Synthesis and biological evaluation of benzimidazole phenylhydrazone derivatives as antifungal agents against phytopathogenic fungi

Article abstract of DOI:10.3390/molecules21111574

A series of benzimidazole phenylhydrazone derivatives (6a-6ai) were synthesized and characterized by ¹H-NMR, ESI-MS, and elemental analysis. The structure of 6b was further confirmed by single crystal X-ray diffraction as (E)-configuration. All the compounds were screened for antifungal activity against Rhizoctonia solani and Magnaporthe oryzae employing a mycelium growth rate method. Compound 6f exhibited significant inhibitory activity against R. solani and M. oryzae with the EC₅₀ values of 1.20 and 1.85 μg/mL, respectively. In vivo testing demonstrated that 6f could effectively control the development of rice sheath blight (RSB) and rice blast (RB) caused by the above two phytopathogens. This work indicated that the compound 6f with a benzimidazole phenylhydrazone scaffold could be considered as a leading structure for the development of novel fungicides.

Full text of DOI:10.3390/molecules21111574

molecules
Article
Synthesis and Biological Evaluation of
Benzimidazole Phenylhydrazone Derivatives as
Antifungal Agents against Phytopathogenic Fungi
Xing Wang 1,2,†, Yong-Fei Chen 1,2,†, Wei Yan 1,2, Ling-Ling Cao 1,2 and Yong-Hao Ye 1,2,
*
1
State & Local Joint Engineering Research Center of Green Pesticide Invention and Application,
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China;
2014102127@njau.edu.cn (X.W.); 2013102122@njau.edu.cn (Y.-F.C.); yanwei@njau.edu.cn (W.Y.);
2015202050@njau.edu.cn (L.-L.C.)
2
Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education,
Nanjing 210095, China
*
†
Correspondence: yeyh@njau.edu.cn; Tel.: +86-25-8439-9753
These authors contributed equally to this paper.
Academic Editors: Daniela Barlocco and Fiorella Meneghetti
Received: 13 October 2016; Accepted: 12 November 2016; Published: 22 November 2016
Abstract: A series of benzimidazole phenylhydrazone derivatives (6a–6ai) were synthesized and
characterized by 1H-NMR, ESI-MS, and elemental analysis. The structure of 6b was further confirmed
by single crystal X-ray diffraction as (E)-configuration. All the compounds were screened for
antifungal activity against Rhizoctonia solani and Magnaporthe oryzae employing a mycelium growth
rate method. Compound 6f exhibited significant inhibitory activity against R. solani and M. oryzae
with the EC50 values of 1.20 and 1.85 µg/mL, respectively. In vivo testing demonstrated that 6f could
effectively control the development of rice sheath blight (RSB) and rice blast (RB) caused by the
above two phytopathogens. This work indicated that the compound 6f with a benzimidazole
phenylhydrazone scaffold could be considered as a leading structure for the development of
novel fungicides.
Keywords: benzimidazole; phenylhydrazone; antifungal activity
1. Introduction
As an important chemical scaffold in medicinal chemistry and agrochemicals [
derivatives have been attracting the interest of organic chemists for their varieties of biological
activities, such as antifungal [ ], anticancer [ ], antibacterial [ ], antioxidant [ ], anti-inflammatory [ ],
and anti-parasitic [ ]. In particular, benzimidazole fungicides, with benzimidazole as the
1], benzimidazole
2
3
4
5
6
7
core substructure (Figure 1), have been widely used throughout the world to fight against
destructive plant pathogens, such as Rhizoctonia solani, Botrytis cinerea, Fusarium graminearum,
and Magnaporthe oryzae [8,9]. However, excessive use of benzimidazole fungicides has led to a
series of negative problems, such as pesticide residues, drug resistance and serious environmental
pollution [10,11]. Therefore, the development of new benzimidazole fungicides with high activity,
good selectivity and eco-friendly properties is extremely urgent.
In our previous work, molecules with phenylhydrazone showed significant antifungal and
antioxidant activities [12,13]. Typically, 1,2,3-triazole phenylhydrazone derivatives displayed
potent antifungal activity in vitro and in vivo. Inspired by these studies, we initiated a study to
synthesize benzimidazole phenylhydrazone derivatives to screen high activity antifungal compounds.
Their antifungal activities against two phytopathogenic fungi (R. solani and M. oryzae) were evaluated
in vitro and in vivo, and their structure-antifungal activity relationships were also discussed.
Molecules 2016, 21, 1574; doi:10.3390/molecules21111574
www.mdpi.com/journal/molecules

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Figure 1. The commercialization of pesticide.
2. Results and Discussion
2.1. Synthesis of Compounds
A total of 35 target compounds were synthesized following the routes outlined in Scheme 1.
The compounds 2a 2d were synthesized by the reaction of o-phenylenediamine with glycolic acid in
75%–85% yield. Then the compounds 2a 2d were further oxidized to aldehydes (3a 3d) using MnO2
in ethyl acetate. The aldehydes were purified by filtration to remove the excess MnO2, then compounds
3a 3d were obtained as white crystals in 60%–76% yield. The anilines 4a 4s were diazotized by NaNO2,
then restored by SnCl2 to obtain substituted phenylhydrazines 5a 5s in 55%–80% yield. Finally, 3a 3d
and 5a 5s were condensed to form the -C=N-NH- bond, giving benzimidazole derivatives 6a 6ai in
45%–80% yield. H-NMR, MS (ESI), and elemental analysis (CHN) data of the target compounds were
in fully accordance with their assigned structures. Among them, compounds 6a 6q 6t 6x, and 6z had
been reported previously [14 15], while the remaining 30 compounds were first reported in this study.
–
–
–
–
–
–
–
–
–
1
,
,
,
,
Scheme 1. The synthetic routes to 6a
–
6ai
.
Reagents and conditions: (
A
) (i) glycolic acid, HCl
◦
◦
(20% aqueous), 100 C, 5 h; (ii) NaOH (15% aqueous), pH 9; (
B) MnO2, EtOAc, 65 C, 1 h; (
C
) (i) NaNO2,
◦
◦
HCl, 0 C, 1 h; (ii) SnCl2, 0 C; r.t., 2 h; (iii) NaOH (40% aqueous), pH 7; (D) MeOH, r.t., 2 h.
2.2. Crystal Structure of Compound 6b
Among these compounds, the crystal structure of compound 6b was determined by X-ray
diffraction analysis. Figure 2 gave a perspective view of 6b with the atomic labelling system.
The X-ray data had been deposited at the Cambridge Crystallographic Data Centre with the CCDC
number 1038194. The result demonstrated that the -C=N-NH- bond bears an (E)-configuration rather
than (Z)-configuration.

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Figure 2. X-ray diffraction structure of 6b, thermal ellipsoids was drawn on the 35% probability.
2.3. Antifungal Activities In Vitro
Different concentrations of compounds 6a
–
6ai were evaluated for their antifungal activities
in vitro against R. solani and M. oryzae. The EC50 values were calculated using linear-regression
analysis, with validamycin A, carbendazim and isoprothiolane as positive controls (Table 1).
Table 1. Antifungal activity of compounds against two phytopathogens a.
EC50 (±SD) µg/mL
Compound
R1
R2
R3
R. solani
M. oryzae
6a
6b b
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
H
H
CH3
CH3
CH3
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-F
3-F
2.86 ± 0.15
2.14 ± 0.12
2.14 ± 0.18
1.88 ± 0.13
1.50 ± 0.08
1.20 ± 0.10
3.98 ± 0.19
3.29 ± 0.17
3.77 ± 0.23
1.00 ± 0.06
2.83 ± 0.15
7.47 ± 0.31
5.85 ± 0.22
>25
4.18 ± 0.21
1.14 ± 0.07
7.02 ± 0.26
7.60 ± 0.23
6.95 ± 0.29
10.04 ± 0.25
>25
11.09 ± 0.92
6.73 ± 0.56
9.08 ± 0.63
8.29 ± 0.28
8.22 ± 0.67
1.85 ± 0.21
12.39 ± 0.89
5.47 ± 0.49
13.66 ± 0.19
8.71 ± 0.55
9.27 ± 0.81
14.66 ± 1.17
12.87 ± 0.76
>25
11.33 ± 1.21
8.90 ± 1.05
16.53 ± 1.24
21.18 ± 1.91
17.62 ± 1.62
>25
6c b
6d b
4-F
6e b
4-OCF3
2,4-2F
2,5-2F
2-Cl
3-Cl
4-Cl
2,4-2Cl
2,5-2Cl
2,6-2Cl
3,5-2Cl
2-Br
6f b
6g b
6h b
6i b
6j b
6k b
6l b
6m b
6n b
6o b
6p b
4-Br
6q
4-OMe
4-CN
3,4-2CH3
H
6r b
H
H
6s b
6t
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
6u b
3-F
>25
6v b
2,4-2F
2,5-2Cl
4-Cl
2,4-2Cl
2-Br
4-OCF3
4-F
4-CN
H
4-Cl
2,4-2F
H
4-Cl
2,4-2F
2.89 ± 0.04
7.89 ± 0.31
6w b
6x
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
6y b
6z
6aa b
6ab b
6ac b
6ad b
6ae b
6af b
6ag b
6ah b
6ai b
carbendazim
validamycin A
isoprothiolane
3.01 ± 0.02
>25
3.84 ± 0.13
1.28 ± 0.09
2.30 ± 0.11
2.65 ± 0.06
0.93 ± 0.04
1.15 ± 0.09
1.84 ± 0.04
5.07 ± 0.28
19.77 ± 0.65
11.85 ± 0.34
6.54 ± 0.19
14.24 ± 0.58
10.40 ± 0.24
5.26 ± 0.14
1.87 ± 0.10
H
H
H
H
H
0.02 ± 0.01
a
The data of the fungicidal activities were statistically analyzed using Excel to give the results of EC50 values.
The results were expressed as the mean
±
SD of triplicate experiments; b Compounds that were first reported.

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Among 35 compounds, 27 compounds showed significant inhibitory activities against R. solani.
6m 6q 6t were slightly weaker than validamycin A (5.07 g/mL), while the other 20 compounds
were more potent than validamycin A. Interestingly, seven compounds were superior to carbendazim
(1.84 g/mL). Taking single halogen substituents (6k 6m) into account, it was obvious that 6m
(2,4-2Cl, 2.83 g/mL) > 6l (2,6-2Cl, 5.85 g/mL) > 6k (2,5-2Cl, 7.47 g/mL) (“>” means more potent).
g/mL) showed the strongest activity, which was much more superior to
g/mL). When R2 was methyl, the activity of compounds (6a and 6t 6c and
6l
–
,
–
µ
µ
–
µ
µ
µ
The compound 6ah (0.93
µ
that of carbendazim (1.84
µ
,
6u) was significantly decreased; H was compared to R2.
Some target compounds showed great inhibitory activities against M. oryzae. Among these
35 compounds, 6f showed potent inhibiting effects with EC50 of 1.85 g/mL, compared to carbendazim
(EC50 of 1.87 g/mL). Taking single halogen substituents (6b 6d) and (6h 6j) into account, it was
obvious that 6b (2F, 6.73 g/mL) > 6d (4F, 8.29 g/mL) > 6c (3F, 9.08 g/mL), 6h (2Cl, 5.47 g/mL)
> 6j (4Cl, 8.71, µg/mL) > 6i (3Cl, 13.66 µg/mL).
µ
µ
–
–
µ
µ
µ
µ
Based on the results of the antifungal activities shown in Table 1, 6f was selected for further
antifungal activity tests in vivo.
2.4. Inhibition of 6f on the Sclerotia Germination of R. solani
As shown in Table 2, when grain dry sclerotia was inoculated, germination could be observed in
the water of the blank control after a certain incubation period (about four days) at 25 ◦C in the dark.
6f had better controlling effects than sclerotia.
Table 2. The inhibition rate of 6f on the germination of sclerotia of R. solani a.
Treatment (µg/mL)
Compound
1
10
50
6f
0
24.4%
52.7%
53.1%
100%
validamycin A
26.3%
a
Values are the average of 3 replicates.
2.5. Protective Activity of 6f against Rice Sheath Blight (RSB) In Vivo
Compound 6f was selected for evaluating the protective activity against RSB caused by R. solani.
As shown in Figure 3 and Table 3, four days after inoculating with R. solani, significant differences
could be observed between the treated and untreated groups. Tan spots appeared on the leaf sheath
of the blank control, and the scab length reached 16.4 mm. When at a concentration of 200
the protective effect of 6f could reach 68.9% in vivo, close to validamycin A (73.8%), which was
commonly used as a fungicide against RSB. When at a concentration of 100 g/mL, the protective
µg/mL,
µ
effect of 6f and validamycin A reached 66.5% and 61.0% respectively. Thus, 6f showed similar activity
with validamycin A against RSB in vivo.

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Figure 3. Protective activity of 6f against rice sheath blight (RSB).
Table 3. Protective activity of 6f against RSB a.
Compound
6f
Treatment (µg/mL)
Lesion Lengtha (mm)
Protection Efficacy (%)
200
100
200
100
5.1
5.5
4.3
6.4
16.4
68.9
66.5
73.8
61.0
validamycin A
blank control
a
Values are the average of 20 replicates.
2.6. Inhibition of 6f on the Conidium Germination of M. oryzae
As shown in Figure 4, conidia of M. oryzae were inoculated after 24 h, germination could be
observed in the water of the blank control under the microscope with a germination rate of 100%,
and 1% DMSO had no effect on the conidium germination. The conidia could not form the germ tube
or appressorium when incubated with 6f at the concentration of 1.0 µg/mL (1% DMSO).
Figure 4. The effect of 6f on the conidium germination rate of M. oryzae.
2.7. Protective Activity of 6f against Rice Blast (RB) In Vivo
The efficacy of the protective activity experiment is shown in Figure 5 and Table 4, fungus spores
were inoculated on leaves, blast lesions could be observed after 7 days. At a concentration of 50
µg/mL,
the protective effect of 6f could reach 21.6%, whereas carbendazim treatment resulted in 88.0%.

Molecules 2016, 21, 1574
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Figure 5. Protective activity of 6f against rice blast (RB).
Table 4. Protective activity of 6f against RB a.
Compound
6f
Treatment (µg/mL)
Blast Lesions
Protection Efficacy (%)
50
100
25
32.7
26
11.3
5.0
21.6
37.6
72.9
88.0
carbendazim
blank control
50
41.7
a
Values are the average of 10 replicates.
3. Materials and Methods
3.1. Reagents and Analysis
All reagents bought from Alfa Aesar (Ward Hill, MA, USA), Aladdin (Shanghai, China) or
Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) were pure analytical grades and used without
further treatments. Reactions were monitored by TLC using silica gel coated glass slides (silica-gel
60 GF254, Qingdao Haiyang Chemical, Qingdao, China). Melting points were measured on WRS-1B
digital melting-point apparatus (SPSIC, Shanghai, China), uncorrected. 1H-NMR spectra were recorded
on a Bruker Avance III 400 NMR spectrometer (Bruker, Stuttgart, Germany). The chemical shifts (δ)
are reported in ppm with reference to internal TMS, and coupling constants (J) are given in Hz.
ESI-MS spectra were recorded on a Bruker UHR-TOF maxis (Bruker, Billerica, MA, USA). X-ray single
crystal diffraction analysis was conducted on a Bruker D8 Venture diffractometer (Bruker, Karlsruhe,
German). Elemental analyses were performed on a Elementar Vario MICRO instrument (Elementar,
Langenselbold, German) and were within ±0.4% of the theoretical values.
3.2. Synthesis
3.2.1. Synthesis and Purification of Compound 2a–2d
The procedures were conducted according to the literature [16]. Glycolic acid (15.24 g, 0.2 mol)
◦
was dissolved in 40 mL HCl (20% aqueous) in the flask. When the temperature reac◦hed 65 C,
compound 1a
–
1d (16.27 g, 0.15 mol) was added at three times, then refluxed at 100 C for 5 h.

Molecules 2016, 21, 1574
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After cooling to room temperature, the solution was basified with NaOH (15%, aqueous) until it
reached pH 9.0 to precipitate the desired compounds 2a–2d (in 75%–85% yield).
3.2.2. Synthesis and Purification of Compound 3a–3d
A solution of 2a–2d (2 mmol), MnO2 (3.48 g, 40 mmol) was added to EtOAc (120 mL), then refluxed
at 65 ◦C for 1 h (monitored by TLC). Afterwards the solution was filtered, and concentrated in vacuo
to give pure compounds 3a–3d (in 60%–76% yield) [17].
3.2.3. Synthesis and Purification of Compound 5a–5s
Substituted aniline 4a–4s (50 mmol) was dissolved in the 50 mL HCl (18%, aqueous) in the ice
bath. NaNO2 (50 mmol) dissolved in 50 mL water was added dropwise. The reaction mixture was
stirred for 1 h to obtain a clear solution. Then the solution of SnCl2 (0.1 mol) in 30 mL of concentrated
HCl was added dropwise at 0 ◦C. The mixture was stirred at room temperature for 2 h. Afterwards,
the mixture was extracted with 50 mL EtOAc and the organic impurities were discarded. Then the
solution was basified with NaOH (40%, aqueous) until it reached pH 7.0. The reaction mass was
extracted with EtOAc three times. Finally, substituted phenylhydrzine 5a–5s was afforded after being
vapored under reduced pressure (in 55%–80% yield) [12].
3.2.4. Synthesis and Purification of Compound 6a–6ai
The equimolar aldehyde 3a–3d (1 mmol) and substituted phenylhydrazine 5a–5s (1 mmol) were
mixed in CH3OH (10 mL) and stirred at room temperature [18]. After about 2 h, the reaction was
completed (monitored by TLC). The residual crude was purified via silica gel column chromatogram
using a gradient mixture of petroleum ether and ethyl acetate to obtain the pure target compounds
6a–6ai (in 45–80% yield).
(E)-2-((2-Phenylhydrazono)methyl)-1H-benzo[d]imidazole (6a): Yellow power, yield 64.2%, m.p.
185.3–186.1 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.52 (s, 1H), 10.90 (s, 1H), 7.88 (s, 1H), 7.53 (s, 2H),
7.31–7.26 (m, 2H), 7.25 (t, J = 4.2 Hz, 2H), 7.18 (d, J = 2.8 Hz, 2H), 6.84 (dd, J = 9.7, 4.2 Hz, 1H). MS (ESI):
237.10 (C14H13N4, [M + H]+). Anal. Calcd. For C14H12N4: C, 71.17; H, 5.12; N, 23.71. Found: C, 70.95;
H, 5.19; N, 23.49.
(E)-2-((2-(2-F◦luorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6b): Sandybrown power, yield 72.8%,
1
201.8–202.4 C. H-NMR (400 MHz, DMSO-d6)
δ 12.64 (s, 1H), 10.89 (s, 1H), 8.18 (s, 1H), 7.83
(t, J = 8.2 Hz, 1H), 7.62–7.49 (m, 2H), 7.25–7.14 (m, 4H), 6.86 (dd, J = 13.0, 7.0 Hz, 1H). MS (ESI):
255.10 (C14H12FN4, [M + H]+). Anal. Calcd. For C14H11FN4: C, 66.13; H, 4.36; N, 22.04. Found: C,
66.34; H, 4.11; N, 21.89.
(E)-2-((2-(3-Fluorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6c): Yellow power, yield 69.3%, m.p.
203.5–205.9 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.61 (s, 1H), 11.09 (s, 1H), 7.90 (d, J = 0.6 Hz, 1H),
7.55 (s, 2H), 7.33–7.26 (m, 1H), 7.26–7.17 (m, 3H), 6.91 (dd, J = 8.1, 1.2 Hz, 1H), 6.63 (td, J = 8.3, 2.0 Hz,
1H). MS (ESI): 255.10 (C14H12FN4, [M + H]+). Anal. Calcd. For C14H11FN4: C, 66.13; H, 4.36; N, 22.04.
Found: C, 66.34; H, 4.11; N, 21.89.
(E)-2-((2-(4-Fluorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6d): Yellow power, yield 76.8%, m.p.
199.2–199.4 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.54 (s, 1H), 10.90 (s, 1H), 7.86 (s, 1H), 7.52 (s, 2H),
7.24 (dt, J = 10.2, 4.1 Hz, 2H), 7.21–7.11 (m, 4H). MS (ESI): 255.10 (C14H12FN4, [M + H]+). Anal. Calcd.
For C14H11FN4: C, 66.13; H, 4.36; N, 22.04. Found: C, 66.34; H, 4.11; N, 21.89.
(E)-2-((2-(4-(Trifluoromethoxy)phenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6e): White power, yield
◦
1
54.1%, m.p. 207.3–207.9 C. H-NMR (400 MHz, DMSO-d6)
δ 12.61 (s, 1H), 11.07 (s, 1H), 7.91
(d, J = 0.6 Hz, 1H), 7.54 (s, 2H), 7.33–7.28 (m, 4H), 7.19 (d, J = 2.8 Hz, 2H). MS (ESI): 321.09
(C15H12F3N4O, [M + H]+). Anal. Calcd. For C15H11F3N4O: C, 56.25; H, 3.46; N, 17.49. Found:
C, 56.27; H, 3.65; N, 17.07.

Molecules 2016, 21, 1574
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(E)-2-((2-(2,4-Difluorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6f): Brown power, yield 65.5%,
◦
m.p. 200.2–200.3 C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.68 (s, 1H), 11.05 (s, 1H), 8.18 (s, 1H), 7.70–7.64
(m, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.29–7.21 (m, 2H), 7.18 (dd, J = 13.4, 5.6 Hz,
1H), 6.64 (ddd, J = 11.9, 8.5, 3.3 Hz, 1H). MS (ESI): 273.09 (C14H11F2N4, [M + H]+). Anal. Calcd. For
C14H10F2N4: C, 61.76; H, 3.70; N, 20.58. Found: C, 61.89; H, 3.83; N, 20.49.
(E)-2-((2-(2,5-Difluorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6g): Pale yellow power, yield 59.6%,
◦
m.p. 247.1–248.2 C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.67 (s, 1H), 11.04 (s, 1H), 8.18 (s, 1H), 7.70–7.63
(m, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.29–7.21 (m, 2H), 7.21–7.14 (m, 1H), 6.67–6.59
(m, 1H). MS (ESI): 273.09 (C14H11F2N4, [M + H]+). Anal. Calcd. For C14H10F2N4: C, 61.76; H, 3.70; N,
20.58. Found: C, 61.89; H, 3.83; N, 20.49.
(E)-2-((2-(2-Chlorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6h): Brown power, yield 72.6%, m.p.
◦
1
198.8–199.3 C. H-NMR (400 MHz, DMSO-d6)
δ 12.65 (s, 1H), 10.54 (s, 1H), 8.35 (s, 1H), 7.88
(d, J = 8.2 Hz, 1H), 7.56 (s, 2H), 7.38 (d, J = 7.9 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 7.21 (s, 2H), 6.88
(dd, J = 10.9, 4.3 Hz, 1H). MS (ESI): 271.05 (C14H12ClN4, [M + H]+). Anal. Calcd. For C14H11ClN4: C,
62.11; H, 4.10; N, 20.70. Found: C, 61.94; H, 4.19; N, 20.29.
(E)-2-((2-(3-Chlorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6i): Pale yellow power, yield 66.7%,
m.p. 212.0–212.2 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.62 (s, 1H), 11.06 (s, 1H), 7.89 (s, 1H), 7.59
(d, J = 7.9 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.43 (s, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.22 (t, J = 7.5 Hz,
1H), 7.16 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 6.86 (dd, J = 7.8, 1.2 Hz, 1H). MS (ESI): 271.05
(C14H12ClN4, [M + H]+). Anal. Calcd. For C14H11ClN4: C, 62.11; H, 4.10; N, 20.70. Found: C, 61.94; H,
4.19; N, 20.29.
(E)-2-((2-(4-Chlorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6j): Yellow power, yield 72.3%, m.p.
◦
1
194.4–194.5 C. H-NMR (400 MHz, DMSO-d6)
δ 12.59 (s, 1H), 11.02 (s, 1H), 7.88 (s, 1H), 7.59
(d, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 2.0 Hz, 1H), 7.33–7.31 (m, 1H), 7.28–7.25
(m, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.24–7.19 (m, 1H), 7.16 (t, J = 7.6 Hz, 1H). MS (ESI): 271.05 (C14H12ClN4,
[M + H]+). Anal. Calcd. For C14H11ClN4: C, 62.11; H, 4.10; N, 20.70. Found: C, 61.94; H, 4.19; N, 20.29.
(E)-2-((2-(2,4-Dichlorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6k): Yellow power, yield 69.4%,
m.p. 212.7–212.8 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.71 (s, 1H), 10.65 (s, 1H), 8.36 (d, J = 0.8 Hz,
1H), 7.89 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 2.3 Hz, 3H), 7.41 (dd, J = 8.9, 2.3 Hz, 1H), 7.21 (s, 2H). MS (ESI):
305.02 (C14H11Cl2N4, [M + H]+). Anal. Calcd. For C14H10Cl2N4: C, 55.10; H, 3.30; N, 18.36. Found: C,
55.58; H, 3.56; N, 17.88.
(E)-2-((2-(2,5-Dichlorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6l): Yellow power, yield 60.1%,
m.p. 215.2–215.3 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.87 (s, 1H), 10.70 (s, 1H), 8.38 (s, 1H), 7.94
(d, J = 2.5 Hz, 1H), 7.58 (dd, J = 5.6, 3.5 Hz, 2H), 7.40 (t, J = 6.1 Hz, 1H), 7.26–7.20 (m, 2H), 6.91
(dd, J = 8.5, 2.5 Hz, 1H). MS (ESI): 305.02 (C14H11Cl2N4, [M + H]+). Anal. Calcd. For C14H10Cl2N4: C,
55.10; H, 3.30; N, 18.36. Found: C, 55.58; H, 3.56; N, 17.88.
(E)-2-((2-(2,6-Dichlorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6m): Pale yellow power, yield
72.3%, m.p. 212.5–212.9 ◦C.1H-NMR (400 MHz, DMSO-d6)
δ 12.38 (s, 1H), 10.13 (s, 1H), 7.87 (s, 1H),
7.56 (d, J = 8.1 Hz, 3H), 7.43 (s, 1H), 7.25 (t, J = 8.1 Hz, 1H), 7.16 (s, 2H). MS (ESI): 305.02 (C14H11Cl2N4,
[M + H]+). Anal. Calcd. For C14H10Cl2N4: C, 55.10; H, 3.30; N, 18.36. Found: C, 55.58; H, 3.56; N, 17.88.
(E)-2-((2-(3,5-Dichlorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6n): Yellow power, yield 55.8%,
m.p. 260.8–261.0 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.71 (s, 1H), 11.20 (s, 1H), 7.91 (s, 1H), 7.56
(s, 2H), 7.24 (d, J = 1.2 Hz, 2H), 7.21 (s, 2H), 6.97 (t, J = 1.8 Hz, 1H). MS (ESI): 305.02 (C14H11Cl2N4,
[M + H]+). Anal. Calcd. For C14H10Cl2N4: C, 55.10; H, 3.30; N, 18.36. Found: C, 55.58; H, 3.56; N, 17.88.
(E)-2-((2-(2-Bromophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6o): Brown power, yield 65.9%, m.p.
◦
1
202.2–202.3 C. H-NMR (400 MHz, DMSO-d6)
δ 12.65 (s, 1H), 10.32 (s, 1H), 8.39 (s, 1H), 7.86

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(dd, J = 8.3, 1.3 Hz, 1H), 7.64–7.50 (m, 3H), 7.39–7.34 (m, 1H), 7.19 (t, J = 12.5 Hz, 2H), 6.83 (td, J = 7.9,
1.5 Hz, 1H). MS (ESI): 315.02 (C14H12BrN4, [M + H]+). Anal. Calcd. For C14H11BrN4: C, 53.35; H, 3.52;
N, 17.78. Found: C, 53.38; H, 3.67; N, 17.28.
(E)-2-((2-(4-Bromophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6p): Brown power, yield: 75.2%, m.p.
202.2–202.5 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.59 (s, 1H), 11.02 (s, 1H), 7.88 (s, 1H), 7.53 (s, 2H),
7.45 (d, J = 8.8 Hz, 2H), 7.23–7.16 (m, 4H). MS (ESI): 315.02 (C14H12BrN4, [M + H]+). Anal. Calcd. For
C14H11BrN4: C, 53.35; H, 3.52; N, 17.78. Found: C, 53.38; H, 3.67; N, 17.28.
(E)-2-((2-(4-Methoxyphenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6q): Brown power, yield: 69.5%,
m.p. 196.8–196.9 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ:12.46 (s, 1H), 10.74 (s, 1H), 7.81 (s, 1H), 7.51
(dd, J = 5.2, 3.6 Hz, 2H), 7.19 (d, J = 2.1 Hz, 1H), 7.17 (d, J = 3.2 Hz, 2H), 7.15 (d, J = 3.2 Hz, 1H), 6.91
(d, J = 2.1 Hz, 1H), 6.90–6.88 (m, 1H), 3.72 (s, 3H). MS (ESI): 267.10 (C15H15N4O, [M + H]+). Anal.
Calcd. For C15H14FN4O: C, 67.65; H, 5.30; N, 21.04. Found: C, 67.38; H, 5.21; N, 21.29.
(E)-4-(2-((1H-Benzo[d]imidazol-2-yl)methylene)hydrazinyl)benzonitrile (6r): Brown power, yield 67.9%,
m.p. 260.1–260.2 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.71 (s, 1H), 11.41 (s, 1H), 7.72 (d, J = 8.9 Hz,
2H), 7.53 (d, J = 35.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.28–7.15 (m, 2H). MS (ESI): 262.09 (C15H12N5,
[M + H]+). Anal. Calcd. For C15H11N5: C, 68.95; H, 4.24; N, 26.80. Found: C, 68.74; H, 4.13; N, 26.99.
(E)-2-((2-(3,4-Dimethylphenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6s): Brown power, yield 56.7%,
◦
m.p. 203.9–204.5 C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.48 (s, 1H), 10.76 (s, 1H), 7.82 (s, 1H), 7.56–7.47
(m, 2H), 7.17 (dd, J = 5.8, 2.9 Hz, 2H), 7.08–7.00 (m, 2H), 6.99–6.94 (m, 1H), 2.22 (s, 3H), 2.16 (s, 3H). MS
(ESI): 265.12 (C16H17N4, [M + H]+). Anal. Calcd. For C16H16N4: C, 72.70; H, 6.10; N, 21.20. Found: C,
72.24; H, 6.19; N, 21.09.
(E)-1-Methyl-2-((2-phenylhydrazono)methyl)-1H-benzo[d]imidazole (6t): Yellow power, yield: 65.2%, m.p.
◦
201.5–201.7 C. 1H-NMR (400 MHz, DMSO-d6)
δ 10.91 (s, 1H), 8.04 (s, 1H), 7.65–7.55 (m, 2H), 7.34–7.25
(m, 3H), 7.21 (t, J = 7.1 Hz, 1H), 7.11 (d, J = 7.7 Hz, 2H), 6.85 (t, J = 7.3 Hz, 1H), 4.14 (s, 3H). MS (ESI):
251.10 (C15H15N4, [M + H]+). Anal. Calcd. For C15H14N4: C, 71.98; H, 5.64; N, 22.38. Found: C, 71.73;
H, 5.31; N, 22.69.
(E)-2-((2-(3-Fluorophenyl)hydrazono)methyl)-1-methyl-1H-benzo[d]imidazole (6u): Yellow power, yield:
◦
1
54.6%, m.p. 219.7–319.8 C. H-NMR (400 MHz, DMSO-d6)
δ 11.09 (s, 1H), 8.06 (s, 1H), 7.64–7.58
(m, 2H), 7.30 (dddd, J = 9.4, 7.2, 5.2, 2.7 Hz, 2H), 7.22 (ddd, J = 8.2, 7.2, 1.2 Hz, 1H), 6.91–6.84 (m, 2H),
6.68–6.61 (m, 1H), 4.13 (s, 3H). MS (ESI): 269.14 (C15H14FN4, [M + H]+). Anal. Calcd. For C15H13FN4:
C, 67.15; H, 4.88; N, 20.88. Found: C, 67.23; H, 4.71; N, 20.69.
(E)-2-((2-(2,4-Difluorophenyl)hydrazono)methyl)-1-methyl-1H-benzo[d]imidazole (6v): Yellow power, yield:
◦
64.1%, m.p. 191.6–191.8 C. 1H-NMR (400 MHz, DMSO-d6)
δ 10.81 (s, 1H), 8.31 (s, 1H), 7.61 (t, J = 8.4 Hz,
2H), 7.47 (td, J = 9.3, 5.9 Hz, 1H), 7.30 (ddd, J = 14.6, 8.2, 2.8 Hz, 2H), 7.23 (dd, J = 11.1, 4.0 Hz, 1H), 7.08
(t, J = 8.6 Hz, 1H), 4.12 (s, 3H). MS (ESI): 287.11 (C15H13F2N4, [M + H]+). Anal. Calcd. For C15H12F2N4:
C, 62.93; H, 4.22; N, 19.57. Found: C, 62.83; H, 4.11; N, 19.66.
(E)-2-((2-(2,5-Dichlorophenyl)hydrazono)methyl)-1-methyl-1H-benzo[d]imidazole (6w): Yellow power, yield:
◦
72.3%, m.p. 206.5–207.2 C. 1H-NMR (400 MHz, DMSO-d6)
δ 10.68 (s, 1H), 8.56 (s, 1H), 7.64 (dd, J = 16.3,
8.0 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 6.93 (dd, J = 8.5,
2.2 Hz, 1H), 4.13 (s, 3H). MS (ESI): 319.06 (C15H13Cl2N4, [M + H]+). Anal. Calcd. For C15H12Cl2N4: C,
56.44; H, 3.79; N, 17.55. Found: C, 56.31; H, 4.01; N, 17.61.
(E)-2-((2-(4-Chlorophenyl)hydrazono)methyl)-1-methyl-1H-benzo[d]imidazole (6x): Yellow power, yield:
67.5%, m.p. 213.2–213.5 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 11.02 (s, 1H), 8.05 (d, J = 1.1 Hz, 1H),
7.63–7.57 (m, 2H), 7.34 (d, J = 2.0 Hz, 1H), 7.33–7.30 (m, 1H), 7.29–7.26 (m, 1H), 7.22 (ddd, J = 8.2, 7.1,
1.3 Hz, 1H), 7.11 (d, J = 2.1 Hz, 1H), 7.09 (d, J = 2.1 Hz, 1H), 4.12 (s, 3H). MS (ESI): 285.09 (C15H14ClN4,
[M + H]+). Anal. Calcd. For C15H13ClN4: C, 63.27; H, 4.60; N, 19.68. Found: C, 63.11; H, 4.69; N, 19.76.

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(E)-2-((2-(2,4-Dichlorophenyl)hydrazono)methyl)-1-methyl-1H-benzo[d]imidazole (6y): Yellow power, yield:
75.4%, m.p. 211.8–212.9 ◦C. 1H-NMR (400 MHz, DMSO-d6)
10.62 (s, 1H), 8.53 (d, J = 1.1 Hz, 1H),
δ
7.67–7.61 (m, 2H), 7.55 (d, J = 2.4 Hz, 1H), 7.52 (d, J = 8.9 Hz, 1H), 7.39 (dd, J = 8.9, 2.4 Hz, 1H), 7.31 (ddd,
J = 8.2, 7.1, 1.2 Hz, 1H), 7.24 (ddd, J = 8.2, 7.2, 1.2 Hz, 1H), 4.13 (s, 3H). MS (ESI): 319.06 (C15H13Cl2N4,
[M + H]+). Anal. Calcd. For C15H12Cl2N4: C, 56.44; H, 3.79; N, 17.55. Found: C, 56.31; H, 4.01; N, 17.61.
(E)-4-(2-((1-Methyl-1H-benzo[d]imidazol-2-yl)methylene)hydrazinyl)benzonitrile (6z): Yellow power, yield:
1
55.8%, m.p. 258.8–259.0 ◦C. H-NMR (400 MHz, DMSO-d6)
δ 11.42 (s, 1H), 8.14 (s, 1H), 7.71
(d, J = 8.6 Hz, 2H), 7.63 (t, J = 8.3 Hz, 2H), 7.31 (t, J = 7.5 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.19
(d, J = 8.5 Hz, 2H), 4.13 (s, 3H). MS (ESI): 329.05 (C15H14BrN4, [M + H]+). Anal. Calcd. For C15H13BrN4:
C, 54.73; H, 3.98; N, 17.02. Found: C, 54.51; H, 4.01; N, 17.11.
(E)-1-Methyl-2-((2-(4-(trifluoromethoxy)phenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6aa): Pale yellow
power, yield: 68.2%, m.p. 196.8–196.9 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 11.07 (s, 1H), 8.07 (s, 1H),
7.61 (t, J = 7.9 Hz, 2H), 7.29 (dd, J = 11.2, 4.7 Hz, 3H), 7.25–7.20 (m, 1H), 7.16 (d, J = 9.0 Hz, 2H), 4.13
(s, 3H). MS (ESI): 335.07 (C16H14F3N4O, [M + H]+). Anal. Calcd. For C16H13F3N4O: C, 57.49; H, 3.92;
N, 16.76. Found: C, 57.17; H, 3.89; N, 16.76.
(E)-2-((2-(4-Fluorophenyl)hydrazono)methyl)-1-methyl-1H-benzo[d]imidazole (6ab): Yellow power, yield:
1
60.8%, m.p. 183.2–184.1 ◦C. H-NMR (400 MHz, DMSO-d6)
δ 10.91 (s, 1H), 8.03 (s, 1H), 7.60
(t, J = 8.5 Hz, 2H), 7.27 (dd, J = 11.1, 4.1 Hz, 1H), 7.22 (dd, J = 11.0, 4.0 Hz, 1H), 7.15(s,1H), 7.14–7.07
(m, 3H), 4.12 (s, 3H). MS (ESI): 269.14 (C15H14FN4, [M + H]+). Anal. Calcd. For C15H13FN4: C, 67.15;
H, 4.88; N, 20.88. Found: C, 67.23; H, 4.71; N, 20.69.
(E)-2-((2-(2-Bromophenyl◦)hydrazono)methyl)-1-methyl-1H-benzo[d]imidazole (6ac): Brown power, yield:
56.6%, m.p. 190.1–190.9 C. 1H-NMR (400 MHz, DMSO-d6)
δ 10.29 (s, 1H), 8.56 (s, 1H), 7.63 (dd, J = 14.4,
8.0 Hz, 2H), 7.53 (ddd, J = 17.2, 8.1, 1.2 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.30 (dd, J = 11.2, 4.0 Hz, 1H),
7.26–7.20 (m, 1H), 6.87–6.81 (m, 1H), 4.13 (d, J = 7.6 Hz, 3H). MS (ESI): 276.13 (C16H14N5, [M + H]+).
Anal. Calcd. For C16H13N5: C, 69.80; H, 4.76; N, 25.44. Found: C, 69.75; H, 4.74; N, 25.23.
(E)-6-Methyl-2-((2-phenylhydrazono)methyl)-1H-benzo[d]imidazole (6ad): Yellow power, yield: 64.3%,
m.p. 204.6–205.3 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.40 (s, 1H), 10.87 (s, 1H), 7.84 (s, 1H), 7.48
(t, J = 7.1 Hz, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.30 (s, 1H), 7.29–7.25 (m, 2H), 7.25–7.20 (m, 2H), 6.84
(t, J = 7.1 Hz, 1H), 2.41 (s, 3H). MS (ESI): 251.10 (C15H15N4, [M + H]+). Anal. Calcd. For C15H14N4: C,
71.98; H, 5.64; N, 22.38. Found: C, 71.73; H, 5.31; N, 22.69.
(E)-2-((2-(4-Chlorophenyl)hydrazono)methyl)-6-methyl-1H-benzo[d]imidazole (6ae): Yellow power, yield:
72.5%, m.p. 216.3–217.5 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.48 (s, 1H), 11.00 (s, 1H), 7.86 (s, 1H),
7.42 (d, J = 8.2 Hz, 1H), 7.34 (s, 1H), 7.31 (s, 2H), 7.24 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.1 Hz, 1H), 2.41
(s, 3H). MS (ESI): 285.09 (C15H13ClN4, [M + H]+). Anal. Calcd. For C15H13ClN4: C, 63.27; H, 4.60; N,
19.68. Found: C, 63.11; H, 4.69; N, 19.76.
(E)-2-((2-(2,4-Difluorophenyl)hydrazono)methyl)-6-methyl-1H-benzo[d]imidazole (6af): Pale yellow power,
yield: 59.4%, m.p. 221.9–222.7 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.51 (s, 1H), 10.81 (s, 1H), 8.13
(s, 1H), 7.80 (td, J = 9.3, 5.9 Hz, 1H), 7.46–7.38 (m, 1H), 7.35–7.23 (m, 2H), 7.10 (tt, J = 8.8, 1.9 Hz,
1H), 7.01 (d, J = 8.2 Hz, 1H), 2.41 (s, 3H). MS (ESI): 287.11 (C15H13F2N4, [M + H]+). Anal. Calcd. For
C15H12F2N4: C, 62.93; H, 4.22; N, 19.57. Found: C, 62.83; H, 4.11; N, 19.66.
(E)-6-Chloro-2-((2-phenylhydrazono)methyl)-1H-benzo[d]imidazole (6ag): Pale yellow power, yield: 72.1%,
◦
m.p. 215.6–215.9 C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.73 (s, 1H), 11.03 (s, 1H), 7.87 (s, 1H), 7.63–7.46
(m, 2H), 7.30 (dd, J = 8.6, 6.9 Hz, 2H), 7.28–7.23 (m, 2H), 7.21 (dd, J = 8.5, 2.1 Hz, 1H), 6.87 (t, J = 7.0,
1.4 Hz, 1H). MS (ESI): 271.05 (C14H12ClN4, [M + H]+). Anal. Calcd. For C14H11ClN4: C, 62.11; H, 4.10;
N, 20.70. Found: C, 61.94; H, 4.19; N, 20.29.

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(E)-6-Chloro-2-((2-(4-chlorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6ah): Brown power, yield:
62.8%, m.p. 235.5–236.9 ◦C. 1H-NMR (400 MHz, DMSO-d6)
12.77 (s, 1H), 11.13 (s, 1H), 7.87 (s, 1H),
δ
7.67–7.46 (m, 3H), 7.35 (d, J = 2.2 Hz, 1H), 7.34 (d, J = 2.3 Hz, 1H), 7.26 (d, J = 2.2 Hz, 1H), 7.25
(d, J = 2.1 Hz, 1H), 7.21 (d, J = 8.8 Hz, 1H). MS (ESI): 305.02 (C14H11Cl2N4, [M + H]+). Anal. Calcd. For
C14H10Cl2N4: C, 55.10; H, 3.30; N, 18.36. Found: C, 55.58; H, 3.56; N, 17.88.
(E)-6-Chloro-2-((2-(2,4-difluorophenyl)hydrazono)methyl)-1H-benzo[d]imidazole (6ai): Yellow power, yield:
68.2%, m.p. 230.2–23.0.7 ◦C. 1H-NMR (400 MHz, DMSO-d6)
δ 12.79 (s, 1H), 10.94 (s, 1H), 8.12 (s, 1H),
7.80 (td, J = 9.3, 5.9 Hz, 1H), 7.67–7.58 (m, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.30 (ddd, J = 11.8, 8.9, 2.8 Hz,
1H), 7.22 (dd, J = 20.5, 8.3 Hz, 1H), 7.12 (tt, J = 8.9, 2.1 Hz, 1H). MS (ESI): 306.99 (C14H10ClF2N4,
[M + H]+). Anal. Calcd. For C14H9ClF2N4: C, 54.83; H, 2.96; N, 18.27. Found: C, 54.61; H, 3.01;
N, 17.98.
3.3. Crystallographic Study
X-ray single-crystal diffraction data for compound 6b were measured on a Bruker SMART APEX
CCD area detector diffractometer using MoKα radiation (λ = 0.71073 Å) by the π and ω scan mode.
The program SAINT was used for integration of the diffraction profiles. The structure was solved
by direct methods using the SHELXS program of the SHELXTL package and refined by full-matrix
least-squares methods with SHELXL. All non-hydrogen atoms of compound 6b were refined with
anisotropic thermal parameters. All hydrogen atoms were placed in geometrically idealized positions
and constrained to ride on their parent atoms.
3.4. Biological Assay
The test fungi R. solani and M. oryzae were provided by the Laboratory of Plant Disease Control at
Nanjing Agricultural University. After removing strains from the storage tube, the strains were
◦
incubated in potato dextrose agar (PDA) at 25 C for a week to develop new mycelia for the
antifungal assay.
3.4.1. Antifungal Activity Assays in Vitro
The in vitro antifungal activities of the target compounds against R. solani and M. oryzae were
evaluated using the mycelium growth rate method [19,20]. The compounds were dissolved in
DMSO and then mixed with 30 mL sterile molten PDA to obtain final concentrations of 0.625, 1.25,
◦
2.5, 5, 10 and 20
µg/mL (containing 4
‰
DMSO) at 50 C, then PDA with different compounds
was poured into 90 mm petri dishes (15 mL/dish), on which 0.5 cm mycelial disks of the two
fungi were planted in the center. Each measurement consisted of at least three replicates. After a
certain incubation period (1.5◦days for R. solani, 5 days for M. oryzae, according to their different
mycelial growth rates) at 25 C in the dark, mycelia growth diameters were measured. DMSO
was served as negative control. The inhibition rate was calculated according to the formula:
Inhibition rate = (A − B)/(A − 0.5 cm) × 100%. Where A is the diameter (cm) of the negative control
and B is the mycelial diameter (cm) in petri dishes with compounds. Carbendazim, validamycin A and
isoprothiolane were co-assayed as the positive control. Those compounds were treated to calculate
their median effective concentration (EC50) values.
3.4.2. Inhibition of 6f on the Sclerotia Germination of R. solani
Sclerotia of R. solani were cultured and dried for germination test [21]. A series of concentrations
of tested samples were prepared, then two layers of filter paper were soaked with the solution.
These filter paper layers were put in culture dishes and each concentration consisted of at least
three replicates. A replicate included 50 sclerotia. Validamycin A and distilled water was served
◦
as the positive control and blank control, respectively. These dishes were incubated at 25 C for
4 days. Then the inhibition rate of the compounds on the germination of sclerotia was calculated

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as: Inhibition rate = (average germination number of blank control
treatment)/average germination number of blank control × 100%.
−
average germination number of
3.4.3. Protective Activity of 6f against RSB In Vivo
For evaluating the antifungal activity of 6f against RSB in vivo, rice cultivar (Shanyou 63) was
sown and grown in plastic pots in the greenhouse [22]. The compound 6f at the concentration
of 200
µg/mL and 100
µg/mL was sprayed on the cultivar, respectively, and inoculated with
R. solani 24 h later. Validamycin A at the concentration of 200
µ
g/mL and 100 g/mL was used
µ
as the positive control. All the treatments were replicated for 20 plants and incubated in the
greenhouse with an average midday relative humidity of 85%. A visual disease assessment was
made 7 days after inoculating with R. solani by measuring the lesion length. The protective efficacy
was calculated as: Protection efficacy = ((average lesion length of control
treated group)/average lesion length of control) × 100%.
−
average lesion length of
3.4.4. Inhibition of 6f on the Conidium Germination of M. oryzae
Abundant spores of M. oryzae were collected and suspended to a concentration of 5
per milliliter in sterile water for conidium germination test. A series of concentrations of tested samples
6f and control) were mixed with conidial suspension, respectively. Aliquots of 10 L of prepared
×
104 spores
(
µ
conidial suspension wer◦e placed on separate glass slides in triplicate, which were incubated in a
moisture chamber at 25 C for 24 h. Each slide was then observed under the microscope, and the
appressorium formation percentage was examined [23].
3.4.5. Protective Activity of 6f against RB In Vivo
Compound 6f was further measured for its antifungal activity in vivo against RB on rice
(Shanyou 63). Different concentrations (50 µg/mL and 100 µg/mL) of 6f (4 mL) were sprayed on
two-week old seedlings of rice leaves. After 24 h, previously prepared spore suspension of M. oryzae
◦
was also sprayed with 0.2% (w/v) gelatin. Plants were kept at 25 C with 90% humidity and in a
12/12 h light/dark cycle. The positive control is carbendazim of 25 and 50 µg/mL. Lesion formation
was observed daily and the protective efficacies were calculated after 7 days [24].
4. Conclusions
In conclusion, a series of benzimidazole derivatives had been synthesized and investigated for the
antifungal activities against R. solani and M. oryzae in vitro and in vivo. The result showed that many
of the compounds had significant antifungal activities. Compound 6f exhibited most potent antifungal
activity with an EC50 value of 1.20
(1.84 g/mL). The protective activity of 6f in vivo reached 66.5% at 100
A was 61.0%. At the same time, 6f displayed moderate activity against M. oryzae with EC50 value
of 1.85 g/mL, and inhibited the conidium germination at 1.0 g/mL. The result indicated that
µg/mL against R. solani, which was better than carbendazim
µ
µg/mL, whereas validamycin
µ
µ
the compound 6f with a benzimidazole phenylhydrazone scaffold could be considered as a leading
structure for the development of novel fungicides.
Acknowledgments: This work was co-supported by the National Natural Science Foundation of China (31572043),
the Jiangsu Agriculture Science and Technology Innovation Fund (CX(15)1054), and the Natural Science
Foundation of Jiangsu Province (BK20151428).
Author Contributions: Y.-H.Y. designed the research; X.W. and Y.-F.C. performed the synthesis, separation
and structure elucidation. W.Y. and L.-L.C. contributed to evaluation of antifungal activities. X.W. and Y.-H.Y.
coordinated writing the paper to which all co-authors contributed.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016, 21, 1574
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References
1.
2.
3.
Woolley, D.W. Some biological effects producted by benzimidazole and their reversal by purines.
J. Biol. Chem. 1944, 152, 225–232.
Madkour, H.; Farag, A.A.; Ramses, S.S.; Ibrahiem, N. Synthesis and fungicidal activity of new imidazoles
from 2-(chloromethyl)-1H-benzimidazole. Phosphorus Sulfur 2006, 181, 255–265. [CrossRef]
Singla, P.; Luxami, V.; Paul, K. Synthesis and in vitro evaluation of novel triazine analogues as anticancer
agents and their interaction studies with bovine serum albumin. Eur. J. Med. Chem. 2016, 117, 59–69.
[CrossRef] [PubMed]
4.
5.
Song, D.; Ma, S.T. Recent development of benzimidazole-containing antibacterial agents. ChemMedChem
2016, 11, 646–659. [CrossRef] [PubMed]
Kus, C.; Ayhan-Kilcigil, G.; Ozbey, S.; Kaynak, F.B.; Kaya, M.; Coban, T.; Can-Eke, B. Synthesis
and antioxidant properties of novel N-methyl-1,3,4-thiadiazol-2-amine and 4-methyl-2H-1,2,4-triazole-3
(4H)-thione derivatives of benzimidazole class. Bioorg. Med. Chem. 2008, 16, 4294–4303. [CrossRef] [PubMed]
Fonseca, T.; Gigantea, B.; Gilchrist, L. A short synthesis of phenanthro[2,3-d]imidazoles from dehydroabietic
acid. Application of the methodology as a convenient route to benzimidazoles. Tetrahedron 2001, 57,
1793–1799. [CrossRef]
Perez-Villanueva, J.; Santos, R.; Hernandez-Campos, A.; Giulianotti, M.A.; Castillo, R.; Medina-Franco, J.L.
Structure-activity relationships of benzimidazole derivatives as antiparasitic agents: Dual activity-difference
(DAD) maps. MedChemComm 2011, 2, 44–49. [CrossRef]
6.
7.
8.
9.
Bi, C.W.; Qiu, J.B.; Zhou, M.G.; Chen, C.J.; Wang, J.X. Effects of carbendazim on conidial germination and
mitosis in germlings of Fusarium graminearum and Botrytis cinerea. Int. J. Pset Manag. 2009, 55, 157–163.
Tanejk, M.; Grover, R.K. Efficacy of benzimidazole and related fungicides against Rhizoctonia solain and
R. bataticola. Ann. Appl. Biol. 1982, 100, 425–432. [CrossRef]
10. Liu, L.X.; Tom, H. Estimating benzimidazole residues in thatch and turfgrass by bioassay. Pestic. Sci. 1996
46, 139–143. [CrossRef]
,
11. Zhu, L.F.; Hou, Z.; Zhou, K.; Tong, Z.B.; Kuang, Q.; Geng, H.L.; Zhou, L. Synthesis, bioactivity
and structure-activity relationships of new 2-aryl-8-OR-3,4-dihydroisoquinolin-2-iums salts as potential
antifungal agents. Bioorg. Med. Chem. Lett. 2016, 26, 2413–2417. [CrossRef] [PubMed]
12. Zhang, M.; Dai, Z.C.; Qian, S.; Liu, J.; Xiao, Y.; Lu, A.; Zhu, H.L.; Wang, J.X.; Ye, Y.H. Design, synthesis,
antifungal, and antioxidant activities of (E)-6-((2-phenylhydrazono)methyl)quinoxaline derivatives. J. Agric.
Food Chem. 2014, 62, 9637–9643. [CrossRef] [PubMed]
13. Dai, Z.C.; Chen, Y.F.; Zhang, M.; Li, S.K.; Yang, T.T.; Shen, L.; Wang, J.X.; Qian, S.; Zhu, H.L.; Ye, Y.H.
Synthesis and antifungal activity of 1,2,3-triazole phenylhydrazone derivatives. Org. Biomol. Chem. 2015, 13,
477–486. [CrossRef] [PubMed]
14. Ellis, G.P.; Wathey, W.B. Benzimidazoles. Part 2. A new synthesis of [1,2,4] triazino [4,5-a] benzimidazol
-1-ones. J. Chem. Res. 1984, 12, 384–385.
15. Le, B.; Marie, T.; Wahl, H. Monoazo dye derivatives of 1,2-dimethylbenzimidazole. C. R. 1957, 245, 2058–2060.
16. Eom, Y.W.; Oh, S.; Woo, H.B.; Ham, J.; Ahn, C.M.; Lee, S. Cytotoxicity of substituted benzimidazolyl curcumin
mimics against multi-drug resistance cancer cell. Bull. Korean Chem. Soc. 2013, 34, 1272–1274. [CrossRef]
17. Hagiwara, H.; Okada, S. A polymorphism-dependent T1/2 shift of 100 K in a hysteretic spin-crossover
complex related to differences in intermolecular weak CH· · · X hydrogen bonds (X = S vs. S and N).
Chem. Commun. 2016, 52, 815–818. [CrossRef] [PubMed]
18. Ayati, A.; Falahati, M.; Irannejad, H.; Emami, S. Synthesis, in vitro antifungal evaluation and in silico study
of 3-azolyl-4-chromanone phenylhydrazones. Daru J. Pharm. Sci. 2012, 20, 46. [CrossRef] [PubMed]
19. Ye, Y.H.; Ma, L.; Dai, Z.C.; Xiao, Y.; Zhang, Y.Y.; Li, D.D.; Wang, J.X.; Zhu, H.L. Synthesis and antifungal
activity of Nicotinamide derivatives as succinate dehydrogenase inhibitors. J. Agric. Food Chem. 2014, 62,
4063–4071. [CrossRef] [PubMed]
20. Xiao, Y.; Li, H.X.; Li, C.; Wang, J.X.; Li, J.; Wang, M.H.; Ye, Y.H. Antifungal screening of endophytic fungi
from Ginkgo biloba for discovery of potent anti-phytopathogenic fungicides. FEMS Microbiol. Lett. 2013
339, 130–136. [CrossRef] [PubMed]
,
21. Tang, Z.H.; Wang, H.C.; Wang, J.X.; Chen, C.J.; Zhou, M.G. Fungicidal activity of propiconazole to
Rhizoctonia. solani and its control efficacy against rice sheath blight. Plant Prot. 2012, 38, 158–161.

Molecules 2016, 21, 1574
14 of 14
22. Peng, D.; Li, S.D.; Wang, J.X.; Chen, C.J.; Zhou, M.G. Integrated biological and chemical control of rice sheath
blight by Bacillus subtilis NJ-18 and jinggangmycin. Pest Manag. Sci. 2014, 70, 258–263. [CrossRef] [PubMed]
23. Cao, L.L.; Zhang, Y.Y.; Liu, Y.J.; Yang, T.T.; Zhang, J.L.; Zhang, Z.G.; Ye, Y.H. Anti-phytopathogenic activity
of sporothriolide, a metabolite from endophyte Nodulisporium. sp. A21 in Ginkgo biloba. Pestic. Biochem. Phys.
2016, 129, 7–13. [CrossRef] [PubMed]
24. Zhang, H.F.; Tang, W.; Liu, K.; Huang, Q.; Zhang, X.; Yan, X. Eight RGS and RGS-like proteins orchestrate
growth, differentiation, and pathogenicity of Magnaporthe oryzae. PLoS Pathog. 2011, 7, e100245012. [CrossRef]
[PubMed]
Sample Availability: Sample Availability: Samples of the compounds are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).