Discovery of Natural Product-Based Fungicides (II): Semisynthesis and Biological Activity of Sarisan Attached 3-Phenylisoxazolines as Antifungal Agents

Article abstract of DOI:10.1002/cbdv.202000763

Many phytopathogenic fungi cause severe damage to crop yields. In continuation of our research aimed at the discovery and development of natural products-based fungicides, a series of thirty-one sarisan attached 3-phenylisoxazolines were synthesized and evaluated for their antifungal activities against five phytopathogenic fungi (B. cinerea, C. lagenarium, A. solani, F. solani, and F. graminearum). Among all title sarisan derivatives, compounds IV2, IV14 and IV23 showed potent antifungal activity against some phytopathogenic fungi. In particular, compound IV2 exhibited a broad-spectrum and more potent antifungal activity against A. solani, F. solani, and F. graminearum than the commercial fungicide Hymexazol. In addition, compounds IV2, IV14 and IV23 also displayed relative low toxicity on normal NRK-52E cells. This work will give some insights into the development of sarisan derivatives as new fungicide candidates in plant protection.

Full text of DOI:10.1002/cbdv.202000763

Title: Natural product-based fungicides discovery (II): Semisynthesis
and biological activity of sarisan attached 3-phenylisoxazolines
as antifungal agents
Authors: Zhiyan Liu, Jiangping Cao, Xiaoting Yan, Wanqing Cheng,
Xiaoguang Wang, Ruige Yang, and Yong Guo
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000763
Link to VoR: https://doi.org/10.1002/cbdv.202000763

10.1002/cbdv.202000763
Chemistry & Biodiversity
Chem. Biodiversity
Natural product-based fungicides discovery (II): Semisynthesis and biological
activity of sarisan attached 3-phenylisoxazolines as antifungal agents
Zhiyan Liu,a,b,1 Jiangping Cao,a,1 Xiaoting Yan,b Wanqing Cheng,b Xiaoguang Wang,b Ruige Yang,b,* and Yong
Guo. *,b
a College of Chemistry and Chemical Engineering, Engineering and Technology Research Center of Liupanshan Resources, Ningxia Normal University, Xueyuan
Road, Guyuan 756000, Ningxia Hui Autonomous Region, PR China.
b School of Pharmaceutical Sciences, Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, Zhengzhou University, No. 100,
KeXue Avenue, Zhengzhou, 450001, Henan Province, PR China.
*Corresponding authors: Tel. /fax: +86 371 67781902, e-mail: yrggg@zzu.edu.cn, guoyong_122@zzu.edu.cn.
1 Zhiyan Liu and Jiangping Cao contributed equally to this work.
Many phytopathogenic fungi cause severe damage to crop yields. In continuation of our research aimed at the discovery and development of natural
products-based fungicides, a series of thirty-one sarisan attached 3-phenylisoxazolines were synthesized and evaluated for their antifungal activities
against five phytopathogenic fungi (B. cinerea, C. lagenarium, A. solani, F. solani, and F. graminearum). Among all title sarisan derivatives, compounds IV2,
IV14 and IV23 showed potent antifungal activity against some phytopathogenic fungi. In particular, compound IV2 exhibited a broad-spectrum and more
potent antifungal activity against A. solani, F. solani, and F. graminearum than the commercial fungicide Hymexazol. In addition, compounds IV2, IV14 and
IV23 also displayed relative low toxicity on normal NRK-52E cells. This work will give some insights into the development of sarisan derivatives as new
fungicide candidates in plant protection.
Keywords: Natural product • Sarisan derivative • Isoxazoline • Antifungal activity • Cytotoxic activity
Introduction
Many phytopathogenic fungi such as Botrytis cinerea, Colletotrichum lagenarium, Alternaria solani, Fusarium solani, and Fusarium graminearum infect crops,
are hard to control and results in severe damages to crop yields.[1-4] Although many synthetic chemical fungicides play an important role in plant diseases
control, repeated use of these agents has led to the increase of drug-resistance and other detrimental effects on the environment and human beings.[5,6]
Thus, there is an urgent need for the discovery of effective, low mammalian toxicity, and ecofriendly fungicide alternatives to address the plant diseases
problems.
Figure 1. Design of the sarisan attached 3-phenylisoxazolines.
Natural products (NPs) have a long history of being used as sources for the development of pesticides due to their low toxicities to non-target organism
and ecofriendly properties.[7,8] Therefore, using natural products or their derivatives to develop new fungicides has been becoming a hot topic in recent
years. Sarisan (1, Figure 1), a naturally occurring lignan compound, is isolated from many plants such as Asarum sieboldii,[9] Piperguineense,[10] Piper
solmsianum C.DC,[11] Beilschmiedia miersii,[12] and Saururus chinensis.[13] Compound 1 has been reported to possess many biological activities including
1
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Chem. Biodiversity
antimicrobial activity, psychopharmacologic effect, and anti-septic activity, but the most noteworthy is its insecticidal and antifungal activities.[14-18] In
addition, isoxazole and isoxazoline are important active N-heterocycles for the development of novel pesticides. Oxathiapiprolin and Fluoxapiprolin (Figure.
1), discovered by Bayer AG and DowDuPont Inc., respectively, are two new isoxazoline fungicides.[19,20] Hymexzaol (Figure. 1) is commercially known as
Tachigaren, and it is a widely used fungicide to control many plant diseases. Based on the above mentioned, and in our continuous efforts to develop natural
product-based pesticides,[21-24] herein, we have prepared a series of novel sarisan attached 3-phenylisoxazolines and evaluated for their antifungal and
cytotoxic activities.
Results and Discussion
Chemistry
Scheme 1. Synthetic route of sarisan attached 3-phenylisoxazolines IV1–31.
As shown in Scheme 1, the key intermediates N-hydroxybenzimidoyl chlorides III1–31 were prepared through two steps according to the previously
reported procedures.[25] First, different substituted benzaldehydes I1–31 reacted with hydroxylamine hydrochloride (NH2OH·HCl), respectively, in the
presence of Na2CO3 in EtOH at reflux to acquire the corresponding benzaldoximes II1–31. Then, chlorination of benzaldoximes II1–31 N-chlorosuccinimide
(NCS) to give the key intermediates III1–31. The sarisan was synthesized by using sesamol as the starting material through etherification, Claisen
rearrangement and methylation as our previous report.[26] Finally, [3+2] cyclization of sarisan (1) and the corresponding N-hydroxybenzimidoyl chlorides
III1–31 with Et3N in anhydrous dichloromethane under N2 to obtain the target derivatives IV1–31 in 21–81% yields. All target derivatives IV1–31 were
determined by melting point (m.p.) IR, 1H/13C NMR, and ESI-MS spectral analyses. Moreover, the stereo structure of IV23 (CCDC 2026211) was
unambiguously identified by X-ray diffraction (Figure 2).
2
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Figure 2. X-ray crystal structure of compound IV23.
Antifungal and Cytotoxic Activities
The antifungal activity of the title compounds IVI–31 against five phytopathogenic fungi (B. cinerea, C. lagenarium, A. solani, F. solani, and F. graminearum)
was preliminarily evaluated at a concentration of 50 μg/mL using the mycelium growth rate method with a commercial fungicide Hymexazol as the positive
control.[21] In general, as the Table 1 displays, we found that most sarisan attached 3-phenylisoxazolines showed more potent antifungal activity against B.
cinerea and C. lagenarium than the precursor sarisan after structural modification. For example, all title sarisan derivatives except compounds IV9, IV12,
IV20, IV22 and IV25 displayed more potent antifungal activity against B. cinerea than sarisan (inhibition rate: 28.5%); towards derivatives IVI–31 against C.
lagenarium, twenty-one target derivatives exhibited more potent antifungal activity against C. lagenarium than sarisan (inhibition rate: 34.4%). Among all
title derivatives IVI–31, compounds IV7, IV23 and IV30 exhibited potent antifungal activity against B. cinerea with the inhibition rates of 67.4%, 67.9% and
61.4%, respectively, which is better than the commercial fungicide Hymexazol (inhibition rate: 55.3%). Compounds IV4, IV10, IV13, IV22, IV23, IV25, IV26
and IV29 displayed more potent antifungal activity against C. lagenarium than the positive control Hymexazol, their inhibition rates were 43.0%, 44.4%,
43.7%, 43.5%, 59.6%, 43.5%, 49.4% and 59.6%, respectively. To our delight, we found that compound IV2 exhibited more potent and broad-spectrum
antifungal activity against three phytopathogenic fungi (A. solani, F. solani, and F. graminearum) with the inhibition rates all greater than 50%. In addition,
compound IV14 showed more promising antifungal activity against both A. solani and F. graminearum with inhibition rates of 55.7%, and 53.1%, respectively.
Compound IV16 exhibited potent antifungal activity against F. graminearum with the inhibition rate of 53.1%.
Table 1. Antifungal activity of IV1–31 against five phytopathogenic fungi at 50 μg/mL[a]
Inhibition rate (% ± SD)
Compound
B. cinerea
C. lagenarium
A. solani
F. solani
F. graminearum
IV1
41.9 ± 4.4
45.5 ± 2.1
41.6 ± 1.7
35.3 ± 1.4
43.2 ± 2.2
32.1 ± 5.1
67.4 ± 3.6
36.2 ± 2.3
19.0 ± 0.6
31.6 ± 3.8
42.4 ± 2.4
20.3 ± 3.8
42.0 ± 1.8
43.7 ± 1.9
37.4 ± 2.0
40.3 ± 1.3
47.5 ± 0.03
45.9 ± 1.8
44.5 ± 2.4
21.1 ± 3.1
35.9 ± 1.6
20.5 ± 3.6
67.9 ± 5.1
43.1 ± 2.2
25.0 ± 0.9
35.7 ± 0.03
38.3 ± 0.02
46.4 ± 2.1
32.1 ± 1.8
61.4 ± 2.1
37.4 ± 1.3
28.5 ± 2.2
55.3 ± 2.4
41.4 ± 0.04
39.2 ± 0.03
35.0 ± 3.0
43.0 ± 5.4
33.7 ± 1.4
31.5 ± 1.6
36.1 ± 1.3
32.2 ± 1.6
30.1 ± 2.3
44.4 ± 1.3
17.7 ± 1.8
39.8 ± 1.3
43.7 ± 1.6
27.6 ± 2.2
36.7 ± 3.6
38.0 ± 1.8
34.5 ± 1.6
39.8 ± 1.4
37.8 ± 1.4
38.0 ± 1.3
28.7 ± 1.3
43.5 ± 3.9
59.6 ± 0.04
32.2 ± 1.6
43.5 ± 3.9
49.4 ± 1.6
39.2 ± 0.02
31.5 ± 1.6
59.6 ± 0.03
40.8 ± 0.04
33.7 ± 1.4
34.4 ± 2.9
42.1 ± 1.4
53.1 ± 1.5
54.3 ± 4.0
41.4 ± 6.0
54.3 ± 2.1
33.8 ± 2.1
20.6 ± 4.5
9.3 ± 0.04
13.2 ± 2.1
32.7 ± 3.1
31.0 ± 2.7
30.0 ± 2.0
33.6 ± 2.7
20.6 ± 4.6
55.7 ± 2.0
16.2 ± 2.0
45.7 ± 0.03
17.6 ± 4.2
27.9 ± 2.1
14.7 ± 0.04
14.7 ± 3.8
13.3 ± 1.9
22.7 ± 0.04
17.5 ± 4.5
14.7 ± 0.02
17.3 ± 0.03
12.7 ± 2.2
19.0 ± 2.4
14.3 ± 0.04
0 ± 0
24.1 ± 1.3
55.0 ± 3.5
22.5 ± 7.1
15.0 ± 3.5
35.0 ± 3.5
19.3 ± 1.6
22.2 ± 1.6
24.7 ± 2.2
22.3 ± 1.3
18.3 ± 4.7
20.0 ± 3.7
28.3 ± 1.3
40.6 ± 4.6
25.0 ± 3.5
37.8 ± 2.2
15.0 ± 0.02
44.1 ± 2.2
42.5 ± 3.5
43.8 ± 1.8
41.5 ± 1.6
22.2 ± 1.2
29.0 ± 1.4
42.2 ± 1.6
26.2 ± 0.04
43.0 ± 1.6
13.6 ± 3.2
22.3 ± 1.6
39.3 ± 2.2
43.1 ± 1.6
45.0 ± 3.5
40.3 ± 1.8
38.5 ± 3.1
46.4 ± 2.0
40.7 ± 1.9
55.9 ± 1.1
43.0 ± 1.1
23.3 ± 4.7
33.1 ± 1.1
IV2
IV3
IV4
IV5
IV6
38.6 ± 2.4
35.3 ± 4.1
IV7
23.7 ± 2.0
23.9 ± 1.7
29.5 ± 1.4
22.1 ± 4.0
30.0 ± 3.5
23.1 ± 2.2
IV8
IV9
IV10
IV11
IV12
IV13
IV14
IV15
IV16
IV17
IV18
IV19
IV20
IV21
IV22
IV23
IV24
IV25
IV26
IV27
IV28
IV29
IV30
IV31
Sarisan
Hym [b]
50.8 ± 1.1
44.6 ± 3.2
53.1 ± 2.2
33.4 ± 0.5
46.0 ± 2.1
46.8 ±1.8
38.5 ± 0.03
27.9 ± 1.5
32.9 ± 1.8
45.5 ± 2.6
16.8 ± 3.4
37.3 ± 1.5
28.3 ± 2.8
16.5 ± 3.2
37.2 ± 2.9
34.0 ± 3.1
47.7 ± 0.02
40.0 ± 1.1
45.3 ± 2.5
54.5 ± 1.1
25.0 ± 2.1
17.6 ± 4.2
35.2 ± 1.3
38.3 ± 1.5
[a] Values are means ± SD of three replicates; [b] Hymexazol was used as a positive control.
Moreover, in order to investigate the antifungal activity of some potent sarisan attached 3-phenylisoxazolines more accurately, EC50 values of compounds
IV2, IV14 and IV23 against five phytopathogenic fungi were further screened. As shown in Table 2, compound IV2 exhibited more potent antifungal activity
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against three phytopathogenic fungi (A. solani, F. solani, and F. graminearum) with the EC50 values of 32.9 ± 1.6, 35.6 ± 0.3, and 33.4 ± 0.4 μg/mL, respectively,
than the commercial fungicide Hymexazol. Compound IV14 showed better antifungal activity against A. solani with EC50 value of 37.0 ± 1.0 μg/mL than the
Hymexazol (EC50: 69.9 ± 2.0 μg/mL). The EC50 values of compound IV23 against B. cinerea and C. lagenarium were 30.1 ± 0.7 and 36.6 ± 0.9 μg/mL,
respectively, whereas, the EC50 values of Hymexazol against B. cinerea and C. lagenarium were 41.2 ± 0.5 and 65.7 ± 1.6 μg/mL, respectively. This result
indicated IV23 had better antifungal activity against B. cinerea and C. lagenarium than Hymexazol. In addition, effects of compound IV2 on the growth of
A. solani, F. solani and F. graminearum at different concentrations were displayed in Figure 3. It is apparent that the inhibition effects of compound IV2
against A. solani, F. solani and F. graminearum increased in a concentration dependent manner. Given the potent activity of compound IV2, it could be used
as a potential antifungal agent to protect some crops that are susceptible to fungi A. solani, F. solani and F. graminearum, such as tomatoes, potatoes and
wheat.
Table 2. The EC50 values of IV2, IV14, IV23 and Hymexazol against five phytopathogenic fungi.
EC50 ± SD (μg/mL) [a]
Compound
B. cinerea
C. lagenarium
A. solani
F. solani
F. graminearum
[b]
IV2
/
/
32.9 ± 1.6
37.0 ± 1.0
/
35.6 ± 0.3
33.4 ± 0.4
36.8 ± 0.5
/
IV14
IV23
Hym [c]
/
/
/
30.1 ± 0.7
41.2 ± 0.5
36.6 ± 0.9
65.7 ± 1.6
/
69.9 ± 2.0
49.6 ± 1.9
34.6 ± 1.2
[a] 50% Effective concentration: concentration of compound that inhibits the fungi growth by 50%; [b] /: not detected; [c] Hymexazol was used as a
positive control.
Figure 3. The inhibition effect of compound IV2 at different concentrations against A. solani (a), F. solani (b) and F. graminearum (c), CK: blank control group.
To explore the detrimental effects of these sarisan attached 3-phenylisoxazolines on normal mammalian cells, three potent compounds IV2, IV14 and IV23
were further investigated for their cytotoxicities on normal NRK-52E cells using CCK-8 method.[27] As displayed in Figure. 4, when NRK-52E cells were
treated with different concentrations of compounds IV2, IV14 and IV23, the NRK-52E cells still remained high viabilities. These resluts indicated that these
sarisan attached 3-phenylisoxazolines showed relative low toxicity on normal NRK-52E cells, and had selectivity between phytopathogenic fungi and
normal mammalian cells.
Figure 4. Relative cell viabilities of NRK-52 cells treated with compounds IV2, IV14 and IV23.
Conclusions
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In summary, we have synthesized a series of thirty-one sarisan attached 3-phenylisoxazolines IV1–31, and determined their structures by different spectral
analyses of 1H/13C NMR, IR and ESI-MS. All title sarisan attached 3-phenylisoxazolines IV1–31 were evaluated for their antifungal activities against five
phytopathogenic fungi (B. cinerea, C. lagenarium, A. solani, F. solani, and F. graminearum). Among all title sarisan derivatives, compounds IV2, IV14 and
IV23 showed more promising antifungal activity against some phytopathogenic fungi than the commercial fungicide Hymexazol. In particular, compound
IV2 exhibited a broad-spectrum and potent antifungal activity against three phytopathogenic fungi (A. solani, F. solani, and F. graminearum) with the EC50
values of 32.9 ± 1.6, 35.6 ± 0.3, and 33.4 ± 0.4 μg/mL, respectively. In addition, compounds IV2, IV14 and IV23 also displayed relative low toxicity on normal
NRK-52E cells. These resluts indicated that the potent sarisan attached 3-phenylisoxazolines IV2, IV14 and IV23 had selectivity between phytopathogenic
fungi and normal mammalian cells. This work will pave the way for the development of sarisan derivatives as fungicide candidates in plant protection.
Experimental Section
Instruments and Reagents
1H NMR/13C NMR spectra of all sarisan derivatives IV1–31 were recorded on a Bruker 400/100 MHz instrument (Avance 400 MHz, Bremerhaven, Germany).
Infrared (IR) spectra were detected by a PE-1710 FT-IR spectrometer. A Microplate Reader (Tecan Infinite Pro series M200) was used to record optical density
and fluorescence. Sesamol was purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Anhydrous solvents were dried and purified
according to standard methods before use. Other chemicals were of analytical grade and all obtained from commercial resources.
Synthesis of Sarisan Attached 3-Phenylisoxazolines IV1–31
Sarisan and the intermediates N-hydroxybenzimidoyl chlorides III1–31 were prepared as our previously reported procedures.[24, 25] Then, to a solution of
sarisan (0.5 mmol, 96.1 mg) in CH2Cl2 (10 mL) under N2, the corresponding N-hydroxybenzimidoyl chlorides III1–31 (0.55 mmol) and trimethylamine (0.55
mmol, 55.7 mg) were added. The mixture was stirred for 6–24 h at room temperature (r.t.). When the reaction was complete, the solvent was concentrated
and then purified by PTLC with developing solvents petroleum ether: ethyl acetate = 3: 1 (v/v) to give the target derivatives IV1–31 in 21–81% yields.
Examples of spectra data of IV1–3 are shown below, whereas data of other derivatives IV4–31 are listed in Supporting Information.
1
Data for IV1: Gray solid, yield: 81%, m.p. 90-92oC; IR cm-1 (KBr): 2916, 1621, 1487, 1354, 1290, 1196, 1031, 923, 845, 757, 690; H NMR (400 MHz CDCl3) δ:
7.65-7.62 (m, 2H, -Ar), 7.39-7.38 (m, 3H, -Ar), 6.72 (s, 1H, -Ar), 6.52 (s, 1H, -Ar), 5.90 (s, 2H, -OCH2O-), 5.01-4.94 (m, 1H, H-5′), 3.76 (s, 3H, -OCH3), 3.30-3.23
(m, 1H, H-4′), 3.09-3.05 (m, 1H, H-6′), 3.03-3.00 (m, 1H, H-4′), 2.85-2.80 (m, 1H, H-6′); 13C NMR (100 MHz CDCl3) δ: 156.5, 152.5, 146.9, 140.9, 129.9, 128.6,
126.6, 110.9, 101.0, 94.6, 80.9, 56.2, 39.2, 35.2, 35.3; MS (ESI) m/z calcd for C18H18NO4 ([M+H]+) 312.1, found 312.1.
1
Data for IV2: Yellow solid, yield: 38%, m.p. 52-54oC; IR cm-1 (KBr): 2924, 1596, 1488, 1456, 1195, 1036, 931, 761; H NMR (400 MHz CDCl3) δ: 7.84-7.80 (m,
1H, -Ar), 7.39-7.33 (m, 1H, -Ar), 7.17-7.13 (m, 1H, -Ar), 7.11-7.06 (m, 1H, -Ar), 6.72 (s, 1H, -Ar), 6.51 (s, 1H, -Ar), 5.89 (s, 2H, -OCH2O-), 5.00-4.93 (m, 1H, H-5′),
3.76 (s, 3H, -OCH3), 3.39-3.32 (m, 1H, H-4′), 3.19-3.13 (m, 1H, H-6′), 3.02-2.97 (m, 1H, H-4′), 2.86-2.81 (m, 1H, H-6′); 13C NMR (100 MHz CDCl3) δ: 161.5, 159.0,
153.3, 152.5, 146.9, 140.9, 131.4, 129.0, 124.3, 117.9, 117.2, 116.4, 116.2, 110.9, 101.0, 94.5, 81.1, 56.2, 41.0, 35.2; MS (ESI) m/z calcd for C18H17FNO4 ([M+H]+)
330.1, found 330.3.
1
Data for IV3: Gray solid, yield: 24%, m.p. 121-123oC; IR cm-1 (KBr): 2936, 1622, 1498, 1486, 1196, 1035, 927, 852, 763; H NMR (400 MHz CDCl3) δ: 7.59-7.56
(m, 1H, -Ar), 7.41-7.39 (m, 1H, -Ar), 7.34-7.27 (m, 2H, -Ar), 6.74 (s, 1H, -Ar), 6.51 (s, 1H, -Ar), 5.90 (s, 2H, -OCH2O-), 5.04-4.96 (m, 1H, H-5′), 3.76 (s, 3H, -OCH3),
3.45-3.38 (m, 1H, H-4′), 3.25-3.19 (m, 1H, H-6′), 3.05-3.00 (m, 1H, H-4′), 2.88-2.83 (m, 1H, H-6′); 13C NMR (100 MHz CDCl3) δ: 156.5, 152.2, 146.9, 140.9, 132.8,
130.4, 129.5, 126.9, 117.1, 111.0, 101.0, 94.5, 81.5, 56.2, 41.7, 35.0; MS (ESI) m/z calcd for C18H1735ClNO4 ([M+H]+) 346.0, found 346.1, C18H1737ClNO4 ([M+H]+)
348.0, found 348.1.
Antifungal and Cytotoxic Activities
The antifungal activity of all title sarisan attached 3-phenylisoxazolines IV1–31 against five phytopathogenic fungi was evaluated by using the mycelium
growth rate method according to our previous report.[21] PDA medium was prepared through using potato dextrose agar and then sterilized at 115 °C for 30
min. Each fungus was incubated in PDA at 28 ± 1 °C for about 5 days to get new mycelium for the antifungal assays, and then using a sterilized puncher to
cut a mycelia disk of approximately 5 mm diameter from culture medium. Subsequently, the mycelia disk was inoculated in the center of the PDA Petri
dishes. The tested derivatives IV1–31 and Hymexazol were dissolved in acetone, then mixed with the medium to set at the final concentration of 50 μg/mL.
The mixed medium was then added to the sterilized Petri dishes. The inoculated Petri dishes were incubated at 28±1 °C for 3 days. Acetone with only PDA
was served as a blank control (CK); the commercial fungicide Hymexazol was used as the positive control. Each treatment was conducted with three
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replicates. Finally, The radial growths of the fungal colonies were measured by a vernier caliper. The cytotoxic activity of compounds IV2, IV14 and IV23
against NRK-52E cells was assessed by the reported CCK-8 (Sigma-Aldrich,Germany) method.[27] In brief, 5 × 103 NRK-52E cells (Shanghai Cell Bank, Chinese
Academy of Sciences) in 100 μL medium were added to a 96-well plates. Subsequently, these cells were incubation at 37 °C for 24 h, a 100 μL of fresh
medium with the compounds IV2, IV14 and IV23 in different concentration was added to remove and replace the used culture medium. The negative control
was only added fresh medium. After incubation for 24 h, the used culture medium was discarded, and then using the new culture medium to wash the used
medium twice. Finally, new medium containing 5% CCK-8 (100 μL) was added to each well. The cells were cultured at 37 °C for a further 4 h and then the
absorbance at 450 nm was measured using a Microplate Reader.
Data and statistical analysis
The Inhibition rates of compounds IV1–31 against five phytopathogenic fungi were calculated by the following formula: Inhibition rate (%) = (C–T) × 100/(C–
5 mm), where C is the diameter of fungi growth on untreated PDA, and T represents the diameter of fungi on treated PDA. The relative cell viability was
calculated with the formula: Cell viability rate (%) = (ODexperiment – ODblank)/(ODnegative control – ODblank) × 100%. All data are expressed as mean ± SD, and each
group was performed three times. Statistical analysis was processed by the SPSS 21.0 software (SPSS Inc., Chicago, USA).
Supplementary Material
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-number.
Acknowledgements
We are thankful to the financial supports from the Ningxia Normal University Research Grant (Grant Number NXSFZDA1812).
Author Contribution Statement
Zhiyan Liu and Xiaoguang Wang prepared the sarisan derivatives. Jiangping Cao provided some phytopathogenic fungi samples and performed part of
antifungal activity assays. Zhiyan Liu, Xiaoting Yan,and Wanqing Cheng also performed part of antifungal and cytotoxic activities assays, and then analyzed
the data. Ruige Yang and Yong Guo conceived the experiments, and wrote the manuscript. All authors approved the final version of the manuscript.
Declaration of competing interest
The authors declare no competing financial interest.
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A series of sarisan attached 3-phenylisoxazolines with potent antifungal activity against against some phytopathogenic fungi.
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