Benzofurazan derivatives as antifungal agents against phytopathogenic fungi

Article abstract of DOI:10.1016/j.ejmech.2014.04.058

A series of benzofurazan derivatives were prepared and evaluated for their biological activities against four important phytopathogenic fungi, namely, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium graminearum and Phytophthora capsici, using the mycelium growth inhibition method. The structures of these compounds were characterized by¹H NMR,¹³C NMR, and HRMS. N-(3-chloro-4-fluorophenyl)-7-nitrobenzo[c][1,2,5] oxadiazol-4-amine (A3) displayed the maximum antifungal activity against R. solani (IC₅₀= 1.91 μg/mL), which is close to that of the positive control Carbendazim (IC₅₀= 1.42 μg/mL). For other benzofurazan derivatives with nitro group at R⁴position (A series), 9 out of 30 compounds exhibited high antifungal effect against strain R. solani, with IC₅₀values less than 5 μg/mL. Most of the derivatives with substituents at R²and R³positions (B series) displayed moderate growth inhibition against S. sclerotiorum (IC₅₀< 25 μg/mL). Also, several benzofuran derivatives with nitro group at R⁴position and another conjugated aromatic ring at the R¹position of the phenyl ring displayed high antifungal capability against strain R. solani. Compounds with substituents at R²and R³position had moderate efficacy against strain S. sclerotiorum.

Full text of DOI:10.1016/j.ejmech.2014.04.058

European Journal of Medicinal Chemistry 80 (2014) 535e542
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Benzofurazan derivatives as antifungal agents against
phytopathogenic fungi
,
,
Lili Wang a 1, Ying-Ying Zhang b 1, Lei Wang c, Feng-you Liu a, Ling-Ling Cao b, Jing Yang a,
Chunhua Qiao a , Yonghao Ye
,
b,
*
*
a College of Pharmaceutical Science, Soochow University, Suzhou 215123, Jiangsu, PR China
b College of Plant Protection, Jiangsu Key Laboratory of Pesticide Science, Nanjing Agricultural University, Nanjing 210095, PR China
c School of Biology & Basic Medical Sciences, Soochow University, Suzhou 215123, Jiangsu, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzofurazan derivatives were prepared and evaluated for their biological activities against
four important phytopathogenic fungi, namely, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium
graminearum and Phytophthora capsici, using the mycelium growth inhibition method. The structures of
these compounds were characterized by 1H NMR, 13C NMR, and HRMS. N-(3-chloro-4-fluorophenyl)-7-
nitrobenzo[c][1,2,5]oxadiazol-4-amine (A3) displayed the maximum antifungal activity against
Received 15 January 2014
Received in revised form
17 April 2014
Accepted 21 April 2014
Available online 23 April 2014
R. solani (IC50 ¼ 1.91
mL). For other benzofurazan derivatives with nitro group at R4 position (A series), 9 out of 30 compounds
exhibited high antifungal effect against strain R. solani, with IC50 values less than 5 g/mL. Most of the
derivatives with substituents at R2 and R3 positions (B series) displayed moderate growth inhibition
m
g/mL), which is close to that of the positive control Carbendazim (IC50 ¼ 1.42
mg/
Keywords:
m
Antifungal activity
Phytopathogenic fungi
Benzofurazan derivatives
against S. sclerotiorum (IC50 < 25 m
g/mL). Also, several benzofuran derivatives with nitro group at R4
position and another conjugated aromatic ring at the R1 position of the phenyl ring displayed high
antifungal capability against strain R. solani. Compounds with substituents at R2 and R3 position had
moderate efficacy against strain S. sclerotiorum.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
candidates displaying significant in vitro and in vivo antifungal ac-
tivity [2]. Unlike furoxan (Fig. 1), the benzofuroxans were consid-
Pathogenic fungi and insect pests caused over 30% yield losses in
major crops [1]. To increase yields and provide healthy crops,
application of agrochemicals has become an established part of
modern agriculture. For complicated plant disease situation, the
combined use of different antifungal agents is common (ref).
Consequently, fungicide-resistant pathogens have been selected
and evolved. Therefore, there is a need for novel antifungal com-
pounds with new mechanism of action and lower application
dosage.
During screening of our in house compound library for biolog-
ically active components targeting plant pathogens, the benzofur-
oxan scaffold (Fig. 1) was identified to exhibit good in vitro activity
against four important phytopathogenic fungi. Further structural
modification revealed benzofuroxan derivatives with nitro at the R4
position and small sized amino group at R1 position resulted in
ered to be devoid of the nitric oxide (NO) releasing capability [3].
Therefore, the reported antifungal activity should not relate to the
NO biological function. Alternatively, the benzofuroxan system is
highly electron deficient and nucleophile sensitive, especially when
the phenyl R4 position contains a nitro group. Nucleophiles like
thiol or hydroxyl would readily attack the nitro para position to
form the so called Meisenheimer-type complex intermediate,
which quickly undergoes elimination reaction to provide a more
stable benzofuroxan [4]. Previous studies have also established that
substituents ortho to nitro would considerably diminish the
Meisenheimer-type complex formation [5]. Whether the com-
pound electrophilicity is involved in the antifungal activity should
be explored and addressed. We anticipate that removal of the
electron negative oxygen atom, transformation to benzofurazan
(Fig. 1), will render the whole conjugated system less electron-
deficient. Alternatively, the benzofurazan derivatives have been
reported to possess a variety of bioactivity, such as anti-protozoa,
antibacterial and calcium channel modulated property [6e10].
Given the structural similarity of benzofuroxan and benzofurazan,
* Corresponding authors.
E-mail addresses: qiaochunhua@suda.edu.cn (C. Qiao), yeyh@njau.edu.cn (Y. Ye).
The authors contributed equally to this paper.
1
http://dx.doi.org/10.1016/j.ejmech.2014.04.058
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

536
L. Wang et al. / European Journal of Medicinal Chemistry 80 (2014) 535e542
3. Antifungal activity
The antifungal activities of the synthetic molecules, expressed
as IC50 (median inhibitory concentration) values, were determined
using the mycelia growth inhibitory rate method. The results are
shown in Tables 1e3.
The benzofurazan derivative A3, with the 3-chloro-4-
fluoroaniline substituent at the R1 position, showed a broad spec-
trum of antifungal activity against all four tested fungi phytopath-
Fig. 1. Chemical structures of furoxan, benzofuroxan and benzofurazan.
ogens. Its IC50 value against R. solani was 1.91
mg/mL, which was
close to that of the positive control Carbendazim (IC50 ¼1.42
m
g/mL).
Most of the other compounds with different aniline substitutions at
the R1 position also displayed high potency against R. solani. Espe-
we investigated the antifungal aspect of benzofurazan. In this pa-
per, series of benzofurazan compounds were prepared, and the
in vitro antifungal activities were evaluated against four important
plant pathogen strains including Rhizoctonia solani, Sclerotinia
sclerotiorum, Fusarium graminearum, and Phytophthora capsici.
These four strains of fungi are representative in their biological
species, and caused a wide range of significant plant disease on
different crops. For example, F. graminearum, also known by the
name of teleomorph Gibberella zeae, is a plant pathogen that causes
fusarium head blight on wheat and barley [11]; R. solani is a plant
pathogenic fungus with a wide host range and worldwide distri-
bution, also one of the fungi responsible for Brown patch (a turf-
grass disease), as well as black scurf of potatoes, bare patch of
cereals, root rot of sugar beet, belly rot of cucumber, sheath blight of
rice, and many other pathogenic conditions [12].
cially, the 4-bromoaniline derivative A4 (IC50 ¼ 2.03
mg/mL) and
g/mL) displayed
corresponding chloro analogue A28 (IC50 ¼ 3.87
m
inhibitory potency similar to that of carbendazim. Compounds
A20eA24, with pyrrolidine, piperidine, piperazine and morpholine
at the R1 position, displayed weak antifungal activity with IC50 value
higher than 25 mg/mL against all four tested fungi phytopathogens.
Replacement of the above aliphatic amine with aromatic triazole
and pyrazole (compounds A7eA8) improved the activity, with IC50
values 3.11e22.29
mg/mL against R. solani, S. sclerotiorum and
F. graminearum Sehw. This suggested that the hetero-aromatic azole
group has a great contribution on compound antifungal activity.
Finally, A10eA12 and A19, with different thiols at position 4
exhibited low IC50 (<5
mg/mL) against R. solani. These findings
indicated that the presence of another conjugated system at the R1
position is favorable for the antifungal activity of type A benzofur-
azan derivatives, and they may serve as new leads for the devel-
opment of potentially useful antifungal agents against R. solani.
In the case of series B of benzofurazan derivatives, with sub-
stituents variation at the R2 and R3 positions, whereas both R1 and R4
positions are hydrogen atoms, seven out of ten compounds (B2eB4
and B6eB9) exhibited potency against S. sclerotiorum (IC50 ¼ 13.18e
2. Chemistry
To investigate whether the nitrogeneoxygen coordinate bond
in benzofuroxan is necessary for antifungal activity, the corre-
sponding benzofurazans were synthesized. Specifically, the ben-
zene ring at the R1 position (Fig. 1) was first chosen for
modification (A18eA26, B2eB4 and B10). Further change with
other conjugated heterocyclics, such as azoles and thiophene [13e
16], which are common structural elements in compounds with
antifungal activity were investigated. All new benzofurazan com-
pounds were prepared using the synthetic routes shown in
Schemes 1 and 2. 4-Chloro-7-nitrobenzo[c][1,2,5]oxadiazole(A1)
was prepared from commercially available 2,6-dichloroaniline
employing the reported synthesis procedure [17]. Then nucleo-
philic substitution reaction of compound A1 with different amines
was carried in a sealed tube at 100 ꢀC to provide A2eA6. To pre-
pare A7eA8, a nitrogen atmosphere and low temperature reaction
condition was used for the pyrazole and 1,2,4-triazole substrates.
Treating A1 with thiols using sodium methoxide as base afforded
A9eA12 in good yield (>90%).
The key intermediate 5-chloro-4-fluoro-2-nitroaniline (1) was
obtained from commercially available 3-chloro-4-fluoroaniline
using the reported procedure [2]. Nucleophilic substitution reac-
tion of compound 1 with sodium methoxide gave compound 2 in
90% yield. Based on 1H NMR analysis, only the C5-substituted
product was obtained. Treatment of compound 2 with sodium
hypochlorite and 0.25% (w/v) KOH in ethanol afforded 5-ethoxy-6-
methoxybenzofuroxan (3) in 53% yield. The other three benzo-
furazans (4-2, 4-3, 4-4) were prepared according to our previously
reported procedure [2]. Reduction of these benzofuroxans with
triphenylphosphine under reflux in dichloromethane afforded the
target compounds B1eB4. The remaining listed compounds in
Tables 1e3 (A13eA30, B5eB10 and C1eC5) were prepared ac-
cording to previously reported methods [18e24,27]. All com-
pounds were analyzed by high-pressure liquid chromatography to
ensure the purity (>95%) before submission for biological
evaluation.
24.41
fungi phytopathogens was weak (IC50 > 25
chlorine atom at R2 position with a fluorine atom increased the IC50
m
g/mL), and their antifungal activity against the other three
mg/mL). Replacement of a
value from 8.54
mg/mL (for B2) to >25 mg/mL (for B4) against
R. solani, indicating a chlorine atom at R2 position was more
appropriate than a fluorine atom. Moreover, the same trend was
confirmed with B3 (IC50 ¼ 16.64
m
g/mL) versus B10 (IC50 > 25
mg/
mL) or B8 (IC50 ¼ 15.71
m
g/mL) versus B9 (IC50 ¼ 24.41
mg/mL)
against S. sclerotiorum. In general, this series of compounds
demonstrated better antifungal activity against S. sclerotiorum as
compared with the other three fungi phytopathogens.
Series C benzofurazan derivatives exhibited weak antifungal
activity (IC50 values >25
against R. solani, S. sclerotiorum and F. graminearum Sehw
(IC50 ¼ 10.07e20.82 g/mL), while compound C1 barely exhibited
antifungal activity. When comparing the activity of C4eC5 with the
mg/mL). Compound A13 had potency
m
corresponding A15 and A18 (IC50 values >25
mg/mL), it was
concluded that substitution of the R4 nitro group with another
electron-withdrawing benzenesulfonyl substituent significantly
reduced the antifungal activity. The above results suggested that
the nitro at R4 position was highly important for the antifungal
activity of the compounds.
To further understand the selectivity of these prepared com-
pounds, the most potent compound A3 was evaluated against
invasive fungal pathogen Candida albicans and the human normal
liver cell line HL-7702. At 25 mg/mL. The percentage of inhibition
against C. albicans CMCC(F)98001 is 48.18 ꢁ 2.53, and 84.8% of
human normal liver cell HL-7702 was inhibited. Considering the
IC50 of A3 against phytopathogenic fungi R. solani is 1.91 ꢁ 0.14
mg/
mL, these results demonstrated that compound A3 exhibited a good
selectivity against the phytopathogenic fungi.

L. Wang et al. / European Journal of Medicinal Chemistry 80 (2014) 535e542
537
Scheme 1. Synthetic routes of compounds A2eA12. a (A2eA6): amines, CH3CN, 100 ꢀC in a sealed tube. b (A7eA8): 1,2,4-triazole or pyrazole, acetone, 60 ꢀC under N2 atmosphere. c
(A9eA12): thiols, CH3ONa, EtOH, room temperature.
Scheme 2. Synthetic route of compounds B1eB4. a: CH3ONa, CH3OH, 100 ꢀC in sealed tube, 3 h, 90%. b: NaClO, 0.25% KOH (w/v), EtOH, 0 ꢀC, 1 h, 53%. c: Ph3P, CH2Cl2, reflux, 1 h.
4. Conclusion
5. Experimental section
In conclusion, we have synthesized a series of benzofurazan
derivatives and evaluated their inhibitory ability against four
phytopathogenic fungi. The selected benzofurazans were struc-
turally related to our reported benzofurozan. Interestingly,
removal of the nitrogeneoxygen coordination bond caused a
decline of antifungal activity for most of compounds. However, an
obvious activity improvement as compared to benzofurozans was
observed.
Among these benzofurazan derivatives, the 3-chloro-4-
fluoroaniline derivative A3 displayed a broad spectrum of anti-
fungal activity against all four tested fungi phytopathogens, the
efficacy against R. solani is especially significant, with an IC50 values
5.1. General
All reagents and solvents were of reagent grade or purified ac-
cording to standard methods. Analytical thin layer chromatography
(TLC) was performed with silica gel plates using silica gel 60 GF254
(Qingdao Haiyang Chemical Co., Ltd.). Column chromatography was
performed over silica gel (200e300 mesh, Qingdao Marine Chem-
ical Ltd.). The 1H NMR and 13C NMR spectra were recorded in
deuterochloroform or other indicated solvents at ambient tem-
perature using a Varian Mercury 400 M or 300 M NMR. The low
resolution MS and HRMS were recorded with an Agilent1260LC and
a Waters-GCT Premier, respectively. The melting points of the
products were determined on a SGW X-4 apparatus (Shanghai
Precision &Scientific Instrument Co., Ltd., Shanghai, China). PE,
petroleum ether (bp 60e90 ꢀC); EA, ethyl acetate; CH2Cl2,
dichloromethane; MeOH, methanol. The compound purity was
analyzed using Prominence UFLC from SHIMADZU. And the column
of 1.91
mg/mL. Eight other series A derivatives exhibited high
antifungal effect against R. solani, with IC50 values less than 5.0 mg/
mL. While the series B compounds, with substituents at the R2 and
R3 positions of the phenyl ring, exhibited antifungal potency only
against S. sclerotiorum (B1, B5 and B10 with IC50 higher than 25
mL). Compounds B2eB4 and B6eB9 also displayed growth inhi-
bition against S. sclerotiorum (IC50 < 25 g/mL). None of these
mg/
was InertsilÒ ODS-SP, 5
mm, 4.6 ꢂ 150 mm. HPLC condition for
m
compound purity determination: methanol and water was used as
mobile phase. Compound A23 was eluted with 15% methanol; and
the mobile phase of A3eA4, A9eA12, A20, A28, B8eB10 and C3eC5
was 60% methanol; and 75% methanol was used for compounds
B2eB4, B6eB7. All the other compounds were analyzed using 50%
methanol.
compounds displayed effective antifungal activity against this
strain. Although the benzofurazan derivatives in general displayed
weak antifungal activity compared to our previously reported
benzofuroxan, we consider the benzofurazan scaffold as a more
stable structural motif and less likely to act as a universal electro-
philic reagent, and therefore possessing lower cytotoxicity. Addi-
tionally, results from activity evaluation against invasive fungal
pathogen C. albicans and the human normal liver cell line HL-7702
demonstrated the compound good selectivity against phytopath-
ogenic fungi. Hopefully, further exploration of the benzofurazan
scaffold would lead to the discovery of even more potent agents
against different phytopathogenic fungi.
5.2. General synthetic procedure for the preparation of compounds
A2eA6
4-Chloro-7-nitrobenzofurazan (A1) [15] (50 mg, 0.25 mmol)
was dissolved in acetonitrile (5 mL). Then amine (2.5 mmol, 1-(4-
aminophenyl)ethanone, 3-chloro-4-fluoroaniline, 4-bromoaniline,

538
L. Wang et al. / European Journal of Medicinal Chemistry 80 (2014) 535e542
Table 1
Antifungal activity of series A benzofurazan derivatives against four phytopathogens.a
Compound
R1
Cl
IC50 ꢁ SD (
m
g/mL)a
Rhizoctonia solani
Sclerotinia sclerotiorum
Fusarium graminearum
Phytophthora capsici
A1
A2
4.20 ꢁ 0.97
9.13 ꢁ 0.13
18.41 ꢁ 0.31
>25
>25
>25
>25
>25
A3
1.91 0.14
8.99 ꢁ 0.00
7.13 ꢁ 0.15
11.41 ꢁ 0.13
A4
A5
2.03 0.17
17.26 ꢁ 0.05
>25
22.64 ꢁ 0.23
>25
>25
>25
21.35 ꢁ 0.12
A6
A7
>25
>25
>25
>25
>25
3.11 0.00
21.38 ꢁ 0.13
22.29 ꢁ 0.15
A8
4.43 ꢁ 0.07
>25
18.67 ꢁ 0.18
>25
13.51 ꢁ 0.05
>25
>25
>25
>25
A9
A10
4.49 ꢁ 0.12
22.91 ꢁ 0.17
>25
A11
A12
3.45 0.15
2.58 0.07
>25
>25
>25
>25
>25
>25
A13
A14
A15
A16
A17
A18
H
N3
CH3O
C2H5O
CH3S
PhSO2
10.07 ꢁ 1.38
>25
>25
20.82 ꢁ 0.84
>25
>25
21.47 ꢁ 0.62
22.64 ꢁ 0.25
>25
14.58 ꢁ 0.07
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
21.65 ꢁ 0.86
>25
>25
>25
A19
4.63 ꢁ 1.86
6.58 ꢁ 0.14
>25
>25
A20
A21
>25
>25
>25
>25
>25
>25
>25
>25
A22
A23
A24
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
A25
A26
A27
A28
A29
6.74 ꢁ 0.42
7.04 ꢁ 0.41
7.41 ꢁ 0.15
3.87 ꢁ 0.07
7.13 ꢁ 0.30
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
24.79 ꢁ 0.15
5.95 ꢁ 0.05
21.32 ꢁ 0.23
15.30 ꢁ 0.15
>25
A30
>25
>25
>25
>25

L. Wang et al. / European Journal of Medicinal Chemistry 80 (2014) 535e542
539
Table 1 (continued )
Compound
R1
IC50 ꢁ SD (
m
g/mL)a
Rhizoctonia solani
Sclerotinia sclerotiorum
Fusarium graminearum
Phytophthora capsici
Control 1
Carbendazim
1.42 ꢁ 0.14
0.15 ꢁ 0.03
0.50 ꢁ 0.08
e
Control 2
Chlorothalonil
e
e
e
4.48 ꢁ 0.40
a
Values are the mean ꢁ standard deviation (SD) of three replicates.
2-chloroaniline and pyridin-3-amine) was added. The solution was
stirred at 100 ꢀC in a sealed tube. The reaction was monitored by
TLC, after completion, the solvent acetonitrile was removed in
vacuo, and the residue was subjected to silica gel column chro-
matography (CH2Cl2/PE: 1/1 and then CH2Cl2) to give the desired
products as red solids.
DMSO-d6)
d
155.2 (d, J ¼ 247.5 Hz), 144.9, 144.0, 141.9, 137.4, 135.1
(d, J ¼ 3.0 Hz), 125.9, 124.7 (d, J ¼ 7.1 Hz), 123.8, 120.3 (d,
J ¼ 18.2 Hz), 117.8 (d, J ¼ 22.2 Hz), 102.3; HRMS (CIꢃ) m/z calcd for
C12H6N4O3ClF 308.0112, found 308.0114.
N-(4-bromophenyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine
(A4): a red solid; 76 mg; yield 91%; Rf 0.2 (CH2Cl2/PE: 1/1); mp:
1-(4-((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)phenyl)etha-
none (A2): a red solid; 65 mg; yield 87%; Rf 0.5 (CH2Cl2); mp: 248e
213e214 ꢀC; 1H NMR (400 MHz, acetone-d6)
d
9.85 (br s, 1H), 8.52
(d, J ¼ 8.8 Hz, 1H), 7.69 (d, J ¼ 8.0 Hz, 2H), 7.56 (d, J ¼ 8.0 Hz, 2H),
250 ꢀC; 1H NMR (400 MHz, DMSO-d6)
d
11.14 (br s, 1H), 8.57 (d,
6.92 (d, J ¼ 8.8 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
d 145.0, 144.1,
J ¼ 8.8 Hz,1H), 8.06 (d, J ¼ 8.4 Hz, 2H), 7.62 (d, J ¼ 8.4 Hz, 2H), 7.00 (d,
141.7, 137.4, 137.3, 132.5, 125.7, 123.6, 118.4, 102.2; HRMS (CIꢃ) m/z
calcd for C12H8N4O3 79Br 334.9780 and C12H8N4O3 81Br 336.9759,
found 334.9758 and 336.9756.
J ¼ 8.8 Hz, 1H), 2.59 (s, 3H); 13C NMR (101 MHz, DMSO-d6)
d 196.6,
145.3, 144.0, 142.6, 140.6, 137.1, 133.5, 129.8, 124.6, 122.2, 103.6, 26.6;
HRMS (CIꢃ) m/z calcd for C14H10N4O4 298.0702, found 298.0709.
N-(3-chloro-4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazol-
4-amine (A3): a red solid; 70 mg; yield 91%; Rf 0.3 (PE/EA: 5/1); mp:
N-(2-chlorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine
(A5): a red solid; 70 mg; yield 96%; Rf 0.3 (PE/EA: 5/1); mp: 194e
196 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.46 (d, J ¼ 8.8 Hz, 1H), 7.76 (br
213e214 ꢀC; 1H NMR (400 MHz, acetone-d6)
d
9.83 (brs, 1H), 8.55
s, 1H), 7.61 (dd, J ¼ 14.0, 8.0 Hz, 2H), 7.44 (dd, J ¼ 7.6 Hz, 1H), 7.32
(d, J ¼ 8.8 Hz, 1H), 7.76 (dd, J ¼ 6.4, 2.4 Hz, 1H), 7.68e7.56 (m, 1H),
(dd, J ¼ 7.6 Hz, 1H), 6.67 (d, J ¼ 8.8 Hz, 1H); 13C NMR (101 MHz,
7.49 (dd, J ¼ 8.8 Hz, 1H), 6.89 (d, J ¼ 8.8 Hz, 1H); 13C NMR (101 MHz,
DMSO-d6) d 144.3, 144.1,143.1, 137.3, 134.7,130.7, 130.6,129.6,129.2,
Table 2
Antifungal activity of series B benzofurazan derivatives against four phytopathogens.a
Compound
R2
R3
IC50 ꢁ SD (
m
g/mL)a
Rhizoctonia solani
Sclerotinia sclerotiorum
Fusarium graminearum Sehw
Phytophthora capsici
B1
B2
CH3O
Cl
CH3CH2O
>25
>25
>25
>25
8.54 ꢁ 0.10
>25
22.19 ꢁ 0.07
16.64 ꢁ 0.05
23.14 ꢁ 0.25
>25
>25
>25
>25
>25
>25
B3
B4
Cl
F
>25
B5
B6
B7
B8
B9
Cl
Cl
Cl
Cl
F
H
Cl
CH3
CH3O
CH3O
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
15.99 ꢁ 1.27
13.18 ꢁ 0.76
19.70 ꢁ 0.23
15.71 ꢁ 0.10
24.41 ꢁ 0.08
>25
>25
>25
B10
F
>25
>25
>25
>25
Control 1
Carbendazim
1.42 ꢁ 0.14
0.15 ꢁ 0.03
0.50 ꢁ 0.08
e
Control 2
Chlorothalonil
e
e
e
4.48 ꢁ 0.40
a
Values are the mean ꢁ standard deviation (SD) of three replicates.

540
L. Wang et al. / European Journal of Medicinal Chemistry 80 (2014) 535e542
Table 3
Antifungal activity of series C benzofurazan derivatives against four phytopathogens.a
Compound
R1
R2
R3
R4
IC50 ꢁ SD (
m
g/mL)a
Rhizoctonia
solani
Sclerotinia
sclerotiorum
Fusarium graminearum
Sehw
Phytophthora
capsici
C1
C2
C3
C4
C5
H
H
H
PhSO2
CH3O
H
H
Cl
H
H
H
Cl
Cl
H
H
H
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
NO2
NO2
PhSO2
PhSO2
a
Values are the mean ꢁ standard deviation (SD) of three replicates.
128.7, 123.4, 102.4; HRMS (CIꢃ) m/z calcd for C12H7N4O3Cl
290.0207, found 292.0208.
156 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.36 (d, J ¼ 8.0 Hz,1H), 7.42 (dd,
J ¼ 8.0, 5.6 Hz, 2H), 7.19 (d, J ¼ 8.0 Hz, 1H), 7.05 (dd, J ¼ 8.4 Hz, 2H),
7-Nitro-N-(pyridin-3-yl)benzo[c][1,2,5]oxadiazol-4-amine (A6):
a red solid; 25 mg; yield 39%; Rf 0.6 (CH2Cl2/MeOH: 20/1); mp: 229e
4.51 (s, 2H); 13C NMR (101 MHz, DMSO-d6)
d 161.66 (d,
J ¼ 245.4 Hz), 148.9, 142.5, 139.0, 132.4, 132.1, 131.1 (d, J ¼ 8.1 Hz),
131.1, 122.7, 115.6 (d, J ¼ 21.2 Hz), 34.1; MS (þESI) m/z 327.6
[MþNa]þ.
230 ꢀC; 1H NMR (400 MHz, acetone-d6)
d 9.92 (br s, 1H), 8.82 (d,
J ¼ 1.6 Hz, 1H), 8.56 (m, 2H), 8.03 (d, J ¼ 8.0 Hz, 1H), 7.56 (dd, J ¼ 8.0,
4.8 Hz, 1H), 6.92 (d, J ¼ 8.8 Hz, 1H); 13C NMR (75 MHz, DMSO-d6)
4-((4-Chlorobenzyl)thio)-7-nitrobenzo[c][1,2,5]oxadiazole
(A10): a yellow solid; 78 mg; yield 97%; Rf 0.3 (PE/EA: 5/1); mp:
d
147.0, 145.2, 144.2, 142.1, 137.5, 135.1, 131.2, 124.3, 123.9, 102.4;
HRMS (CIꢃ) m/z calcd for C11H7N5O3257.0549, found 257.0545.
157e159 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.35 (d, J ¼ 7.6 Hz, 1H),
7.35 (dd, J ¼ 21.6, 8.0 Hz, 4H), 7.18 (d, J ¼ 7.6 Hz, 1H), 4.50 (s, 3H); 13
C
NMR (101 MHz, DMSO-d6)
d 148.9, 142.6, 138.8, 134.1, 132.5, 132.1,
5.3. General synthetic procedure for compounds A7eA8
130.9, 128.7, 122.8, 34.0; MS (þESI) m/z 322.2 [MþH]þ.
4-((4-Bromobenzyl)thio)-7-nitrobenzo[c][1,2,5]oxadiazole
To a solution of 4-chloro-7-nitrobenzofurazan A1 (100 mg,
0.50 mmol) in acetone (5 mL) was added 1,2,4-triazole and pyrazole
(5.00 mmol) respectively. The reaction mixture was stirred under
nitrogen atmosphere at 60 ꢀC for 3 days and then concentrated in
vacuo. The residue was purified by silica gel column chromatog-
raphy (CH2Cl2) to give the desired products as yellow solids.
(A11): a yellowsolid; 85 mg; yield 93%; Rf 0.4 (PE/EA: 5/1); mp: 158e
160 ꢀC; 1H NMR (400 MHz, CDCl3)
8.35 (d, J ¼ 8.0 Hz, 1H), 7.49 (d,
J ¼ 8.0 Hz, 2H), 7.32 (d, J ¼ 7.6 Hz, 2H), 7.18 (d, J ¼ 7.6 Hz,1H), 4.49 (s,
d
2H); 13C NMR (101 MHz, DMSO-d6)
d 148.9,142.5,138.8,134.5,132.5,
132.1, 131.6, 131.2, 122.8, 121.0, 34.1; MS (þESI) m/z 366.0 [MþH]þ.
4-Nitro-7-(thiophen-2-ylthio)benzo[c][1,2,5]oxadiazole (A12):
a yellow solid; 63 mg; yield 90%; Rf 0.5 (PE/EA: 5/1); mp: 174e
4-Nitro-7-(1H-pyrazol-1-yl)benzo[c][1,2,5]oxadiazole (A7):
a
yellow solid; 76 mg; yield 65%; Rf 0.7 ( CH2Cl2); mp: 187e188 ꢀC; 1H
176 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.29 (d, J ¼ 7.6 Hz, 1H), 7.77 (d,
NMR (400 MHz, CDCl3)
d
9.02 (s,1H), 8.64 (d, J ¼ 8.4 Hz,1H), 8.22 (d,
J ¼ 8.4 Hz, 1H), 7.92 (s, 1H), 6.68 (s, 1H); 13C NMR (101 MHz, DMSO-
J ¼ 5.2 Hz, 1H), 7.51 (d, J ¼ 2.8 Hz, 1H), 7.31e7.27 (m, 1H), 6.78 (d,
J ¼ 7.6 Hz, 1H); 13C NMR (101 MHz, DMSO-d6)
d 148.0, 142.7, 140.0,
d6) d 144.8,144.3,144.0,134.0,133.2,132.5,132.3,116.1,110.9; HRMS
139.5, 135.8, 133.2, 132.4, 129.5, 123.4, 121.4; HRMS (CIꢃ) m/z calcd
(CIꢃ) m/z calcd for C9H5N5O3231.0392, found 231.0392.
for C10H5N3O3S2278.9772, found 278.9774.
4-Nitro-7-(1H-1,2,4-triazol-1-yl)benzo[c][1,2,5]oxadiazole (A8):
a yellow solid; 80 mg; yield 69%; Rf 0.5 (CH2Cl2); mp: 156e158 ꢀC;
Synthesis of compounds A13eA30: in general, these compounds
were prepared through reaction of A1 with different nucleophiles
according to previously reported methods [18e24,27].
4-Nitrobenzo[c][1,2,5]oxadiazole (A13): yellow solid; yield 60%;
1H NMR (400 MHz, CDCl3)
d
9.63 (s,1H), 8.68 (d, J ¼ 8.4 Hz,1H), 8.29
(d, J ¼ 8.0 Hz, 1H), 8.27 (s, 1H); 13C NMR (101 MHz, DMSO-d6)
d
153.9,145.9, 144.3,143.8,134.1, 133.6,130.1, 118.5; HRMS (CIꢃ) m/z
Rf 0.3 (PE/EA: 5/1); 1H NMR (300 MHz, Acetone-d6)
d 8.71 (d,
calcd for C8H4N6O3232.0345, found 232.0341.
J ¼ 6.0 Hz, 1H), 8.53 (d, J ¼ 9.0 Hz, 1H), 7.93 (dd, J ¼ 6.0, 9.0 Hz, 2H).
4-Azido-7-nitrobenzo[c][1,2,5]oxadiazole (A14): yellow solid;
5.4. General synthetic procedure for compounds A9eA12
yield 80%; Rf 0.4 (PE/EA: 5/1); 1H NMR (300 MHz, CDCl3)
d 8.51 (d,
J ¼ 8.1 Hz, 1H), 7.06 (d, J ¼ 8.1 Hz, 1H).
To a solution of 4-chloro-7-nitrobenzofurazan (A1) (50 mg,
0.25 mmol) in ethanol (5 mL) was added different thiols, including
4-Methoxy-7-nitrobenzo[c][1,2,5]oxadiazole (A15): yellow
solid; yield 61%; Rf 0.8 (CH2Cl2); 1H NMR (300 MHz, CDCl3)
d 8.56 (d,
thiophene-2-thiol,
(4-fluorophenyl)methanethiol,
(4-
J ¼ 8.1 Hz, 1H), 6.70 (d, J ¼ 7.8 Hz, 1H), 4.24 (s, 3H).
chlorophenyl)methanethiol, and (4-bromophenyl)methanethiol
(0.38 mmol), respectively. Then sodium methoxide (20.3 mg,
0.38 mmol) in ethanol (0.5 mL) was added dropwise. The reaction
mixture was stirred at room temperature for 1 h. The solution was
then extracted with EtOAc (2 ꢂ 50 mL) and washed with brine. The
combined organic layer was dried over Na2SO4 and concentrated
under reduced pressure. The residue was subjected to silica gel
flash chromatography (PE/EA: 5/1 and then CH2Cl2) to afford the
desired products as yellow solids.
4-Ethoxy-7-nitrobenzo[c][1,2,5]oxadiazole (A16): yellow solid;
yield 60%; Rf 0.2 (PE/EA: 5/1); 1H NMR (300 MHz, CDCl3)
d 8.54 (d,
J ¼ 8.4 Hz,1H), 6.68 (d, J ¼ 8.4 Hz,1H), 4.48 (q, J ¼ 7.0 Hz, 2H),1.64 (t,
J ¼ 7.0 Hz, 3H).
4-(Methylthio)-7-nitrobenzo[c][1,2,5]oxadiazole (A17): yellow
solid; yield 43%; Rf 0.4 (PE/EA: 5/1); 1H NMR (400 MHz, CDCl3)
d
8.42 (d, J ¼ 8.0 Hz, 1H), 7.11 (d, J ¼ 8.0 Hz, 1H), 2.77 (s, 3H).
4-Nitro-7-(phenylsulfonyl)benzo[c][1,2,5]oxadiazole (A18):
yellow solid; yield 30%; Rf 0.6 (DCM/PE: 2/1); 1H NMR (300 MHz,
CDCl3) 8.56 (s, 1H), 8.45 (s, 1H), 8.22 (s, 2H), 7.59 (s, 3H).
4-((4-Fluorobenzyl)thio)-7-nitrobenzo[c][1,2,5]oxadiazole (A9):
a yellow solid; 74 mg; yield 97%; Rf 0.4 (PE/EA: 5/1); mp: 154e
d

L. Wang et al. / European Journal of Medicinal Chemistry 80 (2014) 535e542
541
4-(Benzylthio)-7-nitrobenzo[c][1,2,5]oxadiazole (A19): yellow
added dropwise until the red color disappeared. Then the reaction
was stirred for another 0.5 h. The mixture was diluted with water
(50 mL), then extracted with EtOAc (2 ꢂ 50 mL) and washed with
brine. The combined organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was then sub-
jected to silica gel flash chromatography (CH2Cl2/PE: 1/1) to afford
the desired product as yellow solid (60 mg, 53%): Rf 0.4 (PE/EA: 5/
solid; yield 71%; Rf 0.4 (PE/EA: 5/1); 1H NMR (300 MHz, CDCl3)
d
8.35 (s, 1H), 7.33 (t, J ¼ 37.3 Hz, 6H), 4.53 (s, 2H).
N,N-dimethyl-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (A20):
red solid; yield 97%; Rf 0.4 (PE/EA: 5/1); 1H NMR (400 MHz,
Acetone-d6)
(s, 6H).
d
8.47 (d, J ¼ 8.6 Hz, 1H), 6.38 (d, J ¼ 8.4 Hz, 1H), 3.69
4-Nitro-7-(pyrrolidin-1-yl)benzo[c][1,2,5]oxadiazole (A21): red
1); mp: 199e200 ꢀC; 1H NMR (400 MHz, CDCl3)
d 6.62 (brs, 1H),
solid; yield 51%; Rf 0.2 (PE/EA: 5/1); 1H NMR (300 MHz, CDCl3)
6.40 (br s, 1H), 4.14 (s, 2H), 3.95 (s, 3H), 1.53 (t, J ¼ 6.8 Hz, 3H); 13
C
d
8.39 (d, J ¼ 8.9 Hz, 1H), 5.99 (d, J ¼ 9.0 Hz, 1H), 4.28 (s, 2H), 3.66 (s,
NMR (101 MHz, DMSO-d6) d 92.7, 87.8, 65.3, 56.8, 14.1.
2H), 2.23 (s, 4H).
4-Nitro-7-(piperidin-1-yl)benzo[c][1,2,5]oxadiazole (A22): red
5.7. Synthesis of 5-ethoxy-6-methoxybenzo[c][1,2,5]oxadiazole
(B1)
solid; yield 56%; Rf 0.7 (CH2Cl2); 1H NMR (300 MHz, CDCl3)
d
8.42
(d, J ¼ 9.0 Hz, 1H), 6.27 (d, J ¼ 9.0 Hz, 1H), 4.11 (s, 4H), 1.84 (s, 6H).
4-Nitro-7-(piperazin-1-yl)benzo[c][1,2,5]oxadiazole (A23): red
solid; yield 70%; Rf 0.7 (DCM/MeOH: 10/1); 1H NMR (400 MHz,
5-Ethoxy-6-methoxybenzofuroxan (3, 50 mg, 0.24 mmol) was
treated with triphenylphosphine (93.6 mg, 0.36 mmol) at 50 ꢀC for
1 h. Then the solvent was removed in vacuo, and the residue was
purified by the preparation thin layer chromatography (CH2Cl2/PE:
1/1) to give the desired product as a white solid (33 mg, 71%): Rf 0.5
CDCl3)
3.13 (s, 4H).
4-Morpholino-7-nitrobenzo[c][1,2,5]oxadiazole (A24): red
solid; yield 70%; Rf 0.8 (DCM/MeOH: 20/1); 1H NMR (300 MHz,
CDCl3)
d
8.43 (d, J ¼ 9.0 Hz, 1H), 6.30 (d, J ¼ 9.0 Hz, 1H), 4.09 (s, 4H),
(CH2Cl2/PE: 1/1); mp: 173e175 ꢀC; 1H NMR (400 MHz, CDCl3)
d 6.86
d
8.45 (d, J ¼ 8.9 Hz, 1H), 6.33 (d, J ¼ 9.0 Hz, 1H), 4.15e4.05
(s, 1H), 6.84 (s, 1H), 4.18 (q, J ¼ 6.8 Hz, 2H), 3.98 (s, 3H), 1.55 (t,
(m, 4H), 4.03e3.91 (m, 4H).
J ¼ 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3)
d 155.7, 154.8, 146.9,
146.8, 91.1, 90.7, 65.3, 56.7, 14.3; HRMS (CIꢃ) m/z calcd for
7-Nitro-N-phenylbenzo[c][1,2,5]oxadiazol-4-amine (A25): red
solid; yield 93%; Rf 0.4 (PE/EA: 5/1); 1H NMR (300 MHz, CDCl3)
C9H10N2O3194.0691, found 194,0684.
d
8.45 (d, J ¼ 8.6 Hz, 1H), 7.81 (s, 1H), 7.51 (dd, J ¼ 5.6, 3.4 Hz, 2H),
7.40 (m, 3H), 6.73 (d, J ¼ 8.6 Hz, 1H).
5.8. General synthetic procedure for compounds B2eB4
7-Nitro-N-(p-tolyl)benzo[c][1,2,5]oxadiazol-4-amine (A26): red
solid; yield 80%; Rf 0.4 (PE/EA: 5/1); 1H NMR (300 MHz, CDCl3)
Three different benzofuroxans (50 mg) in CH2Cl2 (2 mL) were
treated with triphenylphosphine (1.5 equiv) respectively at 50 ꢀC
for 1 h. Then the solvent was removed in vacuo, and the residue was
purified by the preparation of thin layer chromatography (PE/EA: 5/
1) to give the desired products as yellow solids.
d
8.44 (d, J ¼ 8.1 Hz, 1H), 7.76 (s, 1H), 7.31 (s, 4H), 6.64 (d, J ¼ 8.2 Hz,
1H), 2.42 (s, 3H).
N-(4-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine
(A27): red solid; yield 86%; Rf 0.4 (PE/EA: 5/1); 1H NMR (400 MHz,
CDCl3)
d
8.44 (d, J ¼ 8.5 Hz, 1H), 7.73 (s, 1H), 7.41 (dd, J ¼ 8.2, 4.6 Hz,
5-Chloro-6-(piperidin-1-yl)benzo[c][1,2,5]oxadiazole (B2):
yellow solid; 39 mg; yield 83%; Rf 0.8 (PE/EA: 5/1); mp: 77e79 ꢀC;
1H NMR (400 MHz, CDCl3)
7.85 (s, 1H), 7.07 (s, 1H), 3.05 (s, 4H),
1.92e1.69 (m, 4H), 1.69e1.45 (m, 2H); 13C NMR (75 MHz, CDCl3)
a
2H), 7.24 (dd, J ¼ 15.2, 7.0 Hz, 2H), 6.57 (d, J ¼ 8.5 Hz, 1H).
N-(4-chlorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine
(A28): red solid; yield 96%; Rf 0.4 (PE/EA: 5/1); 1H NMR (400 MHz,
d
CDCl3)
d
8.45 (d, J ¼ 8.5 Hz, 1H), 7.80 (s, 1H), 7.49 (d, J ¼ 8.4 Hz, 2H),
d
153.5, 149.1, 146.9, 138.5, 116.8, 101.2, 53.4, 25.9, 24.0; HRMS (CIꢃ)
7.38 (d, J ¼ 8.4 Hz, 2H), 6.69 (d, J ¼ 8.5 Hz, 1H).
calcd for C11H12ClN3O 237.0669, found 237.0662.
N-(2-fluorophenyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine
(A29): red solid; yield 87%; Rf 0.3 (PE/EA: 5/1); 1H NMR (400 MHz,
5-Chloro-6-morpholinobenzo[c][1,2,5]oxadiazole (B3): a yellow
solid; 40 mg; yield 85%; Rf 0.3 (PE/EA: 5/1); mp: 118e120 ꢀC; 1H
CDCl3)
d
8.46 (d, J ¼ 8.5 Hz, 1H), 7.66 (s, 1H), 7.56 (t, J ¼ 7.3 Hz, 1H),
NMR (400 MHz, CDCl3)
d
7.89 (s, 1H), 7.14 (s, 1H), 4.14e3.69 (m, 4H),
152.1, 148.7, 146.8,
7.42e7.22 (m, 3H), 6.60 (d, J ¼ 8.4 Hz, 1H).
3.34e2.95 (m, 4H); 13C NMR (101 MHz, CDCl3)
d
N-(4-methoxyphenyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-
amine (A30): red solid; yield 95%; Rf 0.3 (PE/EA: 5/1); 1H NMR
137.5, 117.1, 101.9, 66.6, 52.2; HRMS (CIꢃ) m/z calcd for
C10H10N3O2Cl 239.0462, found 232.0464.
(400 MHz, Acetone-d6)
d
9.81 (s,1H), 8.52 (d, J ¼ 8.7 Hz,1H), 7.50 (d,
5-Fluoro-6-(piperidin-1-yl)benzo[c][1,2,5]oxadiazole (B4):
a
J ¼ 8.5 Hz, 2H), 7.10 (d, J ¼ 8.5 Hz, 2H), 6.66 (d, J ¼ 8.6 Hz, 1H), 3.87
yellow solid; 40 mg; yield 86%; Rf 0.5 (PE/EA: 5/1); mp: 66e68 ꢀC;
(s, 3H).
1H NMR (400 MHz, CDCl3)
J ¼ 7.6 Hz, 1H), 3.22e3.05 (m, 4H), 1.84e1.70 (m, 4H), 1.70e1.58 (m,
2H); 13C NMR (101 MHz, CDCl3)
d
7.32 (d, J ¼ 11.6 Hz, 1H), 6.92 (d,
5.5. Synthesis of 4-fluoro-5-methoxy-2-nitroaniline (2) [25]
d
160.5 (d, J ¼ 265.6 Hz),148.0,147.8
(d, J ¼ 16.2 Hz), 146.4 (d, J ¼ 15.2 Hz), 99.3 (d, J ¼ 27.3 Hz), 98.5 (d,
J ¼ 3.0 Hz), 52.1, 52.0, 25.9, 24.1; HRMS (CIꢃ) m/z calcd for
A
mixture of 5-chloro-4-fluoro-2-nitroaniline (200 mg,
1.05 mmol) and sodium methoxide (567 mg, 10.5 mmol) in meth-
anol (10 mL) was heated at 100 ꢀC in a sealed tube for 3 h. Then the
solution was cooled to room temperature, diluted with 20 mL of
water, extracted with EtOAc (3 ꢂ 50 mL) and washed with brine.
The organic layer was dried over Na2SO4 and concentrated under
reduced pressure to give intermediate 2 as a yellow solid (176 mg,
yield 90%): Rf 0.5 (CH2Cl2/MeOH: 50/1); 1H NMR (400 MHz, CDCl3)
C11H12N3OF 221.0964, found 221.0963.
6. Antifungal bioassay
All phytopathogenic fungi were provided by the Laboratory of
Plant Disease Control, Nanjing Agricultural University. The strains
were retrieved from the storage tube of potato dextrose agar (PDA)
slants to sterilized PDA Petri dishes and incubated at 25 ꢀC in the
dark for a week to produce new mycelia for the antifungal assay.
The antifungal activities of the synthetic compound were tested
in vitro against four plant pathogenic fungi (R. solani, S. sclerotiorum,
F. graminearum and P. capsici) using a mycelia growth inhibition
method [27]. Before mixing with molten agar, a stock solution of
10 mg/mL in DMSO was prepared at room temperature. The
d
7.85 (d, J ¼ 11.6 Hz, 1H), 6.21 (d, J ¼ 7.2 Hz, 3H), 3.91 (s, 3H).
5.6. Synthesis of 5-ethoxy-6-methoxybenzofuroxan (3) [26]
4-Fluoro-5-methoxy-2-nitroaniline (compound 2, 100 mg,
0.54 mmol) was dissolved in alcohol (10 mL) with 0.25% (w/v) KOH.
The solution was cooled to 0 ꢀC, and 5% aqueous NaClO solution was

542
L. Wang et al. / European Journal of Medicinal Chemistry 80 (2014) 535e542
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concentration for initial screening was 25 mg/mL. And the medium
furoxans: recent developments, Mini Reviews in Medicinal Chemistry
5
containing compounds was then poured into sterilized Petri dishes.
After 2e4 days (different strains with different growth speed) in-
cubation at 25 ꢀC in the dark, the colony diameter of each strain was
measured with the original mycelial disk diameter (5 mm) sub-
tracted from this measurement. Percentage inhibition (I%) was
calculated with the following equation: I% ¼ (1 ꢃ a/b) ꢂ 100, where
a is the colony diameter in Petri dishes with test compounds, and b
is the mean colony diameter in Petri dishes without tested com-
pounds. DMSO served as negative control, whereas commercially
available agricultural fungicide, Carbendazim and Chlorothalonil,
were used as positive controls. Compounds possessing good ac-
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a diazen-1-ium-1,2-
tivities (inhibitory rate >50% at 25
mg/mL) were needed to be
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R.R. Usgaard, T.J. Ward, H.C. Kistler, Chromosome complement of the fungal
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further evaluated using the above-mentioned method but with
different concentrations. Each measurement consisted of at least
three replicates. The concentration dependent curve was the log-
arithmic values of inhibition rates for the y axis against the test
sample concentrations (mg/mL) for the X axis. The IC50 value was
defined as the concentration required for 50% inhibition of mycelial
growth, as shown in Tables 1e3.
Notes
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J.Z. Yao, Z.Y. Miao, W.N. Zhang, C.Q. Sheng, Discovery of highly potent triazole
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The authors declare no competing financial interest.
Acknowledgement
This work was financially supported by grants from the Priority
Academic Program Development of Jiangsu Higher Education In-
stitutions, and co-supported by National Basic Research Program of
China (2010CB126100) and Special Fund for Agro-scientific
Research in the Public Interest (201303023). The Project is also
co-sponsored by the Scientific Research Foundation for the
Returned Overseas Chinese Scholars, State Education Ministry to
Chunhua Qiao, Jiangsu province 333 project funding.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.058.
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