Design, Synthesis, and Antifungal Activities of 3-Acyl Thiotetronic Acid Derivatives: New Fatty Acid Synthase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.7b05491

Emerging fungal phytodiseases are increasingly becoming a food security threat. Twenty-six new 3-acylthiotetronic acid derivatives were designed, synthesized, characterized, and evaluated for activities against Valsa Mali, Curvularia lunata, Fusarium graminearum, and Fusarium oxysporum f. sp. lycopersici. Among the 26 compounds, 6f was the most effective against V. Mali, C. lunata, F. graminearum, and F. oxysporum f. sp. lycopersici with median effective concentrations (EC₅₀) of 4.1, 3.1, 3.6, and 4.1 μg/mL, respectively, while the corresponding EC₅₀ were 0.14, 6.7, 22.4, and 4.3 μg/mL of the fungicide azoxystrobin; 4.2, 41.7, 0.42, and 0.12 μg/mL of the fungicide carbendazim; and >50, 0.19, 0.43, and BS > 50 μg/mL of the fungicide fluopyram. The inhibitory potency against V. Mali fatty acid synthase agreed well with the in vitro antifungal activity. The molecular docking suggested that the 3-acylthiotetronic acid derivatives targeted the C171Q KasA complex. The findings help understanding the mode of action and design and synthesis of novel potent fungicides.

Full text of DOI:10.1021/acs.jafc.7b05491

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Article
Design, Synthesis and Antifungal Activities of 3-Acyl thiotetronic
Acid Derivatives: New Fatty Acid Synthase Inhibitors
Pei Lv, Yiliang Chen, Zheng Zhao, Taozhong Shi, Xiangwei Wu, Jiaying Xue, Qing X. Li, and Rimao Hua
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05491 • Publication Date (Web): 01 Jan 2018
Downloaded from http://pubs.acs.org on January 1, 2018
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Design, Synthesis and Antifungal Activities of 3-Acyl Thiotetronic Acid
Derivatives: New Fatty Acid Synthase Inhibitors
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Pei Lv,† Yiliang Chen,† Zheng Zhao,‡ Taozhong Shi,† Xiangwei Wu,† Jiaying, Xue,† Qing X. Li,
†
†
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§ Rimao Hua*,
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†
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Key Laboratory of Agriꢀfood Safety of Anhui Province, School of Resource & Environment,
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Anhui Agricultural University, 130 Changjiangxi Road, Hefei, Anhui 230036, China
‡ High Magnetic Field Laboratory, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei,
Anhui 230031, China
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§ Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa,
1955 EastꢀWest Road, Honolulu, Hawaii 96822, United States
*Corresponding author: Tel: +86 551 65786320; Fax: +86 551 65786296; Eꢀmail:
rimaohua@ahau.edu.cn
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ABSTRACT. Emerging fungal phytodiseases are increasingly becoming a food security threat.
Twentyꢀsix new 3ꢀacylthiotetronic acid derivatives were designed, synthesized, characterized,
and evaluated for activities against Valsa mali, Curvularia lunata, Fusarium graminearum and
Fusarium oxysporum f. sp. lycopersici. Among the 26 compounds, 6f was the most effective
against V. mali, C. lunata, F. graminearum and F. oxysporum f. sp. lycopersici with median
effective concentrations (EC50) of 4.1, 3.1, 3.6 and 4.1 ꢁg/mL, respectively, while the
corresponding EC50 were 0.14, 6.7, 22.4, and 4.3 ꢁg/mL of the fungicide azoxystrobin, 4.2, 41.7,
0.42 and 0.12 ꢁg/mL of the fungicide carbendazim, and >50, 0.19, 0.43, >50 ꢁg/mL of the
fungicide fluopyram. The inhibitory potency against V. mali fatty acid synthase agreed well with
the in vitro antifungal activity. The molecular docking suggested that the 3ꢀacylthiotetronic acid
derivatives targeted the C171Q KasA complex. The findings help understanding the mode of
action and design and synthesis of novel potent fungicides.
Keywords: Thiotetronic acid; Fatty acid synthase; Antifungal activity; Fungicide
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INTRODUCTION
Natural products have long been used as pesticides and have often served as a source of
inspiration in the discovery of commercial synthetic pesticides as they could aid in identification
of new mode of action and be used as lead structures to derive new potent pesticides.1ꢀ7 The
heterocyclic cores of tetronic acids (2,4ꢀfuranodione), thiotetronic acid (2,4ꢀthiophenedione) and
tetramic acids (pyrrolidineꢀ2,4ꢀdione) have attracted significant attention over the years due to
their occurrence as naturally bioactive products isolated from bacteria, molds, algae, fungi,
lichens and sponges (Figure 1),8 which display a wide range of biological functions. Tetronic
acids are antioxidant,9 antiepileptic,10 antitumor,11 antiꢀinflammatory,12,13 and insecticidal.14
Tetramic acids exhibit antibacterial,15 antiviral,16 anticancer,17 and herbicidal activities.18
Thiolactomycin is a unique thiolactone and specifically inhibits biosynthesis of fatty acids and
mycolic acid, by which exhibits antiꢀmycobacterial activities.19, 20
Fatty acid synthesis in fungi is compartmentalized in the large multifunctional fatty acid
synthases (FAS). FAS function as molecular assembly lines and increase the time of synthesis by
channeling substrates between the active sites and achieving high local concentrations of
intermediates.21ꢀ23 In fatty acid synthesis, extension of a growing fatty acyl chain by two carbons
at a time is initiated by the coupling of a malonylated acyl carrier protein (ACP) with a thioester
linked acyl group at the active site of βꢀketoacyl synthase (KAS).24 There are two tandem
KASꢀtype enzymes, KasA and KasB. KasA specifically elongates palmitoylꢀCoA to C38ꢀ42
monounsaturated acyl chains, whereas KasB extends up to 54 carbons on average in the presence
of KasA to produce longer chain multi unsaturated hydrocarbons.19, 25 In previous studies, the
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acylated enzyme intermediate was mimicked by a C171Q KasA variant in which Glnꢀ171 forms
hydrogen bonds with the KasA oxyanion hole.26ꢀ28 Considering the importance of fatty acids in
fungal survival, this acyl enzyme mimic could be particularly important for the design of new
antiꢀfungal agents to inhibit KAS by binding preferentially to the acylꢀenzyme.
In our efforts to screen for agricultural fungicides from natural products, attempts have been
made to test whether modulation of the five membered ring systems could lead to high fungicidal
activity. Based on the proven potencies of tetramic acid, tetronic acid, thiotetronic acid natural
products and the mode of action of thiotetronic acid antibiotics inhibiting fattyꢀacid
synthases,19,20 26 novel thiotetronic derivatives were designed and synthesized targeting KasA
and KasB to inhibit the biosynthesis of fatty acids and mycolic acid and to reveal the structure
and fungicidal activity information. In addition, docking simulations were performed on the basis
of the Xꢀray crystallographic structure of the C171Q KasA to explore the binding mode of the
compounds in the active site.
MATERIALS AND METHODS
Chemicals. Azoxystrobin (98%) and carbendazim (98%) were purchased from J&K
Chemical Ltd. (Shanghai, China). Fluopyram (99%) was purchased from Anpel Laboratory
Technologies, Inc. (Shanghai, China). Azoxystrobin, carbendazim and fluopyram are commercial
fungicides and were used as positive controls. The other reagents were all purchased from J&K
Chemical and were analytically or chemically pure. All solvents and liquid reagents were dried
by standard methods in advance and distilled prior to use.
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Fungi. The plant pathogenic fungi, Valsa mali, Curvularia lunata, and Fusarium
graminearum, and Fusarium oxysporum f. sp. lycopersici were kindly provided by the
Department of Plant Pathology, Anhui Agricultural University, China. These fungi were grown
on potato dextrose agar (PDA) plates at 28 °C and maintained at 4 °C with periodic
subꢀcultivations.
Instruments. 1H NMR and 13C NMR spectra were recorded on an Agilent 600M DD2
(Agilent Technologies Co. Ltd., Shanghai, China) spectrometer at 25 °C and referenced to
tetramethylsilane (Me4Si). Chemical shifts were reported in ppm (δ) using the residual solvent
line as an internal standard. The purity was determined on an Agilent LCꢀMS Symmetry 3 under
a gradient elution of 5ꢀ90% aqueous acetonitrile containing 0.05% trifluoacetic acid (TFA) over
8 min (10 min run time) at a flow rate of 2 mL/min. The column used was a 50 mm x 4.6 mm i.d.,
5 µM, C18 column (Agilent). High resolution mass spectrometry (HRMS) data were obtained on
a Varian QFTꢀESI instrument (Varian, Inc., Alto Palo, CA). Melting points were determined on a
XT4 MP apparatus (Taike Corp., Beijing, China) and were unꢀcorrected. Analytical thinꢀlayer
chromatography (TLC) was performed on glassꢀbacked silica gel GF254 sheets (Yantai Jiangyou
Silica Gel Development Co. Ltd., China). All compounds were detected at 254 nm or 365 nm.
Synthesis. The synthetic route of the target compounds 5a-5d, 6a-6h, 7a-7e and 8a-8i was
outlined in Figure 2 using thioacetic acid as a starting material.
Synthesis of 2-(acetylthio)acetic acid, 2. A mixture of pꢀdioxane (200 mL) and Et3N (161 g,
1.58 mol) was chilled in an iceꢀwater bath to ca. 10 °C. To this solution thioacetic acid (120 g,
1.58 mol) was added dropwise while the temperature was maintained between 10ꢀ15 °C. The
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resulting brick red solution of thiolacetic acid and triethylamine (Et3N) was added dropwise to a
solution of bromoacetic acid (220 g, 1.58 mol) in pꢀdioxane (500 mL) with vigorous stirring. A
resulting precipitate of triethylamine hydrobromide slowly formed. During the course of addition,
the reaction mixture became warm and was stirred at ambient temperature for 16 h. The reaction
mixture was filtered to remove Et3NHBr. The filtrate was evaporated and the residue was
dissolved in saturated aqueous K2CO3. The aqueous K2CO3 was washed twice with diethyl ether
(Et2O). The aqueous phase was separated and acidified cautiously to pH 3 by the dropwise
addition of concentrated HCl. The product was extracted several times into Et2O. The combined
Et2O extract was washed with H2O and brine, dried over MgSO4, filtered, and concentrated. The
residue was distilled under reduced pressure (1 Torr), and the fraction boiling between 118 and
122 °C was collected to afford pure 2ꢀ(acetylthio)acetic acid, 2, as a pale yellow oil. Yield, 90%;
yellow oil; 1H NMR (600 MHz, CDCl3) δ: 3.76 (s, 2H), 2.43 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ: 193.9, 174.7, 31.3, 30.1.
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Synthesis of 3-acetyl-4-hydroxythiophen-2(5H)-one, 4. Oxalyl chloride (5.44 g, 55.5 mmol,
1.5 equiv) was added dropwise over 1 h at 0 °C to a solution of 2ꢀ(acetylthio)acetic acid 2 (5.00
g, 37 mmol) and DMF (0.38 mL, 4.82 mmol) in anhydrous CH2Cl2 (100 mL). The mixture was
stirred at ambient temperature for 8 h. After solvent removal, the crude oil was distilled at 7 mbar
to give pure 2ꢀ(acetylthio)acetic chloride as an orange oil. The acyl chloride (5.65 g, 37 mmol)
was then added to a solution of toluene (80 mL) and cooled to 0 °C by means of an ice bath.
Methyl acetoacetate sodium salt (15.33 g, 111 mmol) was then added dropwise, while the
temperature was maintained between 5ꢀ10 °C for 3 h. The final slurry was washed with 1 M
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aqueous HCl (3 × 100 mL). The toluene layer was extracted with dilute Na2CO3 (3 × 100 mL)
aqueous solution. To the aqueous phase was added NaOH (2.40 g, 60 mmol). The solution was
kept overnight. The aqueous phase was then acidified cautiously to pH 3 by the dropwise
addition of concentrated HCl. The precipitated crude product was extracted with Et2O several
times. The combined Et2O extract was concentrated in vacuo and purified by silica gel column
chromatography (EtOAc/MeOH, 15:1, v/v) (Rf 0.4) to give the pure intermediate 4. Yield, 67%;
white solid; m.p. 82ꢀ83 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 4.17 (s, 2H), 2.42 (s, 3H). 13C
NMR (150 MHz, DMSOꢀd6) δ: 197.0, 194.7, 193.2, 110.8, 35.4, 25.4; HRMS [ESI] m/z: calcd
for ([MꢀH]ꢀ): 156.9960; found: 156.9965.
General procedure for the synthesis of compounds, 5a-5d.
To a solution of 158 mg (1 mmol) of the intermediate 4, PPh3 (314 mg, l.2 mmol) and the
corresponding alcohol (1.2 mmol) were dissolved in 10 mL of dry THF, followed by addition of
0.19 ml (1.2 mmol) of diisopropyl azodicarboxylate in 5 mL of THF. The reaction was stirred
overnight at room temperature and then concentrated in vacuo. The residue was purified by a
silica gel column with hexane/EtOAc (2:1, v/v) as the eluant. The crude product was then
recrystallized from hexane to give 5a-5d.
3-Acetyl-4-ethoxythiophen-2(5H)-one, 5a. Yield, 56%; green solid; m.p. 82ꢀ83 °C; 1H NMR
(600 MHz, CDCl3) δ: 4.33 (q, J = 6.9 Hz, 2H), 4.01 (s, 2H), 2.45 (s, 3H), and 1.51 (t, J = 6.9 Hz,
3H). 13C NMR (150 MHz, CDCl3) δ: 193.8, 193.5, 192.2, 118.1, 67.8, 31.4, 30.8, and 14.8;
HRMS [ESI] m/z: calcd for ([M+H]+), 187.0429; found: 187.0432.
3-Acetyl-4-isopropoxythiophen-2(5H)-one, 5b. Yield, 60%; green solid; m.p. 76ꢀ78 °C; 1H
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NMR (600 MHz, CDCl3) δ: 4.84 (m, 1H), 4.02 (s, 2H), 2.43 (s, 3H), and 1.45 (d, J = 6.1 Hz, 6H).
13C NMR (150 MHz, CDCl3) δ: 193.5, 192.0, 182.7, 118.3, 75.8, 31.2, 30.9, and 22.7; HRMS
[ESI] m/z: calcd for ([M+H]+), 201.0585; found: 201.0592.
3-Acetyl-4-propoxythiophen-2(5H)-one, 5c. Yield, 73%; green solid; m.p. 72ꢀ73 °C；1H
NMR (600 MHz, CDCl3) δ: 4.21 (t, J = 6.4 Hz, 2H), 4.01 (s, 2H), 2.44 (s, 3H), 1.91 – 1.84 (m,
2H), and 1.06 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ: 193.5, 192.1, 183.9, 117.9,
73.3, 31.4, 30.8, 22.6, and 10.2; HRMS [ESI] m/z: calcd for ([M+H]+), 201.0585; found:
201.0592.
3-Acetyl-4-(benzyloxy)thiophen-2(5H)-one, 5d. Yield, 45%; green solid; m.p. 94ꢀ95 °C; 1H
NMR (600 MHz, CDCl3) δ: 7.40 (dt, J = 14.0, 7.4 Hz, 5H), 5.34 (s, 2H), 4.03 (s, 2H), and 2.43 (s,
3H). 13C NMR (150 MHz, CDCl3) δ: 193.8, 192.3, 183.1, 134.0, 129.1, 127.2, 118.8, 110.0, 73.1,
31.7, and 30.9; HRMS [ESI] m/z: calcd for ([M+H]+), 249.0585; found: 249.0587.
General Procedure for the Preparation of Compounds 6a-6h
A solution of 1.58 g (10 mmol) of 3ꢀacetyltetrahydrothiopheneꢀ2,4ꢀdione, 4, 11 mmol of the
corresponding aromatic aldehyde, and 30 mg of pꢀtoluenesulfonic acid in 50 mL of toluene was
heated under reflux until water no longer separated as an azeotrope (10ꢀ12 h, TLC). The mixture
was cooled to room temperature. The precipitate (3ꢀacetylꢀ5ꢀbenzylidene derivatives), 6a-6h,
was filtered off and recrystallized from MeOH/EtOAc.
3-Acetyl-5-benzylidene-4-hydroxythiophen-2(5H)-one, 6a. Yield, 42%; yellow solid; m.p.
151ꢀ152 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 7.81 (s, 1H), 7.65 (d, J = 7.6 Hz, 2H), 7.50 (t, J =
7.5 Hz, 2H), 7.45 (t, J = 7.3 Hz, 1H), and 2.47 (s, 3H), 13C NMR (150 MHz, DMSOꢀd6) δ: 195.9,
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187.1, 186.0, 134.2, 131.1 (2C), 130.8, 130.7, 129.7 (2C), 127.3, 108.2, and 26.7; HRMS [ESI]
m/z: calcd for ([MꢀH]ꢀ), 245.0272; found: 245.0275.
3-Acetyl-4-hydroxy-5-(4-nitrobenzylidene)thiophen-2(5H)-one, 6b. Yield, 65%; yellow solid;
m.p. 204ꢀ205 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 8.28 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.8 Hz,
2H), 7.69 (s, 1H), and 2.39 (s, 3H). 13C NMR (150 MHz, DMSOꢀd6) δ: 193.2, 186.2, 185.9,
147.1, 141.6, 135.1, 131.3 (2C), 124.6, 124.5 (2C), 106.8, and 27.7; HRMS HRMS [ESI] m/z:
calcd for ([MꢀH]ꢀ), 290.0123; found: 290.0128.
3-Acetyl-5-(4-fluorobenzylidene)-4-hydroxythiophen-2(5H)-one, 6c. Yield, 53%; yellow
solid; m.p. 181ꢀ183 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 7.79 (s, 1H), 7.71 (dd, J = 8.6, 5.6 Hz,
2H), 7.35 (t, J = 8.8 Hz, 2H), and 2.46 (s, 3H). 13C NMR (150 MHz, DMSOꢀd6) δ: 195.6, 186.9,
186.0, 164.0, 162.4, 133.5, 131.0, 129.4, 127.4, 116.9, 116.7, 108.0, and 26.8; HRMS [ESI] m/z:
calcd for ([MꢀH]ꢀ), 263.0178; found: 263.0180.
3-Acetyl-4-hydroxy-5-(4-methoxybenzylidene)thiophen-2(5H)-one, 6d. Yield, 56%; yellow
solid; m.p. 186ꢀ187 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 7.79 (s, 1H), 7.63 (d, J = 8.7 Hz, 2H),
7.08 (d, J = 8.8 Hz, 2H), 3.81 (s, 3H), and 2.46 (s, 3H). 13C NMR (150 MHz, DMSOꢀd6) δ: 187.1,
185.9, 161.5, 133.3, 131.2, 126.8, 115.4, 108.1, 56.0, and 27.1; HRMS [ESI] m/z: calcd for
([MꢀH]ꢀ), 275.0378; found: 275.0381.
3-Acetyl-5-(4-chlorobenzylidene)-4-hydroxythiophen-2(5H)-one, 6e. Yield, 45%; yellow
solid; m.p. 172ꢀ174 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 7.73 (s, 1H), 7.65 (d, J = 8.5 Hz, 2H),
7.55 (d, J = 8.5 Hz, 2H), and 2.44 (s, 3H). 13C NMR (150 MHz, DMSOꢀd6) δ: 195.0, 186.7,
186.0, 135.0, 133.4, 132.5, 129.7, 129.1, 128.4, 107.8, and 27.0; HRMS [ESI] m/z: calcd for
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([MꢀH]ꢀ), 278.9883; found: 278.9886.
3-Acetyl-4-hydroxy-5-(4-(trifluoromethyl)benzylidene)thiophen-2(5H)-one, 6f. Yield, 55%;
yellow solid; m.p. 162ꢀ164 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 7.84–7.79 (m, 4H), 7.71 (s,
1H), and 2.40 (s, 3H). 13C NMR (150 MHz, DMSOꢀd6) δ: 193.8, 186.5, 186.0, 138.9, 132.8,
131.1, 129.3, 126.3, 126.0, 110.0, 107.2, and 27.5; HRMS [ESI] m/z: calcd for ([MꢀH]ꢀ),
313.0146; found: 313.0149.
4-((4-Acetyl-3-hydroxy-5-oxothiophen-2(5H)-ylidene)methyl)benzonitrile, 6g. Yield, 54%;
yellow solid; m.p. 214ꢀ215 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 7.91 (d, J = 8.2 Hz, 2H), 7.76
(d, J = 8.2 Hz, 2H), 7.70 (s, 1H), and 2.42 (s, 3H). 13C NMR (150 MHz, DMSOꢀd6) δ: 194.0,
186.4, 186.0, 139.3, 133.2, 133.1 (2C), 126.3, 119.0, 111.5, 107.2, and 27.2; HRMS [ESI] m/z:
calcd for ([MꢀH]ꢀ), 270.0225; found: 271.0230.
3-Acetyl-4-hydroxy-5-(4-methylbenzylidene)thiophen-2(5H)-one, 6h. Yield, 57%; yellow
solid; m.p. 182ꢀ183 °C; 1H NMR (600 MHz, DMSOꢀd6) δ: 7.72 (s, 1H), 7.53 (d, J = 7.5 Hz, 2H),
7.31 (d, J = 7.4 Hz, 2H), 2.43 (s, 3H), and 2.33 (s, 3H). 13C NMR (150 MHz, DMSOꢀd6) δ: 186.0,
187.0, 140.6, 131.8, 131.1 (2C), 130.3 (2C), 129.8, 27.3, and 21.5; HRMS [ESI] m/z: calcd for
([MꢀH]ꢀ), 259.0429; found: 259.0431.
General procedure for the synthesis of 7a-7e
A stirred solution of 1 mmol of 6b or 6d, 1.2 mmol of the corresponding alcohol and 314 mg
(l.2 mmol) of PPh3 was dissolved in 10 mL of dry THF, followed by addition of 0.19 mL (1.2
mmol) of diisopropyl azodicarboxylate in 5 mL of THF. The reaction mixture was stirred
overnight at room temperature and then concentrated in vacuo. The residue was purified by a
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silica gel column eluted with hexane/EtOAc (6:1, v/v). The crude product was then recrystallized
from hexane to give 7a-7e.
3-Acetyl-4-methoxy-5-(4-methoxybenzylidene)thiophen-2(5H)-one, 7a. Yield, 65%; yellow
solid; m.p. 103ꢀ104 °C; 1H NMR (600 MHz, CDCl3) δ: 7.58 (s, 1H), 7.50 (d, J = 8.7 Hz, 2H),
6.96 (d, J = 8.8 Hz, 2H), 4.04 (s, 3H), 3.86 (s, 3H), and 2.58 (s, 3H). 13C NMR (150 MHz,
CDCl3) δ: 196.4, 189.5, 175.3, 161.2, 132.6 (2C), 131.3, 127.0, 124.2, 116.7, 114.7 (2C), 63.4,
55.5, and 31.6; HRMS [ESI] m/z: calcd for ([M+H]+), 291.0691; found: 291.0693.
3-Acetyl-4-ethoxy-5-(4-nitrobenzylidene)thiophen-2(5H)-one, 7b. Yield, 57%; yellow solid;
m.p. 156ꢀ158 °C; 1H NMR (600 MHz, CDCl3) δ: 8.28 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz,
2H), 7.61 (s, 1H), 4.25 (q, J = 7.0 Hz, 2H), 2.58 (s, 3H), and 1.49 (t, J = 7.0 Hz, 3H). 13C NMR
(150 MHz, CDCl3) δ: 196.3, 188.2, 172.7, 147.6, 140.5, 132.2, 130.8 (2C), 127.3, 124.1 (2C),
117.6, 72.8, 31.6, and 15.2; HRMS [ESI] m/z: calcd for ([M+H]+), 320.0592; found: 320.0594.
3-Acetyl-5-(4-methoxybenzylidene)-4-propoxythiophen-2(5H)-one, 7c. Yield, 62%; yellow
solid; m.p. 104ꢀ106 °C; 1H NMR (600 MHz, CDCl3) δ: 7.58 (s, 1H), 7.56–7.48 (m, 2H),
7.02–6.93 (m, 2H), 4.11 (t, J = 6.4 Hz, 2H), 3.85 (s, 3H), 2.56 (s, 3H), 1.91 – 1.80 (m, 2H), and
1.05 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ: 196.4, 189.5, 174.7, 161.1, 132.6 (2C),
131.0, 127.0, 124.6, 116.8, 114.6 (2C), 78.2, 55.5, 31.5, 23.1, and 10.4; HRMS [ESI] m/z: calcd
for ([M+H]+), 319.1004; found: 319.1006.
3-Acetyl-4-butoxy-5-(4-nitrobenzylidene)thiophen-2(5H)-one, 7d. Yield, 58%; yellow solid;
m.p. 108ꢀ109 °C; 1H NMR (600 MHz, CDCl3) δ: 8.28 (d, J = 8.6 Hz, 2H), 7.68 (d, J = 8.7 Hz,
2H), 7.59 (s, 1H), 4.18 (t, J = 6.4 Hz, 2H), 2.58 (s, 3H), 1.88 – 1.79 (m, 2H), 1.57 – 1.45 (m, 2H),
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and 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ: 196.3, 188.2, 173.0, 147.6, 140.5,
132.3, 130.8 (2C), 127.2, 124.1 (2C), 117.5, 76.7, 31.6 (2C), 19.1, and 13.7; HRMS [ESI] m/z:
calcd for ([M+H]+), 348.0905; found: 347.0908.
3-Acetyl-4-(benzyloxy)-5-(4-nitrobenzylidene)thiophen-2(5H)-one, 7e. Yield, 63%; yellow
solid; m.p. 101ꢀ102 °C; 1H NMR (600 MHz, CDCl3) δ: 8.27 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 8.7
Hz, 2H), 7.59 (s, 1H), 7.42 (t, J = 6.4 Hz, 3H), 7.38 – 7.33 (m, 2H), 5.29 (s, 2H), and 2.45 (s,
3H). 13C NMR (150MHz, CDCl3) δ: 196.3, 188.2, 172.4, 147.6, 140.4, 134.3, 132.2, 130.9 (2C),
129.9, 129.2, 129.0 (2C), 128.3 (2C), 128.1, 127.8, 124.1 (2C), 118.3, 77.7, and 31.4; HRMS
[ESI] m/z: calcd for ([M+H]+), 382.0749; found: 382.0752.
General procedure for the synthesis of Compounds 8a-8i
A mixture of 6a-6d (1 mmol), the corresponding aromatic amine (1.1 mmol), TsOH (30 mg)
in 40 mL methanol was heated at 65 °C for 4 h. After cooling, the precipitated product was
filtered and recrystallized from a mixture of methanol and chloroform (1:1) to afford 8aꢀ8i.
5-Benzylidene-3-(1-(4-(trifluoromethyl)phenylamino)ethylidene)
thiophene-2,4(3H,5H)-dione , 8a. Yield, 80%; yellow solid; m.p. 202ꢀ203 °C; 1H NMR (600
MHz, CDCl3) δ: 13.76 (s, 1H), 7.76 (d, J = 8.8 Hz, 3H), 7.60 (d, J = 7.8 Hz, 2H), 7.45 (t, J = 7.6
Hz, 2H), 7.37 (d, J = 7.9 Hz, 3H), and 2.66 (s, 3H). 13C NMR (150 MHz, CDCl3) δ: 189.7, 189.2,
168.8, 138.8, 134.5, 130.5, 130.3, 129.4, 129.0, 128.5, 127.0, 126.1, 125.9, 124.4, 122.6, 102.3,
and 16.9. HRMS [ESI] m/z: calcd for ([M+H]+), 390.0775; found: 390.0778.
5-Benzylidene-3-(1-(4-methoxyphenylamino)ethylidene)thiophene-2,4(3H,5H)-dione, 8b.
Yield, 75%; yellow solid; m.p. 182ꢀ184 °C; 1H NMR (600 MHz, CDCl3) δ: 13.76 (s, 1H), 7.73 (s,
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1H), 7.59 (d, J = 7.6 Hz, 2H), 7.44 (t, J = 7.7 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1H), 7.13 (d, J = 8.6
Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H), 3.85 (s, 3H), and 2.57 (s, 3H). 13C NMR (150 MHz, CDCl3) δ:
189.5, 189.2, 169.4, 159.4, 134.8, 130.4, 130.0, 129.1, 128.8, 128.1, 127.7, 126.8, 114.9, 101.6,
55.6, and 16.8; HRMS [ESI] m/z: calcd for ([M+H]+), 352.1007; found: 352.1010.
5-(4-Nitrobenzylidene)-3-(1-(phenylamino)ethyl)thiophene-2,4(3H,5H)-dione, 8c. Yield,
78%; yellow solid; m.p. 254ꢀ256 °C; 1H NMR (600 MHz, CDCl3) δ: 13.79 (s, 1H), 8.27 (d, J =
8.4 Hz, 2H), 7.70 (t, J = 9.3 Hz, 3H), 7.51 (t, J = 7.4 Hz, 2H), 7.44 (t, J = 7.2 Hz, 1H), 7.24 (q, J
= 7.8 Hz, 2H), and 2.63 (s, 3H). 13C NMR (150 MHz, CDCl3) δ: 189.0, 187.8, 169.6, 147.1,
141.2, 135.3, 134.7, 130.7, 129.8, 128.7, 125.6, 125.2, 124.5, 124.0, 101.3, and 16.9; HRMS
[ESI] m/z: calcd for ([M+H]+), 369.0909; found: 369.0911.
5-(4-Fluorobenzylidene)-3-(1-(4-(trifluoromethyl)phenylamino)ethylidene)thiophene-2,4(3H,
5H)-dione, 8d. Yield, 85%; m.p. 181ꢀ183 °C; yellow solid; 1H NMR (600 MHz, CDCl3) δ: 14.05
(s, 1H), 7.76 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 9.3 Hz, 1H), 7.61 – 7.51 (m, 2H), 7.36 (t, J = 7.5
Hz, 2H), 7.13 (t, J = 8.5 Hz, 2H), and 2.66 (s, 3H). 13C NMR (150 MHz, CDCl3) δ: 189.6, 188.8,
168.9, 162.2, 138.8, 132.4, 132.1, 130.8, 129.2, 127.2, 127.0, 125.9, 116.2, 116.0, 102.2, and
16.9; HRMS [ESI] m/z: calcd for ([M+H]+), 408.0681; found: 408.0685.
5-(4-Fluorobenzylidene)-3-(1-(phenylamino)ethylidene)thiophene-2,4(3H,5H)-dione, 8e.
Yield, 88%; yellow solid; m.p. 175ꢀ176 °C; 1H NMR (600 MHz, CDCl3) δ: 13.89 (s, 1H), 7.70
(d, J = 9.5 Hz, 1H), 7.60 – 7.53 (m, 2H), 7.49 (t, J = 7.3 Hz, 2H), 7.41 (t, J = 7.2 Hz, 1H), 7.22 (d,
J = 7.5 Hz, 2H), 7.13 (t, J = 8.3 Hz, 2H), and 2.62 (d, J = 30.8 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δ: 189.5, 188.9, 169.2, 162.1, 135.5, 132.3, 132.1, 131.0, 129.8, 128.4, 126.6, 125.6,
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116.1, 115.9, 101.6, and 16.8; HRMS [ESI] m/z: calcd for ([M+H]+), 340.0807; found: 340.0810.
5-(4-Fluorobenzylidene)-3-(1-(4-fluorophenylamino)ethylidene)thiophene-2,4(3H,5H)-dione,
8f. Yield, 73%; yellow solid; m.p. 175ꢀ177 °C; 1H NMR (600 MHz, CDCl3) δ: 13.80 (s, 1H),
7.68 (d, J = 13.4 Hz, 1H), 7.56 (s, 2H), 7.19 (dd, J =10.3 Hz, 4H), 7.12 (s, 2H), and 2.58 (s, 3H).
13C NMR (150 MHz, CDCl3) δ: 189.5, 188.8, 169.5, 162.5, 132.3, 132.1, 131.5, 131.0, 129.4,
127.5, 126.8, 116.8, 116.0, 101.7, and 16.7; HRMS [ESI] m/z: calcd for ([M+H]+), 358.0713;
found: 358.0716.
5-(4-Fluorobenzylidene)-3-(1-(4-methoxyphenylamino)ethylidene)thiophene-2,4(3H,5H)-dio
ne, 8g. Yield, 65%; yellow solid; m.p. 164ꢀ165 °C; 1H NMR (600 MHz, CDCl3) δ: 13.73 (s, 1H),
7.67 (d, J = 13.4 Hz, 1H), 7.63 – 7.46 (m, 2H), 7.12 (t, J = 9.2 Hz, 4H), 6.97 (d, J = 8.1 Hz, 2H),
3.84 (s, 3H), and 2.58 (s, 3H). 13C NMR (150 MHz, CDCl3) δ: 189.4, 188.8, 169.4, 163.7, 162.0,
159.4, 133.3, 132.1, 131.1, 129.7, 128.1, 127.0, 126.8, 126.4, 116.1, 115.9, 114.9, 101.5, 55.6,
and 16.7; HRMS [ESI] m/z: calcd for ([M+H]+), 370.0913; found: 370.0915.
5-(4-Methoxybenzylidene)-3-(1-(4-methoxyphenylamino)ethylidene)thiophene-2,4(3H,5H)-d
ione, 8h. Yield, 79%; yellow solid; m.p. 156ꢀ157 °C; 1H NMR (600 MHz, CDCl3) δ: 13.95 (s,
1H), 7.71 (d, J = 8.8 Hz, 1H), 7.55 (t, J = 9.8 Hz, 2H), 7.48 (t, J = 7.7 Hz, 2H), 7.40 (t, J = 7.4
Hz, 1H), 7.21 (t, J = 7.0 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 3.85 (s, 3H), and 2.65 (s, 3H). 13C
NMR (150 MHz, CDCl3) δ: 189.6, 189.4, 169.0, 160.4, 135.6, 132.2, 132.0, 129.7, 128.3, 127.9,
127.4, 127.2, 125.8, 125.6, 114.4 (2C), 101.8, 55.4, and 16.8; HRMS [ESI] m/z: calcd for
([M+H]+), 382.1113; found: 382.1116.
5-(4-Methoxybenzylidene)-3-(1-(phenylamino)ethylidene)thiophene-2,4(3H,5H)-dione , 8i.
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Yield, 86%; yellow solid; m.p. 190ꢀ192 °C; 1H NMR (600 MHz, CDCl3) δ: 13.80 (s, 1H), 7.69 (s,
1H), 7.55 (d, J = 7.1 Hz, 2H), 7.13 (d, J = 6.4 Hz, 2H), 6.97 (s, 4H), 3.85 (s, 6H), and 2.59 (s,
3H). 13C NMR (150 MHz, CDCl3) δ: 189.6, 189.3, 169.2, 160.4, 159.3, 132.2, 128.2, 127.7,
127.5, 127.3, 127.0, 126.8, 114.8, 114.4, 101.7, 55.6, 55.4, and 16.7; HRMS [ESI] m/z: calcd for
([M+H]+), 352.1007; found: 352.1009.
Bioassays. Fungicidal activities of 5a-5d, 6a-6h, 7a-7e and 8a-8i were tested in vitro against
V. mali, C. lunata, F. graminearum, and F. oxysporum f. sp. lycopersici. Their inhibition (%) was
determined according to the mycelium growth rate method. The test fungi grown on PDA
medium slants were subcultured for 48 h in Petri dishes prior to testing and used for inoculation
of fungal strains on PDA plates. The test compound was completely dissolved in 0.5 mL of
DMSO and then added to 9.5 mL of sterile water. The resulting solution was mixed with 90 mL
of sterile molten PDA to obtain the final concentrations of 50 ꢁg/mL for the activity screening
test. PDA with different compounds was poured into 90 mm Petri dishes (15 mL/dish), on which
a 5 mm thick and 6 mm diameter disc of fungus cut from previously subcultured Petri dishes was
placed at the center of the semisolid medium after the medium in the plate was partially
solidified. The final concentration of DMSO was 0.5%, which was proven to have no significant
effect on the growth of target fungi. DMSO alone and the widely used commercial fungicides
azoxystrobin, fluopyram, carbendazim (50 ꢁg/mL) dissolved in DMSO were used as a solvent
control and positive controls, respectively. The DMSO concentration in both cases was 0.5% in
the culture medium. The dishes were kept in an incubator at 28 °C for 72 h. Each experiment was
carried out in triplicate. After the mycelia fully covered the control group Petri dishes, the
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diameters (in mm) of fungal colony in the treatment Petri dishes were measured with a caliper in
three different directions. The growth inhibition percentages were calculated according to the
following formula and expressed as means ± standard deviations (SD).
Inhibition rate (%) = [(dc − d0) − (ds − d0)]/(dc − d0) × 100
where d0 is the diameter of the fungus cut; dc is the diameter of a fungal colony in the solvent
control plates; and ds is the diameter of a fungal colony in the compoundꢀtreated plates.
Based on the results of in vitro antifungal activity, highly active compounds were further
determined for their median effective concentrations (EC50) according to the same method
described above. The stock solution was mixed with the autoclaved PDA medium to prepare a
set of media containing 0.625, 1.25, 2.5, 5, 10, and 20 ꢁg/mL of the test compound, while the
media containing 0.0781, 0.312, 1.25, 5, 20, and 50 ꢁg/mL of the positive control fungicide.
Similarly, 0.5% DMSO in culture medium was used as a solvent control. Each test was
performed in triplicate. EC50 values and their confidence intervals at 95% probability (95% CI)
were calculated with DPS software ver. 7.05 (Hangzhou Ruifeng Information Technology Co.,
Ltd., Hangzhou, China).
Fatty Acid Synthase Activity Assay. FAS activity was spectrophotometrically determined
by measuring the malonylcoenzyme A (CoA)–dependent oxidation of nicotinamide adenine
dinucleotide phosphate reduced form (NADPH) according to the method of Nepokroeff et al.29
Mycelia of V. mali were inoculated at 0.05 OD600 nm and grown on a reciprocal shaker (180 rpm,
25 °C) for 5 d in Sabouraud maltose broth (SMB) medium that contained different
concentrations (1.25, 2.5, 5, 10, 20, and 50 ꢁg/mL) of 6a, 6c, 6e, 6f. Mycelia were harvested by
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vacuum filtration and disrupted in liquid nitrogen using a mortar and pestle. The resultant
powder was resuspended to 10% w/v in mitochondrial extraction buffer (10 mM of KHPO4 pH
7.2, 10 mM of KCl, 10 mM of MgCl2, 0.5 M of sucrose, 0.2 mM of EDTA, 2 mM of PMSF).
The extract was centrifuged twice at 5000g, 4 °C and 10 min, and intact mitochondria were then
pelleted at 10000g for 20 min at 4 °C and resuspended in the same buffer. The supernatant was
used to evaluate the FAS activity at 340 nm (before/after malonylꢀCoA). The assay solution
without malonylꢀCoA served as a background for the assay. Each sample was assayed in
triplicate.
X-ray Diffraction. Single crystals of C15H13NO5S (7b) were grown from MeOH. A suitable
crystal was selected and mounted on a SuperNova, Dual, Cu at zero, AtlasS2 diffractometer
(Rigaku Oxford Diffraction, Tokyo, Japan). The crystal was kept at 290 (2) K during data
collection. Using Olex2,30 the structure was solved with the ShelXT31 structure solution program
using Direct Methods and refined with the ShelXL32 refinement package using Least Squares
minimization.
Molecular Modeling. Molecular docking of 6f to the C171Q KasA enzyme was performed
with the software YetiX 8.3.33 The enzyme domain with an active conformation (PDB code: 4c6u)
was used as the receptor structure for the docking experiments. 6f was constructed using BioX
4.6 34 and the atomic partial charges were calculated by AmberTools. The docking modes were
optimized by the directional Yeti force field.35
RESULTS AND DISCUSSION
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Chemistry. The synthetic route for 5a-5d, 6a-6h, 7a-7e and 8a-8i was outlined in Figure 2.
As an initial material, thioacetic acid 1 was reacted with bromoacetic acid to prepare
acetothioacetic acid 2.36 Treatment of acetothioacetic with oxalyl chloride followed by Claisen
condensation with methyl acetoacetate sodium salt produced acetylthio diketoester 3.37
Hydrolysis of acetylthio diketoester formed γꢀmercapto diketoester, followed by cyclization and
acidification to provide 3ꢀacetylꢀ4ꢀhydroxyꢀ5Hꢀfuranꢀ2ꢀones 4. The target compounds 6a-6h
were synthesized via the reaction of 4 with the corresponding substituted benzaldehydes in
toluene under reflux. The compounds 5a-5d and 7a-7e were prepared via the Mitsunobu reaction
of 4 and 6a-6e with appropriate alcohols. The compounds 8a-8i were synthesized by the
nucleophilic reaction of 6a-6e with an amine.
Crystal Structure of 7b. The crystal data of 7b were presented in Table 1. Figure 3 shows a
perspective view of 7b with the atomic labelling system.38 The result demonstrated that 6a-6e
adopt a Zꢀconfiguration rather than Eꢀconformation in the formation of double bond by
dehydration of aldol reaction products. However, crystal structures of 6a-6e could not be
obtained in the present study.
Antifungal Activity. Table 2 shows the in vitro antifungal activity of 5a-5d, 6a-6h, 7a-7e
and 8a-8i at the concentration of 50 ꢁg/mL against the fungi V. mali, C. lunata, F. graminearum,
and F. oxysporum f. sp. Lycopersici. All compounds possess antifungal activities in various
degrees against each of the tested fungi.
The commercial fungicides azoxystrobin, carbenzazim and fluopyram were used as positive
controls to compare the potency of the synthetic chemicals because they are commonly used to
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control the four fungal pathogens. Azoxystrobin is a systemic and broadꢀspectrum fungicide. It
prevents production of ATP by binding tightly to Complex III of the mitochondrial electron
transport chain.39 Carbendazim is also a broadꢀspectrum fungicide. It probably binds to tubulin
and suppresses microtubule assembly dynamic, although the exact mechanism of action is
unclear.40 Fluopyram inhibits the succinate dehydrogenase and is a broad spectrum fungicide
registered for use in USA in 2012 and in the EU in 2013.41 To each fungus, at the concentration
of 50 ꢁg/mL, some compounds were more active than the positive control of azoxystrobin.
Carbenzazim showed high activity against V. mali, C. lunata and F. graminearum except F.
oxysporum f. sp. Lycopersici, while fluopyram showed higher potency to C. lunata and F.
graminearum than V. mali and F. oxysporum f. sp. lycopersici. In order to further explore the
antifungal potential and structureꢀactivity relationship, the compounds with inhibition >50% at
50 ꢁg/mL were further examined to determine their EC50 against the four fungal strains (Table
3).
Tests of fungicidal activity (Table 2) indicated that at the concentration of 50 ꢁg/mL, 4 and
5a-5d showed low inhibitions (approximately < 35%) against V. mali, C. lunata, F. graminearum,
and F. oxysporum f. sp. lycopersici. The compounds 6aꢀ6h exhibited high activities (> 70%
inhibition at 50 ꢁg/mL), with EC50 of 3.1−20.3 ꢁg/mL against V. mali. In contrast, 6a, 6b, 6c and
6e, 6f at 50 ꢁg/mL inhibited greater than 83% against V. mali, while the inhibitions by
azoxystrobin, fluopyram and carbendazim were 81.6%, 6.0% and 97.9%, respectively. For C.
lunata, some of 6a-6h with EC50 of 3.1−42.2 ꢁg/mL showed higher activity than azoxystrobin
(EC50 = 6.7 ꢁg/mL) and carbendazim (EC50 = 41.2 ꢁg/mL) against C. lunata. Fluopyram showed
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the highest potency to C. lunata with an EC50 of 0.18 ꢁg/mL. Considering F. graminearum,
6a-6h had moderate potencies (35%ꢀ70% inhibition) at 50 ꢁg/mL, with EC50 of 3.1−46.6 ꢁg/mL.
6e showed the highest activity with an EC50 of 3.1 ꢁg/mL, while EC50 of fluopyram and
carbendazim were 0.43 and 0.42 ꢁg/mL, respectively. For F. oxysporum f. sp. lycopersici, 6aꢀ6h
exhibited high activities (> 70% inhibition at 50 ꢁg/mL), with EC50 of 4.1−16.0 ꢁg/mL. 6f
showed the highest activity with an EC50 value of 4.1 ꢁg/mL, being similar to azoxystrobin
(EC50 = 4.3 ꢁg/mL) and 33 fold higher than carbendazim (EC50 = 0.123 ꢁg/mL). Table 1 shows
that at a concentration of 50 ꢁg/mL, 7a-7d exhibited low (< 35%) to moderate inhibitions
(35%ꢀ70%) against the target fungi. It was noteworthy that 7e was highly active (73.4%
inhibition at 50 ꢁg/mL) against V. mali, whereas 8a-8i had weak inhibitions (approximately
<35%) against V. mali, C. lunata, F. graminearum and F. oxysporum f. sp. lycopersici.
Tables 2 and 3 show that the type and position of substituents on the 3ꢀacylthiotetronic acid
had significant effects on the activity. The general trend is that the presence of benzylidene at the
5ꢀposition increased the activity against the four target fungi, while 4ꢀalkoxy substituents, 5a-5d,
did not improve the activity in comparison with a hydroxyl moiety. Further optimization of 6, by
alkylation of 3ꢀacylthiotetronic acid at 4ꢀhydroxy and condensation of 3ꢀacetyl with amine of
3ꢀacylthiotetronic acid, led to a decrease of the activity against the four fungi. Studies on the
antifungal activities of 3ꢀacylthiotetronic acid derivatives could provide useful information for
evaluating the structureꢀactivity relationship. The 4ꢀhydroxyl and 3ꢀacetyl groups played an
important role in the fungicidal activity.
Comparison of 6a and 6b-6h, which contained different substituents on the benzene ring,
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indicated that the presence of a fluorine atom (6c) or a trifluoromethyl group (6f) led to higher
activity than 6a against V. mali, C. lunata, F. graminearum, while showing similar activities
against F. oxysporum f. sp. lycopersici. This result suggested that the presence of 4'ꢀF and CF3
improved the activity. The methylꢀsubstitution and methoxyꢀsubstitution reduced the activities
against all four fungi. The presence of ꢀCN group decreased the activities against V. mali, C.
lunata, F. graminearum. The introduction of ꢀCl atom improved the activity against both F.
graminearum and C. lunata, but had weak influence on the activity against V. mali and F.
oxysporum f. sp. lycopersici.
Inhibition of Fungal Fatty Acid Synthase. The synthetic compounds possess the scaffold
of thiolactone as thiolactomycin does, which inhibits fattyꢀacid synthases. The compounds 6a, 6c,
6e and 6f had EC50 of 4.4, 3.1, 4.5, and 4.1 ꢁg/mL, respectively, against V. mali. They were
selected and tested against FAS in vitro. As demonstrated in Figure 4, 6a, 6c, 6e and 6f strongly
inhibited FAS with an half maximum inhibition concentration (IC50) of 6.1, 5.3, 6.2 and 4.9
ꢁg/mL, respectively, and showed the similar inhibition profile as that using the mycelium growth
inhibition assay in vitro. The results proved the hypothesis and design of thiotetronic acid
derivatives as FAS inhibitors.
Molecular docking. Figure 5 shows the binding pose and features of 6f with the target
protein. First, the hydroxyl group on the thiophene ring may form two hydrogen bonds with the
nitrogen atoms from the imidazole of His311 and His345, while another hydrogen bond may be
formed between the 3ꢀacyl group with the nitrogen atom of His345. Therefore, 4ꢀhydroxy group
and 3ꢀacyl group of thiophene are likely the important part of the pharmacophore. Secondly, a
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πꢀπ stacking probably occurs between the thiophene ring and the benzyl ring of Phe404, which
plausibly explains the activity enhancement by the introduction of benzylidene at 5ꢀposition on
the thiophene ring. Thirdly, the hydrophobic benzyl trifluoride group may reside deeply at the
hydrophobic pocket, which provides a strong hydrophobic interaction. Additionally, a πꢀπ
stacking between the benzyl trifluoride group and the benzyl group of Phe402 further stabilized
the interaction. Such specific binding features reasonably made 6f a potent inhibitor to the target.
In summary, a series of novel 3ꢀacylthiotetronic acid derivatives were designed and
synthesized. Those compounds exhibited good to excellent fungicidal activities against the four
fungi. The compound 6f showed the highest fungicidal activity against the four tested fungal
pathogens with EC50 of 3.1ꢀ4.1 ꢁg/mL. The EC50 of the commercial fungicides azoxystrobin,
carbenzazim and fluopyram ranged between 0.12 and >50 ꢁg/mL against V. mali, C. lunata, F.
graminearum, and F. oxysporum f. sp. lycopersici. 6f and its analogs are potent FAS inhibitors.
6f is nonꢀselective to the four fungal species, whereas the commercial fungicides azoxystrobin,
carbenzazim and fluopyram is selective to one or two of the four tested fungal species. The
results revealed that the compounds are potent fungicide candidates, which could be further
optimized and developed as lead compounds.
Supporting Information
Characterization data, 1H NMR, 13C NMR spectra for products 5a-5d, 6a-6h, 7a-7e, 8a-8i and
XꢀRay diffraction data are provided. This material is available free of charge via the Internet at
http://pubs.acs.org.
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Funding
This project was supported in part by the National Key Research and Development Program of
China (No. 2016YFD0200205) and the Public Sector (Agriculture) Research Program of China
(201303088ꢀ11).
Conflicts of Interest
The authors declare no conflict of interest.
Author Contributions
P.L. and R.H. conceived and designed the experiments; Y.C. performed the experiments; Z.Z.
performed the molecular docking. T.S. and X.W. analyzed the data; P.L. wrote the paper. Q.L.
interpreted the data and revised the manuscript. J.X. revised the manuscript.
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