Drug repurposing strategy II: from approved drugs to agri-fungicide leads

Abstract

Epidemic diseases of crops caused by fungi deeply affected the course of human history and processed a major restriction on social and economic development. However, with the enormous misuse of existing antimicrobial drugs, an increasing number of fungi have developed serious resistance to them, making the diseases caused by pathogenic fungi even more challenging to control. Drug repurposing is an attractive alternative, it requires less time and investment in the drug development process than traditional R&D strategies. In this work, we screened 600 existing commercially available drugs, some of which had previously unknown activity against pathogenic fungi. From the primary screen at a fixed concentration of 100 μg/mL, 120, 162, 167, 85, 102, and 82 drugs were found to be effective against Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Phytophthora capsici, Fusarium graminearum and Fusarium oxysporum, respectively. They were divided into nine groups lead compounds, including quinoline alkaloids, benzimidazoles/carbamate esters, azoles, isothiazoles, pyrimidines, pyridines, piperidines/piperazines, ionic liquids and miscellaneous group, and simple structure-activity relationship analysis was carried out. Comparison with fungicides to identify the most promising drugs or lead structures for the development of new antifungal agents in agriculture.

Introduction

Plant diseases and pests lead to reduced yields and quality of crops [1–3], which have a major impact on economic development and food security [4]. Research revealed that more than 19,000 species of fungi could cause plant diseases and some of them could be dormant in dead plants until opportunities were conducive to their proliferation [5]. It is estimated that the average annual economic loss caused by plant pathogenic fungi exceeds $200 billion [6]. Fusarium is one of the most important plant pathogenic fungi, for example, Fusarium graminearum and Fusarium oxysporum cause head blight and root rot. They can produce mycotoxins such as monothiocarbates and fumonisin [7, 8]. Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea and Phytophthora capsici host a wide range of more than 200 crops, including fruits and vegetables [9, 10]. Thus the loss of crops caused by fungi had become a severe issue that cannot be ignored.

Currently, there are several approaches to control plant diseases, such as breeding of resistant varieties, biological control and chemical control. However, the breeding also has many drawbacks, including lengthy breeding cycles, lack of varieties and geographical limitations of breeds [11]. Although biological control is advocated, the development process of biopesticides is slow and easy to deposit, so it is difficult to be widely applied in agricultural production at present [12, 13]. Thus, agrochemicals are still the primary form of control [14]. However, an expanding number of these weed, plant disease and pest insects are no longer effectively controlled by many of the existing agrochemical tools, a trend also observed in the medical community with the rise of antibiotic resistance [15, 16]. In particular, the development of resistance to critical fungicides against major crop fungal diseases, such as benzimidazoles and strobilurins, has had a significant impact on the fungicide market and the discovery of agrochemicals [17]. Therefore, it is necessary to develop novel, practical and resistant fungicidal agents to control plant diseases.

Drug development is an expensive, time-consuming and risky process [18]. In general, it can take up to 20 years for a drug from initial discovery to market. The development process can cost up to $2 billion, with only a 5% chance of successfully completing clinical trials and reaching the market [19, 20]. Likewise, an agrochemical takes 10–12 years from discovery to market and costs an estimated $286 million to develop [15, 21, 22]. Thus, it poses challenges to the development of novel pesticides. Drug repositioning or finding novel indications for known drugs is a way to reduce the time and cost of drug discovery as the toxicity, pharmacokinetic and biological activity of these drugs are well defined. In the medical field, many drugs have been successfully applied through repositioning [23–26]. As such, it has proven to be a preferred and advantageous alternative strategy for the more rapid discovery of new applications for drugs [27].

On this basis, 600 approved drugs with different structures and functions against Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Phytophthora capsici, Fusarium graminearum and Fusarium oxysporum were screened in this study and served as a theoretical basis for pesticide development.

Materials and methods

Fungal strains

Six phytopathogenic fungi, named R. solani, S. sclerotiorum, B. cinerea, P. capsici, F. graminearum and F. oxysporum were isolated, purified and identified from susceptible plants cultivated at the Gansu Academy of Agricultural Sciences, China.

Approved drugs

The 600 approved drugs were purchased from commercial suppliers. Drugs were delivered in centrifugal tube (100 μg ml⁻¹, dissolved in DMSO) and kept at −80 °C until use.

Screening Assay

In vitro, the antifungal activity of the drugs was initially evaluated using mycelial growth inhibition assay [6] with some modifications. The dissolved drug was added to the PDA medium so that the concentration of PDA containing the drug was 100 μg/mL. Zero point five percent DMSO (v/v) was added to the PDA medium as a blank control. The six plant pathogenic fungi were used to evaluate the antifungal activity of the samples. Take the disc (5.00 mm diameter) from the edge of the mycelia of the active colony with a hole punch, and then pick it to the center of the drug-containing plate with the inoculating needle. Lastly, the plates were incubated upside down in the dark at 26 °C. Three replicates per treatment. The diameter of the inhibition zone (mm) was measured by the cross method using digital calipers, and the growth inhibition rate of the samples on the fungal mycelium was calculated according to the following formula.

$$\mathrm{Mycelial}\mathrm{growth}\mathrm{inhibition(\%)}=\frac{C-T}{C-5}\times 100$$

The C and T are the average diameter of fungal colonies in the control and treated groups, respectively.

On the basis of the initial screening for antifungal activity, the highly active drugs were selected for virulence effect determination. The PDA medium was diluted to different solution concentrations (50 μg ml⁻¹, 25 μg ml⁻¹, 10 μg ml⁻¹, etc.), and the plates were incubated upside down in the dark at 26 °C. The inhibition rate as above, and the antifungal activity was indicated as EC₅₀.

Statistical Analysis

The statistical analysis was conducted by SPSS 24.0. The EC₅₀ values were derived from the parameters in the regression curves.

Results

The preliminary screening of 600 approved drugs against six phytopathogenic fungi at 100 μg ml⁻¹ showed that 120, 162, 167, 85, 102 and 82 drugs against R. solani, S. sclerotiorum, B. cinerea, P. capsici, F. graminearum and F. oxysporum, respectively, inhibiting the growth of mycelium of by more than 70% (Fig. 1) . To further determine the antifungal activity of these drugs, they were evaluated using the EC₅₀. We considered drugs with EC₅₀ less than 25 μg ml⁻¹ as candidates. Their original uses and toxicity are shown in Table 1 (https://pubchem.ncbi.nlm.nih.gov/, November 2022). As drug repurposing has gained tremendous popularity in the pharmaceutical field, we divided the candidate drugs into 9 lead series and conducted a brief discussion of structure and activity.

Fig. 1.

A total of 600 drugs were tested against pathogenic fungi at 100 μg/mL

Table 1.

Active drugs“hits”identified from initial screening anti-plant pathogenic fungi^a

No.	Compound name	Chemical structure	Main use	Toxicity	EC50 (μg/mL)
					R. s	S. s	B. c	F. g	F. o	P. c
1	Mefloquine hydrochloride		Anti-malarial	Rat: LD50 = 880 mg/kg (oral)	>25	>25	14.55	>25	>25	>25
2	Bedaquiline (fumarate)		Antituberculotic	Mouse and rat: single oral doses of 800 mg/kg	>25	6.81	9.45	>25	>25	>25
3	Dequalinium chloride		Antiseptic	Mouse: LD50 = 70 mg/kg (subcutaneous)	>25	9.52	>25	>25	>25	>25
4	Cabozantinib		Antineoplastic	-	0.032	>25	>25	>25	>25	>25
5	Quinoxyfen		Fungicides (powdery mildew)	Mouse: LD50 > 500 mg/kg (oral)	2.21	>25	>25	>25	>25	>25
6	Pitavastatin hemicalcium		Lipid-lowering agents	Rat: LDLo = 500 mg/kg (oral)	>25	>25	0.75	6.92	0.78	0.32
7	Telmisartan		Antihypertensive	Mouse: LD50 = 200 mg/kg (intravenous)	>25	5.81	2.19	>25	4.80	3.27
8	Parbendazole		Anthelmintic	Mouse: LD50 = 1700 mg/kg (oral)	0.051	0.16	>25	>25	>25	>25
9	Oxfendazole		Anthelmintic	Rat: LD50 > 6400 mg/kg; Mouse: LD50 > 6400 mg/kg	1.08	10.38	>25	>25	>25	>25
10	Fenbendazole		Anthelmintic	Rat: LD50 > 10 gm/kg (oral); Rat: LD50 > 2 gm/kg (subcutaneous)	0.007	0.097	>25	>25	>25	>25
11	Albendazole		Anthelmintic	Rat: LD50 = 2400 mg/kg (oral); Rat: LD50 = 256 mg/kg (intravenous)	0.12	0.11	>25	>25	2.84	>25
12	Mebendazole		Anthelmintic	Rat: LD50 = 714 mg/kg (oral); Mouse: LD50 = 620 mg/kg (oral)	0.011	0.52	>25	>25	>25	>25
13	Oxibendazole		Anthelmintic	Mouse: LDLo = 32 gm/kg (oral)	0.43	0.103	>25	>25	>25	>25
14	Flubendazole		Anthelmintic	Rat: LD50 = 2560 mg/kg (oral); Mouse: LD50 > 2560 mg/kg (oral)	>25	0.36	>25	>25	>25	>25
15	Astemizole		Antihistaminic	Mouse: LD50 = 2052 mg/kg	>25	>25	>25	22.05	>25	>25
16	Albendazole S-oxide		Anthelmintic	Mouse: LD50 > 800 mg/kg (oral)	11.78	9.35	>25	>25	>25	>25
17	Triclabendazole		Anthelmintic	Rat: LD50 > 8 gm/kg (oral); Rat: LD50 > 4 gm/kg (skin)	4.39	4.85	6.92	11.26	28.90	21.15
18	Econazole		Antifungal drug	Mice: LD50 = 462 mg/kg (oral); Rats: LD50 = 668 mg/kg (oral)	13.75	0.25	0.034	0.093	0.094	0.01
19	Itraconazole		Antifungal drug	Mouse: LD50 > 320 mg/kg (oral); Dog: LD50 > 200 mg/kg (oral)	>25	0.024	0.025	>25	0.044	0.024
20	Posaconazole		Antifungal drug	Clinical trials: some patients received posaconazole up to 1600 mg/day with no adverse events noted	>25	0.053	0.061	>25	0.165	0.11
21	Ketoconazole		Antifungal drug	Rat: LD50 = 227 mg/kg (oral)	>25	0.15	0.34	2.34	0.26	0.12
22	Bifonazole		Antifungal drug	Rat: LD50 = 1463 mg/kg (oral); Mouse: LD50 = 2629 mg/kg (oral)	4.86	4.58	1.26	0.54	0.18	0.17
23	Efinaconazole		Antifungal drug	Rat: daily doses of up to 30 (males) and 40 (females) mg/kg (generally well tolerated)	1.51	0.095	0.058	0.15	0.035	0.026
24	Isoconazole nitrate		Antifungal drug	Rat: LD50 = 5600 mg/kg (oral); Rat: LD50 >10 gm/kg (subcutaneous)	7.42	0.066	0.101	1.17	0.28	0.021
25	Fenticonazole nitrate		Antifungal drug	Rat: LD50 > 3 mg/kg (oral); Rat: LD50 >750 gm/kg (subcutaneous)	7.23	0.12	0.054	0.37	0.15	0.056
26	Isavuconazole		Antifungal drug	Rats:At doses up to 90 mg/kg/day (oral:not affect the fertility)	>25	0.76	0.043	0.35	0.14	0.015
27	Deferasirox		Iron chelating agent	-	17.94	>25	4.73	>25	>25	>25
28	Clotrimazole		Antifungal drug	Rat (male): LD50 = 708 mg/kg (oral); Mouse (male): LD50 = 923 mg/kg (oral)	0.61	0.17	0.32	0.48	0.08	0.061
29	Fluconazole		Antifungal drug	Rat: LD50 = 1271 mg/kg (oral); Rat: LD50 > 941 mg/kg (intraperitoneal)	>25	>25	11.16	>25	>25	3.77
30	Voriconazole		Antifungal drug	Mouse: LD50 = 223.07 mg/kg (intravenous)	1.64	0.26	0.12	0.79	0.078	0.032
31	Sulconazle nitrate		Antifungal drug	Rat: LD50 = 1741 mg/kg (oral); Rat: LD50 = 735 mg/kg (intraperitoneal)	2.47	0.14	0.057	1.30	0.12	0.014
32	Vagistat		Antifungal drug	Mouse: LD50 = 1870 mg/kg (oral); Mouse: LD50 = 508 mg/kg (intraperitoneal)	6.45	0.079	0.058	0.96	0.31	0.015
33	Butoconazole nitrate		Antifungal drug	Rat: LD50 = 1720 mg/kg (oral); Rat: LD50 = 940 mg/kg (intraperitoneal)	5.12	0.20	0.29	3.00	0.11	0.019
34	Terconazole		Antifungal drug	Rat (male): LD50 = 1741 mg/kg (oral); Rat (female): LD50 = 849 mg/kg (oral)	>25	9.99	9.74	>25	20.35	0.37
35	Elubiol		Antifungal drug	-	19.61	0.051	0.11	2.82	0.11	0.025
36	Luliconazole		Antifungal drug	In clinical trials, no serious toxicity was reported	8.83	0.003	0.001	0.003	0.005	0.001
37	Ruxolitinib		Antineoplastic	Rat: LD50 = 250 mg/kg (oral)	>25	>25	10.79	>25	24.42	>25
38	4,5-Dichloro-2-octyl- isothiazolone		Fungicide (mould)	Toxicity of poisoning	0.58	0.80	0.63	0.27	2.45	2.64
39	Octyl-2H- isothiazol-3-one		Fungicide (mould)	Rat: LD50 = 550 mg/kg (oral); Rat: LC > 2 gm/m3 (inhalation)	1.29	0.17	0.13	0.28	0.55	1.10
40	Isothiazolinone chloride		Fungicide (mould)	-	17.92	>25	>25	>25	>25	>25
41	1,2-Benzisothiazol- 3(2H)-one		Fungicide (mould)	Rat: LD50 = 1020 mg/kg (oral); Mouse: LD50 = 1150 mg/kg (oral)	3.51	2.12	3.40	2.96	>25	>25
42	Methyl-1,2- benzothiazol-3(2H)- one		Fungicide (mould)	Water flea: EC50 = 0.92 ppm; Freshwater green algae: EC50 = 0.92 ppm	5.84	13.53	20.22	16.86	19.00	>25
43	Fluoro-1,2- benzoisothiazol- 3(2H)-one		Fungicide (mould)	-	8.94	7.70	10.71	10.58	24.39	>25
44	Isothiazol-3-one		Pharmaceutical intermediates	-	7.95	6.21	9.45	9.90	27.41	19.43
45	5-Chloro-3- hydroxyisothiazole		Pharmaceutical intermediates	-	0.98	1.98	3.80	4.06	3.29	3.70
46	5-Fluorouridine		Pharmaceutical intermediates	Rat: LD50 = 400 mg/kg (intraperitoneal); Mouse: LD50 = 160 mg/kg (intraperitoneal)	>25	0.49	0.83	17.26	>25	>25
47	2'-Deoxyguanosine		Pharmaceutical intermediates	Rat: LD50 > 800 mg/kg (intraperitoneal)	>25	15.13	>25	>25	>25	>25
48	Ganciclovir		Antiviral	Mouse: LD50: > 2 g/kg (oral):	>25	7.56	>25	>25	>25	>25
49	Floxuridine		Antineoplastic	Rat: LD50 = 215 mg/kg (oral)	>25	2.43	1.12	>25	>25	>25
50	5-Fluorouracil		Antineoplastic	Mice: LD50=230 mg/kg (oral)	>25	>25	6.33	9.46	>25	>25
51	Sulfatinib		Antineoplastic	-	>25	>25	3.37	>25	>25	>25
52	Gefitinib		Antineoplastic	Low toxicity	>25	>25	13.52	>25	>25	>25
53	Dabrafenib		Antineoplastic	Rats: LD50 > 2000 mg/kg	>25	2.31	0.63	>25	>25	>25
54	Afatinib		Antineoplastic	-	>25	>25	5.00	>25	>25	>25
55	Ibrutinib		Antineoplastic	-	>25	5.53	2.29	>25	14.02	9.11
56	Nilvadipine		The treatment of hypertension	Rat: LD50 = 1560 mg/kg (oral); Rat: LD50 > 1 gm/kg (subcutaneous)	>25	6.46	5.74	>25	20.44	>25
57	Amlodipine maleate		The treatment of hypertension	-	>25	>25	12.43	>25	>25	>25
58	Liranaftate		Antifungal drug	Rat: LD50 > 2 gm/kg (oral); Rat: LD50 > 2 gm/kg (intraperitoneal)	0.048	0.168	0.004	0.19	0.031	0.27
59	Pyributicarb		Herbicides	Rat: LD50 > 5 gm/kg (oral); Rat: LD50 > 5 gm/kg (intraperitoneal)	0.22	2.07	0.089	0.12	0.30	1.93
60	Amlodipine		The treatment of hypertension	Women: LDLo = 1400 ug/kg (oral)	>25	>25	10.53	>25	>25	>25
61	Nimodipine		Treatment of cerebrovascular	Rat: LD50 = 2738 mg/kg (oral); Mouse: LD50 = 940 mg/kg (oral)	>25	>25	10.27	>25	>25	>25
62	Sodium pyrithione		Antimicrobial agents	Mouse: LD50 = 265 mg/kg (intraperitoneal); Rat: LD50 = 385 mg/kg (intraperitoneal)	5.45	1.42	2.26	4.72	10.72	2.83
63	Zinc pyrithione		Antimicrobial agents	-	7.79	0.43	7.97	0.97	3.80	2.25
64	Copper pyrithione		Fungicides	Fathead minnow: LC50 = 4.3 ppb; Crassostrea virginica: EC50 = 9.2 ppb	6.73	0.36	2.55	1.13	5.28	28.54
65	Bispyrithione		Pesticide intermediates	-	3.31	3.17	2.03	0.37	25.55	1.39
66	Ciclopirox ethanolamine		Antifungal agent	Rat: LD50 = 2350 mg/kg (oral); Rat: LD50 > 2500 mg/kg (subcutaneous);	14.68	1.95	5.37	10.93	14.08	18.66
67	Piroctone olamine		Anti-dandruff agent	Mouse: LD50 = 5 gm/kg (oral) Rat: LD50 = 8100 mg/kg (oral)	18.77	7.29	>25	19.28	17.77	17.08
68	Caprylohydroxamic acid		Preservatives	Rat: LD50 = 10700 mg/kg (oral); Mouse: LD50 = 8820 mg/kg (oral)	27.75	16.12	7.08	>25	>25	>25
69	Flavopiridol		Anti-cancer	-	17.83	22.72	12.81	>25	>25	>25
70	Terfenadine		Antihistamines	Rat: LD50 = 5 gm/kg (oral); Rat: LD50 >1250 mg/kg (subcutaneous)	>25	14.52	12.38	23.03	>25	>25
71	Thioridazine hydrochloride		Antipsychotic	Rat: LD50 = 1060 mg/kg (oral); Rat: LD50 = 71 mg/kg (intravenous)	>25	21.62	>25	>25	18.49	15.90
72	Penfluridol		Antipsychotic	Mouse: LD50 = 87 mg/kg (oral); Rat: LD50 = 160 mg/kg (oral)	>25	6.95	13.53	17.32	>25	>25
73	Trifluoperazine		Antipsychotic	Mouse: LD50 = 1350 mg/kg (oral); Mouse: LD50 = 120 mg/kg (intraperitoneal)	3.66	13.20	4.12	6.17	8.59	8.19
74	Ebastine		Antihistamines	Rat: LD50 > 4 gm/kg (oral); Rat: LD50 = 496 mg/kg (intraperitoneal)	21.04	17.05	13.82	>25	19.64	>25
75	Prochlorperazine maleate		Antipsychotic	Rat: LD50 = 750 mg/kg (oral) Rat: LD50 = 320 mg/kg (subcutaneous)	14.84	20.95	17.53	>25	>25	12.31
76	Perphenazine		Antipsychotic	Rat: LD50 = 318 mg/kg (oral); Rat: LD50 = 146 mg/kg (intraperitoneal)	16.19	14.46	18.21	>25	13.70	15.60
77	Clozapine		Antipsychotic	Rat: LD50 = 251 mg/kg (oral); Rat: LD50 = 251 mg/kg (subcutaneous)	>25	>25	23.13	>25	>25	>25
78	Aripiprazole		Antipsychotic	-	>25	20.25	17.55	24.71	17.91	24.92
79	Ziprasidone hydrochloride		Antipsychotic	-	>25	22.96	>25	>25	>25	>25
80	Loratadine		Antihistamines	Rat: LD50 > 5000 mg/kg (oral)	6.19	>25	24.53	>25	>25	12.18
81	Ponatinib		Antiangiogenic; antineoplastic	Toxicity of poisoning	0.017	>25	11.47	>25	>25	>25
82	Bosutinib		Antineoplastic	-	>25	7.69	13.86	>25	>25	>25
83	Vandetanib		Antineoplastic	Low toxicity	>25	>25	17.32	13.34	>25	>25
84	Avitinib		Antineoplastic	-	>25	11.21	11.80	49.36	>25	>25
85	Crizotinib		Antineoplastic	-	>25	11.85	10.59	>25	>25	>25
86	Ceritinib		Antineoplastic	-	>25	>25	19.57	>25	>25	>25
87	Decyl-3- methylimidazolium chloride		Chemical intermediates	-	>25	22.95	>25	>25	>25	>25
88	1-Dodecyl-3- methylimidazolium chloride		Chemical intermediates	-	>25	6.12	>25	>25	>25	>25
89	Dodecyl dimethyl benzyl ammonium bromide		Disinfectant; preservative	Rat: LD50 = 230 mg/kg (oral); Mouse: LD50 = 277 mg/kg	>25	5.80	8.85	>25	>25	>25
90	Cetalkonium chloride		Preservatives	Rat: LD50 > 500 mg/kg (oral)	>25	8.63	>25	>25	>25	>25
91	Myristalkonium chloride		Germicide and algicide	Mouse: LD50 = 919 mg/kg (oral); Mouse: LD50 = 18 mg/kg (intravenous)	>25	14.28	9.06	>25	>25	>25
92	Benzododecinium chloride		Preservatives	Rat: LD50 = 400 mg/kg (oral); Rat: LD50 = 100 mg/kg (intraperitoneal)	>25	>25	11.40	>25	>25	>25
93	1-Dodecylpyridinium bromide		Surfactant	-	>25	15.34	2.63	16.69	>25	>25
94	1,1'-Di-n-heptyl-4,4'- bipyridinium dibromide		Calcium release inhibitors	-	>25	>25	17.80	>25	>25	>25
95	1-Tetradecylpyridinium chloride		Surfactant	-	>25	23.77	11.35	>25	>25	>25
96	Octenidine dihydrochloride		Antibacterial agents	-	>25	17.57	5.36	>25	>25	>25
97	Chlorhexidine diacetate		Cationic broad-spectrum antimicrobial	Mouse: LD50= 2 gm/kg (oral); Mouse: LD50= 38 mg/kg (intraperitoneal)	12.14	6.66	3.35	10.13	11.19	3.65
98	Domiphen bromide		Disinfectants	Guinea pig:lowest published lethal dose: 10 mg/kg	22.25	6.42	9.96	21.27	>25	>25
99	Diminazene aceturate		Antiparasitic agent; trypanocidal drug	-	>25	6.22	3.07	3.18	>25	>25
100	Pentamidine		Antiinfective agent	Mouse: LD50= 50 mg/kg (intraperitoneal)	>25	5.86	3.89	>25	>25	>25
101	Enebicyanog		Fungicides and preservatives	-	2.89	0.91	0.62	20.94	>25	>25
102	Monensin sodium salt		Antiprotozoal agent	-	8.80	0.076	0.11	>25	0.67	0.36
103	Rifamycin sodium		Antibiotics	Rat: LD50= 2680 mg/kg (oral); Mouse: LD50= 2120 mg/kg (oral)	23.18	18.91	7.53	>25	>25	>25
104	(+)-Griseofulvin		Antibiotics	Rat: LD50 >10 gm/kg (oral); Rat: LD50 = 400 mg/kg (intravenous)	2.77	13.20	0.31	6.17	2.26	3.88
105	Natamycin		Antiseptic; antifungal	Rat: LD50 = 2730 mg/kg (oral); Rat: LD50 = 190 mg/kg (subcutaneous)	0.51	1.18	0.84	2.18	2.32	0.63
106	Naftifine hydrochloride		Antifungal	-	>25	12.47	1.68	13.68	9.40	12.78
107	Terbinafine hydrochloride		Antifungal	Rat: LD50 > 2 gm/kg (skin)	18.05	0.80	0.11	0.17	0.26	0.51
108	Butenafine hydrochloride		Antifungal	Rat: LD50 > 4 gm/kg (oral) Rat: LD50 > 100 gm/kg (intravenous)	2.75	0.22	0.07	0.08	0.29	0.85
109	Tolnaftate		Antifungal	Mouse: LD50 = 4800 mg/kg (intravenous); Mouse: LD50 = 120 mg/kg (intraperitoneal)	0.36	7.90	0.07	>25	0.47	>25
110	Cinacalcet		A calcimimetic and a P450 inhibitor	-	9.89	20.12	10.83	15.25	13.37	10.41
111	Salicylanilide		Fungicides (cosmetics)	Mouse: LD50 = 2400 mg/kg (oral); Mouse: LD50 > 500 mg/kg (intraperitoneal)	24.43	>25	21.90	21.77	14.62	11.64
112	Oxyclozanide		Anthelmintic	Rat: LD50 = 1 gm/kg (oral)	0.71	0.50	0.09	>25	>25	>25
113	Nitazoxanide		Anthelmintic	Mouse: LD50 = 1350 mg/kg (oral); Rat: LD50 > 10 gm/kg (oral)	>25	1.42	1.15	>25	>25	>25
114	Sulfaquinoxaline sodium		Anthelmintic	-	>25	>25	>25	14.47	>25	>25
115	Sulfisomezole sodium		Antibiotics	-	>25	>25	14.20	>25	14.71	>25
116	Bensulfuron-methyl		Herbicides	Rat: LD50 > 5 gm/kg (oral); Rat: LD50 > 2 gm/kg (skin)	>25	12.67	12.40	17.36	>25	>25
117	Chlorimuron-ethyl		Herbicides	Rat: LD50 = 4102 mg/kg (oral)	>25	13.09	11.68	>25	>25	>25
118	Vemurafenib		Antineoplastic	Rat: up to 250 mg/kg/day (no evidence of teratogenicity)	0.42	>25	>25	>25	>25	>25
119	Bardoxolone methyl		Antineoplastic; anti-inflammatory	-	0.56	>25	1.72	>25	>25	>25
120	4,4,4-Trifluoro-1- (4-fluorophenyl)butane-1,3-dione		Pharmaceutical intermediates	-	6.45	13.12	4.76	6.43	31.06	31.75
121	Sodium dehydroacetate		Food additives; preservatives	-	18.02	8.91	16.21	6.59	7.95	24.01
122	(4-Chloropheny)- 4,4,4-trifluoro-1,3- butanedione		Pharmaceutical intermediates	-	2.64	3.28	1.69	1.90	9.51	10.36
123	Triclosan		Preservatives; fungicides	Rat: LD50 = 3700 mg/kg	7.88	10.03	2.24	3.15	4.39	4.96
124	Tamoxifen		Antineoplastic	Rat: LD50 = 4100 mg/kg (oral); Rat: LD50 = 700 mg/kg (intraperitoneal)	>25	11.04	7.37	>25	>25	>25
125	Dronedarone hydrochloride		Anti-arrhythmic	-	>25	4.74	10.21	>25	>25	20.85
126	Nimesulide		Non-steroidal anti-inflammatory	Mouse: LD50 = 216 mg/kg (intraperitoneal); Mouse: LD50 = 392 mg/kg (oral)	>25	>25	1.24	>25	>25	>25
127	Disulfiram		Antineoplastic	Rats: LD50 = 8.6 g/kg (oral)	20.28	15.95	>25	>25	>25	>25
128	Bithionol		Anthelmintic	Mouse: LD50 = 760 mg/kg (oral); Mouse: LD50 = 760 mg/kg (intraperitoneal)	>25	>25	>25	>25	>25	10.35
129	JX06		Selective covalent inhibitors	Mouse: LD50 = 3250 mg/kg (oral)	21.28	>25	>25	>25	>25	>25
130	Iodopropynyl butylcarbamate		Fungicides (wood)	Rat: LD50 = 1.5 g/kg (oral)	0.12	0.14	0.11	0.55	0.47	0.45
131	Leflunomide		Non-steroidal anti-inflammatory	Mouse: LD50 = 445 mg/kg (oral); Mouse: LD50 = 185 mg/kg (intraperitoneal)	>25	>25	15.21	>25	>25	>25
132	Efavirenz		Antivirals	Cynomolgus monkeys:dosages of 60 mg/kg daily (substantial malformations)	8.43	6.78	>25	27.13	16.77	16.93
133	Sunitinib		Anti-tumour	Rat, mouse, and dog: 500 mg/kg (The maximally tolerated dose for when given orally)	>25	>25	21.87	>25	>25	>25
134	Indometacin		Non-hormonal anti-inflammatory	Rats: LD50 = 2.42 mg/kg (oral)	13.85	>25	4.46	>25	22.52	>25
135	Dichloro-1,2- dithiacyclopentenone		Fungicides (multi-disciplinary)	Mouse: LD50 = 13 mg/kg (intravenous)	3.69	6.32	6.66	5.27	5.76	6.26
136	3H-1,2-Benzodithiol-3-one		Pharmaceutical intermediates	-	>25	22.43	18.95	>25	>25	>25
137	Sertraline hydrochloride		Antidepressants	Mouse: LDLo = 336 mg/kg (oral); Women: TDLo = 7 mg/kg/2W-I (oral)	17.48	17.71	8.64	34.65	20.60	14.19
138	Simvastatin		Lipid-lowering drugs	Mouse: LD50 = 798 mg/kg (intraperitoneal); Mouse: LD50 = 3 gm/kg (oral)	15.43	9.21	8.15	>25	7.16	>25
139	Benzbromarone		Lowering uric acid	Rat: LD50 = 1230 mg/kg (subcutaneous); Rat: LD50 = 248 mg/kg (oral); Mouse: LD50 = 618 mg/kg (oral)	>25	10.75	1.95	12.48	9.18	>25
140	Fluvastatin sodium salt		Lipid-lowering drugs	-	>25	0.52	0.21	>25	0.62	2.55
141	Dichlorophen		Anti-microbial agents	Rat:LD50 = 1683 mg/kg (oral)	4.52	7.88	7.55	5.24	7.55	7.04
142	Flufenamic acid		Anti-inflammatory	Mouse: LD50 = 490 mg/kg (oral); Mouse: LD50 = 620 mg/kg (subcutaneous)	8.37	15.28	4.56	45.27	24.54	14.25
143	Carbonyl cyanide 3-chloro- phenylhydrazone		A geroprotector; an antibacterial agent and an ionophore	Rat: LDLo = 50 mg/kg (subcutaneous); Mouse: LDLo = 8 mg/kg (intraperitoneal)	0.53	0.57	0.38	4.33	6.07	4.69
144	Bufexamac		Non-steroidal anti-inflammatory	Rat: LD50 = 3370 mg/kg (oral); Mouse: LD50 = 1195 mg/kg (oral)	>25	21.95	>25	>25	>25	>25
145	Diclofenac		Anti-inflammatory drugs	Mouse: LD50 = 170 mg/kg (oral)	6.63	13.34	3.18	>25	>25	>25
146	Phenylpenta-2,4- dienoic acid		Light sensitive compound	-	>25	17.94	>25	>25	>25	>25
147	Amylmetacresol		Antiseptic; antifungal	Rat: LD50 = 1500 mg/kg (oral)	11.58	18.79	7.29	8.81	>25	>25
148	Tapinarof		Antifungal drug	-	9.55	12.32	14.99	11.95	14.20	14.44
149	Febantel		Anthelmintic	Rat: LD50 = 10605 mg/kg (oral); Mouse: LD50 > 10 gm/kg (oral)	0.15	>25	>25	>25	>25	>25
150	Abafungin		Antifungal drug	-	3.05	0.39	18.66	3.37	0.47	1.80

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Fungicides against plant pathogenic fungi

Plant pathogens can cause crop yield reduction and quality deterioration, and control of plant diseases is still dominated by chemical fungicides. We evaluated the in vitro activity of the fungicide in Fig. 2. Carbendazim and thiophanate-methyl were broad-spectrum fungicides belonging to the benzimidazole and substituted benzene fungicides respectively, with EC₅₀ values in the range of 0.14–22.12 μg ml⁻¹ for pathogenic fungi. They had excellent activity against S. sclerotiorum, with EC₅₀ was 0.68 and 0.53 μg ml⁻¹, respectively. Difenoconazole is a sterol demethylation inhibitor with systemic, prophylactic and therapeutic effects. It had relatively potent activity against five pathogens except for R. solani, especially F. oxysporum, with an EC₅₀ of 0.04 μg ml⁻¹. Boscalid was a novel nicotinamide fungicide with positive action against R. solani, S. sclerotiorum, B. cinerea and F. graminearum, with EC₅₀ < 2 μg ml⁻¹. Azoxystrobin and kresoxim-methyl were strobilurin fungicides with good activity against S. sclerotiorum with EC₅₀ of 4.9 and 4.66 μg ml⁻¹, respectively. Pyrimethanil and thirluzamide belong to the genus of methyl pyrimidine and benzamides, respectively. They were potent pesticides against B. cinerea and R. solani with EC₅₀ was 3.89 and 0.054 μg ml⁻¹, respectively. We evaluated the different classes of fungicides against plant pathogens to provide a basis for the activity level of the drugs screened for this study.

Fig. 2.

The EC₅₀ of fungicides against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Quinoline alkaloids

Alkaloids are a class of alkaline nitrogen-containing organic compounds in plants, marine organisms, microorganisms and insects. They have a wide range of biological activities such as lowering blood pressure, anti-tumor, central nervous system, lowering blood glucose, lowering blood lipids, insect repellent and anti-microbial [28–32]. Therefore they show potential for application in medical treatment and agricultural insecticide. In previous studies, our team designed and synthesized a variety of quinoline alkaloid derivatives based on different structures of natural product alkaloids, and tested their activity against plant pathogenic fungi [33–40] (Table 2), which provided a theoretical basis and laid a solid foundation for the development and application of alkaloids. To further obtain a broader spectrum of effective anti-phytopathogenic fungal alkaloids, the 28 drugs with different biological functions were repositioned (Table 3), and 6 compounds with better anti-phytopathogenic fungal activities were obtained, as shown in Table 1 and Fig. 3. Among them, pitavastatin calcium had a relatively broad spectrum of activity against pathogenic fungi, particularly against B. cinerea, P. capsici and F. oxysporum, with EC₅₀ of less than 1 μg ml⁻¹. However, cabozantinib showed more excellent activity against R. solani, with EC₅₀ of 0.032 μg ml⁻¹, which may be the introduction of 1-methoxy-4-methylbenzene into the quinoline structure to enhance the antifungal activity. In addition, dequalinium chloride, mefloquine hydrochloride and bedaquiline showed potential against B. cinerea or S. sclerotiorum. Therefore, the quinoline alkaloids designed and synthesized in our laboratory, as well as the repositioning of other functional alkaloids, we found that alkaloids have great potential in the field of agricultural disease control.

Table 2.

The EC₅₀ of quinoline alkaloids designed and synthesized in our laboratory against plant pathogenic fungi^a

No.	Chemical structures	EC50(μg/mL)							References
		R. s	S. s	B. c	F. g	M. o	P. c	M. m
1		11.79	5.03	0.08	0.94	-	-	0.98	[33]
2		14.04	1.07	0.09	2.80	-	-	0.83	[33]
3		4.93	2.80	0.07	1.92	-	-	-	[33]
4		5.29	1.93	0.16	1.63	-	-	-	[33]
5		5.36	2.68	0.14	1.48	-	-	-	[33]
6		0.75	2.77	0.10	6.51	-	-	-	[33]
7		å 30	8.44	0.09	19.50	-	-	-	[33]
8		-	-	0.050		-	-	-	[34]
9		-	-	0.037		-	-	-	[34]
10		-	-	0.027		-	-	-	[34]
11		-	-	0.034		-	-	-	[34]
12		-	-	0.040		-	-	-	[34]
13		-	-	0.061		-	-	-	[34]
14		-	-	0.055		-	-	-	[34]
15		-	-	0.086		-	-	-	[34]
16		-	-	0.037		-	-	-	[34]
17		-	-	0.032		-	-	-	[34]
18		-	-	0.035		-	-	-	[34]
19		-	-	0.047		-	-	-	[34]
20		-	-	0.068		-	-	-	[34]
21		-	-	0.071		-	-	-	[34]
22		-	-	1.32		-	-	-	[34]
23		1.52	1.79	1.33	8.37	-	-	-	[35]
24		2.59	1.98	7.91	2.60	-	-	-	[35]
25		2.64	1.20	0.76	3.55	-	-	-	[35]
26		5.41	1.16	1.88	15.72	-	-	-	[35]
27		3.58	1.16	0.73	5.90	-	-	-	[35]
28		1.84	0.52	0.50	21.37	-	-	-	[35]
29		>40	0.83	1.99	32.06	-	-	-	[35]
30		-	-	8.42	27.79	-	-	-	[36]
31		9.45	13.29	6.03	15.38	-	-	-	[36]
32		-	-	6.01	15.93	-	-	-	[36]
33		-	-	4.53	14.76	-	-	-	[36]
34		-	-	6.93	17.37	-	-	-	[36]
35		-	-	7.24	19.60	-	-	-	[36]
36		-	-	9.16	34.92	-	-	-	[36]
37		8.95	10.31			-	-	-	[36]
38		2.28	2.74	9.61	2.21	-	-	-	[37]
39		2.85	0.78	3.62	1.59	-	-	-	[37]
40		2.71	-	4.37	5.11	-	-	-	[37]
41		-	-	1.32	-	-	-	-	[37]
42		1.97	2.87	5.97	-	12.72	-	-	[38]
43		1.770	-	-	5.959	-	-	-	[39]
44		0.098	-	-	-	-	-	-	[39]
45		0.065	-	-	-	-	-	-	[39]
46		0.073	-	-	-	-	-	-	[39]
47		5.550	30.920	24.049	12.282	11.076	9.281	-	[39]
48		5.708	20.971	21.871	27.733	19.796	12.512	-	[39]
49		3.915	1.569	4.999	0.249	0.246	0.505	-	[39]
50		-	-	0.103	-	-	-	-	[40]
51		-	-	0.146	-	-	-	-	[40]
52		-	-	0.321	-	-	-	-	[40]
53		-	-	0.069	-	-	-	-	[40]
54		-	-	0.036	-	-	-	-	[40]
55		-	-	0.050	-	-	-	-	[40]
56		-	-	0.066	-	-	-	-	[40]
57		-	-	0.093	-	-	-	-	[40]
58		-	-	0.191	-	-	-	-	[40]
59		-	-	0.042	-	-	-	-	[40]
60		-	-	0.229	-	-	-	-	[40]
61		-	-	0.047	-	-	-	-	[40]
62		-	-	0.133	-	-	-	-	[40]
63		-	-	0.108	-	-	-	-	[40]
64		-	-	0.245	-	-	-	-	[40]
65		-	-	0.397	-	-	-	-	[40]

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici; M.o, Magnaporthe oryzae; M.m, Mycosphaerella melonis

Table 3.

In vitro antifungal activities (inhibition rate/%) of the quinoline alkaloids against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	Quinine sulfate dihydrate	100	12.85 ± 1.52	2.54 ± 1.47	20.62 ± 1.48	8.99 ± 0.94	12.65 ± 0.62	10.38 ± 1.66
2	Hydroquinidine	100	59.34 ± 1.66	14.43 ± 1.39	24.9 ± 1.37	22.31 ± 0.46	18.48 ± 0.52	16.97 ± 0.11
3	Hydroquinidine 4-chlorobenzoate	100	68.33 ± 0.43	81.23 ± 1.88	88.05 ± 1.74	28.96 ± 1.98	75.01 ± 0.4	70.48 ± 0.82
4	Hydroquinine	100	4.11 ± 1.30	7.55 ± 0.10	18.14 ± 1.85	12.60 ± 1.41	20.11 ± 2.05	16.88 ± 0.99
5	Quinine hydrochloride dihydrate	100	0 ± 0	0 ± 0	36.25 ± 1.69	6.19 ± 0.55	21.35 ± 0.51	17.87 ± 1.52
6	Synephrine	100	6.04 ± 0.32	0 ± 0	0 ± 0	0 ± 0	20.33 ± 1.39	8.95 ± 0.56
7	N-Benzylcinchoninium chloride	100	11.11 ± 2.39	0 ± 0	10.52 ± 0.67	0 ± 0	18.63 ± 1.04	24.22 ± 1.89
8	N-Benzylquininum chloride	100	17.96 ± 0.42	0 ± 0	11.84 ± 1.57	13.68 ± 1.98	31.34 ± 1.05	0 ± 0
9	N-Benzylcinchonidinium chloride	100	13.45 ± 2.54	0 ± 0	5.48 ± 2.51	0 ± 0	28.23 ± 2.15	11.99 ± 1.34
10	Cinchonine hydrochloride	100	15.79 ± 2.26	0 ± 0	8.8 ± 0.45	0 ± 0	32.26 ± 0.48	18.13 ± 0.67
11	Quinine dihydrochloride	100	17.36 ± 0.79	0 ± 0	9.83 ± 0.63	0 ± 0	18.15 ± 1.97	17.45 ± 0.52
12	Hydroquinidine hydrochloride	100	17.47 ± 0.52	15.09 ± 1.9	15.03 ± 1.19	9.23 ± 1.65	24.29 ± 1.25	22.44 ± 0.93
13	Cinchonine	100	0 ± 0	8.77 ± 0.79	6.45 ± 0.82	10.84 ± 1.01	22.83 ± 0.31	21.69 ± 0.18
14	Cinchonidine	100	5.25 ± 1.39	0 ± 0	10.93 ± 1.82	0 ± 0	21.5 ± 0.33	0 ± 0
15	Quinidine	100	0 ± 0	30.52 ± 0.94	25.7 ± 1.18	20.59 ± 1.08	36.44 ± 1.85	20.12 ± 0.19
16	Quinine	100	47.17 ± 0.04	22.98 ± 1.37	17.18 ± 1.52	0 ± 0	17.81 ± 1.59	18.46 ± 0.34
17	Quininne hydrochloride	100	0 ± 0	14.32 ± 2.69	21.95 ± 1.82	8.75 ± 2.29	11.19 ± 0.31	13.03 ± 0.01
18	(9 S) − 10,11-Dihydro-cinchonan-6',9-diol	100	16.57 ± 1.03	0 ± 0	48.42 ± 0.92	30.04 ± 0.01	7.36 ± 0.78	16.26 ± 1.46
19	Plaquenil	100	0 ± 0	0 ± 0	23.73 ± 0.85	38.83 ± 0.17	12.78 ± 2.26	20.79 ± 1.08
20	Mefloquine hydrochloride	100	73.2 ± 2.53	0 ± 0	100 ± 0	60.71 ± 1.47	66.92 ± 0.30	71.23 ± 1.43
21	Bedaquiline (fumarate)	100	38.76 ± 1.73	86.4 ± 0.9	89.72 ± 0.7	30.38 ± 1.26	49.23 ± 0.05	55.99 ± 1.05
22	Dequalinium chloride	100	47.29 ± 0.44	100 ± 0	100 ± 0	37.83 ± 0.29	68.18 ± 0.82	97.11 ± 0.01
23	Lenvatinib	100	66.06 ± 0.39	37.28 ± 1.56	11.86 ± 0.11	21.69 ± 1.02	22 ± 0.76	0 ± 0
24	Cabozantinib	100	100 ± 0	14.81 ± 1.01	58.71 ± 2.27	18.32 ± 0.14	18.28 ± 1.23	23.91 ± 1.95
25	Primaquine diphosphate	100	8.91 ± 0.44	48.57 ± 1.02	35.52 ± 2.75	0 ± 0	15.75 ± 0.51	0 ± 0
26	Quinoxyfen	100	92.45 ± 0.09	59.4 ± 1.16	29.25 ± 1.51	61.41 ± 1.22	34.02 ± 0.02	43.08 ± 0.13
27	Pitavastatin calcium	100	55.22 ± 1.2	68.0 ± 1.24	100 ± 0	74.07 ± 0.87	95.94 ± 0.28	97.4 ± 0.08
28	Decoquinate	100	16.51 ± 0.24	0 ± 0	25.69 ± 0.17	0 ± 0	0 ± 0	0 ± 0

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Fig. 3.

The EC₅₀ of quinoline alkaloids against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Benzoimidazole/carbamate drugs

Benzimidazoles and their derivatives are an essential group of active agents in pesticides and pharmaceuticals with broad-spectrum biological activities, such as anticancer [41], antibacterial [42], antiviral [43] and antiparasitic [44]. Likewise, carbamates are a group of insecticides with outstanding bioactivity, which have properties such as rapid decomposition, short residual period and low bioaccumulation [45, 46]. On this basis, we screened 26 drugs against pathogenic fungi, as shown in Table 4, and screened out 11 drugs with excellent action, as shown in Table 1 and Fig. 4, which laid the foundation for searching for lead compounds with good activity.

Table 4.

In vitro antifungal activities (inhibition rate/%) of the benzimidazole/carbamate drugs against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	Omeprazole	100	0 ± 0	7.07 ± 1.45	30.42 ± 1.88	11.72 ± 0.07	20.91 ± 0.63	28.2 ± 0.19
2	Esomeprazole magnesium	100	7.53 ± 1.11	0 ± 0	21.85 ± 1.25	14.72 ± 2.61	22.89 ± 0.36	24.33 ± 1.42
3	Ufiprazole	100	51.42 ± 1.75	27.93 ± 0.7	27.72 ± 0.25	36.05 ± 1.75	52.05 ± 0.5	63.33 ± 0.52
4	Lansoprazole	100	36.07 ± 1.36	32.07 ± 1.59	24.4 ± 2.55	23.01 ± 2.23	36.26 ± 1.07	49.72 ± 0.73
5	Lansoprazole sulphide	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	10.23 ± 1.83
6	R-(+)-lansoprazole	100	40.35 ± 0.72	0 ± 0	22.89 ± 0.9	5.69 ± 0.49	39.58 ± 1.56	51.81 ± 1.09
7	Ilaprazole (IY 81149)	100	43.74 ± 1.99	22.9 ± 1.09	59.26 ± 0.78	12.95 ± 0.83	43 ± 0.32	49.98 ± 0.91
8	Pantoprazole sodium	100	7.46 ± 1.34	5.90 ± 1.11	18.55 ± 1.81	0 ± 0	24.34 ± 0.44	27.55 ± 1.53
9	Pantoprazole thioether	100	67.27 ± 0.31	11.75 ± 1.86	35.38 ± 0.25	32.94 ± 1.03	53.25 ± 0.68	60.46 ± 1.38
10	Rabeprazole sulphide	100	33.15 ± 1.16	0 ± 0	23.75 ± 1.1	63.97 ± 1.39	54.88 ± 1.81	56.38 ± 1.41
11	Azilsartan	100	0 ± 0	31.25 ± 1.32	19.15 ± 1.58	13.50 ± 2.16	5.33 ± 1.40	35.35 ± 1.16
12	Telmisartan	100	70.28 ± 0.97	82.22 ± 1.08	86.19 ± 0.81	51.44 ± 0.45	80.08 ± 1.94	80.95 ± 1.41
13	Candesartan cilexetil	100	0 ± 0	9.73 ± 0.21	0 ± 0	22.37 ± 0.8	8.77 ± 1.55	6.1 ± 1.14
14	Dabigatran etexilate	100	26.37 ± 1.92	72.09 ± 0.16	76.94 ± 1.16	0 ± 0	13.68 ± 0.7	36.76 ± 1.2
15	Pimobendan	100	0 ± 0	19.52 ± 2.91	0 ± 0	0 ± 0	0 ± 0	11.42 ± 1.41
16	Parbendazole	100	97.34 ± 0.58	97.98 ± 0.38	0 ± 0	31.76 ± 0.7	35.58 ± 0.28	28.5 ± 0.14
17	Oxfendazole	100	100 ± 0	99.02 ± 0.29	54.09 ± 1.77	49.68 ± 1.72	40.01 ± 0.41	42.43 ± 5.07
18	Fenbendazole	100	100 ± 0	98.08 ± 0.33	0 ± 0	0 ± 0	38.82 ± 1.07	17.15 ± 1.13
19	Albendazole	100	100 ± 0	100 ± 0	0 ± 0	54.52 ± 0.06	80.64 ± 0.61	75.77 ± 0.69
20	Mebendazole	100	100 ± 0	100 ± 0	0 ± 0	47.42 ± 0.23	68.64 ± 1.8	55.36 ± 0.27
21	Oxibendazole	100	91.69 ± 0.8	100 ± 0	16.97 ± 2.54	33.15 ± 1.36	68.78 ± 0.57	57.89 ± 0.52
22	Flubendazole	100	30.49 ± 0.34	92.34 ± 1.09	8.35 ± 0.66	26.69 ± 1.43	39.62 ± 0.45	21.51 ± 0.87
23	Albendazole S-oxide	100	96.03 ± 0.47	100 ± 0	14.9 ± 1.51	47.13 ± 1.55	18.35 ± 0.72	19.11 ± 1.45
24	Triclabendazole	100	79.69 ± 0.03	74.19 ± 0.48	92.65 ± 1.17	72.23 ± 0.53	67.35 ± 0.58	67.48 ± 1.91
25	Bilastin	100	0 ± 0	0 ± 0	7.95 ± 0.61	11.95 ± 0.33	0 ± 0	8.15 ± 1.63
26	Selumetinib	100	6.49 ± 0.46	23.33 ± 1.96	39.24 ± 0.67	29.58 ± 0.60	7.38 ± 0.16	10.98 ± 0.47

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Fig. 4.

The EC₅₀ of benzoimidazole/carbamate drugs against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

The structure-activity relationship showed that drugs attached to the benzene ring to n-butyl had positive activity against R. solani and S. sclerotiorum with EC₅₀ of 0.051 μg ml⁻¹ and 0.16 μg ml⁻¹, respectively, while replacing the C atom in n-butyl with an S atom (fenbendazole) or O atom (oxibendazole) had an insignificant effect on activity. However, the S-atom in n-butyl was replaced by sulfur monoxide (albendazole S-oxide), significantly less active against both pathogens. The acetophenone structure (mebendazole) exhibited positive inhibition activity against R. solani and S. sclerotiorum. But the introduction of an F-atom into the acetophenone structure (flubendazole) significantly reduced the inhibition activity against R. solani (EC₅₀ > 25 μg ml⁻¹). Surprisingly, the substitution of the acetophenone with the phenyl sulfane moiety (fenbendazole) showed significant inhibitory activity against R. solani and S. sclerotiorum with EC₅₀ of 0.007 μg ml⁻¹ and 0.097 μg ml⁻¹ respectively. However, the replacement of the S atom by the sulfoxide resulted in significantly reduced activity against both pathogens. By comparing the activity of benzimidazole/carbamate against plant pathogens, we found that iodopropynyl butylcarbamate was effective in expanding the antifungal spectrum and had promising activity against pathogenic fungi. Thus the repositioning of benzoimidazoles/carbamates can be an effective way to expand their application areas.

Azole drugs

Azoles drugs have a wide range of applications in agriculture and medicine, such as low cost, availability and bioavailability, making azoles drugs of choice treating of fungal infections in most HIV/AIDS patients [47]. In agricultural production, triazole fungicides are mainly used to control plant fungal diseases caused by rust and mulberry powdery mildew pathogens due to their high efficiency and low toxicity [48]. The results indicate that azoles have broad antifungal activity as an essential backbone, which offers the possibility of developing new drugs. We screened 46 azole drugs (Table 5) against plant pathogens and obtained 16 drugs with optimal activity, as shown in Table 1 and Fig. 5.

Table 5.

In vitro antifungal activities (inhibition rate/%) of the azole drugs against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	Ronidazole	100	8.24 ± 1.98	12.57 ± 2.29	0 ± 0	14.20 ± 2.25	12.55 ± 1.30	9.98 ± 0.39
2	Benzoylmetronildazole	100	43.63 ± 0.32	21.18 ± 0.45	36.45 ± 1.33	27.64 ± 3.77	0 ± 0	13.05 ± 1.21
3	Daclatasvir	100	0 ± 0	33.05 ± 2.09	26.45 ± 0.25	0 ± 0	0 ± 0	6.01 ± 0.72
4	Econazole	100	89.76 ± 0.81	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
5	Itraconazole	100	25.09 ± 2.31	92.16 ± 0.79	96.0 ± 0.64	17.29 ± 3.32	87.4 ± 0.86	75.9 ± 1.17
6	Posaconazol	100	11.99 ± 0.35	100 ± 0	100 ± 0	66.44 ± 1.41	95.98 ± 0.32	100 ± 0
7	Letrozole	100	14.94 ± 1.23	0 ± 0	0 ± 0	0 ± 0	13.15 ± 0.01	0 ± 0
8	Anastrozole	100	7.62 ± 0.5	47.48 ± 1.96	0 ± 0	0 ± 0	10.03 ± 0.78	0 ± 0
9	Ketoconazole	100	66.19 ± 2.1	100 ± 0	100 ± 0	94.01 ± 0.29	100 ± 0	100 ± 0
10	Bifonazole	100	89.17 ± 0.25	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
11	KP 103	100	88.68 ± 0.93	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
12	Isoconazole nitrate	100	100 ± 0	100 ± 0	100 ± 0	97.83 ± 0.26	96 ± 0.75	100 ± 0
13	Fenticonazole nitrate	100	89.38 ± 0.04	97.18 ± 2.01	96.71 ± 0.46	76.8 ± 0.28	88.36 ± 0.41	94.72 ± 0.21
14	Isavuconazole	100	68.78 ± 0.49	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
15	Atipamezole hydrochloride	100	0 ± 0	22.85 ± 0.25	0 ± 0	13.28 ± 0.16	7.84 ± 1.13	35.77 ± 0.75
16	Valsartan	100	0.91 ± 0.21	0.70 ± 0.1	1.30 ± 0.3	3.09 ± 0.72	1.01 ± 0.02	0.98 ± 0.2
17	Deferasirox	100	94.51 ± 0	72.91 ± 0.62	89.16 ± 0.93	38.23 ± 0.44	80.91 ± 0.33	81.06 ± 0.67
18	Topiroxostat	100	10.82 ± 1.87	36.38 ± 1.65	0 ± 0	0 ± 0	0 ± 0	0 ± 0
19	Ribavirin	100	40.83 ± 0.48	20.75 ± 1.49	0 ± 0	0 ± 0	7.23 ± 0.24	10.86 ± 1.54
20	Levamisole hydrochloride	100	13.37 ± 1.48	-	0 ± 0	0 ± 0	0 ± 0	0 ± 0
21	Temozolomideacid	100	0 ± 0	0 ± 0	0 ± 0	5.05 ± 0.71	0 ± 0	0 ± 0
22	Imiquimod	100	16.28 ± 0.81	13.87 ± 1.43	52.68 ± 0.77	30.09 ± 3	17.03 ± 2.04	23.49 ± 1.41
23	Miconazole	100	23.60 ± 1.48	13.03 ± 1.52	8.60 ± 0.71	25.13 ± 1.49	17.79 ± 1.96	11.85 ± 1.02
24	Atipamezole hydrochloride	100	0 ± 0	22.85 ± 0.25	0 ± 0	13.28 ± 0.16	7.84 ± 1.13	35.77 ± 0.75
25	Levamisole hydrochloride	100	13.37 ± 1.48	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
26	Temozolomide	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	8.21 ± 1.86	0 ± 0
27	(+)-Pilocarpine hydrochloride	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	3.03 ± 1.05
28	Metronidazole	100	4.66 ± 1.79	12.02 ± 2.01	0 ± 0	13.67 ± 1.97	7.90 ± 0.59	11.08 ± 0.91
29	Ornidazole	100	2.87 ± 0.38	5.37 ± 1.47	0 ± 0	7.14 ± 3.41	0.00 ± 0.00	11.87 ± 0.6
30	Tinidazole	100	7.04 ± 0.98	28.46 ± 1.86	0 ± 0	17.15 ± 2.81	19.13 ± 0.89	0 ± 0
31	Clotrimazole	100	91.15 ± 1.05	100 ± 0	100 ± 0	99.65 ± 0.09	100 ± 0	100 ± 0
32	Fluconazole	100	59.15 ± 0.13	92.03 ± 0.01	94.14 ± 0.29	0 ± 0	67.88 ± 0	100 ± 0
33	Voriconazole	100	87.55 ± 0.84	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
34	Sulconazle nitrate	100	85.21 ± 2.1	100 ± 0	100 ± 0	85.69 ± 0.73	93.27 ± 0.53	100 ± 0
35	Vagistat	100	88.03 ± 1.71	100 ± 0	100 ± 0	97.06 ± 0.54	100 ± 0	100 ± 0
36	Butoconazole nitrate	100	95.27 ± 0.02	100 ± 0	100 ± 0	82.53 ± 0.1	87.75 ± 0.17	100 ± 0
37	Terconazole	100	51.4 ± 1.55	98.69 ± 0.01	82.17 ± 1.29	39.07 ± 0.08	72.57 ± 0	96.08 ± 0.29
38	Elubiol	100	74.86 ± 1.21	100 ± 0	100 ± 0	75.93 ± 0.75	100 ± 0	100 ± 0
39	Luliconazole	100	95.48 ± 0.27	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
40	Deracoxib	100	39.23 ± 1.39	48.42 ± 1.72	60.7 ± 1.71	55.03 ± 1.32	20.31 ± 1.27	48.38 ± 0.23
41	Cilostazol	100	17.94 ± 0.54	0 ± 0	0 ± 0	0 ± 0	27.18 ± 2.23	16.35 ± 1.76
42	Ruxolitinib	100	79.21 ± 1.87	42.33 ± 0.47	79.79 ± 0.01	53.7 ± 0.69	70.78 ± 0.97	72.32 ± 0.45
43	Baricitinib	100	18.12 ± 2.51	24.48 ± 1.49	14.74 ± 2.84	54.43 ± 0.99	48.29 ± 1.21	33 ± 0.61
44	Cimetidine	100	11.20 ± 0.92	8.73 ± 2.54	10.76 ± 1.73	3.70 ± 0.33	7.47 ± 0.79	6.24 ± 0.68
45	Pemirolast potassium	100	8.78 ± 0.63	15.20 ± 1.22	25.73 ± 1.46	18.05 ± 2.53	14.51 ± 0.32	9.01 ± 0.17
46	Aprepitant	100	60.09 ± 3.08	26.51 ± 1.65	62.67 ± 1.75	30.45 ± 1.11	0 ± 0	6.16 ± 1.27

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Fig. 5.

The EC₅₀ of azole drugs against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

The activity of bifonazole and clotrimazole showed that clotrimazole was more active than bifonazole against R. solani and S. sclerotiorum, which may be related to 1-benzyl-1H-imidazole. Econazole, vagistat, isoconazole nitrate and fenticonazole nitrate shared the basic structure (1-(2-(2,4-dichlorophenyl)-methoxy-2-ethyl)−1H-imidazole) and had comparable activity against all pathogens. All the compounds showed excellent activity against P. capsici with EC₅₀ < 0.06 μg ml⁻¹, indicating that this basic structure played a vital role in anti-pathogenic fungi. Replacing the O atom in the basic structure above with an S atom (sulconazle nitrate) had little effect on the activity against the plant pathogens, suggesting that the basic structure was still the key to activity. Voriconazole, efinaconazole and isavuconazole had similar basic structures, but efinaconazole showed better activity than the other two drugs with EC₅₀ of 0.095 μg ml⁻¹ and 0.035 μg ml⁻¹ against S. sclerotiorum and F. oxysporum, respectively. The activity of ketoconazole against plant pathogens was significantly higher than that of terconazole, and the EC₅₀ was in the range of 0.12–2.34 μg ml⁻¹, which showed that 1-methyl-1H-imidazole was more effective than 1-methyl-1H − 1,2,4-triazole in this type of drug. However, not all drugs containing 1-methyl-1H − 1,2,4-triazole structures were less active against pathogens than 1-methyl-1H-imidazole. Itraconazole and posaconazole showed the strongest inhibitory activity against pathogens with EC₅₀ < 0.17 μg ml⁻¹. In summary, the azole backbone is the main active group against plant pathogenic fungi with a view to repositioning old drugs for plant disease control.

Isothiazolinone drugs

Isothiazolinone is a major industrial bactericide, antiseptic and anti-enzyme agent, with outstanding inhibition of mold, algae and other microorganisms [49]. Recently, a series of derivatives with anti-tuberculosis and lipase inhibitors have been designed and synthesized [50, 51]. We selected 26 isothiazolinones (Table 6) for screening against phytopathogenic fungi and obtained 8 drugs with good activity, which were briefly analysed in Table 1 and Fig. 6.

Table 6.

In vitro antifungal activities (inhibition rate/%) of the isothiazolinone drugs against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	Methazolamide	100	69.21 ± 0.02	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
2	Acetazolamide	100	0 ± 0	0 ± 0	63.74 ± 2.25	0 ± 0	0 ± 0	4.45 ± 0.34
3	Nizatidine	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
4	Famotidine	100	0 ± 0	0.45 ± 0.64	0 ± 0	0 ± 0	0 ± 0	0 ± 0
5	2-Mercaptobenzothiazolyl	100	65.82 ± 0.96	83.59 ± 1.46	65.24 ± 1.22	62.11 ± 0.01	69.98 ± 0.31	74.35 ± 0.08
6	Ethyl 2-(2-aminothiazol-4-yl)glyoxylate	100	16.99 ± 2.38	0 ± 0	16.1 ± 1.63	6.67 ± 2.95	0 ± 0	0 ± 0
7	Ceftazidime intermediate	100	7.08 ± 0.34	0 ± 0	41.25 ± 0.11	0 ± 0	0 ± 0	0 ± 0
8	6-Aminopenicillanic acid	100	0 ± 0	11.02 ± 2.58	0 ± 0	0 ± 0	0 ± 0	11.12 ± 0.13
9	Aztreonam	100	0 ± 0	16.04 ± 1.4	0 ± 0	0 ± 0	0 ± 0	11.11 ± 0.15
10	4,5-Dichloro-2-octyl-isothiazolone	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
11	2-Octyl-2H-isothiazol-3-one	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
12	Isothiazolinone chloride	100	79.46 ± 1.97	93.27 ± 0.27	38.58 ± 1.98	41.23 ± 1.33	51.44 ± 1.12	-
13	2-Methyl-4-isothiazolin-3-one	100	72.09 ± 1.30	72.63 ± 1.77	54.44 ± 1.20	44.23 ± 1.92	42.64 ± 2.00	-
14	1,2-Benzisothiazol-3(2H)-one	100	83.43 ± 0.73	90.98 ± 0.55	84.89 ± 2.28	86.18 ± 1.50	75.43 ± 0.40	100 ± 0
15	2-Methyl-1,2-benzothiazol-3(2H)-one	100	92.23 ± 0.28	98.84 ± 0.11	66.18 ± 0.18	58.19 ± 0.86	75.3 ± 0.08	54.07 ± 0.53
16	6-Fluoro-1,2-benzoisothiazol-3(2H)-one	100	85.62 ± 0.66	99.33 ± 0.26	85.92 ± 2.97	66.57 ± 1.78	60.23 ± 0.93	42.70 ± 0.22
17	Benzo[D]isoxazol-3-ol	100	23.85 ± 1.65	87.8 ± 0.47	48.81 ± 0.32	53.79 ± 1.68	87.5 ± 0.24	-
18	6-Chlorobenzo[D]isoxazol-3-ol	100	42.85 ± 1.56	37.05 ± 1.08	22.18 ± 1.18	37.61 ± 0.23	43.75 ± 0.47	-
19	3-Indazolinone	100	17.2 ± 1.1	0 ± 0	3.60 ± 0.57	0 ± 0	15.27 ± 1.12	-
20	6-Bromo-1H-indazol-3-ol	100	24.83 ± 1.41	26.94 ± 1.12	8.63 ± 1.21	0 ± 0	11.06 ± 0.43	-
21	Saccharin	100	6.45 ± 1.19	19.07 ± 1.80	0 ± 0	0 ± 0	0 ± 0	-
22	6-Nitro-1,2-benzisothiazolin-3-one 1,1-dioxide	100	9.43 ± 1.17	27.63 ± 1.90	0 ± 0	0 ± 0	0 ± 0	-
23	3-(1-Piperazinyl)-1,2-benzisothiazole	100	12.18 ± 1.49	39.65 ± 1.57	7.89 ± 0.77	18.18 ± 1.92	13.91 ± 1.33	31.8 ± 0.2
24	Isothiazol-3-one	100	95.80 ± 0.05	100 ± 0	100 ± 0	100 ± 0	85.07 ± 0.64	100 ± 0
25	5-Chloro-3-hydroxyisothiazole	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
26	Febuxostat	100	79.65 ± 1.17	75.35 ± 0.05	63.28 ± 1.73	44.01 ± 1.5	73.85 ± 0.22	60.55 ± 1.12

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Fig. 6.

The EC₅₀ of isothiazolinone drugs against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

The 5-chloro-3-hydroxyisothiazole was the introduction of a Cl atom to the isothiazol-3-one structure, which significantly increased the activity against plant fungi with an EC₅₀ in the range of 0.98–4.06 μg ml⁻¹, but the introduction of a methyl group to 5-chloro-3-hydroxyisothiazole decreased the antifungal activity. The introduction of a Cl atom and octane on the 5-chloro-3-hydroxyisothiazole structure resulted in increasing activity against phytopathogenic fungi with an EC₅₀ in the range of 0.27–2.64 μg ml⁻¹. However, 2-octyl-2H-isothiazol-3-one showed comparable activity against plant pathogens compared to 4,5-dichloro-2-octyl-isothiazolone. Therefore, the introduction of octane in this structure may enhance the activity of phytopathogenic fungi, compared to 1,2-benzisothiazol-3(2H)-one, 2-methyl-1,2-benzothiazol-3(2H)-one and 6-fluoro-1,2-benzoisothiazol-3(2H)-one showed reduced antifungal activity, indicating that the introduction of substituents in this structure (benzoisothiazole) reduced the antifungal activity. Overall, the isothiazolinone structure is a potential lead compound against phytopathogenic fungi.

Pyrimidine drugs

Pyrimidine derivatives play an important role in insecticide, fungicide, weed control, antiviral, anticancer, etc. [52, 53], and have been the focus of attention of major pesticide companies in the world. In this study, we screened 65 drugs (Table 7) against agropathogenic fungi and obtained 10 highly active drugs, as shown in Table 1 and Fig. 7.

Table 7.

In vitro antifungal activities (inhibition rate/%) of the pyrimidine drugs against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	5-Fluorouridine	100	93.47 ± 0.92	100 ± 0	100 ± 0	81.11 ± 1.22	88.73 ± 0.23	89.6 ± 0.18
2	Doxifluridine	100	92.06 ± 0.44	91.33 ± 0.41	100 ± 0	77.2 ± 1.56	79.73 ± 0.39	74.97 ± 0.5
3	Avanafil	100	29.26 ± 0.52	18.75 ± 1.42	36.75 ± 1.65	56.86 ± 0.47	15.27 ± 1.6	22.08 ± 0.85
4	Uridine	100	83.38 ± 0.81	92.21 ± 1.97	89.26 ± 1.19	51.65 ± 1.02	58.24 ± 1.48	57.18 ± 0.08
5	2'-Fluoro-2'-deoxyuridine	100	0 ± 0	13.48 ± 2.85	0.94 ± 0.05	25.39 ± 1.21	0.73 ± 0.26	3.8 ± 2.69
6	1-(2-Deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil	100	15.47 ± 0.58	70.6 ± 0.39	6.4 ± 0.44	30.52 ± 2	5.9 ± 0.83	6.2 ± 0.41
7	1-Beta-D-Arabinofuranosyluracil	100	17.04 ± 0.14	100 ± 0	100 ± 0	38.05 ± 1.65	79.19 ± 0.01	88.56 ± 0.01
8	Trifluorothymine	100	66.42 ± 0.95	100 ± 0	100 ± 0	62.18 ± 0.57	90.12 ± 0.68	90.62 ± 0.74
9	Broxuridine	100	69.6 ± 0.57	91.57 ± 0.84	90.95 ± 0.23	62.53 ± 0.8	72.06 ± 0.17	63.3 ± 0.83
10	5-Bromouridine	100	19.95 ± 0.62	8.89 ± 1.23	9.49 ± 2.54	2.09 ± 0.3	0.19 ± 0.03	4.81 ± 1.02
11	5-Iodouridine	100	9.38 ± 0.99	17.47 ± 0.01	6.46 ± 2.59	6.35 ± 0.48	2.3 ± 0.17	6.53 ± 0.66
12	Carmofur	100	2.48 ± 0.11	9.85 ± 1.38	9.95 ± 0.57	2.04 ± 0.23	1.03 ± 0.04	3.16 ± 2.23
13	Tegafur	100	1.93 ± 0.73	14.48 ± 0.98	9.22 ± 1.58	1.73 ± 0.15	3.91 ± 0.02	6.04 ± 0.83
14	Cytidine	100	1.94 ± 0.71	1.03 ± 1.01	2.00 ± 0.06	6.96 ± 1.26	2.08 ± 0.02	1.03 ± 0.05
15	5-Fluorocytidine	100	1.94 ± 0.38	2.40 ± 0.03	35.9 ± 1.12	3.47 ± 0.17	1.04 ± 0.04	1.06 ± 0.02
16	5-Azacytidine	100	1.97 ± 0.82	51.98 ± 1.21	1.79 ± 0.29	5.74 ± 0.49	2.73 ± 0.22	1.55 ± 0.36
17	Lamivudine	100	74.77 ± 0.31	69.04 ± 0.21	69.78 ± 0	88.88 ± 2.4	84.74 ± 0	39.57 ± 2.3
18	Trifluridine	100	44.17 ± 1.98	25.52 ± 0.78	33.36 ± 0.21	18.62 ± 1.79	19.55 ± 0	8.09 ± 0.69
19	Guanosine	100	2.08 ± 0.02	2.71 ± 0.02	2.01 ± 0.10	14.12 ± 1.44	1.39 ± 0.03	5.77 ± 0.42
20	2'-Deoxyguanosine	100	64.42 ± 0.14	72.56 ± 0.12	100 ± 0	95.08 ± 0.00	94.05 ± 0.89	75.7 ± 0.02
21	Dideoxyinosine	100	1.80 ± 0	9.53 ± 0.79	1.92 ± 0.01	10.19 ± 0.75	1.35 ± 0.63	6.16 ± 1.11
22	Stavudine	100	0.83 ± 0.09	2.74 ± 0.03	7.95 ± 0.85	1.03 ± 0.04	3.71 ± 0.7	4.09 ± 0.16
23	Abacavir	100	1.88 ± 0.63	2.47 ± 0.92	0.03 ± 0.01	0.21 ± 0.01	0.21 ± 0.04	7.04 ± 1.12
24	Acyclovir	100	2.81 ± 0.17	2.93 ± 0.06	0.30 ± 0.01	2.30 ± 2.1	3.89 ± 0.1	1.00 ± 0.4
25	Famciclovir	100	0 ± 0	18.31 ± 2.66	4.01 ± 0.01	2.71 ± 0.03	5.01 ± 0.71	2.5 ± 1.84
26	Penciclovir	100	1.92 ± 0.81	4.90 ± 0.01	2.28 ± 0.02	4.08 ± 0.92	2.01 ± 0.08	3.51 ± 0.73
27	Ganciclovir	100	97.92 ± 0.11	96.2 ± 0.52	97.48 ± 0	75.04 ± 0.81	87.89 ± 1.33	91.68 ± 0.25
28	Brivudine	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
29	Cytarabine	100	3.81 ± 0.47	0 ± 0	11.74 ± 0.41	14.15 ± 1.46	7.10 ± 1.02	5.59 ± 0.49
30	Vidarabine monophosphate	100	21.73 ± 0.98	15.67 ± 1.03	0 ± 0	0 ± 0	0 ± 0	0 ± 0
31	Idoxuridine	100	7.95 ± 0.66	11.8 ± 0.04	14.95 ± 1.39	15.71 ± 0.24	20.73 ± 0.15	15.60 ± 0.98
32	Thymidine	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
33	Floxuridine	100	51.75 ± 2.84	83.70 ± 2.28	96.42 ± 0.38	81.14 ± 0.78	38.48 ± 0.01	31.11 ± 2.37
34	5-Fluorouracil	100	74.98 ± 1.98	48.82 ± 1.16	82.15 ± 0	81.39 ± 1.49	54.46 ± 0.92	34.62 ± 0.12
35	Fluorocytosine	100	0 ± 0	56.34 ± 1.69	75.94 ± 0.73	7.78 ± 0.95	3.55 ± 0.66	0 ± 0
36	Emtricitabine	100	0 ± 0	13.02 ± 1.05	0 ± 0	0 ± 0	0 ± 0	0 ± 0
37	6-Thioguanine	100	5.09 ± 2.48	0 ± 0	8.77 ± 1.51	0 ± 0	5.48 ± 0.19	5.54 ± 0.04
38	Zidovudine	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	9.16 ± 1.74	5.76 ± 1.02
39	Capecitabine	100	12.26 ± 1.51	24.62 ± 1.09	0 ± 0	0 ± 0	0 ± 0	0 ± 0
40	Dasatinib	100	38.91 ± 0.29	74.83 ± 1.62	89.72 ± 0.3	63.88 ± 1.94	76.26 ± 0.28	55.91 ± 0.22
41	Lapatinib	100	28.91 ± 1.96	69.92 ± 1.2	80.1 ± 0.69	48.92 ± 0.49	45.96 ± 0.22	42.33 ± 0.69
42	Nilotinib	100	0 ± 0	0 ± 0	10.93 ± 1.78	7.78 ± 0.78	0 ± 0	8.74 ± 0.7
43	Pazopanib hydrochloride	100	12.43 ± 0.79	28.21 ± 0.65	43.95 ± 1.46	34.76 ± 1.97	30.28 ± 0.05	20.39 ± 0.46
44	Sulfatinib	100	66.17 ± 7.04	77.12 ± 0.29	76.75 ± 1.92	62.24 ± 1.62	49.76 ± 0.13	32.5 ± 0.99
45	CAL-101	100	48.6 ± 2.85	0 ± 0	47.66 ± 0.09	32.02 ± 0.07	20.3 ± 0.58	16.82 ± 1.29
46	Gefitinib	100	23.29 ± 0.31	75.68 ± 2.59	92.22 ± 1	72.28 ± 0.63	67.35 ± 0.54	57.22 ± 0.24
47	Erlotinib	100	11.69 ± 1.62	0 ± 0	42.3 ± 0.41	12.17 ± 0.61	9.68 ± 0.96	15.94 ± 0.98
48	Dabrafenib	100	64.51 ± 0.9	83.7 ± 0.84	89.72 ± 1.23	64.2 ± 0.36	62.51 ± 0.47	65.69 ± 0.74
49	Nilotinib hydrochloride monohydrate	100	0 ± 0	47.15 ± 1.28	16.78 ± 0.46	10.76 ± 1.58	0 ± 0	9.46 ± 0.22
50	Tofacitinib	100	9.85 ± 1.96	0 ± 0	5.54 ± 0.36	11.9 ± 0.13	3.14 ± 0.13	2.62 ± 0.77
51	Afatinib	100	44.53 ± 0.76	78.99 ± 0.03	88.16 ± 0.24	52.59 ± 0.36	70.81 ± 0.21	45.23 ± 0.08
52	Ibrutinib	100	75.18 ± 2.12	93.23 ± 0.15	87.82 ± 0.01	67 ± 0.25	73.96 ± 1.33	79.25 ± 0.6
53	Mereletinib	100	56.8 ± 2.61	83.79 ± 0.91	55.15 ± 3.53	65.29 ± 1.14	81.29 ± 0.77	59.2 ± 0.85
54	Gemcitabine	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
55	Pyrimethamine	100	32.89 ± 0.59	36.34 ± 3.86	47.49 ± 0.16	43.67 ± 2.67	27.08 ± 0.02	32.67 ± 0.93
56	Thiamine chloride	100	0 ± 0	13.82 ± 1.32	0 ± 0	0 ± 0	0 ± 0	2.88 ± 0.96
57	Thiamine nitrate	100	0 ± 0	29.26 ± 1.32	0 ± 0	0 ± 0	0 ± 0	0 ± 0
58	Trimethoprim	100	13.36 ± 0.90	58.24 ± 2.37	13.00 ± 0.82	7.12 ± 3.05	17.35 ± 2.67	12.73 ± 2.25
59	Methotrexate	100	0 ± 0	29.19 ± 3.11	0 ± 0	0 ± 0	0 ± 0	0 ± 0
60	Triamterene	100	0 ± 0	0 ± 0	0 ± 0	19.77 ± 1.85	0 ± 0	0 ± 0
61	Revaprazan hydrochloride	100	10.18 ± 3.65	0 ± 0	6.63 ± 0.35	16.63 ± 2.06	0 ± 0	4.93 ± 0.63
62	Thiamine hydrochloride	100	6.24 ± 2.19	11.51 ± 1.01	0 ± 0	0 ± 0	0 ± 0	0 ± 0
63	Etravirine	100	7.45 ± 1.99	14.73 ± 2.98	0 ± 0	0 ± 0	0 ± 0	0 ± 0
64	Arprinocide	100	82.28 ± 2.14	29.35 ± 1.6	23.81 ± 0.47	13.37 ± 1.71	0 ± 0	0 ± 0
65	Amprolium	100	6.01 ± 0.81	0 ± 0	19.1 ± 0.62	5.3 ± 1.8	0 ± 0	0 ± 0

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Fig. 7.

The EC₅₀ of pyrimidine drugs against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Taking 5-fluorouracil as a backbone, a molecule ((2R,3S,4R,5S)−2-(hydroxymethyl)-tetrahydrofuran-3,4-diol) was introduced to become 5-fluorouridine, which significantly enhanced its activity against plant pathogenic fungi. Compared with 5-fluorouridine, the structure of floxuridine was one less OH group, but it was slightly less active against S. sclerotiorum and B. cinerea. It showed that the introduction of this moiety directly affected the anti-pathogenic fungal activity of the compound. Compared to ganciclovir, 2'-deoxyguanosine was less active against S. sclerotiorum. The pyrimidine-4-amine-based compounds showed inhibitory activity against B. cinerea with an EC₅₀ range of 2.29–13.52 μg ml⁻¹. Both dabrafenib and sulfatinib contain N-methylmethanesulfonamide, which were active against S. sclerotiorum and B. cinerea, and had superior antifungal activity to sulfamitinib. Thus, the activity of pyrimidine analogues against phytopathogenic fungi are based on the pyrimidine structure with other moieties, which are beneficial to improve the activity and can be used as candidate lead compounds against plant pathogenic fungi.

Pyridine drugs

In agriculture, pyridines are used as insecticides, herbicides and plant growth regulators. In particular, in herbicides, a number of highly effective and low-toxicity varieties have been developed, such as pyrimethanesulfuron, pirimicarb and acetamiprid [54]. In this study, 31 drugs that have not yet been applied against plant pathogenic fungi were screened, as shown in Table 8, and 12 drugs with application potential were finally screened out, as shown in Table 1. We aim to obtain lead structures or drugs with triple action of insecticide, herbicide, and disease control.

Table 8.

In vitro antifungal activities (inhibition rate/%) of the pyridines drugs against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	Nilvadipine	100	47.94 ± 1.46	89.46 ± 0.01	87.47 ± 0.01	59.50 ± 1.98	77.12 ± 0.42	57.97 ± 0.16
2	Roflumilast	100	6.9 ± 1.92	19.74 ± 0.9	0 ± 0	0 ± 0	0 ± 0	0 ± 0
3	Acalabrutinib	100	0 ± 0	41.65 ± 0.9	11.8 ± 2.02	29.67 ± 2.46	0 ± 0	0 ± 0
4	Nvp-lde225	100	71.65 ± 0.64	44.78 ± 1.29	33.58 ± 0.85	28.33 ± 0.21	0 ± 0	8.5 ± 1.08
5	Sorafenib tosylate	100	14.85 ± 0.95	25.01 ± 0.8	56.98 ± 0.36	22.5 ± 0.45	12.12 ± 0.47	22.05 ± 0.37
6	Abemaciclib	100	0 ± 0	54.13 ± 1.44	79.46 ± 0.73	14.97 ± 1.24	16.32 ± 1.71	13.87 ± 1.68
7	Axitinib	100	37.77 ± 0.14	0 ± 0	4.36 ± 0.82	11.76 ± 0.25	0 ± 0	5.02 ± 0.5
8	Regorafenib hydrate	100	27.25 ± 1.11	12.65 ± 0.44	53.24 ± 0.17	5.23 ± 0.07	5.12 ± 0.7	31.69 ± 0.51
9	Pheniramine maleate	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
10	Chlorpheniramine maleate	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	16.65 ± 1.63
11	Amlodipine maleate	100	42.06 ± 0.97	90.1 ± 0.33	93.27 ± 0.21	60.98 ± 1.05	66.89 ± 0.61	69.91 ± 1.11
12	Clopidol	100	9.94 ± 2.26	0 ± 0	15.9 ± 1.99	6.25 ± 2.75	0 ± 0	0 ± 0
13	Liranaftate	100	97.07 ± 0.34	100 ± 0	95.94 ± 0.27	81.72 ± 0.43	88.1 ± 0.10	81.48 ± 1.00
14	Rosiglitazone	100	23.44 ± 2.32	0 ± 0	16.17 ± 2.03	45.43 ± 1.26	61.95 ± 0.07	45.06 ± 0.23
15	Pioglitazone hydrochloride	100	0 ± 0	3.54 ± 0.06	5.72 ± 0.08	16.85 ± 0.12	9.75 ± 0.09	3.43 ± 3.41
16	Pyributicarb	100	92.93 ± 0.49	98.27 ± 0.11	92.06 ± 0.76	77.56 ± 0.01	81.45 ± 0.12	87.06 ± 0.09
17	Acrivastine	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
18	Milrinone	100	14.34 ± 1.36	0 ± 0	0 ± 0	0 ± 0	10.23 ± 1.19	0 ± 0
19	Tropicamide	100	9.13 ± 0.69	11.71 ± 0.88	21.34 ± 0.36	8.92 ± 2.01	0 ± 0	0 ± 0
20	Protionamide	100	17.5 ± 1.27	0 ± 0	34.9 ± 3.34	10.86 ± 0.44	5.21 ± 1.26	14.43 ± 1.16
21	Ethionamide	100	36.73 ± 0.92	0 ± 0	49.06 ± 0.96	24.68 ± 3.44	0 ± 0	9.06 ± 0.44
22	Amlodipine	100	62.82 ± 0.83	92 ± 0.37	96.1 ± 0.07	53.2 ± 1.29	76.95 ± 1.55	70.47 ± 2.13
23	Nimodipine	100	53.05 ± 2.65	61.5 ± 0.04	74.06 ± 1.61	31.96 ± 1.26	75.39 ± 0.81	67.49 ± 0.92
24	Isoniazid	100	8.18 ± 2.55	0 ± 0	11.98 ± 0.63	7.4 ± 1.93	0 ± 0	9.45 ± 0.28
25	Sodium pyrithione	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
26	Zinc pyrithione	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
27	Copper pyrithione	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	63.52 ± 1.33
28	Bispyrithione	100	100 ± 0	100 ± 0	82.36 ± 1.01	100 ± 0	59.39 ± 0.84	88.41 ± 1.99
29	Ciclopirox ethanolamine	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
30	Piroctone olamine	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
31	Caprylohydroxmic acid	100	91.58 ± 0.93	98.02 ± 0.57	100 ± 0	37.50 ± 1.31	58.76 ± 2.70	47.39 ± 1.19

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

As shown in Fig. 8, nilvadipine, nimodipine, amlodipine and amlodipine maleate belonged to the dihydropyridine group and showed activity against B. cinerea, among which nilvadipine had the strongest activity with an EC₅₀ of 5.74 μg ml⁻¹. This may be related to the electron-absorbing groups attached to the pyridine ring. Amlodipine maleate was a salt form of amlodipine with a slightly increased activity against B. cinerea. Liranaftate and pyributicarb had broad-spectrum and excellent activity against plant phytopathogens. Compared with liranaftate and pyributicarb, the activity of benzene ring-linked the cyclohexane ring with benzene ring-linked tert-butyl ring was one order of magnitude higher against five pathogenic fungi except F. oxysporum, among which the activity against B. cinerea was the best, with EC₅₀ of 0.004 μg ml⁻¹. The results suggest that pyridines, especially liranaftate and pyributicarb are promising for repositioning as fungicides for the control of plant pathogens.

Fig. 8.

The EC₅₀ of pyridine drugs compounds against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Piperidine/Piperazine drugs

Piperidine ring and piperazine group are often introduced into many drug molecules to improve the pharmacokinetic properties by effectively adjusting the ratio of lipid-water distribution and acid-base balance of drugs, which improves the bioavailability of drug molecules and drug efficacy [55–58]. In this study, mainly 65 antipsychotics were used to screen agricultural fungi, as shown in Table 9, and 18 drugs with relatively good activity were obtained as shown in Table 1.

Table 9.

In vitro antifungal activities (inhibition rate/%) of the piperidine/piperazine drugs against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	Piperazine	100	10.13 ± 1.09	0 ± 0	0 ± 0	0 ± 0	8.07 ± 0.4	0 ± 0
2	N-Aminoethylpiperazine	100	7.23 ± 0.89	26.08 ± 1.92	0 ± 0	0 ± 0	12.98 ± 0.87	0 ± 0
3	4-Methyl-1-piperazineethanamine	100	9.38 ± 1.28	0 ± 0	0 ± 0	0 ± 0	10.4 ± 1.09	0 ± 0
4	Buclizine dihydrochloride	100	33.46 ± 1.33	59.92 ± 0.15	66.63 ± 1.18	29.69 ± 2.47	23.08 ± 0.44	17.17 ± 1.25
5	Flavopiridol	100	89.93 ± 0.21	91.42 ± 0.81	89.23 ± 1.08	44.95 ± 2.38	56.25 ± 1.29	62.24 ± 1.14
6	Terfenadine	100	65.52 ± 0.22	100 ± 0	93.81 ± 0.8	75.97 ± 2.41	80.95 ± 0.9	69.96 ± 0.26
7	Thioridazine hydrochloride	100	91.41 ± 0.12	95.3 ± 0.04	81.65 ± 1.32	77.6 ± 1.14	87.61 ± 0.46	86.22 ± 0.82
8	Pimozide	100	52.60 ± 1.99	80.59 ± 0.77	87.66 ± 0.9	26.66 ± 2.29	74.42 ± 0.24	76.53 ± 1.29
9	Penfluridol	100	85.13 ± 0.01	91.7 ± 1.32	90.53 ± 0.58	87.27 ± 0.29	64.9 ± 0.82	49.98 ± 3.41
10	Loperamide hydrochloride	100	20.07 ± 2.39	50.38 ± 1.76	61.69 ± 2.72	54.23 ± 0.33	28.53 ± 0.05	41.3 ± 0.42
11	Benzhexol hydrochloride	100	28.46 ± 0.81	14.3 ± 4.44	33.66 ± 0.53	27.88 ± 1.3	0 ± 0	16.35 ± 0.77
12	Trifluoperazine	100	86.9 ± 0.43	94.95 ± 0.49	95.93 ± 0.26	82.95 ± 2.51	77.85 ± 1.61	84.87 ± 1.02
13	Paroxetine hydrochloride	100	66.8 ± 1.53	76.83 ± 0.28	88.59 ± 0.49	62.77 ± 1.72	64.5 ± 0.33	67.08 ± 1.15
14	Ebastine	100	88.1 ± 0.24	89.73 ± 0.12	90.82 ± 0.22	47.57 ± 2.44	87.09 ± 0.42	74.72 ± 0.56
15	Haloperidol	100	44.75 ± 1.84	64.38 ± 0.42	84.53 ± 0	56.68 ± 2.24	42.75 ± 1.23	42.42 ± 0.17
16	Mizolastine	100	6.85 ± 0.35	58.66 ± 0.77	86.42 ± 1.92	50.32 ± 2.94	45.1 ± 0.7	48.11 ± 2.81
17	Vortioxetine	100	1.50 ± 0.01	0.70 ± 0.20	3.0 ± 0.12	2.45 ± 0.19	1.82 ± 0.71	6.25 ± 0.54
18	Sildenafil	100	0 ± 0	16.68 ± 1.1	34.92 ± 0.01	22.88 ± 0	15.85 ± 0.04	25.12 ± 1.12
19	Prochlorperazine maleate	100	90.84 ± 0.07	85.45 ± 0.75	84.67 ± 1.33	62.29 ± 1.61	70.01 ± 0.34	84.52 ± 0.24
20	Perphenazine	100	100 ± 0	94.53 ± 0.9	85.08 ± 1.33	84.54 ± 2.13	77.46 ± 0.44	86.13 ± 1.34
21	Clozapine	100	44.03 ± 2.74	44.32 ± 0.07	74 ± 0.58	67.7 ± 1.11	56.56 ± 0.5	50.4 ± 2.67
22	Olanzapine	100	0 ± 0	22.77 ± 1.16	33.84 ± 1.37	33.69 ± 1.15	16.16 ± 1.36	44.43 ± 0.46
23	Ranolazine	100	9.64 ± 1.43	42.17 ± 0.68	14.1 ± 0.12	0 ± 0	7.15 ± 1.33	18.79 ± 0.05
24	Amoxapine	100	46.5 ± 0.34	78.43 ± 0.14	59.03 ± 0.01	59.22 ± 1.16	60.67 ± 0.01	60.26 ± 0.28
25	Mirtazapine	100	0 ± 0	47.18 ± 0.11	7.87 ± 0.27	0 ± 0	11.43 ± 0.44	26.08 ± 0.45
26	Sitagliptin	100	0 ± 0	43.52 ± 2.16	9.92 ± 0.04	0 ± 0	1.01 ± 0.06	9.55 ± 0.1
27	Brexpiprazole	100	23.77 ± 0.18	59.61 ± 1.75	43.04 ± 2.47	52.28 ± 1.98	65.19 ± 0.27	29.94 ± 0.98
28	Aripiprazole	100	84.8 ± 2.57	97.46 ± 0.57	96.66 ± 0.32	86.81 ± 0.74	82.19 ± 0.46	74.18 ± 0.17
29	Ziprasidone hydrochloride	100	27.61 ± 0.91	86.92 ± 1.14	22.6 ± 1.05	21.25 ± 2.1	37.33 ± 1	31.94 ± 0.33
30	Cinnarizine	100	5.28 ± 0.36	34.77 ± 1.43	32.62 ± 0.38	16.65 ± 1.34	10.8 ± 0.79	20.15 ± 1.89
31	Cetirizine	100	0 ± 0	0 ± 0	11.88 ± 1.63	0 ± 0	0 ± 0	12.93 ± 0.15
32	Domperidone	100	0 ± 0	0 ± 0	53.03 ± 0.43	16.05 ± 1.27	32.58 ± 0.62	33.23 ± 2.01
33	Bilastin	100	0 ± 0	0 ± 0	7.95 ± 0.61	11.95 ± 0.33	0 ± 0	8.15 ± 1.63
34	Risperidone	100	0 ± 0	0 ± 0	17.48 ± 1.01	21.52 ± 1.87	14.93 ± 2.33	16.71 ± 1.8
35	Terazosin hydrochloride	100	9.93 ± 1.09	0 ± 0	46.52 ± 1.49	22.21 ± 2.14	21.79 ± 1.37	21.64 ± 1.13
36	Donepezil	100	15.4 ± 0.43	43.31 ± 2.81	48.97 ± 2.58	31.26 ± 1.11	7.94 ± 2.31	19.61 ± 1.56
37	Droperidol	100	0 ± 0	0 ± 0	7.88 ± 1.62	5.53 ± 0.50	22.79 ± 1.34	25.66 ± 1.47
38	Flibaserin	100	39.16 ± 0.42	51.1 ± 0.82	38.81 ± 0.56	58.49 ± 1.53	61.42 ± 0.01	49.12 ± 1.83
39	Piperaquinoline	100	0 ± 0	0 ± 0	12.59 ± 0.85	34.6 ± 0.07	34.55 ± 0.01	34.86 ± 0.01
40	Desloratadine	100	25.73 ± 1.98	75.43 ± 0.08	87.24 ± 0.14	36.5 ± 0.64	67.43 ± 2.63	62.52 ± 0.41
41	Loratadine	100	91.93 ± 0.01	80.7 ± 1.62	86.37 ± 1.3	62.28 ± 1.28	74.13 ± 0.66	79.88 ± 1.34
42	Fexofenadine	100	0 ± 0	7.72 ± 1.26	0 ± 0	0 ± 0	0.6 ± 0.89	2.65 ± 1.61
43	Vardenafil hydrochloride	100	0 ± 0	69.92 ± 1.2	28.12 ± 2.91	14.7 ± 0.36	18.83 ± 0.71	21.06 ± 1.17
44	Quetiapine fumarate	100	0 ± 0	0 ± 0	0 ± 0	10.78 ± 1.1	35.58 ± 2.3	24.12 ± 0.35
45	Imatinib	100	21.94 ± 2.34	37.25 ± 1.04	55.81 ± 0.26	40.84 ± 0.26	23.07 ± 1.14	24.15 ± 0.72
46	Alectinib	100	19.82 ± 1.35	11.63 ± 0.29	50.06 ± 0.67	17.83 ± 1	8.67 ± 1.14	8.07 ± 0.21
47	Venclexta	100	13.15 ± 1.06	0 ± 0	22.31 ± 0.32	0 ± 0	0 ± 0	0 ± 0
48	Ponatinib	100	100 ± 0	69.58 ± 0.4	85.41 ± 0.3	67.65 ± 2.46	51.95 ± 0.4	35.42 ± 0.27
49	Bosutinib	100	35.3 ± 1.3	86.18 ± 0.01	83.58 ± 0.68	67.06 ± 0.22	56.99 ± 0.31	42.85 ± 0.62
50	Vandetanib	100	78.56 ± 0.44	84.25 ± 2.61	91.72 ± 0.09	90.26 ± 0.22	79.7 ± 1.49	67.36 ± 0.72
51	Avitinib	100	37.78 ± 1.3	92 ± 0.42	75.26 ± 1.09	73.36 ± 0.49	66.97 ± 0.14	35.2 ± 1.02
52	Tandutinib	100	16.89 ± 0.19	53.1 ± 0.95	60.7 ± 0.45	47.66 ± 0.37	44.74 ± 0.16	34.73 ± 0.82
53	Palbociclib	100	0 ± 0	78.56 ± 0.67	78.39 ± 1.01	35.62 ± 2.13	63.32 ± 0.97	29.8 ± 1.09
54	Ribociclib	100	11.36 ± 2.6	76.33 ± 0.24	56.63 ± 1.19	38.34 ± 2.14	54.85 ± 0.11	28.86 ± 0.97
55	Brigatinib	100	24.11 ± 1.03	68.01 ± 0.95	42.23 ± 2.91	68.85 ± 1.18	37.45 ± 0.38	27.48 ± 0.62
56	Olaparib	100	0 ± 0	26.69 ± 1.16	54.44 ± 0.96	10.87 ± 1.9	7.7 ± 0.79	13.04 ± 2.02
57	Crizotinib	100	48.83 ± 1.08	100 ± 0	96.02 ± 0.02	85.98 ± 0.21	86.35 ± 0.54	63.94 ± 0.59
58	Niraparib	100	11.13 ± 0.11	84.17 ± 0.63	81.11 ± 0.48	40.9 ± 2.55	53.85 ± 0.75	31.13 ± 0.8
59	Nintedanib	100	7.51 ± 0.89	47.64 ± 0.62	45.98 ± 0.93	13.23 ± 0.18	9.94 ± 0.56	17.71 ± 1.78
60	Ceritinib	100	83.03 ± 0.22	52.37 ± 0.19	82.9 ± 1.21	33.97 ± 0.03	41.12 ± 1.53	31.26 ± 0.93
61	Cetirizine hydrochloride	100	0 ± 0	0 ± 0	14.1 ± 1.99	8.6 ± 0.04	2.88 ± 0.4	6.03 ± 1.4
62	Trazodone hydrochloride	100	20.46 ± 1.71	43.6 ± 0.62	17.85 ± 1.56	14.73 ± 0.01	26.78 ± 0.02	37.01 ± 1.7
63	Alogliptin	100	5.61 ± 2.05	31.87 ± 3.26	0 ± 0	0 ± 0	0 ± 0	0 ± 0
64	Ticlopidine	100	24.3 ± 2.74	44.33 ± 2.2	0 ± 0	56.11 ± 3.26	20.28 ± 1.53	16.77 ± 3.05
65	Celecoxib	100	31.09 ± 1.44	0 ± 0	39.62 ± 1.54	21.03 ± 0.01	23.32 ± 0.54	33.99 ± 0.89

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

As shown in Fig. 9, the drugs with piperazine and piperidine structures include two forms of N-methyl group on the outside and inside, and the two forms of piperazine drugs have little difference against antifungal activity. But loratadine and penfluridol had excellent activity with EC₅₀ of 6.19 μg ml⁻¹ and 6.59 μg ml⁻¹ against R. solani and S. sclerotiorum, respectively. Compared with the piperazine structure with the N-methyl position on the outside, the piperidine structure had better anti-phytopathogenic activity. Among them, trifluoperazine not only had significant antifungal activity but also expanded the antifungal spectrum. In addition, ponatinib had the best activity against R. solani. The EC₅₀ was 0.017 μg ml⁻¹. Therefore, piperazine and piperidine compounds have the potential to develop drugs against agricultural pathogenic fungi.

Fig. 9.

The EC₅₀ of piperidine/piperazine drugs against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Ionic liquids

Ionic liquids are considered a friendly solvent and commonly used in the extraction of natural products. They are mainly classified as quaternary ammonium ionic liquids, pyridine ionic liquids, quaternary phosphate ionic liquids and imidazole ionic liquids [59, 60]. This study used 37 ionic liquids to inhibit plant pathogens as shown in Table 10. According to Table 1 and Fig. 10, 15 potential drugs were briefly analyzed in order to apply them to agriculture.

Table 10.

In vitro antifungal activities (inhibition rate/%) of the ionic liquids against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	3-Methyl-1-octylimidazolium chloride	100	8.69 ± 2.59	69.77 ± 1.81	32.53 ± 2.77	51.93 ± 1.64	9.88 ± 0.39	19.73 ± 0.21
2	1-Hexyl-3-methylimidazolium chloride	100	0 ± 0	24.87 ± 2.52	11.14 ± 1.41	21.89 ± 1.42	0 ± 0	13.17 ± 0.37
3	1-Decyl-3-methylimidazolium chloride	100	21.81 ± 1.06	72.67 ± 1.16	55.45 ± 1.28	88.51 ± 0.49	46.57 ± 0.71	55.45 ± 0.04
4	1-Dodecyl-3-methylimidazolium chloride	100	47.13 ± 0.72	100 ± 0	97.77 ± 0.02	77.56 ± 0.68	63.94 ± 2.95	45.16 ± 1.33
5	1-Hexyl-3-methylimidazolium chloride	100	69.61 ± 0.95	55.87 ± 2.85	95.05 ± 0.02	54.85 ± 1.87	36.67 ± 0.96	21.65 ± 0.02
6	1-Decyl-3-methylimidazolium bromide	100	15.73 ± 0.87	58.88 ± 0.72	61.76 ± 0.13	85.96 ± 1.82	50.68 ± 1.86	42.96 ± 1.24
7	Dodecyl dimethyl benzyl ammonium bromide	100	73.33 ± 0.43	100 ± 0	95.11 ± 0.01	60.63 ± 0.39	55.92 ± 1.14	61.1 ± 0.55
8	Benzyldimethylhexadecylammonium chloride	100	58.29 ± 1.08	100 ± 0	92.78 ± 0.76	44.26 ± 2.12	29.23 ± 2.75	39.2 ± 1.61
9	Myristalkonium chloride	100	68.55 ± 0.76	100 ± 0	95.23 ± 0.01	62.15 ± 1.21	33.25 ± 0.52	38.36 ± 2.01
10	Benzyldimethylstearylammonium Chloride	100	31.81 ± 2.36	84.1 ± 1.35	92.45 ± 0.01	27.37 ± 0.94	28.19 ± 0.35	15.53 ± 1.22
11	Benzododecinium chloride	100	80.22 ± 0.65	6.1 ± 0.55	95.2 ± 0.22	60.48 ± 0.39	50.79 ± 2.96	34.15 ± 1.15
12	1-Butylpyridinium bromide	100	0 ± 0	0 ± 0	5.38 ± 0.62	5.64 ± 0.35	0 ± 0	0 ± 0
13	N-butyl-4-methylpyridinium chloride	100	0 ± 0	5.13 ± 0.47	0 ± 0	10.65 ± 0.55	7.4 ± 0.62	0 ± 0
14	1-Hexadecylpyridinium bromide	100	79.49 ± 1.56	76.72 ± 1.19	91.56 ± 0.21	56.61 ± 1.49	27.58 ± 2.46	11.43 ± 0.53
15	1-Dodecylpyridinium bromide	100	76.02 ± 0.42	100 ± 0	100 ± 0	78.72 ± 1.21	79.39 ± 0.29	41.32 ± 0.84
16	3-Methyl-1-octylimidazolium chloride	100	4.77 ± 0.84	19.9 ± 2.25	4.14 ± 0.61	24.56 ± 1.21	14.11 ± 3.02	13.49 ± 0.83
17	1,1'-Di-n-heptyl-4,4'-bipyridinium dibromide	100	25.87 ± 0.88	97.12 ± 0.42	86.62 ± 0.35	85.75 ± 0.27	81.14 ± 1.29	52.12 ± 0.59
18	1-Tetradecylpyridinium chloride	100	74.55 ± 0.59	96.45 ± 0.87	96.88 ± 0.72	73.76 ± 0.54	56.25 ± 1.16	21.25 ± 2.06
19	1-Hexadecyl-3-methylimidazolium chloride monohydrate	100	4.13 ± 0.67	4.64 ± 0.18	0 ± 0	23.73 ± 3.16	0 ± 0	0 ± 0
20	1-Butyl-3-methylimidazolium chloride	100	0 ± 0	0 ± 0	0 ± 0	8.21 ± 0.74	0 ± 0	0 ± 0
21	1-Propyl-3-methyl imidazolium	100	0 ± 0	0 ± 0	0 ± 0	6.68 ± 0.02	7.55 ± 0.82	3.55 ± 0.74
22	Octenidine dihydrochloride	100	87.13 ± 0.62	89.21 ± 1.56	93.45 ± 0.06	86.47 ± 0.64	81.53 ± 0.48	79.33 ± 0.61
23	Miltefosine	100	75.63 ± 1.35	52.49 ± 0.85	92.30 ± 0.12	25.08 ± 5.06	22.27 ± 1.87	35.73 ± 1.94
24	Chlorhexidine diacetate	100	94.33 ± 0.18	100 ± 0	93.72 ± 0.46	97.84 ± 0.17	76.48 ± 1.18	71.91 ± 0.31
25	Hexadecyl trimethyl ammonium bromide	100	74.22 ± 2.27	67.42 ± 0.66	93.73 ± 1.62	54.38 ± 1.27	46.29 ± 0.35	15.02 ± 1.14
26	Cetalkonium chloride	100	70.43 ± 1.91	80.75 ± 1.56	94.66 ± 0.55	56.15 ± 1.12	39.23 ± 0.85	12.8 ± 1.31
27	Domiphen bromide	100	86.76 ± 0.49	100 ± 0	94.47 ± 0.7	83.74 ± 0.86	71.25 ± 0.04	46.21 ± 0.82
28	Cetylpyridinium chloride	100	70.53 ± 0.25	81.05 ± 0.9	91.4 ± 0.82	47.44 ± 1.05	32.82 ± 1.26	12.63 ± 0.37
29	Diminazene aceturate	100	8.23 ± 0.21	100 ± 0	96.23 ± 0.39	100 ± 0	59.38 ± 1.65	24.99 ± 0.25
30	Pentamidine	100	10.48 ± 1.00	100 ± 0	100 ± 0	-	57.11 ± 2.45	28.65 ± 0.37
31	N-Octadecyl-4-stilbazole bromide	100	0 ± 0	12.4 ± 1.85	20.02 ± 1.31	0 ± 0	0 ± 0	7.59 ± 1.16
32	Enebicyanog	100	80.68 ± 1.90	98.06 ± 0.36	100 ± 0	87.43 ± 1.86	46.73 ± 0.39	39.45 ± 1.93
33	Perifosine	100	51.28 ± 1.23	60.74 ± 1.92	93.29 ± 0.32	14.42 ± 2.95	32.64 ± 0.69	22.24 ± 2.74
34	Pralidoxime chloride	100	6.95 ± 1.65	29.13 ± 1.98	0 ± 0	34.52 ± 1.29	0 ± 0	0 ± 0
35	Bephenium hydroxynaphthoate	100	24.28 ± 1.36	67.72 ± 0.09	53.65 ± 1.35	5.88 ± 2.12	0 ± 0	7.74 ± 1.11
36	Benzethonium chloride	100	1.00 ± 0.02	2.09 ± 0.20	1.20 ± 0.65	2.91 ± 0.56	1.92 ± 0.07	10.33 ± 0.64
37	Potassium sorbate	100	16.4 ± 0.19	26.29 ± 1.55	0 ± 0	0 ± 0	8.23 ± 0.57	-

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Fig. 10.

The EC₅₀ of ionic liquids against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

In all ionic liquids, we found that the longer the carbon chain of the drug, the better the activity against S. sclerotiorum. 1-Dodecyl-3-methylimidazolium chloride products better active with EC₅₀ of 6.12 μg ml⁻¹ against S. sclerotiorum. Compared to 1-dodecylpyridinium bromide, 1-tetradecylpyridinium chloride was less active against phytopathogenic fungi despite the carbon-chain length, so the effect on antifungal activity may be ion-related. 1-Dodecylpyridinium with the bromine ion increased the activity against S. sclerotiorum, B. cinerea and F. oxysporum. Compared to myristalkonium chloride, the carbon chain increased and the activity was enhanced against S. sclerotiorum with cetalkonium chloride EC₅₀ of 8.36 μg ml⁻¹, but significantly decreased activity against B. cinerea. Compared with benzyldodecyldimethylammonium bromide, chloride ion replaced by bromine ion dodecyl dimethyl benzyl ammonium bromide increased the activity of S. sclerotiorum and B. cinerea. The EC₅₀ values were 5.80 μg ml⁻¹ and 8.85 μg ml⁻¹, respectively. In summary, the carbon chain length of the ionic liquid drugs had a significant effect on the resistance to phytopathogenic fungi. Compared to chlorohexidine diacetate, enebicyanog had a narrower spectrum of activity against phytopathogenic fungi, but it had better activity against S. sclerotiorum and B. cinerea, with EC₅₀ of 0.91 μg ml⁻¹ and 0.62 μg ml⁻¹ respectively. Therefore, ionic liquids are expected to be used in the control of plant resistant pathogenic fungi.

Miscellaneous group drugs

Miscellaneous group drugs against plant pathogenic fungi are shown in Table 11 and Table 1. Some drugs with relatively broad anti-pathogenic activity were selected for a brief analysis as shown in Fig. 11.

Table 11.

In vitro antifungal activities (inhibition rate/%) of the miscellaneous group against phytopathogenic fungi^a

No.	Compounds	Concentration(μg/mL)	Inhibition rate/%
			R. s	S. s	B. c	F. g	F. o	P. c
1	Chloramphenicol	100	12.82 ± 2.10	34.51 ± 0.63	11.54 ± 0.57	10.69 ± 1.73	16.80 ± 1.63	9.15 ± 1.03
2	Thiamphenicol	100	15.15 ± 0.43	40.83 ± 1.71	13.35 ± 2.69	3.09 ± 0.12	14.31 ± 1.26	5.12 ± 0.88
3	Monensin sodium salt	100	73.63 ± 2.79	92.9 ± 1.24	100 ± 0	47.12 ± 1.5	88.0 ± 0.42	91.3 ± 1.03
4	Novobicin sodium salt	100	40.85 ± 0.43	58.64 ± 3.27	83.95 ± 1.81	29.3 ± 2.92	17.05 ± 0.91	45.99 ± 2.39
5	Rifapentine	100	25.83 ± 2.61	0 ± 0	53.84 ± 0.65	20.36 ± 2.19	20.77 ± 1.95	46.25 ± 1.82
6	Rifaximin	100	9.78 ± 0.65	0 ± 0	65.32 ± 1.46	14.03 ± 0.67	0 ± 0	21.41 ± 1.24
7	Rifamycin sodium	100	78.25 ± 1.61	79 ± 2.85	83.72 ± 0.27	52.35 ± 2.81	40 ± 4.66	43.76 ± 1.27
8	Rifampicin	100	9.23 ± 0.28	0 ± 0	49.54 ± 0.03	29.38 ± 0.82	0 ± 0	31.73 ± 1.29
9	(+)-Griseofulvin	100	89.85 ± 0.82	84.15 ± 1.19	100 ± 0	87.69 ± 0.81	80.26 ± 0.86	85.98 ± 0.79
10	Natamycin	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
11	Tiamulin	100	4.77 ± 0.69	65.13 ± 0.2	36.58 ± 0.67	37.56 ± 0.17	12.3 ± 0.14	79.63 ± 0.23
12	Retapamulin	100	17.21 ± 0.83	72.23 ± 0.42	58.09 ± 0.74	25.97 ± 0.39	41.9 ± 0.27	48.57 ± 1.51
13	Naftifine hydrochloride	100	56.59 ± 1.34	100 ± 0	100 ± 0	100 ± 0	96.30 ± 0.08	100 ± 0
14	Terbinafine hydrochloride	100	68.44 ± 2.20	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
15	Butenafine hydrochloride	100	91.83 ± 0.65	100 ± 0	100 ± 0	98.27 ± 0.20	100 ± 0	100 ± 0
16	N-(2-Hydroxynaphthoyl)-2,4-dimethoxy-5-chloroanilide	100	53.35 ± 0.84	28.33 ± 1.01	42.56 ± 0.44	32.48 ± 1.75	17.19 ± 1.31	36.23 ± 2.45
17	Tolnaftate	100	84.8 ± 0.12	98.27 ± 0.32	91.63 ± 0.54	64.33 ± 1.02	79.2 ± 0.41	66.81 ± 0.33
18	Pyrantel pamoate	100	16.05 ± 1.72	50.15 ± 2.7	62.71 ± 1.24	25.75 ± 1.1	0 ± 0	13.6 ± 1.42
19	Propranolol hydrochloride	100	0 ± 0	8.18 ± 1.5	48.94 ± 2.03	23.36 ± 0.24	17.44 ± 1.45	25.23 ± 1.36
20	Cinacalcet	100	100 ± 0	100 ± 0	100 ± 0	89.51 ± 1.09	93.07 ± 0.92	95.54 ± 1.12
21	1,7-Dlaminoheptane	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	11.92 ± 1.28	14.85 ± 0.66
22	Tris(2-aminoethyl)amine	100	0 ± 0	0 ± 0	16.88 ± 0.69	2.04 ± 1.57	0 ± 0	4.49 ± 0.36
23	N,N'-bis(3-aminopropyl)ethylenediamine	100	0 ± 0	0 ± 0	47.34 ± 1.69	0 ± 0	0 ± 0	6.53 ± 0.54
24	Tetraethyenepentamine	100	0 ± 0	0 ± 0	12.92 ± 1.84	0 ± 0	0 ± 0	8.25 ± 0.51
25	Diethylenetriamine	100	0 ± 0	0 ± 0	13.12 ± 1.48	3.21 ± 0.97	0 ± 0	3.27 ± 0.38
26	N1,N1'-(butane-1,4-diyl)bis(ethane-1,2-diamine)	100	0 ± 0	12.27 ± 2.69	98.53 ± 0.45	27.21 ± 2.51	3.97 ± 0.55	3.56 ± 2.23
27	N- (3-Dimethylaminopropyl)-1,3-propanediamine	100	0 ± 0	0 ± 0	11.05 ± 1.19	12.87 ± 3.58	0 ± 0	4.98 ± 1.09
28	Cyclen	100	26.59 ± 1.61	18.36 ± 1.15	0 ± 0	0 ± 0	12.93 ± 0.18	0 ± 0
29	1,4,7,10,13,16-hexazacyclooctadecane	100	12.98 ± 3.11	12.94 ± 0.37	0 ± 0	0 ± 0	8.34 ± 0.39	0 ± 0
30	Triethylenetetramine	100	4.94 ± 0.66	16.65 ± 1.53	0 ± 0	0 ± 0	12.17 ± 0.94	6.41 ± 0.84
31	Ethylenediamine	100	9.07 ± 0.01	0 ± 0	0 ± 0	0 ± 0	6.37 ± 0.78	0 ± 0
32	2-Aminoethyl(ethyl)amine	100	6.53 ± 0.15	18.35 ± 1.92	14.56 ± 2.77	11.04 ± 0.98	0 ± 0	0 ± 0
33	1,3-Diaminopropane	100	6.01 ± 0.65	13.88 ± 1.21	18.3 ± 0.87	9.53 ± 2.16	0 ± 0	0 ± 0
34	1,4-Butylenediamine	100	10.73 ± 0.63	12.73 ± 0.53	22.97 ± 2.95	0 ± 0	0 ± 0	0 ± 0
35	1,6-Diaminohexane	100	11.16 ± 0.24	19.06 ± 0.43	18.05 ± 2.41	0 ± 0	0 ± 0	0 ± 0
36	1,5-Diaminopentane	100	7.85 ± 0.11	17.55 ± 4.31	17.09 ± 1.72	0 ± 0	0 ± 0	0 ± 0
37	N-octyl-N'-[2-(octylamino)ethyl]ethylenediamine	100	63.46 ± 0.96	86.94 ± 0.67	87.71 ± 0.38	55.59 ± 1.31	68.6 ± 0.47	38.35 ± 0.98
38	Ethambutol	100	17.45 ± 1.19	11.25 ± 1.80	6.37 ± 0.24	15.67 ± 0.56	5.43 ± 1.76	9.27 ± 0.29
39	Diethylenetriaminepentaacetic acid	100	1.92 ± 0.01	6.25 ± 1.25	1.03 ± 0.04	5.66 ± 0.81	1.91 ± 0.18	8.24 ± 1.92
40	Eflornithine hydrochloride hydrate	100	0 ± 0	37 ± 0.23	28.45 ± 1.5	0 ± 0	81.45 ± 2.72	0 ± 0
41	N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine	100	62.30 ± 0.56	30.58 ± 1.39	86.93 ± 0.96	64.83 ± 0.39	62.70 ± 0.49	48.66 ± 0.31
42	Rimantadine hydrochloride	100	0 ± 0	19.9 ± 1.83	0 ± 0	14.44 ± 0.07	0 ± 0	20.45 ± 0.54
43	Amantadine	100	0 ± 0	20.42 ± 2.1	0 ± 0	0 ± 0	0 ± 0	0 ± 0
44	Vildagliptin	100	0 ± 0	13.51 ± 0.27	11.64 ± 2.8	0 ± 0	0 ± 0	0 ± 0
45	Strontium ranelate	100	0 ± 0	23.47 ± 1.01	7.96 ± 1.07	0 ± 0	0 ± 0	0 ± 0
46	1-Adamantanamine hydrochloride	100	0 ± 0	8.66 ± 1.49	0 ± 0	0 ± 0	0 ± 0	5.87 ± 0.88
47	Taurolidine	100	18.3 ± 0.96	14.65 ± 0.82	34.09 ± 3.32	16.69 ± 1.54	0 ± 0	0 ± 0
48	(5 R)-3-(4-Bromo-3-fluorophenyl)-5-hydroxy methyloxazolidin-2-one	100	57.19 ± 0.05	0 ± 0	22.9 ± 0.03	68.54 ± 0.04	36.49 ± 2.42	23.43 ± 0.21
49	Intermediate of linezolid	100	0 ± 0	0 ± 0	6.98 ± 0.06	0 ± 0	0 ± 0	0 ± 0
50	Linezolid related compound	100	0 ± 0	0 ± 0	0 ± 0	14.74 ± 1.73	19.97 ± 0.97	16.68 ± 0.79
51	Rivaroxaban intermediate	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
52	Rivaroxaban	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	7.65 ± 1.14	6.41 ± 2.67
53	Linezolid	100	24.9 ± 1.41	35.76 ± 2.54	0 ± 0	61.78 ± 0.91	31.44 ± 0.37	29.94 ± 0.77
54	Furaltadone hydrochloride	100	37.07 ± 3.77	18.53 ± 1.23	36.08 ± 1.17	17.28 ± 2.55	24.66 ± 0.01	36.83 ± 1.97
55	Furazolidone	100	63.35 ± 0.67	73.99 ± 1.6	54.45 ± 2.2	61.51 ± 1.51	43.28 ± 1.08	39.72 ± 0.92
56	Nifuratel	100	9.96 ± 2.01	50.16 ± 0.01	13.9 ± 1.03	48.52 ± 0.94	63.98 ± 1.18	55.51 ± 0.45
57	L-Cycloserine	100	16.64 ± 0.97	0 ± 0	51.98 ± 0.61	26.34 ± 2.06	0 ± 0	0 ± 0
58	Acetylsalicylic acid	100	4.17 ± 0.92	18.14 ± 0.16	7.68 ± 1.09	0 ± 0	8.35 ± 0.03	23.98 ± 1.32
59	2-Hydroxy-N-(4-hydroxyphenyl)-benzamide	100	62.28 ± 0.56	23.18 ± 0.91	30.14 ± 0.59	41.74 ± 0.97	34.35 ± 1.04	46.4 ± 0.26
60	2,4-Dihydroxybenzoic acid	100	0 ± 0	9.48 ± 0.13	12.68 ± 0.59	0 ± 0	8.25 ± 0.01	29.26 ± 1.29
61	4-Methoxysalicylic acid	100	48.36 ± 0.72	34.95 ± 0.48	39.6 ± 1.69	12.81 ± 1.01	29.36 ± 0.31	38.01 ± 1.82
62	Salicylanilide	100	100 ± 0	66.6 ± 1.14	100 ± 0	100 ± 0	95.21 ± 0.58	100 ± 0
63	4-Aminosalicylic acid	100	0 ± 0	14.92 ± 0.01	9.45 ± 0.88	8.7 ± 0.06	14.03 ± 0.03	32.08 ± 1.77
64	4-Fluoro-2-hydrxybenzoic acid	100	5.35 ± 0.81	21.45 ± 0.93	15.89 ± 0.27	15.99 ± 0.69	23.85 ± 0.05	30.04 ± 0.12
65	Ethyl 2-hydroxybenzoate	100	0 ± 0	11.1 ± 1.13	0 ± 0	6.11 ± 0.2	5.86 ± 1.46	7.47 ± 0.89
66	Sasapyrine	100	28.57 ± 1.5	51.14 ± 0.77	53.73 ± 1.66	5.98 ± 0.51	14.88 ± 1.72	26.91 ± 2.02
67	Benorilate	100	20.38 ± 0.03	50.08 ± 0.67	11.13 ± 0.99	22.93 ± 2.54	9.6 ± 0.14	8.39 ± 1.02
68	Thiazolidine	100	0 ± 0	24.43 ± 0.29	33.64 ± 2.24	8.03 ± 0.53	1.48 ± 0.01	10.96 ± 0.44
69	Salicylhydroxamic acid	100	5.91 ± 0.18	42.9 ± 0.01	7.25 ± 0.44	34.34 ± 0.93	32.18 ± 0.4	25.48 ± 0.01
70	Labetalol hydrochloride	100	0 ± 0	15.73 ± 1.24	10.6 ± 0.27	10.43 ± 0.21	9.52 ± 0.48	18.26 ± 0.88
71	Mosapride	100	18.56 ± 1.34	34.73 ± 0.16	9.61 ± 0.15	49.02 ± 1.14	40.9 ± 0.56	39.17 ± 0.72
72	Xipamide	100	69.76 ± 1.08	52.3 ± 1.79	39.06 ± 0.55	14.88 ± 3.04	0.59 ± 0.06	0.87 ± 0.09
73	Salicylamide	100	27.33 ± 0.71	10.73 ± 1.26	0 ± 0	20.75 ± 0.54	22.76 ± 1.03	26.25 ± 1.74
74	Niclosamide	100	35.9 ± 0.82	49.39 ± 0.42	-	19.26 ± 0.8	8.95 ± 1.5	30.62 ± 0.07
75	Salicylic acid	100	15.05 ± 2.25	57.11 ± 2.27	31.44 ± 0.98	0 ± 0	10.49 ± 0.78	30.08 ± 0.04
76	Oxyclozanide	100	92.41 ± 1.06	99.21 ± 0.53	83.85 ± 0.4	55.87 ± 0.74	62.78 ± 1.03	64.47 ± 1.97
77	Closantel	100	8.75 ± 0.65	10.71 ± 1.16	0 ± 0	19.77 ± 0.65	5.23 ± 1.87	0 ± 0
78	Closantel sodium	100	17.2 ± 0.6	10.46 ± 2.37	26.32 ± 0.97	42.18 ± 3.89	0 ± 0	0 ± 0
79	Rafoxanide	100	8.75 ± 0.65	10.71 ± 1.16	0 ± 0	19.77 ± 0.65	5.23 ± 1.87	0 ± 0
80	Nitazoxanide	100	73.03 ± 1.51	100 ± 0	100 ± 0	34.04 ± 0.16	18.41 ± 0.08	38.06 ± 0.26
81	Otilonium bromide	100	2.82 ± 0.37	0.23 ± 0.04	2.94 ± 0.27	7.23 ± 2.79	2.28 ± 0.31	7.69 ± 0.02
82	Diflunisal	100	1.57 ± 0.32	3.0 ± 0.18	2.21.±0.03	7.59 ± 1.38	5.35 ± 3.97	6.73 ± 1.54
83	Sulfasalazine	100	0 ± 0	5.13 ± 0.35	0 ± 0	15.58 ± 1.47	9.55 ± 0.01	24.04 ± 3.33
84	Enzalutamide	100	58.87 ± 0.98	52.21 ± 0.83	54.04 ± 1.95	11.39 ± 1.82	25.17 ± 0.95	29.94 ± 1.28
85	Olsalazine sodium	100	11.38 ± 0.4	25.42 ± 0.13	15.17 ± 0.71	-	-	-
86	Tranilast	100	13.82 ± 1.5	63.23 ± 1.41	66.9 ± 1.81	0 ± 0	19.58 ± 0.58	43.75 ± 1.03
87	Metoclopramide hydrochloride	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
88	Sulfasalazine	100	0 ± 0	5.13 ± 0.35	0 ± 0	15.58 ± 1.47	9.55 ± 0.01	24.04 ± 3.33
89	Sulfaquinoxaline sodium	100	50.37 ± 0.89	39.96 ± 2.97	69.26 ± 1.06	89.19 ± 0.84	86.95 ± 0.31	83.33 ± 1.59
90	Sodium N-(6-chloropyrazinyl)sulphanilamidate	100	21.53 ± 0.89	13.04 ± 1.27	64.72 ± 1.17	38.93 ± 0.74	76.13 ± 0.57	11.4 ± 1.07
91	Sulfamethazine	100	7.78 ± 1.08	0 ± 0	44.55 ± 0.64	6.05 ± 1.35	0 ± 0	0 ± 0
92	Sulfamonomethoxine	100	26.8 ± 4.44	0 ± 0	55.82 ± 1.52	52.15 ± 1.51	68.05 ± 1.65	10.19 ± 3.72
93	Sulfachloropyridazine	100	11.93 ± 0.92	0 ± 0	59.16 ± 0.5	28.5 ± 2.09	7.23 ± 0.61	0 ± 0
94	Sodium N-(5-methylisoxazol-3-yl)sulphanilamidate	100	30.78 ± 1.37	25.33 ± 0.65	90.8 ± 1.05	84.63 ± 0.09	86.75 ± 0.01	62.85 ± 0.02
95	Sulfamethoxypyridazine	100	5.98 ± 1.61	0 ± 0	5.13 ± 0.98	13.44 ± 0.44	11.16 ± 2.24	2.39 ± 0.41
96	Sulfisoxazole	100	0 ± 0	54.52 ± 1.29	59.39 ± 3.07	51.54 ± 3.01	0 ± 0	76.85 ± 0.66
97	Bensulfuron-methyl	100	50.42 ± 1.71	96.7 ± 0.78	89.04 ± 0.64	75.41 ± 1.32	44.46 ± 0.37	47.11 ± 0.55
98	Chlorimuron-ethyl	100	73.12 ± 1.23	91.36 ± 0.51	92.16 ± 0.17	76.78 ± 0.62	64.46 ± 2.29	61.42 ± 1.2
99	Fasudil hydrochloride	100	0 ± 0	23.79 ± 3.65	8.44 ± 3.47	38.43 ± 1.29	5.79 ± 1.90	5.18 ± 0.92
100	Vemurafenib	100	93.29 ± 0	15.48 ± 2.71	3.82 ± 0.08	10.74 ± 0.14	0 ± 0	4.58 ± 0.47
101	Brinzolamide	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	9.18 ± 2.75	0 ± 0
102	Dexamethasone	100	1.73 ± 0.07	69.93 ± 1.36	14.13 ± 0.72	8.15 ± 0.63	3.92 ± 0.99	6.77 ± 1.11
103	Spironolactone	100	33.88 ± 1.58	48.54 ± 0.37	52.52 ± 1.23	42.73 ± 1.89	24.83 ± 1.41	38.08 ± 0.66
104	Triamcinolone acetonide	100	0 ± 0	0 ± 0	5.8 ± 2.03	0 ± 0	0 ± 0	0 ± 0
105	Betamethasone	100	0 ± 0	7.65 ± 0.94	0 ± 0	0 ± 0	35.41 ± 1.12	0 ± 0
106	Hydrocortisone	100	0 ± 0	0 ± 0	0 ± 0	10.42 ± 2.34	0 ± 0	0 ± 0
107	Prednisolone	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
108	Fluticasone propionate	100	0 ± 0	0 ± 0	31.48 ± 1.52	0 ± 0	0.79 ± 0.56	1.95 ± 1.54
109	Bardoxolone methyl	100	75.15 ± 1.32	36.5 ± 2.81	88.74 ± 0.82	61.27 ± 0.54	57.32 ± 0.39	26.27 ± 1.17
110	Megestrol	100	0 ± 0	16 ± 2.63	0 ± 0	10.4 ± 1.73	6.98 ± 0.13	13.07 ± 0.89
111	Trilostane	100	7.98 ± 1.23	36.02 ± 1.96	22.35 ± 2.87	0 ± 0	0 ± 0	0 ± 0
112	Stanozolol	100	2.92 ± 1.03	19.45 ± 2.16	20.18 ± 0.36	33.94 ± 1.22	48.6 ± 0.35	45.57 ± 1.96
113	Megestrol acetate	100	1.94 ± 0.5	2.1 ± 0.1	1.28 ± 0.29	5.98 ± 0.76	3.00 ± 0.1	1.81 ± 0.04
114	Thiacetazone	100	9.13 ± 0.55	0 ± 0	21.38 ± 0.23	13.33 ± 1.25	12.33 ± 0.36	18.86 ± 0.47
115	Imidurea	100	17.07 ± 0.74	18.11 ± 2.84	18.56 ± 1.04	6.63 ± 2.15	0 ± 0	0 ± 0
116	Imidocarb dipropionate	100	19.28 ± 0.94	41.27 ± 1.37	63.31 ± 1.08	73.21 ± 1.01	55.28 ± 2.53	22.58 ± 0.93
117	Glimepiride	100	15.73 ± 0.2	32.37 ± 2.33	43.39 ± 1.11	0 ± 0	16.34 ± 0.98	0 ± 0
118	Triclocarban	100	31.08 ± 1.2	39.37 ± 0.83	0 ± 0	7.22 ± 0.75	9.79 ± 0.54	27.36 ± 0.62
119	Ripretinib	100	0 ± 0	27.57 ± 1.15	13.33 ± 0.72	17.56 ± 1.24	16.8 ± 1.43	0 ± 0
120	Allantoin	100	0 ± 0	63.06 ± 0.03	0 ± 0	22.05 ± 0.31	11.7 ± 1.76	30.96 ± 0.42
121	Dichloroisocyanyric acid	100	0 ± 0	31.03 ± 1.27	7.31 ± 2.97	17.73 ± 0.32	10.65 ± 0.03	25.63 ± 0.59
122	1,3-Dichloro-5,5-dimethylhydantoin	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	-
123	Thalidomide	100	12.18 ± 3.2	5.74 ± 0.33	0 ± 0	0 ± 0	0 ± 0	23.83 ± 1.43
124	Apremilast	100	0 ± 0	59.24 ± 1.36	16.61 ± 1.79	0 ± 0	16.81 ± 0.44	0 ± 0
125	Primidone	100	0 ± 0	21.07 ± 1.99	0 ± 0	0 ± 0	0 ± 0	0 ± 0
126	4,4,4-Trifluoro-1-(4-fluorophenyl)butane-1,3-dione	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	85.48 ± 0.31	94.74 ± 0.71
127	Sodium dehydroacetate	500	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
128	1-(4-Chlorophenyl)-4,4,4-trifluoro-1,3-butanedione	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	97.11 ± 0.66	100 ± 0
129	2H-1,3-Benzoxazine-2,4(3H)-dione	100	33.45 ± 1.7	0 ± 0	3.63 ± 0.93	24.83 ± 1.28	29.28 ± 0.55	21.69 ± 0.29
130	Diclazuril	100	25.02 ± 0.39	0 ± 0	68.33 ± 0.98	7.78 ± 1.78	30.48 ± 2.34	50.68 ± 1.09
131	Avobenzone	100	24.84 ± 0.09	0 ± 0	10.03 ± 0.62	12.19 ± 2.2	0 ± 0	0 ± 0
132	N,N-methylenebis N'-1-(hydroxymethyl)-2,5-dioxo-4-imidazolidinyl urea	100	17.07 ± 0.74	18.11 ± 2.84	18.56 ± 1.04	6.63 ± 2.15	0 ± 0	0 ± 0
133	Theophylline	100	15.4 ± 1.68	0 ± 0	22.36 ± 0.99	4.87 ± 0.39	0 ± 0	18.21 ± 1.55
134	Bumetanide	100	0 ± 0	33.73 ± 0.56	5.51 ± 0.28	23.32 ± 0.57	8.78 ± 0.97	76.71 ± 2.02
135	Triclosan	100	88.87 ± 0.57	92.01 ± 0.17	100 ± 0	100 ± 0	93.26 ± 0.17	89.18 ± 0.46
136	Tamoxifen	100	53.7 ± 2.67	100 ± 0	100 ± 0	41.21 ± 0.97	69.21 ± 0.33	62.77 ± 0.01
137	Fluoxetine hydrochloride	100	49.9 ± 1.4	67.16 ± 3.81	80.15 ± 0.53	65.05 ± 1.54	56.21 ± 1.27	60.84 ± 0.06
138	Benztropine mesylate	100	28.64 ± 1.36	48.56 ± 2.36	80.4 ± 0.3	33.24 ± 1.44	23.88 ± 0.75	41.18 ± 0.67
139	Dronedarone hydrochloride	100	86.92 ± 0.01	78.44 ± 0.53	98.97 ± 0.22	68.88 ± 0.01	79.75 ± 0.42	76.4 ± 0.78
140	Bazedoxifene acetate	100	0 ± 0	64.9 ± 1.86	60.5 ± 2.82	17.93 ± 2.12	29.53 ± 3.07	36.82 ± 0.43
141	Rolipram	100	17.01 ± 0.25	21.2 ± 1.04	0 ± 0	0 ± 0	17.75 ± 0.56	13.61 ± 0.65
142	Ranitidine hydrochloride	100	17.86 ± 2.59	10.71 ± 2.08	16.61 ± 1.09	0 ± 0	14.67 ± 0.36	7.65 ± 0.19
143	Nimesulide	100	67.47 ± 0.55	66.11 ± 0.73	97.2 ± 0.87	55.85 ± 2.09	34.62 ± 2.45	40.55 ± 0.92
144	Orphenadrine citrate	100	28.84 ± 2.05	12.99 ± 2.93	45.9 ± 1.41	23.88 ± 1.29	0 ± 0	13.14 ± 1.59
145	Diphenhydramine hydrochloride	100	27.53 ± 0.3	11.56 ± 1.05	31.72 ± 0.56	21.66 ± 2.1	0 ± 0	12.29 ± 0.95
146	Diphenhydramine	100	0 ± 0	0 ± 0	12.22 ± 3.82	22.11 ± 3.99	0 ± 0	10.46 ± 1.16
147	5,5′-Dithiobis(2-nitrobenzoic acid)	100	17.1 ± 7.15	12.02 ± 0.65	28.88 ± 2.52	6.63 ± 1.22	0 ± 0	6.14 ± 0.28
148	Toltrazuril	100	20.55 ± 0.6	0 ± 0	44.98 ± 1.48	9.36 ± 0.8	0 ± 0	14.5 ± 0.74
149	Arbidol hydrochloride	100	66.22 ± 0.13	84.81 ± 0.34	65.49 ± 0.53	57.95 ± 1.44	66.27 ± 0.49	59.6 ± 0.5
150	Disulfiram	100	91.86 ± 0.79	100 ± 0	58.48 ± 1.12	71.27 ± 1.45	71.51 ± 0.88	66.57 ± 0.01
151	Probucol	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
152	Benzydamine hydrochloride	100	40.63 ± 2.31	33.35 ± 0.71	64.5 ± 0.91	35.25 ± 1.39	17.95 ± 1.12	31.62 ± 1.49
153	Bithionol	100	71.66 ± 2.32	70.57 ± 0.41	57.32 ± 0.76	60.02 ± 0.78	65.85 ± 0.7	75.17 ± 0.51
154	Fluoxetine	100	48.91 ± 1.22	63.73 ± 0.15	80.02 ± 3.45	63.55 ± 2.95	37.25 ± 1.93	-
155	Carvedilol	100	22.17 ± 1.27	65.81 ± 2.18	79.24 ± 0.96	63.44 ± 1.94	28.83 ± 1.10	-
156	JX06	100	89.95 ± 0.73	12.35 ± 0.26	39.71 ± 0.84	59.5 ± 0.65	52.79 ± 0.14	56.32 ± 0.06
157	Iodopropynyl butylcarbamate	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	92.05 ± 0.14	100 ± 0
158	2- Cyano-3-hydroxy-N-(4'-trifluoromethylphenyl)-crotone amide	100	35.29 ± 1.18	79.74 ± 1.91	83.26 ± 1.46	6.87 ± 1.24	8.86 ± 1.31	21.17 ± 0.6
159	Vorinostat	100	0 ± 0	0 ± 0	43.4 ± 1.34	10.04 ± 0.38	8.23 ± 1.84	19.21 ± 0.4
160	Florfenicol	100	14.31 ± 1.95		16.61 ± 0.96	11.95 ± 0.63	22.82 ± 0.68	6.37 ± 0.78
161	Bicalutamide	100	37.05 ± 0.7	53.43 ± 0.43	64.66 ± 1.02	32.35 ± 2.56	30.94 ± 1.68	28.01 ± 2.13
162	Leflunomide	100	96.77 ± 0.25	90.45 ± 0.46	86.69 ± 0.34	50.27 ± 2.01	78.5 ± 0.28	70.53 ± 1.6
163	Itopride hydrochloride	100	0 ± 0	0 ± 0	2.39 ± 0.99	0 ± 0	0 ± 0	5 ± 1.8
164	Entacapone	100	9.8 ± 1.5	69.31 ± 2.32	7.98 ± 0.05	0 ± 0	21.8 ± 1.28	0 ± 0
165	Favipiravir	100	12.44 ± 1.83	68.29 ± 0.27	61.55 ± 1.07	0 ± 0	9.43 ± 1.63	-
166	1,2,4-Triazolo[4,3-a]pyridin-3(2H)-one	100	0 ± 0	49.48 ± 1.81	0 ± 0	0 ± 0	7.72 ± 0.73	11.88 ± 0.57
167	Tadalafil	100	23.09 ± 1.73	0 ± 0	9.15 ± 0.77	0 ± 0	12.71 ± 0.87	12.46 ± 1.17
168	Efavirenz	100	94.85 ± 0.01	96.37 ± 0.04	62.6 ± 2.31	73.4 ± 1.52	78.25 ± 0.41	82.11 ± 1.37
169	Levosimendan	100	27.63 ± 1.15	58.5 ± 0.68	58.59 ± 0.62	16.13 ± 1.38	13.98 ± 0.34	13.91 ± 0.59
170	Azelastine hydrochloride	100	43.78 ± 1.45	75.59 ± 1.13	78 ± 0.93	45.1 ± 1.29	58.94 ± 0.39	51.33 ± 0.6
171	Sunitinib	100	59.79 ± 0.86	75.71 ± 0.81	87.45 ± 0.5	55.94 ± 2.6	64.79 ± 0.87	46.12 ± 0.36
172	Upadacitinib	100	0 ± 0	26.86 ± 1.92	12.32 ± 2.05	28.42 ± 3.65	17.41 ± 0.49	0 ± 0
173	PF01367338 phosphate	100	86.26 ± 0.73	90.65 ± 0.37	73.85 ± 1.23	38.88 ± 1.45	53.65 ± 1.66	43.16 ± 0.37
174	Trametinib	100	18.3 ± 2.6	0 ± 0	9.95 ± 0.49	10.79 ± 0.27	3.34 ± 0.61	0 ± 0
175	Valnemulin hydrochloride	100	0 ± 0	63.1 ± 0.45	70.73 ± 2.11	12.03 ± 0.31	25.23 ± 1.03	57.88 ± 1.74
176	Nifuroxazide	100	14.82 ± 1.61	14.05 ± 1.28	15.81 ± 0.55	16.29 ± 0.64	0 ± 0	0 ± 0
177	Iproniazid	100	0 ± 0	4.44 ± 0.75	8.06 ± 1.4	0 ± 0	0 ± 0	5.27 ± 0.06
178	Cyanoacetohydrazide	100	12.49 ± 1.81	24.16 ± 3.75	17.81 ± 0.84	0 ± 0	0 ± 0	0 ± 0
179	Praziquantel	100	61.69 ± 1.82	22.65 ± 1.10	38.58 ± 2.56	31.59 ± 0.37	28.65 ± 1.06	24.92 ± 0.64
180	Pyrazinamide	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
181	Ebselen	100	75.99 ± 0.93	24.38 ± 0.92	65.36 ± 0.91	-	30.09 ± 0.80	49.65 ± 0.18
182	Temozolomideacid	100	4.82 ± 0.78	0 ± 0	16.2 ± 1.03	11.03 ± 1.79	0 ± 0	12.02 ± 2.68
183	Atorvastatin	100	7.1 ± 1.11	16.44 ± 0.68	1.67 ± 0.38	28.2 ± 0.03	10.14 ± 2.01	8.06 ± 0.42
184	Indometacin	100	90.33 ± 0.45	65.23 ± 0.83	81.24 ± 1.09	57.2 ± 0.61	78.02 ± 0.41	59.86 ± 2.63
185	Trimethobenzamide hydrochloride	100	11.39 ± 2.65	27.93 ± 1.18	20.55 ± 1.55	18.71 ± 1.94	0 ± 0	0 ± 0
186	Levetiracetam	100	10.95 ± 1	0 ± 0	11.9 ± 1.01	18.2 ± 1.85	0 ± 0	0 ± 0
187	7-Aminodesacetoxycephalosporanic acid	100	5.55 ± 0.58	17.58 ± 1.13	0 ± 0	0 ± 0	0 ± 0	9.94 ± 0.64
188	(2S-trans)-3-Amino-2-methyl-4-oxoazetidine-1-sulphonic acid	100	3.77 ± 0.42	17.93 ± 2.42	0 ± 0	0 ± 0	0 ± 0	11.82 ± 0.25
189	Dichloro-1,2-dithiacyclopentenone	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
190	Anethole trithione	100	41.44 ± 0.74	49.75 ± 1.18	0 ± 0	10.92 ± 0.44	7.34 ± 0.74	13.82 ± 0.2
191	3H-1,2-Benzodithiol-3-one	100	100 ± 0	100 ± 0	100 ± 0	62.89 ± 1.13	19.59 ± 1.51	62.44 ± 1.47
192	2,3-Dimercapto-1-propanol	100	10.9 ± 0.89	5.93 ± 0.14	0 ± 0	15.44 ± 0.46	0 ± 0	23.24 ± 0.92
193	Oltipraz	100	33.89 ± 0.43	41.34 ± 1.50	46.53 ± 2.43	4.49 ± 1.16	4.51 ± 0.69	12.98 ± 2.2
194	Sertraline hydrochloride	100	100 ± 0	100 ± 0	94.11 ± 0.79	77.6 ± 0.45	88.74 ± 0.66	88.23 ± 0.71
195	Ethyl bromopyruvate	100	18.87 ± 1.3	9.17 ± 0.56	0 ± 0	20.65 ± 0.43	16.84 ± 0.44	20.51 ± 0.29
196	Atovaquone	100	0 ± 0	10.5 ± 1.33	15.98 ± 0.68	36.04 ± 1.85	14.46 ± 0.92	29.8 ± 0.04
197	Clorprenaline hydrochloride	100	0 ± 0	0 ± 0	4.72 ± 1.73	0 ± 0	0 ± 0	5.78 ± 0.23
198	Simvastatin	100	83.39 ± 0.04	74.06 ± 0.23	86.2 ± 0.68	56.73 ± 0.01	73.68 ± 2.4	68.46 ± 0.25
199	Clomipramine hydrochloride	100	62.01 ± 1.98	82.57 ± 1.33	88.14 ± 0.83	63.92 ± 0.31	75.11 ± 1.97	69.94 ± 0.71
200	Benzbromarone	100	57.66 ± 0.01	82.36 ± 1.45	98.63 ± 0.15	82.28 ± 2.17	92.04 ± 0.08	51.79 ± 0.04
201	Nortaiptyline hydrochloride	100	0 ± 0	2.01 ± 0.96	3.1 ± 0.05	0.19 ± 0.93	2.98 ± 0.01	2.60 ± 0.1
202	Fluvastatin sodium salt	100	86.78 ± 1.28	100 ± 0	100 ± 0	54.06 ± 0.74	100 ± 0	100 ± 0
203	Tulobuterol hydrochloride	100	12.47 ± 2.49	0 ± 0	17.18 ± 0.92	0 ± 0	0 ± 0	0 ± 0
204	Tilorone dihydrochloride	100	10.3 ± 0.64	0 ± 0	61.23 ± 2.09	28.14 ± 1.93	19.48 ± 1.07	16.02 ± 0.26
205	Dichlorophen	100	96.01 ± 1.31	100 ± 0	87.6 ± 0.49	84.2 ± 0.45	87.62 ± 0.49	83.1 ± 0.99
206	Clofazimine	100	41.89 ± 1.84	18.72 ± 0.89	55.46 ± 2.2	30.52 ± 0.88	34.86 ± 0.02	32.45 ± 1.97
207	Ethopabate	100	19.63 ± 1.42	0 ± 0	31.73 ± 1.15	6.81 ± 1.92	9.45 ± 1.39	9.24 ± 1.75
208	(E,E)-Farnesol	100	76.7 ± 0.33	59.79 ± 1.57	72.95 ± 0.32	52.13 ± 1.03	16.28 ± 0.91	29.52 ± 0.9
209	Chlorphenesin	100	25.04 ± 1.93	54.53 ± 0.06	29.76 ± 0.7	34.94 ± 2.77	0 ± 0	36.21 ± 1.34
210	2-Benzoxazolinone	100	20.15 ± 2.33	9.08 ± 0.62	26.61 ± 3.17	37.21 ± 1.24	0 ± 0	8.93 ± 0.67
211	Triacetin	100	8.4 ± 1.03	15.82 ± 3.67	18 ± 2.33	5.69 ± 0.81	0 ± 0	0 ± 0
212	Bronopol	100	71.19 ± 1.64	50 ± 1.53	58.31 ± 0.96	82.6 ± 1.08	53.32 ± 2.05	39.32 ± 0.68
213	Flufenamic acid	100	94.95 ± 0.12	97.95 ± 0.26	91.7 ± 0.19	77.13 ± 1.49	84.31 ± 0.54	82.43 ± 0.24
214	Pyrantel tartrate salt	100	5.08 ± 0.67	0 ± 0	5.18 ± 0.2	21.76 ± 1.58	1.89 ± 0.34	6.26 ± 1.3
215	Carbonyl cyanide 3-chloro-phenylhydrazone	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0	100 ± 0
216	Atropine sulfate monohydrate	100	5.52 ± 1.64	0 ± 0	7.76 ± 0.82	2.31 ± 0.93	0 ± 0	5.17 ± 0.26
217	Monomyristitin	100	0 ± 0	0 ± 0	5.31 ± 1.58	3.79 ± 0.76	0 ± 0	0.87 ± 0.5
218	Lumefantrine	100	0 ± 0	22.51 ± 0.38	24.36 ± 2.37	0 ± 0	0 ± 0	0 ± 0
219	4-(2-Aminoethyl)benzenesulfonylfluoride hydrochloride	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	15.96 ± 0.86
220	Bufexamac	100	19.1 ± 1.94	92.13 ± 0.21	57.14 ± 0.4	34.26 ± 0.81	0 ± 0	42.07 ± 0.68
221	1-(2,6-Dichlorophenyl)-2-indolinone	100	83.65 ± 0.41	91.79 ± 0.86	78.64 ± 0.04	39.33 ± 0.22	74.43 ± 0.79	80.63 ± 1.16
222	5-Phenylpenta-2,4-dienoic acid	100	68.76 ± 0.35	100 ± 0	93.72 ± 0.46	47.14 ± 0.21	33.08 ± 1.56	47.11 ± 2.43
223	Hydroxyurea	100	50.19 ± 0.55	23.08 ± 1.13	0 ± 0	0 ± 0	0 ± 0	0 ± 0
224	Acetylcysteine	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	12.22 ± 1.17
225	Escitalopram oxalate	100	0 ± 0	11.52 ± 1.51	0 ± 0	17.36 ± 1.29	0 ± 0	11.25 ± 1.5
226	Ezetimibe	100	62.77 ± 1.32	68.85 ± 0.73	63.88 ± 0.52	48.58 ± 0.66	44.76 ± 0.43	60.89 ± 0.51
227	Venlafaxine hydrochloride	100	0 ± 0	12.48 ± 0.65	0 ± 0	0 ± 0	0 ± 0	25.47 ± 1.76
228	(+/-)-Verapamil hydrochloride	100	0 ± 0	2.82 ± 0.65	31.6 ± 2.72	27.8 ± 2.09	5.03 ± 0.84	9.41 ± 2.1
229	Mecarbinate	100	0 ± 0	0 ± 0	0 ± 0	5.86 ± 2.28	7.78 ± 1.61	11.26 ± 1.24
230	Ipratropium bromide	100	0 ± 0	0 ± 0	4.03 ± 0	4.45 ± 0.62	0 ± 0	4.82 ± 1.62
231	Ketotifen fumarate	100	24.23 ± 3.25	21.97 ± 0.4	36.68 ± 1.72	44.51 ± 1.46	0 ± 0	11.93 ± 1.04
232	Verapamil	100	4.9 ± 2.23	45.33 ± 0.97	17.69 ± 1.7	18.51 ± 1.85	6.73 ± 0.38	0 ± 0
233	RU-58841 (72)	100	4 ± 0.38	43.44 ± 1.04	0 ± 0	12.31 ± 1.77	10.25 ± 1.38	0 ± 0
234	Amylmetacresol	100	100 ± 0	100 ± 0	100 ± 0	100 ± 0	70.15 ± 1.79	54.78 ± 0.6
235	Nisin	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	7.53 ± 0.16
236	Silver	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
237	Combretastatin A-4	100	69.75 ± 0.04	42.08 ± 0.27	19.06 ± 2.17	11.96 ± 0.11	29.35 ± 0.3	64.04 ± 0.48
238	Procaine	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	14.13 ± 0.28	13.34 ± 0.87
239	Phenformin hydrochloride	100	9.35 ± 1.75	36.91 ± 0.71	28.82 ± 0.54	10.65 ± 1.83	6.75 ± 0.34	20.3 ± 1.02
240	Moroxydine hydrochloride	100	13.39 ± 1.09	0 ± 0	22.41 ± 0.04	0 ± 0	0 ± 0	0 ± 0
241	Febantel	100	100 ± 0	38.88 ± 1.59	68.78 ± 0.37	36.27 ± 1.81	6.36 ± 0.12	13.28 ± 0.43
242	3,4-dihydroquinolin-2(1H)-one	100	21.01 ± 2.51	52.19 ± 1.99	0 ± 0	16.05 ± 0.52	13.9 ± 1	24.9 ± 1.4
243	6-Bromo--3,4-dihydro-1H-Quinolin-2-one	100	84.91 ± 1.44	62.48 ± 0.39	61.03 ± 1.12	49.67 ± 2.81	64.05 ± 0.39	46.4 ± 0.01
244	7-Hydroxy-3,4-dihydro-2(1H)-quinolinone	100	12.75 ± 0.01	45.63 ± 0.34	23.65 ± 3	0 ± 0	9.58 ± 0.02	21.5 ± 0.45
245	6-Hydroxy-1,2,3,4-Tetrahydro--2-Quinolinone	100	8.89 ± 0.46	45.68 ± 0.57	5.6 ± 1.32	0 ± 0	7.95 ± 1.43	13.99 ± 1.28
246	5-Hydroxy-3,4-dihydro-2(1H)-quinolinone	100	12.4 ± 1.16	45.85 ± 0.02	0 ± 0	0 ± 0	8.75 ± 0.39	15.05 ± 0.41
247	Oxolinc acid impurity B	100	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0	5.37 ± 0.47
248	Levofloxacin hydrochloride	100	0 ± 0	0 ± 0	61.22 ± 1.05	24.08 ± 0.50	0 ± 0	25.68 ± 0.35
249	Norfloxacin	100	0 ± 0	0 ± 0	25.55 ± 0.17	39.69 ± 0.28	0 ± 0	10.73 ± 0.51
250	Gatifloxacin	100	26.47 ± 0.59	0 ± 0	46.60 ± 0.36	10.95 ± 0.70	6.95 ± 0.32	20.50 ± 0.53
251	Fleroxacin	100	0 ± 0	42.88 ± 0.44	42.03 ± 0.57	22.37 ± 0.17	00 ± 0	13.50 ± 0.83
252	Ciprofloxacin	100	0 ± 0	4.30 ± 0.26	48.90 ± 0.40	42.93 ± 0.24	4.42 ± 0.34	16.58 ± 0.30
253	Enrofloxacin	100	0 ± 0	0 ± 0	59.99 ± 0.45	8.50 ± 0.31	33.35 ± 0.30	59.60 ± 0.43
254	Moxifloxacin	100	0 ± 0	17.78 ± 0.44	45.00 ± 0.63	8.25 ± 0.44	18.42 ± 0.13	25.91 ± 0.24
255	Enoxacin	100	0 ± 0	0 ± 0	6.90 ± 0.57	4.43 ± 0.50	4.45 ± 0.29	10.50 ± 0.26
256	Nalidixic acid	100	9.87 ± 0.53	71.98 ± 0.60	5.88 ± 0.46	15.68 ± 0.26	69.57 ± 0.22	69.95 ± 0.54
257	Ofloxacin	100	0 ± 0	48.03 ± 0.55	58.39 ± 0.53	20.78 ± 0.23	0 ± 0	17.07 ± 0.63
258	Marbofloxacin	100	0 ± 0	30.02 ± 0.34	50.31 ± 0.46	11.5 ± 0.20	0 ± 0	18.94 ± 0.52
259	2-Hydroxyacetophenone	100	42.43 ± 2.66	75.59 ± 0.79	26.67 ± 1.69	10.18 ± 0.44	8.51 ± 0.18	18.88 ± 0.96
260	4'-Hydroxyacetophenone	100	36.62 ± 2.69	18.76 ± 0.43	27.33 ± 1.56	8.93 ± 0.68	12.22 ± 2.17	0 ± 0
261	3'-Hydroxyacetophenone	100	10.08 ± 0.93	27.95 ± 2.21	41.13 ± 1.93	0 ± 0	6.58 ± 0.68	13.38 ± 0.54
262	3,4-Dihydroxyacetophenone	100	0 ± 0	19.83 ± 0.34	0 ± 0	0 ± 0	0 ± 0	0 ± 0
263	3,4-Dimethoxyacetophenone	100	14 ± 2.59	0 ± 0	45.41 ± 0.68	7.67 ± 0.16	21.64 ± 1.53	17.23 ± 0.1
264	Acetovanillone	100	26.54 ± 0.69	21.08 ± 1.63	12.05 ± 0.41	0 ± 0	16.78 ± 0.12	9.42 ± 0.01
265	2',6'-Dihydroxyacetophenone	100	80.59 ± 0.01	31.46 ± 1.81	78.41 ± 1.25	65.73 ± 0.36	58.92 ± 0.01	61.48 ± 0.02
266	2,4-Dihydroxyacetophenone	100	62.94 ± 2.1	23.2 ± 1.27	58.31 ± 0.5	23.23 ± 1.93	41.25 ± 0.32	34 ± 0.28
267	2,4-Dihydroxyacetophenone	100	25.95 ± 2.93	34.27 ± 0.46	38.9 ± 0.85	35.35 ± 1.47	25.16 ± 1.14	25.4 ± 0.57
268	2',3',4'-Trihydroxyacetophenone	100	27.04 ± 1.19	43.18 ± 0.70	0 ± 0	15.02 ± 0.78	14.58 ± 0.4	15.16 ± 1.57
269	2',4',6'-Trihydroxyacetophenone monohydrate	100	7.81 ± 0.49	13.53 ± 0.8	0 ± 0	14.24 ± 1.41	10.79 ± 0.19	15.32 ± 1.91
270	4,6-Diacetylresorcinol	100	79.69 ± 1.15	96.55 ± 0.87	50.85 ± 1.58	71.88 ± 3.12	72.74 ± 0.46	63.2 ± 1.03
271	Flopropione	100	45.45 ± 0.6	45.25 ± 4.05	33.04 ± 1.03	24.38 ± 3.29	25.52 ± 0.67	46.37 ± 0.25
272	2',3',4'-Trimethoxyacetophenone	100	6.91 ± 1.46	12.44 ± 0.38	19.16 ± 3.27	8.91 ± 1.98	9.81 ± 0.06	14.19 ± 2.51
273	Acetosyringone	100	4.4 ± 0.93	13.4 ± 2.03	0 ± 0	0 ± 0	9.42 ± 0.84	17.29 ± 0.64
274	2'-Hydroxy-4',5'-dimethoxyacetophenone	100	77.92 ± 1.28	18.52 ± 1.61	60.32 ± 1.61	24.19 ± 2.44	24.11 ± 1.51	34.6 ± 1.63
275	2',4'-Dihydroxypropiophenone	100	87.09 ± 0.49	55.05 ± 2.65	76.33 ± 0.63	62.05 ± 0.42	71.54 ± 0.69	68.29 ± 2.32
276	Abafungin	100	95.31 ± 0.38	100 ± 0	93.63 ± 0.42	76.34 ± 1.38	92.71 ± 0.04	88.33 ± 0.92

^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Fig. 11.

The EC₅₀ of miscellaneous drugs against phytopathogenic fungi^a. ^aR.s, Rhizoctonia solani; S.s, Sclerotinia sclerotiorum; B.c, Botrytis cinerea; F.g, Fusarium graminearum; F.o, Fusarium oxysporum; P.c, Phytophthora capsici

Monensin, natamycin and griseofulvin are antibiotics, but they have different effects, which monensin inhibits the growth of coccidia, gram-positive bacteria, algae and protozoa [61]. Natamycin is commonly used as a preservative to prevent mould contamination in food [62, 63]. Griseofulvin is widely used in clinical medicine to treat skin and stratum corneum fungal infections, and also in the prevention and treatment of fungal diseases in agriculture [64]. Monensin sodium salt, natamycin and griseofulvin had broad-spectrum activity against plant pathogenic fungi, with EC₅₀ ranging from 0.076 to 13.20 μg ml⁻¹. Butenafine hydrochloride, terbinafine hydrochloride and tolnaftate are a group of antifungal drugs, which are applied to the treatment of tinea capitis and other tinea diseases [65, 66]. In this screening, butenafine hydrochloride, terbinafine hydrochloride and tolnaftate were also found to have excellent activity against pathogenic fungi, with EC₅₀ in the range of 0.07–18.05 μg ml⁻¹. It was worth noting that they had significant activity in B. cinerea, with EC₅₀ of 0.07, 0.11 and 0.07 μg ml⁻¹, respectively. Oxyclozanide is the drug of choice for clinical anti-helminth infections, which has the characteristics of broad spectrum, low toxicity and low residue [67]. Through drug repositioning strategy, we found that oxyclozanide also had excellent activity against phytopathogenic fungi with EC₅₀ in the range of 0.09–0.71 μg ml⁻¹. Carbonyl cyanide 3-chloro-phenylhydrazone (CCCP) is an inhibitor of oxidative phosphorylation that disrupts the mitochondrial membrane potential [68]. The evaluation of the in vitro activity of CCCP against pathogenic fungi revealed a broad antifungal spectrum and potent activity with EC₅₀ in the range of 0.38–6.07 μg ml⁻¹. Although this group of drugs was not analyzed by activity and structure, these results provided a structure-based screening approach to repurpose commercially available drugs with the expectation of discovering broad-spectrum, effective drugs against plant pathogens.

Discussion and conclusion

In an era of emerging drug resistance, there is an increasing need to optimise old drugs or develop new ones to alleviate the problem. In this worrying situation, drug repurposing is a promising approach as a way to obtain effective drugs or lead structures to solve the problem. In the field of medicine, studies have been carried out with drug repurposing strategies to re-screen approved library for potential anti-tumour, anti-inflammatory, antituberculous, antimicrobial drugs [23, 69–74]. Similarly, in the agricultural field, the potential of halofuginone, kaempferol, honokiol and tavaborole against agricultural pathogens has also been identified [75–78]. In addition, novel lead structures can also be found through drug repurposing. Antibacterial conversion of neamine aminoglycosides through alkyl modification could turn old drugs into agricultural fungicides [79]. In this paper, we can obtain some potential drugs or lead structures against agricultural pathogenic fungi by screening. However, studies have found that for some repurposed drugs, the original mechanism of action may become a negative side effect of the new indication. Thus, maintaining the positive effects of the new indication while eliminating the original mechanism of the drug is a more attractive study. Conversely, the study show that several AHAS inhibitors developed as commercial herbicides are powerful accumulative inhibitors of C. albicans AHAS [80]. This provides two different directions for our subsequent research.

We have obtained 150 drug candidates through activity screening and many of the compounds had low acute toxicity. Surprisingly, we found that benzoimidazole/carbamate drugs (parbendazole, fenbendazole, mebendazole) (Fig. 4) and azole drugs (econazole, isoconazole nitrate, clotrimazole) (Fig. 5) showed excellent activity against plant pathogenic fungi and low toxicity. In this article benzoimidazole/carbamate drugs (parbendazole, fenbendazole, mebendazole) are mainly used as anthelmintics. The original use of halofuginone was found to be an anticoccidial. Through a drug repurposing strategy, it was found to have excellent activity against Phytophthora [78]. Therefore, we hope to obtain potential drugs or lead structures against plant pathogenic fungi through this strategy, which will provide the possibility for the development of agricultural fungicides.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.