 (d,
2H, J = 8 Hz, H-30, 50), 7.01 (d, 2H, J = 8 Hz, H-20,
60), 9.21 (s, 1H, 40-OH), 10.37 (s, 1H, 4-OH), 11.54 (s,
1H, 2-OH), 12.50 (s, 1H, 6-OH). ESI-MS C15H15O6
[M+H]+ 291. Anal. Calcd for C15H14O6: C, 62.07; H,
4.86. Found: C, 62.11; H, 4.82.
4.5.17. Tectorigenin (20) and w-tectorigenin (18)..37
A
similar treatment described for 17 of compound 15
(203 mg, 0.7 mmol) in anhydrous DMF (1.6 mL) with
CH3SO2Cl (0.3 mL, 3 mmol) in anhydrous DMF
(0.7 mL) afforded a yellow precipitate. The precipitate
was separated and purified by a flash chromatography
with EtOAc–petroleum ether (1:1) to give two fractions.
The first fraction gave compound 18 as pale yellow nee-
dles (76 mg, 36%). Mp 230–232 ꢁC (lit., 228–230 ꢁC). 1H
NMR d: 3.75 (s, 1H, OCH3), 6.50 (s, 1H, H-6), 6.81 (d,
2H, J = 8 Hz, H-30, 50), 7.37 (d, 2H, J = 8 Hz, H-20, 60),
8.34 (s, 1H, H-2), 9.60 (s, 1H, 40-OH), 10.78 (s, 1H, 7-
OH), 13.06 (s, H, 5-OH). ESI-MS C16H13O6 [M+H]+
301. Anal. Calcd for C16H12O6: C, 64.00; H, 4.03.
Found: C, 63.96; H, 4.05. The second fraction gave com-
pound 19 as pale yellow needles (85 mg, 40%). Mp 241–
4.5.14. 4-(p-Hydroxyphenethyl)pyrogallol (15). NaBH4
(304 mg, 8 mmol) was added to a solution of pyrogallol
(260 mg, 1 mmol) in 1.22 M NaOH (1.64 mL, 2 mmol)
and water (5 mL), and the mixture was heated under
reflux for 3 h. After cooling, 5 M hydrochloric acid was
added to decompose the excess NaBH4 on an ice-water
bath. The mixture was extracted with EtOAc. After
removal of the solvent, the resulting residue was purified
over a silica gel column eluting with isopropanol–petro-
leum ether (1:4) to give compound 15 as yellowish oil
which was crystallized from 5 M hydrochloric acid (pale
brown needles, 210 mg, 85%). Mp 181–183 ꢁC (begin to
decompose above 160 ꢁC). 1H NMR d: 2.62 (s, 4H,
2ArCH2), 6.17 (d, 1H, J = 8 Hz, ArH-6), 6.30 (d, 1H,
J = 8 Hz, H-5), 6.64 (d, 2H, J = 8 Hz, H-30, 50), 6.97 (d,
2H, J = 8 Hz, H-20, 60), 8.04 (br s, 2H, 2,3-OH), 8.74
(s, 1H, 1-OH), 9.06 (s, 1H, 40-OH). ESI-MS C14H14O4Na
[M+Na]+ 269. Anal. Calcd for C14H14O4: C, 68.28; H,
5.73. Found: C, 68.23; H, 5.69.
1
243 ꢁC (lit., 240 ꢁC). H NMR d: 3.76 (s, 1H, OCH3),
6.31 (s, 1H, H-8), 6.81 (d, 2H, J = 8 Hz, H-30, 50), 7.37
(d, 2H, J = 8 Hz, H-20, 60), 8.41 (s, 1H, H-2), 9.60 (s,
1H, 40-OH), 10.83 (s, 1H, 7-OH), 12.65 (s, H, 5-OH).
ESI-MS C16H13O6 [M+H]+ 301. Anal. Calcd for
C16H12O6: C, 64.00; H, 4.03. Found: C, 64.05; H, 4.01.
4.5.15.
6-(1-Hydroxy-2-(4-methoxyphenyl)ethyl)-2,3-
Acknowledgment
dimethoxyphenol (16). Acetic acid (0.1 mL) is added with
cooling to NaBH4 (25 mg, 0.6 mmol) in 1 mL THF.
After the evolution of hydrogen has ceased (about 3 h)
compound 12 is added at room temperature with stir-
ring for 4 h. The reduction is interrupted by adding
1 mL of water, the mixture neutralized with aqueous
NaHCO3, and the reaction product isolated by extrac-
tion with EtOAc. After drying and evaporation, com-
pound 16 is afforded as pale yellow solid (83 mg,
91%). Mp 87–89 ꢁC. 1H NMR d: 2.60 (d · d, 1H,
J = 14 Hz, J = 8Hz ArCH2), 2.83 (d · d, 1H,
J = 14 Hz, J = 4Hz, ArCH2), 3.66 (s, 3H, 2-OCH3),
3.70 (s, 3H, 1-OCH3), 3.75 (s, 3H, 40-OCH3), 4.90 (q,
1H, Ar C(OH)H, J = 8 Hz, J = 4Hz), 6.46 (d, 1H,
J = 9 Hz, H-5), 6.79 (d, 2H, J = 9 Hz, H-30, 50), 6.95
(d, 1H, J = 9 Hz, H-6), 7.07 (d, 2H, H-20,60, J = 9 Hz),
8.76 (s, H, 3-OH). ESI-MS C17H19O4 [MꢀH2O+H]+
287. Anal. Calcd for C17H20O5: C, 67.09; H, 6.62.
Found: C, 667.11; H, 5.59.
The work was financed by Grant (Project 30672516)
from National Natural Science Foundation of China.
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