The synthesis, structure and activity evaluation of pyrogallol and catechol derivatives as Helicobacter pylori urease inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.08.015

Some pyrogallol and catechol derivatives were synthesized, and their urease inhibitory activity was evaluated by using acetohydroxamic acid (AHA), a well known Helicobacter pylori urease inhibitor, as positive control. The assay results indicate that many compounds have showed potential inhibitory activity against H. pylori urease. 4-(4-Hydroxyphenethyl)phen-1,2-diol (2a) was found to be the most potent urease inhibitor with IC₅₀s of 1.5 ± 0.2 μM for extracted fraction and 4.2 ± 0.3 μM for intact cell, at least 10 times and 20 times lower than those of AHA (IC₅₀ of 17.2 ± 0.9 μM, 100.6 ± 13 μM), respectively. This finding indicate that 2a would be a potential urease inhibitor deserves further research. Molecular dockings of 2a into H. pylori urease active site were performed for understanding the good activity observed.

Full text of DOI:10.1016/j.ejmech.2010.08.015

European Journal of Medicinal Chemistry 45 (2010) 5064e5070
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The synthesis, structure and activity evaluation of pyrogallol and catechol
derivatives as Helicobacter pylori urease inhibitors
,
a
a
a
a
a,b,
**
Zhu-Ping Xiao a , Tao-Wu Ma , Wei-Chang Fu , Xiao-Chun Peng , Ai-Hua Zhang , Hai-Liang Zhu
*
a Key Laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou University, Jishou 416000, PR China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Some pyrogallol and catechol derivatives were synthesized, and their urease inhibitory activity was
evaluated by using acetohydroxamic acid (AHA), a well known Helicobacter pylori urease inhibitor, as
positive control. The assay results indicate that many compounds have showed potential inhibitory
activity against H. pylori urease. 4-(4-Hydroxyphenethyl)phen-1,2-diol (2a) was found to be the most
Received 6 May 2010
Received in revised form
30 July 2010
Accepted 6 August 2010
Available online 12 August 2010
potent urease inhibitor with IC50s of 1.5 ꢀ 0.2
m
M for extracted fraction and 4.2 ꢀ 0.3
m
M for intact cell, at
M), respectively.
least 10 times and 20 times lower than those of AHA (IC50 of 17.2 ꢀ 0.9
m
M, 100.6 ꢀ 13
m
This finding indicate that 2a would be a potential urease inhibitor deserves further research. Molecular
dockings of 2a into H. pylori urease active site were performed for understanding the good activity
observed.
Keywords:
Urease inhibitor
Helicobacter pylori
Pyrogallol derivatives
Catechol derivatives
Autodock
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
between two nickel ions. In the course of enzymatic reaction, urea
replaces these three water molecules and bridges the two metal
Urease is widely distributed in nature and is found in a variety of
plants, algae, fungi and bacteria [1,2]. This enzyme is directly
involved in the formation of infection of stones and contributes to
the pathogenesis of urolithiasis, pyelonephritis, ammonia and
hepatic encephalopathy, hepatic coma and urinary catheter
encrustation [3,4].
Urease (urea amidohydrolase EC3.5.1.5) is a large hetero-
polymeric enzyme with the active site containing two nickel (II)
atoms [5] and accelerates hydrolysis of urea by at least 1014 over the
spontaneous reaction [6]. The amino acid sequences of the active
site are principally conserved in all known ureases, and the cata-
lytic mechanism of their action is believed to be the same. On the
basis of several crystal structures of complexes of Bacillus pasteurii
urease, Ciurli and his co-workers proposed the most reliable
enzymatic reaction mechanism [7]. The active site of the native
enzyme binds three water molecules and a hydroxide ion bridged
ions [8]. The bidentated urea molecule is anchored tightly by
a hydrogen-bonding network. This arrangement strongly activates
the inert urea molecule, which is subsequently attacked by the Ni-
bridging hydroxide ion, forming a tetrahedral transition state. As
a result, ammonia is released from the active site followed by the
negatively charged carbamate. The latter decomposes rapidly and
spontaneously, yielding a second molecule of ammonia. These
reactions in return cause significant increase of solution pH, which
is known as a major cause of pathologies induced by Helicobacter
pylori (H. pylori) and allows bacteria to survive at acidic pH of the
stomach during colonization. The urease activity of H. pylori
therefore plays an important role in the pathogenesis of gastric and
peptic ulcer (including cancer) [6].
Several classes of compounds show significant inhibitory
activity against urease with hydroxamic acids being the best
recognized inhibitors [9,10] and with phosphoramidates being the
most active [11,12]. Degradation of phosphoramidates at low pH
[13] and teratogenicity of hydroxamic acids in rats [14] prevented
them from using in vivo. Current efforts are therefore focused on
seeking novel urease inhibitors with good bioavailability and low
toxicity. Meanwhile, the studies on novel urease inhibitors are
essential not only for the basic research on urease biochemistry but
also for the possible development of a highly needed therapy for
urease mediated bacterial infections.
* Corresponding author. Tel./fax: þ86 743 8563911.
** Corresponding author. Key Laboratory of Hunan Forest Products and Chemical
Industry Engineering, Jishou University, Jishou 416000, PR China. Tel.: þ86 25 8359
2572; fax: þ86 25 83592672.
E-mail addresses: xiaozhuping2005@163.com (Z.-P. Xiao), zhuhl@nju.edu.cn
(H.-L. Zhu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.015

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 45 (2010) 5064e5070
5065
O
OH
O
R
R
OH
OH
R
OH
NaBH4
NaOH
B
HO
HO
O
MeO
MeO
MeO
PPA
OMe
OMe
OMe
OH
R2
OH
Scheme 3. Synthetic route of 3ge6g.
R1
(+)-Gallocatechin
OH
OH
1,2-diarylethanes
modifications. All newly reported compounds were fully charac-
terized by spectroscopic methods and elemental analysis together
with a crystal structure (1b). Out of the compounds, fifteen (1be1h,
1m, 2be2c, 2ee2g and 5ge6g) were reported for the first time.
O
OH
OH
O
quercetin
2.2. Description of the crystal structure and determination of
central bond
Scheme 1. The evolution process of the aimed urease inhibitors.
Compound 1b was determined by X-ray diffraction analysis. The
crystal data are presented in Table 1, and Fig. 1 gave perspective
views of compound 1b with the atomic labeling system. The bond
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
studies and in vitro experiments with polyphenols have indicated
their broad variety of biological activities, including anticancer [15],
anti-inflammatory [16], antibacterial [17], cardioprotective [18],
anti-osteoporotic [19] and enzyme-inhibitory [20] activities.
Recently, some polyphenols from green tea ((þ)-gallocatechin) [21]
and several naturally occurring flavonoids (quercetin) [22] were
shown to be H. pylori urease inhibitors in micromolar range. On the
basis of these researches, we have reported some urease inhibitors
derived from flavonoid by splitting C-ring (Scheme 1), in which 4-
(4-hydroxyphenethyl)phen-1,2,3-triol (1a) showed the highest
inhibitory activity against H. pylori urease [23]. In view of these
positive results, we recently focused our efforts to develop urease
inhibitors based on 1a. In this paper, we present the synthesis of 1a
analogues and their evaluation on H. pylori urease inhibitory
activity. Obtained results showed high activity of the synthesized
ꢀ
length of C7eC8 (1.416(6) A) is significantly shorter than that in
other reported 1,2-diphenylethane derivatives [25], and much
longer than that in stilbenes [26]. However, four proton signals at dH
2.84 strongly indicated that C7eC8 is a single bond. In fact, the
shortening is commonly observed for the central bond in 1,2-
diphenylethane derivatives [27,28]. Harada and Ogawa attributed
the shortening to an artifact caused by the torsional vibration of the
CePh bonds in crystals [29]. Rings A and B are consequently linked
by an eCH2eCH2e group, which is consistent with the target
structure described in Table 2. Based on this result, the structure of
compounds 1ce1h were distinctly determined to have
eCH2eCH2e linkage.
In the crystal structure of compound 1b, C1eC6 (ring A) form
ꢀ
a plane with the mean deviation of 0.0020 A, and C9eC14 (ring B)
ꢀ
form an other plane with the mean deviation of 0.0026 A. The two
planes make a dihedral angle of 55.8(1)ꢁ. The torsion angle of C4,
C7, C8 and C9 is ꢂ178.5(4)ꢁ, which clearly indicated ring A and ring
B are in the antiperiplanar conformation. There are two classical
intramolecular hydrogen bonds occurring the three hydroxyl
groups, and there is a non-classical intramolecular hydrogen bonds
between C7 and O3 (Fig 1).
structures, with IC50 of 1.5 ꢀ 0.2
mM for the most active compound.
2. Results and discussion
2.1. Chemistry
Using 4-(4-hydroxyphenethyl)phen-1,2,3-triol (1a) as lead
compound, twenty-nine compounds were designed and synthe-
sized for H. pylori urease inhibitors by the routes outlined in
Schemes 2 and 3. In our previous work [23], we found that the two
ortho-hydroxyl groups were essential for urease inhibitory activity
of a polyphenol. Consequently, the inhibitor design was based on
catechol and pyrogallol derivatives, and the reforming focused on
the B-ring. Structure-activity relationship (SAR) studies were also
performed by replacement of B-ring with alkyl chains to determine
if the subunits displayed urease inhibitory activity. The catechol
and pyrogallol derivatives were all synthesized by the method
described elsewhere [23] and compounds 3g to 6g were synthe-
sized by the method reported by Percec et al. [24] with some
2.3. Urease inhibitory activity
The inhibitory potential of the synthesized compounds was
assessed against urease from H. pylori with some also against in
intact cell. Urease activity was evaluated using Berthelot color
reaction procedure, as other methods of ammonia quantification
based on pH changes (e.g., phenol red assay) lead to artificial results
due to strong buffering properties of the studied inhibitors [30].
The results for all assayed compounds are given in Table 2. Aceto-
hydroxamic acid (AHA) was used as the reference compound for
the assay, and its value is also included in Table 2. Originally
designed lead compound 1a exhibited moderate activity with IC50
of 32 ꢀ 7
mM. Further modification of this structure caused
O
O
R
R
R
OH
NaBH4
NaOH
A
HO
R'
BF3 Et2O
HO
R'
HO
R'
OH
OH
OH
R'= OH, pyrogallol derivatives
R'=H, catechol derivatives
Scheme 2. Synthetic route of catechol and pyrogallol derivatives.

5066
Table 1
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 45 (2010) 5064e5070
Table 2
Crystal structure data for 1b.
Inhibitory activity (IC50) of the synthetic compounds against H. pylori urease.
Compound
Formula
Mr
1b
C14H13FO3
248.24
R
A
IC50, mM
Crystal size/mm3
Crystal system
Space group
0.30 ꢃ 0.20 ꢃ 0.20
R'''
R'
Triclinic
R''
P-1
5.981(3)
7.447(3)
14.287(6)
86.810(10)
84.165(12)
73.009(10)
605.2(4)
ꢀ
Entry
R
R0
R00
R000
Cell-free
urease
Urease in
intact cell
a/A
ꢀ
b/A
ꢀ
c/A
ꢁ
ꢁ
a
/
OH
b
/
B
1a
OH
OH
OH
32 ꢀ 7
ND
ꢁ
/
g
3
ꢀ
V/A
Z
2
Dc/(g/cm3)
1.362
0.105
m
/mmꢂ1
F
B
1b
1c
1d
OH
OH
OH
OH
OH
OH
OH
OH
OH
43 ꢀ 5
47 ꢀ 11
48 ꢀ 9
ND
ND
ND
F(000)
260
Max. and min. trans.
0.9793 and 0.9691
1.43/25.49
ꢂ7/6, ꢂ8/9, ꢂ15/17
3230/2219
2219/0/160
0.0217
q
range/ꢁ
Index range (h, k, l)
Reflections collected/unique
Data/restraints/parameters
Rint
Cl
Br
B
B
Goodness-of-fit on F2
R1, wR2 [I > 2
R1, wR2
1.047
s
(I)]
0.0753/0.2110
0.1313/0.2453
e
Extinction coefficient
3
ꢀ
(
Dr)
max, (Dr
)
(e/A )
0.335/ꢂ0.425
min
P
P
R ¼ kFojꢂjFcj/ jFoj.
P
P
wR ¼ [ [w(F2o ꢂ F2c)2]/ [w(F2o)2]]1/2
.
B
1e
1f
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
50 ꢀ 14
52 ꢀ 10
71 ꢀ 16
69 ꢀ 13
ND
ND
ND
ND
considerable activity improvement for compounds 2a, 2b and 2i
toward chosen enzyme (with IC50 ¼ 1.5 ꢀ 0.2, 6.2 ꢀ 0.7 and
Cl
4.9 ꢀ 0.4
mM, respectively). Five of the 25 catechol and pyrogallol
derivatives, 2a, 2b, 2c, 2i and 2j, significantly decreased the mean
activity of H. pylori urease with IC50 below 10 M. 4ge6g, Without
m
B
pyrogallol or catechol moiety, show very low inhibitory activity
against H. pylori urease, which is consistent with our previous work
[23].
Br
In general, the compounds with 1,2,3-trihydroxy groups
(1ae1o) in A-ring are much less active than those analogues with
1,2-dihydroxy groups against H. pylori urease. The dropped urease
inhibitory activity of compound 1a as compared to structurally
related compounds 2a may be due to the presence of a hydroxyl
group at position 3 of A-ring which may provide some resistance to
A-ring to bind at the active site. This was supported by the
molecular docking study, which disclosed that the binding energy
of 1a (ꢂ9.89 kcal/mol) with H. pylori urease is higher than that of 2a
(ꢂ11.48 kcal/mol). Out of the compounds, 2a was found to show the
most potent activity, with IC50 values up to 10-fold lower than the
reference compound, AHA. Substitution of hydroxyl group (1a and
B
OMe
1g
1h
OMe
OEt
OEt
B
1i
1j
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
34 ꢀ 6
79 ꢀ 11
91 ꢀ 15
103 ꢀ 18
124 ꢀ 20
168 ꢀ 23
250 ꢀ 27
ND
ND
ND
ND
ND
ND
ND
n-ethyl
n-butyl
n-hexyl
n-octyl
n-decyl
1k
1l
1m
1n
1o
n-dodecyl
OH
B
2a
2b
H
H
OH
OH
OH
OH
1.5 ꢀ 0.2
6.2 ꢀ 0.7
4.2 ꢀ 0.3
32.4 ꢀ 2.5
F
B
Fig. 1. Molecular structure of compound 1b, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 30% probability level. Solid dashed lines
indicate hydrogen bonds.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 45 (2010) 5064e5070
5067
Table 2 (continued)
R
A
IC50, mM
R'''
R'
R''
Entry
R
R0
H
R00
R000
Cell-free
urease
Urease in
intact cell
Cl
B
2c
OH
OH
8.3 ꢀ 0.5
50.1 ꢀ 4.9
94.7 ꢀ 14
B
2e
2f
H
H
OH
OH
OH
OH
15.7 ꢀ 1.0
Cl
Fig. 2. The active site residues of the AHA-inhibited H. pylori urease, and the molecule
of AHA is labeled with HAE800.
B
16.9 ꢀ 1.2
88.9 ꢀ 11
position in B-ring led to some drop in H. pylori urease inhibitory
potency. Compounds 1g, 1h and 2g, bearing two methoxy groups
(or ethoxy groups) at positions 3 and 4 in B-ring, show the lowest
activity in their own analogues.
Br
Kubo and his co-workers reported anacardic acids, salicylic acid
analogues with a long alkyl chain, as Jack bean urease inhibitors [31].
Based on this discovery, we substituted a series of alkyl chains for B-
ring to probe the available space around the catechol or pyrogallol
moiety. The results clearly showed that changing the length of the
alkyl chain could significantly affect the activity of the molecule
against H. pylori urease. The longer the side chain, the lower the
activity. It is noted that compounds having 1e3 methylene units
such as 1i and 1j show inhibitory activity compared to 1,2-diaryl-
ethane derivatives (1a to 1h) as seen by same range of IC50 values. In
comparison with 1k to 1o, the enhanced potency of 1a to 1h may be
due to a decrease in conformational flexibility, suggesting that the
space around the catechol or pyrogallol is limited.
B
OMe
OMe
2g
H
OH
OH
22.7 ꢀ 2.6
ND
2i
2j
2k
2l
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
4.9 ꢀ 0.4
7.9 ꢀ 0.6
12.3 ꢀ 0.9
58 ꢀ 9
17.3 ꢀ 1.2
38.1 ꢀ 3.4
77.8 ꢀ 6.7
ND
n-ethyl
n-butyl
n-hexyl
B
OMe
OMe
3g
4g
5g
OH
H
H
Cl
H
227 ꢀ 19
204 ꢀ 28
235 ꢀ 25
ND
ND
ND
B
B
B
OMe
OMe
H
OH
OMe
OMe
H
H
OMe
OMe
6g
H
H
Cl
254 ꢀ 32
ND
AHA
e
e
e
e
17.2 ꢀ 0.9
100.6 ꢀ 13
Note: ND ¼ no determination.
2a) with fluorine (1b and 2b) at 4-position in B-ring resulted in
a slight decrease in inhibitory activity, while the activity was
maintained when the fluorine is replaced by bromine and chlorine,
respectively. Shifting the halogen atom from the para- to meta-
Fig. 3. Binding mode of compound 2a with H. pylori urease. For clarity, only interacting
residues are displayed. The hydrogen bond is displayed as green dashed line.

5068
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 45 (2010) 5064e5070
dockings of the most potent compound 2a into H. pylori urease
active site were performed. The binding pattern of 2a may explain
its inhibitory activity against H. pylori urease.
4. Experimental section
4.1. Biological activity
4.1.1. Materials
Protease inhibitors (Complete mini EDTA-free) were purchased
from Roche Diagnostics GmbH (Mannheim, Germany) and Brucella
broth was from BectoneDickinson (Cockeysville, MD). Horse serum
was from Hyclone (Utah, American).
4.1.2. Bacteria
Fig. 4. Binding mode of compound 2a with H. pylori urease. The enzyme is shown as
surface; while 2a docked structures are shown as sticks and close contacts are shown
as wireframe sphere.
H. pylori (ATCC 43504; American Type Culture Collection,
Manassas, VA) was grown in Brucella 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
[32].
Compounds 2aec, 2e,f, 2iek and AHA, with relatively low IC50
values against cell-free urease, were selected for inhibiting urease
activity in intact H. pylori. As shown in Table 2, the IC50 values of the
test compounds against urease in intact cell were 3- to 6-fold
higher than those for extracted urease. The higher IC50s may reflect
the permeability barrier of the cell surface to the compounds by H.
pylori. Another interesting finding of this assay is that 2a also
significantly inhibited the urease activity in intact cell. This indi-
cates that 2a would be a potential urease inhibitor deserves further
research.
4.1.3. Preparation of H. pylori urease
For urease inhibition assays, 50 mL broth cultures
(2.0 ꢃ 108 CFU/mL) were centrifuged (5000 g, 4 ꢁC) to collect the
bacteria, and after washing twice with phosphate-buffered saline
(pH 7.4), the H. pylori precipitation was stored at ꢂ80 ꢁC. H. pylori
was returned to room temperature, and after addition of 3 mL of
distilled water and protease inhibitors, sonication was performed
for 60 s. Following centrifugation (15,000 g, 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.
2.4. Molecular docking study
In the X-ray structure available for the native H. pylori urease
(entry 1E9Z in the Protein Data Bank), the two nickels were coor-
dinated by His136, His138, Kcx219, His248, His274, Asp362 and
water molecules, while in the AHA-inhibited H. pylori urease (entry
1E9Y in the Protein Data Bank), these water molecules were
replaced by AHA (Fig. 2, labeled with HAE800). In order to give an
explanation and understanding of good activity observed, molec-
ular docking of the most potent inhibitor 2a into AHA binding site
of H. pylori urease was performed on the binding model based on
the H. pylori urease complex structure (1E9Y.pdb). The binding
model of compound 2a and H. pylori urease was depicted in Fig. 3
and the enzyme surface model was showed in Fig. 4, which
revealed that the molecular is well filled in the active pocket. There
are two novel interactions, in comparison to AHA-urease complex,
anchored the ligand to this enzyme, namely two hydrogen bonds
formed by 2-hydroxyl group in A-ring and 4-hydroxyl group in B-
ring of 2a with Asn168 and Glu222, respectively. The docking
calculations revealed that compound 2a has lower free energy of
binding (ꢂ11.48 kcal/mol) than that of AHA (ꢂ10.01 kcal/mol),
which may explain the excellent inhibitory activity of 2a against H.
pylori urease.
4.1.4. Measurement of urease activity
The assay mixture, containing 25 mL (10U) of H. pylori urease
which was replaced by 25
for the urease assay of intact cells and 25 mL of the test compound,
m
L of cell suspension (4.0 ꢃ 107 CFU/mL)
was pre-incubated for 1.5 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 Weath-
erburn [30].
4.2. Protocol of docking study
The automated docking studies were carried out using Auto-
Dock version 4.2. First, AutoGrid component of the program pre-
calculates a three-dimensional grid of interaction energies based
on the macromolecular target using the AMBER force field. The
ꢀ
ꢀ
cubic grid box of 60 A size (x, y, z) with a spacing of 0.375 A and
grid maps were created representing the catalytic active target
site region where the native ligand was embedded. Then auto-
mated docking studies were carried out to evaluate the binding
free energy of the inhibitor within the macromolecules. The GALS
search algorithm (genetic algorithm with local search) was
chosen to search for the best conformers. The parameters were
set using the software ADT (AutoDockTools package, version
1.5.4) on PC which is associated with AutoDock 4.2. Default
settings were used with an initial population of 50 randomly
placed individuals, a maximum number of 2.5 ꢃ106 energy
evaluations, and a maximum number of 2.7 ꢃ 104 generations.
A mutation rate of 0.02 and a crossover rate of 0.8 were chosen.
3. Conclusions
Some catechol and pyrogallol derivatives were prepared and
tested for their inhibitory activity against H. pylori urease.
Compound 2a, 4-(4-hydroxyphenethyl)phen-1,2-diol, showed the
most potent inhibitory activity with IC50 of 1.5 ꢀ 0.2
mM against this
enzyme. Substitution of hydroxyl group with halogen at 4-position
in B-ring resulted in a drop in inhibitory activity. On the other hand,
replacement B-ring with a long alkyl chain significantly weakened
the activity of the molecule against H. pylori urease. Molecular
ꢀ
Results differing by less than 0.5 A in positional root-mean-square
deviation (RMSD) were clustered together and the results of the

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 45 (2010) 5064e5070
5069
most favorable free energy of binding were selected as the
resultant complex structures.
5.01 (s, 1H); 5.19 (s, 1H); 6.36 (d, J ¼ 8.2 Hz, 1H); 7.46 (d, J ¼ 8.4 Hz,
1H); 7.04 (d, J ¼ 8.4 Hz, 2H); 7.37 (d, J ¼ 8.2 Hz, 2H); EIMS m/z 308
(Mþ). Anal. Calcd for C14H13BrO3: C, 54.39; H, 4.24; Found: C, 54.31;
H, 4.27.
4.3. Crystallographic studies
X-ray single-crystal diffraction data for compound 1b was
4.4.1.4. 4-(3-Chlorophenethyl)phen-1,2,3-triol (1e). White powder,
58%, mp 108e110 ꢁC, 1H NMR (CDCl3): 2.85 (s, 4H); 4.92e5.50
(bs, 3H); 6.37 (d, J ¼ 8.2 Hz, 1H); 7.48 (d, J ¼ 8.2 Hz, 1H); 7.05 (dd,
J ¼ 8.3 Hz, J ¼ 1.9 Hz, 1H); 7.12e7.23 (m, 3H); EIMS m/z 264 (Mþ).
Anal. Calcd for C14H13ClO3: C, 63.52; H, 4.95; Found: C, 63.57; H, 4.94.
collected on a Bruker SMART APEX CCD diffractometer at 296(2)
ꢀ
K using MoK
a
radiation (
l
¼ 0.71073 A) by the
u scan mode. The
program SAINT was used for integration of the diffraction
profiles. All the structures were solved by direct methods using
the SHELXS program of the SHELXTL package and refined by full-
matrix least-squares methods with SHELXL [33]. All non-
hydrogen atoms of compound 1b were refined with anisotropic
thermal parameters. All hydrogen atoms were generated theo-
retically onto the parent atoms and refined isotropically with
fixed thermal factors.
4.4.1.5. 4-(3-Bromophenethyl)phen-1,2,3-triol (1f). White powder,
69%, mp 112e114 ꢁC, 1H NMR (CDCl3): 2.84 (s, 4H); 4.90e5.51
(bs, 3H); 6.36 (d, J ¼ 8.4 Hz, 1H); 7.48 (d, J ¼ 8.2 Hz, 1H); 7.03 (dd,
J ¼ 8.2 Hz, J ¼ 2.0 Hz, 1H); 7.15e7.27 (m, 3H); EIMS m/z 308 (Mþ).
Anal. Calcd for C14H13BrO3: C, 54.39; H, 4.24; Found: C, 54.30; H,
4.28.
4.4. Chemistry
4.4.1.6. 4-(3,4-Dimethoxyphenethyl)phen-1,2,3-triol
(1g). White
All chemicals (reagent grade) used were purchased from Aldrich
(U.S.A.) and Sinopharm Chemical Reagent Co., Ltd (China). Melting
points (uncorrected) were determined on a XT4 MP apparatus
(Taike Corp., Beijing, China). EI mass spectra were obtained on
a Waters GCT mass spectrometer, and 1H NMR spectra were
recorded on a Bruker AV-300 spectrometer at 25 ꢁC with TMS and
solvent signals allotted as internal standards. Chemical shifts were
powder, 61%, mp 144e145 ꢁC, 1H NMR (CDCl3): 2.83 (s, 4H); 3.80
(s, 3H); 3.85 (s, 3H); 4.83 (s, 1H); 5.07 (s, 1H); 5.27 (s, 1H); 6.40
(d, J ¼ 8.2 Hz, 1H); 6.52 (d, J ¼ 8.4 Hz, 1H); 6.63 (d, J ¼ 1.8 Hz, 1H);
6.72 (dd, J ¼ 8.1 Hz, J ¼ 1.8 Hz, 1H); 6.82 (d, J ¼ 7.9 Hz, 1H); EIMS m/z
290 (Mþ). Anal. Calcd for C16H18O5: C, 66.19; H, 6.25; Found: C,
66.25; H, 6.22.
reported in ppm (
d
). Elemental analyses were performed on a CHN-
4.4.1.7. 4-(3,4-Diethoxyphenethyl)phen-1,2,3-triol
(1h). White
O-Rapid instrument and were within ꢀ0.4% of the theoretical
powder, 77%, mp 122e123 ꢁC, 1H NMR (CDCl3): 1.39 (t, J ¼ 7.0 Hz,
3H); 1.42 (t, J ¼ 7.0 Hz, 3H); 2.81 (s, 4H); 3.98e4.09 (m, 4H);
4.40e5.61 (bs, 3H); 6.38 (d, J ¼ 8.4 Hz, 1H); 6.50 (d, J ¼ 8.2 Hz, 1H);
6.64 (d, J ¼ 2.0 Hz, 1H); 6.69 (dd, J ¼ 8.2 Hz, J ¼ 2.0 Hz, 1H); 6.80
(d, J ¼ 8.0 Hz, 1H); EIMS m/z 318 (Mþ). Anal. Calcd for C18H22O5: C,
67.91; H, 6.97; Found: C, 67.85; H, 6.97.
values.
4.4.1. General procedure for the Preparation of catechol and
pyrogallol derivatives
5 mmol of pyrogallol (catechol) and 5 mmol of appropriately
substituted phenylacetic acid (aliphatic acid) were dissolved into
10 mL of fresh distilled BF3$Et2O. The mixture was stirred and
heated on the oil-bath at 80e85 ꢁC for about 3 h. After cooling, the
contents were poured into 150 mL of ice-cold aqueous sodium
acetate (w% ¼ 10%) with stirring. Then, the precipitate was filtered
and washed thrice with water. Crystallization from methanol-water
gave the products, ketones. For some cases, the products were
necessary to be purified over a silica gel column.
4.4.1.8. 4-Decylphen-1,2,3-triol (1m). White powder, 86%, mp
110e112 ꢁC, 1H NMR (CDCl3): 0.88 (t, J ¼ 7.0 Hz, 3H); 1.18e1.38
(m, 16H); 2.53 (t, J ¼ 7.5 Hz, 2H); 4.88 (s, 1H); 5.08 (s, 1H); 5.20
(s,1H); 6.39 (d, J ¼ 8.2 Hz,1H); 6.54 (d, J ¼ 8.2 Hz,1H); EIMS m/z 266
(Mþ). Anal. Calcd for C16H26O3: C, 72.14; H, 9.84; Found: C, 72.06; H,
9.87.
The resulting ketone (3 mmol) was dissolved in 0.33 M NaOH
(21 mL), and 24 mmol of NaBH4 was subsequently added. 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 EtOAcepetroleum ether.
4.4.1.9. 4-(4-Fluorophenethyl)phen-1,2-diol (2b). White powder,
65%, mp 83e85 ꢁC, 1H NMR (CDCl3): 2.74e2.87 (m, 4H); 5.09
(bs, 2H); 6.57 (dd, J ¼ 8.0 Hz, J ¼ 1.8 Hz, 1H); 6.66 (d, J ¼ 1.8 Hz, 1H);
6.76 (d, J ¼ 8.0 Hz, 1H); 6.94 (t, J ¼ 8.7 Hz, 2H); 7.08 (dd, J ¼ 8.4 Hz,
J ¼ 5.5 Hz, 2H); EIMS m/z 232 (Mþ). Anal. Calcd for C14H13FO2: C,
72.40; H, 5.64; Found: C, 72.46; H, 5.62.
4.4.1.10. 4-(4-Chlorophenethyl)phen-1,2-diol (2c). White powder,
71%, mp 134e136 ꢁC,1H NMR (CDCl3): 2.74e2.87 (m, 4H); 4.51e5.92
(bs, 2H); 6.56 (dd, J ¼ 8.0 Hz, J ¼ 1.8 Hz, 1H); 6.66 (d, J ¼ 1.8 Hz, 1H);
6.75 (d, J ¼ 8.0 Hz, 1H); 7.05 (d, J ¼ 8.2 Hz, 2H); 7.22 (d, J ¼ 8.0 Hz,
2H); EIMS m/z 248 (Mþ). Anal. Calcd for C14H13ClO2: C, 67.61; H, 5.27;
Found: C, 67.54; H, 5.28.
4.4.1.1. 4-(4-Fluorophenethyl)phen-1,2,3-triol (1b). White powder,
yield 62%, mp 132e133 ꢁC,1H NMR (CDCl3): 2.84 (s, 4H); 4.91 (s,1H);
4.99 (s, 1H); 5.20 (s, 1H); 6.37 (d, J ¼ 8.2 Hz, 1H); 7.48 (d, J ¼ 8.2 Hz,
1H); 6.94 (t, J ¼ 8.6 Hz, 2H); 7.11 (t, J ¼ 8.4 Hz, 2H); EIMS m/z 248
(Mþ). Anal. Calcd for C14H13FO3: C, 67.73; H, 5.28; Found: C, 67.79;
H, 5.26.
4.4.1.11. 4-(3-Chlorophenethyl)phen-1,2-diol (2e). White powder,
63%, mp 59e61 ꢁC, 1H NMR (CDCl3): 2.74e2.87 (m, 4H); 4.53e5.47
(bs, 2H); 6.56 (dd, J ¼ 8.0 Hz, J ¼ 1.8 Hz, 1H); 6.66 (d, J ¼ 1.8 Hz, 1H);
6.75 (d, J ¼ 8.0 Hz,1H); 7.04 (dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz,1H); 7.12e7.23
(m, 3H); EIMS m/z 248 (Mþ). Anal. Calcd for C14H13ClO2: C, 67.61; H,
5.27; Found: C, 67.57; H, 5.26.
4.4.1.2. 4-(4-Chlorophenethyl)phen-1,2,3-triol (1c). White powder,
yield 80%, mp 148e149 ꢁC,1H NMR (CDCl3): 2.84 (s, 4H); 4.86 (s,1H);
5.03 (s, 1H); 5.18 (s, 1H); 6.36 (d, J ¼ 8.4 Hz, 1H); 7.46 (d, J ¼ 8.4 Hz,
1H); 7.08 (d, J ¼ 8.2 Hz, 2H); 7.22 (d, J ¼ 8.4 Hz, 2H); EIMS m/z 264
(Mþ). Anal. Calcd for C14H13ClO3: C, 63.52; H, 4.95; Found: C, 63.53;
H, 4.97.
4.4.1.12. 4-(3-Bromophenethyl)phen-1,2-diol (2f). White powder,
59%, mp 62e64 ꢁC, 1H NMR (CDCl3): 2.76e2.86 (m, 4H);
4.65e5.38 (bs, 2H); 6.58 (dd, J ¼ 8.0 Hz, J ¼ 1.9 Hz, 1H); 6.67 (d,
4.4.1.3. 4-(4-Bromophenethyl)phen-1,2,3-triol (1d). White powder,
yield 60%, mp 147e149 ꢁC,1H NMR (CDCl3): 2.83 (s, 4H); 4.87 (s,1H);

5070
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 45 (2010) 5064e5070
J ¼ 1.9 Hz, 1H); 6.76 (d, J ¼ 8.2 Hz, 1H); 7.05 (d, J ¼ 8.0 Hz, 1H); 7.13
(t, J ¼ 8.0 Hz, 1H); 7.29e7.36 (m, 2H); EIMS m/z 292 (Mþ). Anal.
Calcd for C14H13BrO2: C, 57.36; H, 4.47; Found: C, 57.29; H, 4.48.
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powder, 79%, mp 60e62 ꢁC, 1H NMR (CDCl3): 2.74e2.85 (m, 4H);
3.83 (s, 3H); 3.85 (s, 3H); 6.54 (dd, J ¼ 8.8 Hz, J ¼ 1.5 Hz, 1H); 6.60
(d, J ¼ 1.7 Hz, 1H); 6.73 (d, J ¼ 8.6 Hz, 1H); 7.06 (dd, J ¼ 8.6 Hz,
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powder, 75%, mp 63e64 ꢁC, 1H NMR (CDCl3): 2.73e2.87 (m, 4H);
3.82 (s, 3H); 3.84 (s, 3H); 6.55 (dd, J ¼ 8.6 Hz, J ¼ 1.8 Hz, 1H); 6.63
(d, J ¼ 1.9 Hz, 1H); 6.71 (d, J ¼ 8.8 Hz, 1H); 7.06 (d, J ¼ 8.8 Hz, 2H);
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12.81.
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Acknowledgements
The work was financed by Scientific Research Fund of Hunan
Provincial Education Department (Project 09B083) of China and
Key Laboratory of Hunan Forest Products and Chemical Industry
Engineering (Project JDZ200904) of China.
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