Engineering >

2020 , Vol. 6 >Issue 5: 560 - 568

DOI: https://doi.org/10.1016/j.eng.2019.10.016

Synthesis, Characterization, and Antifungal Evaluation of Thiolactomycin Derivatives

  • Pei Lv a, * ,
  • Yiliang Chen a, * ,
  • Dawei Wang b ,
  • Xiangwei Wu a ,
  • Qing X. Li b, c ,
  • Rimao Hua a, *
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  • a Key Laboratory of Agri-Food Safety of Anhui Province, School of Resource & Environment, Anhui Agricultural University, Hefei quaternion Fourier transform (QFT)–electrospray ionization 230036, China
  • b State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China
  • c Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road, Honolulu, HI 96822, USA

Received date: 23 Dec 2018

Published date: 24 Jan 2020

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Abstract

5-Substituted benzylidene 3-acylthiotetronic acids are antifungal. A series of 3-acylthiotetronic acid derivatives with varying substitutions at the 5-position were designed, synthesized, and characterized, based on the binding pose of 3-acyl thiolactone with the protein C171Q KasA. Fungicidal activities of these compounds were screened against Valsa Mali, Curvularia lunata, Fusarium graminearum, and Fusarium oxysporum f. sp. lycopersici. Most target compounds exhibited excellent fungicidal activities against target fungi at the concentration of 50 μg·mL−1. Compounds 11c and 11i displayed the highest activity with a broad spectrum. The median effective concentration (EC50) values of 11c and 11i were 1.9–10.7 and 3.1–7.8 μg·mL−1, respectively, against the tested fungi, while the EC50 values of the fungicides azoxystrobin, carbendazim, and fluopyram were respectively 0.30, 4.22, and > 50 μg·mL−1 against V. Mali; 6.7, 41.7, and 0.18 μg·mL−1 against C. lunata; 22.4, 0.42, and 0.43 μg·mL−1 against F. graminearum; and 4.3, 0.12, and > 50 μg·mL−1 against F. oxysporum f. sp. Lycopersici. The structures and activities of the target compounds against C. lunata were analyzed to obtain a statistically significant comparative molecular field analysis (CoMFA) model with high prediction abilities (q2 = 0.9816, r2 = 0.8060), and its reliability was verified. The different substituents on the benzylidene at the 5-position had significant effects on the activity, while the introduction of a halogen atom at the benzene ring of benzylidene was able to improve the activity against the tested fungi.

Key words： 3-Acylthiotetronic acid; Fungicide; Quantitative structure-activity relationship; Antifungal activity

Cite this article

Pei Lv , Yiliang Chen , Dawei Wang , Xiangwei Wu , Qing X. Li , Rimao Hua . Synthesis, Characterization, and Antifungal Evaluation of Thiolactomycin Derivatives[J]. Engineering, 2020 , 6(5) : 560 -568 . DOI: 10.1016/j.eng.2019.10.016

1. Introduction

Fungal diseases are increasingly recognized as a worldwide threat to food security, the devastation of agricultural crops, and altered forest ecosystem dynamics[1–3]. To guard against fungal pathogens, a large number of synthetic fungicides that are both economical and efficient have been provided for crop protection since the 1960s, and have played an indispensable role in meeting the soaring food demand due to rapid population growth. However, as a result of the repeated use of fungicides with identical or similar modes of action, a rapid increase in fungicide resistance has appeared, leading to the failure of fungal disease control in crops[4–5]. Meanwhile, non-target and environmental hazards have emerged along with fungicide utilization[6–7]. Therefore, there is a continuing need to develop newer fungicides for fungal disease control in crops.
Fatty acids are essential to fungal survival and are one of the most abundant components of the cell wall in fungi; they function as an ample supply of lipids for membrane biosynthesis, which involves regulating substrates between active sites and increasing local concentrations of intermediates[8–10]. There are two distinct fatty acid biosynthetic pathways of long-chain C60–90 α-alkyl-β-hydroxy fatty acids (mycolic acids) [11]. Most bacteria, fungi, and plants possess a fatty acid synthase (FAS) type-II system with dissociated enzymes encoded by separate genes, whereas in mammals, the process of fatty acid synthesis is carried out by a highly integrated FAS type-I multienzyme system that differs significantly from the FAS II enzyme complex [12]. Therefore, FAS II enzymes are an attractive target for the development of new anti-mycobacterial and antimalarial drugs.
Thiolactomycin (TLM), a thiolactone antibiotic, is a known inhibitor of dissociable FAS II enzymes through the inhibition of β-ketoacyl-acyl carrier protein (ACP) synthases (Kases)[13,14]. In recent years, many compounds containing a moiety of thiolactone have been found to exhibit noticeable biological activity against many pathogenic bacteria and mycobacteria[15,16]. It has also been found that TLM and its derivatives show antimalarial activity[17,18] and anti-tuberculosis activity [19]. In addition, we have previously demonstrated that 3-acyl thiolactone acts as a potent anti-phytopathogen agent by inhibiting FAS activity [20]. As shown in Fig. 1, the chemical structure that provides the three hydrogen bonds and the hydrophobic tail interacting with the specific amino acid residues in the Kas is crucial for inhibitory potency.
Fig. 1. TLM (left), 3-acetyl thiotetronic acid lead (middle), and predicted docking pose (right) of 3-acetyl thiotetronic acid in a complex with C171Q KasA enzyme (protein data bank (PDB): 4c6u).
In view of such molecular interactions, it is worth developing new 3-acyl thiolactone derivatives by modifying the substituents of thiolactone for screening highly effective fungicides. Hence, 28 new 3-acylthiotetronic acid derivatives with different substituents of benzylidene at the 5-position were designed and synthesized (See Supplementary data Fig. S1 for compounds 1–12a); these derivatives were expected to exhibit fungicidal activity due to the binding pose of 3-acyl thiolactone with the protein C171Q KasA (Fig. 1) and the proven active group of thiolactone[13–20]. In addition, the comparative molecular field analysis (CoMFA) method implemented in the SYBYL software packages was used to develop predictive three-dimensional (3D) quantitative structure–activity relationship (QSAR) models [21]. This study also predicted the substituents of new thiotetronic acid derivatives with potential antifungal activities based on 3D-QSAR analysis.

2. Materials and methods

2.1. General information
All anhydrous solvents were dried and purified by standard techniques before use. 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were acquired on an Agilent DD2 NMR spectrometer (600 MHz, Agilent Technologies, Inc., USA) at 25 °C with tetramethylsilane as internal standards. Chemical shifts were measured relative to the residual solvent line as an internal standard in ppm (δ ). When peak multiplicities are reported, the following abbreviations are used: singlet (s), doublet (d), doublet of doublets (dd), triplet (t), multiplet (m), quarter (q). High-resolution mass spectrometry (HR-MS) data were determined using a Varian quaternion Fourier transform (QFT)–electrospray ionization (ESI) instrument. The melting points (m.p.) of the products were taken on an XT4 MP apparatus (Taike Corp., China) and the thermometer was not corrected. Analytical thin-layer chromatography (TLC) was performed on silica gel GF 254. Column chromatographic purification was performed using silica gel.
2.2. Synthesis
2.2.1. General procedure for the synthesis of compounds 6a–6i
4-Hydroxybenzaldehyde (0.5 g, 0.0041 mol), acids (0.0045 mol), and 4-dimethylaminopyridine (DMAP; 0.5 g, 0.0041 mol) were dissolved in dichloromethane (30 mL) at 0 °C. To this solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 1.2 g, 0.0062 mol) in dichloromethane (10 mL) was added dropwise. The mixture was then stirred at room temperature for 12 h, detected by TLC. Upon completion of the reaction, the dichloromethane was removed by rotary evaporation. The crude product was dissolved with ethyl acetate, washed with saturated aqueous NaHCO3 and H2O, dried over MgSO4, filtered, and then concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (PE/EA) = 8:1) to obtain 6a–6i:
• 4-Formylphenyl acetate (6a). Colorless oil; yield 80.4%; 1H NMR (600 MHz, CDCl3) δ : 9.98 (s, 1H), 7.93–7.89 (m, 2H), 7.26 (t, coupling constant J = 5.9 Hz, 2H), and 2.32 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 190.89, 168.68, 155.30, 133.96, 131.19, 122.35, and 21.14; HR-MS (ESI): mass-to-charge ratio (m/z) calculated for C9H8O3 ([M+H]+ ): 165.0552; found: 165.0554.
• 4-Formylphenyl propionate (6b). Colorless oil; yield 85.6%; 1H NMR (600 MHz, CDCl3) δ : 9.97 (s, 1H), 7.90 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.6 Hz, 2H), 2.61 (q, J = 7.5 Hz, 2H), and 1.26 (t, J = 7.5 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 190.92, 172.22, 155.46, 133.87, 131.17, 122.32, 27.75, and 8.92; HR-MS (ESI): m/z calculated for C10H10O3 ([M+H]+ ): 179.0708; found: 179.0710.
• 4-Formylphenyl hexanoate (6c). Colorless oil; yield 83.4%; 1H NMR (600 MHz, CDCl3) δ : 9.97 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.3 Hz, 2H), 2.57 (t,J = 7.5 Hz, 2H), 1.81–1.62 (m, 2H), 1.46–1.21 (m, 4H), and 0.92 (t, J = 6.8 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 190.84, 171.52, 155.48, 133.89, 131.13, 122.32, 34.32, 31.18, 24.46, 22.25, and 13.85; HR-MS (ESI): m/z calculated for C13H16O3 ([M+H]+ ): 221.1178; found: 221.1180.
• 4-Formylphenyl 4-fluorobenzoate (6d). White solid; yield 85.4%; m.p. 99–100 °C; 1H NMR (600 MHz, CDCl3) δ : 10.02 (s, 1H), 8.28–8.17 (m, 2H), 7.97 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), and 7.19 (t, J = 8.3 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ : 190.82, 167.21, 165.51, 163.47, 155.49, 134.14, 132.91, 131.24, 125.15, 122.45, 116.03, and 115.88; HR-MS (ESI): m/z calculated for C14H9FO3 ([M+H]+ ): 245.0614; found: 245.0618.
• 4-Formylphenyl 4-chlorobenzoate (6e). White solid; yield 86.7%; m.p. 111–112 °C; 1H NMR (600 MHz, CDCl3) δ : 10.02 (s, 1H), 8.13 (d, J = 8.6 Hz, 2H), 7.97 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), and 7.40 (d, J = 8.5 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ : 190.83, 163.63, 155.40, 140.62, 134.17, 131.61, 131.27, 129.10, 127.33, and 122.43; HR-MS (ESI): m/z calculated for C14H9ClO3 ([M+H]+ ): 261.0318; found: 261.0322.
• 4-Formylphenyl 4-bromobenzoate (6f). White solid; yield 91.5%; m.p. 112–113 °C; 1H NMR (600 MHz, CDCl3) δ : 10.02 (s, 1H), 8.05 (d, J = 8.5 Hz, 2H), 7.97 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), and 7.40 (d, J = 8.5 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ : 190.81, 163.78, 155.39, 134.18, 132.10, 131.68, 131.26, 129.34, 127.80, and 122.42; HR-MS (ESI): m/z calculated for C14H9BrO3 ([M+H]+ ): 304.9813; found: 304.9814.
• 4-Formylphenyl 4-methylbenzoate (6g). White solid; yield 86.9%; m.p. 112–113 °C; 1H NMR (600 MHz, CDCl3) δ : 10.01 (s, 1H), 8.08 (d, J = 8.2 Hz, 2H), 7.96 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 7.9 Hz, 2H), and 2.46 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 190.97, 164.53, 155.78, 144.97, 133.95, 131.23, 130.30, 129.42, 126.10, 122.56, and 21.79; HR-MS (ESI): m/z calculated for C15H12O3 ([M+H]+ ): 241.0865; found: 241.0868.
• 4-Formylphenyl 4-(trifluoromethyl)benzoate (6h). White solid; yield 79.8%; m.p. 85–86 °C; 1H NMR (600 MHz, CDCl3) δ : 10.03 (s, 1H), 8.32 (d, J = 8.1 Hz, 2H), 7.99 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), and 7.42 (d, J = 8.4 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ : 190.83, 163.32, 155.21, 135.50, 135.29, 134.31, 132.13, 131.33, 131.11, 130.66, 125.75, 124.34, 122.53, and 122.34; HR-MS (ESI): m/z calculated for C15H9F3O3 ([M+H]+ ): 295.0582; found: 295.0587.
• 4-Formylphenyl 4-methoxybenzoate (6i). White solid; yield 89.8%; m.p. 95–96 °C; 1H NMR (600 MHz, CDCl3) δ : 10.01 (s, 1H), 8.14 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 6.99 (d, J = 8.7 Hz, 2H), and 3.89 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 190.95, 164.18, 155.85, 133.89, 132.42, 131.20, 122.57, 121.10, 113.97, and 55.54; HR-MS (ESI): m/z calculated for C15H12O4 ([M+H]+ ): 257.0814; found: 257.0816.
2.2.2. General procedure for the preparation of compounds 8a–8g
4-Formylbenzoic acid (0.5 g, 0.0033 mol), alcohols (0.0030 mol), and DMAP (0.4 g, 0.0033 mol) were dissolved in dichloromethane (30 mL) at 0 °C. To this solution, EDC (1.0 g, 0.0050 mol) in dichloromethane (10 mL) was added dropwise. The mixture was then stirred at room temperature for 12 h, detected by TLC. Upon completion of the reaction, dichloromethane was removed by rotary evaporation. The crude product was dissolved with ethyl acetate, washed with saturated aqueous NaHCO3 and H2O, dried over MgSO4, filtered, and then concentrated. The residue was purified by silica gel column chromatography (PE/EA = 15:1) to obtain 8a–8g:
• Methyl 4-formylbenzoate (8a). White solid; yield 91.1%; m.p. 58–59 °C; 1H NMR (600 MHz, CDCl3) δ : 10.08 (s, 1H), 8.18 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), and 3.94 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 191.61, 166.02, 139.09, 135.04, 130.15, 129.48, and 52.55; HR-MS (ESI) m/z calculated for C9H8O3 ([M +H]+ ): 165.0552; found: 165.0551.
• Ethyl 4-formylbenzoate (8b). Colorless oil; yield 81.9%; 1H NMR (600 MHz, CDCl3) δ : 10.08 (s, 1H), 8.20–8.10 (m, 2H), 7.93 (d, J = 8.2 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), and 1.40 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 191.69, 165.55, 139.02, 135.41, 130.08, 129.50, 61.59, and 14.23; HR-MS (ESI) m/z calculated for C10H10O3 ([M+H]+ ): 179.0708; found: 179.0709.
• Isopropyl 4-formylbenzoate (8c). Colorless oil; yield 83.7%; 1H NMR (600 MHz, CDCl3) δ : 10.08 (s, 1H), 8.16 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 8.4 Hz, 2H), 5.29–5.22 (m, 1H), and 1.37 (d, J = 6.3 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ : 191.65, 165.00, 138.97, 135.85, 130.02, 129.39, 69.19, and 21.84; HR-MS (ESI) m/z calculated for C11H12O3 ([M+H]+ ): 193.0865; found: 193.0868.
• Propyl 4-formylbenzoate (8d). Colorless oil; yield 83.1%; 1H NMR (600 MHz, CDCl3) δ : 10.09 (s, 1H), 8.20–8.11 (m, 2H), 7.94 (d, J = 8.3 Hz, 2H), 4.31 (t, J = 6.7 Hz, 2H), 1.83–1.76 (m, 2H), and 1.03 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 191.68, 165.60, 139.03, 135.45, 130.11, 129.47, 67.14, 22.01, and 10.46; HR-MS (ESI) m/z calculated for C11H12O3 ([M+H]+ ): 193.0865; found: 193.0868.
• Butyl 4-formylbenzoate (8e). Colorless oil; yield 89.4%; 1H NMR (600 MHz, CDCl3) δ : 10.08 (s, 1H), 8.17 (d, J = 8.2 Hz, 2H), 7.93 (d, J = 8.1 Hz, 2H), 4.34 (t, J = 6.6 Hz, 2H), 1.79–1.72 (m, 2H), 1.51–1.43 (m, 2H), and 0.97 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 191.67, 165.60, 139.02, 135.44, 130.10, 129.46, 65.45, 30.65, 19.21, and 13.71; HR-MS (ESI) m/z calculated for C12H14O3 ([M+H]+ ): 207.1021; found: 207.1022.
• Octyl 4-formylbenzoate (8f). Colorless oil; yield 79.6%; 1H NMR (600 MHz, CDCl3) δ : 10.09 (s, 1H), 8.18 (d, J = 8.2 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H), 4.34 (t, J = 6.7 Hz, 2H), 1.81–1.72 (m, 2H), 1.50–1.20 (m, 10H), and 0.87 (t, J = 6.6 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 191.58, 165.58, 139.06, 135.49, 130.10, 129.44, 65.75, 31.74, 29.16, 28.62, 25.97, 22.59, and 14.03; HRMS (ESI): m/z calculated for C16H22O3 ([M+H]+ ): 263.1647; found: 263.1650.
• Benzyl 4-formylbenzoate (8g). White solid; yield 73.8%; m.p. 43–44 °C; 1H NMR (600 MHz, CDCl3) δ : 10.09 (s, 1H), 8.22 (d, J = 8.1 Hz, 2H), 7.94 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 7.3 Hz, 2H), 7.40 (t, J = 7.4 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 5.39 (s, 2H). 13C NMR (150 MHz, CDCl3) δ : 191.60, 165.37, 139.18, 135.51, 135.06, 130.29, 129.50, 128.68, 128.49, 128.33, and 67.30; HR-MS (ESI) m/z calculated for C15H12O3 ([M+H]+ ): 241.0865; found: 241.0866.
2.2.3. General procedure for compounds 9a–9i, 10a–10g, 11a–11m, and 12a
A solution of 158 mg (1 mmol) of 3-acetylthiophene-2,4 (3H,5H )-dione (4) and appropriate substituted aromatic aldehyde (1.1 mmol) in 50 mL of toluene containing p -TsOH (30 mg, 0.17 mmol) was refluxed with the azeotropic removal of water and detected by TLC. The mixture was cooled to room temperature, and the precipitate 5-substuted benzylidene 3-acylthiotetronic acid (i.e., 9a–9i, 10a–10g, 11a–11m, and 12a) was filtered off and recrystallized from the MeOH-ethyl acetate:
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl acetate (9a). Yellow solid; yield 70.9%; m.p. 192– 193 °C; 1H NMR (600 MHz, CDCl3) δ : 7.81 (s, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 2.61 (s, 3H), 2.32 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.57, 187.14, 168.94, 152.17, 133.50, 132.31, 131.62, 131.29, 126.07, 122.45, 116.34, 108.59, 25.69, and 21.15; HR-MS (ESI) m/z calculated for C15H12O5S ([M +H]+ ): 305.0484; found: 305.0485.
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl propionate (9b). Yellow solid; yield 65.7%; m.p. 167–168 °C; 1H NMR (600 MHz, CDCl3) δ : 7.80 (s, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 2.64–2.58 (m, 5H), and 1.26 (q, J = 7.7 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.58, 187.14, 172.46, 152.33, 132.31, 131.70, 131.16, 125.95, 122.43, 108.59, 27.76, 25.70, and 8.96; HR-MS (ESI) m/z calculated for C16H14O5S ([M+H]+ ): 319.0640; found: 319.0642.
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl hexanoate (9c). Yellow solid; yield 66.8%; m.p. 124–125 °C; 1H NMR (600 MHz, CDCl3) δ : 7.81 (s, 1H), 7.61 (d, J = 8.5 Hz, 2H), 7.20 (d, J = 8.5 Hz, 2H), 2.61 (s, 3H), 2.57 (t, J = 7.5 Hz, 2H), 1.81–1.71 (m, 2H), 1.39 (d, J = 3.3 Hz, 4H), and 0.93 (t, J = 6.8 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.54, 187.12, 171.77, 152.35, 132.28, 131.67, 131.16, 125.98, 122.45, 108.59, 34.34, 31.21, 25.66, 24.51, 22.27, and 13.87; HR-MS (ESI) m/z calculated for C19H20O5S ([M+H]+ ): 361.1110; found: 361.1112.
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl 4-fluorobenzoate (9d). Yellow solid; yield 65.4%; m.p. 191–192 °C; 1H NMR (600 MHz, CDCl3) δ : 8.22 (dd, J = 8.4, 5.5 Hz, 2H), 7.84 (s, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 7.20 (t, J = 8.5 Hz, 2H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.53, 187.12, 167.17, 165.47, 163.69, 152.35, 132.89, 132.36, 131.51, 126.25, 125.28, 122.54, 116.00, 115.85, 108.59, and 25.64; HR-MS (ESI) m/z calculated for C20H13FO5S ([M+H]+ ): 385.0546; found: 385.0547.
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl 4-chlorobenzoate (9e). Yellow solid; yield 69.9%; m.p. 188–189 °C; 1H NMR (600 MHz, CDCl3) δ : 7.83 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.60, 187.13, 163.86, 152.25, 140.53, 132.41, 131.60, 131.55, 131.51, 129.07, 127.42, 126.23, 122.53, 108.59, and 25.73; HR-MS (ESI) m/z calculated for C20H13ClO5S ([M+H]+ ): 401.0250; found: 401.0251.
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl 4-bromobenzoate (9f). Yellow solid; yield 65.1%; m.p. 184–185 °C; 1H NMR (600 MHz, CDCl3) δ : 8.05 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 7.67 (d, J = 7.9 Hz, 4H), 7.34 (d, J = 8.5 Hz, 2H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.60, 187.14, 164.03, 152.23, 132.41, 132.08, 131.70, 131.54, 129.27, 127.88, 126.25, 122.52, 108.60, and 25.73; HR-MS (ESI) m/z calculated for C20H13BrO5S ([M+H]+ ): 444.9745; found: 444.9746.
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl 4-methylbenzoate (9g). Yellow solid; yield 69.3%; m.p. 181–182 °C; 1H NMR (600 MHz, CDCl3) δ : 8.08 (d, J = 8.1 Hz, 2H), 7.85 (s, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.33 (dd, J = 15.2, 8.3 Hz, 4H), and 2.62 (s, 3H), and 2.46 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.58, 187.14, 164.72, 152.65, 144.85, 132.36, 131.75, 131.25, 130.28, 129.38, 126.24, 126.01, 122.65, 108.62, 25.71, and 21.78; HR-MS (ESI) m/z calculated for C21H16O5S ([M+H]+ ): 381.0797; found: 381.0796.
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl 4-(trifluoromethyl)benzoate (9h). Yellow solid; yield 67.4%; m.p. 195–196 °C; 1H NMR (600 MHz, CDCl3) δ : 8.32 (d, J = 8.2 Hz, 2H), 7.85 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H), and 2.63 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.53, 187.12, 163.51, 152.07, 132.40, 131.74, 131.38, 130.63, 126.48, 125.70, 122.44, 108.59, and 25.63; HR-MS (ESI) m/z calculated for C21H13F3O5S ([M+H]+ ): 435.0514; found: 435.517.
• 4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene) methyl)phenyl 4-methoxybenzoate (9i). Yellow solid; yield 63.9%; m.p. 155–156 °C; 1H NMR (600 MHz, CDCl3) δ : 8.14 (d, J = 8.2 Hz, 2H), 7.84 (s, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.3 Hz, 2H), 3.90 (s, 3H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.64, 187.25, 187.05, 164.39, 164.13, 152.70, 132.40, 131.83, 131.14, 125.88, 122.70, 121.20, 113.93, 108.61, 55.56, and 25.77; HR-MS (ESI) m/z calculated for C21H16O6S ([M+H]+ ): 397.0746; found: 397.0747.
• Methyl 4-((4-acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene)- methyl)benzoate (10a). Yellow solid; yield 63.6%; m.p. 177–178 °C; 1H NMR (600 MHz, CDCl3) δ : 8.10 (d, J = 8.4 Hz, 2H), 7.82 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 3.94 (s, 3H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.33, 187.35, 186.81, 166.15, 137.70, 131.25, 130.92, 130.71, 130.14, 128.71, 108.55, 52.40, and 25.46; HR-MS (ESI) m/z calculated for C15H12O5S ([M+H]+ ): 305.0484; found: 305.0486.
• Ethyl 4-((4-acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene)- methyl)benzoate (10b). Yellow solid; yield 64.5%; m.p. 147– 148 °C; 1H NMR (600 MHz, CDCl3) δ : 8.12 (d, J = 8.4 Hz, 2H), 7.83 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 4.39 (q, J = 7.1 Hz, 2H), 2.62 (s, 3H), and 1.40 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.36, 187.33, 186.87, 165.68, 137.59, 131.63, 131.03, 130.68, 130.11, 128.60, 108.57, 61.36, 25.49, and 14.28; HR-MS (ESI) m/z calculated for C16H14O5S ([M+H]+ ): 319.0640; found: 319.0640.
• Isopropyl 4-((4-acetyl-3-hydroxy-5-oxothiophen-2(5H )- ylidene)methyl)benzoate (10c). Yellow solid; yield 70.1%; m.p. 157–158 °C; 1H NMR (600 MHz, CDCl3) δ : 8.10 (d, J = 8.3 Hz, 2H), 7.83 (s, 1H), 7.63 (d, J = 8.2 Hz, 2H), 5.29–5.22 (m, 1H), 2.62 (s, 3H), and 1.37 (d, J = 6.3 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ : 197.34, 187.31, 186.86, 165.14, 137.48, 132.08, 131.08, 130.63, 130.07, 128.52, 108.56, 68.89, 25.47, and 21.90; HR-MS (ESI) m/z calculated for C17H16O5S ([M+H]+ ): 333.0797; found: 333.0800.
• Propyl 4-((4-acetyl-3-hydroxy-5-oxothiophen-2(5H )- ylidene)methyl)benzoate (10d). Yellow solid; yield 70.6%; m.p. 142–143 °C; 1H NMR (600 MHz, CDCl3) δ : 8.12 (d, J = 8.3 Hz, 2H), 7.83 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 4.30 (t, J = 6.7 Hz, 2H), 2.62 (s, 3H), 1.84–1.77 (m, 2H), and 1.03 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.34, 187.34, 186.84, 165.72, 137.60, 131.67, 131.01, 130.68, 130.10, 128.62, 108.57, 66.91, 25.46, 22.06, and 10.48; HR-MS (ESI) m/z calculated for C17H16O5S ([M +H]+ ): 333.0797; found: 333.0800.
• Butyl 4-((4-acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene)- methyl)benzoate (10e). Yellow solid; yield 66.7%; m.p. 112– 113 °C; 1H NMR (600 MHz, CDCl3) δ : 8.11 (d, J = 8.3 Hz, 2H), 7.82 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 4.34 (t, J = 6.6 Hz, 2H), 2.62 (s, 3H), 1.79–1.72 (m, 2H), 1.52–1.43 (m, 2H), and 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.34, 187.33, 186.84, 165.72, 137.58, 131.66, 131.01, 130.69, 130.10, 128.60, 108.55, 65.23, 30.70, 25.47, 19.25, and 13.74; HR-MS (ESI) m/z calculated for C18H18O5S ([M+H]+ ): 347.0953; found: 347.0954.
• Octyl 4-((4-acetyl-3-hydroxy-5-oxothiophen-2(5H )-ylidene)- methyl)benzoate (10f). Yellow solid; yield 65.4%; m.p. 113– 114 °C;1H NMR (600 MHz, CDCl3) δ : 8.11 (d, J = 8.3 Hz, 2H), 7.83 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 4.33 (t, J = 6.7 Hz, 2H), 2.62 (d, J = 7.9 Hz, 3H), 1.80–1.73 (m, 2H), 1.47–1.40 (m, 2H), 1.39–1.23 (m, 8H), and 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.32, 187.34, 186.82, 165.72, 137.59, 131.69, 131.01, 130.68, 130.10, 128.63, 108.56, 65.54, 31.76, 29.18, 28.67, 26.00, 25.44, 22.61, and 14.06; HR-MS (ESI) m/z calculated for C22H26O5S ([M +H]+ ): 403.0579; found: 403.1580.
• Benzyl 4-((4-acetyl-3-hydroxy-5-oxothiophen-2(5H)- ylidene)methyl)benzoate (10g). Yellow solid; yield 69.8%; m.p. 168–169 °C; 1H NMR (600 MHz, CDCl3) δ : 8.14 (d, J = 8.4 Hz, 2H), 7.82 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 7.1 Hz, 2H), 7.40 (t, J = 7.3 Hz, 2H), 7.35 (dd, J = 8.5, 6.0 Hz, 1H), 5.38 (s, 2H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.31, 187.35, 186.78, 165.49, 137.82, 135.69, 131.25, 130.89, 130.70, 130.26, 128.79, 128.64, 128.39, 128.25, 108.55, 67.07, and 25.44; HR-MS (ESI) m/z calculated for C21H16O5S ([M+H]+ ): 381.0797; found: 381.0796.
• 3-Acetyl-5-(2-bromobenzylidene)-4-hydroxythiophen-2 (5H)-one (11a). Yellow solid; yield 66.9%; m.p. 152–153 °C; 1H NMR (600 MHz, CDCl3) δ : 8.14 (s, 1H), 7.66 (dd, J = 11.3, 8.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 1H), 7.26–7.24 (m, 1H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.50, 187.17, 186.97, 133.65, 131.39, 130.25, 128.99, 127.79, 126.69, 108.94, and 25.63; HRMS (ESI) m/z calculated for C13H9BrO3S ([M+H]+ ): 324.9534; found: 324.9555.
• 3-Acetyl-5-(3-bromobenzylidene)-4-hydroxythiophen-2 (5H)-one (11b). Yellow solid; yield 69.7%; m.p. 150–151 °C; 1H NMR (600 MHz, CDCl3) δ : 7.72 (d, J = 7.2 Hz, 2H), 7.52 (dd, J = 11.8, 8.2 Hz, 2H), 7.33 (t, J = 7.9 Hz, 1H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.32, 187.25, 186.74, 135.66, 133.64, 133.26, 130.56, 129.14, 127.85, 123.20, 108.55, and 25.45; HRMS (ESI) m/z calculated for C13H9BrO3S ([M+H]+ ): 324.9534; found: 324.9555.
• 3-Acetyl-5-(4-bromobenzylidene)-4-hydroxythiophen-2(5H)- one (11c). Yellow solid; yield 78.6%; m.p. 176–177 °C; 1H NMR (600 MHz, CDCl3) δ : 7.74 (s, 1H), 7.59 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), and 2.61 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.42, 187.18, 186.80, 132.47, 132.22, 131.17, 126.90, 125.28, 108.53, and 25.54; HR-MS (ESI) m/z calculated for C13H9BrO3S ([M+H]+ ): 324.9534; found: 324.9555.
• 3-Acetyl-5-(2-fluorobenzylidene)-4-hydroxythiophen-2(5H)- one (11d). Yellow solid; yield 67.5%; m.p. 165–166 °C; 1H NMR (600 MHz, DMSO-d6) δ : 7.75 (s, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.48 (d, J = 6.2 Hz, 1H), 7.39–7.29 (m, 2H), and 2.44 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ : 194.31, 186.63, 185.78, 161.98, 160.31, 132.37, 129.74, 125.60, 122.50, 119.47, 116.43, 107.54, and 27.07; HR-MS (ESI) m/z calculated for C13H9FO3S ([M+H]+ ): 265.0335; found: 265.0336.
• 3-Acetyl-5-(3-fluorobenzylidene)-4-hydroxythiophen-2(5H)- one (11e). Yellow solid; yield 63.8%; m.p. 151–152 °C; 1H NMR (600 MHz, DMSO-d6) δ : 7.75 (s, 1H), 7.53 (dd, J = 14.2, 7.4 Hz, 1H), 7.45 (t, J = 8.9 Hz, 2H), 7.27 (t, J = 8.4 Hz, 1H), and 2.45 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ : 195.17, 186.65, 186.02, 163.47, 161.85, 136.73, 131.59, 129.58, 128.67, 126.73, 117.42, 117.27, 117.12, 107.91, and 26.68; HR-MS (ESI) m/z calculated for C13H9FO3S ([M+H]+): 265.0335; found: 265.0336.
• 3-Acetyl-5-(2-chlorobenzylidene)-4-hydroxythiophen-2(5H)- one (11g). Yellow solid; yield 64.9%; m.p. 157–158 °C; 1H NMR (600 MHz, CDCl3) δ : 8.20 (s, 1H), 7.68–7.65 (m, 1H), 7.47 (dd, J = 7.3, 1.4 Hz, 1H), 7.38–7.31 (m, 2H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.49, 187.08, 136.26, 131.97, 131.33, 130.30, 130.13, 128.84, 128.65, 127.19, 108.87, and 25.62; HRMS (ESI) m/z calculated for C13H9ClO3S ([M+H]+ ): 281.0039; found: 281.0040.
• 3-Acetyl-5-(3-chlorobenzylidene)-4-hydroxythiophen-2(5H)- one (11h). Yellow solid; yield 62.9%; m.p. 142–143 °C; 1H NMR (600 MHz, CDCl3) δ : 7.73 (s, 1H), 7.55 (s, 1H), 7.47 (d, J = 6.7 Hz, 1H), 7.42–7.36 (m, 2H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.34, 187.26, 186.76, 135.38, 135.17, 130.71, 130.33, 128.77, 127.82, 108.55, and 25.46; HR-MS (ESI) m/z calculated for C13H9ClO3S ([M+H]+ ): 281.0039; found: 281.0040.
• 3-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H)-ylidene) methyl)benzonitrile (11j). Yellow solid; yield 64.8%; m.p. 171– 172 °C; 1H NMR (600 MHz, CDCl3) δ : 7.84 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.76 (s, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), and 2.63 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.18, 187.47, 186.20, 134.93, 134.27, 133.98, 133.13, 130.00, 129.35, 129.20, 117.87, 113.64, 108.38, and 25.29; HR-MS (ESI) m/z calculated for C14H9NO3S ([M+H]+ ): 272.0381; found: 272.0382.
• 3-Acetyl-4-hydroxy-5-(3-(trifluoromethyl)benzylidene) thiophen-2(5H)-one (11k). Yellow solid; yield 70.8%; m.p. 125– 126 °C; 1H NMR (600 MHz, DMSO-d6) δ : 7.96 (s, 1H), 7.89 (d, J = 6.8 Hz, 1H), 7.79 (s, 1H), 7.72 (dd, J = 16.0, 8.7 Hz, 2H), and 2.43 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ : 194.35, 186.41, 186.02, 135.83, 133.93, 131.26, 130.66, 127.24, 127.03, 126.20, 107.45, and 27.18; HR-MS (ESI) m/z calculated for C14H9F3O3S ([M+H]+ ): 315.0303; found: 315.0305.
• 3-Acetyl-4-hydroxy-5-(3-methoxybenzylidene)thiophen-2 (5H)-one (11l). Yellow solid; yield 67.2%; m.p. 141–143 °C; 1H NMR (600 MHz, CDCl3) δ : 7.79 (s, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.10 (s, 1H), 6.97 (dd, J = 8.2, 1.5 Hz, 1H), 3.85 (s, 3H), and 2.61 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.57, 187.32, 187.07, 159.95, 134.88, 132.76, 130.09, 126.25, 123.83, 116.94, 115.46, 108.68, 55.35, and 25.70; HR-MS (ESI) m/z calculated for C14H12O4S ([M+H]+ ): 277.0535; found: 277.0538.
• 3-Acetyl-5-(biphenyl-4-ylmethylene)-4-hydroxythiophen2(5H)-one (11m). Yellow solid; yield 64.4%; m.p. 123–124 °C; 1H NMR (600 MHz, CDCl3) δ : 7.87 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 7.3 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.3 Hz, 1H), and 2.62 (s, 3H). 13C NMR (150 MHz, CDCl3) δ : 197.67, 187.35, 186.96, 143.32, 139.64, 132.48, 131.69, 128.98, 128.20, 127.70, 127.09, 125.71, 108.65, and 25.80; HR-MS (ESI) m/z calculated for C19H14O3S ([M+H]+ ): 323.0742; found: 323.0742.
• 3-Acetyl-4-hydroxy-5-((E)-3-phenylallylidene)thiophen-2 (5H)-one (12a). Yellow solid; yield 63.1%; m.p. 138–139 °C; 1H NMR (600 MHz, DMSO-d6) δ : 7.64 (d, J = 7.2 Hz, 2H), 7.58 (d, J = 11.4 Hz, 1H), 7.39 (t, J = 7.2 Hz, 2H), 7.36 (d, J = 6.9 Hz, 1H), 7.35–7.28 (m, 1H), 6.98 (dd, J = 15.0, 11.6 Hz, 1H), and 2.45 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ : 196.63, 186.34, 183.88, 144.37, 136.10, 132.33, 130.32, 129.42, 128.34, 124.52, 110.07, and 27.09; HR-MS (ESI) m/z calculated for C15H12O3S ([M+H]+ ): 273.0585; found: 273.0586.
2.3. Screening of antifungal activity in vitro
Each bioassay was performed in triplicate at (25 ± 1) C. According to the mycelium growth rate method, 9a–9i, 10a–10g, 11a– 11m, and 12a were screened for antifungal activities in vitro against four phytopathogenic fungi, Valsa mali, Curvularia lunata, Fusarium graminearum, and Fusarium oxysporum f. sp. lycopersici, at 50 μg·mL-1 . Activity results were estimated according to a percentage scale of 0–100. Detailed bioassay procedures for fungicidal activity have been described previously [20].
2.4. CoMFA calculation
The CoMFA method was used to investigate the QSAR of the synthesized compounds [21]. A total of 27 compounds were selected for the QSAR study, based on their chemical diversity and C. lunata inhibitory bioactivity. The 3D structures of 27 compounds were constructed using the default settings of SYBYL 7.3 software (TriposTM, Certara Inc., USA), and optimized with the steepest-descent algorithm to a convergence criterion of 0.005 kcal·mol-1 (1 kcal = 4184 J). The CoMFA descriptors, steric, and electrostatic field energies were calculated by the SYBYL default parameters: an sp3 carbon probe atom with +1 charge, 2.0 Å rid points spacing, the energy cutoff of 30.0 kcal·mol-1 , and a minimum column filtering (σ ) of 2.0 kcal·mol-1[22–24].

3. Results and discussion

Compounds 4, 11f, 11i, and 11n were prepared in three steps, as previously described [20]. Compounds 6 and 8 were prepared by the esterification of the corresponding acid and alcohol in one step. The target compounds 9a–9i, 10a–10g, 11a–11n, and 12a were obtained by condensation with intermediate 4. Compound 11c' was obtained by the reaction of 11c with ethanol.
The structures of all of the target compounds were characterized by 1H NMR, 13C NMR, and HR-MS spectra. In addition, the crystal structure of 11c' was determined by X-ray diffraction analyses. As shown in Fig. 2, the double bond formed by the dehydration of the aldol reaction product adopts a Z-configuration rather than an E-conformation.
Fig. 2. X-ray single-crystal structure of 11c' .
3.1. Fungicidal activity
The preliminary determination of the inhibition rates of compounds 9a–9i, 10a–10g, 11a–11n, and 12a (50 μg·mL-1 ) against four plant-pathogenic fungi (V. mali, C. lunata, F. graminearum, and F. oxysporum f. sp. lycopersici ) is shown in Table 1. The data suggested that most of the target compounds displayed moderate to good fungicidal activities against all the tested fungi at a dose of 50 μg·mL-1 . In order to compare the potency of the synthetic chemicals, azoxystrobin, carbendazim, and fluopyram were used as positive controls.
Table 1 Comparison of fungicidal activities of compounds 9a–9i, 10a–10g, 11a–11n, and 12a at a concentration of 50 μg·mL-1 against V. mali, C. lunata, F. graminearum, and F. oxysporum f. sp. lycopersici.
To further explore the antifungal potential and structure–activity relationship (SAR), the compounds with inhibition rates greater than 70% at a concentration of 50 μg·mL-1 were used to further determine their regression equations and median effective concentration (EC50 values) toward the four tested fungi (Table 2).
Table 2 EC50 values (μg·mL-1) of compounds 10b, 10c, 11a–11l, and 12a against V. mali, C. lunata, F. graminearum, and F. oxysporum f. sp. lycopersici.
a 95% CI: confidence intervals at 95% probability; b average of three replicates.
Table 1 shows that compounds 9a–9i displayed weak inhibition (< 50% inhibition rate) against the target fungi. Compounds 10a and 10d–10g displayed different degrees of fungicidal activity, ranging from 5.25% to 82.7% inhibition against the tested fungi at a dose of 50 μg·mL-1 . It was interesting that compounds 10b, 10c, and 10d were highly active (79.4%–82.7% inhibition at 50 μg·mL-1 ) against F. oxysporum f. sp. lycopersici, while compounds 11a–11m and 12a exhibited moderate (35%–70%) to good activities (> 70% inhibition at 50 μg·mL-1 ) against V. mali. In contrast (See Table 2), the EC50 values of compounds 11a, 11c, 11f, 11g, 11i, and 11k ranged from 3.1 to 18.7 μg·mL-1 , while compound 11f displayed a roughly similar level of antifungal activity to that of carbendazim (EC50 = 4.2 μg·mL-1 ) against V. mali, which was superior to that of fluopyram (EC50 > 50 μg·mL-1 ) and ten-fold greater than that of azoxystrobin (EC50 = 0.3 μg·mL-1 ). In regard to C. lunata, the EC50 of compounds 11a–11k and 12a ranged from 1.9 to 8.96 μg·mL-1 , with some of these compounds exhibiting more fungicidal activity than the positive controls azoxystrobin (EC50 = 6.7 μg·mL-1 ) and carbendazim (EC50 = 41.2 μg·mL-1 ); these values can be compared with the EC50 of fluopyram, which is 0.18 μg·mL-1 . It was interesting that compound 11j had low inhibition against C. lunata. In regard to F. graminearum, compounds 11a–11m and 12a had low (< 35% inhibition) to moderate potencies (35%–70% inhibition) at 50 μg·mL-1 ; compound 11i exhibited the highest fungicidal activity (EC50 = 3.1 μg·mL-1 ), which was 7.2- and 7.4-fold greater than those of fluopyram (EC50 = 0.43 μg·mL-1 ) and carbendazim (EC50 = 0.42 μg·mL-1 ), respectively. In regard to F. oxysporum f. sp. lycopersici, compounds 11a–11m and 12a inhibited F. oxysporum f. sp. lycopersici with an inhibition higher than 50% with the exception of compound 11m, which had an EC50 of 4.5–16.5 μg·mL-1 ; compound 11c displayed the highest fungicidal activity. The EC50 values of compound 11c, azoxystrobin, and carbendazim were 4.5, 4.3, and 0.123 μg·mL-1 , respectively. The results showed that compound 11c exhibited a roughly similar level of antifungal activity to azoxystrobin and a level that was 37- fold greater than that of carbendazim.
3.2. CoMFA studies
3D-QSAR is widely used in the drug and pesticide discovery process to describe the SARs of compounds. To investigate the substituent effect on C. lunata inhibitory activity, a CoMFA model for the 28 compounds was developed. The conventional coefficient r 2 of the CoMFA model was 0.9816, the cross-validated coefficient q2 was 0.8060, and the predicted noncross-validated coefficient r 2 (pred) was 0.9693. The plots of the predicted inhibitory activities against C. lunata versus the experimental values are shown in Fig. 3(a), and the alignment result of the 22 training set compounds is shown in Fig. 3(b). The steric field contour maps are shown in green and yellow (Fig. 3(c)). The yellow polyhedra shows that bulky substituents at these sites were detrimental to activity. For example, when an ester group or alkoxycarbonyl was introduced, compounds 9a–9i showed lower activity than their parent compound 11n; the same trend can be found in compounds 10a– 10g. In comparison, the green polyhedra indicates that small substituents are favorable to activity. The electrostatic contour maps are shown in Fig. 3(d), where the blue contours indicate that positive charges in these areas will increase inhibitory activity against C. lunata. For example, compounds with a halogen atom at the 4- position of the benzene ring displayed higher activity than 11n; thus, introducing a bromine atom to 11n would significantly increase its bioactivity (i.e., 11c > 11n, 11f > 11n, and 11i > 11n). The red contours indicate that negative charges in these areas are unfavorable to activity, which supported the finding that compound 11j, which contains cyano groups, would display decreased activity (i.e., 11n > 11j).
Fig. 3. CoMFA calculations. (a) CoMFA model predicted values versus experimental inhibitory values. (b) Structural alignment of 22 training set compounds. (c) Contour map of steric contribution, where compound 11c is shown inside the field. The yellow polyhedra indicates that sterically bulky substituents are detrimental to activity, while the green polyhedra shows that sterically bulkier substituents are favorable to activity. (d) Contour map of electrostatic contribution, where compound 11c is shown inside the field. Blue contours indicate that positive charges in these areas will increase the activity; red contours indicate that negative charges in these areas will decrease the activity.

4. Conclusions

In summary, a series of 3-acylthiotetronic acid derivatives 9a– 9i, 10a–10 g, 11a–11 m, and 12a were designed and synthesized in the present study. The biological assay results indicated that most of the target compounds possessed in vitro antifungal activities toward the fungal pathogens V. mali, C. lunata, F. graminearum, and F. oxysporum f. sp. lycopersici. Compounds 11c and 11i displayed broad-spectrum fungicidal activity with respective EC50 values of 1.9–10.7 and 3.1–7.8 μg·mL-1 against the tested four fungal species. The results of the bioassays and QSAR studies indicated that bulky substituents at para-position of benzene ring could significantly decrease the antifungal activities of the target compounds. It was noteworthy that the introduction of a halogen atom at the benzene ring of benzylidene could improve the activity against the tested fungi. Further studies on the structural optimization and biological evaluation of the compounds are in progress.

Acknowledgements

This work was financially supported in part by the National Natural Science Foundation of China (31901906), the Opening Project of Shanghai Key Laboratory of Chemical Biology, the Natural Science Foundation of Anhui Province, China (1808085QC71), the Natural Science Foundation of Anhui Education Department (KJ2016A834), and the US Department of Agriculture (USDA: HAW5032-R).

Compliance with ethics guidelines

Pei Lv, Yiliang Chen, Dawei Wang, Xiangwei Wu, Qing X. Li, and Rimao Hua declare that they have no conflict of interest or financial conflicts to disclose.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.eng.2019.10.016.

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