Introduction

Plant pathogenic bacteria are considered as an important factor affecting crop yield and quality, leading to huge economic losses to farmers around the world each year [1,2,3]. For example, the Gram-negative bacteria of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac) are the causal agents of bacterial leaf blight (BLB) of rice and citrus canker diseases, respectively. BLB is one of the most destructive rice diseases, since the pathogen Xoo invades through the vascular system and then occupies the intercellular spaces of the parenchyma tissue, triggering annual yield losses of 10 ~ 50% in many rice-planting countries [4, 5]. In addition, the bacterium of Xac can be spreaded by the wind, rain, and touch, this pathogen enters plants via the stomata in leaves, the lenticels, and wounds [6, 7]. Furthermore, tobacco bacterial wilt is caused by the bacterium Ralstonia solanacearum (Rs), which is a soil-borne pathogen that enters the tobacco plant and spreads rapidly through the vascular system and gives rise to the browning of the xylem and lethal wilting [8, 9]. The employment of antibacterial agents to cope with these pathogens was regarded as a cost-effective approach to achieving the crop protection [10]. Bismerthiazol, a thiadiazole-thione derivative, has long been used as an agrobactericide for controlling rice leaf blight and citrus canker diseases since the 1970s. However, the long-term use of Bismerthiazol led to the reduced efficacies and ever-growing resistance of the pathogenic bacteria [11]. Taking these into consideration, the development of new agrobactericides with novel structure, high efficacy, and good environmental compatiblitiy has become an urgent task in the field of crop protection.

The quinazolinone skeleton is one of the most important nitrogen-containing heterocycles, which is discovered in more than 200 natural compounds [12]. Many quinazolinone derivatives exhibited a range of bioactivities, including antibacterial [13], antifungal [14], antiviral [15], antitumor [16], antimalarial [17], and anti-inflammatory effects [18] et al. Additionally, some of them have been successfully launched into the market as the commercialized pesticides and drugs, such as agrofungicide Fluquinconazole (Fig. 1), soporific drug Cloroqualone, muscle relaxant Afloqualone, anticancer drug Nolatrexed, antimalarial agent Febrifugine, and antiprotozoal agent Halofuginone. On the other hand, the isopropanol linkage is often utilized for constructing bioactive molecules, which can improve the biocompatibility of small molecules and easily establish extra hydrogen-bonding interactions with the relevant target proteins [19]. A few commercialized agents contain this linkage, including antifungal drugs Albaconazole/Fluconazole/Voriconazole and agrofungicide Prothioconazole. Furthermore, some compounds incorporating the linkage also demonstrated other biological activities, like antibacterial [3, 20, 21] and antiviral efficacies [22].

Fig. 1
figure 1

Some of the representative bioactive molecules containing the quinazolinone moiety, the isopropanol linkage, or the 1,2,4-triazole Schiff base moiety

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Compared with the 1,3,4-oxadiazole and 1,3,4-thiadiazole rings, the 1,2,4-triazole heterocycle has an unique advantage in the number of modifiable positions, not only in the 3- and 5-positions, but also in the 4-position. The introduction of substituents into 4-position of the 1,2,4-triazole ring can tune the bioactivity and physiochemical properties of the formed molecules [23]. For example, the 3,5-disubstituted-4-amino-1,2,4-triazole could be tranformed into the corresponding Schiff-base derivatives, displaying favorable antibacterial & antifungal effects [24,25,26]. Based on the above-mentioned considerations, we synthesized a series of new quinazolinone derivatives containing both 1,2,4-triazole Schiff base moiety and an isopropanol linkage (Fig. 2) using the molecular hybridization approach, and assessed their inhibitory activites in vitro against some important phytopathogenic bacteria and fungi in agriculture.

Fig. 2
figure 2

Design strategy for target compounds in this work

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Experimental

Instruments

All the chemicals were purchased from commercial suppliers and used without further purification (unless stated otherwise). Melting points were uncorrected and determined on a XT−4 binocular microscope (Beijing Tech Instrument Co., China). 1 H and 13 C NMR spectra were recorded on a JEOL-ECX 500 NMR spectrometer in DMSO-d6 at room temperature using TMS as an internal standard (s = singlet; d = doublet; t = triplet; m = multiple), chemical shift (δ) was reported in parts per million (ppm), and coupling constants were expressed in Hertz (Hz). HRMS-ESI spectra were measured by a Thermo Scientific Q Exactive series. Scanning electron microscopy (SEM) images were visualized and obtained using a Nova NanoSEM 450. The X-ray crystallographic data were collected based on an Agilent SuperNova area detector diffractometer with Mo-Ka radiation. All the strains of bacteria and fungi were provided by the Laboratory of Plant Disease Control at Guizhou University.

Synthesis of intermediate B

To an acetone solution (80 mL) containing quinazolin-4-one A [27] (1.0 g, 6.8 mmol) and anhydrous K2CO3 (1.9 g, 13.7 mmol), epichlorohydrin (4.1 mL, 51.3 mmol) was added dropwise. Next, the above mixture was heated under reflux for 24 h. After the removal of acetone under reduced pressure, the resultant residues were dissolved in ethyl acetate, washed with water, dried over anhydrous Na2SO4, and then evaporated to afford the white intermediate B. Yield: 61%, m.p. 99 − 100 °C. 1 H NMR (500 MHz, DMSO-d6) δ: 8.24 (s, 1 H), 8.14 (d, J = 10.0 Hz, 1 H), 7.80 (t, J = 5.0 Hz, 1 H), 7.65 (d, J = 10.0 Hz, 1 H), 7.53 (t, J = 5.0 Hz, 1 H), 4.28 (d, J = 10.0 Hz, 1 H), 4.05 (d, J = 10.0 Hz, 1 H), 2.78–2.76 (m, 1 H), 2.55 (s, 1 H), 2.47 (s, 1 H). 13 C NMR (125 MHz, DMSO-d6) δ: 160.8, 148.5, 148.4, 135.0, 127.8, 127.7, 126.7, 121.9, 49.7, 47.4, 45.7. ESI-HRMS m/z: [M + H]+ calcd for C11H11N2O2: 203.0815; found: 203.0810.

General procedures for the synthesis of target compounds E1−E31

Intermediate B (0.28 g, 1.4 mmol) and anhydrous NaHCO3 (0.14 g, 1.7 mmol) were added into an ethanol solution (10 mL) and stirred for 10 min, and then the appropriate 1,2,4-triazole Schiff base D [28,29,30] (1.0 mmol) was introduced. The above reaction mixture was continuously stirred at room temperature for 12 h. Next, the mixture was poured into cold water, and the formed precipitate was filtered, washed with water and dried to generate target compounds E1 − E31.

3-(3-((4-((3-bromobenzylidene)amino)-5-methyl-4H-1,2,4-triazol-3-yl)thio)-2-hydroxypropyl)quinazolin-4(3H)-one (E12). Yield: 95%, light-yellow solid, m.p. 155–157 °C. 1 H NMR (500 MHz, DMSO-d6) δ: 8.84 (s, 1 H), 8.19 (s, 1 H), 8.12–8.08 (m, 2 H), 7.91 (d, J = 10.0 Hz, 1 H), 7.81–7.77 (m, 2 H), 7.63 (d, J = 10.0 Hz, 1 H), 7.52–7.49 (m, 2 H), 5.65 (d, J = 5.0 Hz, 1 H), 4.27–4.24 (m, 1 H), 4.08 (s, 1 H), 3.84–3.79 (m, 1 H), 3.36–3.33 (m, 1 H), 3.28–3.23 (m, 1 H), 2.44 (s, 3 H). 13 C NMR (125 MHz, DMSO-d6) δ: 163.0, 161.0, 149.6, 149.2, 148.5, 147.8, 135.9, 134.8, 131.9, 131.8, 130.9, 128.2, 127.6, 127.4, 126.6, 122.9, 122.1, 67.3, 51.1, 37.2, 11.8. ESI-HRMS m/z: [M + H]+ calcd for C21H20N6O2BrS: 499.0546; found: 499.0551.

Results and discussion

Synthesis and spectral analysis

Synthetic routes of target compounds E1−E31 were depicted in Scheme 1. In brief, quinazolinone A was firstly treated with epichlorohydrin in acetone with anhydrous K2CO3 as a catalyst to afford the oxirane-appended quinazolinone B in 61% yield. Once intermediate B in hand, it was then reacted with the appropriate 1,2,4-triazole Schiff base D in the EtOH-NaHCO3 system to give the desired compounds E1−E31 in 52−95% yields. All the target compounds were fully characterized by 1 H NMR, 13 C NMR, and HRMS. Taking compound E12 as an example (dissolved in DMSO-d6), a singlet at 8.84 ppm in its 1 H NMR spectrum was assigned to the imine CH proton signal of the Schiff base moiety. Additionally, 2-position CH signal of the quinazolinone backbone was observed at 8.19 ppm as a well-defined singlet. The resonance at δ = 5.65 ppm was attributed to the hydroxyl proton from the isopropanol linkage. Furthermore, the signal of the methyl group on the 1,2,4-triazole ring appeared at 2.44 ppm. In its 13 C NMR spectrum, four of the diagnostic aliphatic carbon signals were found at 67.3, 51.1, 37.2, and 11.8 ppm, respectively. Finally, compound E12 exhibited an intense peak at m/z = 499.0551 in its mass spectrometry, assigned to the [M + H]+ species.

Scheme 1
scheme 1

Synthetic route for target compounds E1−E31

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Crystal structure analysis

Fortunately, single crystals of compound E12 suitable for X-ray diffraction analysis (Fig. 3) were grown by slow evaporation of its methanolic solution at room temperature. Clearly, the imine bond adopted a trans-conformation in the solid state. Crystallographic parameters for this compound: colorless crystal, C21H19BrN6O2S, Mr = 499.4, monoclinic, space group C1c1; a = 5.0265 (6) Å, b = 15.7022 (17) Å, c = 26.915 (3) Å, α = 90º, β = 90.916 (10)º, γ = 90º, V = 2124.0 (4) Å3, T = 100 K, Z = 4, Dc = 1.562 g/cm3, F (000) = 1016.0, reflections collected/independent reflections = 8308/3497, goodness of fit on F2 = 1.076, R1 = 0.0550, wR2 = 0.1002.

Fig. 3
figure 3

Crystal structure of compound E12 (CCDC 1831397)

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In vitro antibacterial activity

In vitro antibacterial effects of compounds E1 − E31 were evaluated against three bacteria Xac, Xoo, and Rs using the classical turbidimetric method [31,32,33]. Meanwhile, the commercialized bactericides Bismerthiazol (BMT) and Thiodiazole copper (TDC) were used as the positive control agents. As listed in Table 1, compounds E10, E13, E20, and E23 exhibited similar anti-Xac efficacies to BMT at 200 µg/mL, having the inhibition rates of 94.3%, 100%, 100%, and 100%, respectively. In addition, some compounds were found to possess higher antibacterial effects against Xoo, relative to BMT. For example, compounds E4, E10, and E13 demonstrated the inhibition rates exceeding 90% towards this pathogen at 200 µg/mL, much better than BMT (69.2%).

Table 1 In vitro antibacterial activities of compounds E1−E31 against Xac, Xoo, and Rs
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Inspired by the preliminary antibacterial results, EC50 (half-maximal effective concentration) values of some of the compounds were further measured according to the serial dilution method (namely 200, 100, 50, 25, and 12.5 µg/mL). As listed in Table 2, compounds E2, E10, E13, E20, E22, and E23 had EC50 values of 61.1, 31.9, 31.5, 47.7, 44.8, and 45.5 µg/mL towards Xac, respectively, better than control BMT (62.8 µg/mL). Moreover, compounds E3, E4, E10, E13, and E22 displayed EC50 values of 55.4, 39.5, 49.5, 53.5, and 57.4 µg/mL against Xoo (Table 3), respectively, superior to control BMT (94.5 µg/mL). Notably, compound E4 was 2.4-fold more effective than BMT in inhibiting the bacterium Xoo in terms of their EC50 values. As far as the pathogen Rs was concerned, compounds E2, E4, E7, E8, and E20 showed better inhibition effects than control TDC (217.4 µg/mL), having EC50 values of 81.5, 83.8, 91.6, 75.4, and 94.2 µg/mL (Table 4), respectively.

Table 2 EC50 values of some compounds against the pathogen Xac
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Table 3 EC50 values of some compounds against the pathogen Xoo
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Table 4 EC50 values of some compounds against the pathogen Rs
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SEM Observation of the cell morphology

To examinethe effects of compound E4 on the morphology of Xoo cells, scanning electron microscopy (SEM) experiments were carried out. After comparison with the blank control group (Fig. 4a), the Xoo cells changed from the plump and regular appearances into the contracted and corrugated surfaces after treatment with 50 µg/mL of compound E4 (Fig. 4b). Whilst the tested concentration was further increased to 100 µg/mL, the significant cell surface deformation even cell rupture were observed (Fig. 4c). These findings showed that this compound probably exerted its anti-Xoo effects via causing the cell membrane damage.

Fig. 4
figure 4

SEM images of the pathogen Xoo after treatment with different concentrations of compound E4: (a) 0 µg/mL, (b) 50.0 µg/mL, and (c) 100.0 µg/mL

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In vitro antifungal activity

In vitro antifungal activities of compounds E1−E31 were also assessed against six phytopathogenic fungi using the mycelial growth rate method [34, 35], including Gibberella zeae, Pellicularia sasakii, Phytophthora infestans, Verticillium dahliae, Fusarium oxysporum, and Sclerotinia sclerotiorum. As summarized in Table 5, some compounds exhibited moderate fungicidal effects against certain fungi at 50 µg/mL. For instance, compounds E22 and E30 possessed the inhibition rates of 52.7% and 54.6% against G. zeae, respectively, higher than control agent Hymexazol (48.4%). Moreover, the inhibition rates of compounds E8, E22, and E30 against S. sclerotiorum were all higher than 50%. Last, compounds E22 and E30 showed an inhibitory rate greater than 55% towards the fungus P. sasakii.

Table 5 In vitro antifungal activities of compounds E1−E31 at 50 µg/mL
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Structure-activity relationships (SAR) analysis of target compounds against the pathogenic bacteria

Based on the antibacterial results of target compounds listed in Tables 1, 2, 3 and 4, substitution patterns (including the type, position, and numbers of the substituents) of the terminal phenyl ring had a profound effect on their antibacterial effects. Some preliminary conclusions could be drawn as follow: (a) For the inhibition of the pathogen Xoo, the substitution at the 4-position of the phenyl group was conducive to the activity (relative to their 2-/3-position isomers), as exemplified by several best-performing compounds E4 (4-CH3, EC50 = 39.5 µg/mL), E10 (4-Cl, EC50 = 49.5 µg/mL), and E13 (4-Br, EC50 = 53.5 µg/mL). The same rules also held true in the case of inhibiting the pathogen Xac, like compounds E10 (4-Cl, EC50 = 31.9 µg/mL) and E13 (4-Br, EC50 = 31.5 µg/mL); (b) Among all the substituents, compound E4 bearing a weakly electron-donating 4-CH3 group was identified as the optimal compound for inhibiting the bacterium Xoo, being 2.4-fold more effective than control BMT; (c) Moreover, mono-substituted compounds exhibited higher antibacterial effects than di- and tri-substituted compounds. Three of the most active compounds towards the tested three bacteria (namely compounds E13, E4, and E8 from Tables 2, 3 and 4) all conformed to this rule.

Conclusions

To summarize, a class of new quinazolinone derivatives bearing the 1,2,4-triazole Schiff base moiety and an isopropanol linker were prepared and assessed for their agricultural antimicrobial activities. The bioassay results indicated that many compounds displayed good antibacterial activities in vitro against the tested phytopathogenic bacteria. In particular, compounds E4 and E10 could significantly inhibit the bacterium Xoo (with EC50 values of 39.5 and 49.5 µg/mL, respectively), far better than the commercialized Bismerthiazol (EC50 = 94.5 µg/mL). Additionally, some compounds also exhibited moderate fungicidal activities in vitro against the fungus G. Zeae, such as compounds E22 and E30. In a word, this class of compounds can be considered as the promising lead compounds for developing more effective agricultural bactericides in the future.