Synthesis and antifungal activity of novel α-alkoxyimino-(1H-benzoimidazol-2-yl)acetonitriles containing piperazine moiety

Article abstract of DOI:10.1007/s11164-014-1853-9

A series of α-alkoxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-benzimidazol-2-yl] acetonitriles were synthesized from o-phenylenediamine, ethyl cyanoacetate, chloroacetyl piperazine, and haloalkane/benzyl chloride by multi-step reactions. The structures of the target compounds were elucidated by IR, ¹H NMR, MS, and elemental analysis. All the target compounds were tested for in vitro antifungal activities against Sclerotinia sclerotiorum and Botrytis cinerea by the mycelium growth rate method, and the results indicated that some compounds displayed higher antifungal activity as compared to carbendazim.

Full text of DOI:10.1007/s11164-014-1853-9

Res Chem Intermed
DOI 10.1007/s11164-014-1853-9
Synthesis and antifungal activity of novel
a-alkoxyimino-(1H-benzoimidazol-2-yl)
acetonitriles containing piperazine moiety
•
Qingdong Jin Fubo Li Ying Li Fei Jin Lin Jiang
•
•
•
Received: 23 August 2014 / Accepted: 17 October 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract A series of a-alkoxyimino-[1-(4-methylpiperazino-1-yl)carbonylmeth-
yl-1H-benzimidazol-2-yl] acetonitriles were synthesized from o-phenylenediamine,
ethyl cyanoacetate, chloroacetyl piperazine, and haloalkane/benzyl chloride by
multi-step reactions. The structures of the target compounds were elucidated by IR,
1H NMR, MS, and elemental analysis. All the target compounds were tested for
in vitro antifungal activities against Sclerotinia sclerotiorum and Botrytis cinerea by
the mycelium growth rate method, and the results indicated that some compounds
displayed higher antifungal activity as compared to carbendazim.
Keywords Benzimidazole ꢀ Alkoxyimine ꢀ Piperazine ꢀ Synthesis ꢀ Antifungal
activity
Introduction
Benzimidazole is an important heterocyclic ring, and its derivatives have a wide
spectrum of biological activity, such as antifungal [1], antibacterial [2], antiviral [3],
antiparasite [4], anti-inflammatory [5], and antihypertensive [6] activities, so
benzimidazole derivatives are extensively used in both pesticide and medicine. In
the field of pesticides, many benzimidazole fungicides including carbendazim and
benomyl, are usually used to control Botrytis cinerea, Blumeria graminis,
Sclerotinia sclerotiorum, and Cladospor iumcucumerinum [7]. However, their
Q. Jin ꢀ F. Li
College of Plant Protection, Shandong Agricultural University, Tai’an 271018, China
Y. Li ꢀ F. Jin ꢀ L. Jiang (&)
College of Chemistry and Material Science, Shandong Agricultural University, Tai’an 271018,
China
e-mail: jiangl@sdau.edu.cn
123

Q. Jin et al.
OH
N
NH2
NH2
N
N
NaNO2 , HOAc
20 o
o-C6H4Cl2
NC
COOEt
170 o
C
C
N
H
N
H
CN
CN
1
2
O
OH
R
N
N
N
N
N
N
O
N
N
CH3
CN
CN
ClH2C
R
X
O
O
K2CO3, acetone, r.t.
K2CO3, acetone, r.t.
N
N
N
N
CH3
CH3
3
4a ~ i
4a: R = CH3, 4b: R = C2H5, 4c: R = n-C3H7, 4d: R = n-C4H9, 4e: R = C6H5CH2,
4f: R = 4-FC6H4CH2, 4g: R = 4-ClC6H4CH2, 4h: R = 4-IC6H4CH2, 4i: R = 4-NCC6H4CH2
Scheme 1 Synthetic route for the target compounds 4a–i
antifungal activities are decreased because of the serious resistance of plant
pathogenic fungi [8]. Thus, it is necessary to develop some novel benzimidazole
fungicides that can overcome the resistance.
Meanwhile, alkoxyimines, also known as oxime-ethers, have attracted much
attention because of their significant biological activity such as fungicidal [9],
insecticidal [10], and herbicidal [11] activities. For example, pyrifenox and
dimoxystrobin are two efficient alkoxyimine fungicides [12, 13]. In addition, some
compounds containing piperazine moiety exhibit excellent bioactivity, and are often
used as fungicides and antibacterial drugs, e.g., triforine and ciprofloxacin [14]. In
view of these observations, and in continuation of our interest in the synthesis and
bioactivity of benzimidazole and alkoxyimine [15, 16], we have attached
alkoxyimine and piperazine moieties to a benzimidazole ring and synthesized nine
novel a-alkoxyimino-(1H-benzoimidazol-2-yl)acetonitriles containing piperazine
moiety (4a–i), and evaluated their antifungal activity against two plant pathogenic
fungi. The synthetic pathway is shown in Scheme 1.
Experimental
Chemicals and instruments
Melting points were measured on a Tech X-5 microscopic melting point apparatus
and are uncorrected. IR spectra were recorded on a Shimadzu IR-440 infrared
1
spectrophotometer using KBr discs. HNMR spectra were determined on a Bruker
AM-400 spectrometer with CDCl3 as solvent and TMS as internal standard,
Chemical shifts were reported in d (parts per million) values. ESI–MS spectra were
acquired on an Agilent 1200 spectrometer. The elemental analyses were measured
by an Elementary Vario EL III analyzer.
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Synthesis and antifungal activity
2-Cyanobenzimidazole (1) was synthesized from o-phenylenediamine and ethyl
cyanoacetate in accordance with a procedure reported elsewhere [17]. The key
intermediate a-hydroxyimino-(1H-benzimidazol-2-yl) acetonitrile (2) was prepared
in accordance with Ref. [18]. Other chemical reagents were purchased from J&K
Chemical Ltd. or Aladdin Reagent Co. Ltd., and are of analytical grade.
Synthesis of a-hydroxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-
benzimidazol-2-yl]acetonitrile 3
A mixture of a-hydroxyimino-(1H-benzimidazol-2-yl)acetonitrile (1.90 g, 10 mmol)
and K2CO3 (2.07 g, 15 mmol) in acetone (25 ml) was added 1-chloroacetyl-4-
methylpiperazine (1.80 g, 10 mmol), then the reaction mixture was stirred at room
temperature for 7 h. After completion of the reaction, the solid was removed by
filtration and the filtrate was evaporated under vacuum, giving a dark brown solid.
Further purification was accomplished by silica gel column chromatography, using
ethyl acetate/methanol (9:1 v/v) as eluent.
Light yellow solid, 1.98 g, yield 57 %, m.p. 186.7–187.3 °C. 1H NMR (DMSO-
d6, 400 Hz): d 2.27 (s, 3H, NCH3), 2.36–2.56 (m, 4H, Pip–H), 3.39–3.56 (m, 4H,
Pip–H), 5.24 (s, 2H, CH2–C(=O)), 7.28–7.74 (m, 4H, Ar–H); IR (KBr): m 3,222,
2,238, 1,660, 1,589, 1,468 cm-1; ESI–MS: m/z 349.5 (M ? Na)?; Anal. calcd for
C16H18N6O2: C 58.88, H 5.56, N 25.75; found: C 58.60, H 5.42, N 25.97.
General procedure for synthesis of a-alkoxyimino-[1-(4-methylpiperazino-1-
yl)carbonylmethyl-1H-benzimidazol-2-yl]acetonitriles 4a–i
To a mixture of intermediate 3 (0.98 g, 3 mmol) and K2CO3 (0.71 g, 5 mmol) in
acetone (15 ml) was added KI (0.17 g, 1 mmol) and stirred at room temperature for
0.5 h, followed by dropwise addition of haloalkane or benzyl chloride (3.6 mmol) in
acetone (10 ml) within 20 min. The reaction mixture was then stirred at ambient
temperature for 8–10 h until 3 disappeared (monitored by TLC analysis). The solid
in the reaction mixture was removed by filtration and the filtrate was evaporated
under vacuum, yielding a brown residue. The crude product was purified by silica
gel column chromatography, using ethyl acetate/methanol (10:1 v/v) as luent.
a-Methoxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-benzimidazol-2-
yl]acetonitrile 4a
White solid, 0.52 g, yield 51 %, m.p. 139.8–140.4 °C. 1H NMR (DMSO-d6,
400 Hz): d 2.32 (s, 3H, NCH3), 2.40–2.48 (m, 4H, Pip–H), 3.55–3.68 (m, 4H, Pip–
H), 4.00 (s, 3H, OCH3), 5.11 (s, 2H, CH2–C(= O)), 7.35–7.92 (m, 4H, Ar–H); IR
(KBr): m 2,235, 1,653, 1,586, 1,462, 1,074 cm-1; ESI–MS: m/z 363.5 (M ? Na)?;
Anal. calcd for C17H20N6O2: C 59.99, H 5.92, N 24.69; found: C 60.28, H 5.81, N
24.94.
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Q. Jin et al.
a-Ethoxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-benzimidazol-2-
yl]acetonitrile 4b
White solid, 0.65 g, yield 61 %, m.p. 149.8–150.6 °C. 1H NMR (DMSO-d6,
400 Hz): d 1.39 (t, J = 7.2 Hz, 3H, OCH2–CH3), 2.31 (s, 3H, NCH3), 2.39–2.48
(m, 4H, Pip–H), 3.56–3.67 (m, 4H, Pip–H), 4.48 (q, J = 7.2 Hz, 2H, OCH2–CH3),
5.10 (s, 2H, CH2–C(= O)), 7.33–7.92 (m, 4H, Ar–H); IR (KBr): m 2,232, 1,662,
1,604, 1,464, 1,068 cm-1; ESI–MS: m/z 377.6 (M ? Na)?; Anal. calcd for
C18H22N6O2: C 61.00, H 6.26, N 23.71; found: C 60.72, H 5.91, N 23.58.
a-Propoxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-benzimidazol-2-
yl]acetonitrile 4c
White solid, 0.63 g, yield 57 %, m.p. 158.6–159.2 °C. 1H NMR (DMSO-d6,
400 Hz): d 0.95 (t, J = 7.2 Hz, 3H, O(CH2)2–CH3), 1.78–1.84 (m, 2H, OCH2CH2–
CH3), 2.32 (s, 3H, NCH3), 2.39–2.48 (m, 4H, Pip–H), 3.56–3.67 (m, 4H, Pip–H),
4.38 (q, J = 7.2 Hz, 2H, OCH2–C2H5), 5.09 (s, 2H, CH2–C(= O)), 7.32–7.92 (m,
4H, Ar–H); IR (KBr): m 2,229, 1,663, 1,484, 1,457, 1,070 cm-1; ESI–MS: m/z 391.5
(M ? Na)?; Anal. calcd for C19H24N6O2: C 61.94, H 6.57, N 22.81; found: C
62.18, H 6.19, N 22.50.
a-Butoxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-benzimidazol-2-
yl]acetonitrile 4d
White solid, 0.55 g, yield 48 %, m.p. 144.2–144.9 °C. 1H NMR (DMSO-d6,
400 Hz): d 0.95 (t, J = 7.2 Hz, 3H, O(CH2)3–CH3), 1.35–1.41 (m, 2H, O(CH2)2-
CH2–CH3), 1.71–1.79 (m, 2H, OCH2CH2–C2H5), 2.32 (s, 3H, NCH3), 2.39–2.47
(m, 4H, Pip–H), 3.54–3.67 (m, 4H, Pip–H), 4.42 (q, J = 7.2 Hz, 2H, OCH2–C3H7),
5.09 (s, 2H, CH2–C(= O)), 7.32–7.92 (m, 4H, Ar–H); IR (KBr): m 2,232, 1,664,
1,556, 1,464, 1,068 cm-1; ESI–MS: m/z 405.7 (M ? Na)?; Anal. calcd for
C20H26N6O2: C 62.81, H 6.85, N 21.97; found: C 63.14, H 6.70, N 22.16.
a-Benzyloxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-benzimidazol-2-
yl]acetonitrile 4e
White solid, 0.85 g, yield 68 %, m.p. 127.0–127.7 °C. 1H NMR (DMSO-d6,
400 Hz): d 2.30 (s, 3H, NCH3), 2.34–2.42 (m, 4H, Pip–H), 3.42–3.60 (m, 4H, Pip–
H), 4.91 (s, 2H, CH2–C(= O)), 5.67(s, 2H, OCH2–Ph), 7.00–7.96 (m, 9H, Ar–H); IR
(KBr): m 2,229, 1,666, 1,469, 1,454, 1,076 cm-1; ESI–MS: m/z 439.6 (M ? Na)?;
Anal. calcd for C23H24N6O2: C 66.33, H 5.81, N 20.18; found: C 66.08, H 5.96, N
20.45.
123

Synthesis and antifungal activity
a-(4-Fluorobenzyloxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-
benzimidazol-2-yl]acetonitrile 4f
1
Light yellow solid, 0.83 g, yield 64 %, m.p. 128.9–131.0 °C. H NMR (DMSO-d6,
400 Hz): d 2.31 (s, 3H, NCH3), 2.34–2.42 (m, 4H, Pip–H), 3.47–3.62 (m, 4H, Pip–
H), 4.93 (s, 2H, CH2–C(= O)), 5.64 (s, 2H, OCH2–Ph), 6.95–7.97 (m, 8H, Ar–H);
IR (KBr): m 2,235, 1,656, 1,607, 1,509, 1,457, 1,072 cm-1; ESI–MS: m/z 457.6
(M ? Na)?; Anal. calcd for C23H23FN6O2: C 63.58, H 5.34, N 19.34; found: C
63.92, H 5.61, N 19.11.
a-(4-Chlorobenzyloxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-
benzimidazol-2-yl]acetonitrile 4g
1
Light yellow solid, 0.68 g, yield 59 %, m.p. 135.8–136.6 °C. H NMR (DMSO-d6,
400 Hz): d 2.32 (s, 3H, NCH3), 2.37–2.40 (m, 4H, Pip–H), 3.43–3.58 (m, 4H, Pip–
H), 4.91(s, 2H, CH2–C(=O)), 5.64 (s, 2H, OCH2–Ph), 6.95–7.96 (m, 8H, Ar–H); IR
(KBr): m 2,232, 1,655, 1,493, 1,454, 1,076 cm-1; ESI–MS: m/z 474.1 (M ? Na)?;
Anal. calcd for C23H23ClN6O2: C 61.26, H 5.14, N 18.64; found: C 60.95, H 5.36, N
18.80.
a-(4-Iodobenzyloxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-
benzimidazol-2-yl]acetonitrile 4h
1
Light yellow solid, 1.14 g, yield 70 %, m.p. 145.7–146.4 °C. H NMR (DMSO-d6,
400 Hz): d 2.31 (s, 3H, NCH3), 2.33–2.40 (m, 4H, Pip–H), 3.42–3.58 (m, 4H, Pip–
H), 4.91(s, 2H, CH2–C(=O)), 5.61 (s, 2H, OCH2–Ph), 6.76–7.97 (m, 8H, Ar–H); IR
(KBr): m 2,235, 1,663, 1,484, 1,458, 1,071 cm-1; ESI–MS: m/z 565.6 (M ? Na)?;
Anal. calcd for C23H23IN6O2: C 50.93, H 4.27, N 15.49; found: C 51.24, H 4.15, N
15.18.
a-(4-Cyanobenzyloxyimino-[1-(4-methylpiperazino-1-yl)carbonylmethyl-1H-
benzimidazol-2-yl]acetonitrile 4i
White solid, 0.86 g, yield 65 %, m.p. 149.4–151.1 °C. 1H NMR (DMSO-d6,
400 Hz): d 2.30 (s, 3H, NCH3), 2.32–2.40 (m, 4H, Pip–H), 3.40–3.59 (m, 4H, Pip–
H), 4.87(s, 2H, CH2–C(=O)), 5.72(s, 2H, OCH2–Ph), 7.12–7.98 (m, 8H, Ar–H); IR
(KBr): m 2,227, 1,660, 1,609, 1,458, 1,073 cm-1; ESI–MS: m/z 464.7 (M ? Na)?;
Anal. calcd for C24H23IN7O2: C 65.29, H 5.25, N 22.21; found: C 65.52, H 5.10, N
22.07.
Bioassays of antifungal activity
The in vitro fungicidal activities of the target compounds 4a–i against Sclerotia
sclerotium and Botrytis cinerea were evaluated using the mycelium growth rate
method [19]. The culture media, with known concentrations (10, 50, 100, and
200 lg/ml) of the test compounds, were obtained by mixing the 1 % water
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Q. Jin et al.
Table 1 Antifungal activities for the target compounds 4a–i
Compounds
R1
EC50 (lg/ml), 95 % CLa
S. sclerotium
B. cinerea
4a
CH3
37.33 (30.98–44.98)
80.69 (66.80–97.45)
77.84 (60.25–100.55)
79.38 (63.25–99.61)
32.06 (28.78–35.71)
14.74 (12.98–16.73)
29.02 (25.83–32.60)
35.47 (32.79–38.36)
39.12 (36.12–42.38)
41.02 (32.85–51.21)
32.65 (28.23–37.76)
58.24 (44.38–76.43)
39.31 (19.07–81.02)
66.50 (53.75–82.28)
39.12 (36.12–42.37)
7.14 (6.11–8.34)
4b
C2H5
4c
n-C3H7
4d
n-C4H9
4e
C6H5CH2
4-FC6H4CH2
4-ClC6H4CH2
4-IC6H4CH2
4-NCC6H4CH2
4f
4g
13.99 (12.52–15.63)
32.31 (29.82–35.01)
30.77 (26.56–35.64)
18.71 (13.98–25.04)
4h
4i
Carbendazim
a
EC50: Concentration that inhibited mycelium growth by 50 %; 95 % CL: 95 % confidence limits of
EC50
suspension (1 ml) of 4 with potato dextrose agar (PDA, 9 ml) at 50 °C. The medium
was then poured into a 9-cm Petri dish and cooled to room temperature, which was
inoculated with 5-mm mycelium of S. sclerotium or B. cinerea. The Petri dish was
then placed in a light incubator at 25 1 °C for 72 h. A commercial fungicide
carbendazim was used as the positive control, and sterile water was used as the
blank. Three replications were employed for each treatment. The inhibition rate was
expressed as the mean of values obtained in three independent experiments.
Effective concentration (EC50) was obtained using log-probit analysis (Table 1).
The inhibition rate was calculated according to the formula:
Inhibition rate ð%Þ ¼ ½ðD0ꢁD1Þ=D0ꢂ ꢃ 100 %
where D0 is the expansion diameter of mycelia in the blank test, and D1 is the
expansion diameter of mycelia in the presence of tested compounds.
Results and discussion
Spectra
1
In the H NMR spectra of target compounds 4a–i, the singlet signal of the CH3
protons connected to the piperazine ring was observed at d 2.30–2.32 ppm, and the
signal of the CH2 between benzimidazole ring and carbonyl at 4.87–5.11 ppm.
Moreover, because of the different substituents connected to the two nitrogen atoms
of piperazine, the proton chemical shifts of piperazine were of much difference. The
signals of the four protons near to methyl were observed at 2.34–2.48 ppm, while
the other four protons near to carbonyl at 3.40–3.68 ppm. In their IR spectra, the
C : N stretching vibrations appeared at 2,227–2,235 cm-1 and the carbonyl
123

Synthesis and antifungal activity
absorption at 1,653–1,666 cm-1. The bands around 1,070 cm-1 were attributed to
the N–O–C absorption of alkoxyimio moiety.
Antifungal activity
As shown in Table 1, the EC50 values of the target compounds ranged from 14.74 to
80.69 lg/ml against S. sclerotium. Except for 4b–d, the EC50 values of the other six
compounds were less than carbendazim (EC50 = 41.02 lg/ml), which means these
compounds displayed good antifungal activity. Especially, 4f and 4 g showed high
activity. On the other hand, the EC50 values of 4f and 4g against B. cinerea were
7.14 and 13.99 lg/ml, respectively, much less than carbendazim (EC50 = 18.71 lg/
ml), suggesting 4f and 4g exhibited excellent antifungal activities.
The structural analysis of the target compounds indicated that the compounds
bearing benzyl possessed higher antifungal activities in general as compared to the
analogues bearing alkyl group. The two compounds especially bearing 4-fluorob-
enzyl or 4-chlorobenzyl (4f and 4g) exhibited the best activity, indicating that the
introduction of the two substituents onto the target molecule can improve antifungal
activity.
Conclusions
In summary, we have synthesized nine a-alkoxyimino-[1-(4-methylpiperazino-1-
yl)carbonylmethyl-1H-benzimidazol-2-yl]acetonitriles by multi-step reactions in
yields of 48–70 %. All these compounds were tested for in vitro antifungal activities
against Sclerotia sclerotium and Botrytis cinerea by the mycelium growth rate
method. The results indicated that two compounds (4f and 4g) displayed high
antifungal activity against both S. sclerotium and B. cinerea, and their activities
were higher than carbendazim. This study is a useful reference in the search for
novel benzimidazole antifungal agents.
Acknowledgments This project was supported by Shandong Province Science and Technology
Development Plan (No. 2013GZX20109).
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