Design , Synthesis and Antifungal Activity of Novel Benzoylcarbamates Bearing a Pyridine Moiety

Many natural and synthetic pyridine derivatives have good biological activity, and are widely used in the fields of pesticides and medicines. On the other hand, carbamate fungicides possess some unique properties, such as high efficiency, strong selectivity, low toxicity, and environmental friendliness, and are often used to control many plant diseases. Therefore, discovering novel pyridine-based carbamates is of great significance. In this paper, we chose the excellent fungicides tolprocarb and picarbutrazox as lead compounds, integrating benzoyl, carbamate, and pyridinyl moieties into a molecule. Thus, we designed and synthesized a series of substituted benzoyl carbamates containing a pyridine ring, and evaluated the in vitro antifungal activity. The target compounds exhibited moderate to strong bioactivity against Botrytis cinerea, among which the compounds 4d, 4f, 4g, and 4h exhibited significant activity with EC50 values (the concentration resulting in a 50% inhibition) of 6.45–6.98 μg/mL, and their activities were near or superior to that of chlorothalonil. Additionally, 4h exhibited moderate activity against Sclerotinia sclerotiorum with an EC50 value of 10.85 μg/mL.


Introduction
Sclerotinia sclerotiorum and Botrytis cinerea are two common plant pathogenic fungi that greatly influence the yield and quality of fruits and vegetables.The effective method for controlling such fungi is to employ chemical fungicides such as carbendazim, thiophanate-methyl, boscalid, and tebuconazole [1,2].However, the unreasonable use of fungicides has resulted in serious resistances in recent years.Therefore, developing novel and effective antifungal agents is critical to the control of plant diseases.
Pyridine derivatives are important nitrogen-based heterocyclic compounds and are widely distributed in nature, e.g., nicotine, anabasine, pyridoxine, nicotinic acid, and nicotinamide.Many natural and synthetic pyridine derivatives show broad biological activity, including antifungal [3], anticancer [4], antivirus [5], anti-inflammatory [6], antitubercular [7], insecticidal [8], and herbicidal [9] activities.Additionally, pyridine is the bioisotere of benzene, but its electron density and hydrophobic constant are lower than those of benzene, thus pyridine derivatives often have high bioactivity and a notable systemic character [10].Therefore, pyridine derivatives often serve as medicines and pesticides.Recently, two novel pyridine-based carbamate fungicides, picarbutrazox and pyribencarb, were respectively developed by Nippon Soda Co., Ltd. and Kumiai Chemical Industry Co., Ltd.[11,12].In addition, diethofencarb, tolprocarb, and benthiavalicarb-isopropyl are also carbamate fungicides [13][14][15].These carbamate fungicides possess some unique properties such as strong selectivity, low toxicity, and environmental friendliness, and are commonly used to control many plant diseases, e.g., Sclerotinia sclerotiorum, Botrytis cinerea, Plasmopara viticola, and Phytophthora capsici.On the other hand, some benzoyl carbamates exhibit high antifungal activity.For example, Li et al. designed and synthesized a series of aryl carbamic acid-5-aryl-2-furanmethyl esters [16], and three of them displayed notable antifungal activity against four fungi; the morphologic results suggested that the compound can disturb the cell wall formation of fungi.
Inspired by the above findings, and based on our previous studies on the synthesis and bioactivity of pyridine-based compounds [17,18], we herein chose tolprocarb and picarbutrazox as lead compounds, integrated benzoyl, carbamate, and pyridinyl moieties into one molecule by the method of active substructure combination, and designed and synthesized ten substituted benzoyl carbamates containing a pyridine ring.In the target molecule, benzoyl is directly linked to the carbamate moiety, whereas pyridinyl is linked to the carbamate via two carbon atoms, which can enhance the flexibility of the molecule.Thus, the affinity between the target molecule and fungus might be increased [19].Moreover, we also evaluated their antifungal activity against Sclerotinia sclerotiorum and Botrytis cinerea.Figure 1 shows the design strategy of the target compounds.

Synthesis
Melting points were measured on an X-4 melting point apparatus (Beijing Tech Instrument Co., China). 1 H NMR and 13 C NMR spectra were obtained on a Bruker AM-400 spectrometer (Bruker Co., Switzerland) with tetramethylsilane as an internal standard ( 1 H NMR at 400 MHz, 13 C NMR at 100 MHz).IR spectra were performed on a Nicolet 380 infrared spectrophotometer (Nicolet Co., America) using potassium bromide pellets.Elemental analyses were carried out with an Elementary Vario EL III analyzer (Elementar Co., Germany).
To a solution of 3,3-Dimethyl-1-(pyridin-3-yl)butan-2-one (1.77 g, 10 mmol) in methanol (10 mL), sodium borohydride (0.74 g, 20 mmol) was added in portions under stirring conditions at 0 • C. The resulting suspension was stirred constantly for 2 h at the same temperature.Then, water (5 mL) was added for extraction with ethyl acetate (3 × 10 mL).The combined organic phase was dried over sodium sulphate and the solvent was removed to yield a white solid, which was purified by silica gel chromatography with ethyl acetate/petroleum ether (V:V = 1:3) as eluent to generate intermediate 2 (1.52A solution of appropriate substituted benzamide (10 mmol) in anhydrous1,2-Dichoroethane (6 mL) was cooled to 0 • C, and oxalyl dichloride (2.05 g, 16 mmol) was slowly added under stirring.The reaction proceeded under stirring at room temperature for 1 h, followed by refluxing for 3-4 h.The solvent was distilled off under vacuum, to yield corresponding benzoyl isocyanate [21,22].Crude isocyanates were directly used for the next reaction.

General Procedure for the Synthesis of Substituted Benzoylcarbamates (4a-4j)
To a mixture of benzoyl isocyanate freshly prepared as above (9 mmol) in dry tetrahydrofuran (12 mL), a solution of 3,3-Dimethyl-1-(pyridin-3-yl)butan-2-ol (1.62 g, 9 mmol) in dry tetrahydrofuran (20 mL) was slowly added under stirring.After stirring at room temperature for 3 h, the solvent was evaporated to yield a crude product.Further purification was performed by silica gel chromatography with ethyl acetate/petroleum ether (V:V = 1:2) as eluent, and target compounds were obtained as white solids.

Biological Assay
The in vitro fungicidal activity of compounds 4a-4j against Sclerotinia sclerotiorum and Botrytis cinerea were measured by the mycelium growth rate method [23].On the basis of pre-test results, PDA (potato glucose agar) cultures containing five concentrations (5, 10, 25, 50, and 100 µg/mL) of tested compound were prepared, and then inoculated with 5 mm plugs of the tested fungus.These cultures were incubated at 25 ± 1 • C, and the incubation times were 48 h for S. sclerotiorum, and 72 h for B. cinerea.Moreover, three fungicides, diethofencarb, carbendazim, and chlorothalonil, were provided as the positive controls, and sterile water was treated as the blank control.The bioassay was performed three times.The inhibition rate was calculated by the following formula.Lastly, the EC 50 value (the concentration resulting in a 50% inhibition) was obtained by DPS Software (Table 1).In which D 0 is the expansion diameter of the mycelia in the blank test, and D 1 is that in the presence of the tested compound.
Target compounds were identified by 1 H NMR, 13 C NMR, IR, and elemental analysis.In 1 H NMR spectra, the singlet of methyl protons of t-butyl moiety was observed at δ 1.02-1.07ppm.The methine proton can form an ABX system with its two neighboring methylene protons, thus its signal was split into a quartet around δ 5.0 ppm, while the signals of methylene protons were split into two groups of multiplet near δ 2.80 and 2.98 ppm, respectively.A broad peak in the range of δ 7.90 to 9.40 ppm was ascribed to the presence of the NH proton.The signals of protons of pyridinyl and benzyl rings appeared in the range of δ 6.86-8.45ppm.In 13 C NMR spectra, the signal for the carbonyl carbon of benzoyl was observed at δ 164.38 to 166.32 ppm, whereas that of carbamate occurred at around δ 151.00 ppm.The peak at δ 82.91-83.47ppm corresponded to the methine carbon, whereas the peak near δ 35.00 ppm corresponded to the methylene carbon.The signal of the methyl carbon of t-butyl was observed at δ 25.90-26.19ppm.In their IR spectra, the absorption peak around 3250 cm −1 could be assigned to the N-H stretching vibration; the strong absorption peaks of carbamate and benzoyl C=O were observed at 1740-1776 cm −1 and 1679-1702 cm −1 , respectively.The bending vibration of tert-butyl displayed two peaks near 1390 and 1365 cm −1 [24].Scheme 1. Synthetic route for target compounds 4a-4j.

Antifungal Activity
The in vivo antifungal activities of target compounds against S. sclerotiorum and B. cinerea are summarized in Table 1.Compounds 4a-4j demonstrated moderate antifungal activities against S. sclerotiorum, with EC 50 values of 10.85-15.89µg/mL.Comparing their EC 50 values with diethofencarb (2.95 µg/mL), chlorothalonil (9.97 µg/mL), and carbendazim (0.24 µg/mL),it might be found that their activity was lower than those of the controls.On the other hand, the synthesized compounds displayed moderate to strong antifungal activities toward B. cinerea, the EC 50 values of which were in the range of 6.45-13.03µg/mL.Interestingly, compounds 4d, 4f, 4g, and 4h elicited significant activities with EC 50 values of 6.45-6.98µg/mL, indicating that their activities were near or superior to that of chlorothalonil (EC 50 , 6.56 µg/mL), and much higher than that of carbendazim (EC 50 , 19.20 µg/mL).However, their activities were lower than that of diethofencarb (EC 50 , 4.72 µg/mL).In general, compound 4h possessed the best activity against both S. sclerotiorum and B. cinerea, and the EC 50 values were 10.85 and 6.45 µg/mL, respectively.
We analyzed the relationship between the activity and structure of the target compounds and found that the types of substituent at the benzene ring (R), including the electron-donating group (CH 3 or OCH 3 ) and the electron-withdrawing group (Cl or Br), had an inconspicuous effect on antifungal activity.However, the positions of the substituent had a notable effect on the activity, and the compounds, and compounds with the substituent at the 4-position possessing higher activity than those with the substituent at the 2-or 3-position.Compounds 4d (R = 4-CH 3 ), 4h (R = 4-Cl), and 4j (R = 4-Br), for example, exhibited stronger activity against the two tested fungi in comparison to their 2or 3-position analogues.On the other hand, due to the introduction of a chlorine atom at the 4-position of the benzene ring, compound 4h had a suitable oil-water partition coefficient (Log P), therefore, it might easily access the cell wall of fungi and play an effective role in antifungal activity [16,25].
Generally, compound 4h displayed the best activity against both tested fungi.Due to its favorable pharmacophore (4-chlorophenyl) in the molecule and sufficient antifungal activity, 4h possesses value for further structural optimization and bioactivity research.