The Discovery of Novel Ferulic Acid Derivatives Incorporating Substituted Isopropanolamine Moieties as Potential Tobacco Mosaic Virus Helicase Inhibitors

Target-based drug design, a high-efficiency strategy used to guide the development of novel pesticide candidates, has attracted widespread attention. Herein, various natural-derived ferulic acid derivatives incorporating substituted isopropanolamine moieties were designed to target the tobacco mosaic virus (TMV) helicase. Bioassays demonstrating the optimized A19, A20, A29, and A31 displayed excellent in vivo antiviral curative abilities, affording corresponding EC50 values of 251.1, 336.2, 347.1, and 385.5 μg/mL, which visibly surpassed those of commercial ribavirin (655.0 μg/mL). Moreover, configurational analysis shows that the R-forms of target compounds were more beneficial to aggrandize antiviral profiles. Mechanism studies indicate that R-A19 had a strong affinity (Kd = 5.4 μM) to the TMV helicase and inhibited its ability to hydrolyze ATP (50.61% at 200 μM). Meanwhile, A19 could down-regulate the expression of the TMV helicase gene in the host to attenuate viral replication. These results illustrate the excellent inhibitory activity of A19 towards the TMV helicase. Additionally, docking simulations uncovered that R-A19 formed more hydrogen bonds with the TMV helicase in the binding pocket. Recent studies have unambiguously manifested that these designed derivatives could be considered as promising potential helicase-based inhibitors for plant disease control.


Introduction
The ever-increasing malignant tobacco mosaic virus (TMV) is a cardinal agricultural and horticultural plant pathogenic virus that leads to enormous yield losses and cripples the quality of crops [1][2][3][4]. Pesticides are consistently viewed to be one of the most effective tools for preventing and controlling causative agents while reducing labor costs. Nonetheless, with the evolution and resistance development of viruses, obsolete pesticides are incapable of maintaining their efficacy sustainably [5,6]. Therefore, it is highly imperative and urgent to continuously exploit novel pesticidal candidates which empower distinct mechanisms of action to address this complex dilemma.
Helicases are present in virtually all living organisms, including eukaryotes, yeast, bacteria, and viruses. Typically, they act as molecular motors, employing the energy generated by hydrolyzing nucleoside triphosphates to break the hydrogen bonds between base pairs, thereby unwinding double-strand nucleic acid in preparation for replication [7][8][9][10]. Based on substrate specificity, helicases can be divided into two categories: DNA helicases and RNA helicases. DNA helicases are essential for DNA replication, recombination, transcription, and repair. RNA helicases regulate RNA structure and are involved in various aspects of RNA metabolism, such as transcription, translation, RNA splicing, ribosome assembly, RNA editing, and RNA degradation [11][12][13][14]. As a result, helicases-targeted 2 of 23 drug design has attracted growing interest from researchers. Currently, the design of viral inhibitors is mainly based on RNA helicases. Frick and colleagues have screened a series of antiviral candidates with potential application prospects targeting virus helicase [15][16][17][18]. Shah et al. reviewed the nature-derived phyllaemblicin with inhibitory activity toward helicase NSp13 activity in SARS-CoV-2 [19]. Greger reported promising flavonoids that inhibit the synthesis of proteins by interacting with an ATP-dependent DEAD-box RNA helicase [20]. While the overwhelming majority of studies mainly focused on the research of animal viruses, inspired by these investigations, targeting plant viral RNA helicase may provide valuable orientations for the discovery of potential viral inhibitors.
Naturally occurring products possess structural diversity, low mammalian toxicity, environmental friendliness, specificity to target species, unique modes of action, and can be deployed as ideal pesticide scaffolds [21,22]. Ferulic acid (3-methoxy-4-hydroxycinnamic acid), as an active ingredient, is widely distributed in Ferula ferulae, Angelica sinensis, Ligusticum wallichii, Cimicifuga foetida, and other Chinese herbal medicine with a wide range of medicinal value. Undoubtedly, ferulic acid (FA), together with its analogs (Figure 1), exhibits broad-spectrum medicinal or pesticide bioactivities, such as antifungal, antibacterial, antioxidant, and herbicidal activity [23][24][25][26][27]. More importantly, research showed that FA [28][29][30] and FA analogues [31,32] possess potential virus helicase inhibitory activity, along with an excellent antiviral profile (cold viruses, respiratory syncytial virus, plant viruses, etc.) [28,33]. On the other hand, privileged versatile isopropylamine fragments have the functions of facilitating target scaffold flexibility, transforming water solubility, and enhancing ligand-receptor H-bond interactions, and they have been wildly applicable in pesticide design [34,35]. tory activity toward helicase NSp13 activity in SARS-CoV-2 [19]. Greger reported promising flavonoids that inhibit the synthesis of proteins by interacting with an ATP-dependent DEAD-box RNA helicase [20]. While the overwhelming majority of studies mainly focused on the research of animal viruses, inspired by these investigations, targeting plant viral RNA helicase may provide valuable orientations for the discovery of potential viral inhibitors.
To excavate novel plant viral helicase inhibitors in view of the above considerations, a series of FA derivatives containing different substitutions of isopropanolamine fragments were successfully synthesized and screened for anti-TMV activities. Subsequently, the circular dichroism (CD) spectrum, microscale thermophoresis (MST), and ATPase activity analysis were applied to explore the preliminary mechanism of target compounds with the TMV helicase and simulated by molecular docking and density functional theory (DFT) calculation. Additionally, quantitative real-time polymerase chain reaction (RT-qPCR) experiments were conducted to assess potential inhibition of title compounds for TMV helicase gene expression. Moreover, pot experiments were conducted to further investigate the effect of target compounds on TMV propagation in the plant Nicotiana benthamiana. To explore the multifunctional applications of these compounds in agriculture, corresponding bioassays against phytopathogenic bacteria were also evaluated. To excavate novel plant viral helicase inhibitors in view of the above considerations, a series of FA derivatives containing different substitutions of isopropanolamine fragments were successfully synthesized and screened for anti-TMV activities. Subsequently, the circular dichroism (CD) spectrum, microscale thermophoresis (MST), and ATPase activity analysis were applied to explore the preliminary mechanism of target compounds with the TMV helicase and simulated by molecular docking and density functional theory (DFT) calculation. Additionally, quantitative real-time polymerase chain reaction (RT-qPCR) experiments were conducted to assess potential inhibition of title compounds for TMV helicase gene expression. Moreover, pot experiments were conducted to further investigate the effect of target compounds on TMV propagation in the plant Nicotiana benthamiana. To explore the multifunctional applications of these compounds in agriculture, corresponding bioassays against phytopathogenic bacteria were also evaluated.

Chemistry and Antiviral Activity In Vivo
In view of pesticidal candidate exploration, nature-derived low-cost structures containing elevated potency biological activity with a brief synthetic route can be seen as practical. The designed synthetic route of title derivatives is depicted in Figure 2. In short, the starting material FA was esterified by methanol to provide intermediate 1. Then, through substitution with epibromohydrin, the crucial intermediate 2 was successfully prepared. After that, through a typically ring-opening reaction between intermediate 2 with substituents containing different heteroatoms, title FA-originated derivatives incorporating disparate substitutions of isopropanolamine fragments (A 1 -A 19 ) were afforded. All molecular structures were confirmed by NMR and HRMS (Supporting Information). In this bioassay, a classical half leaf-inoculation approach was employed to validate the antiviral profile of A 1 -A 19 toward TMV in vivo, with the starting material FA and commercial virucide ribavirin (Ri) used as controls for comparison. The synthesized target compounds (A 1 -A 19 ) of antiviral results are shown in Table 1. It could be found that various heteroatom substituted molecules exhibited divergent degrees of biological activities (focused on curative effect). Among them, A 3 and A 4 displayed higher and the highest antiviral efficacy, respectively, as follows: A 4 (R = 4-(4-chlorophenoxy)anilino, 43.8%) > Ri (42.1%) > A 3 (R = 3-phenoxyanilino, 38.6%) > A 11 (R = (2-chlorophenyl)thio, 36.1%) > A 1 (R = 2-hydroxypropylamino, 32.7%) ≈ FA (31.4%) > A 5 (R = methoxyl, 10.3%) at 500 µg/mL, indicating that the antiviral capacity of target compounds obtained by the ring opening of nitrogen-containing substituents was evidently higher than that substituted by oxygen or sulfur. Meaningfully, compared with the starting material, target FA derivatives formed by the ring opening of the nitrogen-containing group had the potential to confer enhanced biological activity. Based on this outcome, the noncyclic amine (A 13 ) and cyclic amine (A 14 ) fragments were introduced into the target structure to further evaluate the effect of the substituents on the biological activity of the molecular backbone. The therapeutic activity of piperidine substitution was generally higher than that of aniline and fatty amine, such as A 14 (R = 2-methylpiperidyl, 41.5%) > A 13 (R = diethylin, 28.3%) > A 2 (R = 4-chlorophenylamino, 19.2%) at 500 µg/mL. This result shows that the introduction of cyclic amine may refurbish the designed molecular antiviral potency. Therefore, some target compounds containing six-membered heterocyclic structures were derived (A 15 -A 19 ). As shown in Table 1, compared with commercial Ri, the applausive antiviral abilities of A 16 and A 19 were obtained after incorporating methylpiperidyl or morpholine into the target structure. Moreover, the position of the methyl group on the piperidine ring also possessed a definite effect on the final anti-TMV effectiveness. The order is as follows: A 16 (R = 4-methylpiperidyl, 46.5%) > A 15 (R = 3-methylpiperidyl, 43.1%) > Ri (42.1%) ≈ A 14 (R = 2-methylpiperidyl, 41.5%) at 500 µg/mL, suggesting that the methyl group at the fourth position could more efficiently facilitate empower bioactivity. Additionally, when replacing the methyl of piperidyl by the ethyl formate group, the relevant A 17 and A 18 (21.7% and 27.1% at 500 µg/mL, respectively) had a significantly declined ability against TMV, demonstrating that the ester group with relatively steric hindrance and dipole interactions was unconducive to activity. Interestingly, when the heteroatom on the six-membered ring was increased, i.e., when piperidine was replaced by morpholine, the obtained A 19 showed the strongest antiviral competence (62.7% at 500 µg/mL).   To explore the structural diversity of substituents based on piperidine structure and its influence on activity, different substituted piperazines were incorporated into the target structure under similar synthesis conditions ( Figure 3). Bioassay results indicate that the ultimate activities (focused on curative effect) showed a marked divergence ( Table 2). When the alkyl substituted piperazine was introduced to the molecule, the corresponding A 20 (R = 1-ethylpiperazinyl, 56.9%) and A 21 (R = 1-isopropylpiperazinyl, 45.2%) showed comparable antiviral potential to Ri at 500 µg/mL. This variation caused an increase in the hydrophobic property and sterically hindered the group of alkyl-substituted piperazine with slightly quenched antiviral activity. When the phenyl-substituted piperazine was constructed on the target structure, A 22 and A 23 (20.1% and 31.6% at 500 µg/mL) only showed weak efficacy, demonstrating that a rigid, steric, and unsaturated alkane directly substituted with nitrogen on piperazine was unfavorable for molecular bioactivity. On the other hand, the introduction of electron-donating groups at the ortho or meta position of the benzyl group in piperazine was beneficial to curative activity, e.g., A 31 (R = 2-methylbenzyl, 61.7%) > A 30 (R = 3-methylbenzyl, 51.1%) > A 24 (R = benzyl, 44.6%) at 500 µg/mL. Additionally, after introducing electron-withdrawing groups (-NO2, -F, -CI) at the ortho or meta position of the benzyl group in piperazine, the obtained A 25 -A 28 displayed moderate to weak activity (28.7-47.3%), suggesting that the electron-donating group located at the ortho or meta position of the benzyl group could block or scarcely contribute to target compound bioactivity. However, when introducing an electron-withdrawing group at the para position of the benzyl group, it could endow enhanced antiviral capacity, such as 28.7%) at 500 µg/mL. To accurately assess the antiviral efficacy of these compounds, typical compounds with moderate to strong primary screening activity were selected for different concentrations in the in vivo test (Figure 4  Since these compounds all have a unique chiral center, the differences in configuration may also often affect molecule biological activity in the host. As a result, to investigate the influence of absolute configuration of title molecules on overall antiviral activity or target receptor bioactivity, different antiviral levels of derivatives R-A 16 , S-A 16 , R-A 19 , S-A 19 , R-A 20 , S-A 20 , R-A 29 , and S-A 29 were synthesized ( Figure 5). Their antiviral curative activities (Table 3) presented the following order: S-forms (45.7%, S-A 16 ) < S-forms (49.9%, S-A 20 ) < R-forms (55.1%, R-A 16 ) ≈ S-forms (55.4%, S-A 19 ) < R-forms (58.0%, R-A 20 ) < R-forms (67.5%, R-A 19 ) at 500 µg/mL, suggesting that the R-forms of target compounds were more beneficial to aggrandize antiviral activity ( Figure 6).

Circular Dichroism Spectroscopic Study
Spectroscopic techniques have gained a wide reputation in the study of protein-ligand interactions [36][37][38]. Hence, in this bioassay, CD spectroscopy was engaged to identify the influence of the pattern of target compounds on the helicase secondary structure (190-260 nm). As seen in Figure 7, the purely helicase CD spectrum (black line) exhibited typical

Circular Dichroism Spectroscopic Study
Spectroscopic techniques have gained a wide reputation in the study of protein-ligand interactions [36][37][38]. Hence, in this bioassay, CD spectroscopy was engaged to identify the influence of the pattern of target compounds on the helicase secondary structure (190-260 nm). As seen in Figure 7, the purely helicase CD spectrum (black line) exhibited typical positive Cotton at 196 nm (β-sheet) and negative Cotton at 208 nm (α-helix) and 217 nm (β-sheet). However, after treated with FA or target compounds, the α-helix peak position of helicase was shifted, such as the FA-treated group (207 nm) and the A 19 -treated group (210 nm). Moreover, the β-sheet of helicase declined or disappeared, especially for the A 19 -or A 31 -treated group. These transitions may illustrate that the designed FA derivative had considerable capacity to bind to helicase and drive its secondary structure transfer.

Interaction Analysis
Helicases represent a class of molecular motors with crucial roles in gene replication, transcription, translation, recombination, and repair. These features make it a potential molecular target for the drug design and mechanism of action studies [13,14]. To determine whether there was an evident interaction between the designed molecule and helicase, R-A19, S-A19, and A31 were selected to interact with the TMV helicase for binding analysis. Besides exploring the influence of different substituents of FA on receptor affinity, it was also observed whether different chiral configurations had a significant influence on the interaction of helicase (Figure 8). The MST test results showed that R-A19 bound to helicase had a Kd value of 5.4 μM and S-A19 bound to helicase had a Kd value of 22.8 μM. Moreover, A31 bound to helicase had a Kd value of 13.5 μM, while Ri had no apparent interaction with the TMV helicase. In contrast, R-A19 possessed the strongest binding ability to helicase. This result demonstrates that the R configuration may be more favorable for entering the active pocket of helicase and firmly interacting with the corresponding residues.

Interaction Analysis
Helicases represent a class of molecular motors with crucial roles in gene replication, transcription, translation, recombination, and repair. These features make it a potential molecular target for the drug design and mechanism of action studies [13,14]. To determine whether there was an evident interaction between the designed molecule and helicase, R-A 19 , S-A 19 , and A 31 were selected to interact with the TMV helicase for binding analysis. Besides exploring the influence of different substituents of FA on receptor affinity, it was also observed whether different chiral configurations had a significant influence on the interaction of helicase (Figure 8). The MST test results showed that R-A 19 bound to helicase had a K d value of 5.4 µM and S-A 19 bound to helicase had a K d value of 22.8 µM. Moreover, A 31 bound to helicase had a K d value of 13.5 µM, while Ri had no apparent interaction with the TMV helicase. In contrast, R-A 19 possessed the strongest binding ability to helicase. This result demonstrates that the R configuration may be more favorable for entering the active pocket of helicase and firmly interacting with the corresponding residues.

Inhibitory Activity of Helicase ATPase
Based on antiviral activity and MST consequences, ATPase activity assays were performed using R-A19, S-A19, and A31 to explore the effect of different levels of antiviral activity and different chiral configurations of the compounds on helicase ATPase activity. As seen in Figure 9, compared with commercial Ri and FA, these target compounds empowered excellent blocking of TMV helicase ATPase activity. Illustrative examples are: R-A19 (50.61%) > A31 (47.03%) > S-A19 (44.50%) > FA (26.35%) > Ri (16.43%) at 200 μM, which suggested that A19 and A31 had enhanced inhibitory effects targeting the TMV helicase, and the R configuration possessed stronger enzyme inhibition competence. The above results indicated that the designed FA-based inhibitors could restrain the ability of the helicase to hydrolyze ATP, thereby potentially interfering with its physiological function during virus replication.

Inhibitory Activity of Helicase ATPase
Based on antiviral activity and MST consequences, ATPase activity assays were performed using R-A 19 , S-A 19 , and A 31 to explore the effect of different levels of antiviral activity and different chiral configurations of the compounds on helicase ATPase activity. As seen in Figure 9, compared with commercial Ri and FA, these target compounds empowered excellent blocking of TMV helicase ATPase activity. Illustrative examples are: R-A 19 (50.61%) > A 31 (47.03%) > S-A 19 (44.50%) > FA (26.35%) > Ri (16.43%) at 200 µM, which suggested that A 19 and A 31 had enhanced inhibitory effects targeting the TMV helicase, and the R configuration possessed stronger enzyme inhibition competence. The above results indicated that the designed FA-based inhibitors could restrain the ability of the helicase to hydrolyze ATP, thereby potentially interfering with its physiological function during virus replication.

Docking Analysis
To profoundly understand the absolute configuration interaction (R-form or S-form) of A19 on the TMV helicase, the receptor-ligand binding simulation was performed using SYBYL-X 2.0. As shown in Figure 10, the interactions of A19 (R/S) and TMV helicase apparently occurred in the binding pocket defined by six residues (Gly10, Lys11, Thr12, Arg40, Leu139, Arg140, and Gly216). R-A19 displayed strong binding to helicase residues, including hydrogen-bond interactions with Gly10, Lys11, Thr12, Arg40, and Gly216 (2.1, 2.7, 2.5, 2.7, and 2.2 Å), polar interactions with Arg140, and hydrophobic interactions with Leu139 ( Figure 10A). Comparably, the hydrogen bond of S-A19 with amino acid residues in the binding pocket was significantly reduced, mainly reflected in the morpholine ring and the methoxy group ( Figure 10B), suggesting that the heteroatom of morpholine, the methoxy group of the benzene ring, and the hydroxyl group of the chiral carbon in R configuration were more conducive to forming hydrogen bonds in space. Moreover, docking results show that the C score value (the total scores of Surflex-Dock represent a -logKd value) of R-A19 with the helicase (6.77) was higher than S-A19 (6.59). Collectively, the results of the molecular docking studies were basically consistent with the MST measurements, which could theoretically illustrate that R-A19 had higher helicase ATPase inhibitory activity compared to S-A19.

Docking Analysis
To profoundly understand the absolute configuration interaction (R-form or S-form) of A 19 on the TMV helicase, the receptor-ligand binding simulation was performed using SYBYL-X 2.0. As shown in Figure 10, the interactions of A 19 (R/S) and TMV helicase apparently occurred in the binding pocket defined by six residues (Gly10, Lys11, Thr12, Arg40, Leu139, Arg140, and Gly216). R-A 19 displayed strong binding to helicase residues, including hydrogen-bond interactions with Gly10, Lys11, Thr12, Arg40, and Gly216 (2.1, 2.7, 2.5, 2.7, and 2.2 Å), polar interactions with Arg140, and hydrophobic interactions with Leu139 ( Figure 10A). Comparably, the hydrogen bond of S-A 19 with amino acid residues in the binding pocket was significantly reduced, mainly reflected in the morpholine ring and the methoxy group ( Figure 10B), suggesting that the heteroatom of morpholine, the methoxy group of the benzene ring, and the hydroxyl group of the chiral carbon in R configuration were more conducive to forming hydrogen bonds in space. Moreover, docking results show that the C score value (the total scores of Surflex-Dock represent a -logK d value) of R-A 19 with the helicase (6.77) was higher than S-A 19 (6.59). Collectively, the results of the molecular docking studies were basically consistent with the MST measurements, which could theoretically illustrate that R-A 19 had higher helicase ATPase inhibitory activity compared to S-A 19 .

Effect of Target Compounds on GFP-Labeled TMV and Relative Expression Levels of Helicase In Vivo
As is vividly depicted in Figure 11, after 7 days, the plants inoculated with 1% DMSO could hardly observe green fluorescence under UV light in either the inoculated leaves or upper uninoculated leaves (Figure 11a). Inversely, when inoculated with TMV-GFP, the

Effect of Target Compounds on GFP-Labeled TMV and Relative Expression Levels of Helicase In Vivo
As is vividly depicted in Figure 11, after 7 days, the plants inoculated with 1% DMSO could hardly observe green fluorescence under UV light in either the inoculated leaves or upper uninoculated leaves (Figure 11a). Inversely, when inoculated with TMV-GFP, the apical fresh leaves of plant showed nearly the entire area of fluorescence, intuitively showing that the virus had completely invaded the top of the tobacco (Figure 11b). Comparatively, the positive control (the Ri-treated group) showed obvious green fluorescence in the upper uninoculated leaves, along with a large-area diffused tendency on the newly top leaves (Figure 11c). However, when the plants were injected with TMV-GFP containing 0.25 mM A 19 (Figure 11d), green fluorescence was still visible but weak in the upper uninoculated leaves, and the distribution range was dramatically smaller than that of the positive control, which indicated that the virus only had a limited amount of expression in the host.

Computational Analysis
To reveal the skeleton structure characters of these family compounds and the potential surface variations resulting from different replacements, A19 and A31 were typically analyzed by DFT calculations with the B3LYP method (Gaussian view 6.0, 6-311G(d) and 6-311G(d, p) basis sets). The corresponding molecular total energy (MTE), the frontier molecular orbital (FMO) energy, the energy gap between the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO), and the additional calculated parameters of A19 and A31 are listed in Table 4. In the light of frontier molecular orbital theory, MTE, HOMO, and LUMO play a predominant role in affecting pesticide physicochemical properties or binding with target receptors. Generally, HOMO could donate electrons, while LUMO could accept electrons, and lower pesticides in the HOMO−LUMO gap (ΔE) could result in higher antiphytopathogenic activity [39,40]. Figure 12 shows that the LUMOs of A19 were primarily located on the FA skeleton, and the HOMOs of A19 were mainly distributed on the benzene ring, the isopropanolamine bridge, and the morpholine ring fragments. Therefore, the total electron transition of A19 was initiated from heterocyclic ring to the FA skeleton through the isopropanolamine bridge, and the total energy was −1207.326 hartree. For A31, the LUMOs were primarily The relative quantitative gene expression of the TMV helicase in Nicotiana tabacum cv. K326 may indirectly shed light on the replication status of the virus in the host. To explore whether title compounds restrain the TMV helicase gene, different antiviral levels of A 19 , A 20 , and A 31 were used in pot experiments (500 µg/mL) and validated by RT-qPCR. Compared with the CK group, the relative expression of the TMV helicase gene in the A 19 -treated group, the A 20 -treated group, or the A 31 -treated group declined to 29%, 41%, or 32% within 3 days, respectively. Moreover, compared with the positive control (78%) or the FA-treated group (83%), these designed FA derivatives displayed an enhanced ability to down-regulate helicase gene relative expression in tobacco (Figure 11e). In addition, to further identify the inhibition of the helicase gene by A 19 , different concentrations of A 19 (100, 250, 500, 750, and 1000 µg/mL) were assessed. Subsequent results show a clear dose-dependent inhibition relationship between A 19 and the helicase gene from 100 to 750 µg/mL (18-84%). However, the relative expression of the TMV helicase gene barely changed when the concentration of A 19 was continuously raised from 750 µg/mL (Figure 11f). These results demonstrated that A 19 was able to repress the expression of the TMV helicase gene, disrupt helicase biosynthesis, and thereby delaying virus replication.

Computational Analysis
To reveal the skeleton structure characters of these family compounds and the potential surface variations resulting from different replacements, A 19 and A 31 were typically analyzed by DFT calculations with the B3LYP method (Gaussian view 6.0, 6-311G(d) and 6-311G(d, p) basis sets). The corresponding molecular total energy (MTE), the frontier molecular orbital (FMO) energy, the energy gap between the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO), and the additional calculated parameters of A 19 and A 31 are listed in Table 4. In the light of frontier molecular orbital theory, MTE, HOMO, and LUMO play a predominant role in affecting pesticide physicochemical properties or binding with target receptors. Generally, HOMO could donate electrons, while LUMO could accept electrons, and lower pesticides in the HOMO−LUMO gap (∆E) could result in higher antiphytopathogenic activity [39,40]. Figure 12 shows that the LUMOs of A 19 were primarily located on the FA skeleton, and the HOMOs of A 19 were mainly distributed on the benzene ring, the isopropanolamine bridge, and the morpholine ring fragments. Therefore, the total electron transition of A 19 was initiated from heterocyclic ring to the FA skeleton through the isopropanolamine bridge, and the total energy was −1207.326 hartree. For A 31 , the LUMOs were primarily located on the FA skeleton, and the HOMOs were mainly concentrated on the FA skeleton, the isopropanolamine bridge, and the piperazine ring. Hence, the total electron transition of A 31 was different from A 19 , and its total energy was lower (−1207.326 hartree). Moreover, A 19 exhibited a lower HOMO−LUMO gap (0.15 hartree) range than A 31 (0.152 hartree). These results suggested that A 19 may have a higher capacity to form ligand-receptor complexes, consistent with the enzyme activity assay described above. Energy calculations and electronic transitions provided a particularly meaningful perspective to distinguish and illustrate the different levels of the antiviral competence of A 19 and A 31 . Molecular lipophilic potential (MLP) and electrostatic potential (ESP) have been wildly applied to identify H-bonding interactions or hydrophobic effects, visualize molecularly charged regions, and predict reactions or ligand-receptor interactions. Therefore, A 19 and A 31 , with different antiviral profile levels, were selected to perform MLP and ESP simulations. As depicted in Figure 13, the positively charged regions (blue) were mainly located in the FA ring and substituted in the heterocycle or phenyl group, while the negatively charged regions (red or yellow) were concentrated on the ester group and the methoxy group of the FA skeleton, the morpholine ring, as well as the hydroxyl group of the isopropanolamine bridge. These extremely charged structural regions may be considered to exhibit a significant role in binding to the target receptor. For A 31 , the domain of the positive charges on the substituent of 1-(2-methylbenzyl)piperazine ring displayed more positive and higher lipophilicity compared with A 19 , which presumably helped to hypothesize why A 19 had a stronger binding capacity than A 19 . These results were in accordance with the molecular docking analysis presented above.  Molecular lipophilic potential (MLP) and electrostatic potential (ESP) have been wildly applied to identify H-bonding interactions or hydrophobic effects, visualize molecularly charged regions, and predict reactions or ligand-receptor interactions. Therefore, A19 and A31, with different antiviral profile levels, were selected to perform MLP and ESP simulations. As depicted in Figure 13, the positively charged regions (blue) were mainly located in the FA ring and substituted in the heterocycle or phenyl group, while the negatively charged regions (red or yellow) were concentrated on the ester group and the methoxy group of the FA skeleton, the morpholine ring, as well as the hydroxyl group of the isopropanolamine bridge. These extremely charged structural regions may be considered to exhibit a significant role in binding to the target receptor. For A31, the domain of the positive charges on the substituent of 1-(2-methylbenzyl)piperazine ring displayed more positive and higher lipophilicity compared with A19, which presumably helped to hypothesize why A19 had a stronger binding capacity than A19. These results were in accordance with the molecular docking analysis presented above.

Antibacterial Activity
To exploit the multiple potentialities of the target molecules, all synthesized derivatives were also evaluated for antibacterial activities, with the commercial agents bismerthiazol (BT) and thiodiazole copper (TC) as positive controls. Bioactivity screening shows (Tables 5-7) that most target compounds had weak antibacterial activity against R. solanacearum, except A1 (70.5% at 100 μg/mL), which demonstrated comparable activity to the control agent TC (40.6% at 100 μg/mL). It is worth mentioning that a great number of target compounds showed moderate to strong antibacterial activity against Xanthomonas oryzae pv oryzae. Among them, A28, A29, and A30 exhibited considerable antibacterial activities to suppress the growth of pathogenic bacteria. In order to further evaluate the antibacterial activity of the target compound against Xoo, we conducted an EC50 test for the compound whose activity exceeded 60% at 50 μg/mL. The results of bioassays indicate that compounds A28 (12.65 μg/mL), A29 (12.38 μg/mL), and A30 (10.87 μg/mL) exhibited the best antibacterial activity compared to those commercial agents, i.e., BT (43.25 μg/mL) and

Instruments and Chemicals
The melting points of the target products were measured using a WRX-4 micro-meltingpoint apparatus (Shanghai Yice Apparatus & Equipment Co., Ltd., Shanghai, China). 1 H and 13 C nuclear magnetic resonance (NMR) spectral analyses were performed on a JEOL-ECX 500 NMR spectrometer using CDCl 3 or DMSO-d6 as the solvent and tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry (HRMS) was conducted on a LTQ Orbitrap (Thermo Scientific, Missouri, USA). The CD spectrum of helicase was performed through a JASCO J-1500 spectropolarimeter from JASCO (Tokyo, Japan) Co., Ltd. Binding studies were performed on a nanotemper monolith NT.115 instrument (Nanotemper, Munich, Germany) for microscale thermophoresis (MST). The ATPase activity experiment used enzyme assay reagent kits purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All analytical reagents were used in the experiment obtained from Energy Chemical (Shanghai Saen Chemical Technology Co., Ltd., Shanghai, China), without further drying or purification.

General Procedures for Preparing Intermediate
Intermediate 1 was prepared using FA (10.0 g, 60 mmol) and methanol (50 mL) into a 150 mL three-necked flask equipped with a thermometer and a condenser, and concentrated sulfuric acid (60 mmol) was slowly added dropwise at room temperature. The temperature was raised to reflux and the reaction was completed after 8 h (indicated by thin-layer chromatography). Next, excess alcohol was removed under reduced pressure and enough saturated sodium bicarbonate solution was added to wash until no gas was generated. Afterwards, 30

Biological Assay
The antiviral activities against tobacco mosaic virus (TMV) and the antibacterial activities against Ralstonia solanacearum (R. s.), Xanthomonas oryzae pv. oryzae (Xoo), and X. axonopodis pv. citri (Xac) of all synthesized target compounds were tested by exerting a half-leaf inoculation approach and the classical turbidimetric method. All the detailed operations follow our group's previously reported methods [44,45]. Measurements were performed in triplicate.

Secondary Structural Analyzation
To identify the effect of title compounds upon target protein secondary structure, the TMV helicase (10.0 µg) was incubated with or without title compounds (5.0 µM) in a buffer solution (20 mM phosphate, 150 mM KCI, pH = 7.4) for 10 min. Subsequently, a JASCO J-1500 spectropolarimeter was used to detect the corresponding treated samples [36].

Binding Analysis between Antiviral Compounds and TMV Helicase
Microscale thermophoresis was performed on a Monolith NR 115 to compute the binding energy between the helicase and the target compound. The compound concentration was isocratically diluted with PBS (pH 7.2) from 2 mM to 0.1 mM in a different tube. Then, 10 µL of the 0.1 mM soluble labeled helicase was added into every diluted compound tube. After that, all tubes were incubated for 5 min and the samples were loaded into a special NanoTemper glass capillary. The LED power was modulated to 40% to perform microthermophoresis. The dissociation constant value (K d ) was calculated from NanoTemper software [37].

TMV Helicase ATPase Activity
To validate the inhibition effect of TMV by target compounds on ATPase activity, a classical phospholipid malachite green assay was implemented by measuring the amount of Pi released in the mixed system. Hence, the commonly used phosphate buffer was replaced by deionized water for this assay. Firstly, the TMV helicase (10 µg) was incubated for 10 min without or with different ligands (20 µM), and centrifuged for 10 min (4000 g). Then, the supernatant (20 µL) was taken into the sample tube, and the phosphorus fixing reagent (200 µL) was added. After mixing and heating in a water bath (40 • C) for 10 min, the absorbance value of solution was recorded at 660 nm [38].

Homologous Modeling of TMV Helicase 3D Structure and Docking
The TMV helicase sequence (V829-T1085) was obtained from the protein NCBI database. Employing a sequence similarity search with BLAST in Protein Data Bank to find the ToMV helicase (PDB code: 3VKW) had a FASTA sequence identity (up to 90%) with the TMV helicase ( Figure S1). Therefore, the TMV helicase homology model was built by SWISS-MODEL based on the crystal structure of the ToMV helicase ( Figures S2-S5). All parameter settings used default values. The receptor protein was prepared by the SYBYL-X 2.0 software biopolymer module, according to the modeled TMV helicase structure. After