Synthesis and evaluation of N-analogs of 1,2-diarylethane as Helicobacter pylori urease inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.06.014

Therapies based on urease inhibition are now seriously considered as the first line of treatment for infections caused by Helicobacter pylori. However, the present inhibitors are ineffective or unstable in highly acidic gastric juice. Here, we report a series of benzylanilines as effective inhibitors of H. pylori urease. Out of the obtained twenty-one compounds, N-(3,4-dihydroxybenzyl)-4-nitroaniline (4) was evaluated in detail and shows promising features for development as anti-H. pylori agent. Excellent potency against urease in both cell-free extract and intact cell was observed at low concentrations of 4 (IC₅₀ = 0.62 ± 0.04 and 1.92 ± 0.09 μM), which showed over 29- and 54-fold increase in potency with respect to the positive control AHA. The SAR analysis revealed that protection of 3,4-dihydroxy group of 4 as methoxy or changes of 4-NO₂ will result in a moderate to dramatic decrease in potency.

Full text of DOI:10.1016/j.bmc.2015.06.014

Bioorganic & Medicinal Chemistry 23 (2015) 4508–4513
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of N-analogs of 1,2-diarylethane as
Helicobacter pylori urease inhibitors
a
b
a
a
a
Zhu-Ping Xiao a,b, , Wei-Kang Shi , Peng-Fei Wang , Wei Wei , Xiao-Tong Zeng , Ji-Rong Zhang
⇑
Na Zhu a, Miao Peng a, Bin Peng a, Xiao-Yi Lin a, Hui Ouyang a, , Xiao-Chun Peng a, Guang-Cheng Wang a,
⇑
Hai-Liang Zhu b,
⇑
a College of Chemistry and Chemical 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:
Therapies based on urease inhibition are now seriously considered as the first line of treatment for infec-
tions caused by Helicobacter pylori. However, the present inhibitors are ineffective or unstable in highly
acidic gastric juice. Here, we report a series of benzylanilines as effective inhibitors of H. pylori urease. Out
of the obtained twenty-one compounds, N-(3,4-dihydroxybenzyl)-4-nitroaniline (4) was evaluated in
detail and shows promising features for development as anti-H. pylori agent. Excellent potency against
Received 11 April 2015
Revised 14 May 2015
Accepted 4 June 2015
Available online 14 June 2015
urease in both cell-free extract and intact cell was observed at low concentrations of
4
Keywords:
(IC50 = 0.62 0.04 and 1.92 0.09 M), which showed over 29- and 54-fold increase in potency with
l
Benzylaniline
Urease inhibitor
Peptic ulcer
respect to the positive control AHA. The SAR analysis revealed that protection of 3,4-dihydroxy group
of 4 as methoxy or changes of 4-NO2 will result in a moderate to dramatic decrease in potency.
Ó 2015 Elsevier Ltd. All rights reserved.
Molecular docking
1. Introduction
According to recent statistics, at least half of the world popula-
tion is estimated to be infected by H. pylori, and potent inhibitors of
It is well known that Helicobacter pylori infection causes various
gastric diseases, including chronic gastritis, gastric lymphoma,
peptic ulcer, and stomach cancer.1 With the help of a constitutive
urease production, H. pylori create a protective ammonium cloud
from urea, which allows the bacteria to survive at the low pH of
the stomach during colonization.2 Urease therefore plays an
important role in the pathogenesis of gastric diseases induced by
H. pylori and is known to be a major virulence factor,3 and strate-
gies based on urease inhibition are now essential lines of treatment
for H. pylori infection.1 Noticeably, ureases have also been isolated
from a variety of other pathogenic bacteria such as Proteus mir-
abilis, Brucella abortus and Yersinia enterocolitica.4 In infections with
these bacteria, urease is considered to be implicated in urolithiasis
(stone formation) and be directly involved in the development of
acute pyelonephritis and infection-induced reactive arthritis.5–7
Thus urease inhibition is of very great therapeutic and pharmaco-
logical significances.8
H. pylori urease have increasingly attracted the interests of medic-
inal chemists. In the past decades, many synthetic compounds,
natural products and metal complexes were determined as urease
inhibitors,9–13 with phosphoramidates being the most potent com-
pounds. However, direct treatment of H. pylori infection with phos-
phoramidates has been limited by the rapid hydrolysis in the low
pH of gastric juice.14 Therefore, it is of great urgency to discover
and develop novel chemical entities with satisfactory stability
and effective activity against H. pylori urease.
Recently, some novel urease inhibitors15–17 were determined by
our group with 1,2-diarylethane (1, Scheme 1) to be noted.18 This
specific inhibitor shows good potency against H. pylori urease in
both cell-free system and intact cell.19 It is well known that a rel-
ative simple molecular skeleton and low molecular weight indicate
a large of optimization space. Based on this consideration, com-
pound 1 was selected as a lead in this work. Molecular docking
studies of 1 revealed that the linkage of ethylene does not involve
in any interactions with residues in active site of urease. In this
docking complex, however, a relative short contact was observed
between the ethylene group in 1 and the sulfhydryl group in
Cys-321.19 Structural studies of the enzyme disclosed that
⇑
Corresponding authors. Tel./fax: +86 743 8563911 (Z.-P.X. and H.O.); tel.: +86
25 8359 2572; fax: +86 25 83592672 (H.-L.Z.).
Cys-321 is located on a flexible flap composed of residues
a
a313-
E-mail addresses: xiaozhuping2005@163.com (Z.-P. Xiao), oyhmail@163.com
(H. Ouyang), zhuhl@nju.edu.cn (H.-L. Zhu).
346,20 and is an essential residue for the catalytic activity of
http://dx.doi.org/10.1016/j.bmc.2015.06.014
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

Z.-P. Xiao et al. / Bioorg. Med. Chem. 23 (2015) 4508–4513
4509
Table 1
OH
In vitro inhibitory activity data of the synthesized compounds against H. pylori urease
HO
HO
R2
1
IC50 (lM)
N
H
R1
Compound
Scheme 1. Structure of compound 1.
R1
R2
Cell free
urease
Urease in intact
cell
H. pylori urease.21 Motivated by these findings and in continuation
of our ongoing efforts on the discovery of new urease inhibitors, a
series of benzylanilines, bioisosteres of 1 with substitution of NH
for a CH2, were herein reported, and some showe good potency.
4
3,4-(OH)2
4-OH
4-
NO2
4-
NO2
4-
NO2
4-
NO2
3-
NO2
3-
NO2
3-
NO2
3-
NO2
3-
NO2
3-
NO2
3-
NO2
2-
0.62 0.04
8.26 0.48
19.5 1.0
26.3 1.2
2.33 0.11
50.4 5.0
13.1 0.9
16.8 1.1
57.5 3.3
64.9 5.8
85.0 7.1
45.1 3.6
1.92 0.09
28.1 1. 8
83.8 1.6
126.2 13.1
9.52 0.60
ND
5
6
4-Cl
7
4-NO2
2. Results and discussion
2.1. Chemistry
8
3,4-(OH)2
3,4-(OMe)2
3,4-
9
The general synthetic route for benzylaniline derivatives
(4–24), N-analogs of 1,2-arylethanes, is shown in Scheme 2. The
synthesis of all benzylanilines begins from aldehydes (2), which
coupled with anilines (3) under Borch reaction conditions to yield
target compounds (4–24).
10
11
12
13
14
15
60.3 5.2
72.4 6.5
ND
Methylenedioxy
4-NMe2
4-Cl
4-NO2
3-NO2
3,4-(OH)2
ND
2.2. Urease inhibitory activity
ND
To understand the SAR for urease inhibition, structurally
diverse benzylanilines were profiled in an in vitro H. pylori urease
assay (Table 1). The results revealed that many compounds were
active in assays, and 4 was the most potent compound with an
ND
NO2
4-Cl
4-Cl
16
17
3,4-(OMe)2
3,4-
Methylenedioxy
4-NMe2
2-NO2
4-NO2
3-NO2
4-NMe2
>500
>500
ND
ND
IC50 of 0.62 0.04 lM, showing a >2- and 30-fold upward shift in
potency with respect to that of the lead compound 1 and the
positive control AHA (acetohydroxamic acid), respectively. This is
consistent with our earlier observations where the 3,4-dihydrox-
yphenyl pattern is very important to activity.19 Movement of the
nitro group of 4 from 4- to 3-position resulted in compound 8,
showing moderate (about 4-fold) decrease in potency, while move-
ment to 2-potion rendered the molecule (15) significant loss of
activity. In comparison with compound 4, removal of 3-OH led to
the compound 5 with a 13-fold decrease in potency. The activity
further decreased when 4-OH of 5 was replaced by Cl (6) or NO2
(7). These findings indicate that 3,4-dihydroxyl group of benzyl
moiety and 4-nitro group of aniline moiety are very essential
to urease inhibition, which were confirmed by a partial tolerance
of 3,4-dihydroxyl group being protected as methyl ether or
1,3-dioxolane (8 vs 9 or 10), and by an intolerance of 4-nitro group
being further replaced (4 vs 16 or 17).
18
19
20
21
22
4-Cl
4-Cl
3-Cl
3-Cl
4-
OMe
4-
OMe
4-
>500
ND
ND
ND
ND
ND
238.7 14.4
236.5 20.9
410.3 39.8
>500
23
4-Cl
>500
ND
24
3-NO2
319.2 18.3
18.4 1.3
ND
OMe
HAH
104.8 9.7
One should be noted that 4 also shows substantial intact-cell
urease assay activity, with over 54-fold improvement in compar-
ison to positive control AHA, indicating that 4 deserves further
research as a potential agent to treat diseases caused by H. pylori
infection.
Similar to what was observed for 3,4-disubstituted derivatives,
the 4-substituted analog with 3-nitro group in aniline moiety (12)
showed a moderate decrease in potency when compared to the
compound with 4-nitro group (6). In para–meta series (11–13),
conversion of the chlorine atom in compound 12 to a N,N-dimethy-
lamino group enhanced potency, while to a nitro group reduced
potency. These data together with those from para–para series
(5–7) confidently indicated the requirement of an electron-donat-
ing group for compounds with 4-monosubstitution pattern in ben-
zyl moiety. Regarding aniline moiety, the nitro group seems to be
optimal at both meta- and para-positions, and any variations of
the substitution caused significant degradation of activity or ren-
dered the molecule inactive (16–24).
2.3. Molecular docking
To further understand the results of biological measures, we
performed computational docking studies on the top active com-
pound 4. A detailed representation of the 4-urease active site is
shown in Figures 1 and 2. It is observed that the NH moiety of 4
is involved in an interaction with the key residue Cys-321 on the
flex flap, forming a N–Hꢀ ꢀ ꢀS hydrogen bond with a H-S distance
of 2.863 Å, which confirms our design conception for benzylani-
lines. In comparison with the previous reported compound 1,
compound 4 docked the urease active site in a similar pose with
3,4-dihydroxyphenyl moiety being oriented toward the entrance
cavity and the other phenyl moiety toward the bottom. However,
4-nitrophenyl moiety is more buried in the urea binding pocket
with respect to 4-hydroxyphenyl group of 1. The nitro group drops
completely into the bottom and chelates the metal doublet,
forming a tetrahedron with distances in a rang of 1.868–2.331 Å.
CHO
H2N
R2
NaBH4
EtOH
R1
+
R2
N
H
R1
2
3
4-24
In addition,
a hydrophobic interaction between the ring of
Scheme 2. Synthesis of benzylanilines.
4-nitrophenyl moiety and the backbone CH3 of Ala-365 (3.297 Å)

4510
Z.-P. Xiao et al. / Bioorg. Med. Chem. 23 (2015) 4508–4513
interactions with the enzyme provided by NO2, NH and 3,4-dihy-
droxy groups. The SAR analysis also confirmed that any changes
of NO2 or 3,4-dihydroxy group of compound 4, including substitu-
tion and shift, will lead to a moderate to dramatic decrease in
potency. Further investigations are in progress to determine the
activity in vivo and optimize the pharmacokinetics profile of the
compounds.
4. Experimental section
4.1. Materials
Protease inhibitors (Complete mini EDTA-free) were purchased
from Roche Diagnostics GmbH (Mannheim, Germany) and Brucella
broth was from Becton-Dickinson (Cockeysville, MD). Horse serum
was from Hyclone (Utah, American).
4.2. Bacteria
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 microaer-
obic conditions (5% O2, 10% CO2, and 85% N2), as our previously
described.22,23
Figure 1. Binding mode of compound 4 with H. pylori urease. The enzyme is shown
as surface, while 4 docked structure is shown as stick. The figure was made using
PyMol.
4.3. Preparation of H. pylori urease
For urease inhibition assays, 50 mL broth cultures
(2.0 ꢁ 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
were returned to room temperature, and after addition of 3 mL of
distilled water and protease inhibitors, sonication was performed
for 60 s. Following centrifugation (15,000g, 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.
4.4. Urease activity determination
One unit of urease activity is defined as the amount of enzyme
needed to liberate 1.0 lmol of NH3 from urea per min at pH 7.0 at
25 °C. According to the enzymatic assays of Sigma–Aldrich,24 the
activity of the obtained H. pylori urease was determined. Briefly,
one mL of 0.5 M urea with 0.05% (w/v) bovine serum albumin solu-
tion was pipetted into a test tube and warmed to 25 °C. Then,
0.20 mL of urease solution containing 0.02 M sodium phosphate
buffer (pH 7.0 at 25 °C) was added. The obtained mixture was
incubated at 25 °C for exactly 5 minutes with magnetic stirring,
and followed by addition of 0.20 mL of indicator (4.0 mg/mL of
p-nitrophenol solution), which was titrated immediately with
0.1 M standardized HCl until the color turns from yellow to color-
less. As for blank, only difference is that the urease solution was
added after incubation. The urease activity was calculated based
on the following formula:
Figure 2. Binding mode of compound 4 with H. pylori urease. For clarity, only
interacting residues were labeled. Hydrogen bonding interactions are shown in
dash. Ni atoms are shown as yellow balls. The figure was made using PyMol.
is observed. It is important to be noted that 3,4-dihydroxyphenyl
moiety is involved in three hydrogen bonding interactions
with the backbone amino group of Asn-168 and carbonyl group
of Asp 165. Additional interactions provided by NH and NO2
may give a rational explanation for the higher potency of 4
(IC50 = 0.62 0.04 lM) than 1 (IC50 = 1.5 0.2 lM).
3. Conclusion
0:1 mmol=mL ꢁ
D
VHCl mL ꢁ 1000
lmol=mmol
Benzylanilines, the bioisostere of 1,2-diarylethane with substi-
tution of NH for CH2, were determined as H. pylori urease inhibi-
tors. Several derivatives showed excellent activity against urease
in both cell-free extract and intact cell, with 4-((4-nitropheny-
lamino)methyl)benzene-1,2-diol (4) being the most active, which
has the potential to be developed for treatment of gastritis and gas-
tric ulcer caused by H. pylori infection. Molecular dockings dis-
closed that its potency may be attributed to the tight
U=mL urease ¼
5 min ꢁ0:2 mL
4.5. Measurement of urease inhibitory activity
The assay mixture, containing 25
and 25 L of the test compound, was pre-incubated for 1.5 h at
room temperature in a 96-well assay plate. Urease activity was
lL (10U) of H. pylori urease
l

Z.-P. Xiao et al. / Bioorg. Med. Chem. 23 (2015) 4508–4513
4511
determined by measuring ammonia production using the indophe-
nol method as described by Weatherburn.25
244 (M+). Anal. Calcd for C13H12N2O3: C, 63.93; H, 4.95; N, 11.47.
Found: C, 63.98; H, 4.95; N, 11.45.
4.6. Protocol of docking study
4.7.1.3. N-(4-Chlorobenzyl)-4-nitroaniline (6).
Yellow pow-
der, yield 48%, mp 197–199 °C, 1H NMR (400 MHz, CDCl3
+
The automated docking studies were carried out using
AutoDock 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 56 Å size (x, y, z) with a spacing of 0.375 Å
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 ꢁ 106 energy evaluations, and a maxi-
mum number of 2.7 ꢁ 104 generations. A mutation rate of 0.02
and a crossover rate of 0.8 were chosen. Results differing by less
than 0.5 Å in positional root-mean-square deviation (RMSD) were
clustered together and the results of the most favorable free energy
of binding were selected as the resultant complex structures.
DMSO-d6): 4.32 (s, 2H); 6.52 (d, J = 8.6 Hz, 2H); 7.17 (bs, 1H);
7.24 (s, 4H); 7.91 (d, J = 8.7 Hz, 2H); EIMS m/z 262 (M+). Anal.
Calcd for C13H11ClN2O2: C, 59.44; H, 4.22; Cl, 13.50; N, 10.66.
Found: C, 59.48; H, 4.22; Cl, 13.49; N, 10.65.
4.7.1.4. 4-Nitro-N-(4-nitrobenzyl)aniline (7).
Light yellow
powder, yield 41%, mp 187–188 °C, 1H NMR (300 MHz, DMSO-
d6): 4.19 (d, J = 5.6 Hz, 2H); 6.52 (t, J = 5.5 Hz, 1H); 7.48 (d,
J = 8.4 Hz, 2H); 6.57 (d, J = 8.8 Hz, 2H); 7.94 (d, J = 8.9 Hz, 2H);
8.27 (d, J = 8.4 Hz, 2H); EIMS m/z 273 (M+). Anal. Calcd for
C13H11N3O4: C, 57.14; H, 4.06; N, 15.38. Found: C, 57.19; H, 4.05;
N, 15.39.
4.7.1.5.
(8).
4-((3-Nitrophenylamino)methyl)benzene-1,2-diol
Orange powder, yield 77%, mp 126–128 °C, 1H NMR
(300 MHz, DMSO-d6): 4.15 (s, 2H); 6.61 (d, J = 8.0 Hz, 1H); 6.68
(d, J = 8.0 Hz, 1H); 6.73 (s, 1H); 6.84 (bs, 1H); 6.97 (d, J = 7.0 Hz,
1H); 7.25–7.33 (m, 3H); 8.72 (bs, 1H); 8.80 (bs, 1H); EIMS m/z
260 (M+). Anal. Calcd for C13H12N2O4: C, 60.00; H, 4.65; N, 10.76.
Found: C, 59.94; H, 4.67; N, 10.77.
4.7. Chemistry
4.7.1.6. N-(3,4-Dimethoxybenzyl)-3-nitroaniline (9).
Yellow
powder, yield 37%, mp 146–148 °C, 1H NMR (300 MHz, DMSO-d6):
3.72 (s, 3H); 3.74 (s, 3H); 4.26 (d, J = 5.9 Hz, 2H); 6.86–6.92 (m,
3H); 6.96–7.02 (m, 2H); 7.29 (t, J = 7.9 Hz, 1H); 7.32 (d, J = 1.8 Hz,
1H); 7.37 (d, J = 2.2 Hz, 1H); EIMS m/z 288 (M+). Anal. Calcd for
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 or 400 spectrometer at 25 °C with
TMS and solvent signals allotted as internal standards. Chemical
shifts were reported in ppm (d). Elemental analyses were per-
formed on a CHN-O-Rapid instrument and were within 0.4% of
the theoretical values.
C15H16N2O4: C, 62.49; H, 5.59; N, 9.72. Found: C, 62.41; H, 5.60;
N, 9.73.
4.7.1.7.
(10).
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-3-nitroaniline
Yellow powder, yield 63%, mp 146–148 °C, 1H NMR
(300 MHz, DMSO-d6): 4.25 (d, J = 5.9 Hz, 2H); 6.12 (s, 2H); 6.84–
6.93 (m, 3H); 6.98 (s, 1H); 7.00 (d, J = 8.1 Hz, 1H); 7.29 (t,
J = 7.9 Hz, 1H); 7.32 (s, 1H); 7.37 (s, 1H); EIMS m/z 272 (M+).
Anal. Calcd for C14H12N2O4: C, 61.76; H, 4.44; N, 10.29. Found: C,
61.69; H, 4.44; N, 10.30.
4.7.1. General procedure for the preparation of compounds 4-24
A solution of an aniline 3 (10 mmol) and an appropriate substi-
tuted benzaldehyde 2 (10 mmol) in methanol (10 mL) was refluxed
for 3–5 h. After cooling and addition of NaBH4 (15 mmol), the mix-
ture was further stirred at room temperature for 3–6 h and concen-
trated under reduced pressure. The residue was dissolved in AcOEt
(80 mL), and 40 mL of water was added. The aqueous layer was
separated and extracted with AcOEt (2 ꢁ 40 mL). The organic layer
and extracts were combined, dried over MgSO4, and concentrated
under reduced pressure, and the residue was purified by flash col-
umn chromatography (silica gel, EtOAc/petroleum ether) to give
desired compounds 4–24.
4.7.1.8. N,N-Dimethyl-4-((3-nitrophenylamino)methyl)aniline
(11).
Light yellow powder, yield 51%, mp 104–106 °C, 1H
NMR (300 MHz, DMSO-d6): 2.85 (s, 6H); 4.19 (d, J = 5.7 Hz, 2H);
6.69 (d, J = 7.5 Hz, 2H); 6.83 (t, J = 5.5 Hz, 1H); 6.98 (d, J = 7.0 Hz,
1H); 7.18 (d, J = 7.7 Hz, 2H); 7.25–7.35 (m, 3H); EIMS m/z 271
(M+). Anal. Calcd for C15H17N3O2: C, 66.40; H, 6.32; N, 15.49.
Found: C, 66.46; H, 6.33; N, 15.47.
4.7.1.9. N-(4-Chlorobenzyl)-3-nitroaniline (12).
Light yel-
low powder, yield 83%, mp 108–110 °C, 1H NMR (400 MHz,
CDCl3): 4.30 (d, J = 5.3 Hz, 2H); 4.36 (bs, 1H); 6.79 (dd, J = 7.9 Hz,
J = 1.9 Hz, 1H); 7.18–7.27 (m, 5H); 7.34 (t, J = 1.9 Hz, 1H); 7.47
(dd, J = 7.9 Hz, J = 1.4 Hz, 1H); EIMS m/z 262 (M+). Anal. Calcd for
4.7.1.1.
(4).
4-((4-Nitrophenylamino)methyl)benzene-1,2-diol
Yellow powder, yield 85%, mp 182–183 °C, 1H NMR
(300 MHz, DMSO-d6): 4.21 (d, J = 5.3 Hz, 2H); 6.59 (dd, J = 8.0 Hz,
J = 1.8 Hz, 1H); 6.65 (d, J = 9.7 Hz, 2H); 6.68 (d, J = 8.0 Hz, 1H);
6.71 (d, J = 1.8 Hz, 1H); 7.69 (t, J = 5.5 Hz, 1H); 7.97 (d, J = 9.3 Hz,
2H); 8.78 (s, 1H); 8.84 (s, 1H); EIMS m/z 260 (M+). Anal. Calcd for
C13H11ClN2O2: C, 59.44; H, 4.22; Cl, 13.50; N, 10.66. Found: C,
59.49; H, 4.22; Cl, 13.48; N, 10.65.
C
13H12N2O4: C, 60.00; H, 4.65; N, 10.76. Found: C, 59.92; H, 4.65;
4.7.1.10. 3-Nitro-N-(4-nitrobenzyl)aniline (13).
Orange
N, 10.78
powder, yield 69%, mp 129–131 °C, 1H NMR (400 MHz, CDCl3):
4.55 (d, J = 4.6 Hz, 2H); 4.6 (bs, 1H); 6.86 (d, J = 7.9 Hz, 1H); 7.29
(t, J = 8.0 Hz, 1H); 7.40 (s, 1H); 7.54 (d, J = 8.3 Hz, 2H); 7.58 (d,
J = 8.0 Hz, 1H); 8.23 (d, J = 8.4 Hz, 2H); EIMS m/z 273 (M+). Anal.
Calcd for C13H11N3O4: C, 57.14; H, 4.06; N, 15.38. Found: C,
57.08; H, 4.07; N, 15.40.
4.7.1.2. 4-((4-Nitrophenylamino)methyl)phenol (5).
Light
yellow powder, yield 53%, mp 141–143 °C, 1H NMR (300 MHz,
DMSO-d6): 4.37 (d, J = 5.2 Hz, 2H); 6.47 (t, J = 5.2 Hz, 1H); 7.06 (s,
4H); 7.42 (d, J = 8.8 Hz, 2H); 8.21 (d, J = 8.9 Hz, 2H); EIMS m/z

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Z.-P. Xiao et al. / Bioorg. Med. Chem. 23 (2015) 4508–4513
4.7.1.11. 3-Nitro-N-(3-nitrobenzyl)aniline (14).
Light yellow
4.7.1.19. 4-((4-Methoxyphenylamino)methyl)-N,N-dimethylani-
line (22).
White powder, yield 64%, mp 86–87 °C, 1H NMR
(400 MHz, DMSO-d6): 2.84 (s, 6H); 3.60 (s, 3H); 4.05 (d,
J = 6.1 Hz, 2H); 5.56 (t, J = 5.9 Hz, 1H); 6.52 (d, J = 9.0 Hz, 2H);
6.66 (d, J = 9.0 Hz, 2H); 6.67 (d, J = 8.8 Hz, 2H); 7.16 (d, J = 8.8 Hz,
2H); EIMS m/z 256 (M+). Anal. Calcd for C16H20N2O: C, 74.97; H,
7.86; N, 10.93. Found: C, 74.89; H, 7.87; N, 10.95.
powder, yield 79%, mp 130–131 °C, 1H NMR (400 MHz, CDCl3):
4.54 (d, J = 4.2 Hz, 2H); 6.88 (d, J = 7.8 Hz, 1H); 7.30 (t, J = 8.0 Hz,
1H); 7.41 (s, 1H); 7.54 (d, J = 7.8 Hz, 1H); 7.58 (d, J = 6.9 Hz, 1H);
7.71 (d, J = 7.2 Hz, 1H); 8.17 (d, J = 7.9 Hz, 1H); 8.24 (s, 1H); EIMS
m/z 273 (M+). Anal. Calcd for C13H11N3O4: C, 57.14; H, 4.06; N,
15.38. Found: C, 57.19; H, 4.06; N, 15.37.
4.7.1.12.
(15).
4-((2-Nitrophenylamino)methyl)benzene-1,2-diol
4.7.1.20. N-(4-Chlorobenzyl)-4-methoxyaniline (23).
White
Yellow powder, yield 58%, mp 109–111 °C, 1H NMR
powder, yield 75%, mp 70–72 °C, 1H NMR (400 MHz, DMSO-d6):
3.74 (s, 3H); 3.84 (bs, 1H); 4.26 (s, 2H); 6.57 (d, J = 8.6 Hz, 2H);
6.70 (d, J = 8.6 Hz, 2H); 7.30 (s, 4H); EIMS m/z 247 (M+). Anal.
Calcd for C14H14ClNO: C, 67.88; H, 5.70; Cl, 14.31; N, 5.65. Found:
C, 67.82; H, 5.71; Cl, 14.33; N, 5.65.
(300 MHz, DMSO-d6): 4.41 (d, J = 5.9 Hz, 2H); 6.59–6.69 (m, 3H);
6.72 (d, J = 2.0 Hz, 1H); 6.92 (d, J = 8.8 Hz, 1H); 7.44 (t, J = 7.5 Hz,
1H); 8.05 (dd, J = 8.6 Hz, J = 1.5 Hz, 1H); 8.48 (t, J = 5.7 Hz, 1H);
8.80 (bs, 2H); EIMS m/z 260 (M+). Anal. Calcd for C13H12N2O4: C,
60.00; H, 4.65; N, 10.76. Found: C, 60.08; H, 4.64; N, 10.74.
4.7.1.21. 4-Methoxy-N-(3-nitrobenzyl)aniline (24).
Light
4.7.1.13.
(16).
4-Chloro-N-(3,4-dimethoxybenzyl)aniline
yellow powder, yield 49%, mp 76–78 °C, 1H NMR (300 MHz,
CDCl3): 3.76 (s, 3H); 4.44 (s, 2H);6.61 (d, J = 9.0 Hz, 2H); 6.80 (d,
J = 8.9 Hz, 2H); 7.52 (t, J = 7.9 Hz, 1H); 7.74 (d, J = 7.7 Hz, 1H);
8.14 (d, J = 8.2 Hz, 1H); 8.27 (s, 1H); EIMS m/z 258 (M+). Anal.
Calcd for C14H14N2O3: C, 65.11; H, 5.46; N, 10.85. Found: C,
65.17; H, 5.45; N, 10.83.
White powder, yield 75%, mp 119–121 °C, 1H NMR
(300 MHz, DMSO-d6): 3.71 (s, 2H); 3.76 (s, 3H); 3.72 (s, 3H); 4.15
(d, J = 5.9 Hz, 2H); 6.32 (t, J = 6.0 Hz, 1H); 6.57 (d, J = 9.0 Hz, 2H);
6.84 (dd, J = 8.4 Hz, J = 1.5 Hz, 1H); 6.88 (d, J = 8.0 Hz, 1H); 6.95
(d, J = 1.3 Hz, 1H); 7.04 (d, J = 8.8 Hz, 2H); EIMS m/z 277 (M+).
Anal. Calcd for C15H16ClNO2: C, 64.87; H, 5.81; Cl, 12.76; N, 5.04.
Found: C, 64.82; H, 5.80; Cl, 12.78; N, 5.05.
Acknowledgements
4.7.1.14.
(17).
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-4-chloroaniline
White powder, yield 71%, mp 112–114 °C, 1H NMR
The work was financed by grants from National Natural Science
Foundation (Grant No. 81273382) of China, by a Project supported
by Hunan Provincial Natural Science Foundation of China (Grant
No. 2015JJ2116 and 13JJ2030), by aid program (Environment and
Energy Materials and deep processing of mineral resources in
Wuling Mountain) for Science and Technology Innovative
Research Team in Higher Educational Institutions of Hunan
Province, by aid program from Science and Technology
Innovative Research Team in Jishou University for Improving
Drug-like Properties of Active Components from Medicinal Plants
in Wulin Montains.
(300 MHz, DMSO-d6): 4.15 (d, J = 5.9 Hz, 2H); 6.09 (s, 2H); 6.31
(t, J = 5.8 Hz, 1H); 6.57 (d, J = 8.9 Hz, 2H); 6.84 (d, J = 8.2 Hz, 1H);
6.88 (d, J = 8.4 Hz, 1H); 7.05 (d, J = 8.9 Hz, 2H); EIMS m/z 261
(M+). Anal. Calcd for C14H12ClNO2: C, 64.25; H, 4.62; Cl, 13.55; N,
5.35. Found: C, 64.19; H, 4.61; Cl, 13.57; N, 5.36.
4.7.1.15. 4-((4-Chlorophenylamino)methyl)-N,N-dimethylani-
line (18).
White powder, yield 46%, mp 93–95 °C, 1H NMR
(300 MHz, DMSO-d6): 2.84 (s, 6H); 4.09 (d, J = 5.9 Hz, 2H); 6.23
(t, J = 5.9 Hz, 1H); 6.56 (d, J = 8.8 Hz, 2H); 6.68 (d, J = 8.6 Hz, 2H);
7.01 (d, J = 8.8 Hz, 2H); 7.15 (d, J = 8.4 Hz, 2H); EIMS m/z 260
(M+). Anal. Calcd for C15H17ClN2: C, 69.09; H, 6.57; Cl, 13.60; N,
10.74. Found: C, 69.14; H, 6.55; Cl, 13.59; N, 10.75.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.06.014.
4.7.1.16. 4-Chloro-N-(2-nitrobenzyl)aniline (19).
Yellow
powder, yield 72%, mp 83–84 °C, 1H NMR (300 MHz, DMSO-d6):
4.59 (d, J = 6.0 Hz, 2H); 6.48–6.59 (m, 3H); 7.05 (d, J = 8.8 Hz,
2H); 7.52 (t, J = 7.5 Hz, 1H); 7.58 (d, J = 7.7 Hz, 1H); 7.69 (d,
J = 7.5 Hz, 1H); 8.05 (d, J = 8.0 Hz, 1H); EIMS m/z 262 (M+). Anal.
Calcd for C13H11ClN2O2: C, 59.44; H, 4.22; Cl, 13.50; N, 10.66.
Found: C, 59.48; H, 4.21; Cl, 13.48; N, 10.67.
References and notes
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Light yel-
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d6): 4.41 (d, J = 5.2 Hz, 2H); 6.20 (t, J = 4.8 Hz, 1H); 6.42 (d,
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