Synthesis and biological activity of novel benzimidazole derivatives as potential antifungal agents

Abstract

In the present study, a series of novel benzimidazole derivatives containing chrysanthemum acid moieties was designed and synthesized. Preliminary investigation of biological activity indicated that all of the compounds exhibited lower activity than that of beta-cypermethrin against Plutella xylostella and Lipaphis erysimi; meanwhile, they showed good inhibitory activity against Botrytis cinerea and Sclerotinia sclerotiorum in vitro. The fungicidal activity of compound 8a against B. cinerea was approximately equal to that of thiabendazole and was twice as active against S. sclerotiorum as was thiabendazole. In addition, compound 9e displayed the most potent inhibitory activity against both fungi and was almost twice as potent as thiabendazole.

Introduction

In recent years, benzimidazole compounds have emerged as a hot research topic due to their varied biological activities. Indeed, benzimidazole is a privileged scaffold in medicinal chemistry and agrochemistry. Several commercial fungicides containing the benzimidazole scaffold have been launched or announced (Fig. 1A). On the other hand, because of their high efficiency, low toxicity, and low residue,^1,2) pyrethroid pesticides are widely used in agriculture to protect crops and in households to control insect pests. Structure-activity relationship (SAR) studies have suggested that the outstanding activity of pyrethroid compounds may be attributed primarily to the chrysanthemum acid moiety^3,4) (Fig. 1B). To mitigate crop damage caused by pests and diseases, we must utilize various crop agrochemicals, such as insecticides, herbicides, and fungicides, to control harmful insects, weed species, and plant diseases that afflict crops. However, the heavy usage of crop-protection chemicals has resulted in a number of negative impacts, such as pest resistance to pesticides,^5–8) pest resurgence,⁹⁾ and environmental pollution.¹⁰⁾ Benzimidazoles (especially carbendazim) have been and are still widely used to control varieties of fungal diseases, but their resistance problems may result in failure to control disease.^8,11) Studies have also proved that not only carbendazim but also thiabendazole shows serious resistance to some diseases.⁷⁾ Therefore, it is necessary to discover and develop novel agrochemicals that could overcome or minimize the side effects of agrochemicals that are currently used. In the present work, our strategy is to design a new type of pesticide via a combination of the benzimidazole scaffold and the chrysanthemum acid moiety in a single structure to control both harmful insects and fungi. In addition, this type of hybrid pesticide might also possess better activity against pesticide-resistant insects and fungi. Based on this strategy, we have designed and synthesized a number of new compounds that combine the benzimidazole scaffold and the chrysanthemum acid moiety and have systematically investigated their insecticidal and fungicidal activities.

Fig. 1. Benzimidazoles (A) and pyrethroids (B) pesticides.

Materials and Methods

1. General

¹H-NMR spectra were recorded on a Bruker DPX300 NMR instrument with tetramethylsilane as an internal standard and CDCl₃ or DMSO-d₆ as a solvent. Mass spectroscopy analyses were performed with an Agilent 6200 Series TOF or 6500 Series Q-TOF LC/MS mass spectrometer. Melting points were obtained with RY-1G melting point apparatus and were uncorrected. All commercial reagents were used as received. TLC analyses were performed using silica gel plates and visualized under ultraviolet light (254 nm).

2. Synthesis

2.1. General procedure for preparation of the key intermediates

2-(2-hydroxyphenyl)-1H-benzimidazole, 2-(3-hydroxyphenyl)-1H-benzimidazole, 2-(4-hydroxyphenyl)-1H-benzimidazole and 2-(4-hydroxy-3-methoxyphenyl)-1H-benzimidazole were prepared according to literatures.^12–14) Briefly, 0.05 mol of hydroxybenzaldehyde and 0.05 mol of sodium bisulfite were dissolved in 30 mL of ethanol and water, respectively, and then mixed and stirred for 20 min at room temperature. The resulting reaction mixture was filtered, and sodium hydroxy(hydroxyphenyl)methanesulfonate salt was obtained. The resulting salt (0.02 mol) and o-phenylenediamine (0.02 mol) in 60 mL dimethylformamide were refluxed for 3 hr in an oil bath. After the reaction mixture was poured onto crushed ice, intermediates 4 were separated by filtration and recrystallized from a methanol/water mixture (Fig. 2). The NMR data of intermediates are shown in the supplemental information.

Fig. 2. Synthesis of benzimidazole derivatives (7a–d, 8a–d, 9a–l).

2.2. General procedure for the synthesis of compounds 7–9

Benzimidazole derivatives 7a–d, 8a–d, and 9a–l were synthesized by the reaction of intermediates 4 and chrysanthemoyl chloride (6a–c). The general route for the synthesis of title compounds is depicted in Fig. 2. Briefly, one intermediate was dissolved in dichloromethane, and the solution was cooled to −5°C. The corresponding chrysanthemoyl chloride (6a–c) that was obtained by the reported methods¹⁵⁾ was dropped into the flask and stirred at this temperature for 1 hr; the reaction mixture was then warmed to room temperature and stirred overnight. The reaction was monitored by TLC analyses until the starting material was consumed. The resulting solution was washed with saturated brine and then dried with anhydrous MgSO₄. The solvent was removed by rotary evaporation under reduced pressure. Pure product was obtained by column chromatography. The title compounds are shown in Table 1. The NMR spectral data are shown in the supplemental information.

Table 1. The structure, melting point and yield of title compounds.

Comp.	The linking position	R1	R2	Appearance	Yield	Mp. (°C)
7a	2	H	H	white solid	63%	182–183
7b	3	H	H	yellow solid	68%	182–183
7c	4	H	H	white solid	72%	182–183
7d	4	OCH3	H	white solid	69%	240–241
8a	2	H	H	white solid	55%	169–170
8b	3	H	H	white solid	58%	171–172
8c	4	H	H	white solid	63%	169–170
8d	4	OCH3	H	white solid	67%	155–156
9a	2	H	H	gray solid	62%	208–209
9b	3	H	H	white solid	61%	209–210
9c	4	H	H	white solid	68%	208–209
9d	4	OCH3	H	white solid	63%	159–160
9e	2	H	CH3	brown solid	60%	186–187
9f	3	H	CH3	brown solid	67%	187–188
9g	4	H	CH3	brown solid	55%	187–188
9h	4	OCH3	CH3	brown solid	58%	197–198
9i	2	H	Cl	white solid	55%	170–171
9j	3	H	Cl	gray solid	53%	171–172
9k	4	H	Cl	gray solid	57%	170–171
9l	4	OCH3	Cl	white solid	47%	192–193

3. Biological assay

3.1. Insecticidal activity in vivo

All tested insects were provided by the Biological Activity Evaluation and Testing Laboratory at the Institute of Plant Protection of Agricultural Sciences. The insecticidal activity of compounds against Plutella xylostella and Lipaphis erysimi was tested using the leaf-dip method that was reported previously.¹⁶⁾ Chinese cabbage leaves containing a certain number of apterous adults were dipped in the diluted Triton X-100 (0.5 g/L) solution for 10 sec. The leaves were then placed on a plastic dish until they were dry. The dried leaves were transported to a conditioned room (25±1°C, 50±5% RH). An aqueous solution of Triton X-100 (0.5 g/L) was used as the control. Beta cypermethrin was also evaluated against P. xylostella and L. erysimi and utilized as a positive control. Each test had three repetitions. Mortality was assessed after 24 hr for L. erysimi and 48 hr for P. xylostella.

3.2. Fungicidal activities in vitro

Tested strains (Botrytis cinerea and Sclerotinia sclerotiorum) were provided by the Pesticide Toxicology and Applied Technology Laboratory, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. Evaluation of the fungicidal activities of compounds against B. cinerea and S. sclerotiorum was performed by the radial growth inhibition method.^17,18) Each compound was dissolved in dimethylsulfoxide and then mixed with sterile molten potato dextrose agar medium (PDA) to obtain a series of concentrations (from 6.25 mg/L to 100 mg/L). PDA with different compounds was poured into 90-mm Petri dishes, on which 5-mm mycelial disks of the two fungi were planted in the center. The disks were obtained from a pure PDA culture plate by punching at the edge of the actively growing mycelia colony. Three replicates were conducted for each treatment. Sterile water was added to the PDA medium as a control; meanwhile thiabendazole and azoxystrobin were tested as positive controls. After a certain incubation period at 25°C in the dark, the mycelial growth diameters were measured. The diameters of the colonies were measured after the colonies in the control treatments had covered two-thirds of the Petri dishes. The colony diameter was measured by the cross bracketing method, and the percentage of mycelial growth inhibition was then calculated.

Results and Discussion

1. Chemistry

Given that the designed compounds contain both the benzimidazole scaffold and the chrysanthemum acid moiety, benzimidazole derivatives containing phenolic hydroxyl groups were selected as key intermediates. The key intermediates were synthesized in accordance with the published procedures.¹³⁾ These benzimidazole intermediates reacted with chrysanthemoyl chloride to produce ester compounds. Specifically, where R² is –H, intermediates 4 reacted with chrysanthemoyl chloride to give esters 7a–d, 8a–d, or 9a–d, respectively. Similarly, where R² is –CH₃, the reaction of compounds 4 and chrysanthemoyl chloride afforded the desired products 9e–h; where R² is –Cl, the reaction of 4 and chrysanthemoyl chloride afforded the desired products 9i–l.

2. Biological activity

2.1. Insecticidal activity in vivo

The insecticidal activity of the synthesized benzimidazole compounds was tested against both Lipaphis erysimi and Plutella xylostella at a single concentration using beta-cypermethrin as a positive control. The mortality zone against the selected insects for the compounds is provided in Table 2. As shown in Table 2, the insecticidal activity of the title compounds at a concentration of 400 mg/L was lower than that of beta-cypermethrin at the concentration of 40 mg/L. Based on the insecticidal activity of compounds 7a–c, 9a–c, 9e–g, and 9i–k against L. erysimi, we might conclude that the linking position of the chrysanthemum acid moiety in the title compounds played a significant role in the insecticidal activity. The compound with the chrysanthemum acid moiety at the ortho position (of the benzene ring) exhibited optimal insecticidal activity. For example, the insecticidal activity of 9a was more potent than that of 9b–c, and that of 9e was more potent than that of 9f–g. In addition, the introduction of R² groups also affected the observed insecticidal activity; howerer, there was no regularity.

Table 2. Insecticidal activity against Plutella xylostella and Lipaphis erysimi in vivo.

Comp.	Mortality (%)		Comp.	Mortality (%)
	P. xylostella	L. erysimi		P. xylostella	L. erysimi
7a	6.67±2.71	57.89±4.96	9d	9.09±2.59	50.00±4.26
7b	23.08±4.99	39.39±5.33	9e	9.09±2.59	56.82±5.39
7c	23.08±3.99	32.26±4.87	9f	5.88±2.28	48.48±5.10
7d	13.33±3.63	55.56±4.75	9g	10.00±3.50	53.13±4.92
8a	20.00±5.07	41.67±5.97	9h	14.29±3.59	47.22±4.40
8b	20.00±6.81	42.55±4.95	9i	30.77±5.03	65.52±5.49
8c	7.69±3.16	45.45±4.32	9j	21.05±3.88	32.50±4.63
8d	18.18±2.84	41.82±6.11	9k	33.33±4.42	48.00±5.82
9a	7.69±3.04	69.23±4.87	9l	5.26±2.74	45.00±5.88
9b	7.14±2.68	39.39±5.35	Beta-cypermethrin	52.38±5.11	98.85±1.64
9c	23.08±3.48	48.72±4.32	Control	0	2.63±1.60

Values were means±SD of three replicates, only Triton X-100 was used in the control. The concentration of compounds was 400 mg/L, but the concentration of beta-cypermethrin was 40 mg/L.

2.2. Fungicidal activity in vitro

The concentration of an inhibitor that is required to give 50% of the maximum effect was taken as the EC₅₀ value. The EC₅₀ values of the title compounds against the Botrytis cinerea and Sclerotinia sclerotiorum strains were evaluated and are presented in Table 3; thiabendazole and azoxystrobin were used as positive controls. It was found that compounds 8a, 9a, 9e, and 9i showed high inhibitory activity against both tested strains. Compounds 7c, 8a, 9a, 9e, and 9i exhibited good inhibitory activity against B. cinerea, while compounds 7a, 8a, 8b, 9a, 9b, 9e, 9i, and 9k displayed good inhibitory activity against S. sclerotiorum. Based on the fungicidal activity of compounds 7a–c, 8a–c and 9a–c against S. sclerotiorum, we concluded that compounds with the chrysanthemum acid moiety at the ortho position displayed good fungicidal activity. Comparing the fungicidal activity of compounds 7c and 7d, 8c and 8d, 9c and 9d, and 9k and 9l against S. sclerotiorum, we found that the introduction of the methoxy group in the R¹ position significantly reduced fungicidal activity, suggesting that the bulky group is not tolerant in the R¹ position. In addition, the introduction of R², such as methyl and chloride, also significantly affected the observed fungicidal activity. Specifically, the introduction of the methyl group significantly enhanced fungicidal activity. For example, the fungicidal activity of 9e (EC₅₀=18.27 mg/L) was more potent than that of 9a (EC₅₀=25.76 mg/L). However, the introduction of chlorine atom decreased the fungicidal activity. As shown in Table 3, compound 9i (EC₅₀=36.12 mg/L) having a chlorine atom in the position of R², had inferior fungicidal potency as compared to compound 9a. Therefore, the fungicidal activities of these compounds closely depended on both the core structure and their substituents, such as chloride and methyl groups.

Table 3. Fungicidal activity against Botrytis cinerea and Sclerotinia sclerotiorum in vitro.

Comp.	B. cinerea EC50±SE (mg/L)	S. sclerotiorum EC50±SE (mg/L)	Comp.	B. cinerea EC50±SE (mg/L)	S. sclerotiorum EC50±SE (mg/L)
7a	>200	25.57±0.20	9d	>200	173.44±0.20
7b	43.89±0.20	50.75±0.21	9e	9.75±0.23	18.27±0.22
7c	34.05±0.19	39.95±0.22	9f	>200	>200
7d	>200	>200	9g	>200	>200
8a	15.38±0.21	14.18±0.21	9h	>200	158.39±0.19
8b	>200	24.64±0.21	9i	12.77±0.20	36.12±0.22
8c	>200	111.61±0.19	9j	>200	281.43±0.19
8d	>200	195.61±0.19	9k	>200	30.71±0.22
9a	17.38±0.20	25.76±0.27	9l	>200	>200
9b	>200	25.52±0.22	Thiabendazole	14.16±0.20	39.43±0.23
9c	>200	84.91±0.19	Azoxystrobin	39.22±0.26	30.37±0.28

Values are means±SE of three replicates.

Conclusion

In conclusion, a series of novel benzimidazole derivatives designed on the basis of the core structure of existing pyrethroids was synthesized. The title compounds 7a–d, 8a–d, and 9a–l were evaluated as insecticidal and fungicidal agents. Preliminary biological evaluation indicated that most of the title compounds showed potent fungicidal activities against Botrytis cinerea and Sclerotinia sclerotiorum. Specifically, compounds 8a, 9a, and 9e displayed significant fungicidal activities against B. cinerea and S. sclerotiorum. Among them, compound 9e displayed more potent fungicidal activity against both fungi than did thiabendazole. The results of this antifungal evaluation indicated that these compounds are a promising type of potential antifungal agents against B. cinerea and S. sclerotiorum for controlling plant diseases. Further evaluation of their fungicidal properties, particularly in field studies designed to examine their biological efficacy, crop safety, and toxicity, is required before they can be adopted for widespread use.

The online version of this article contains supplementary material (characterization data for the new compounds), which is available at http://www.jstage.jst.go.jp/browse/jpestics/.