2.1 Instruments and chemicals

The target compounds were characterized, by using JEOL-ECX500 nuclear magnetic resonance spectrometer (Tokyo, Japan) and Thermo Scientic Q Exactive mass spectrometer (Missour, America). The K_d values of compounds to TMV CP were determined by using NanoTemper Monolith NT.115 microscale thermophoresis instrument (München, Germany). All solvents were purchased from Tianjin Zhi Yuan Regent Co., Ltd. (Tianjin, China). All reagents were purchased from Shanghai Titan chemical Co., Ltd. (Shanghai, China) and Adamas Reagent, Ltd. (Shanghai, China). Enzyme activity kits were purchased from Suzhou Keming Biotechnology Co., Ltd (Jiangsu, China).

2.2 Synthesis

2.2.3

2.2.3 General synthesis of target compounds T1 − T25

As shown in Scheme 1, the target compounds T1 − T25 were synthesized with the method, which had been modified by us later, from the literature (Hagar, et al., 2016). Intermediate 2 (1.96 mmol), intermediate 3 (2.36 mmol), and K₂CO₃ (3.93 mmol) were added to 20 mL of DMF, reacted at 90 ℃ for 6 h. The reaction system was poured into 500 mL of ice water, and a white solid was precipitated. After being held stationarily for 2 h, it was filtered under reduced pressure and dried to obtain a crude product, which was separated and purified by column chromatography later (ethyl acetate: petroleum ether = 2:1, v/v).

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Scheme 1

Synthetic route of target compounds T1 − T25.

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2.3 Biological assays

2.4 Microscale thermophoresis experiment

The K_d values of some target compounds and NNM to TMV CP were determined with the method reported in the literature. (Li et al., 2019; Wu, et al., 2021). DMSO-dissolved drug was formulated into 16 concentration gradients at room temperature. Compounds were tested for K_d values on NanoTemper Monolith NT.115 after adding equal amounts of TMV CP to groups of each concentration.

2.5 Molecular docking

Molecular docking is a process in which receptors and substrates recognize each other through energy matching and geometric matching. Molecular docking is very important in both supramolecular architecture and drug design (José, et al., 2020; Gabriel, et al., 2021). The molecular docking of compound T15 and NNM to TMV CP were carried out with the method reported in the literature (Chen, et al., 2021). Molecular docking of the TMV CP (PDB code: 1EI7) with the structure of the selected compound was performed on Discovery Studio.

2.6 Determination of chlorophyll content

According to the method in the literature, the compound T15 with better protective activity was selected for the determination of the change of chlorophyll content (Gan, et al., 2017). The virus-inoculated tobacco leaves were ground in liquid nitrogen to pulverize. A mixed solution of ethanol and acetone was added and allowed to be held stationarily for 1 h in the dark. The absorbance at 645 nm and 663 nm were measured separately. The chlorophyll content was calculated according to the following formula. $$C_a\left(\mathit{mg}/L\right)=9.784O{D}_{663}-0.990O{D}_{645}$$ $$C_b\left(\mathit{mg}/L\right)=21.426O{D}_{645}-4.650O{D}_{663}$$ $$C_t\left(\mathit{mg}/L\right)=C_a+C_b=5.134O{D}_{663}-20.643O{D}_{645}$$ $$\mathit{chlorophyll}content\left(\mathit{mg}/g\right)=\frac{C\left(\mathit{mg}/L\right)\times thevolumeofextracts\left(L\right)}{\mathit{sample}freshweight\left(g\right)}$$

2.7 Determination of defense enzyme activity

Peroxidases (POD) are involved in plant development, stress responses and hormone signaling. It is closely related to the photosynthesis and respiration of plants (Wu, et al., 2019). The compound T15 was tested for the activity of the defense enzyme according to the method in the literature (Zhou, et al., 2021). The virus-inoculated tobacco leaves were added to liquid nitrogen and ground to pulverize. The POD content of the leaves was determined with the method of enclosed instructions of testing kit.

3.1 Structural characterization of some target compounds

2-((3-((5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-chromen-3-yl)oxy)propyl)thio)-6-fluoro-3-phenylquinazolin-4(3H)-one (T5). White solid, m.p. 185.6–187.3 °C, yield, 27%; ¹H NMR (500 MHz, Chloroform-d) δ 7.83 (dd, J = 8.1, 2.9 Hz, 1H, Ph − H), 7.55 – 7.53 (m, 1H, Ph − H), 7.53 – 7.51 (m, 3H, Ph − H), 7.41 (td, J = 8.3, 2.9 Hz, 1H, Ph − H), 7.28 (s, 2H, Ph − H), 7.28 – 7.25 (m, 2H, Ph − H), 6.48 (d, J = 2.2 Hz, 1H, Ph − H), 6.34 (d, J = 2.3 Hz, 1H, Ph − H), 4.11 (t, J = 6.2 Hz, 2H, −O − CH₂CH₂CH₂ − S − ), 3.95 (s, 3H, Ph − OCH₃), 3.89 (s, 3H, Ph − OCH₃), 3.88 (s, 3H, Ph − OCH₃), 3.87 (s, 6H, Ph − OCH₃), 3.26 – 3.22 (m, 2H, −O − CH₂CH₂CH₂ − S − ), 2.11 (p, J = 6.3 Hz, 2H, −O − CH₂CH₂CH₂ − S − ); ¹³C NMR (126 MHz, Chloroform-d) δ 174.02, 167.85, 164.11, 161.07, 158.87, 156.67, 153.04, 152.77, 144.65, 140.48, 139.92, 135.78, 126.05, 123.24, 123.05, 120.97 (d, J = 8.4 Hz), 112.17, 111.99, 109.44, 105.85, 95.94, 92.48, 71.09, 61.11, 56.54, 56.39, 55.94, 29.73, 29.43; ¹⁹F NMR (471 MHz, Chloroform-d) δ −114.11; HRMS (ESI) calcd for C₃₇H₃₄O₉N₂FS [M + H]⁺: 701.19636, found 701.19629.

2-((4-((5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-chromen-3-yl)oxy)butyl)thio)-6-methyl-3-phenylquinazolin-4(3H)-one (T15). White solid, m.p. 196.9–198.2 °C, yield, 56%; ¹H NMR (500 MHz, Chloroform-d) δ 7.98 (s, 1H, Ph − H), 7.53 – 7.48 (m, 4H, Ph − H), 7.45 (d, J = 8.3 Hz, 1H, Ph − H), 7.31 (s, 2H, Ph − H), 7.29 – 7.26 (m, 2H, Ph − H), 6.47 (d, J = 2.3 Hz, 1H, Ph − H), 6.33 (d, J = 2.3 Hz, 1H, Ph − H), 4.04 – 4.00 (m, 2H, −O − CH₂CH₂CH₂CH₂ − S − ), 3.94 (s, 3H, Ph − OCH₃), 3.90 – 3.87 (m, 12H, Ph − OCH₃), 3.19 – 3.14 (m, 2H, −O − CH₂CH₂CH₂CH₂ − S − ), 2.44 (s, 3H, Ph − CH₃), 1.83 (p, J = 3.0, 2.6 Hz, 4H, −O − CH₂CH₂CH₂CH₂ − S − ); ¹³C NMR (126 MHz, Chloroform-d) δ 174.07, 164.04, 162.06, 161.09, 158.85, 156.30, 153.00, 152.60, 145.98, 140.68, 139.88, 136.21, 136.07, 135.80, 129.90, 129.71, 129.27, 126.63, 126.15, 119.58, 109.47, 105.84, 95.87, 92.46, 71.82, 61.12, 56.52, 56.38, 55.92, 32.26, 29.54, 25.43, 21.35; HRMS (ESI) calcd for C₃₉H₃₉O₉N₂S [M + H]⁺: 711.23708, found 711.23706.

3.2 Antibacterial activity of the target compounds T1 − T25

The results of Table 1 show that most of the compounds had a certain inhibitory effect on Xac and Xoo when the concentration of compound was 100 μg/mL. In particular, compounds T1 and T5 exhibited good inhibitory activity against Xac, with the inhibition rates of 87.8 and 89.1% respectively, which were superior than those of commercial drugs BC (60.2 %) and TC (53.6 %). The inhibitory activity against Xoo of compounds T2, T5, T15 and T16, with inhibition rates of 79.6, 82.1, 89.1 and 77.7% respectively, had better advantages than those of BT (54.0%) and TC (54.2%).

Based on the activity screening results, the EC₅₀ values of some target compounds were tested, whose results are displayed in Table 2. The EC₅₀ values of compounds T1 and T5 against Xac were 23.6 and 22.4 μg/mL respectively, which were less than the control drugs BT (62.2 μg/mL) and TC (97.5 μg/mL). The EC₅₀ values of compounds T5 and T15 against Xoo were 18.5 and 21.6 μg/mL respectively, which were much better than BT (63.7 μg/mL) and TC (86.1 μg/mL). In particular, compound T5 has good inhibitory activity against both plant bacteria.

3.3 Antiviral activity of the target compounds T1 − T25

The test results of antiviral activity of the target compounds T1 − T25 are shown in Table 3. When the compound concentration was 500 μg/mL, compounds T2 and T15 had good curative activity against TMV, and the inhibition rates were 68.1 and 67.3% respectively, which were higher than the commercial drug NNM (54.1%). The protective activity against TMV of compounds T2, T11 and T15, with the inhibition rates of 64.3, 68.2 and 64.3 % respectively, were better than the commercial drug NNM (57.1%). Fig. 4 reveals the morphology of compound T15 against TMV in vivo.

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Fig. 4

Tobacco leaf morphology effects of T15 and NNM against TMV in vivo. The left side of each leaf was drug-treated, the right side of each leaf was untreated with drug.

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The EC₅₀ values of compounds T2, T11, T15 and NNM are summarized in Table 4. Compounds T2, T11 and T15 have good curative activity against TMV, with EC₅₀ values of 190.0, 227.3 and 166.9 μg/mL respectively, which were lower than the commercial drug NNM of 319.7 μg/mL. In terms of protective activity, the EC₅₀ values of compounds T2, T11 and T15 against TMV were 228.7, 178.9 and 130.5 μg/mL respectively, which were lower than NNM (341.3 μg/mL).

3.4 Structure-activity relationship

Referring to the previous research reports on quinazolinones, most of the changes of substituents are carried out at the 6-position. In addition, electron withdrawing groups were often used as substituents. (Zu, et al., 2021). From the results of antibacterial activity, the compounds with − CH₃ and − F at the 6th substitution position have better antibacterial activity. For example, compounds T1, T5 and T15 exhibited inhibitory activities more than 80% on Xac and Xoo at the tested concentration of 100 μg/mL. The results of antiviral activity showed that some compounds containing − Cl and − CH₃ had better inhibitory activity on TMV. Compared with the previously reported compounds (Liu, et al., 2021), the antibacterial activity of some of the compounds with n = 3 was improved after the introduction of thioether, and the anti-TMV activity was also generally improved.

3.5 Microscale thermophoresis

The K_d value shows the affinity of the interaction between two molecules. The smaller the value is, the stronger the affinity of the two molecules is (Wang, et al., 2020). Fig. 5 shows that the binding constants (K_d) of compounds T2, T11, T15 and NNM to TMV CP. Among them, the K_d value of compound T15 to TMV CP was 0.024 ± 0.006 μM, which was smaller than that of compounds T2 (0.052 ± 0.016 μM) and T11 (0.054 ± 0.023 μM). However, the above data are extremely small compared with NNM (8.491 ± 2.027 μM). It shows that the binding abilities of compounds T2, T11 and T15 to TMV CP are much greater than that of the control drug NNM. These results were positively correlated with EC₅₀ values.

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Fig. 5

The K_d values of componds T2, T11, T15 and NNM to TMV CP.

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3.6 Molecular docking

The length of hydrogen bonds is negatively correlated with the binding force, and the number of hydrogen bonds is positively correlated with the binding force (Wang, et al., 2021).The pictures in Fig. 6 validate the binding ability of the compound T15 and NNM to TMV CP. Compound T15 (A, a) interacted with surrounding residues SER A:49, SER A:15, ARG A:46, GLN A:45, GLU A:50 and TRP A:52 in the TMV CP active pocket via conventional hydrogen bond, carbon hydrogen interacts with π-alkyl. Compound T15 formed two strong hydrogen bonds with residues SER A:49 (1.92 Å) and SER A:15 (2.20 Å). The commercial drug NNM (B, b), interacted with surrounding residues THR B:237, ALA B:240 and GLN B:234 in the active pocket of TMV CP via π-donor hydrogen bond, π-alkyl and conventional hydrogen bond. The naked amino group in the NNM structure formed a traditional hydrogen bond (2.30 Å) with residue SER B:215. Based on the above analysis, it was found that compound T15 was superior to commercial drug NNM in terms of the number of action sites and hydrogen bond length. Compared with NNM, T15 has a stronger binding ability than that of TMV CP, which has the potential for being a more effective antiviral agent.

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Fig. 6

Molecular docking of compound T15 (A, a) and NNM (B, b) to TMV CP.

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3.7 Effects of compound T15 on chlorophyll content in tobacco

Chlorophyll is an important substance for plants to carry out photosynthesis. It is reported that plant diseases induce a decrease in chlorophyll content in plants, resulting in reduced crop yields (Mandal, et al., 2009). The following conclusions could be drawn from Fig. 7: firstly, the chlorophyll content in CK group increased slightly on the third day, but decreased gradually in the later period as time went by. Secondly, CK + TMV group showed a slight increase in chlorophyll content after the TMV inoculation on first day, followed by a decreasing trend. Thirdly, the chlorophyll content in T15 group increased significantly on the first day after TMV inoculation, and decreased in the later period. Finally, the overall change trend of the T15 + TMV group was similar to that of the CK group, but its chlorophyll content increased significantly on the third day, which showed that the compound T15 caused the chlorophyll content in the plant to reach its peak at this time, and the chlorophyll content gradually decreased with plant aging at a later stage.

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Fig. 7

Changes of chlorophyll content in tobacco after compound T15 treatment.

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3.8 Effects of compound T15 on peroxidase (POD) content in tobacco

The elevated POD content helps to scavenge free radicals in plants to improve disease resistance (Gan, et al., 2021). The changes in peroxidase (POD) content in tobacco after treatment with compound T15 are displayed in Fig. 8. The POD content of each group gradually increased over time during the 7 d of TMV inoculation. In general, POD content in CK + TMV and T15 + TMV groups increased significantly on the fifth and seventh days compared with CK and T15 groups. In addition, POD contents of T15 group and T15 + TMV group were higher than that of group without compound T15 treatment. The above analysis showed that compound T15 could make peroxidase increase to a certain extent and enhance plant disease resistance.

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Fig. 8

Effects of compound T15 on POD content in tobacco.

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