Synthesis and biological evaluation of novel benodanil-heterocyclic carboxamide hybrids as a potential succinate dehydrogenase inhibitors

Article abstract of DOI:10.3390/molecules25184291

In order to discover new antifungal agents, twenty novel benodanil-heterocyclic carboxamide hybrids were designed, synthesized, and characterized by ¹H NMR and HRMS. In vitro, their antifungal activities against four phytopathogenic fungi were

Full text of DOI:10.3390/molecules25184291

molecules
Article
Synthesis and Biological Evaluation of Novel
Benodanil-Heterocyclic Carboxamide Hybrids
as a Potential Succinate Dehydrogenase Inhibitors
Jian Yang, Yongtian Zhao, Jun Wan, Mingfang Jiang, Hong Jin * , Ke Tao and Taiping Hou
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences,
Sichuan University, Chengdu 610064, China; YJian_1210@163.com (J.Y.); zyttian@163.com (Y.Z.);
junwan88888888@163.com (J.W.); 18080776976@163.com (M.J.); taoke@scu.edu.cn (K.T.);
houtplab@scu.edu.cn (T.H.)
* Correspondence: jinhong@scu.edu.cn; Tel.: +86-28-85415611
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 August 2020; Accepted: 15 September 2020; Published: 18 September 2020
Abstract: In order to discover new antifungal agents, twenty novel benodanil-heterocyclic
1
carboxamide hybrids were designed, synthesized, and characterized by H NMR and HRMS.
In vitro, their antifungal activities against four phytopathogenic fungi were evaluated, as well as
some of the target compounds at 50 mg/L demonstrated significant antifungal activities against
Rhizoctonia solani. Especially, compounds 17 (EC50 = 6.32 mg/L) and 18 (EC50 = 6.06 mg/L) exhibited
good antifungal activities against R. solani and were superior to the lead fungicide benodanil
(a succinate dehydrogenase inhibitor, SDHI) (EC50 = 6.38 mg/L). Furthermore, scanning electron
microscopy images showed that the mycelia on treated media with the addition of compound 17 grew
abnormally as compared with the negative control with tenuous, wizened, and overlapping colonies,
and compounds 17 (IC50 = 52.58 mg/L) and 18 (IC50 = 56.86 mg/L) showed better inhibition abilities
against succinate dehydrogenase (SDH) than benodanil (IC50 = 62.02 mg/L). Molecular docking
revealed that compound 17 fit in the gap composed of subunit B, C, and D of SDH. Furthermore,
it was shown that the main interaction, one hydrogen bond interaction, was observed between
compound 17 and the residue C/Trp-73. These studies suggested that compound 17 could act as a
potential fungicide to be used for further optimization.
Keywords: benodanil-heterocyclic carboxamide hybrids; antifungal activity; molecular docking;
succinate dehydrogenase inhibitor
1. Introduction
Plant diseases always seriously affect production and quality of crops in global agriculture, and thus
result in serious economic losses in crop yields and food security [
of control methods of plant diseases, chemical controls using fungicides are one of the most effective
methods [ ]. As one of the most important categories of agrochemical fungicides, aromatic amide
1–3]. At present, among the varieties
4–6
fungicides (a kind of succinate dehydrogenase inhibitors, SDHIs) have been intensively employed all
over the world to fight against destructive plant pathogens, such as Rhizoctonia solani, Botrytis cinerea,
and Sclerotinia sclerotiorum [
fungicides, they involve benodanil (BASF), mepronil (Kumiai), flutolanil (Nihon Nohyaku),
fenfuram (Shell), thifluzamide (Monsanto), boscalid (BASF), bixafen (Bayer), and so on (Figure 1) [ ].
However, with the frequent and extensive application of these aromatic amide fungicides, several
resistant fungi have been reported [10 12]. Therefore, it is necessary to develop new fungicides with
7–9]. From the first SDHI carboxin to subsequent commercial agrochemical
5
–
novel molecular frameworks to effectively control plant diseases [13–15].
Molecules 2020, 25, 4291; doi:10.3390/molecules25184291
www.mdpi.com/journal/molecules

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During previous researches for SDHIs, we found that SDHIs all had a common prototypical
pharmacophoric scheme, which included a conserved amide function, a structurally diverse carboxyl
“core” [16–21]. For example, fenfuram has been used as a lead molecule and diarylamines were
introduced to design and synthesize a class of fenfuram-diarylamine hybrids with better antifungal
activity against R. solani than fenfuram [17]. Therefore, in this paper, on the basis of the molecular
structure of benodanil, a series of novel benodanil-heterocyclic carboxamides hybrids containing an
active skeleton, ortho-replaced benodanil, were designed and the iodine atom on the ortho position
of benzene ring was replaced by an aromatic amide group (Figure 2). This modification kept the
benodanil scaffold and contained two aromatic amides in the molecular structure of one compound,
which was important to the compound’s antifungal activity, and ensured pharmacological properties
of benodanil. All of the benodanil-heterocyclic carboxamide hybrids 1–
20 were identified by 1H NMR,
13C NMR, and HRMS. In vitro, their antifungal activities were evaluated against four plant pathogenic
fungi (R. solani, Fusarium oxysporum, Alternaria tenuissima, and Alternaria solani), and enzyme inhibitory,
mycelium morphology, and molecular docking were also reported. To the best of our knowledge,
this is the first time that the bioactivities of the benodanil-heterocyclic carboxamide hybrids have
been studied.
Figure 1. Some commercial aromatic amide fungicides.
Figure 2. Design strategy of novel benodanil-heterocyclic carboxamide hybrids.

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2. Results and Discussion
2.1. Chemistry
Schemes 1 and 2 and Tables 1 and 2 summarize the synthesis and chemical structures of the
target compounds
were obtained by classic amidation reaction with compound
aniline), and reduction with Fe/NH4Cl [19
in good yield, by condensation reaction with aromatic acid
1
–
10 and 11
–
20. The key intermediates
d
b
(or
and 3-chloroaniline (or substituted
10 (or 11 20) were obtained,
), following reported procedures [21].
f), shown in Scheme 1 (or 2),
,20]. The target compounds
1
–
–
d
(or
f
Scheme 1. General synthesis of the target compounds 1–10. Reaction conditions: (i) SOCl2/CH2Cl2,
70 ◦C, 3 h; (ii) CH2Cl2, Et3N, 0 ◦C r.t., 3 h; (iii) Fe/NH4Cl, 75 % C2H5OH, 90 ◦C 3 h; (iv) EDCI/DMAP,
DCM, r.t., 6 h.
Scheme 2. General synthesis of the target compounds 11–20. Reaction conditions: (i) SOCl2/CH2Cl2,
70 ◦C, 3 h; (ii) CH2Cl2, Et3N, 0 ◦C r.t., 3 h; (iii) Fe/NH4Cl, 75 % C2H5OH, 90 ◦C, 3 h; (iv) EDCI/DMAP,
DCM, r.t., 6 h.

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Table 1. Antifungal activities of the target compounds 1–10 against four fungi at 50 mg/L.
Inhibition Rate (%)
No.
1
Ar
R. solani
F. oxysporum A. tenuissima
A. solani
34.33 ± 0.8
28.81 ± 1.3
33.55 ± 2.0
30.00 ± 1.1
19.22 ± 1.7
21.55 ± 1.4
2
31.34 ± 0.7
11.06 ± 0.7
3
4
5
35.82 ± 1.9
50.75 ± 2.2
10.45 ± 0.8
27.45 ± 2.1
12.84 ± 1.6
20.53 ± 2.1
22.30 ± 0.9
18.59 ± 0.8
19.68 ± 1.1
15.47 ± 1.5
22.53 ± 2.3
10.80 ± 2.0
6
7
8
32.84 ± 1.5
83.58 ± 1.6
62.69 ± 1.0
11.49 ± 1.8
47.59 ± 2.2
39.87 ± 1.3
21.21 ± 1.5
33.96 ± 1.4
20.58 ± 1.5
22.201.4
40.78 ± 1.9
29.96 ± 2.4
9
68.66 ± 2.2
88.81 ± 0.9
42.73 ± 0.6
50.60 ± 1.2
37.45 ± 1.4
43.27 ± 1.9
33.44 ± 1.7
47.21 ± 1.1
10
Table 2. EC50 values of some compounds with excellent effect against R. solani.
No. EC50 (mg/L)
No.
EC50 (mg/L)
7
10
17
20.37 ± 0.4
10.34 ± 0.7
6.32 ± 0.4
18
6.06 ± 0.7
6.38 ± 0.6
benodanil
2.2. Antifungal Activities
The antifungal activities of the synthetic compounds against R. solani, F. oxysporum, A. tenuissima,
and A. solani were tested, according to our previous reported procedure [21].
Firstly, the results of the in vitro antifungal activities of compounds 1–10 at a dosage of 50 mg/L
against R. solani, F. oxysporum, A. tenuissima, and A. solani were listed in Table 1. Here, their
antifungal activities were expressed as the inhibition percentage [21]. Although it seemed impossible
to find an obvious structure activity relationship, it was found that compounds
antifungal activities to varying extents against the four fungi and most of compounds
some antifungal activities against R. solani. Especially compounds (Ar = 2-chloro-3-pyridyl) and
1
–10 exhibited
1–
10 showed
7

Molecules 2020, 25, 4291
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10 (Ar = 2-difluoromethyl-4-methyl-pyrazolyl) had good antifungal activity against R. solani and their
inhibition rates were 83.58 and 88.81%, respectively. In addition, compound 10 displayed a much higher
antifungal activity against R. solani (88.81%) than against F. oxysporum, A. tenuissima, and A. solani (50.60,
43.27, and 47.21%). To further analyze the antifungal activities of the compounds, compounds 7 and 10
that exhibited stronger antifungal activities against R. solani were evaluated by determination of EC50
values. The corresponding EC50 values are listed in Table 2. Compound 10 (EC50 = 10.34 mg/L) had
better antifungal activity against R. solani than compound
that compound 10 containing a 3-difluoromethyl-1-methyl-4-pyrazolyl moiety had the best antifungal
activity as compared with other compounds containing a different aromatic heterocyclic moiety.
7 (EC50 = 20.37 mg/L). The results showed
1–9
It was seen that compound 10 could be the novel potential lead compound against R. solani.
Thus, in order to further study antifungal activities of the novel benodanil-heterocyclic
carboxamide hybrids, on the basis of compound 10 as the lead compound by bioisosteric modification,
some substituted groups were introduced, such as fluorine atom, bromine atom, and so on, to replace
the 3-chloro atom in benzene ring. Antifungal activities of the novel target compounds 11
evaluated by the growth rate method [21]. Preliminary in vitro antifungal activities of the novel target
compounds 11 20 against R. solani are summarized in Table 3. In compounds 11 20, compounds
17 (R = 3-CH(CH3)2) and 18 (R = 3-CF3) displayed promising antifungal activities (inhibition rate
80%) against R. solani at a dosage of 50 mg/L, and the inhibition rate of compound 17 (90.30%)
–20 were
–
–
≥
was found to be higher than that of compound 10 (88.81%). Their EC50 values also displayed that
compounds 17 (EC50 = 6.32 mg/L) and 18 (EC50 = 6.06 mg/L) had better antifungal activities against
R. solani than the lead compound 10 (R = 3-Cl, EC50 = 10.34 mg/L) and the commercial SDHI benodanil
(EC50 = 6.38 mg/L), as shown in Table 2.
Table 3. Antifungal activities of the target compounds 11–20 against R. solani at 50 mg/L.
No.
R
Inhibition Rate (%) No.
R
Inhibition Rate (%)
11
12
13
14
15
3-F
3-Br
4-F
4-Cl
4-Br
40.30 ± 1.2
58.21 ± 1.1
62.69 ± 1.6
61.19 ± 0.9
34.33 ± 1.1
16
17
18
19
20
3-CH3
3-CH(CH3)2
3-CF3
3,4-Cl2
3–5-Cl2
5.97 ± 0.7
90.30 ± 1.5
83.58 ± 1.1
77.61 ± 1.8
79.10 ± 0.7
2.3. Effects on the Mycelium Morphology
Scanning electron microscope (SEM) images (Figure 3) showed that R. solani cultivated on media,
without the addition of any compound, featured dense, sturdy, and smooth mycelia with a fine
morphology. In contrast, the morphology of the mycelia of R. solani changed when it was cultured
on media with the addition of compound 17 (10 mg/L) and the mycelia grew abnormally with a
comparatively tenuous, wizened, and overlapping colony.
(A)
(B)
Figure 3. Scanning electron microscopes (SEM). (
A
) Negative control; (
B) Treated colony with compound
17 at 10 mg/L.

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2.4. Fungal SDH Inhibition Activities
Furthermore, because the lead fungicide benodanil was a succinate dehydrogenase inhibitor,
in order to investigate whether the novel benodanil-heterocyclic carboxamide hybrids had inhibitory
activity against succinate dehydrogenase (SDH) or not, inhibition of the fungal SDH was performed.
Compounds 17 and 18 were selected and tested against SDH, in vitro, from mitochondria of R. solani.
As demonstrated in Table 4, the selected compounds 17 (IC50 = 52.58 mg/L) and 18 (IC50 = 56.86 mg/L)
showed better inhibition abilities against SDH than benodanil (IC50 = 62.02 mg/L), proving that the
SDH probably was a potent target of benodanil-heterocyclic carboxamide hybrids.
Table 4. IC50 (mg/L) values of compounds 17 and 18 against SDH of R. solani.
No.
IC50 (mg/L)
17
18
52.58 ± 1.2
56.86 ± 1.7
62.02 ± 1.2
benodanil
2.5. Molecular Docking
To further survey whether the SDH is a potential target enzyme of benodanil-heterocyclic
carboxamide hybrids or not, compound 17 was docked into the binding site of the SDH. The theoretical
binding mode between 17 and SDH is shown in Figure 4. Compound 17 fit into the gap composed of
subunit B, C, and D of SDH. The phenyl group in the middle of compound 17 occupied the hydrophobic
pocket, which was composed of the residues B/Pro-202, B/Ile-251, C/Ile-77, and C/Trp-73, while the
3-(difluoromethyl)-1-methyl-1H-pyrazole scaffold of compound 17 located at another hydrophobic
pocket, surrounded by the residues B/Trp-205, B/Trp-206, C/Phe-64, and C/Trp-73, formed a stable
hydrophobic binding. A detailed analysis showed that a
between the pyrazole group in compound 17 and the sidechain of the residue C/Phe-64. In addition,
the isopropylphenyl group in compound 17 formed a cation– interaction with C/Arg-80. Importantly,
π–π stacking interaction was observed
π
one hydrogen bond interaction was observed between compound 17 and the residue C/Trp-73
(bond length 3.4 Å), which was the main interaction between them (Figure 4). All these interactions
helped compound 17 to anchor in the binding site of SDH.
Figure 4. The theoretical binding mode between compound 17 and SDH by PyMoL 1.7.6.

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3. Materials and Methods
3.1. Reagents and Instruments
Some ingredients were purchased from commercial sources unless otherwise specified. All solvents
and liquid reagents were dried by standard methods in advance and distilled before use. All fungi
1
were obtained from the Institute of Pesticide and Crop Protection, Sichuan University. H NMR
spectra were recorded in DMSO-d6 solution on a Bruker 400 MHz (Bruker Corporation, Billerica,
MA, USA), using tetramethylsilane (TMS) as an internal standard, and chemical shift values (δ) were
given in parts per million (ppm). The following abbreviations were used to designate chemical
shift multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, and m = multiple. Thin-layer
chromatography (TLC) was performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd., Qingdao,
China). Column chromatography (CC) purification was performed over silica gel (200–300 mesh,
Qingdao Marine Chemical Ltd.). MS data were obtained using a MALDI-TOF/TOF mass spectrometer
(Shimadzu Corporation, Kyoto, Japan). Scanning electron microscopy was performed using a SU3500
Hitachi (Hitachi Group, Tokyo, Japan)
3.2. General Synthetic Procedure for Key Intermediates
3.2.1. Synthesis of the Intermediate c
Compound a (20 mmol) was dissolved in dichloromethane (20 mL) at room temperature, then,
N,N-dimethylformamide (5–6 drops) and thionyl chloride (15 mL) were added, in that order, and the
resulting mixture was stirred at 70 ◦C, for 3 h. The remaining solvents dichloromethane and thionyl
chloride were removed by distillation under reduced pressure to yield the corresponding compound
To a solution of 3-chloroaniline (0.02 mol) in triethylamine (5 mL) and dichloromethane (15 mL)
in an ice bath, freshly prepared compound was added dropwise, and the resulting mixture was
stirred at room temperature for 3 h. Then, the mixture was filtered, and successively washed with
aqueous sodium hydroxide (5%, 3 15 mL) and water (2 15 mL). The organic phase was, then, dried
b.
b
×
×
over anhydrous sodium sulfate, filtered, and evaporated in vacuo to remove dichloromethane. Finally,
the residue was purified by column chromatography to yield the intermediate c.
3.2.2. Synthesis of Intermediate d
Compound c (15 mmol), reductive iron powder (15 mmol), NH4Cl (45 mmol), and ethanol aqueous
solution (75%, 60 mL) were added in a flask. The reaction proceeded with refluxing for 3 h, at 90 ◦C.
When the reaction finished, the mixtures were cooled to room temperature and extracted with CH2Cl2
(3 × 20 mL) and the organic phase was evaporated in a vacuum to obtain compound d.
3.3. General Synthetic Procedures for Target Compounds 1–20
Intermediate
were dissolved in DCM (CH2Cl2, 30 mL). The mixture was stirred at room temperature for 6 h.
The target compounds 10 were purified via column chromatography. Similarly, the intermediate
, and target compounds 11 20 were synthesized by using the same methods. The spectrogram of
d (10 mmol), EDCI (10 mmol), DMAP (0.1 mmol), and aromatic acid (10 mmol)
1–
b,
e,
f
–
target compounds 1–20 are presented as Supporting Information.
1
Compound
1
: Yield, 83.6%; white solid; H NMR (400 MHz, DMSO)
δ
11.52 (s, 1H), 10.71 (s, 1H),
8.49–8.45 (m, 1H), 8.02 (dd, J = 1.7, 0.7 Hz, 1H), 7.92 (dd, J = 7.9, 1.4 Hz, 1H), 7.90 (t, J = 2.1 Hz, 1H),
7.70–7.66 (m, 1H), 7.66–7.61 (m, 1H), 7.43 (t, J = 8.1 Hz, 1H), 7.32 (dd, J = 7.6, 1.0 Hz, 1H), 7.29–7.28 (m, 1H),
7.22 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H), 6.74 (dd, J = 3.5, 1.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
δ
167.88, 156.10, 147.77, 146.59, 140.44, 138.41, 133.37, 132.92, 130.82, 129.50, 124.38, 123.79, 122.83, 121.64,
120.84, 119.80, 115.90, 113.08; ESI-HRMS: m/z [M + Na]+ calcd. For [C18H13ClN2NaO3]: 363.0512;
found: 363.0480.

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1
Compound
2
: Yield, 75.5%; white solid; H NMR (400 MHz, DMSO)
δ
11.01 (s, 1H), 10.64 (s, 1H),
8.35 (s, 1H), 8.22 (d, J = 8.1 Hz, 1H), 7.90 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.85–7.80 (m, 1H),
7.67 (d, J = 8.2 Hz, 1H), 7.61 (dd, J = 11.4, 4.2 Hz, 1H), 7.40 (t, J = 8.1 Hz, 1H), 7.30 (t, J = 7.3 Hz, 1H),
7.19 (dd, J = 8.0, 1.2 Hz, 1H), 6.86 (d, J = 1.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 167.73, 160.58,
146.39, 145.23, 140.63, 138.14, 133.35, 132.58, 130.78, 129.37, 124.53, 124.18, 124.06, 123.42, 122.57, 120.62,
119.58, 108.98; ESI-HRMS: m/z [M + Na]+ calcd. For [C18H13ClN2NaO3]: 363.0512; found: 363.0481.
1
Compound
3
: Yield, 69.8%; grey solid; H NMR (400 MHz, DMSO)
δ
11.03 (s, 1H), 10.65 (s, 1H),
8.36 (d, J = 7.6 Hz, 1H), 7.89 (t, J = 1.9 Hz, 1H), 7.87 (d, J = 1.2 Hz, 1H), 7.73–7.66 (m, 1H),
7.65 (d, J = 2.0 Hz, 1H), 7.60 (s, 1H), 7.41 (t, J = 8.1 Hz, 1H), 7.29 (dd, J = 7.6, 0.9 Hz, 1H), 7.24–7.17 (m, 1H),
6.77 (d, J = 2.0 Hz, 1H), 2.56 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 167.96, 161.71, 157.45, 141.93,
140.52, 138.76, 133.34, 132.72, 130.80, 129.37, 124.28, 123.63, 123.36, 121.92, 120.74, 119.69, 116.45, 109.04,
13.66; ESI-HRMS: m/z [M + Na]+ calcd. For [C19H15ClN2NaO3]: 377.0669; found: 377.0633.
Compound 4 δ 11.42 (s, 1H), 10.67 (s, 1H),
: Yield, 77.9%; yellow solid; 1H NMR (400 MHz, DMSO)
8.27 (dd, J = 8.2, 0.7 Hz, 1H), 7.91 (d, J = 1.2 Hz, 1H), 7.90–7.89 (m, 1H), 7.88 (d, J = 1.3 Hz, 1H),
7.79 (dd, J = 3.8, 1.1 Hz, 1H), 7.71–7.65 (m, 1H), 7.65–7.55 (m, 1H), 7.41 (t, J = 8.1 Hz, 1H),
7.31 (td, J = 7.7, 1.1 Hz, 1H), 7.26 (dd, J = 5.0, 3.8 Hz, 1H), 7.20 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H);
13C NMR (100 MHz, DMSO-d6)
δ 167.84, 159.92, 140.52, 139.86, 139.75, 138.27, 133.35, 132.73, 132.68,
130.79, 129.43 129.35, 128.86, 124.29, 124.08, 122.35, 120.76, 119.71; ESI-HRMS: m/z [M + Na]+ calcd.
for [C18H13ClN2NaO2S]: 379.0284; found: 379.0335.
1
Compound
5
: Yield, 68.7%; white solid; H NMR (400 MHz, DMSO)
δ
11.26 (s, 1H), 10.66 (s, 1H),
8.32 (d, J = 8.1 Hz, 1H), 8.26 (dd, J = 2.9, 1.3 Hz, 1H), 7.89 (t, J = 1.8 Hz, 1H), 7.87 (d, J = 1.3 Hz, 1H),
7.71 (dd, J = 5.1, 2.9 Hz, 1H), 7.67 (dd, J = 8.2, 1.0 Hz, 1H), 7.64–7.59 (m, 1H), 7.52 (dd, J = 5.1, 1.3 Hz, 1H),
7.40 (t, J = 8.1 Hz, 1H), 7.30 (td, J = 7.7, 1.1 Hz, 1H), 7.20 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) δ 167.89, 160.85, 140.56, 138.56, 138.00, 133.34, 132.70, 130.79, 130.45, 129.43, 128.33, 126.65,
124.25, 123.97, 123.90, 122.26, 120.73, 119.70; ESI-HRMS: m/z [M + Na]+ calcd. for [C18H13ClN2NaO2S]:
379.0284; found: 379.0369.
1
Compound
6
: Yield, 70.2%; white solid; H NMR (400 MHz, DMSO)
δ
11.37 (s, 1H), 10.67 (s, 1H),
9.09 (d, J = 2.0 Hz, 1H), 8.78 (dd, J = 4.8, 1.6 Hz, 1H), 8.24 (ddd, J = 15.6, 8.9, 4.4 Hz, 2H),
7.89 (d, J = 1.7 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.66–7.58 (m, 3H), 7.39 (d, J = 8.1 Hz, 1H),
7.36–7.31 (m, 1H), 7.22–7.13 (m, 1H); 13C NMR (100 MHz, DMSO-d6)
δ 168.82, 160.22, 149.78, 146.29,
143.85, 142.01, 140.79, 138.80, 136.32, 135.45, 134.14, 131.43, 129.80, 126.64, 124.19, 122.41, 121.19, 119.81,
118.34; ESI-HRMS: m/z [M + Na]+ calcd. For [C19H14ClN3NaO2]: 374.0672; found: 374.0638.
1
Compound
7
: Yield, 82%; orange solid; H NMR (400 MHz, DMSO)
δ
10.92 (s, 1H), 10.63 (s, 1H),
8.68–8.48 (m, 1H), 8.25 (dt, J = 7.7, 1.9 Hz, 1H), 8.09–8.00 (m, 1H), 8.00–7.86 (m, 1H), 7.77 (d, J = 7.6 Hz, 1H),
7.59 (ddd, J = 10.2, 7.7, 5.0 Hz, 2H), 7.36 (td, J = 8.0, 1.7 Hz, 1H), 7.24–7.05 (m, 1H), 6.82–6.54 (m, 1H),
6.37 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
δ 167.70, 160.13, 145.34, 141.66, 137.58, 135.72, 134.36,
133.03, 132.51, 129.31, 125.45, 124.35, 123.52, 123.25, 119.10, 116.49, 114.37, 112.29, 110.09; ESI-HRMS:
m/z [M + Na]+ calcd. For [C19H13Cl2N3NaO2]: 408.0283; found: 408.0257.
1
Compound
8
: Yield, 78.5%; grey solid; H NMR (400 MHz, DMSO)
δ
10.90 (d, J = 8.8 Hz, 1H),
10.63 (d, J = 3.6 Hz, 1H), 8.53 (d, J = 4.7 Hz, 1H), 8.12–7.97 (m, 2H), 7.90 (d, J = 1.9 Hz, 1H),
7.76 (dd, J = 5.1, 2.5 Hz, 1H), 7.59 (ddd, J = 12.4, 9.6, 6.2 Hz, 3H), 7.36 (t, J = 8.0 Hz, 2H),
7.15 (dd, J = 7.7, 1.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
δ 168.35, 149.95, 147.05, 143.83,
141.21, 135.22, 133.26, 132.83, 130.63, 129.18, 123.44, 121.82, 120.19, 119.14, 117.11, 115.54, 112.02, 101.88;
ESI-HRMS: m/z [M + Na]+ calcd. For [C19H13BrClN3NaO2]: 451.9777; found: 452.0810.
1
Compound
9
: Yield, 81.9%; white solid; H NMR (400 MHz, DMSO)
δ
11.15 (s, 1H), 10.65 (s, 1H),
7.94 (dd, J = 16.2, 8.2 Hz, 2H), 7.85–7.75 (m, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.40–7.34 (m, 2H),
7.20–7.12 (m, 1H), 2.75 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 168.69, 167.04, 141.16, 140.77, 136.31,

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133.31, 132.25, 130.75, 130.64, 129.36, 129.17, 125.48, 124.02, 123.48, 120.33, 119.27, 119.16, 117.23, 31.11;
ESI-HRMS: m/z [M + Na]+ calcd. For [C19H13ClFN3NaO2S]: 462.0267; found: 462.0248.
1
Compound 10: Yield, 67%; white solid; H NMR (400 MHz, DMSO)
δ 10.94 (s, 1H), 10.65 (s, 1H),
8.39 (s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.91 (s, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.63–7.55
(m, 1H), 7.40 (t, J = 8.1 Hz, 1H), 7.30 (dd, J = 6.1, 1.4 Hz, 1H), 7.25–7.20 (m, 1H), 7.20–7.14 (m, 1H),
3.99 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 167.66, 160.05, 138.05, 136.58, 132.85, 132.49, 130.72,
130.62, 129.33, 124.56, 124.07, 122.72, 116.97, 116.95, 116.61, 110.06, 108.13, 107.87, 31.11; ESI-HRMS:
m/z [M + Na]+ calcd. For [C19H15ClF2N4NaO2]: 427.0749; found: 427.0728.
Compound 11: Yield, 68.6%; white solid; 1H NMR (400 MHz, DMSO)
δ 10.95 (s, 1H), 10.67 (s, 1H),
8.71 (s, 1H), 8.37 (s, 1H), 8.22–8.16 (m, 1H), 7.85 (dd, J = 7.8, 1.3 Hz, 1H), 7.70 (dt, J = 11.7, 2.2 Hz, 1H),
7.63–7.55 (m, 1H), 7.50 (dd, J = 8.2, 0.9 Hz, 1H), 7.40 (ddd, J = 16.3, 7.5, 1.9 Hz, 1H), 7.35–7.27 (m, 1H),
7.01–6.91 (m, 1H), 3.98 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 167.72, 160.10, 157.02, 139.96, 137.78,
136.67, 132.97, 132.44, 130.31, 129.34, 125.80, 125.18, 124.80, 124.24, 123.04, 120.75, 117.33, 110.03, 31.11;
ESI-HRMS: m/z [M + Na]+ calcd. For [C19H15F3N4NaO2]: 411.1045; found: 411.1022.
Compound 12: Yield, 70.5%; white solid; 1H NMR (400 MHz, DMSO)
δ 10.89 (s, 1H), 10.62 (s, 1H),
8.71 (s, 1H), 8.45–8.30 (m, 1H), 8.16 (d, J = 6.9 Hz, 1H), 8.02 (s, 1H), 7.84 (d, J = 7.8 Hz, 1H),
7.68 (d, J = 4.0 Hz, 1H), 7.64–7.55 (m, 1H), 7.35–7.31 (m, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.24–7.08 (m, 1H),
3.97 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 167.62, 163.36, 160.06, 140.75, 137.98, 136.58, 132.90,
132.48, 131.05, 129.37, 127.10, 124.71, 124.11, 123.56, 121.80, 120.04,112.58, 109.12, 30.14; ESI-HRMS:
m/z [M + Na]+ calcd. For [C19H15BrF2N4NaO2]: 471.0244; found: 471.0211.
Compound 13: Yield, 77.7%; white solid; 1H NMR (400 MHz, DMSO)
δ 11.16 (s, 1H), 10.56 (s, 1H),
8.71 (s, 1H), 8.34 (s, 1H), 8.27 (d, J = 7.4 Hz, 1H), 7.89 (d, J = 6.8 Hz, 1H), 7.73 (s, 2H),
7.58 (d, J = 6.5 Hz, 1H), 7.29 (s, 1H), 7.26–7.02 (m, 2H), 3.98 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ
167.50, 159.99, 145.28, 138.39, 136.58, 135.29, 135.26, 132.73, 132.50, 129.27, 123.89, 123.77, 123.50,
123.42, 122.32, 116.68, 115.79, 115.576, 31.11; ESI-HRMS: m/z [M + Na]+ calcd. For [C19H15F3N4NaO2]:
411.1045; found: 411.1013.
1
Compound 14: Yield, 79%; yellow solid; H NMR (400 MHz, DMSO)
δ 11.03 (d, J = 10.4 Hz, 1H),
10.62 (s, 1H), 8.35 (s, 1H), 8.26–8.18 (m, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H),
7.63–7.54 (m, 1H), 7.47–7.43 (m, 1H), 7.43–7.38 (m, 1H), 7.24 (dt, J = 50.7, 5.8 Hz, 2H), 3.97 (s, 3H);
13C NMR (100 MHz, DMSO-d6)
δ 167.56, 160.01, 156.02, 145.88, 138.19, 138.03, 132.79, 132.49, 129.33,
128.97, 128.23, 124.25, 123.99, 122.93, 122.56, 116.81, 110.05, 108.34, 31.12; ESI-HRMS: m/z [M + Na]+
calcd. For [C19H15ClF2N4NaO2]: 427.0749; found: 427.0729.
Compound 15: Yield, 59.7%; white solid; 1H NMR (400 MHz, DMSO)
δ 11.02 (s, 1H), 10.62 (s, 1H),
8.72 (s, 1H), 8.35 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H), 7.92–7.81 (m, 1H), 7.71 (d, J = 8.9 Hz, 2H),
7.58 (ddd, J = 8.9, 7.7, 1.6 Hz, 2H), 7.32–7.26 (m, 1H), 7.37–7.04 (m, 1H), 3.98 (s, 3H); 13C NMR
(100 MHz, DMSO-d6)
δ 167.57, 163.36, 160.01, 138.45, 138.18, 136.58, 132.79, 132.49, 131.88, 129.33,
124.26, 123.99, 123.29, 122.56, 116.65, 116.35, 112.33, 110.00, 31.10; ESI-HRMS: m/z [M + Na]+ calcd.
For [C19H15BrF2N4NaO2]: 471.0244; found: 471.0210.
Compound 16: Yield, 80.4%; white solid; 1H NMR (400 MHz, DMSO)
δ 11.16 (s, 1H), 10.44 (s, 1H),
8.35 (s, 1H), 8.26 (d, J = 7.5 Hz, 1H), 7.90–7.84 (m, 1H), 7.61–7.55 (m, 1H), 7.49 (dd, J = 23.8, 15.8 Hz, 2H),
7.32–7.11 (m, 3H), 6.96 (d, J = 7.5 Hz, 1H), 3.97 (s, 3H), 2.31 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ
167.55, 160.00,148.92, 145.28, 138.81, 138.37, 138.27, 132.74, 132.43, 129.32, 128.91, 125.39, 123.95, 123.89,
122.31, 122.11, 118.80, 110.07, 31.12, 21.59; ESI-HRMS: m/z [M + Na]+ calcd. For [C20H18F2N4NaO2]:
407.1296; found: 407.1268.
Compound 17: Yield, 79.4%; white solid; 1H NMR (400 MHz, DMSO)
δ 11.17 (s, 1H), 10.44 (s, 1H),
8.35 (s, 1H), 8.26 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.63–7.54 (m, 1H), 7.52 (s, 1H),
7.43–7.16 (m, 1H), 7.32–7.27 (m, 1H), 7.03 (d, J = 7.7 Hz, 1H), 3.97 (s, 3H), 2.93–2.80 (m, 1H),

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1.21 (d, J = 6.9 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)
δ
167.51, 160.00, 149.34, 144.94, 138.85, 138.33,
132.78, 132.40, 129.32, 128.96, 124.04, 123.89, 122.76, 122.34, 119.59, 119.27, 116.71, 110.52, 40.12, 33.88,
24.27; 19F NMR (225 MHz, DMSO-d6) δ −114.10 (d, J = 31.5Hz, 2F); ESI-HRMS: m/z [M + Na]+ calcd.
For [C22H22F2N4NaO2]: 435.1609; found: 435.1590.
1
Compound 18: Yield, 66.9%; white solid; H NMR (400 MHz, DMSO)
δ 10.84 (s, 1H), 10.76 (s, 1H),
8.38 (s, 1H), 8.12 (d, J = 6.1 Hz, 2H), 7.99 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.66–7.55 (m, 2H),
7.48 (d, J = 7.8 Hz, 1H), 7.40–7.13 (m, 1H), 7.34–7.29 (m, 1H), 3.97 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ
167.59, 163.90, 152.82, 148.67, 140.72, 137.85, 135.59, 133.33, 132.48, 130.78, 130.20, 129.46, 125.58, 124.58,
124.32, 124.10, 123.00, 120.50, 119.46, 31.12; 19F NMR (225 MHz, DMSO-d6)
δ -114.10 (d, J = 33.8Hz, 2F),
−61.27 (s, 3F); ESI-HRMS: m/z [M + Na]+ calcd. For [C20H15F5N4NaO2]: 461.1013; found: 461.0971.
Compound 19: Yield, 73.4%; white solid; 1H NMR (400 MHz, DMSO)
δ 10.84 (s, 1H), 10.74 (s, 1H),
8.37 (d, J = 20.5 Hz, 1H), 8.10 (dd, J = 12.0, 5.1 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 2.2 Hz,
1H), 7.62 (dd, J = 9.7, 5.6 Hz, 2H), 7.42–7.11 (m, 2H), 5.77 (d, J = 1.6 Hz, 1H), 3.98 (s, 3H); 13C NMR
(100 MHz, DMSO-d6)
δ 167.62, 163.35, 160.09, 139.36, 137.81, 136.58, 132.96, 132.49, 131.27, 130.95,
129.35, 125.91, 125.01, 124.20, 123.02, 122.35, 121.15, 110.04, 31.11; ESI-HRMS: m/z [M + Na]+ calcd.
For [C19H14Cl2F2N4NaO2]: 461.0360; found: 461.0328.
Compound 20: Yield, 77.2%; dark grey solid; 1H NMR (400 MHz, DMSO) δ 10.73 (s, 2H), 8.41 (s, 1H),
8.09 (s, 1H), 7.81 (s, 2H), 7.59 (d, J = 6.7 Hz, 1H), 7.49–7.04 (m, 3H), 5.77 (s, 1H), 3.98 (s, 3H); 13C NMR
(100 MHz, DMSO-d6)
δ 167.04, 164.62, 152.65, 151.28, 148.20, 146.89, 140.94, 140.68, 138.69, 133.30,
133.01, 130.72, 130.66, 129.32, 127.07, 123.67, 120.16, 119.09, 32.26; ESI-HRMS: m/z [M + Na]+ calcd.
For [C19H14Cl2F2N4NaO2]: 461.0360; found: 461.0334.
3.4. Antifungal Bioassay
All synthesized compounds 1–20 were screened for their in vitro antifungal activities against
four fungi, including R. solani, F. oxysporum, A. tenuissima, and A. solani. by the mycelium growth
rate method. All screened compounds were dissolved in acetone (1 mL), and the solutions were
diluted with aqueous 1% Tween 60, and then were added to sterile potato dextrose agar (PDA, 49 mL).
For primary screening, the compounds were tested at a concentration of 50 mg/L. All fungi species
were cultivated in PDA at 28
were conducted. Next, mycelia dishes of about 8 mm diameter were cut from the culture medium and
were picked up with a sterilized inoculating needle and inoculated in the center of the PDA plates.
±
1 ◦C, for 2 days, to produce new mycelium before the antifungal assays
The inoculated plates were incubated at 28
±
1 ◦C, for 24 h. Acetone in sterile aqueous 1% Tween 60
served as the negative control, whereas thifluzamide served as the positive control. Each sample was
screened in three replicates and each colony diameter of the three replicates was measured four times
by the cross-bracketing method. The data was statistically analyzed and the corrected inhibitory rates
(I) were calculated using the following formula: I (%) = [(C-T)/(C-8 mm)]
×
100, where C represents the
average diameter of mycelia in the blank test, T represents the average diameter of mycelia on treated
PDA media. The results of the antifungal tests are summarized in Tables 1 and 3, and the EC50 values
of some compounds were calculated and are listed in Table 2.
3.5. Scanning Electron Microscopy
The effect of compound 17 on mycelial morphology was observed, according to the previously
reported method [22]. Five 0.7 cm cakes of R. solani were put into PDB liquid medium and cultured at
28 ◦C, 160 r/min, for 3 days. Compound 17 dissolved in DMSO was added into 50 mL PDB liquid
culture medium, the final concentrations of the agent were 10 mg/L, and the control was DMSO of the
same volume, and the culture continued for 24 h. The mycelium was taken out, 2.5% glutaraldehyde
was fixed at 4 ◦C, for 4–6 h, the mycelium was washed with PBS buffer for 2–3 times, then dehydrated
with ethanol gradient (30%, 50%, 70%, 85%, and 90% once respectively, 100% twice, 20 min each time);

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the dehydrated mycelium was dried in vacuum, finally gold plating was carried out. The mycelia
were observed and photographed on a SU3500 Hitachi scanning electron microscope.
3.6. Assay of Enzyme Inhibitory
3.6.1. Isolation of R. solani
Fungus mitochondria were isolated, according to a previously reported method. Cultures were
inoculated at 0.05 OD600 nm and grown on a reciprocal shaker (180 rpm, 25 ◦C) for 5 days in Sabouraud
maltose broth (SMB) medium. Cells were harvested by vacuum filtration and disrupted in liquid
nitrogen using a mortar and pestle. The resultant powder was resuspended to10 % w/v in mitochondrial
extraction buffer (10 mM KH2PO4, pH 7.2, 10 mM KCl, 10 mM MgCl2, 0.5 M sucrose, 0.2 mM EDTA,
2 mM PMSF). The extract was clarified by centrifugation (5000 g, 4 ◦C, for 10 min, 2 times), and then
intact mitochondria were pelleted at 10,000 g for 20 min, at 4 ◦C, and resuspended in the same buffer.
Mitochondrial suspensions were brought to a concentration of 10 mg/mL and stored at
use. The activity of SDH was found to remain stable for months.
−
80 ◦C until
3.6.2. Succinate:Ubiquinone/DCPIP Activity Inhibition
◦
Mitochondrial suspensions were diluted 1/20 in extraction buffer and preactivated at 30 C
for 30 min in the presence of 10 mM succinate. Succinate:ubiquinone/DCPIP activity inhibition
measurements were performed by adding 10
(50 mM phosphate/sodium, pH 7.2, 250 mM sucrose, 3 mM NaN3, 10 mM succinate) supplemented
with 140 M dichlorophenolindophenol (DCPIP) and 1 mM 2,3-dimethoxy-5-methyl-1,4-benzoquinone
(Q0). Inhibitor concentrations ranged between 0.0050 and 15 mg/L, with uniform 5 dilution factor
µL of preactivated mitochondria to 200 µL of assay buffer
µ
×
steps (six inhibitor concentrations + DMSO control). A total of 96 well plates were pre-equilibrated
at reaction temperature (30 ◦C) for 10 min before the reactions were started by the addition of 10
of preactivated R. solani mitochondrial suspension. DCPIP reduction was conducted at 30 C and
monitored at 595 nm. Calculated absorbance slopes (OD/h) were used for half-inhibitory concentration
(IC50) calculations using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).
µL
◦
3.7. Homology Modeling and Molecular Docking
3.7.1. Homology Modeling
The NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/) was used to search the SDH
amino acid sequence of R. solani. The employed protein sequence was CUA72490.1, CUA71217.1,
CUA73421.1, and CUA73959.1 reported by Wibberg. The BLAST server (http://blast.ncbi.nlm.nih.gov)
was used to search a template for the chain. We applied SDH from avian (PDB ID:1YQ3) as the template,
and the homology of amino acid sequence was aligned. Homology modeling of SDH from R. solani
was carried out using MODELER 9.15 (http://salilab.org/modeller/).
3.7.2. Molecular Docking
Molecular docking studies were performed to investigate the binding mode of compound 17 to
SDH using Autodock vina 1.1.2 (Scripps Research, San Diego, CA, USA) The three-dimensional (3D)
structures of compound 17 were drawn by ChemBioDraw Ultra 14.0 (PerkinElmer, Waltham, MA, USA)
and ChemBio3D Ultra 14.0 softwares (PerkinElmer, Waltham, MA, USA) The AutoDock Tools 1.5.6
package (http://mgltools.scripps.edu) was employed to generate the docking input files. The search
grid of SDH was identified as center_x: 86.459, center_y: 65.6, and center_z: 85.537 with dimensions
size_x: 15, size_y: 15, and size_z: 15. The value of exhaustiveness was set to 20. For Vina docking,
the default parameters were used if it was not mentioned. The best-scoring pose, as judged by the Vina
docking score, was chosen and visually analyzed using PyMOL1.7.6 software (http://www.pymol.org/).

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4. Conclusions
In summary, a series of novel benodanil-heterocyclic carboxamide hybrids were designed,
synthesized, and screened for their antifungal activity against four phytopathogenic fungi. Compounds
17 (R = 3-CH(CH3)2, EC50 = 6.32 mg/L) and 18 (R = 3-CF3, EC50 = 6.06 mg/L) exhibited better antifungal
activities against R. solani than the lead benodanil (EC50 = 6.38 mg/L). In addition, compounds 17
(IC50 = 52.58 mg/L) and 18 (IC50 = 56.86 mg/L) showed better inhibition abilities against SDH than
benodanil (IC50 = 62.02 mg/L). In the SEM images, the marked changes on the treated media with the
addition of compound 17 displayed that the mycelia grew abnormally with a comparatively tenuous,
wizened, and overlapping colony. This molecular docking study provided further insights into the
interactions between compound 17 and SDH. The benodanil-heterocyclic carboxamide hybrids could
be a class of promising lead compounds for the development of SDHIs.
Supplementary Materials: The spectrogram of title compounds 1–20 were presented as Supporting Information.
Author Contributions: H.J., K.T. and T.H. conceived and designed the experiments; J.Y. realized the synthetic
work, homology modeling, and molecular docking; Y.Z. and M.J. evaluated antifungal activities, scanning electron
microscopy, assay of enzyme inhibitory; J.Y. and J.W. analyzed the data and wrote the paper. All authors have
read and agreed to the published version of the manuscript.
Funding: This study was supported by National Key Research and Development Program of China
(grant no. 2016YFC0502004) and (grant no. 2017YFD0200503) for financial support.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).