Development of 3,5-dinitrobenzoate-based 5-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.03.008

Human 5-lipoxygenase (5-LOX) is a well-validated target for anti-inflammatory therapy. Development of novel 5-LOX inhibitors with higher activities is highly demanded. In previous study, we have built a model for the active conformation of human 5-LOX, and identified naphthalen-1-yl 3,5-dinitrobenzoate (JMC-4) as a 5-LOX inhibitor by virtual screening. In the present work, 3,5-dinitrobenzoate-based 5-lipoxygenase inhibitors were developed. Twenty aryl 3,5-dinitrobenzoates, N-aryl 3,5-dinitrobenzamides and analogues were designed and synthesized. Several of them were found with significantly increased activities according to cell-free assay and human whole blood assay. The structure-activity relationship study may provide useful insights for designing effective 5-LOX inhibitors.

Full text of DOI:10.1016/j.bmc.2014.03.008

Bioorganic & Medicinal Chemistry 22 (2014) 2396–2402
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of 3,5-dinitrobenzoate-based 5-lipoxygenase inhibitors
⇑
Erchang Shang, Ying Liu , Yiran Wu, Wei Zhu, Chong He, Luhua Lai
BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Center for Quantitative Biology,
Peking University, Beijing 100871, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Human 5-lipoxygenase (5-LOX) is a well-validated target for anti-inflammatory therapy. Development of
novel 5-LOX inhibitors with higher activities is highly demanded. In previous study, we have built a
model for the active conformation of human 5-LOX, and identified naphthalen-1-yl 3,5-dinitrobenzoate
(JMC-4) as a 5-LOX inhibitor by virtual screening. In the present work, 3,5-dinitrobenzoate-based
5-lipoxygenase inhibitors were developed. Twenty aryl 3,5-dinitrobenzoates, N-aryl 3,5-dinitrobenza-
mides and analogues were designed and synthesized. Several of them were found with significantly
increased activities according to cell-free assay and human whole blood assay. The structure–activity
relationship study may provide useful insights for designing effective 5-LOX inhibitors.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 10 February 2014
Revised 5 March 2014
Accepted 6 March 2014
Available online 17 March 2014
Keywords:
3,5-Dinitrobenzoate
5-Lipoxygenase inhibitors
Cell-free assay
Human whole blood assay
SAR study
1. Introduction
pound and the enzyme. As shown in Figure 1, one nitro group
makes a hydrogen bond with His600 of 5-LOX; the naphthyl group
5-Lipoxygenase (5-LOX) is a key enzyme in the metabolism of
arachidonic acid (AA) to produce leukotrienes. It catalyzes the
two-step conversion from AA to leukotriene A4 (LTA4).1 LTA4 is fur-
ther metabolized by leukotriene A4 hydrolase to leukotriene B4, or
by leukotriene C4 synthase to leukotriene C4 and then to leukotri-
ene D4 and E4.2 Leukotrienes are important mediators of inflamma-
tory and allergic diseases, and also play roles in cardiovascular
diseases and cancers.3–6 The upstream enzyme of leukotrienes,
5-LOX, has been validated as a drug target for inflammation and
related disorders therapy.7 Several types of 5-LOX inhibitors have
been reported, including redox, iron ligand, nonredox, and compet-
itive inhibitors (for review see Ref. 8).8 Up to now, only one 5-LOX
inhibitor, zileuton, entered the market.9 However, weak potency
and short duration are the therapeutic problems of zileuton.10
Development of safe and effective 5-LOX inhibitors is highly
demanded.
is crucial to form hydrophobic interaction with the pocket around
Leu414. The ester linker of JMC-4 is close to Tyr181 and Gln363,
but no hydrogen bond is formed for the relative position. Guided
by this interaction model, in the present work, we tried to improve
the potency of JMC-4. Firstly, phenyls with different substituent
We previously reported a model for the active conformation of
human 5-LOX and new inhibitors identified by virtual screening
using this model.11 Among these inhibitors, naphthalen-1-yl 3,
5-dinitrobenzoate (JMC-4), shows activity in the micromolar range
and possesses novel structure compared to known 5-LOX inhibi-
tors. Through analyzing of the docking conformation of JMC-4 with
5-LOX, we recognized several key interactions between the com-
⇑
Corresponding author. Tel.: +86 10 62751490; fax: +86 10 62751725.
E-mail address: liuying@pku.edu.cn (Y. Liu).
Figure 1. Binding mode of JMC-4 with 5-LOX substrate binding site (predicted by
molecular docking).
http://dx.doi.org/10.1016/j.bmc.2014.03.008
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

E. Shang et al. / Bioorg. Med. Chem. 22 (2014) 2396–2402
2397
groups were introduced to search a more active hydrophobic moi-
ety. Secondly, amide bond was used to replace the ester bond to
test a linker with hydrogen bond donor. Finally, as the binding con-
formation shows only one nitro group makes a key interaction
with the target, the mono-nitro group counterpart of JMC-4 and
the compounds with carboxyl group instead of nitro group were
tested.
(10a–10c). Then compounds (11a–11c) were obtained by cleavage
of the benzyl ester of 10a–10c.
2.2. Inhibition assay of 5-LOX in cell-free system
A fluorescence-based enzyme assay was used to test inhibition
of the target compounds to human 5-LOX.13 The fluorescent signal
is measured during oxidation of 2,7-dichlorodihydrofluorescein
diacetate (H2DCFDA) by human 5-LOX product. All compounds
were dissolved in DMSO, and the inhibition values were tested at
2. Results and discussion
2.1. Chemistry
the concentration of 50 lM in preliminary test. Zileuton was used
as the positive control and DMSO (4.5%, v/v) was used as vehicle
control. The compounds of inhibition over 50% in the preliminary
test were subjected to the determination of IC50 values (see
Table 1).
To prepare compounds 3a–3f and 4a, 3,5-dinitrobenzoic acid or
3-nitrobenzoic acid was reacted with different substituted phe-
nols.
Using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI) as coupling agent and 4-dimethylaminopyri-
dine (DMAP) as catalyst, the target compounds were obtained in
moderate to high yields (Scheme 1). The amide analogs (7a–7i)
were prepared using a two-step synthetic method, as shown in
Scheme 2. The overall yields ranged from 60% to 80%. Preparation
of the target compounds (11a–11c) were outlined in Scheme 3. The
monobenzyl ester (9) was synthesized starting from isophthalic
acid (8) according to the literature.12 Acid chloride formation step
followed by amidation or esterification gave the intermediates
2.3. Measuring LTB4 generation in human whole blood
All the compounds were also evaluated in a human whole blood
(HWB) assay, in which calcium ionophore A23187 was used to in-
duce the 5-LOX pathway of AA metabolic network.14 A LTB4 ELISA
kit was used to monitor the production of LTB4 in HWB assay. As a
preliminary test, all the compounds were tested at 50 lM concen-
tration. Compounds with inhibition activity over 50% were then
subjected to the determination of IC50 values. Zileuton was used
as the positive control and DMSO (4.5%, v/v) was used as vehicle
control.
2.4. Structure–activity relationship studies
Among the newly prepared ester analogs with different
hydrophobic groups, some compounds show improved activities.
The introduction of 3-Me-phenyl as a hydrophobic moiety leads
to
a
highly active compound 3-tolyl 3,5-dinitrobenzoate, 3a
M in HWB assay).
(IC50 = 6 nM in cell-free assay and IC50 = 0.5
l
Phenyl group at the same position leads to compound 3b with
activity comparable to the parent compound, but 3-propyl-phenyl
(compound 3c) results in a 5-fold loss of potency. Furthermore,
replacement of phenyl with 2,3-dichlorophenyl (3d) loses the
activity. Introduction of phenyl at the 3-position of phenyl (3e) re-
sults in about 9-fold and 4-fold loss of potency in cell-free and
HWB assays, respectively. This tendency of potency reducing is en-
hanced while the phenyl group is substituted at the 4-position (3f).
These results indicate that the shape of the hydrophobic moiety is
crucial for inhibition activity: phenyl with small substituent group
at 3-position performs best, while 2- or 4-substitution is less
optimal.
Scheme 1. Synthesis of ester linker analogs of JMC-4. Reagents and conditions: (a)
R1OH, EDCI, DMAP, THF, rt.
Most amide analogs of the parent compound show enhanced
inhibition activities to 5-LOX in cell-free assay. Interestingly, only
one atom change from oxygen to nitrogen leads to the significant
potency improvement of compound N-(naphthalene-1-yl)-3,5-
dinitrobenzamide, 7a (from 1.0 lM of JMC-4 to 4 nM of 7a). Be-
Scheme 2. Synthesis of amide linker analogs of JMC-4. Reagents and conditions: (a)
SOCl2, reflux; (b) R3NH2, Et3N, THF, rt.
sides, a series of phenyl analogues (7c, 7d, 7f, and 7i) also show
nanomolar inhibition. The significant potency increasing of amide
analogs may due to the relatively rigid amide bond, which makes
the two moieties maintain quasi-planar geometries. However, in
HWB assay, only compounds N-phenyl 3,5-dinitrobenzamide
(7b),
N-(4-methoxyphenyl)-3,5-dinitrobenzamide
(7f)
and
N-(3-bromobenzyl)-3,5-dinitrobenzamide (7h) can inhibit the
production of LTB4, while others cannot.
The mono-nitro derivative naphthalene-1-yl 3-nitrobenzoate
(4a) has inhibition potency (IC50 = 7.1
with the parent compound in cell-free assay, and is also active in
HWB (IC50 = 24.6 M). This result is in good agreement with the
prediction that only one nitro group is essential in the binding. A
series of carboxyl analogs of the mono-nitro compound 4a were
lM) in the same magnitude
l
Scheme 3. Synthesis of carboxyl derivatives of JMC-4. Reagents and conditions: (a)
(i) Et3N, MeOH, rt; (ii) BnBr, DMF, 100 °C; (b) (i) SOCl2, reflux; (ii) R2YH, THF, rt; (c)
H2, Pd/C, MeOH, rt.

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E. Shang et al. / Bioorg. Med. Chem. 22 (2014) 2396–2402
Table 1
Bio-evaluation of compounds
O
X
Z
R
Y
Compd
Structure
Z
Cell-freeb IC50
(l
M)
HWBb IC50
(lM)
X
Y
R
Zileutona,11
1.1 0.5
1.0 0.2
3.7 0.4
8.6 0.9
JMC-411
NO2
NO2
NO2
NO2
NO2
O
O
O
O
3a
3b
3c
NO2
NO2
NO2
0.006 0.001
0.5 0.2
15.8 1.2
6.5 1.4
CH3
1.6 0.1
7.4 0.6
CH3
Cl
Cl
3d
3e
NO2
NO2
NO2
NO2
O
O
>50
>50
8.7 0.4
38.0 4.5
3f
NO2
NO2
NO2
H
O
O
>50
>50
4a
7.1 1.6
24.6 2.2
7a
7b
NO2
NO2
NO2
NO2
NH
NH
0.004 0.002
>50
1.8 0.7
15.2 4.0
7c
NO2
NO2
NO2
NO2
NH
NH
0.01 0.01
>50
>50
CH3
7d
0.033 0.005
Cl

E. Shang et al. / Bioorg. Med. Chem. 22 (2014) 2396–2402
2399
M)
Table 1 (continued)
Compd
Structure
Cell-freeb IC50
(l
M)
HWBb IC50
(l
X
Y
Z
R
7e
7f
NO2
NO2
NH
>50
>50
Br
OCH3
NO2
NO2
NO2
NO2
NH
NH
0.034 0.004
2.7 0.7
7g
7h
1.1 0.1
>50
OCH3
OCH3
NO2
NO2
NH
83.6 6.4
14.4 7.6
Br
7i
9
NO2
NO2
NH
0.030 0.003
>50
>50
COOH
H
O
>50
11a
11b
11c
COOH
COOH
COOH
H
H
H
O
>50
>50
>50
>50
>50
>50
NH
NH
a
Positive control.
Data are given as mean SE of n P 3 determinations.
b
also synthesized and evaluated for inhibition activity. However, all
the carboxylic acid derivatives, 9 and 11a–11c, have no inhibition
activity to 5-LOX. The potency loss of these compounds may due to
their undesirable pKa profiles.
3,5-Dinitrobenzoate is always used as organic synthesis inter-
mediate for its important scaffold. Some structures of our newly
designed 5-LOX inhibitors were listed in Reaxys database.15 Fortu-
nately, we do not found any bio-activity report of the prepared
compounds through an extensive literature search. Consequently,
this is the first research about anti-inflammatory activities of these
compounds.
To explain the increasing activities of the newly synthesized
compounds, we docked ester analog 3a and amide analog 7c into
the binding site of 5-LOX (Fig. 2). The two compounds share
the same conformation of the linker, and both form an addi-
tional hydrogen bonds to Tyr181 of 5-LOX (3a, the ester oxygen
atom; 7c, the amide oxygen atom). Also for the hydrogen bond
between nitro group and His600 of 5-LOX, which exists in 3a
(2.77 Å) and 7c (2.59 Å) is stronger than that of JMC-4 (3.11 Å).
These interactions are likely to enhance the activities of the two
compounds. Additionally, the relatively rigid amide bond may
play key roles in the significant potency increasing of most amide
analogs.
3. Conclusion
Twenty compounds with 3,5-dinitrobenzoate scaffold were
synthesized and evaluated using cell-free and HWB assays for their
5-LOX inhibition activities. Compared to the parent compound, six
compounds show significantly increased enzyme inhibition activi-
ties. The best compound, 3-tolyl 3,5-dinitrobenzoate (3a), showed
high potency in cell free assay (IC50 = 6 nM) and in human whole
blood assay (IC50 = 0.5
lM). SAR studies gave insights to the design
of better 5-LOX inhibitors.

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E. Shang et al. / Bioorg. Med. Chem. 22 (2014) 2396–2402
Figure 2. Inhibitors in 5-LOX substrate binding site (predicted by molecular docking). (A) Compound 3a (green). (B) Compound 7c (magenta).
3-Propylphenyl 3,5-dinitrobenzoate (3c). The compound 3c was
prepared according to the general procedure described above as a
gray lamellar crystal in 36% yield. Mp: 83–85 °C (ethyl acetate/hex-
ane). 1H NMR (300 MHz, CDCl3): d 9.30 (m, 3H), 7.39 (m, 1H), 7.17
(d, J = 9.0 Hz, 1H), 7.08 (d, J = 3.0 Hz, 2H), 2.65 (t, J = 6.0 Hz, 2H),
1.67 (m, J = 6.0 Hz, 2H), 0.97 (t, 3H); HRMS (ESI): calcd for C16H14-
N2NaO6 [(M+Na)+] 353.0744, found 353.0744.
4. Experiments
4.1. Chemistry
4.1.1. General conditions
The reagents and solvents were commercially available and
purified according to conventional methods. Melting points were
determined on an X6 microscopic melting point apparatus and
were uncorrected. All new compounds gave satisfactory 1H NMR,
1H NMR spectra are represented as follows: chemical shift, multi-
plicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad,
m = multiplet), integration. Representative compounds were mea-
sured their 13C NMR spectra. 1H (300 MHz) and 13C (100 MHz)
NMR spectra were recorded on a Bruker Ascend 300/400 MHz
spectrometer. Chemical shifts (d) are relative to tetramethylsilane
(0 ppm). HRMS were recorded on a Bruker Apex IV FTMS mass
spectrometer using ESI (electrospray ionization). Compound 3d
and 7i did not show the molecular ion peak in ESI test due to
hardly ionization.
2,3-Dichlorophenyl 3,5-dinitrobenzoate (3d). The compound 3e
was prepared according to the general procedure described above
as a light yellow needle crystal in 68% yield. Mp: 154–156 °C (ethyl
acetate/hexane). 1H NMR (300 MHz, CDCl3): d 9.13 (m, 3H), 7.72
(m, 1H), 7.64 (m, 1H), 7.56 (m, 1H).
Biphenyl-3-yl 3,5-dinitrobenzoate (3e). The compound 3f was
prepared according to the general procedure described above as
a light yellow solid in 70% yield. Mp: 176–177 °C (ethyl acetate/
hexane). 1H NMR (300 MHz, DMSO): d 9.12 (m, 3H), 7.72 (m,
4H), 7.61 (t, 1H), 7.50 (t, 2H), 7.41 (d, 2H); HRMS (ESI): calcd for
C
19H12N2NaO6 [(M+Na)+] 387.0588, found 387.0596.
4.1.2. Synthesis of 3a–3g, and 4a
Biphenyl-4-yl 3,5-dinitrobenzoate (3f). The compound 3g was
prepared according to the general procedure described above as
a light yellow solid in 78% yield. Mp: 229–230 °C (ethyl acetate/
hexane). 1H NMR (300 MHz, DMSO): d 9.12 (m, 3H), 7.83 (d,
J = 9.0 Hz, 2H), 7.80 (d, J = 9.0 Hz, 2H), 7.50 (m, 4H), 7.40 (t,
J = 9.0 Hz, 1H); HRMS (ESI): calcd for C19H12N2NaO6 [(M+Na)+]
387.0588, found 387.0579.
General method for the preparation of the dinitrobenzo-
ates. To a stirred solution of 3,5-dinitrobenzoic acid (1.1 mmol)
in THF (10 mL), phenol (1.0 mmol), EDCI (1.2 mmol) and DMAP
(cat.) were added. The mixture was stirred at room temperature
for 8 h. The solvent was eliminated in vacuo and the resulting
residual was recrystallised from ethyl acetate/hexane to furnish
the dinitrobenzoate.
Naphthalene-1-yl 3-nitrobenzoate (4a). The compound 4a was
prepared according to the general procedure described above as
a maple solid in 46% yield. Mp: 111–113 °C (ethyl acetate/hexane).
1H NMR (300 MHz, DMSO): d 8.90 (d, J = 3.0 Hz, 1H), 8.62 (m, 2H),
8.00 (d, J = 9.0 Hz, 1H), 7.97 (m, 3H), 7.60 (m, 4H); 13C NMR
(100 MHz, DMSO-d6) dC: 164.0, 148.8, 147.0, 136.9, 135.1, 131.9,
131.2, 129.4, 129.0, 127.9, 126.6, 125.3, 121.8, 119.5; HRMS
(ESI): calcd for C17H11NNaO4 [(M+Na)+] 316.0580, found 316.0583.
3-Tolyl 3,5-dinitrobenzoate (3a). The compound 3a was pre-
pared according to the general procedure described above as a light
yellow needle crystal in 40% yield. Mp/Lit.:16 169–171 °C (ethyl
acetate/hexane)/165.4 °C. 1H NMR (300 MHz, CDCl3): d 9.32 (m,
3H), 7.37 (t, J = 6.0 Hz, 1H), 7.16 (d, J = 6.0 Hz, 1H), 7.07 (d,
J = 6.0 Hz, 1H), 2.43 (s, 3H); 13C NMR (100 MHz, DMSO-d6) dC:
162.5, 151.1, 149.3, 140.4, 133.5, 130.3, 130.2 Â 3, 128.1, 123.9,
122.9, 119.5, 21.7; HRMS (ESI): calcd for
[(M+Na)+] 325.0431, found 325.0429.
C14H10N2NaO6
4.1.3. Synthesis of 7a–7i
General method for the preparation of the dinitrobenza-
mides. A solution of 3,5-dinitrobenzoic acid (1 mmol) in SOCl2
(2 mL) was stirred and refluxed for 4 h. The solvent was evaporated
under reduced pressure and added a solution of Et3N (3 mmol) in
THF (5 mL) to the resulting residual. After stirring for several min-
utes, a solution of aromatic amine (1 mmol) in THF (2 mL) was
Phenyl 3,5-dinitrobenzoate (3b). By use of the above-described
procedure, the target compound was obtained as a gray needle
crystal in 42% yield. Mp/Lit.:16 147–148 °C (ethyl acetate/hex-
ane)/145.8 °C (enthanol). 1H NMR (300 MHz, DMSO): d 9.10 (m,
3H), 7.52 (t, J = 8.0 Hz, 2H), 7.39 (m, 3H); HRMS (ESI): calcd for C13-
H8N2NaO6 [(M+Na)+], 311.0275, found 311.0280.

E. Shang et al. / Bioorg. Med. Chem. 22 (2014) 2396–2402
2401
added. The mixture was allowed to stir at room temperature for 8–
12 h. The solvent was removed under reduced pressure and the
resulting crude residual was recrystallised from ethyl acetate/hex-
ane to furnish the dinitrobenzamide.
3H), 3.76 (s, 3H); HRMS (ESI): calcd for C15H13N3O7 [M+] 347.0748,
found 347.0740.
N-(3-Bromobenzyl)-3,5-dinitrobenzamide (7h). The compound
7h was prepared according to the general procedure described
above as a light yellow needle crystal in 72% yield. Mp: 181–
182 °C (ethyl acetate/hexane). 1H NMR (300 MHz, DMSO): d 9.78
(t, J = 5.7 Hz, 1H), 9.10 (d, J = 2.1 Hz, 2H), 8.97 (t, J = 2.1 Hz, 1H),
7.57 (s, 1H), 7.49 (m, 1H), 7.37 (m, 2H), 4.55 (d, J = 5.7 Hz, 2H);
13C NMR (100 MHz, DMSO-d6) dC: 163.1, 149.1 Â 2, 142.5, 137.5,
131.5, 131.2, 130.8, 128.5 Â 2, 127.6, 122.6, 121.8, 43.5; HRMS
(ESI): calcd for C14H11BrN3O5 [(M+H)+] 379.9877 found 379.9869.
N-(Naphthalene-1-yl)-3,5-dinitrobenzamide (7a). The com-
pound 7a was prepared according to the general procedure de-
scribed above as a gray needle crystal in 68% yield. Mp: 265–
267 °C (ethyl acetate/hexane). 1H NMR (300 MHz, DMSO):
d
11.13 (s, 1H), 9.29 (d, J = 2.1 Hz, 2H), 9.06 (t, J = 2.1 Hz, 1H), 8.00
(m, 2H), 7.94 (d, J = 9.0 Hz, 1H), 7.60 (m, 4H); HRMS (ESI): calcd
for C17H11N3NaO5 [(M+Na)+] 360.0591, found 360.0598.
N-Phenyl 3,5-dinitrobenzamide (7b). The compound 7b was
prepared according to the general procedure described above as
a light yellow needle crystal in 60% yield. Mp/Lit.:17 241–242 °C
(ethyl acetate/hexane)/235.8 °C. 1H NMR (300 MHz, DMSO): d
10.87 (s, 1H), 9.14 (d, J = 2.1 Hz, 2H), 9.01 (t, J = 2.1 Hz, 1H), 7.80
(d, J = 8.4 Hz, 2H), 7.42 (t, J = 8.4 Hz, 2H), 7.19 (t, J = 8.4 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) dC: 162.1, 149.0 Â 2, 139.2, 138.3,
129.7 Â 2, 128.9 Â 2, 125.4, 122.0, 121.6 Â 2; HRMS (ESI): calcd
for C13H9N3NaO5 [(M+Na)+] 310.0434, found 310.0431.
N-(Biphenyl-3-yl)-3,5-dinitrobenzamide (7i). The compound 7i
was prepared according to the general procedure described above
as a gray solid in 67% yield. Mp: 228–229 °C (ethyl acetate/hexane).
1H NMR (300 MHz, DMSO): d 10.96 (s, 1H), 9.21 (d, J = 2.1 Hz, 2H),
9.02 (t, J = 2.1 Hz, 1H), 8.10 (s, 1H), 7.84 (m, 1H), 7.68 (m, 2H), 7.51
(m, 4H), 7.41 (m, 1H).
4.1.4. Synthesis of 9 and 11a–11c
3-(Benzyloxycarbonyl)benzoic acid (9). To a suspension of iso-
phthalic acid (4.0 g, 24 mmol) in methanol (50 mL) and water
(5 mL), the solution of triethylamine (2.5 g, 24 mmol) in methanol
(25 mL) was added. The mixture was stirred at rt overnight. The
solvent was evaporated under reduced pressure and the residual
was dissolved in DMF (60 mL). Benzyl bromide (4.5 g, 27 mmol)
was added to the DMF solution and the resulting mixture was
heated to 100 °C for 2 h. After cooling to room temperature, the
mixture was poured into NaHCO3(aq) (5%, 120 mL). The pH was ad-
justed to around 3 by progressively dropping 1 M HCl(aq) and the
resulting mixture was extracted with EtOAc (100 mL Â 3). The
combined organic phases were washed with saturated NaCl(aq)
and dried over anhydrous Na2SO4. The solvent was evaporated un-
der reduced pressure and the residual was purified by chromatog-
raphy on silica gel (MeOH/DCM) to provide a white solid 9 (2.1 g,
34%). 1H NMR (300 MHz, DMSO): d 13.37 (s, 1H), 8.50 (s, 1H),
8.22 (m, 2H), 7.69 (t, 1H), 7.45 (m, 5H), 5.39 (s, 2H); HRMS (ESI):
calcd for C15H12NaO4 [(M+Na)+] 279.0628, found 279.0630.
N-3-Tolyl 3,5-dinitrobenzamide (7c). The compound 7c was
prepared according to the general procedure described above as
a yellow solid in 55% yield. Mp/Lit.:18 281–283 °C (ethyl acetate/
hexane)/263 °C. 1H NMR (300 MHz, DMSO): d 10.80 (s, 1H), 9.17
(d, J = 2.1 Hz, 2H), 9.01 (t, J = 2.1 Hz, 1H), 7.61 (d, 2 J = 7.8 Hz, H),
7.30 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 2.27 (s, 3H); HRMS
(ESI): calcd for
324.0591.
C
14H11N3NaO5 [(M+Na)+] 324.0591, found
N-(3-Chlorophenyl)-3,5-dinitrobenzamide (7d). The compound
7d was prepared according to the general procedure described
above as a white needle crystal in 80% yield. Mp/Lit.:18 233–
234 °C (ethyl acetate/hexane)/229 °C. 1H NMR (300 MHz, DMSO):
d 10.99 (s, 1H), 9.15 (d, J = 2.1 Hz, 2H), 9.00 (t, J = 2.1 Hz, 1H),
7.95 (t, J = 2.1 Hz, 1H), 7.72 (m, 1H), 7.44 (t, J = 8.4 Hz, 1H), 7.24
(m, 1H); HRMS (ESI): calcd for
344.0045, found 344.0044.
C
13H8ClN3NaO5 [(M+Na)+]
3-((Naphthalene-1-yloxy)carbonyl)benzoic acid (11a). A solu-
tion of EDCI (0.38 g, 2.0 mmol) and 3-(benzyloxycarbonyl)benzoic
acid (9) (0.38 g, 1.5 mmol) in THF (20 mL) was stirred at rt. After
0.5 h, DMAP (0.0018 g, 0.15 mmol) and 1-naphthol (0.22 g,
1.5 mmol) were added and the resulting mixture was stirred
for 12 h at same temperature. The solvent was evaporated
under reduced pressure and the residual was dissolved in water.
The mixture was brought to pH = 2 with 2 M HCl, filtered and
washed with ice ethyl acetate, then dried in a high vacuum oven
N-(3-Bromophenyl)-3,5-dinitrobenzamide (7e). The compound
7e was prepared according to the general procedure described
above as a white needle crystal in 76% yield. Mp/Lit.:18 220–
221 °C (ethyl acetate/hexane)/220 °C. 1H NMR (300 MHz, DMSO):
d 10.97 (s, 1H), 9.17 (d, J = 2.1 Hz, 2H), 9.02 (t, J = 2.1 Hz, 1H),
8.10 (s, 1H), 7.79 (m, 1H), 7.39 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) dC: 162.4, 149.1 Â 2, 140.8, 138.0, 131.7, 129.0 Â 2,
128.1, 123.9, 122.4, 122.2, 120.3; HRMS (ESI): calcd for C13H8BrN3-
NaO5 [(M+Na)+] 387.9540, found 387.9548.
to give
a yellow solid benzyl naphthalene-1-yl isophthalate
(0.40 g, 70% yield).
N-(4-Methoxyphenyl)-3,5-dinitrobenzamide (7f). The com-
pound 7f was prepared according to the general procedure de-
scribed above as a yellow solid in 60% yield. Mp/Lit.:18 244–
246 °C (ethyl acetate/hexane)/244 °C. 1H NMR (300 MHz, DMSO):
d 10.76 (s, 1H), 9.17 (d, J = 2.1 Hz, 2H), 9.00 (t, J = 2.1 Hz, 1H),
7.70 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 0.0 Hz, 2H), 3.77 (s, 3H); HRMS
The solution of benzyl naphthalene-1-yl isophthalate (0.19 g,
0.50 mmol) and Pd/C (0.04 g) in 10 mL of methanol was put into
a
hydrogenation device. Setting the hydrogen pressure at
0.4 MPa, the solution was stirred at room temperature for 5 h.
The mixture was then filter through Celite and the solvent was re-
moved under reduced pressure. The resulting crude residual was
recrystallised from ethyl acetate/hexane to give a gray solid
3-((naphthalene-1-yloxy)carbonyl)benzoic acid (11a) (0.11 g, 75%
yield). Mp: 220–222 °C (ethyl acetate/hexane). 1H NMR
(300 MHz, DMSO): d 13.51 (s, 1H), 8.77 (s, 1H), 8.51 (d, J = 8.1 Hz,
1H), 8.49 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 7.2 Hz, 1H), 7.87 (m, 3H),
7.60 (m, 4H); 13C NMR (100 MHz, DMSO) dC: 167.3, 165.1, 147.2,
135.6, 135.1, 135.0, 132.6, 131.4, 130.7, 130.1, 129.0, 127.9,
127.7, 127.2, 127.2, 126.7, 121.8, 119.6; HRMS (ESI): calcd for C18-
H12NaO4 [(M+Na)+] 315.0628, found 315.0629.
(ESI): calcd for
340.0539.
C
14H11N3NaO6 [(M+Na)+] 340.0540, found
N-(3,4-Dimethoxyphenyl)-3,5-dinitrobenzamide (7g). The com-
pound 7g was prepared according to the general procedure de-
scribed above as a yellow solid in 75% yield. Mp: 255–257 °C
(ethyl acetate/hexane). 1H NMR (300 MHz, DMSO): d 10.74 (s, 1H),
9.17 (d, J = 2.1 Hz, 2H), 9.00 (t, J = 2.1 Hz, 1H), 7.44 (d, J = 2.4 Hz,
1H), 7.38 (dd, J = 8.7 Hz, 2.4 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 3.78 (s,

2402
E. Shang et al. / Bioorg. Med. Chem. 22 (2014) 2396–2402
3-(Naphhthalen-1-ylcarbmoyl)benzoic acid (11b). The com-
pound 11b was prepared according to the procedure described
above as a gray solid in 80% yield. Mp: 251–254 °C (ethyl ace-
tate/hexane). 1H NMR (300 MHz, DMSO): d 13.29 (s, 1H), 10.67
(s, 1H), 8.67 (s, 1H), 8.33 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 7.8 Hz,
1H), 7.99 (m, 2H), 7.87 (d, J = 6.3 Hz, 1H), 7.71 (t, J = 7.8 Hz, 1H),
4.4. Molecular docking
Molecular docking with flexible ligands and rigid receptor was
performed with the program AutoDock 4.00.19 Lamarckian genetic
algorithm was used with following parameters: number of individ-
uals in population, 300; maximum number of energy evaluations,
25,000,000; maximum of generations, 27,000; number of runs,
100. Docked conformations of the 100 runs were clustered with a
rms tolerance of 2.0 Å, and the lowest energy conformation from
the largest cluster was taken as docking result. The crystal structure
of human 5-LOX is used in molecular docking (PDB ID: 3O8Y).20
7.59 (m, 4H); HRMS (ESI): calcd for
292.0968, found 292.0967.
C
18H13NO3 [(M+H)+]
3-(Phenylcarbamoyl)benzoic acid (11c). The compound 11c was
prepared according to the procedure described above as a gray so-
lid in 70% yield. Mp: 258–260 °C (ethyl acetate/hexane). 1H NMR
(300 MHz, DMSO): d 13.31 (s, 1H), 10.47 (s, 1H), 8.53 (s, 1H),
8.18 (m, 2H), 7.80 (d, J = 7.5 Hz, 2H), 7.70 (t, J = 7.8 Hz, 1H), 7.37
(t, J = 7.8 Hz, 2H), 7.12 (t, J = 7.5 Hz, 1H); 13C NMR (100 MHz,
DMSO) dC: 167.8, 165.7, 139.9, 136.3, 133.1, 132.9, 132.0, 129.8,
129.6 Â 2, 129.4, 124.8, 121.4 Â 2; HRMS (ESI): calcd for
Acknowledgments
We thank Mr. Hu Meng and Mr. Jianshu Hu for experimental
technical assistance.
This research was supported, in part, by the Ministry of Science
and Technology of China (Grant Numbers: 2012AA020308 and
2009CB918503) and the Natural Science Foundation of China
(Grant Numbers: 20873003 and 11021463).
C
14H12NO3 [(M+H)+] 242.0812, found 242.0811.
4.2. Inhibition assay of 5-LOX in cell-free system
According to our reported protocol, a fluorescence-based en-
zyme assay was used to test the target compounds’ inhibition to
human 5-LOX. In 96-well microtiter plates (Costar, Corning Inc.),
the compounds were incubated with enzyme and H2DCFDA
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.03.008.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
(10
dithiothreitol, 0.1 mM EDTA, 0.5 mM CaCl2) for 10 min. After prein-
cubation, the reaction was initiated by the addition of AA (25 M)
lM) in assay buffer (50 mM Tris–HCl, 0.2 mM ATP, 0.1 mM
l
and quickly monitored by excitation at 500 nm and emission at
520 nm using a multiwall fluorometer (Synergy4, BIOTEK). A kinet-
ics mode program was used to record the fluorescence signals and
the initial reaction rate was used to calculate the inhibition value.
IC50 was calculated with a four parameter logistical model of the
GraphPad using inhibition value at different inhibitor
concentrations.
References and notes
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tubes (Greiner, containing lithium heparin) and was used within
1 h after collection. Fresh blood were aliquot into 200
of 96-well plate preloaded with either 1 L of DMSO (total activity)
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15 min at 37 °C. 1 L calcium ionophore A23187 (12.5 mM in
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l
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