# Design, Synthesis, and Antifungal Activity of Novel Benzimidazole Derivatives Bearing Thioether and Carbamate Moieties

Authors: Lei Yang
Journal: Journal of Chemistry
 (2022)

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## Abstract

In this study, a series of benzimidazole derivatives bearing thioether and carbamate moieties have been synthesized and evaluated for theirin vitro antifungal activity against Cytospora mandshurica, Thanatephorus cucumeris, Botrytis cinerea, Verticillium daliae, Phytophthora infestans, and Gibberella zeae by the mycelium growth rate method. The result of bioassay demonstrated that most of the compounds had certain antifungal activity. Especially, compound E11 revealed better antifungal activity against Verticillium daliae and Phytophthora infestans at 50 μg/mL, with the inhibition rates of 70% and 75%, respectively, than those of albendazole (38% and 61%, respectively). To the best of our knowledge, it is the first study on the synthesis and antifungal activity of benzimidazole derivatives containing thioether and carbamate moieties.


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## Body

## 1. Introduction

In several decades, the fungal diseases are caused by fungal pathogens, leading to severe losses to agriculture and horticulture crop production worldwide and constitute an emerging threat to the global food security [1–5]. Among the drugs to treat fungal infection, benzimidazole derivatives as potent and safe antifungal agents have attracted more and more attention for a long time [6–8]. Carbendazol, Benomyl, Thiabendazole, etc., have been widely used in antifungal therapy. However, the extensive use of those antifungal drugs has led to the resistance to drug treatment. Therefore, development of new benzimidazole drugs for antifungal treatment is pressing need.Benzimidazole derivatives have received much attention in synthesis and bioassay investigations as a result of their extremely important role in medicinal chemistry and agrochemicals [9–12]. Many investigations on the structures and bioactivities of benzimidazole compounds have been carried out. In 2006, Madkour et al. synthesized a series of 2-substituent benzimidazole derivatives, and these compounds demonstrated good inhibitory effect against Botrytis cinerea and Fusarium solani [13]. In 2010, Mobinikhaledi et al. investigated 2-substituted benzimidazole derivatives through the mycelium growth rate method test in vitro, and the result indicated that most compounds displayed good inhibitory effect antifungal bioactivities [14]. In 2012, Zhang et al. found a series of novel 1H-benzimidazol-1-yl acetates and 1H-benzimidazol-1-yl propionates containing 1H-1,2,4-triazole moiety. The antifungal activities of the target compounds against Botrytis cinerea and Sclerotinia sclerotiorum were evaluated by mycelial growth rate method. All the target compounds exhibited higher activities against Botrytis cinerea, with the EC50 values of 7.96–21.74 mg/mL, than that of carbendazim [15]. In 2013, Bai et al. found that 2-chloromethyl-1H-benzimidazole derivatives showed strong growth inhibition of Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani, and Fusarium solani [16]. In 2016, a series of novel N-alkylated benzimidazole derivatives were synthesized and bioassay results showed that the target compounds revealed excellent antifungal activity against Bacillus subtilis and Bacillus proteus [17].Herein, inspired by those description facts above and in connection of our work on benzimidazole derivatives, using albendazole as the lead compound, we design and synthesize a series of benzimidazole derivatives containing thioether and carbamate moieties and evaluated for their in vitro antifungal activity againstCytospora mandshurica (C. mandshurica), Thanatephorus cucumeris (T. cucumeris), Botrytis cinerea (B. cinerea), Verticillium daliae (V. daliae), Phytophthora infestans (P. infestans), and Gibberella zeae (G. zeae).


## 2. Materials and Methods

### 2.1. Synthesis

Melting points were determined using an XT-4 binocular microscope (Beijing Tech Instrument Co., China) and left uncorrected. The1H and 13C NMR spectra (solvent DMSO-d6) were measured with a JEOL-ECX 500 NMR spectrometer operating at room temperature with using TMS as an internal standard. The course of the reactions was monitored by thin-layer chromatography analysis on silica gel GF254. All solvents were freshly distilled or purified according to standard procedures. All reagents were purchased from Accela ChemBio Co., Ltd (Shanghai, China).


### 2.2. Preparation Procedure of Intermediate D

A mixture of 2-amino benzimidazole (0.01 mol), diethyl carbonate (10 mL), and EtONa (0.02 mol) was stirred in ethyl acetate (20 mL), and the reaction was reacted at 90°C and monitored by TLC. After complication of the reaction, the resulting mixture was filtrated and washed with ethyl acetate and dried under vacuum to get intermediate B.A mixture of intermediate B (0.01 mol) and sulfonic acid chloride (5 mL) was stirred in ethylene dichloride (20 mL), and the reaction was reacted for 0.5 h at 0°C and then reacted for 3 h at 60°C and monitored by TLC. After complication of the reaction, the resulting mixture was washed with alcohol, filtrated, and dried under vacuum to get intermediate C.A mixture of intermediate C (0.01 mol) and Fe (0.02 mol) was stirred in a mixture of HCl (5 mL), HCOOH (5 mL), and H2O (10 mL). The reactions were reacted for 10 h at 60°C. Then, the pH values of the reaction mixture were regulated to 4-5 by 5% NaOH, solid precipitation, and filtration and dried under vacuum to obtain intermediate D.


### 2.3. Preparation Procedure of the Target Compounds E1-E15

A mixture of intermediate D (0.01 mol), K2CO3 (0.02 mol), and RX (0.012 mol) was stirred in DMF (20 mL). The reactions were reacted for 5 h at room temperature. Then, the residue was filtered, dried under vacuum, and recrystallized from ethanol to get the target compounds E1-E15.


### 2.4. Bioactivity Assay

All the target compounds E1-E15 were evaluated for their in vitro antifungal activity againstC. mandshurica, T. cucumeris, B. cinerea, V. daliae, P. infestans, and G. zeae by the growth rate method in comparison with albendazole at 50 μg/mL [18]. Each treatment was repeated 3 times. Inhibition rate I (%) is calculated by the following formula, where C represents the diameter of control group (not treated with compound) and T represents the diameter of treatment group.
(1)I%=C−TC−0.4×100.


## 2.1. Synthesis

Melting points were determined using an XT-4 binocular microscope (Beijing Tech Instrument Co., China) and left uncorrected. The1H and 13C NMR spectra (solvent DMSO-d6) were measured with a JEOL-ECX 500 NMR spectrometer operating at room temperature with using TMS as an internal standard. The course of the reactions was monitored by thin-layer chromatography analysis on silica gel GF254. All solvents were freshly distilled or purified according to standard procedures. All reagents were purchased from Accela ChemBio Co., Ltd (Shanghai, China).


## 2.2. Preparation Procedure of Intermediate D

A mixture of 2-amino benzimidazole (0.01 mol), diethyl carbonate (10 mL), and EtONa (0.02 mol) was stirred in ethyl acetate (20 mL), and the reaction was reacted at 90°C and monitored by TLC. After complication of the reaction, the resulting mixture was filtrated and washed with ethyl acetate and dried under vacuum to get intermediate B.A mixture of intermediate B (0.01 mol) and sulfonic acid chloride (5 mL) was stirred in ethylene dichloride (20 mL), and the reaction was reacted for 0.5 h at 0°C and then reacted for 3 h at 60°C and monitored by TLC. After complication of the reaction, the resulting mixture was washed with alcohol, filtrated, and dried under vacuum to get intermediate C.A mixture of intermediate C (0.01 mol) and Fe (0.02 mol) was stirred in a mixture of HCl (5 mL), HCOOH (5 mL), and H2O (10 mL). The reactions were reacted for 10 h at 60°C. Then, the pH values of the reaction mixture were regulated to 4-5 by 5% NaOH, solid precipitation, and filtration and dried under vacuum to obtain intermediate D.


## 2.3. Preparation Procedure of the Target Compounds E1-E15

A mixture of intermediate D (0.01 mol), K2CO3 (0.02 mol), and RX (0.012 mol) was stirred in DMF (20 mL). The reactions were reacted for 5 h at room temperature. Then, the residue was filtered, dried under vacuum, and recrystallized from ethanol to get the target compounds E1-E15.


## 2.4. Bioactivity Assay

All the target compounds E1-E15 were evaluated for their in vitro antifungal activity againstC. mandshurica, T. cucumeris, B. cinerea, V. daliae, P. infestans, and G. zeae by the growth rate method in comparison with albendazole at 50 μg/mL [18]. Each treatment was repeated 3 times. Inhibition rate I (%) is calculated by the following formula, where C represents the diameter of control group (not treated with compound) and T represents the diameter of treatment group.
(1)I%=C−TC−0.4×100.


## 3. Results and Discussion

### 3.1. Chemistry

Using 2-amino benzimidazole as the starting material, as shown in Scheme1, a series of novel benzimidazole derivatives bearing thioether and carbamate moieties were synthesized with the yields of 16.7%-36.4%. Their structures were characterized by nuclear magnetic resonance (1H NMR and 13C NMR), and the physical characteristics 1H NMR, and 13C NMR data for all the target compounds E1-E15 can be found in the Supplemental Materials (available here).Scheme 1
Synthetic pathway for the target compounds.High product yield, good molar equivalent, suitable reaction temperature, and short reaction time encouraged us to apply previously reported methods under the optimized reaction conditions with the aim of expanding their applications in synthesis and medicine field. Thus, we used the synthesis of intermediates C and D as the model reactions with some modifications about the reaction solvent, reaction reduction and acid, reaction temperature and time, and molar equivalent of intermediate C : reduction. As shown in Table1, the highest yield (100%) of intermediate C was obtained when the reaction solvent, reaction temperature, and reaction time were CH2Cl2, 0°C, and 3 h, respectively. Meanwhile, Table 2 shows that the highest yield (27.4%) of intermediate D was obtained when the reduction, acid, and reaction temperature were Fe, HCl, and 60°C, respectively, at a reaction time of 8 h and a molar equivalent of intermediate C : reduction of 1 : 4.Table 1
Effect of different reaction condition on the yields of intermediate C.
EntrySolventTemp (°C)Time (h)Yield (%)1Ethyl acetate08102DMF0803CH3CN08<104Acetone08<105THF08<106CH2Cl203100Table 2
Effect of different reaction condition on the yields of intermediate D.
EntryReductionAcidTemp (°C)Time (h)Molar equivalent (C : reduction)Yield (%)1ZnHCl6081 : 4-2MgHCl6081 : 4-3FeHCl6081 : 4274FeHCl2081 : 4135FeH2SO46081 : 4186FeHNO36081 : 412


### 3.2. Antifungal Activity Test

The results of the preliminary bioassays, as listed in Table3, indicated that most of the compounds had certain in vitro antifungal activity against C. mandshurica, T. cucumeris, B. cinerea, V. daliae, P. infestans, and G. zeae at 50 μg/mL. Table 3 shows that compounds E11 and E15 showed better in vitro antifungal activity against V. daliae, with the inhibition rates of 70% and 69%, respectively, than albendazole (38%). Meanwhile, compound E11 showed superior in vitro antifungal activity against P. infestans (75%) to albendazole (61%). In addition, compound E15 showed good in vitro antifungal activity against C. mandshurica (44%), which were superior to albendazole (32%).Table 3
The in vitro antifungal activity of the target compounds againstC. mandshurica, T. cucumeris, B. cinerea, V. daliae, P. infestans, and G. zeae at 50 μg/mL.
CompoundsInhibition rate (%)T. cucumerisV. daliaeP. infestansG. zeaeC. mandshuricaE117±1.539±0.73±2.225±1.426±1.9E219±3.219±2.50.004±1.634±1.4E323±1.001±0.010±3.018±1.0E442±1.012±0.927±0.328±2.99±3.1E57±0.75±0.98±1.812±5.529±3.1E66±0.88±1.424±3.09±0.623±0.9E717±2.513±1.87±1.120±2.510±1.4E810±0.614±4.014±3.120±2.816±2.3E915±2.69±0.92±0.04±0.71±1.3E102±1.519±1.57±0.919±3.130±1.9E119±2.870±0.175±0.812±0.611±2.7E123±8.936±1.222±1.408±2.3E1314±2.843±2.50.003±1.72±2.8E1417±1.103±1000E1541±2.969±3.322±3.415±0.744±1.8Albendazole61±1.338±0.161±2.432±1.432±1.4


## 3.1. Chemistry

Using 2-amino benzimidazole as the starting material, as shown in Scheme1, a series of novel benzimidazole derivatives bearing thioether and carbamate moieties were synthesized with the yields of 16.7%-36.4%. Their structures were characterized by nuclear magnetic resonance (1H NMR and 13C NMR), and the physical characteristics 1H NMR, and 13C NMR data for all the target compounds E1-E15 can be found in the Supplemental Materials (available here).Scheme 1
Synthetic pathway for the target compounds.High product yield, good molar equivalent, suitable reaction temperature, and short reaction time encouraged us to apply previously reported methods under the optimized reaction conditions with the aim of expanding their applications in synthesis and medicine field. Thus, we used the synthesis of intermediates C and D as the model reactions with some modifications about the reaction solvent, reaction reduction and acid, reaction temperature and time, and molar equivalent of intermediate C : reduction. As shown in Table1, the highest yield (100%) of intermediate C was obtained when the reaction solvent, reaction temperature, and reaction time were CH2Cl2, 0°C, and 3 h, respectively. Meanwhile, Table 2 shows that the highest yield (27.4%) of intermediate D was obtained when the reduction, acid, and reaction temperature were Fe, HCl, and 60°C, respectively, at a reaction time of 8 h and a molar equivalent of intermediate C : reduction of 1 : 4.Table 1
Effect of different reaction condition on the yields of intermediate C.
EntrySolventTemp (°C)Time (h)Yield (%)1Ethyl acetate08102DMF0803CH3CN08<104Acetone08<105THF08<106CH2Cl203100Table 2
Effect of different reaction condition on the yields of intermediate D.
EntryReductionAcidTemp (°C)Time (h)Molar equivalent (C : reduction)Yield (%)1ZnHCl6081 : 4-2MgHCl6081 : 4-3FeHCl6081 : 4274FeHCl2081 : 4135FeH2SO46081 : 4186FeHNO36081 : 412


## 3.2. Antifungal Activity Test

The results of the preliminary bioassays, as listed in Table3, indicated that most of the compounds had certain in vitro antifungal activity against C. mandshurica, T. cucumeris, B. cinerea, V. daliae, P. infestans, and G. zeae at 50 μg/mL. Table 3 shows that compounds E11 and E15 showed better in vitro antifungal activity against V. daliae, with the inhibition rates of 70% and 69%, respectively, than albendazole (38%). Meanwhile, compound E11 showed superior in vitro antifungal activity against P. infestans (75%) to albendazole (61%). In addition, compound E15 showed good in vitro antifungal activity against C. mandshurica (44%), which were superior to albendazole (32%).Table 3
The in vitro antifungal activity of the target compounds againstC. mandshurica, T. cucumeris, B. cinerea, V. daliae, P. infestans, and G. zeae at 50 μg/mL.
CompoundsInhibition rate (%)T. cucumerisV. daliaeP. infestansG. zeaeC. mandshuricaE117±1.539±0.73±2.225±1.426±1.9E219±3.219±2.50.004±1.634±1.4E323±1.001±0.010±3.018±1.0E442±1.012±0.927±0.328±2.99±3.1E57±0.75±0.98±1.812±5.529±3.1E66±0.88±1.424±3.09±0.623±0.9E717±2.513±1.87±1.120±2.510±1.4E810±0.614±4.014±3.120±2.816±2.3E915±2.69±0.92±0.04±0.71±1.3E102±1.519±1.57±0.919±3.130±1.9E119±2.870±0.175±0.812±0.611±2.7E123±8.936±1.222±1.408±2.3E1314±2.843±2.50.003±1.72±2.8E1417±1.103±1000E1541±2.969±3.322±3.415±0.744±1.8Albendazole61±1.338±0.161±2.432±1.432±1.4


## 4. Conclusion

In this study, 15 new benzimidazole derivatives bearing thioether and carbamate moieties were synthesized. Bioassay results showed some of the target compounds revealed superior antifungal activity to that of albendazole. It was demonstrated that the benzimidazole derivatives bearing thioether and carbamate moieties can be used to develop potential agrochemicals.



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*Source: 8646557-2022-05-04.xml*