Synthesis and biological evaluation of novel inhibitors against 1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea

Article abstract of DOI:10.1016/j.bmc.2016.01.053

1,3,8-Trihydroxynaphthalene reductase (3HNR) is an essential enzymes that is involved in fungal melanin biosynthesis. Based on the structural informations of active site of 3HNR, a series of β-nitrostyrene compounds were rationally designed and synthesized. The enzymatic activities of these compounds showed that most of them exhibited high inhibitory activities (<5.0 μM) against 3HNR; compound 3-2 exhibit the highest inhibitory activity (IC₅₀ = 0.29 μM). In particular, some of these compounds had moderate fungicidal activity against Magnaporthe grisea. Compound 3-4 showed high in vivo activities against M. grisea (EC₅₀ = 9.5 ppm). Furthermore, compound 3-2 was selected as a representative molecule, and the probable binding mode of this compound and the surrounding residues in the active site of 3HNR was elucidated by using molecular dock. The positive results suggest that β-nitrostyrene derivatives are most likely to be promising leads toward the discovery of novel agent of rice blast.

Full text of DOI:10.1016/j.bmc.2016.01.053

Bioorganic & Medicinal Chemistry 24 (2016) 1225–1230
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel inhibitors against
1,3,8-trihydroxynaphthalene reductase from Magnaporthe grisea
Haifeng Chen a,b,y, Xinya Han a,y, Nian Qin a,y, Lin Wei a, Yue Yang a, Li Rao a, Bo Chi a, Lingling Feng a,
Yanliang Ren a, , Jian Wan a,
⇑
⇑
a Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079, PR China
b Ocean College, Qinzhou University, Qinzhou 535099, Guangxi, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
1,3,8-Trihydroxynaphthalene reductase (3HNR) is an essential enzymes that is involved in fungal mela-
nin biosynthesis. Based on the structural informations of active site of 3HNR, a series of b-nitrostyrene
compounds were rationally designed and synthesized. The enzymatic activities of these compounds
Received 24 December 2015
Revised 26 January 2016
Accepted 27 January 2016
Available online 29 January 2016
showed that most of them exhibited high inhibitory activities (<5.0
lM) against 3HNR; compound 3-2
exhibit the highest inhibitory activity (IC50 = 0.29 M). In particular, some of these compounds had mod-
l
erate fungicidal activity against Magnaporthe grisea. Compound 3-4 showed high in vivo activities against
M. grisea (EC50 = 9.5 ppm). Furthermore, compound 3-2 was selected as a representative molecule, and
the probable binding mode of this compound and the surrounding residues in the active site of 3HNR
was elucidated by using molecular dock. The positive results suggest that b-nitrostyrene derivatives
are most likely to be promising leads toward the discovery of novel agent of rice blast.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
1,3,8-Trihydroxynaphthalene reductase
Novel inhibitors
Magnaporthe grisea
1. Introduction
1,3,6,8-Tetrahydroxynaphthalene (4HN) is reduced to scytalone
by tetrahydroxynaphthalene reductase (4HNR), followed by
Magnaporthe grisea (M. grisea) is known as a typical causal agent
of rice blast, which is one of the most destructive diseases of culti-
vated rice worldwide. Rice blast causes significant annual crop
losses and has major economic impacts. The amount of rice
reduced by the disease every year is estimated to be sufficient to
feed 60 million people.1–4
The management of rice blast is based on the use of fungicides
to reduce progression of disease. Chemical compounds with
diverse modes of action have been used to manage rice blast,
and two most used are: sterol demethylase inhibitors (DMIs) and
melanin biosynthesis inhibitors (MBIs).5 DMIs inhibit ergosterol
biosynthesis by inhibiting demethylation of precursor sterols at
position 14, in a reaction catalyzed by CYP51.6 Diniconazole is a
typical commercial compound of DMIs. MBIs interfere with mela-
nin production in the appressoria of M. grisea, which results in a
lack of turgor pressure and consequent unsuccessful host infec-
tion.7 The fungal melanin biosynthesis proceeds through a multi-
hydroxynaphthalene route (Scheme 1).
dehydration of scytalone to 1,3,8-trihydroxynaphthalene (3HN),
which is catalyzed by scytalone dehydratase (SD). An important
intermediate step is the 1,3,8-trihydroxynaphthalene reductase
(3HNR)-catalyzed reduction of 3HN to vermelone. 1,8-Dihydroxy-
naphthalene (2HN) is generated from dehydration of vermelone,
which is also catalyzed by scytalone dehydratase (SD). 2HN is
considered to be the ultimate precursor of melanin.8 MBIs have
mostly been studied in the rice blast pathogen M. grisea.9 Tricycla-
zole (5-methyl-1,2,4-triazolo[3,4-b]benzothiazole) is the most
important representative compound among MBIs.10 It has been
demonstrated to specifically inhibit the 3HNR in the melanin
biosynthetic pathway.11
In recent years, resistance of M. grisea has become increasingly
evident because of the heavy-use of commercial inhibitors. Since
the 1980s, field fungal populations resistant to DMIs have been
found in powdery mildews of barley, cucumber, grape and in other
several important plant diseases.12,13 However, except for labora-
tory tricyclazole-resistant mutants of M. grisea described in
2006,14 there are no confirmed reports of field resistance to mela-
nin biosynthesis inhibitor-reductases (MBI-Rs).15 Thus, MBR-Rs are
regard as the most promising target to overcome the resistance of
this rice disease.16 To date, only a few MBIs were reported, such as
commercially agents including tricyclazole, pyroquilon, and
⇑
Corresponding authors. Tel./fax: +86 27 67862022.
E-mail addresses: renyl@mail.ccnu.edu.cn (Y. Ren), jianwan@mail.ccnu.edu.cn
(J. Wan).
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2016.01.053
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

1226
H. Chen et al. / Bioorg. Med. Chem. 24 (2016) 1225–1230
OH
OH OH
O
OH OH
4HNR
SD
HO
OH
HO
SD
OH
OH
1,3,6,8-tetrahydroxy-
naphthalene (4HN)
scytalone
1,3,8-trihydroxy-
naphthalene (3HN)
OH
O
OH OH
3HNR
melanin
OH
vermelone
1,8-dihydroxy-
naphthalene (2HN)
Scheme 1. Melanin biosynthetic pathway of Magnaporthe grisea.
phthalide,17–19 several natural compounds, like biochanin A, api-
genin, naringenin;20 and some benzo-heterocyclic compouds.21
Hence, on the basis of the structural information of the active site
of 3HNR, a series of b-nitrostyrene derivatives that could poten-
tially target into 3HNR were found. Indeed, various biological
activities of b-nitrostyrene derivatives, such as antiplatelet activ-
ity,22 antiproliferative activity,23 pro-apoptotic effect,24 antibacte-
rial activity,25 and anti-HIV activity,26 have been reported
previously. The antifungal activities of b-nitrostyrene derivatives,
such as Candida albicans,27 Trichophyton mentagrophytes,28 and
Aspergillus niger,29 have also been documented. Herein, a series of
b-nitrostyrene derivatives have been prepared though condensa-
tion reaction of the corresponding benzaldehyde and nitro-
methane. Their enzymatic activity against 3HNR was determined,
and their structure–activity relationships were further explored.
Furthermore, the fungicidal activities of these compounds against
M. grisea were performed.
CHO
NO2
NO2
a
b
R
R
R
Br
2
3
1
Scheme 2. Synthetic pathway of b-nitrostyrene 2 and 3. Reagents and conditions:
(a) CH3NO2, NH4OAc, AcOH, 90 °C, 5 h; (b) Br2, NaOAc, CHCl3, reflux, 5–8 h.
R2 substituents to H, CH3, C2H5, SCH3, benzyloxy, halogen (F and
Br), CF3, OH and COOH, such as compounds 2-1, 2-8–2-16. In com-
parison, compounds 2-1, 2-8–2-14 almost exhibited similar 3HNR
activities (IC50 = 1.2–5.0 lM). However, when OH and COOH were
introduced to the R2 position, the 3HNR activities of hit compounds
(2-15 and 2-16) significantly decreased than others.
Taken together, except for the compounds 2-6, 2-7, 2-15 and
2-16, altering the R1/R2 groups of the phenyl ring of the hit com-
pounds did not obviously affect the 3HNR activities. Similarly, we
also found from Table 1 that, when the R1 groups of hit compounds
were changed to F and the R2 groups were changed to CN and F,
such as compounds 2-17 and 2-18, the corresponding 3HNR
activities of these compounds did not exhibit remarkable changes
compared with those of compounds with H atoms at the R1 and R2
positions. Nevertheless, when the R3 substituents were changed to
Br, the corresponding 3HNR activities of the hit compound
increased remarkably. As seen in Table 1, when R3 was changed
2. Results and discussion
2.1. Chemistry
Substituted 2-nitrovinyl benzene (2) was prepared using corre-
sponding aromatic aldehyde (1) and nitromethane. (Scheme 2) The
condensation reactions were carried out in a solution of acetic acid
with ammonium acetate as catalyst. The mixture was refluxed for
5 h to obtain 2 in 62–88% yield.
Substituted 2-bromo-2-nitrovinyl benzene (3) was synthesized
by a bromination of corresponding 2-nitrovinyl benzene (2) with
Br2 (Scheme 2). In these reactions, Br2 was added to the reaction
mixture dropwise at 0 °C, and the resulting solution was refluxed
for 5–8 h. The final products were obtained in 64–69% yield.
to Br, the IC50 values of compounds 3-1 (0.56
and 3-3 (0.91 M) decreased approximately 5-fold compared to
those of compounds 2-8 (2.7 M), 2-9 (1.2 M) and 2-12
(5.0 M), respectively. These results indicate that the Br sub-
lM), 3-2 (0.29 lM)
l
l
l
l
stituent at the R3 position is important for the inhibitory activities
of the hit compounds.
To identify the proposed binding-modes of the hit compounds
with 3HNR, compound 3-2 was docked in the active site of 3HNR
as a representative compound by using Surflex-Dock module in
Sybyl-X 1.3.30 The probable binding mode of compound 3-2 and
3HNR is illustrated in Figure 1. As seen from Figure 1, the nitro
group of compound 3-2 could form the hydrogen bonds with the
OH group of Tyr178, and the benzene ring of compound 3-2 could
2.2. Enzymatic activity against 3HNR and structure–activity
relationships
To evaluate the inhibitory potencies of the hit compounds syn-
thesized in this study, the half-maximal inhibitory concentration
(IC50) values of the test compounds were determined at the 3HNR
recombinant protein level, as listed in Table 1. As shown in Table 1,
most of the b-nitrostyrene derivatives showed high inhibition
form a
p stack with the phenol ring of Tyr233. These two important
interactions could lead to the high 3HNR activities of the hit com-
pounds. In particular, when Br was introduced to the R3 position of
the hit compounds, such as in the case of 3-1, 3-2 and 3-3, a halo-
gen bond between the Br atoms of the hit compounds and the OH
of Tyr233 was observed. These theoretical results qualitatively
explain our experimental findings that the compounds with Br at
the R3 positions exhibited greater inhibitory potencies against
3HNR, whereas the compounds with H atoms at the R3 position
have lower 3HNR activities.
(IC50 < 5.0 lM) against 3HNR. We then fixed the R2 and R3 sub-
stituents to hydrogen and changed the R1 substituents to H, CH3,
F, Br, CF3, OH and COOH, such as compounds 2-1, 2-2–2-7. Gener-
ally, most of these compounds exhibited high inhibition against
3HNR (IC50 < 5.0 lM). Unexpectedly, the compounds (2-6 and 2-7)
with an OH and a COOH at their R1 positions exhibited weak
3HNR activities compared to the other derivatives.
It is should be noticed from Figure 1 that the left side of the
active site is a hydrophobic region comprising two residues
To examine the effects of the R2 position of benzene ring moi-
ety, we also fixed R1 and R3 as hydrogen atoms and changed the

H. Chen et al. / Bioorg. Med. Chem. 24 (2016) 1225–1230
1227
Table 1
Substitution patterns of nitroalkene derivatives and their inhibition activity against 3HNR and M. grisea
R1
R2
R3
NO2
Compound
R1
R2
R3
IC50 (3HNR,
lM)
Inhibition rate (M. grisea, %)a
EC50 (M. grisea, ppm)
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
H
CH3
F
Br
CF3
OH
COOH
H
H
H
H
H
H
H
H
H
F
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
3.8 0.2
2.4 0.1
3.4 0.2
1.1 0.1
2.2 0.1
7.2 0.2
26.9 1.2
2.7 0.1
1.2 0.1
2.4 0.1
1.2 0.1
5.0 0.2
1.5 0.1
2.4 0.1
60.3
98.2
53.2
34.1
40.8
41.1
1.6
67.7
79.0
34.5
6.8
59.2
19.4
63.0
70.4
14.9
34.1
51.3
97.0
97.5
98.2
99.3
/
14.6 0.8
/
/
/
/
/
25.4 1.2
22.7 0.7
/
/
/
/
/
22.0 2.0
/
/
/
15.4 0.5
14.8 0.9
13.6 0.5
9.5 0.4
CH3
C2H5
SCH3
Benzyloxy
F
Br
CF3
OH
COOH
CN
F
CH3
C2H5
F
2-9
2-10
2-11
2-12
2-13
2-14
2-15
2-16
2-17
2-18
3-1
3-2
3-3
3-4
Tricyclazole
Diniconazole
28
1
15.2 0.3
3.6 0.1
1.2 0.1
0.56 0.02
0.29 0.02
0.91 0.09
0.58 0.02
0.23 0.08
H
F
H
1.5 0.2
a
The inhibition rates were determined at 50 ppm tested compounds.
Figure 2. The inhibitions of M. grisea cultures by control and nitroalkene deriva-
tives (inhibition rate > 50%).
Figure 1. The proposed binding-mode of compound 3-2 into the active site of
3HNR. The ribbons represent the helix and sheet of protein, the creamy white stick
represent the hit compound 3-2, and the surrounding important residues were
represented by the green line. The gray dashed lines represent the important
interactions of the compound 3-2 and 3HNR.
of these compounds at wb97xd/6–31G⁄-smd theory level by using
Gaussian 09 software package,31 as listed in Table S1. Consist with
our experimental findings, the solvation energies of compounds
2-6, 2-7, 2-15 and 2-16, were 9.2–9.7 kcal/mol, which are larger
(>2.0 kcal/mol) than others (1.6–7.6 kcal/mol).
(Met283 and Trp243), and these two residues are far from the ben-
zene ring of the hit compounds. To some extent, the larger distance
between these residues and the benzene rings explain why the
substituents, either large (i.e., Benzyloxy and SCH3) or small
(i.e., F, Br and CH3), introduced at the R1/R2 positions of the
benzene rings of the hit compounds only slightly affected their
3HNR activities. However, when OH and COOH groups were intro-
duced to the R1/R2 positions of the hit compounds, the correspond-
ing 3HNR activities were significantly decreased, as described
previously. A possible explanation of this observation is that
compounds with OH and COOH substituents had larger energies
of solvation, which was unfavorable for the binding of the target
compounds with 3HNR. Thus, we calculated the solvation effects
2.3. Fungicidal activity against M. grisea
The inhibition potencies of the hit compounds against M. grisea
at 50 ppm were determined, as also summarized in Table 1. The
fungicidal (M. grisea) activities of these compounds (inhibition
rate > 50%) are also illustrated in Figure 2. It is observed that most
of these compounds exhibited moderate inhibition potencies (inhi-
bition rate > 50%) against M. grisea. Notably, when CH3 was at the
R1 position (2-2) and Br was introduced at R3 position (3-1–3-4),
the corresponding hit compounds exhibited the highest potencies
at 50 ppm level against M. grisea. As also seen from Figure 2, no
growth of M. grisea was observed for compounds 2-2, 3-1–3-4.

1228
H. Chen et al. / Bioorg. Med. Chem. 24 (2016) 1225–1230
NMR solvent signals as an internal reference. Coupling constants (J)
are reported in Hz. Standard abbreviations s, d, t and m refer to sin-
glet, doublet, triplet and multiplet.
100
80
60
40
20
0
4.1. General procedure for the preparation of substituted 2-
nitrovinyl benzene derivatives18
An aromatic aldehyde (5 mmol) was dropped into a solution of
ammonium acetate (12 mmol) in dry nitromethane (10 mL) and
acetic acid (20 mL) at 90 °C with stirring. The mixture was then
refluxed for 5 h, and the mixture was poured into water and
extracted with ethyl acetate (3 ꢀ 50 mL). The extracts were
washed with a saturated NaCl solution, dried over Na2SO4, filtered
and evaporated under reduced pressure. The residue was recrystal-
lized from methanol or purified by column chromatography on
silica gel (ethyl acetate/petroleum ether) to afford the target
2-nitrovinyl benzene derivative.
0
5
10
15
3-4 (ppm
20
25
30
)
Figure 3. The dose response for 3-4 in the inhibition of M. grisea growth.
To further evaluate the inhibitory potencies of the hit com-
pounds, the half-maximal effective concentration (EC50) values of
the compounds with high inhibition rate (>65%) were determined,
as summarized in Table 1. As evident in Table 1, when Br was
introduced to the R3 position of the hit compounds, the corre-
sponding compounds (3-1–3-4) not only have higher 3HNR activ-
4.1.1. (E)-(2-Nitrovinyl)benzene (2-1)
Yield 88%. Yellow solid. Mp: 57–58 °C. 1H NMR (400 MHz,
CDCl3) d: 8.01 (d, J = 13.7 Hz, 1H), 7.59 (d, J = 13.7 Hz, 1H), 7.55
(d, J = 7.1 Hz, 2H), 7.50 (d, J = 7.0 Hz, 1H), 7.48–7.42 (m, 2H). 13C
NMR (101 MHz, CDCl3) d: 133.81, 131.89, 126.89, 124.84, 124.16,
123.89. MS (ESI) m/z: 149.1 [M]+.
ities (0.29–0.91 lM), but also exhibit lower EC50 values against
M. grisea (9.5–15.4 ppm). In particular, compound 3-4 exhibited
the lowest EC50 value (9.5 ppm), which is of the same order of mag-
nitude as the EC50 value of the commercial inhibitors diniconazole
(1.5 ppm). The dose response of 3-4 in the inhibition of M. grisea
growth is also illustrated in Figure 3.
4.1.2. (E)-1-Methyl-3-(2-nitrovinyl)benzene (2-2)
Yield 81%. Yellow liquid. 1H NMR (600 MHz, CDCl3) d: 7.97 (d,
J = 13.7 Hz, 1H), 7.58 (d, J = 13.7 Hz, 1H), 7.37–7.29 (m, 4H), 2.40
(s, 3H). 13C NMR (151 MHz, CDCl3) d: 134.13, 134.06, 131.73,
127.88, 124.80, 124.55, 124.09, 121.22, 16.11. MS (ESI) m/z:
163.1 [M]+.
3. Conclusion
In this work, a series of b-nitrostyrene compounds, including
eighteen substituted 2-nitrovinyl benzene and four substituted 2-
bromo-2-nitrovinyl benzene compounds were designed and syn-
thesized on basis of the structural information of active site of
3HNR. Their enzymatic activities against 3HNR were further deter-
mined at the recombinant protein level. The results showed that
most of the b-nitrostyrene compounds exhibited high inhibitory
4.1.3. (E)-1-Fluoro-3-(2-nitrovinyl)benzene (2-3)
Yield 72%. Yellow solid. Mp: 45–47 °C. 1H NMR (600 MHz,
CDCl3) d: 7.97 (d, J = 13.6 Hz, 1H), 7.57 (d, J = 13.7 Hz, 1H), 7.45
(q, J = 7.5 Hz, 1H), 7.35 (d, J = 7.7 Hz, 1H), 7.25 (d, J = 9.2 Hz, 1H),
7.21 (t, J = 8.2 Hz, 1H). 13C NMR (151 MHz, CDCl3) d: 163.75,
162.10, 138.06, 137.69, 132.13, 132.08, 131.10, 131.05, 125.17,
119.14, 119.00, 115.45, 115.30. MS (ESI) m/z: 167.0 [M]+.
activities (<5.0 lM) against 3HNR. Especially, when Br was intro-
duced to the R3 positions, the 3HNR activities of the corresponding
hit compounds (3-1–3-4) increased approximately 5-fold com-
pared to the activities of compounds without Br substituent at R3
position. In comparison, compound 3-2 exhibited the highest inhi-
4.1.4. (E)-1-Bromo-3-(2-nitrovinyl)benzene (2-4)
Yield 72%. Yellow solid. Mp: 61–62 °C. 1H NMR (600 MHz,
CDCl3) d: 7.93 (d, J = 13.7 Hz, 1H), 7.70 (s, 1H), 7.63 (d, J = 7.8 Hz,
1H), 7.56 (d, J = 13.7 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.34 (t,
J = 7.9 Hz, 1H). 13C NMR (151 MHz, CDCl3) d: 132.86, 132.20,
129.68, 126.88, 126.51, 125.71, 122.56, 118.21. MS (ESI) m/z:
226.9 [Mꢁ1]+, 228.9 [M+1]+.
bitory activity (IC50 = 0.29 lM). Furthermore, compound 3-2 was
selected as a representative molecule, and the probable binding
mode of this compound and the surrounding residues in the active
site of 3HNR was elucidated by using molecular dock. The obtained
docking result showed the presence of halogen bonds between the
Br substituents of the hit compounds and the OH group of Tyr233,
which explained the activities of compounds 3-1–3-4. Further-
more, the fungicidal activities of these compounds against M. grisea
were also determined. Compound 3-4 exhibited high in vivo activ-
ity against M. grisea (EC50 = 9.5 ppm). The positive results suggest
that b-nitrostyrene derivatives are most likely to be promising
leads for the discovery of novel agents for inhibiting the growth
of rice blast.
4.1.5. (E)-1-(2-Nitrovinyl)-3-(trifluoromethyl)benzene (2-5)
Yield 69%. Yellow solid. Mp: 75–76 °C. 1H NMR (600 MHz,
CDCl3) d: 8.04 (d, J = 13.7 Hz, 1H), 7.81 (s, 1H), 7.76 (d, J = 7.9 Hz,
2H), 7.65 (d, J = 13.6 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H). 13C NMR
(151 MHz, CDCl3) d: 138.42, 137.27, 132.03, 131.82, 130.87,
130.04, 128.40, 125.65, 124.32, 122.52. MS (ESI) m/z: 216.9 [M]+.
4.1.6. (E)-3-(2-Nitrovinyl)phenol (2-6)
Yield 85%. Yellow solid. Mp: 135–136 °C. 1H NMR (600 MHz,
CDCl3) d: 7.95 (d, J = 13.7 Hz, 1H), 7.56 (d, J = 13.6 Hz, 1H), 7.33
(t, J = 7.9 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.02 (s, 1H), 6.98 (d,
J = 8.1 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) d: 158.10, 139.79,
138.20, 131.79, 130.49, 120.97, 119.55, 116.52. MS (ESI) m/z:
115.1 [M]+.
4. Experimental
All reagents were obtained from commercial suppliers and used
without further purification. TLC analysis was performed using
pre-coated glass plates. Column chromatography was performed
using silica gel (200–300 mesh). 1H NMR and 13C NMR spectra
were obtained on Varian Mercury-Plus400 and Varian NMR System
600 spectrometer. Chemical shifts are reported in ppm (d) with the
4.1.7. (E)-3-(2-Nitrovinyl)benzoic acid (2-7)
Yield 85%. Yellow solid. Mp: 194–195 °C. 1H NMR (600 MHz,
CDCl3) d: 8.30 (s, 1H), 8.23 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 13.7 Hz,

H. Chen et al. / Bioorg. Med. Chem. 24 (2016) 1225–1230
1229
1H), 7.80 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 13.7 Hz, 1H), 7.61 (t,
J = 7.7 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) d: 166.84, 139.10,
138.49, 133.42, 132.55, 132.08, 131.00, 129.65. MS (ESI) m/z:
193.2 [M]+.
4.1.17. (E)-2-Fluoro-5-(2-nitrovinyl)benzonitrile (2-17)
Yield 66%. Yellow solid. Mp: 130–132 °C. 1H NMR (600 MHz,
CDCl3) d: 7.97 (d, J = 13.7 Hz, 1H), 7.87 (dd, J = 5.9, 2.3 Hz, 1H),
7.84 (ddd, J = 7.3, 4.9, 2.3 Hz, 1H), 7.59 (d, J = 13.8 Hz, 1H), 7.37
(t, J = 8.5 Hz, 1H). 13C NMR (151 MHz, CDCl3) d: 165.40, 163.64,
138.67, 135.35, 135.26, 135.20, 134.13, 127.52, 118.06, 117.93,
112.71, 103.25. MS (ESI) m/z: 192.0 [M]+.
4.1.8. (E)-1-Methyl-4-(2-nitrovinyl)benzene (2-8)
Yield 88%. Yellow solid. Mp: 101–103 °C. 1H NMR (400 MHz,
CDCl3) d: 7.97 (d, J = 13.7 Hz, 1H), 7.56 (d, J = 13.7 Hz, 1H), 7.44
(d, J = 7.7 Hz, 2H), 7.25 (d, J = 7.6 Hz, 2H), 2.40 (s, 3H). 13C NMR
(101 MHz, CDCl3) d: 143.05, 139.10, 136.20, 130.07, 129.13,
127.20, 21.60. MS (ESI) m/z: 163.2 [M]+.
4.1.18. (E)-1,2-Difluoro-4-(2-nitrovinyl)benzene (2-18)
Yield 63%. Yellow solid. Mp: 48–49 °C. 1H NMR (600 MHz,
CDCl3) d: 7.93 (d, J = 13.7 Hz, 1H), 7.53 (d, J = 13.7 Hz, 1H), 7.40
(ddd, J = 10.0, 7.4, 2.0 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.31–7.24
(m, 1H). 13C NMR (151 MHz, CDCl3) d: 151.67, 151.60, 149.93,
149.84, 137.74, 136.85, 127.14, 126.26, 118.61, 118.49, 117.57,
117.45. MS (ESI) m/z: 185.0 [M]+.
4.1.9. (E)-1-Ethyl-4-(2-nitrovinyl)benzene (2-9)
Yield 86%. Yellow solid. Mp: 80–81 °C. 1H NMR (400 MHz,
CDCl3) d: 7.98 (d, J = 13.4 Hz, 1H), 7.56 (d, J = 13.7 Hz, 1H), 7.46
(d, J = 7.9 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 2.70 (q, J = 7.6 Hz, 2H),
1.26 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, CDCl3) d: 149.26,
139.11, 136.23, 129.25, 128.89, 127.42, 28.87, 15.07. MS (ESI)
m/z: 177.2 [M]+.
4.2. General procedure for preparation of substituted 2-bromo-
2-nitrovinyl benzene derivatives32
To a stirred solution of a substituted 2-nitrovinyl benzene
(10.0 mmol) in sodium acetate (12.0 mmol) and chloroform
(10 mL) was added neat Br2 (12.0 mmol) dropwise over 5 min at
0 °C. The cloudy yellow reaction mixture was then heated to reflux
and stirred for 5–8 h (monitored by TLC). The excess Br2 was
removed by washing the reaction mixture with a saturated aque-
ous solution of Na2S2O3. The aqueous solution was then extracted
with CH2Cl2 (3 ꢀ 20 mL). The combined CH2Cl2 layers were dried
over anhydrous Na2SO4. The solvent was removed by evaporation
under reduced pressure to give a crude solid that was purified by
silica gel column chromatography using ethyl acetate and hexane
as eluent.
4.1.10. (E)-Methyl(4-(2-nitrovinyl)phenyl)sulfane (2-10)
Yield 80%. Yellow solid. Mp: 84–86 °C. 1H NMR (400 MHz,
CDCl3) d: 7.94 (d, J = 13.6 Hz, 1H), 7.56 (d, J = 13.6 Hz, 1H), 7.44
(d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 2.51 (s, 3H). 13C NMR
(101 MHz, CDCl3) d: 145.11, 138.69, 136.00, 129.42, 126.12,
125.86, 14.78. MS (ESI) m/z: 195.2 [M]+.
4.1.11. (E)-1-(Benzyloxy)-4-(2-nitrovinyl)benzene (2-11)
Yield 78%. Yellow solid. Mp: 119–120 °C. 1H NMR (400 MHz,
CDCl3) d: 7.95 (d, J = 13.6 Hz, 1H), 7.53–7.45 (m, 3H), 7.42–7.30
(m, 5H), 7.01 (d, J = 8.8 Hz, 2H), 5.11 (s, 2H). MS (ESI) m/z:
255.1 [M]+.
4.2.1. 1-(2-Bromo-2-nitrovinyl)-4-methylbenzene (3-1)
Yield 69%. Yellow solid. Mp: 67–68 °C. 1H NMR (600 MHz,
CDCl3) d: 8.62 (s, 1H), 7.81 (d, J = 7.4 Hz, 2H), 7.30 (d, J = 7.6 Hz,
2H), 2.42 (s, 3H). 13C NMR (151 MHz, CDCl3) d: 137.89, 131.44,
125.99, 124.57, 122.07, 121.81, 16.59. MS (ESI) m/z: 241.0
[Mꢁ1]+, 242.9 [M+1]+.
4.1.12. (E)-4-Fluoro-1-(2-nitrovinyl)benzene (2-12)
Yield 78%. Yellow solid. Mp: 100–101 °C. 1H NMR (400 MHz,
CDCl3) d: 7.98 (d, 1H), 7.65–7.49 (m, 3H), 7.20–7.06 (m, 4H). 13C
NMR (101 MHz, CDCl3) d: 166.28, 163.75, 137.94, 136.95, 131.45,
131.36, 126.41, 116.96, 116.74. MS (ESI) m/z: 167.2 [M]+.
4.2.2. 1-(2-Bromo-2-nitrovinyl)-4-ethylbenzene (3-2)
Yield 67%. Yellow liquid. 1H NMR (600 MHz, CDCl3) d: 8.64
(s, 1H), 7.84 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 2.72
(q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H). 13C NMR (151 MHz,
CDCl3) d: 144.05, 131.45, 126.12, 123.38, 122.29, 121.83, 23.83,
9.95. MS (ESI) m/z: 255.0 [Mꢁ1]+, 257.0 [M+1]+.
4.1.13. (E)-1-Bromo-4-(2-nitrovinyl)benzene (2-13)
Yield 79%. Yellow solid. Mp: 151–153 °C. 1H NMR (400 MHz,
CDCl3) d: 7.95 (d, J = 13.7 Hz, 1H), 7.64–7.52 (m, 3H), 7.42
(d, J = 8.5 Hz, 1H). 13C NMR (101 MHz, CDCl3) d: 137.92, 137.60,
132.87, 130.52, 129.07, 126.91, 77.48, 77.16, 76.84. MS (ESI) m/z:
227.1 [Mꢁ1]+, 229.1 [M+1]+.
4.2.3. 1-(2-Bromo-2-nitrovinyl)-4-fluorobenzene (3-3)
Yield 68%. Yellow solid. Mp: 52–53 °C. 1H NMR (600 MHz,
CDCl3) d: 8.63 (s, 1H), 7.94 (dd, J = 8.7, 5.4 Hz, 2H), 7.20 (t,
J = 8.6 Hz, 2H). 13C NMR (151 MHz, CDCl3) d: 165.38, 163.69,
135.33, 133.35, 127.77, 126.28, 116.42. MS (ESI) m/z: 244.8
[Mꢁ1]+, 246.9 [M+1]+.
4.1.14. (E)-1-(2-Nitrovinyl)-4-(trifluoromethyl)benzene (2-14)
Yield 67%. Yellow solid. Mp: 90–92 °C. 1H NMR (600 MHz,
CDCl3) d: 8.03 (d, J = 13.7 Hz, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.68 (d,
J = 8.1 Hz, 2H), 7.62 (d, J = 13.7 Hz, 1H). 13C NMR (151 MHz, CDCl3)
d: 138.81, 137.13, 133.42, 129.25, 126.34. MS (ESI) m/z: 217.2 [M]+.
4.2.4. 1-(2-Bromo-2-nitrovinyl)-3-fluorobenzene (3-4)
Yield 64%. Yellow solid. Mp: 41–42 °C. 1H NMR (600 MHz,
CDCl3) d: 8.58 (s, 1H), 7.67 (d, J = 9.6 Hz, 1H), 7.60 (d, J = 7.8 Hz,
1H), 7.48 (q, J = 7.8 Hz, 1H), 7.23 (t, J = 8.2 Hz, 1H). 13C NMR
(151 MHz, CDCl3) d: 163.33, 161.68, 135.11, 132.14, 130.62,
129.20, 127.23, 118.79, 116.79, 94.82. MS (ESI) m/z: 244.9
[Mꢁ1]+, 246.9 [M+1]+.
4.1.15. (E)-4-(2-Nitrovinyl)phenol (2-15)
Yield 69%. Yellow solid. Mp: 163–165 °C. 1H NMR (600 MHz,
DMSO-d6) d: 10.45 (s, 1H), 8.07 (s, 2H), 7.73 (d, J = 8.5 Hz, 2H),
6.87 (d, J = 8.5 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) d: 161.99,
140.34, 135.23, 132.75, 121.53, 116.61. MS (ESI) m/z: 165.2 [M]+.
4.1.16. (E)-4-(2-Nitrovinyl)benzoic acid (2-16)
Yield 79%. Yellow solid. Mp: > 250 °C. 1H NMR (600 MHz,
DMSO-d6) d: 13.38 (s, 1H), 8.32 (d, J = 13.6 Hz, 1H), 8.20
(d, J = 13.6 Hz, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.98 (d, J = 8.2 Hz, 2H).
13C NMR (151 MHz, DMSO-d6) d: 167.11, 139.93, 138.30, 134.59,
134.25, 130.19, 130.16. MS (ESI) m/z: 193.1 [M]+.
4.3. Inhibition assay for 3HNR
The expression and purification of 3HNR from M. grisea was
performed as previously described.33 Oxidation of the synthesized
substrate
2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one

1230
H. Chen et al. / Bioorg. Med. Chem. 24 (2016) 1225–1230
(DDBO) to 4,5-dihydroxy-2H-benzopyran-2-one (DBO) with the
accompanying reduction of NADP+ to NADPH was detected as pre-
viously described.9,34 by using a microplate reader (Bioteck Syner-
gy2, USA) equipped with a temperature controller accessory. The
difference in NADPH absorbance was measured at 340 nm and
30 °C. Assay were carried out in 0.1 M PBS buffer contained
Municipality (No. 201150530150), and the self-determined
research funds of CCNU from the colleges’ basic research and oper-
ation of MOE (No. CCNU14A05006).
Supplementary data
4.5 lg/mL 3HNR, 200 lM DDBO, and 400 l
M NADP+, at pH 7.0
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.01.053.
and 30 °C. The half maximal inhibitory concentration (IC50) values
of hit compounds were determined from the inhibition curves
which plot the inhibition rate against various concentrations of
test compounds. The inhibition rate (I%) was calculated using the
following formula:
References and notes
1. Baker, B.; Zambryski, P.; Staskawicz, B.; Dinesh-Kumar, S. P. Science 1997, 276,
726.
V0 ꢁ V
2. Tani, H.; Koshino, H.; Sakuno, E.; Nakajima, H. J. Nat. Prod. 2005, 68, 1768.
3. Ueno, M. J. Gen. Plant Pathol. 2011, 77, 364.
4. Howard, R. J.; Valent, B. Annu. Rev. Microbiol. 1996, 50, 491.
5. Kunova, A.; Pizzatti, C.; Cortesi, P. Pest Manag. Sci. 2013, 69, 278.
6. Yan, X.; Ma, W.-B.; Li, Y.; Wang, H.; Que, Y.-W.; Ma, Z.-H.; Talbot, N. J.; Wang,
Z.-Y. Fungal Genet. Biol. 2011, 48, 144.
7. Kurahashi, Y. Pestic. Outlook 2001, 12, 32.
8. Thompson, J. E.; Fahnestock, S.; Farrall, L.; Liao, D. I.; Valent, B.; Jordan, D. B. J.
Biol. Chem. 2000, 275, 34867.
I% ¼
ꢀ 100%
V0
where V0 and V represent the maximum velocity ([I] = 0 lM) and
the velocity when inhibitor was added, respectively. The curves
were fitted by nonlinear regression using logistic equation in origin
7.0 software.
ˇ
9. Brunskole, M.; Štefane, B.; Zorko, K.; Anderluh, M.; Stojan, J.; Lanišnik Rizner, T.;
4.4. Antifungal activity tests in vivo
Gobec, S. Bioorg. Med. Chem. 2008, 16, 5881.
10. Hsieh, C.-H.; Chung, W.-C.; Chen, Y.-N.; Chung, W.-H. J. Pestic. Sci. 2013, 38, 194.
11. Woloshuk, C. P.; Sisler, H. D.; Tokousbalides, M. C.; Dutky, S. R. Pestic. Biochem.
Physiol. 1980, 14, 256.
12. Karaoglanidis, G. S.; Loannidis, P. M.; Thanassoulopoulos, C. C. Plant Pathol.
2002, 51, 55.
13. Leroux, P.; Albertini, C.; Gautier, A.; Gredt, M.; Walker, A. S. Pest Manag. Sci.
2007, 63, 688.
14. Zhang, C. Q.; Zhu, G. N.; Ma, Z. H.; Zhou, M. G. J. Phytopathol. 2006, 154, 392.
15. Zhang, C.-Q.; Huang, X.; Wang, J.-X.; Zhou, M.-G. Pestic. Biochem. Physiol. 2009,
94, 43.
16. Helliwell, E. E.; Yang, Y. Methods Mol. Biol. 2013, 956, 285.
17. Jordan, D. B.; Basarab, G. S.; Liao, D.-I.; Johnson, W. M. P.; Winzenberg, K. N.;
Winkler, D. A. J. Mol. Graphics Modell. 2001, 19, 434.
18. Liao, D.-I.; Thompson, J. E.; Fahnestock, S.; Valent, B.; Jordan, D. B. Biochemistry
2001, 40, 8696.
Potato dextrose agar medium (200 g potatoes, 20 g glucose, 20 g
agar and 1 L water) was used to incubate M. grisea. Quantitative
medium were prepared in 10-cm Petri dishes without inhibitors
(DMSO), with 50 ppm tricyclazole and diniconazole (positive con-
trol), and with 50 ppm compounds, respectively. Then 5 mm myce-
lia disks taken from a 72 h agar culture of M. grisea were inoculated
in the center of the Petri dish. The plates were incubated at 27 °C
without light, and the color and growth of the inoculated mycelia
were followed for 7 days. The inhibition rate (I%) was calculated
using the following formula:
19. Liao, D.-I.; Basarab, G. S.; Gatenby, A. A.; Valent, B.; Jordan, D. B. Structure 2001,
9, 19.
20. Brunskole, M.; Zorko, K.; Kerbler, V.; Martens, S.; Stojan, J.; Gobec, S.; Lanišnik
ðDDMSO ꢁ DMÞ ꢁ ðDI ꢁ DMÞ
I% ¼
ꢀ 100%
ðDDMSO ꢁ DMÞ
ˇ
Rizner, T. Chem. Biol. Interact. 2009, 178, 259.
21. Brunskole Švegelj, M.; Turk, S.; Brus, B.; Lanišnik Rizner, T.; Stojan, J.; Gobec, S.
J. Chem. Inform. Model. 2011, 51, 1716.
where DDMSO, DI, DM, represent the diameter ([I] = 0 lM), the diam-
ˇ
eter when inhibitor was added, and the diameter of mycelia disk
(DM = 5 mm), respectively. Three replicated plates were conducted
for each compound and each concentration. The half maximal effec-
tive concentration (EC50) values of hit compounds were determined
from the inhibition curves which plot the inhibition rate against
various concentrations of test compounds. The curves were fitted
by nonlinear regression using logistic equation in origin 7.0
software.
22. Wang, W.-Y.; Hsieh, P.-W.; Wu, Y.-C.; Wu, C.-C. Biochem. Pharmacol. 2007, 74,
601.
23. Mohan, R.; Rastogi, N.; Namboothiri, I. N. N.; Mobin, S. M.; Panda, D. Bioorg.
Med. Chem. 2006, 14, 8073.
24. Kaap, S.; Quentin, I.; Tamiru, D.; Shaheen, M.; Eger, K.; Steinfelder, H. J.
Biochem. Pharmacol. 2003, 65, 603.
25. Milhazes, N.; Calheiros, R.; Marques, M. P. M.; Garrido, J.; Cordeiro, M. N. D. S.;
Rodrigues, C.; Quinteira, S.; Novais, C.; Peixe, L.; Borges, F. Bioorg. Med. Chem.
2006, 14, 4078.
26. Cheng, P.; Jiang, Z.-Y.; Wang, R.-R.; Zhang, X.-M.; Wang, Q.; Zheng, Y.-T.; Zhou,
J.; Chen, J.-J. Bioorg. Med. Chem. Lett. 2007, 17, 4476.
27. Lo, K.; Cornell, H.; Nicoletti, G.; Jackson, N.; Hügel, H. Appl. Sci. 2012, 2, 114.
28. Mikami, Y.; Yazawa, K.; Maeda, A.; Uno, J.; Kubo, A.; Saito, N.; Kawakami, N. J.
Antibiot. 1991, 44, 1454.
4.5. Molecular docking
A rigorous surflex docking study had been performed by using
Sybyl-X 1.3.30 The X-ray crystallographic coordinates of 3HNR
(PDB ID: 1G0N) was obtained from RCSB Protein Data Bank. Hydro-
gen atoms were added to the protein allowing for appropriate ion-
ization at physiological pH. The whole protein was optimized using
amber force field. The b-nitrostyrene derivatives were constructed
and optimized with Tripos force field and Gasteiger-Huckel
charges using Sybyl-X 1.3. And they were docked into 3HNR active
site by using Surflex-Dock.35
29. Latif, N.; Asaad, F. M.; Hosni, H. Liebigs Ann. Chem. 1987, 1987, 495.
30. Jain, A. N. J. Comput.-Aided Mol. Des. 2007, 21, 281.
31. Frisch, M. J. T., G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J.
R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.;
Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.;
Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery, Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.;
Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.;
Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.;
Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;
Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas,
Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian Inc, Wallingford
CT, 2013.
32. Bharathiraja, G.; Sakthivel, S.; Sengoden, M.; Punniyamurthy, T. Org. Lett. 2013,
15, 4996.
33. Andersson, A.; Jordan, D.; Schneider, G.; Valent, B.; Lindqvist, Y. Proteins: Struct.
Funct. Genet. 1996, 24, 525.
34. Thompson, J. E.; Jordan, D. B. Anal. Biochem. 1998, 256, 7.
35. Jain, A. N. J. Med. Chem. 2003, 46, 499.
Acknowledgements
This work was supported by the National Basic Research
Program of China (973 Program, No. 2010CB126100), the Natural
Science Foundation of China (Nos. 21572077, 21472061,
21373094, 21202056, 21272089 and 21172089), PCSIRT
(No. IRT0953), Natural Science Foundation of Hubei Province
(No. 2010CDB01202), the Program for Academic leader in Wuhan