Introduction

Plant diseases caused by phytopathogenic fungi are the primary factor causing global agricultural losses, which can seriously affect the normal growth, transportation, and storage of grains, vegetables, and fruits [1]. Taking the filamentous fungi Botrytis cinerea and Fusarium graminearum as examples, Botrytis cinerea is the major fungal species that triggers gray mold, which can infect over 200 plant species and cause considerable yield loss during agricultural production [2]. Fusarium graminearum is commonly found on wheat, barley, and corn, infected kernels appear shrunken and have white-to-pink colored mold, which leads to a reduction in grain yield and nutritional value by producing various mycotoxins [3]. Currently, the application of chemical fungicides has given a satisfactory effect on the prevention and control of these devastating diseases. Nevertheless, the long-term overuse and misuse of a single chemical fungicide are responsible for the disadvantages of resistance, residue, and resurgence (“3R” problems) [4]. Therefore, it is urgent to search for small molecules with high efficiency and low toxicity to expand the selection range of fungicides.

Nitrogen-containing heterocyclic compounds are currently considered a promising framework for the development of new pesticides [5, 6], especially compounds containing quinoxaline rings that exhibit various biological properties including antitumor, antimalarial, antiviral, antibacterial, and anti-inflammatory [7, 8]. Meanwhile, imidazoquinoxaline has attracted much attention owing to its striking antitumor [9,10,11,12], antiepileptic (LU 73068) [13], and antiallergic (Dazoquinast) activity [14], while there are rare reports on the utilization of the imidazoquinoxaline backbone as a pesticide. Therefore, to explore the application value of the imidazolequinoxaline skeleton in the prevention and control of plant diseases, this paper adopts homologous derivative and molecular hybridization strategy to design and synthesize various kinds of imidazo[1,2-a]quinoxaline derivatives via available and inexpensive reagents, and their antifungal properties against ten phytopathogenic fungi of agricultural relevance were evaluated firstly (Fig. 1). Finally, the antifungal mechanism of the compounds with excellent inhibitory activity was preliminarily explored by observing the mycelial morphology changes and spore germination via electron microscopy.

Fig. 1
figure 1

The design strategy of target compounds in this work

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Results and discussion

Chemistry

Initially, the construction method of the imidazo[1,2-a]quinoxaline skeleton is shown in Scheme 1. 2,3-dichloroquinoxaline (1a) was reacted with 2,2-dimethoxyethanamine, prop-2-yn-1-amine or ammonium hydroxide in different solvents to obtain the respective 2-amino-3-chloroquinoxaline (2c) and its derivatives (2a and 2b), followed by treatment of compounds 2a and 2b with strong acids (HBr and H2SO4) to afford cyclization products 3a and 3b, which were converted to key intermediate compounds 4a, b via chlorination with POCl3 [15]. Meanwhile, compound 4d can be obtained directly by condensation of compound 2c with ethyl 3-bromo-2-oxopropanoate, but the resulting yield was relatively low (15.2%) [14]; the preparation procedure of compound 4c includes reduction, cyclization, hydrolysis, decarboxylation and chlorination five synthetic steps [16]. Chlorinated (4e) and brominated (4f) derivatives were synthesized by reacting intermediate 4a with N-chlorosuccinimide (NCS) and N-bromosuccinimide (NBS), respectively [17].

Scheme 1
scheme 1

Synthetic route for the preparation of compounds 4a–f

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To investigate the effect of substituents on the antifungal activity, the B ring of imidazo[1,2-a]quinoxalines (4a–c) was modified by the introduction of the corresponding amine, ether, sulfonamide, and hydrazine functionalities as depicted in Schemes 2, 3. Compounds 5a–c and 6a–c (by-products) were obtained by ammonolysis of intermediate 4a–c with NH3–CH3OH at 100 °C, and methylamino (5d–f), dimethylamino (5g–i), and tryptamino (5j–l) substituted compounds were also formed under similar conditions (100/80 °C, tube sealing). Compounds 5m–o were prepared by replacing the chlorine atom of compounds 4a–c with morpholine. In addition, etherification of compounds 4a–c with various alcohols or phenols under basic conditions afforded ether derivatives 6d–i. Finally, as depicted in Scheme 3, direct coupling of compounds 4a, b with various sulfonamides in Pd(OAc)2/xantphos/Cs2CO3/1,4-dioxane system at 100 °C gave compounds 7a–d. Compound 4a was hydrolyzed with 85% hydrazine hydrate and subsequently acylated with acyl chloride to afford 8a, b. Spectroscopic data (HRMS, 1H NMR, and 13C NMR) of the target compounds were consistent with their structures.

Scheme 2
scheme 2

Synthetic route for the preparation of target compounds 5a–o and 6a–i

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Scheme 3
scheme 3

Synthetic route for the preparation of compounds 7a–d and 8a–b

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Antifungal activity

Ten phytopathogenic fungi, including Fusarium solani (FS), Fusarium oxysporum (FO), Botryosphaeria dothidea (BD), Fusarium graminearum (FG), Sclerotinia sclerotiorum (SS), Valsa mali (VM), Alternaria alternata (AA), Pyricularia oryzae (PO), Alternaria brassicae (AB), and Botrytis cinerea (BC), that frequently occur in the Chinese agroecosystem were selected as the test strains. The in vitro antifungal activities of the target compounds were investigated through the classical mycelium linear growth rate method [18]. Hymexazol (Hym) and chlorothalonil (Chl), two commercial fungicides served as positive controls.

The results of the preliminary antifungal activity of all the target compounds are summarized in Table 1. The obtained data revealed that the synthesized compounds displayed good to excellent antifungal activity at 50 μg/mL. Among them, eight compounds (including compounds 4b, 4c, 4e, 5c, 5f, 6e, 6 h, and 7d) showed satisfactory antifungal effects against F. solani with inhibition rates of > 80%, which was superior to the positive controls hymexazol (63.9%) and chlorothalonil (76.6%); eleven compounds exhibited better antifungal activity (> 50%) against F. oxysporum than hymexazol (46.2%), especially compounds 4e (73.9%), 5c (84.1%), and 7d (72.1%), which gave higher activity than chlorothalonil (69.2%). Toward B. dothidea, compounds 4e (81.9%), 5a (98.4%), 6 g (91.9%), and 7d (89.6%) exerted more promising antifungal effects than the two commercial fungicides. For F. graminearum, seven compounds (4b, 4c, 4e, 5c, 5f, 5i, and 7c) displayed slightly higher antifungal activity (75.8–86.7%) than chlorothalonil (70.7%) and hymexazol (63.8%). Regretfully, only five compounds 4e, 5c, 5i, 7c, and 7d possessed comparable antifungal activities (67.9–74.5%) to hymexazol (65.6%) against A. alternata, and no noticeable inhibitory efficacies were observed for all compounds toward S. sclerotiorum, P. oryzae, and A. brassicae in comparison with the positive control hymexazol. However, it is worth mentioning that eighteen compounds demonstrated satisfactory inhibitory effects (> 60%) against V. mali, and thirteen compounds revealed better antifungal activity (> 80%) against B. cinerea than hymexazol (72.7%), especially the inhibition rates of compounds 4a, 4e, 5i, 6a, 6c, 6d, and 6f reached over 90%, which could almost completely suppress the growth of mycelium. Furthermore, the preliminary structure–activity relationship of these compounds is summarized in Fig. 2.

Table 1 Fungicidal activity of the target compounds against ten phytopathogenic fungi (50 μg/mL)
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Fig. 2
figure 2

The preliminary structure–activity relationships of the compounds against tested fungi

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Inspired by the preliminary antifungal activity results, the median effective concentration (EC50) values of some selected compounds were further determined at six different concentrations (50, 25, 12.5, 6.25, 3.125, and 1.5625 μg/mL). As displayed in Fig. 3, many compounds exhibited obvious inhibitory effects against the eight tested fungi. For instance, compound 5f (5.1 μg/mL) exhibited the best antifungal activity against F. solani; compounds 4c, 7c, and 7d had anti-A. brassicae EC50 values of 15.4, 18.9, and 19.7 μg/mL, respectively, superior to hymexazol (26.6 μg/mL) and chlorothalonil (> 50 μg/mL); the EC50 range values of compounds 4b, 4e, 5a, 6 g, 7c, and 7d against B. dothidea were 10.3–24.9 μg/mL, which was better than hymexazol (> 50 μg/mL) but lower than chlorothalonil (7.9 μg/mL); the inhibitory effects of 4e (28.8 μg/mL) and 7c (23.7 μg/mL) on P. oryzae were equivalent to that of hymexazol (24.9 μg/mL). For A. alternata, eight compounds possessed more pronounced antifungal activity than chlorothalonil, especially compound 7c (11.2 μg/mL), which was better than hymexazol (16.7 μg/mL). Regarding the F. graminearum strain, seven compounds exhibited higher activity than hymexazol, but failed to exceed chlorothalonil. Furthermore, it is fortunate that twelve compounds demonstrated more promising potential in controlling V. mali, especially compounds 5c and 5f with EC50 values lower than 6.0 μg/mL; thirteen compounds (1.4–15.2 μg/mL) displayed obvious antifungal activity against B. cinerea, particularly compounds 4a, 4e, 6a, 6c, and 6d exhibited 3.0–8.5 folds more potent activities than hymexazol. In addition, the concentration-dependent suppression of the mycelial growth of F.solani, V.mali, and B. cinerea by compounds 5f or 6a could also be clearly observed in Fig. 4.

Fig. 3
figure 3

EC50 values (μg/mL) of selected compounds against eight phytopathogenic fungi

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Fig. 4
figure 4

Effects of compound 5f or 6a on the growth of F.solani, V.mali, and B. cinerea at different concentrations (CK: blank control group)

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Effects of compounds on mycelial growth and spore germination [19]

To elucidate the preliminary mechanism of the antifungal activity of these compounds, light microscopy was used to investigate the influence of compound 5c (6.25 μg/mL) on the hyphal growth and spore germination of V. mali (VM) fungi. As shown in Fig. 5, in the blank control, the mycelium had a smooth surface, much-branched and abundant attachment of spindle-shaped spores. In contrast, the mycelium of the compound 5c treatment group appeared obvious shrinkage and no spore formation. Furthermore, inhibiting spore germination is an important means to prevent fungal regeneration and infection in plants. Figure 6 shows that the spore germination and germ tube elongation were both significantly suppressed in the presence of compound 5c at different concentrations, and the inhibition rates at concentrations of 25, 12.5, and 6.25 μg/mL were 94.4%, 70.3%, and 54.1%, respectively. This phenomenon demonstrated that compound 5c likely exerted antifungal effects by disrupting hyphal differentiation, spore germination, and germ tube growth.

Fig. 5
figure 5

Effects of compound 5c on the mycelial morphology of V. mali at 6.25 μg/mL. (VM-CK represented the normal mycelial morphology of VM, and VM-5c represented the mycelial morphology of VM after treatment with compound 5c)

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Fig. 6
figure 6

Inhibitory effect of different concentrations of compound 5c on the spore germination of V. mali. (400 × ; scale bar: 50 μm)

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Cell cytotoxicity

Finally, the cell cytotoxicity of the six compounds (4a, 4e, 5c, 6a, 6c, and 6d) with promising antifungal activity against mouse microglia (BV2) cells was further investigated in vitro using the CCK-8 assay [20, 21]. From Fig. 7, we can see that the cell viability of the tested compounds was more than 82.7% on BV2 cells at the high concentration of 100 μg/mL, and the current results suggested that the tested compounds showed low toxicities.

Fig. 7
figure 7

In vitro cytotoxicity of some compounds to BV2 cells

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Conclusions

In summary, thirty-six imidazo[1,2-a]quinoxaline derivatives were synthesized and evaluated for their fungicidal activity against ten common phytopathogenic fungi. The results showed that some compounds exhibited more excellent and broad-spectrum fungicidal activity in vitro than the positive controls chlorothalonil and hymexazol, particularly V. mali and B. cinerea strains exhibited the highest susceptibility with an EC50 values of 1.4–27.0 μg/mL for more than ten compounds. Among them, compounds 5c and 5f displayed the most promising antifungal activity against V. mali and F. solani, with an EC50 values of 5.6 and 5.1 μg/mL, respectively, which can be considered as the potential candidate compounds for controlling specific fungi. SAR analysis showed that the type of substituents on the imidazo[1,2-a]quinoxaline skeleton significantly effects the antifungal activity. Preliminary studies on the mechanism of action indicated that these compounds likely exerted their antifungal effects by disrupting hyphal differentiation, spore germination, and germ tube growth. Moreover, the cell experiment results indicated that the significantly bioactive compounds possessed good safety to BV2 cells. It is worth pointing out that this is the first report on the application of an imidazo[1,2-a]quinoxaline skeleton as agricultural antifungal agent, and further studies on the structural optimization and target exploration are still underway in our laboratory. Overall, our findings may provide a theoretical basis for the future utilization of imidazo[1,2-a]quinoxaline scaffolds as novel fungicides in agriculture.

Experimental

All starting materials were obtained from commercial sources and used without further purification. Melting points were determined by the X-4 digital display micro melting point apparatus (Beijing Tech Instrument Co., Ltd). 1H NMR and 13C NMR spectra were recorded on Bruker Avance NEO 600 MHz and 150 MHz instruments, respectively, using TMS as the internal standard and CDCl3 or DMSO-d6 as the solvent. High-resolution mass spectra (HRMS) were carried out with an APEX II Bruker 4.7 T AS instrument.

Synthesis

See the “Supporting information” section for the synthetic methods of the target compounds.

Antifungal activity and spore germination assay

Antifungal activity assay [18]

The target compounds were screened in vitro for their antifungal activity against ten phytopathogenic fungi (Fusarium solani, Fusarium oxysporum, Botryosphaeria dothidea, Fusarium graminearum, Sclerotinia sclerotiorum, Valsa mali, Alternaria alternata, Pyricularia oryzae, Alternaria brassicae, and Botrytis cinerea) by using the mycelial growth rate method. Potato dextrose agar (PDA) medium was prepared in the flasks and sterilized. The target compounds were dissolved in DMSO before mixing with PDA, and the concentration of test compounds in the medium was fixed at 50 μg/mL. The medium was then poured into sterilized Petri dishes. The mycelia disks (4 mm) were inoculated in the center of the Petri dishes (three replicates for each treatment) and incubated at 27 ± 1 °C for 4 days. DMSO without any compounds mixed with PDA served as a control (the final concentration of DMSO < 0.5%). Hymexazol and chlorothalonil were used as positive controls. The radial growth of the fungal colonies were measured and the data were statistically analyzed. The inhibitory rate was calculated by the following formula: inhibition rate (%) = (C–T) × 100/(C–4 mm), where C represents the diameter of fungi growth on untreated PDA, and T represents the diameter of fungi on treated PDA. Finally, the linear regressions of inhibition rates (%) versus seven concentrations of some selected compounds, were obtained, and the EC50 values were calculated. Statistical analyses of the data were performed with GraphPad Prism 5.0.

Spore germination assay [19]

The V. mali was retrieved from the storage tube and cultured for 2 weeks at 27.5 °C on potato dextrose agar (PDA, Difco). Plates were then flooded with sterile distilled water, and conidia were scraped with a glass stick. Mycelial debris was removed by filtration through double-layer cheesecloth, and the spores were harvested and suspended in sterile distilled water containing 0.1% (v/v) Tween 20. Spores were counted using a hemocytometer and adjusted to 1.0 × 106 spores/mL.

Three concentrations (6.25 µg/mL, 12.5 µg/mL, and 25 µg/mL) of compound 5c and the control (0.5% DMSO) were separately tested for spore germination of V. mali. The samples were inoculated with spore suspension of V. mali containing 1.0 × 106 spores/mL. Aliquots of 10 μL of prepared spore suspension were placed on 96-hole plate in six copies. 96-hole plate containing the spores was incubated in a moisture chamber at 25 °C for 48 h. Each hole was then observed under the microscope for spore germination. The spore-generated germ tubes were enumerated, and the percentage of spore germination was calculated.

Cytotoxicity activity [21]

The cytotoxicity of the target compounds was detected by Cell Counting CCK-8 kit (CCK-8 assay). CCK-8 was based on the water-soluble tetrazolium salt WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-dinitrophenyl)-2H-tetrazole monosodium salt). The BV2 cells were seeded at a density of 1.5 × 104 cells per well in the 96-hole plate and incubated at 37 °C in an atmosphere of 5% CO2 for 24 h. After incubation, different concentrations of the target compounds were added and incubated for 24 h. 10 μL of CCK-8 reagent was added to each well and incubated for 1 h in the dark. The absorbance at 450 nm was measured by microplate reader. The untreated group was considered as the control. The data were analyzed by GraphPad Prism 5.0.

Supporting information

Spectral images of 1H-NMR, 13C-NMR and HRMS are provided in the Supporting Information Section.