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

Abstract

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.

1. 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.

2. Materials and Methods

2.1. Synthesis

Melting points were measured on an X-4 melting point apparatus (Beijing Tech Instrument Co., China). ¹H NMR and ¹³C NMR spectra were obtained on a Bruker AM-400 spectrometer (Bruker Co., Switzerland) with tetramethylsilane as an internal standard (¹H NMR at 400 MHz, ¹³C 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).

All reagents and solvents were commercially available; 1,2-dichloroethane and tetrahydrofuran were dried and redistilled before use.

2.1.1. Synthesis of 3,3-Dimethyl-1-(pyridin-3-yl)butan-2-one (1)

The 3,3-Dimethyl-1-(pyridin-3-yl)butan-2-one (1) was prepared as a pale yellow oil (yield 83%) according to a previous method [20].

2.1.2. Synthesis of 3,3-Dimethyl-1-(pyridin-3-yl)butan-2-ol (2)

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 (1.52 g). Yield 85%, m.p. 113–114°C. ¹H NMR (CDCl₃) δ 1.01 (s, 9H, C(CH₃)₃), 2.46–2.54 (m, 1H, CH₂), 2.87 (d, J = 14 Hz, 1H, CH₂), 3.40 (d, J = 10 Hz, 1H, CH), 4.85 (bs, 1H, OH), 7.20–8.43 (m, 4H, Py-H). IR (KBr)v_max 3350 (O–H), 2961(C–H), 1386 and 1365 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₁₁H₁₇NO: C 73.70, H 9.56, N 7.81; found: C 73.92, H 9.25, N 7.58%.

2.1.3. General Procedure for the Synthesis of Substituted Benzoyl Isocyanate (3)

A 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.

2.1.4. 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.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl benzoylcarbamate (4a). Yield 82%, m.p. 153–154°C. ¹H NMR (CDCl₃) δ 1.07 (s, 9H, C(CH₃)₃), 2.78–2.84 (m, 1H, CH₂), 2.98 (d, J = 14.0 Hz, 1H, CH₂), 5.01 (q, J = 8.0 Hz, 1H, CH), 7.19–7.72 (m, 7H, Ar-H), 8.38 (bs, 1H, NH), 8.45 (s, 2H, Py-H). ¹³C NMR (CDCl₃) δ 165.50, 151.35, 150.36, 147.77, 136.72, 133.67, 133.28, 132.69, 128.61, 127.82, 123.48, 83.02, 35.04, 33.35, 25.96. IR (KBr)v_max 3285 (N–H), 1753 (C=O of carbamate), 1683 (C=O of benzoyl), 1397 and 1371 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₁₉H₂₂N₂O₃: C 69.92, H 6.79, N 8.58; found: C 69.65, H 6.54, N 8.72%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (2-methylbenzoyl)carbamate (4b). Yield 87%, m.p. 157–158°C. ¹H NMR (CDCl₃) δ 1.03 (s, 9H, C(CH₃)₃), 2.31 (s, 3H, CH₃), 2.72–2.79 (m, 1H, CH₂), 2.97 (dd, J = 2.4, 11.6 Hz, 1H, CH₂), 4.98 (q, J = 8.4 Hz, 1H, CH), 7.18–7.61 (m, 6H, Ar-H), 8.17 (bs, 1H, NH), 8.31 (s, 1H, Py-H), 8.43 (s, 1H, Py-H). ¹³C NMR (CDCl₃) δ 168.26, 150.83, 150.22, 147.65, 136.74, 136.29, 134.72, 133.42, 130.99, 126.91, 125.71, 123.58, 82.38, 35.02, 33.23, 25.95, 19.98. IR (KBr)v_max 3285 (N–H), 1770 (C=O of carbamate), 1700 (C=O of benzoyl), 1395 and 1365 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₂₀H₂₄N₂O₃: C 70.57, H 7.11, N 8.23; found: C 70.75, H 7.04, N 8.12%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (3-methylbenzoyl)carbamate (4c). Yield 84%, m.p. 147–148°C. ¹H NMR (CDCl₃) δ 1.07 (s, 9H, C(CH₃)₃), 2.38 (s, 3H, CH₃), 2.78–2.84 (m, 1H, CH₂), 2.98 (dd, J = 2.4, 12.0 Hz, 1H, CH₂), 5.01 (q, J = 8.0 Hz, 1H, CH), 7.19–7.64 (m, 6H, Ar-H), 8.31 (bs, 1H, NH), 8.39 (d, J = 4.8 Hz, 1H, Py-H), 8.45 (d, J = 1.6 Hz, 1H, Py-H). ¹³C NMR (CDCl₃) δ 164.93, 151.34, 150.77, 148.19, 143.87, 136.79, 130.45, 129.61, 127.93, 123.65, 83.47, 35.28, 33.61, 26.19, 21.75. IR (KBr)v_max 3273 (N–H), 1757 (C=O of carbamate), 1679 (C=O of benzoyl), 1396 and 1365 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₂₀H₂₄N₂O₃: C 70.57, H 7.11, N 8.23; found: C 70.81, H 7.14, N 8.06%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (4-methylbenzoyl)carbamate (4d). Yield 82%, m.p. 151–153°C. ¹H NMR (CDCl₃) δ 1.06 (s, 9H, C(CH₃)₃), 2.40 (s, 3H, CH₃), 2.78–2.84 (m, 1H, CH₂), 2.98 (dd, J = 2.4, 12.0 Hz, 1H, CH₂), 5.00 (q, J = 8.0 Hz, 1H, CH), 7.22–7.62(m, 6H, Ar-H), 8.11 (bs, 1H, NH), 8.42–8.46 (m, 2H, Py-H). ¹³C NMR (CDCl₃) δ 164.75, 151.16, 150.59, 148.01, 143.69, 136.61, 130.27, 129.43, 127.75, 123.47, 83.29, 35.10, 33.43, 26.01, 21.57. IR (KBr)v_max 3280 (N–H), 1740 (C=O of carbamate), 1678 (C=O of benzoyl), 1396 and 1368 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₂₀H₂₄N₂O₃: C 70.57, H 7.11, N 8.23; found: C 70.24, H 7.35, N 8.29%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (2-methoxybenzoyl)carbamate (4e). Yield 80%; m.p. 98.9–99.4°C. ¹H NMR (CDCl₃) δ 1.07 (s, 9H, C(CH₃)₃), 2.78–2.84 (m, 1H, CH₂), 2.99 (dd, J = 2.8, 11.6 Hz, 1H, CH₂), 3.85 (s, 3H, OCH₃), 5.01 (q, J = 8.0 Hz, 1H, CH), 7.09–7.63 (m, 6H, Ar-H), 7.90 (bs, 1H, NH), 8.43–8.48 (m, 2H, Py-H). ¹³C NMR (CDCl₃) δ 164.69, 151.07, 150.49, 147.95, 136.64, 134.92, 133.56, 129.74, 123.45, 123.25, 119.39, 119.16, 112.89, 83.29, 55.48, 35.06, 33.35, 25.97. IR (KBr)v_max 3307(N–H), 1770 (C=O of carbamate), 1697 (C=O of benzoyl), 1394 and 1363 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₂₀H₂₄N₂O₄: C 67.40, H 6.79, N 7.86; found: C 67.65, H 6.86, N 7.54%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (2-chlorobenzoyl)carbamate (4f). Yield 87%, m.p. 119–120 °C. ¹H NMR (CDCl₃) δ 1.03 (s, 9H, C(CH₃)₃), 2.72–2.78 (m, 1H, CH₂), 2.96 (dd, J = 2.4, 12.0 Hz, 1H, CH₂), 4.95 (q, J = 8.4 Hz, 1H, CH), 7.19–7.59 (m, 6H, Ar-H), 8.29 (s, 1H, Py-H), 8.42 (s, 1H, Py-H) 8.75 (bs, 1H, NH). ¹³C NMR (CDCl₃) δ 166.32, 150.61, 150.19, 147.58, 136.73, 134.59, 133.41, 131.57, 130.54, 129.78, 129.20, 126.91, 123.54, 82.91, 35.01, 33.25, 25.90. IR (KBr)v_max 3225(N–H), 1759 (C=O of carbamate), 1683 (C=O of benzoyl), 1395 and 1365 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₁₉H₂₁ClN₂O₃: C 63.24, H 5.87, N 7.76; found: C 62.94, H 5.71, N 7.98%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (3-chlorobenzoyl)carbamate (4g). Yield 84%, m.p. 117–118°C. ¹H NMR (CDCl₃) δ 1.02 (s, 9H, C(CH₃)₃), 2.71–2.77 (m, 1H, CH₂), 2.96 (dd, J = 2.4, 11.6 Hz, 1H, CH₂), 4.93 (q, J = 8.4 Hz, 1H, CH), 6.86–7.57 (m, 6H, Ar-H), 8.32 (s, 1H, Py-H), 8.41 (s, 1H, Py-H), 9.40 (bs, 1H, NH). ¹³C NMR (CDCl₃) δ 164.38, 151.39, 150.34, 147.76, 136.78, 134.99, 134.72, 133.63, 132.62, 129.87, 128.11, 125.99, 123.47, 83.41, 35.02, 33.34, 25.93. IR (KBr)v_max 3265(N–H), 1748 (C=O of carbamate), 1683 (C=O of benzoyl), 1395 and 1366 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₁₉H₂₁ClN₂O₃: C 63.24, H 5.87, N 7.76; found: C 63.10, H 5.96, N 7.88%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (4-chlorobenzoyl)carbamate (4h). Yield 82%, m.p. 129–131°C. ¹H NMR (CDCl₃) δ 1.06 (s, 9H, C(CH₃)₃), 2.77–2.83 (m, 1H, CH₂), 2.98 (d, J = 14.4 Hz, 1H, CH₂), 4.99 (q, J = 8.4 Hz, 1H, CH), 7.21–7.64 (m, 6H, Ar-H), 8.41–8.44 (m, 2H, Py-H), 8.77 (bs, 1H, NH). ¹³C NMR (CDCl₃) δ 164.82, 151.44, 150.36, 147.75, 139.11, 136.85, 133.70, 131.57, 129.38, 128.85, 123.52, 83.32, 35.02, 33.36, 25.94. IR (KBr)v_max 3369 (N–H), 1776 (C=O of carbamate), 1689 (C=O of benzoyl), 1390 and 1367 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₁₉H₂₁ClN₂O₃: C 63.24, H 5.87, N 7.76; found: C 63.65, H 5.83, N 7.98%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (2-bromobenzoyl)carbamate (4i). Yield 84%; m.p. 114–115°C. ¹H NMR (CDCl₃) δ 1.03 (s, 9H, C(CH₃)₃), 2.72–2.79 (m, 1H, CH₂), 2.97 (d, J = 13.6 Hz, 1H, CH₂), 4.98 (q, J = 8.4 Hz, 1H, CH), 7.17–7.60 (m, 6H, Ar-H), 8.42 (s, 1H, Py-H), 9.35 (bs, 1H, NH). ¹³C NMR (CDCl₃) δ 150.45, 150.37, 147.89, 136.77, 136.62, 133.35, 132.97, 131.64, 128.96, 127.43, 123.31, 118.86, 83.23, 35.03, 33.29, 25.93. IR (KBr)v_max 3260 (N–H), 1769 (C=O of carbamate), 1702 (C=O of benzoyl), 1394 and 1363 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₁₉H₂₁BrN₂O₃: C 56.31, H 5.22, N 6.91; found: C 56.68, H 5.20, N 7.14%.

3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl (4-bromobenzoyl)carbamate (4j). Yield 87%, m.p. 132–134°C. ¹H NMR (CDCl₃) δ 1.06 (s, 9H, C(CH₃)₃), 2.77–2.83 (m, 1H, CH₂), 2.98 (dd, J = 2.4, 12.0 Hz, 1H, CH₂), 4.99 (q, J = 8.4 Hz, 1H, CH), 7.21–7.63 (m, 6H, Ar-H), 8.39 (bs, NH), 8.43 (s, 2H, Py-H). ¹³C NMR (CDCl₃) δ 164.76, 151.24, 150.44, 147.86, 136.77, 133.63, 132.02, 131.90, 129.42, 127.71, 123.50, 83.44, 35.03, 33.89, 25.96. IR (KBr)v_max 3230 (N–H), 1770 (C=O of carbamate), 1690 (C=O of benzoyl), 1393 and 1365 (C–H of t-butyl) cm⁻¹. Analysis calculated for C₁₉H₂₁BrN₂O₃: C 56.31, H 5.22, N 6.91; found: C 56.12, H 5.26, N 6.98%.

2.2. 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 EC₅₀ value (the concentration resulting in a 50% inhibition) was obtained by DPS Software (

Table 1. Antifungal activities of compounds 4a–4j (EC₅₀).

Compd.	R	EC50 (μg/mL), 95% CL
		S. sclerotiorum	B. cinerea
4a	H	12.67 (8.98–17.89)	13.03 (12.02–14.11)
4b	2-CH3	14.96 (10.42–19.48)	10.50 (8.49–14.03)
4c	3-CH3	15.89 (13.19–19.40)	8.04 (6.54–11.68)
4d	4-CH3	13.18 (9.06–18.21)	6.70 (4.03–11.02)
4e	2-OCH3	14.06 (10.82–19.74)	7.84 (5.50–10.90)
4f	2-Cl	11.84 (8.04–17.90)	6.98 (6.24–7.79)
4g	3-Cl	12.41 (8.19–18.80)	6.81 (5.39–9.60)
4h	4-Cl	10.85 (6.57–17.92)	6.45 (5.44–7.65)
4i	2-Br	12.98 (9.26–16.64)	9.80 (6.48–13.16)
4j	4-Br	11.25 (7.40–17.78)	7.95 (5.21–12.15)
Diethofencarb		2.95 (1.24–5.08)	4.72 (2.49–7.15)
Chlorothalonil		9.97 (7.51–13.22)	6.56 (4.95–9.70)
Carbendazim		0.24 (0.20–0.28)	19.20 (13.27–27.89)

EC₅₀: Concentration resulting in a 50% inhibition; 95% CL: 95% confidence interval of EC₅₀.

).

$$\mathrm{Inhibition} \mathrm{rate}=\frac{D_0-D_1}{D_0}\times 100\%$$

(1)

In which D₀ is the expansion diameter of the mycelia in the blank test, and D₁ is that in the presence of the tested compound.

3. Results and Discussion

3.1. Chemistry

Target compounds 4a–4j were synthesized according to the method depicted in Scheme 1. The starting material, 3-methylpyridine lost a proton under the catalysis of lithium diisopropylamide (LDA) and gave 3-pyridylmethyl lithium, followed by the treatment with ethyl 3,3-dimethylpropionate to yield 3,3-Dimethyl-1-(pyridin-3-yl) butan-2-one (1). Then, 1 was reduced by sodium borohydride to provide the key intermediate 3,3-dimethyl-1-(pyridin-3-yl)butan-2-ol (2). Finally, 2 reacted with freshly prepared substituted benzoyl isocyanate (3) in anhydrous tetrahydrofuran to yield the substituted benzoylcarbamate (4).

Target compounds were identified by ¹H NMR, ¹³C NMR, IR, and elemental analysis. In ¹H NMR spectra, the singlet of methyl protons of t-butyl moiety was observed at δ 1.02–1.07 ppm. 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 6.86–8.45 ppm. In ¹³C 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 82.91–83.47 ppm 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 25.90–26.19 ppm. 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].

3.2. 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 EC₅₀values of 10.85–15.89 μg/mL. Comparing their EC₅₀values 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 EC₅₀values of which were in the range of 6.45–13.03 μg/mL. Interestingly, compounds 4d, 4f, 4g, and 4h elicited significant activities with EC₅₀ values of 6.45–6.98 μg/mL, indicating that their activities were near or superior to that of chlorothalonil (EC₅₀, 6.56 μg/mL), and much higher than that of carbendazim (EC₅₀, 19.20 μg/mL). However, their activities were lower than that of diethofencarb (EC₅₀, 4.72 μg/mL). In general, compound 4h possessed the best activity against both S. sclerotiorum and B. cinerea, and the EC₅₀ 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 (CH₃ or OCH₃) 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-CH₃), 4h (R = 4-Cl), and 4j (R = 4-Br), for example, exhibited stronger activity against the two tested fungi in comparison to their 2- or 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].

4. Conclusions

We designed and synthesized ten 3,3-Dimethyl-1-(pyridin-3-yl)butan-2-yl-substituted benzoylcarbamates and evaluated their antifungal activities against S. sclerotiorum and B. cinerea. Some compounds, such as 4d, 4f, 4g, and 4h, exhibited significant activity against B. cinerea. 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.